WO2025226726A1

WO2025226726A1 - Surface-functionalized microgels and uses thereof for t cell expansion

Info

Publication number: WO2025226726A1
Application number: PCT/US2025/025840
Authority: WO
Inventors: Junzhe Lou; David J. Mooney; Charlotte MEYER; Einat Bleiberg VITNER
Original assignee: Harvard University
Current assignee: Harvard University
Priority date: 2024-04-22
Filing date: 2025-04-22
Publication date: 2025-10-30
Anticipated expiration: 2026-10-22

Abstract

The present disclosure relates, at least in part, to compositions comprising microgels and/or granular hydrogels with tailored surface biochemical properties that can serve, e.g., as artificial antigen-presenting cells (aAPCs) to mediate, e.g., T cell activation and expansion. Such microgels and/or granular hydrogels can be used to manufacture T cells for adoptive therapy in cancer treatment and tissue regeneration, and can be configured to serve, e.g., as a readily tunable and modular system to enable both rapid T cell expansion and control over T cell phenotype. Some aspects of the present disclosure provide methods and compositions for fabricating microgels and/or granular hydrogels, and modulating the surface properties of microgels and/or granular hydrogels by functionalizing, e.g., via layer- by-layer coating.

Description

SURFACE-FUNCTIONALIZED MICROGELS AND USES THEREOF FOR T CELL EXPANSION

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 63/637,308, filed on April 22, 2024, the contents of which are hereby incorporated by reference in their entirety.

GOVERNMENT SUPPORT

This invention was made with government support under 2011754 awarded by National Science Foundation (NSF) and under CA276459 awarded by National Institutes of Health (NIH). The government has certain rights in this invention.

BACKGROUND

Adoptive cell therapy (ACT) of T cells, in which isolated T cells are manipulated and expanded ex vivo before infusing into patients, has proven to be an effective treatment for certain cancers. The activation and expansion of T cells involves signals for T cell receptor (TCR) stimulation and co-stimulation together with growth factors such as interleukin 2 (IL2) to stimulate isolated T cells ex vivo, which in the body are provided by antigen-presenting cells (APCs). Technologies that allow rapid T cell expansion and tune T cell phenotype in a controlled manner provide powerful tools to generate functional therapeutic T cells. (S. A. Rosenberg et al. Curr. Opin. Immunol. 2009, 21 , 233; S. A. Rosenberg et al. Science 2015, 348, 62; A. D. Waldman et al. Nat. Rev. Immunol. 2020, 20, 651 ; J. B. Huppa et al. Nat. Rev. Immunol. 2003, 3, 973; B. L. Levine et al. Mol. Ther. Methods Clin. Dev. 2017, 4, 92).

Biomaterials have served as artificial APCs (aAPCs) by locally providing required stimulatory cues for T cell activation to mimic the endogenous T cell-APC interaction and improve the therapeutic efficacy of ACT. Leveraging the flexible design in various material properties allows biomaterials to modulate T cell proliferation, function, and phenotype. Inorganic, polymeric, liposomal, and lipid-modified particles conjugated with stimulatory ligands for TCR stimulation and costimulation have been explored for T cell activation, and provide various advantages owing to their preparation process and physical properties. The size, morphology, ligand composition, and mobility of aAPCs have profound effects on T cell expansion and phenotype. In addition to particle-based materials, APC mimetic scaffolds assembled from carbon nanotube bundles or lipid coated mesoporous silica rods provide a 3D niche with a large surface area for clustering of ligands and cells, resulting in efficient expansion of T cells. Extracellular matrix-mimetic hydrogels incorporating bioactive ligands are also capable of activating T cells and regulating their functions in a manner dependent on the mechanics of the hydrogel. (Y. Xue et al. Chem. Soc. Rev. 2022, 51 , 1766; A. Isser et al. Biomaterials 2021, 268, 120584; M. Oelke et al. Nat. Med. 2003, 9, 619; K. Perica et al. ACS Nano 2014, 8, 2252; E. R. Steenblock et al. Mol. Ther. 2008, 16, 765; E. R. Steenblock et al. J. Biol. Chem. 2011 , 286, 34883; V. H. Engelhard et al. Proc. Natl. Acad. Sci. U. S. A. 1978, 75, 5688; S. H. Herrmann et al. Proc. Natl. Acad. Sci. U. S. A. 1981 , 78, 2488; Y. Jiang et al. Adv. Mater. 2020, 32, 2001808; M. F. Mescher et al. J. Immunol. 1992, 149, 2402; J. C. Sunshine et al. Biomaterials 2014, 35, 269; J. W. Hickey et al. Nano Lett. 2017, 17, 7045; B. R. Olden et al. Adv. Healthcare Mater. 2019, 8, 1801188; L. J. Eggermont et al. Trends Biotechnol. 2014, 32, 456; T. R. Fadel et al. Nat. Nanotechnol. 2014, 9, 639; A. S. Cheung et al. Nat. Biotechnol. 2018, 36, 160; D. K. Y. Zhang et al. Nat. Commun. 2023, 14, 506; K. AduBerchie et al. Nat. Commun. 2023, 14, 3546; P. Agarwalla et al. Nat. Biotechnol. 2022, 40, 1250; J. W. Hickey et al. Adv. Mater. 2019, 31, 1807359; K. AduBerchie et al. Nat. Biomed. Eng. 2023, 7, 1374; F. S. Majedi et al. Biomaterials 2020, 252, 120058).

However, despite the development of aAPCs, those with flexibly tunable mechanical properties are still underexplored. Considering the importance of various biochemical and physical properties in T cell activation, a biomaterial system with multiple layers of tunability is of interest for research and T cell manufacturing.

SUMMARY

The present disclosure relates, at least in part, to compositions comprising a microgel and/or a granular hydrogel with tailored surface biochemical properties that can serve, e.g., as artificial antigen-presenting cells (aAPCs) to mediate, e.g., T cell activation and expansion. Such microgels and/or granular hydrogels can be used to manufacture immune cells, such as T cells, for adoptive therapy in cancer treatment. In some embodiments, the microgels and/or granular hydrogels can be configured to serve as a readily tunable and modular system, e.g., to enable independent modulation of its physiological properties (e.g., surface concentration, ratio, and distribution of active agents, stiffness, and viscoelasticity) to achieve both spatial and temporal control over cellular behaviors, such as cell phenotype, morphology, spreading, proliferation, differentiation, activation, and expansion. In some embodiments, the compositions comprising a microgel and/or a granular hydrogel can be used to achieve rapid T cell expansion and control over T cell phenotype. In some embodiments, the compositions comprising a microgel and/or a granular hydrogel can be used to present RGD peptides and growth factors on the surface to promoted spreading, proliferation, and differentiation of stem cells, such as mesenchymal stem cells (MSCs).

Some aspects of the present disclosure provide methods and compositions for fabricating and using microgels (e.g., granular hydrogels assembled from microgels) as aAPCs via surface functionalization of microgels using, e.g., layer-by-layer coating. The present disclosure provides experimental data demonstrating, unexpectedly, that sequentially adsorbing oppositely charged polymers formed a thin but dense layer on the surface of the microgel with high stability. In some embodiments, the strategy for surface functionalization of microgels using, e.g., layer-by-layer coating, described herein can be applied to a variety of microgel and coating polymers, and can allow for the introduction of versatile chemistry (e.g., functional groups) for further modification, thus providing a convenient means to modulate microgel surface properties independent of the mechanical properties. As demonstrated herein, efficient conjugation of active agents, such as stimulatory ligands, specifically to the microgel surface promoted, e.g., polyclonal and antigen-specific T cell expansion. The present disclosure provides experimental data demonstrating, unexpectedly, that modulating the concentration, ratio, and distribution of active agents, such as stimulatory ligands, on microgel surfaces as well as the stiffness and/or viscoelasticity of microgels allows control over the expansion, function, and phenotype of cells, e.g., immune cells and/or stem cells.

In one aspect, the present disclosure provides a microgel, comprising (i) a core microgel comprising a crosslinked polymer comprising a functional group selected from the group consisting of a crosslinked tetrazine (Tz) polymer, a crosslinked norbornene (Nb) polymer, and combinations thereof, (ii) a non-covalent polymer coating comprising a positively charged polymer applied to the surface of the core microgel, and (iii) a surface coating comprising a functionalized polymer applied to the surface of the coated core microgel to introduce a functional group for surface-specific conjugation of an active agent. In some embodiments, the core microgel is characterized by a degree of substitution (DS) of about 5 to about 15 functional groups per polymer chain. In some embodiments, the positively charged polymer comprises poly(D-lysine) (PDL). In some embodiments, the core microgel comprises at least one polymer selected from the group consisting of an alginate polymer, a hyaluronic acid (HA) polymer, a collagen polymer, a gelatin polymer, and combinations thereof. In some embodiments, the core microgel comprises a gelatin and alginate-type I collagen interpenetrating network. In some embodiments, the functionalized polymer comprises a click reaction moiety selected from the group consisting of an azide moiety, a dibenzocyclooctyne (DBCO) moiety, a transcyclooctene moiety, a tetrazine (Tz) moiety, a norbornene (Nb) moiety, and variants thereof, optionally wherein the functionalized polymer comprises an azide-modified alginate polymer. In some embodiments, the non- covalent polymer coating and the surface coating are independently characterized by a thickness of about 0.1 pm to about 2 pm, optionally wherein the non-covalent polymer coating and the surface coating are characterized by a combined thickness of about 0.5 pm to about 1.5 pm. In some embodiments, the microgel is characterized by a diameter of about 25 pm to about 250 pm, optionally wherein the microgel is characterized by a diameter of about 50 pm to about 100 pm. In some embodiments, (i) the microgel is characterized by a porosity (e.g., void space %) of about 1% to about 20%; (ii) the microgel is characterized by a zeta potential of about -19.62 mV when uncoated, the microgel is characterized by a zeta potential of about +10.32 when coated with poly(D-lysine) (PDL), and/or the microgel is characterized by a zeta potential of about -13.17 mV when coated with poly(D-lysine) (PDL) and an azide-modified alginate polymer; and/or (iii) the microgel is characterized by an elastic modulus of about 0.5 kPa to about 10 kPa. In some embodiments, the microgel further comprises an active agent comprising a complementary functional group conjugated to the functional group of the surface coating, optionally wherein the active agent comprises a click reaction moiety selected from the group consisting of an azide moiety, a dibenzocyclooctyne (DBCO) moiety, a transcyclooctene moiety, a tetrazine (Tz) moiety, a norbornene (Nb) moiety, and variants thereof, optionally wherein the active agent is modified with an average of about 1 to about 10 click reaction moieties per active agent. In some embodiments, the surface coating comprises an azide-modified polymer to allow surfacespecific conjugation of a dibenzocyclooctyne (DBCO)-modified active agent through strain- promoted azide-alkyne cycloaddition (SPAAC), optionally wherein the active agent is modified with an average of about 3 to about 6 dibenzocyclooctyne (DBCO) moieties per active agent. In some embodiments, the active agent is selected from the group consisting of an antibody or an antigen binding fragment thereof, a peptide, a protein, and combinations thereof. In some embodiments, the active agent is capable of modifying a cellular behavior selected from the group consisting of cell phenotype, morphology, spreading, proliferation, differentiation, activation, expansion and combinations thereof. In some embodiments, the active agent is capable of binding to a T cell surface receptor to promote T cell activation and expansion. In some embodiments, the active agent is selected from the group consisting of a aCD3 antibody, a aCD28 antibody, and combinations thereof. In some embodiments, the active agent comprises a peptide, optionally wherein the active agent comprises a Arg- Gly-Asp peptide (RGD). In some embodiments, the active agent comprises an antigen. In some embodiments, the active agent comprises a major histocompatibility complex (MHC) class I molecule and/or a MHC class II molecule, optionally wherein the active agent comprises a MHC class I molecule and/or a MHC class II molecule presenting a peptide. In some embodiments, the microgel comprises an active agent at a predefined density (e.g., ligand density) of about 3 pg/cm² to about 10 pg/cm². In some embodiments, the surface coating layer of functionalized polymer allows incorporation of an active agent only on the surface to mediate biological functions without introducing functional groups throughout the entire microgel that are not available to cell surface receptors.

In one aspect, the present disclosure provides a composition comprising a microgel described herein and a continuous aqueous phase. In one aspect, the present disclosure provides a granular hydrogel comprising the microgel described herein.

In one aspect, the present disclosure provides a pharmaceutical composition comprising the microgel described herein, and/or the granular hydrogel described herein, and a pharmaceutically acceptable carrier.

In one aspect, the present disclosure provides a method of preparing a microgel, comprising: (i) providing a core microgel comprising a crosslinked polymer comprising a functional group selected from the group consisting of a crosslinked tetrazine (Tz) polymer, a crosslinked norbornene (Nb) polymer, and combinations thereof, optionally wherein the core microgel is characterized by a degree of substitution (DS) of about 5 to about 15 functional groups per polymer chain; (ii) applying a non-covalent polymer coating comprising a positively charged polymer to the surface of the core microgel, optionally wherein the positively charged polymer comprises poly(D-lysine) (PDL); (iii) applying a surface coating comprising a functionalized polymer to the surface of the coated core microgel to introduce a functional group for surface-specific conjugation of an active agent; and (iv) optionally conjugating an active agent comprising a complementary functional group conjugated to the functional group of the surface coating.

In one aspect, the present disclosure provides a method of preparing a granular hydrogel, comprising: (i) providing a composition comprising a plurality of microgels and a continuous aqueous phase; (ii) concentrating the microgels into a pellet via centrifugation; (iii) loading the pellet onto a membrane filter and removing the continuous aqueous phase or a portion thereof via centrifugation, thereby forming a granular hydrogel.

In one aspect, the present disclosure provides a method of activating and expanding a population of T cells, comprising contacting the population of T cells with the microgel and/or the granular hydrogel, as described herein.

In one aspect, the present disclosure provides a method of promoting polyclonal and antigen-specific T cell expansion, comprising contacting the population of T cells with the microgel and/or the granular hydrogel, as described herein.

In one aspect, the present disclosure provides a method of enhancing antigen-specific enrichment of a subpopulation of T cells, comprising contacting the population of T cells with the microgel and/or the granular hydrogel, as described herein.

In one aspect, the present disclosure provides a method of controlling T cell proliferation and T cell phenotype, comprising contacting the population of T cells with the microgel and/or the granular hydrogel, as described herein.

In one aspect, the present disclosure provides a method of regulating the proliferation and differentiation of stem cells, optionally mesenchymal stem cells (MSCs), comprising contacting the population of T cells with the microgel and/or the granular hydrogel, as described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGs. 1A-1E show the fabrication and characterization of microgels and granular hydrogels. FIG. 1A shows a schematic representation of microgel preparation using microfluidic emulsion. Alginate microgels were crosslinked by norbornene-tetrazine click chemistry. FIG. 1B shows a phase-contrast image of alginate microgels crosslinked by norbornene-tetrazine click chemistry. Scale bar: 100 pm. FIG. 1C shows elastic moduli of 2 wt% alginate microgels containing different Nb/Tz ratios as measured by AFM. All the data sets are significantly different ⁽“* P< 0.0001) except the two compared in the figure. FIG. 1D shows a schematic representation of microgel assembly and jamming. Concentrated microgels were loaded on a membrane filter to remove continuous phase by centrifugation, which resulted in jamming of microgels. FIG. 1E shows void space in granular hydrogel calculated from 2D confocal slices as a function of increasing centrifugation time. In FIG. 1C and FIG. 1E, values represent mean ± s.d. an ordinary one-way ANOVA with post hoc Tukey’s multiple comparisons was used. *P< 0.05, **P< 0.01 , ***P< 0.001, *"*P< 0.0001 and NS, not significant.

FIGs. 2A-2J show surface functionalization of microgels. FIG. 2A shows a schematic representation of PDL and subsequent alginate coating on the surface of microgels. FIG. 2B shows confocal images of both PDL and alginate coatings. Scale bar: 100 pm. FIG. 2C shows a confocal image of alginate-Rhodamine B coating on microgel surface at 100x magnification; thickness = 0.74 ± 0.11 pm. Scale bar: 20 pm. FIG. 2D shows change in quantity of alginate coating on microgel surface over 3 weeks in beads buffer. FIG. 2E shows change in quantity of alginate coating on microgel surface over 1 week in T cell culture media. FIG. 2F shows density of alginate polymer coated on microgel surface as a function of alginate concentration in coating solutions. FIG. 2G shows a confocal image of azide-coated microgels labelled with Rhodamine-DBCO. Scale bar: 200 pm. FIG. 2H shows confocal images of alginate-Rhodamine B coated on the surface of microgels formed from hyaluronic acid, gelatin and an alginate/collagen interpenetrating network. Scale bar: 100 pm. FIG. 2I shows confocal images of alginate microgel presenting tetrazine functional groups coated with alginate-sulfoCy5. Microgel core in red, free tetrazine in green and polymer coating in blue. Scale bar: 20 pm. FIG. 2J shows quantification of fluorescent intensity of Rhodamine B (red), FITC (green) and sulfoCy5 (blue) as a function of distance from microgel surface. In FIG. 2D-FIG. 2F, values represent mean ± s.d. an ordinary one- way ANOVA with post hoc Tukey’s multiple comparisons was used. *P< 0.05, **P< 0.01, ***P< 0.001 and ****P< 0.0001.

FIGs. 3A-3J show polyclonal and antigen-specific activation of primary mouse T cells. FIG. 3A shows a schematic representation of modification of aCD3 and aCD28 antibodies on microgel surface by coating microgels with azide-modified alginate and conjugating antibodies using azide-DBCO click chemistry. FIG. 3B shows UV-vis absorption spectra of unmodified aCD3, DBCO-modified aCD3 and DBCO model compounds. FIG. 3C shows carboxifluorescein diacetate succinimidyl ester (CFSE) histogram evaluated using FACS flow cytometry indicating the proliferation profile of stimulated CD4+ T cells. FIG. 3D shows percentage of proliferating CD4+ T cells when cultured with Dynabeads or microgels of different formulations. FIG. 3E shows representative phase contrast images of primary mouse CD4+ T cells cultured with blank microgels, microgels conjugated with anti CD3/CD28 over the entire microgel and microgels functionalized with anti CD3/CD28 on the surface as shown by representative images using phase contrast. Scale bar: 100 pm. FIG. 3F shows a schematic representation of modification of MHC-I and aCD28 on microgel surface. Microgels were first coated with biotin-modified alginate, reacted with streptavidin and then conjugated with ligands using biotin-streptavidin interaction. FIG. 3G shows carboxifluorescein diacetate succinimidyl ester (CFSE) histogram evaluated using FACS flow cytometry indicating the proliferation profile of stimulated antigen-specific CD8+ T cells. FIG. 3H shows representative plots and FIG. 3I shows quantification showing enrichment of live CD8+ cells specific for SIINFEKL peptides when mixed CD8+ T cells were cultured with Dynabeads or MHC-I /antigen functionalized microgels. FIG. 3J shows fold expansion of CD8+ T cells specific for SIIFEKL peptide cultured with Dynabeads or MHC-I functionalized microgels. In FIG. 3D, FIG. 3I and FIG. 3J, values represent mean ± s.d. an ordinary oneway ANOVA with post hoc T ukey’s multiple comparisons was used. *P < 0.05, **P < 0.01 , ***P < 0.001, **** P < 0.0001 and NS, not significant.

FIGs. 4A-4M show polyclonal mouse T cell expansion (CD4+ and CD8+ co-culture) by varying biochemical properties of microgels. FIG. 4A shows expansion of primary mouse T cells (inlcuding CD4+ and CD8+ T cells), FIG. 4B shows CD4/CD8 ratio of CD4+ and CD8+ single-positive cells, FIG. 4C shows CD44 and CD62L expression by live CD4+ (left) or CD8+ (right) T cells that were co-cultured with microgels as a function of overall surface antibody density or Dynabeads on Day 3. aCD3/ aCD28 ratio = 1, CD4/CD8 ratio = 1 on Day 0. FIG. 4D shows quantification of in vitro killing of OVA-expressing B16-F10 target cells by CD8+ OT-I T cells that were co-cultured with microgels as a function of overall surface antibody density. Effector/target cell ratios = 10:1. FIG. 4E shows expansion of primary mouse T cells (inlcuding CD4+ and CD8+ T cells), FIG. 4F shows CD4/CD8 ratio of CD4+ and CD8+ single-positive cells, FIG. 4G shows CD44 and CD62L expression by live CD4+ (left) or CD8+ (right) T cells that were co-cultured with microgels as a function of aCD3/ aCD28 ratio on Day 3. Overall antibody density = 0.4 pg/cm2, CD4/CD8 ratio = 1 on Day 0. FIG. 4H shows a schematic representation of a single type microgel coated with antibodies (medium purple, left) and a mixture of microgels coated with antibodies (dark purple, right) and without antibodies (light purple, right). FIG. 4I shows expansion of primary mouse T cells, FIG. 4J shows CD4/CD8 ratio of CD4+ and CD8+ single-positive cells that were cocultured with a single type microgel or mixed microgels at the same overall surface antibody density on Day 3. aCD3/ aCD28 ratio = 1, CD4/CD8 ratio = 1 on Day 0. FIG. 4K shows a schematic representation of modification of aCD3 and aCD28 antibodies and IL-2 on microgel surface. FIG. 4L shows expansion of primary mouse T cells, FIG. 4M shows CD4/CD8 ratio of CD4+ and CD8+ single-positive cells that were co-cultured with microgels as a function of IL-2 density on Day 3. Overall antibody density = 0.8 pg/cm2, aCD3/ aCD28 ratio = 1, CD4/CD8 ratio = 1 on Day 0. In FIG. 4D- FIG. 4F, FIG. 4I, FIG. 4J, FIG. 4L and FIG. 4M, values represent mean ± s.d. an ordinary one-way ANOVA with post hoc Tukey’s multiple comparisons was used. *P < 0.05, **P < 0.01 , ***P < 0.001 , **** P < 0.0001 and NS, not significant.

FIGs. 5A-5D show polyclonal mouse T cell expansion (CD4+ and CD8+ co-culture) while varying the physical properties of microgels. FIG. 5A shows expansion of primary mouse T cells and FIG. 5Bshows CD44/CD62L expression by live CD4+ (left) or CD8+ (right) T cells that were co-cultured with microgels as a function of stiffness for 3 days. FIG. 5C shows expansion of primary mouse T cells and FIG. 5Dshows CD44/CD62L expression by live CD4+ (left) or CD8+ (right) T cells that were co-cultured with elastic or viscoelastic microgels for 3 days. Overall antibody density = 0.4 pg/cm², aCD3/ aCD28 ratio = 1 in all studies.

FIGs. 6A-6E show polyclonal human T cell expansion (mixture of CD4+ and CD8+) by varying biochemical properties of microgels. FIG. 6A shows expansion of primary human T cells that were co-cultured with microgels as a function of overall surface antibody density on Day 6. FIG. 6B shows CD25 expression by live CD4+ (left) or CD8+ (right) T cells that were co-cultured with microgels as a function of overall surface antibody density on Day 6. FIG. 6C shows CD4/CD8 ratio of cells cultured with microgels as a function of overall surface antibody density on Day 6. FIG. 6D shows CD45RA and CCR7 expression, FIG. 6E shows CD39 expression by live CD4+ (left) or CD8+ (right) T cells that were cultured with microgels as a function of overall surface antibody density on Day 6.

FIG. 7 shows ¹H NMR spectrum (D2O containing 0.02 wt% potassium hydrogen phthalate) of alginate modified with norbornene (Alg-Nb).

FIG. 8 shows ¹H NMR spectrum (D2O containing 0.02 wt% potassium hydrogen phthalate) of alginate modified with tetrazine (Alg-Tz). FIG. 9 shows 3D reconstructions of the porous network in granular hydrogel using FITC-dextran (2 MDa) and confocal imaging. Scale bar: 200 pm.

FIG. 10 shows individual 2D confocal slice imaging FITC dextran between microgels was thresholded and processed by Imaged to calculate the porosity. Scale bar: 100 pm.

FIG. 11 shows void space in granular hydrogel as a function of the stiffness of microgels used to assemble the bulk gel.

FIG. 12 shows confocal 3D reconstructions of Pan T cells (green by Calcein staining) 2 days after seeding on granular hydrogels composed of microgels coated with alginate RGD.

FIG. 13 shows confocal image of alginate-Rhodamine B coating on microgel surface at 100x magnification. Scale bar: 20 pm.

FIG. 14 shows phase-contrast images of alginate microgels before and after polymer coating. The mean size of microgels slightly decreased from 77 to 72 pm after coating process. Scale bar: 200 pm.

FIG. 15 shows Confocal image of alginate-Rhodamine B coating on microgel surface after 10 months at 4°C in beads buffer. Magnification: 100x; thickness = 0.91 ± 0.22 pm; scale bar: 20 pm.

FIG. 16 shows confocal image of alginate-FITC-coated microgels with varying DS of FITC on the alginate. Scale bar: 100 pm.

FIG. 17 shows confocal images of alginate-Rhodamine B coating on microgel surface at 100x magnification using coating solutions of different alginate concentrations. Scale bar: 20 pm.

FIG. 18 shows confocal image of HA-FITC coating on the surface of alginate microgels surface. Scale bar: 100 pm.

FIG. 19 shows erosion kinetics of granular hydrogels after 21 days. Non-click granular hydrogels were formed from microgels not capable of crosslinking, composing of microgels without excess Nb or Tz (Nb/Tz = 1/1), with polymer coating. Click granular hydrogels were formed from microgels capable of interparticle crosslinking, composing of microgels with excess Nb (Nb/Tz = 2/1) and excess Tz (Nb/Tz = 1/2) at 1:1 ratio, with and without coating.

FIG. 20 shows porosity (Void space %) in granular hydrogels at time of formation (Day 0, black) and after Day 3 (grey). Microgels used to form granular hydrogels were either in the absence of (green border) or in the presence of (orange border) alginate polymer coating.

FIG. 21 shows fluorescent image of PDL coating (MW 1-5 kDa). Scale bar: 50 pm.

FIG. 22 shows standard calibration curve of DBCO-PEG12-maleimide in 1X PBS buffer at pH 7.4 at 280 nm and 310 nm. Figure 23A shows quantification of fluorescent intensity of fluorescently labelled antibodies on the surface of microgels using different conjugation strategies. Confocal image of 23B shows alginate-azide coated microgels (surface specific) and FIG. 23C shows alginate microgel presenting tetrazine with Rhodamine-B labelled antibodies (bulk modification throughout entire microgels) at 100x magnification. Scale bar: 10 pm.

FIG. 24 shows confocal image of biotin-coated microgels labelled with streptavidin- FITC. Scale bar: 100 pm.

FIG. 25 shows carboxifluorescein diacetate succinimidyl ester (CFSE) histogram evaluated using FACS flow cytometry indicating the proliferation profile of stimulated antigen-specific CD8+ T cells using in a separate experiment.

FIG. 26 shows percentage of proliferating antigen-specific CD8+ mouse T cells when cultured with Dynabeads or microgels modified with MHC-I.

FIG. 27 shows quantification showing enrichment of live CD8+ cells specific for SIINFEKL from unvaccinated or vaccinated wild type mice when isolated CD8+ T cells were cultured with Dynabeads or MHC-l/antigen functionalized microgels.

FIG. 28 shows representative plots showing enrichment of live CD8+ cells from unvaccinated or vaccinated wild type mice specific for SIINFEKL peptides when mixed CD8+ T cells were cultured with Dynabeads or MHC-l/antigen functionalized microgels.

FIG. 29 shows expansion of primary mouse CD4+ (left) and CD8+ (right) T cells when CD4+ and CD8+ T cells were co-cultured with microgels as a function of overall surface antibody density or Dynabeads on Day 3. aCD3/ aCD28 ratio = 1, CD4/CD8 ratio = 1 on Day 0.

FIG. 30 shows CD25 and OX-40 expression by live CD4+ (left) or CD8+ (right) mouse T cells that were cultured with microgels as a function of overall surface antibody density or Dynabeads on Day 3.

FIG. 31 shows quantification of in vitro killing of B16-F10 cells not expressing OVA by CD8+ OT-I T cells that were co-cultured with microgels as a function of overall surface antibody density. Effector/target cell ratios = 10:1.

FIG. 32 shows expansion of primary mouse CD4+ (left) and CD8+ (right) T cells when CD4+ and CD8+ T cells were co-cultured with microgels, as a function of aCD3/ aCD28 ratio, on Day 3. Overall antibody density = 0.4 pg/cm², CD4/CD8 ratio = 1 on Day 0.

FIG. 33 shows CD25 and OX-40 expression by live CD4+ (left) or CD8+ (right) mouse T cells that were cultured with microgels as a function of aCD3/ aCD28 ratio on Day 3.

FIG. 34 shows CD25 and OX-40 expression by live CD4+ (left) or CD8+ (right) mouse T cells that were cultured with a single type microgel or mixed microgels at the same overall surface antibody density on Day 3. FIG. 35 shows CD44 and CD62L expression by live CD4+ (left) or CD8+ (right) mouse T cells that were cultured with a single type microgel or mixed microgels at the same overall surface antibody density on Day 3.

FIG. 36 shows CD25 and OX-40 expression by live CD4+ (left) or CD8+ (right) mouse T cells that were co-cultured with microgels as a function of IL-2 density on Day 3.

FIG. 37 shows CD44 and CD62L expression by live CD4+ (left) or CD8+ (right) mouse T cells that were co-cultured with microgels as a function of IL-2 density on Day 3.

FIG. 38 shows FOXP3 and CD25 expression by live CD4+ mouse T cells that were co-cultured with microgels as a function of IL-2 microgel surface density on Day 3.

FIG. 39 shows expansion of primary mouse T cells that were cultured with microgels in different culture conditions on Day 3. Co-culture indicates both CD4+ and CD8+ T cells were present in the culture. Single population, only CD4+ or only CD8+ cells were present in the culture. Overall antibody density = 0.4 pg/cm², aCD3/ aCD28 ratio = 1.

FIG. 40 shows CD44 and CD62L expression by live CD4+ or CD8+ mouse T cells that were cultured with microgels in different culture conditions on Day 3. Co-culture, both CD4+ and CD8+ T cells. Single population, only CD4+ or only CD8+. Overall antibody density = 0.4 pg/cm², aCD3/ aCD28 ratio = 1.

FIG. 41 shows CD25 and OX-40 expression by live CD4+ or CD8+ mouse T cells that were cultured with microgels in different culture conditions on Day 3. Co-culture, both CD4+ and CD8+ T cells. Single population, only CD4+ or only CD8+. Overall antibody density = 0.4 pg/cm², aCD3/ aCD28 ratio = 1.

FIG. 42 shows I FNy, TNFa and IL-2 expression by CD8+ mouse T cells that were cultured with microgels in different culture conditions on Day 3. Co-culture, both CD4+ and CD8+ T cells. Single population, only CD4+ or only CD8+. Overall antibody density = 0.4 pg/cm², aCD3/ aCD28 ratio = 1.

FIG. 43 shows CD25 and OX-40 expression by live CD4+ (left) or CD8+ (right) mouse T cells that were co-cultured with microgels as a function of stiffness on Day 3.

FIG. 44 shows CD4/CD8 ratio of CD4+ and CD8+ single-positive cells that were co- cultured with microgels as a function of stiffness on Day 3. CD4/CD8 ratio = 1 on Day 0.

FIG. 45 shows phase-contrast image of alginate microgels crosslinked by calcium ions. Scale bar: 200 pm

FIG. 46 shows elastic moduli of elastic and viscoelastic alginate microgels.

FIG. 47 shows CD25 and OX-40 expression by live CD4+ (left) or CD8+ (right) mouse T cells that were co-cultured with elastic or viscoelastic microgels on Day 3.

FIG. 48 shows CD4/CD8 ratio of CD4+ and CD8+ single-positive cells that were co- cultured with elastic or viscoelastic microgels on Day 3. CD4/CD8 ratio = 1 on Day 0. FIG. 49 shows phase-contrast image of alginate microgels crosslinked by norbornene-tetrazine click chemistry. Scale bar: 100 pm

FIG. 50 shows expansion of primary mouse T cells that were co-cultured with microgels as a function of size on Day 3. Overall antibody density = 0.4 pg/cm², aCD3/ aCD28 ratio = 1.

FIG. 51 shows CD4/CD8 ratio of CD4+ and CD8+ single-positive cells that were cocultured with microgels as a function of size on Day 3. CD4/CD8 ratio = 1 on Day 0.

FIG. 52 shows CD44/CD62L expression by live CD4+ (left) or CD8+ (right) T cells that were co-cultured with microgels as a function of size on Day 3.

FIG. 53 shows CD25 and OX-40 expression by live CD4+ (left) or CD8+ (right) mouse T cells that were co-cultured with microgels as a function of size on Day 3.

FIG. 54 shows expansion of primary mouse T cells that were cultured on antibody- modified tissue culture plate as a function of overall surface antibody density on Day 3. aCD3/ aCD28 ratio = 1.

FIG. 55 shows CD4/CD8 ratio of CD4+ and CD8+ single-positive cells that were cultured on antibody-modified tissue culture plate as a function of overall surface antibody density on Day 3. aCD3/ aCD28 ratio = 1, CD4/CD8 ratio = 1 on Day 0.

FIG. 56 shows CD44 and CD62L expression by live CD4+ (left) or CD8+ (right) T cells that were cultured on antibody-modified tissue culture plate as a function of overall surface antibody density on Day 3.

FIG. 57 shows CD25 and 0X40 expression by live CD4+ (left) or CD8+ (right) T cells that were cultured on antibody-modified tissue culture plate as a function of overall surface antibody density on Day 3.

FIG. 58 shows CD45RA and CD62L expression by live CD4+ (left) or CD8+ (right) human T cells (from Donor #1) that were co-cultured with microgels as a function of overall surface antibody density on Day 6.

FIG. 59 shows PD-1 and Lag-3 expression by live CD4+ (left) or CD8+ (right) human T cells (from Donor #1) that were cultured with microgels as a function of overall surface antibody density on Day 6.

FIG. 60 shows expansion of primary human T cells (from Donor #2) that were co- cultured with microgels as a function of overall surface antibody density on Day 6.

FIG. 61 shows CD4/CD8 ratio of CD4+ and CD8+ single-positive human cells (from Donor #2) that were co-cultured with microgels as a function of overall surface antibody density on Day 6.

FIG. 62 shows CD25 expression by live CD4+ (left) or CD8+ (right) human T cells (from Donor #2) that were co-cultured with microgels as a function of overall surface antibody density on Day 6. FIG. 63 shows CD45RA and CCR7 expression by live CD4+ (left) or CD8+ (right) human T cells (from Donor #2) that were co-cultured with microgels as a function of overall surface antibody density on Day 6.

FIG. 64 shows CD45RA and CD62L expression by live CD4+ (left) or CD8+ (right) human T cells (from Donor #2) that were co-cultured with microgels as a function of overall surface antibody density on Day 6.

FIG. 65 shows CD39 expression by live CD4+ (left) or CD8+ (right) human T cells (from Donor #2) that were co-cultured with microgels as a function of overall surface antibody density on Day 6.

FIG. 66 shows PD-1 and Lag-3 expression by live CD4+ (left) or CD8+ (right) human T cells (from Donor #2) that were co-cultured with microgels as a function of overall surface antibody density on Day 6.

FIG. 67 shows confocal images of alginate coatings and FIG. S61B shows quantification of remaining alginate coatings after injection through 27G and 30G needle. Scale bar: 200 pm.

FIGs. 68A-68G show fabrication and characterization of microgels and granular hydrogels. FIG. 68A shows schematic representation of microgel preparation using microfluidic emulsion. Alginate microgels were crosslinked by norbornene-tetrazine click chemistry. FIG. 68B shows phase-contrast image of alginate microgels crosslinked by norbornene-tetrazine click chemistry. Scale bar: 100 pm. FIG. 68C shows elastic moduli of 2 wt% alginate microgels containing different Nb/Tz ratios as measured by AFM. All the data sets are significantly different ⁽“* P< 0.0001) except the two compared in the figure. FIG. 68D shows schematic representation of microgel assembly and jamming. Concentrated microgels were loaded on a membrane filter to remove continuous phase by centrifugation, which resulted in jamming of microgels. FIG. 68E shows 3D reconstructions of the porous network in granular hydrogel using FITC-dextran (2 MDa) and confocal imaging. Scale bar: 200 pm. FIG. 68F shows void space in granular hydrogel calculated from 2D confocal slices as a function of increasing centrifugation time. FIG. 68G shows void space in granular hydrogel as a function of the stiffness of microgels used to assemble the bulk gel. In FIG. 68C, FIG. 68F and FIG. 68G, values represent mean ± s.d. an ordinary one-way ANOVA with post hoc Tukey’s multiple comparisons was used. *P< 0.05, **P< 0.01 , ***P< 0.001, **** P< 0.0001 and NS, not significant.

FIGs. 69A-69E show surface functionalization of microgels. FIG. 69A shows schematic representation of PDL and subsequent alginate coating on the surface of microgels. FIG. 69B shows confocal images of both PDL and alginate coatings. Scale bar: 100 pm. FIG. 69C shows confocal image of alginate-Rhodamine B coating on microgel surface at 100x magnification; thickness = 0.77 pm. Scale bar: 20 pm. FIG. 69D shows density of alginate polymer coated on microgel surface as a function of alginate concentration in coating solutions. FIG. 69E shows Change in quantity of alginate coating on microgel surface over time as microgels are maintained in suspension. FIG. 69F shows confocal images of alginate-Rhodamine B coated on the surface of microgels formed from hyaluronic acid, gelatin and an alginate/collagen interpenetrating network. Scale bar: 100 pm. FIG. 69G shows confocal images of microgels presenting tetrazine (Tz) after mixing with FITC-TCO for 1 min. Red color indicates the microgel fabricated from alginate labelled with Rhodamine B and green color shows homogeneous distribution of FITC across the microgel. Scale bar: 200 pm. In FIG. 69D and FIG. 69E, values represent mean ± s.d. an ordinary one-way ANOVA with post hoc Tukey’s multiple comparisons was used. *P< 0.05, **P< 0.01, ***P< 0.001 and ****P< 0.0001.

FIGs. 70A-70D show influence of polymer coating on interparticle crosslinking. FIG. 70A shows erosion kinetics of granular hydrogels measured over 21 days. Granular hydrogels were formed either from microgels not capable of crosslinking (non-click) with polymer coating or from microgels capable of interparticle crosslinking (click) with and without coating. FIG. 70B shows porosity (Void space %) in granular hydrogels as a function of centrifugation time, at time of formation (Day 0, dark grey) and after Day 3 (light grey). Microgels used to form granular hydrogels were either absence of (green border) or in the presence of (orange border) alginate polymer coating. FIG. 70C shows confocal images of alginate microgel presenting tetrazine functional groups coated with alginate-sulfoCy5. Microgel core in red, free tetrazine in green and polymer coating in blue. Scale bar: 20 pm. FIG. 70D shows quantification of fluorescent intensity of Rhodamine B (red), FITC (green) and sulfoCy5 (blue) as a function of distance from microgel surface. In FIG. 70B, values represent mean ± s.d. an ordinary one-way ANOVA with post hoc Tukey’s multiple comparisons was used. *P< 0.05, **P< 0.01, ***P< 0.001, ****P< 0.0001 and NS, not significant.

FIGs. 71A-71H show regulation of MSC behaviors. FIG. 71 A shows representative examples of DAPI and phalloidin staining of D1 MSCs growing in granular hydrogels composed of microgels coated with alginate no RGD (DS0), alginate with DS2 and 20. DAPI in blue and phalloidin in green. Scale bar: 50 pm. Quantification of FIG. 71 B shows cell area and FIG. 71C shows circularity in granular hydrogels composed of microgels coated with alginate RGD DS0, 2 and 20. FIG. 71 D shows quantification of Edll positive cells in granular hydrogels composed of microgels coated with alginate RGD DS0, 2 and 20. FIG. 71 E shows representative examples of DAPI and phalloidin staining of D1 MSCs cultured in granular hydrogels composed of microgels coated with alginate RGD DS20 (red) and those coated with alginate with no RGD (yellow ring). DAPI in blue and phalloidin in green. Scale bar: 50 pm. FIG. 71F shows quantification of D1 MSCs in contact with microgels coated with RGD or without RGD in granular hydrogels. FIG. 71G shows representative images of alkaline phosphatase (ALP) staining of D1 MSCs growing in granular hydrogels composed of microgels coated with and without BMP-2. ALP in blue. Scale bar: 200 pm. FIG. 71 H shows quantification of ALP positive cells. In FIG. 71B-FIG. 71 D, FIG. 71 F and FIG. 71 H, values represent mean ± s.d. an ordinary one-way ANOVA with post hoc Tukey’s multiple comparisons was used. *P< 0.05, **P< 0.01, ***P< 0.001 and ****P< 0.0001.

FIG. 72A-72E show regulation of T cell behaviors. FIG. 72A shows representative phase contrast images of primary mouse CD4+ T cells cultured with granular hydrogels composed of blank microgels, microgels conjugated with anti CD3/CD28 throughout the entire microgel, and microgels functionalized with anti CD3/CD28 on the surface. Scale bar: 100 pm. FIG. 72B shows CellTrace yellow histogram evaluated using flow cytometry indicating the proliferation profile of stimulated CD4+ T cells. CD4+ T cells were cocultured with Dynabeads or granular hydrogels composed of blank microgels, microgels conjugated with anti CD3/CD28 throughout the entire microgel, coated microgels without antibody on the surface and microgels functionalized with anti CD3/CD28 on the surface. FIG. 72C shows percentage of proliferating CD4+ T cells when cultured with Dynabeads or granular hydrogels of different formulations. FIG. 72D shows expansion of CD4+ and CD8+ T cells that were co-cultured with Dynabeads or granular hydrogels. FIG. 72E shows CD4/CD8 ratio of co-cultures with Dynabeads or surface functionalized granular hydrogels. In FIG. 72C- FIG. 72E, values represent mean ± s.d. an ordinary one-way ANOVA with post hoc Tukey’s multiple comparisons was used. *P< 0.05, **P< 0.01 , ***P< 0.001, ****P< 0.0001 and NS, not significant.

DETAILED DESCRIPTION Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains.

As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the content clearly dictates otherwise.

The articles “a” and “an” are used herein to refer to one or to more than one (/.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element, e.g., a plurality of elements.

The use of the alternative (e.g., “or”) should be understood to mean either one, both, or any combination thereof of the alternatives. The term “or” is used herein to mean, and is used interchangeably with, the term “and/or,” unless context clearly indicates otherwise. As used herein, the term “about,” when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20%, or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

As used herein, any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated.

As used herein, “comprise,” “comprising,” “comprises,” and “comprised of” are meant to be synonymous with “include,” “including,” “includes,” or “contain,” “containing,” “contains” and are inclusive or open-ended terms that specifies the presence of what follows, e.g., component and do not exclude or preclude the presence of additional, non-recited components, features, element, members, steps, known in the art or disclosed therein. By way of example, the term “including” is used herein to mean, and is used interchangeably with, the phrase “including but not limited to.”

As used herein, the terms “such as,” “for example,” and the like are intended to refer to exemplary embodiments and not to limit the scope of the present disclosure.

As used herein, the term “contacting” includes the physical contact of at least one substance to another substance, either directly or indirectly.

As used herein, the term “sufficient amount” and “sufficient time” includes an amount and time needed to achieve the desired result or results.

As used herein the terms “preventing” or “prevention” refer to a reduction in risk of acquiring a disease or disorder (/.e., causing at least one of the clinical symptoms of the disease not to develop in a patient that may be exposed to or predisposed to the disease but does not yet experience or display symptoms of the disease).

As used herein, the terms “treating” or “treatment” refer to a beneficial or desired result including, but not limited to, alleviation or amelioration of one or more symptoms, diminishing the extent of infection, stabilized (/.e., not worsening) state of infection, amelioration or palliation of the infectious state, whether detectable or undetectable. "Treatment" can also mean prolonging survival as compared to expected survival in the absence of treatment.

As used herein, the term “prophylactically effective amount,” is intended to include the amount of an active agent that, when administered to a subject who does not yet experience or display symptoms of a condition, disease, and/or disorder, but who may be predisposed to the condition, disease, and/or disorder, is sufficient to prevent or ameliorate the condition, disease, and/or disorder or one or more symptoms of the condition, disease, and/or disorder. Ameliorating the condition, disease, and/or disorder includes slowing the course of the condition, disease, and/or disorder or reducing the severity of later-developing condition, disease, and/or disorder. The “prophylactically effective amount” may vary depending on the active agent, how the active agent is administered, the degree of risk of condition, disease, and/or disorder, and the history, age, weight, family history, genetic makeup, the types of preceding or concomitant treatments, if any, and other individual characteristics of the patient to be treated.

A “therapeutically effective amount” or “prophylactically effective amount” also includes an amount of an active agent that produces some desired local or systemic effect at a reasonable benefit/risk ratio applicable to any treatment. Active agents employed in the methods of the present disclosure may be administered in a sufficient amount to produce a reasonable benefit/risk ratio applicable to such treatment.

As used herein, the term “administering” to a subject includes dispensing, delivering or applying a composition as described herein to a subject by any suitable route for delivery of the composition to the subject, including delivery by injection. Exemplary modes of administration include, but are not limited to, injection, infusion, instillation, inhalation, or ingestion. “Injection” includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal, intracerebro spinal, and intrasternal injection and infusion. In preferred embodiments, the compositions are administered by injection, e.g., subcutaneous injection.

As used herein, the term “immune cells” generally refer to resting and/or activated cells of the immune system involved in defending a subject against both infectious disease and foreign materials. Examples of immune cells include, without limitations, white blood cells including, e.g., neutrophils, eosinophils, basophils, lymphocytes e.g., B-cells, T-cells, and natural killer cells), monocytes, macrophages (including, e.g., resident macrophages, resting macrophages, and activated macrophages); as well as Kupffer cells, histiocytes, dendritic cells, Langerhans cells, mast cells, microglia, and any combinations thereof. In some embodiment, immune cells include derived immune cells, for example, immune cells derived from lymphoid stem cells and/or myeloid stem cells. In some embodiment, immune cells include white blood cells (leukocytes) which are derived from hematopoietic stem cells (HSC) and/or hematopoietic progenitor cells (HPC). In some embodiment, immune cells include hematopoietic stem cells (HSC) and/or hematopoietic progenitor cells (HPC). In some embodiment, immune cells include lymphocytes (T cells, B cells, natural killer (NK) cells) and/or myeloid-derived cells (neutrophil, eosinophil, basophil, monocyte, macrophage, dendritic cells). In some embodiments, the immune cell is a leukocyte, such as a myeloid cell or a lymphoid cell. Exemplary myeloid cells (also referred to as “myelocytes”) include, without limitation, neutrophils, eosinophils, mast cells, basophils, and monocytes (e.g., dendritic cells and macrophages). Exemplary lymphoid cells (also referred to as “lymphocytes”) include, without limitation, T cells (e.g., helper CD4+ T cells, cytotoxic CD8+ T cells, memory T cell, regulatory CD4+ T cells, innate-like T cells, natural killer T (NKT) cells, mucosal associated invariant T (MAIT) cells, and gamma delta (yb) T cells), B cells (e.g., plasmablasts, plasma cells, lymphoplasmacytoid cells, memory B cells, follicular (FO) B cells (also known as “B-2 cells”), marginal-zone (MZ) B cells, B-1 cells, and regulatory B (Breg) cells), and natural killer (NK) cells.

In some embodiments, the cell is a T cell (e.g., a helper CD4+ T cell, a cytotoxic CD8+ T cell, a memory T cell, a regulatory CD4+ T cell, an innate-like T cell, a natural killer T (NKT) cell, a mucosal associated invariant T (MAIT) cell, and/or a gamma delta (yb) T cell).

In a some embodiments, the immune cell comprises an autologous immune cell or an allogeneic immune cell. In a some embodiments, the immune cell comprises a phagocyte, a macrophage, a dendritic cell, a monocyte, a B cell, a T cell, a natural killer cell (NK) cell, and combinations thereof. In a some embodiments, the immune cell is a T cell. In a some embodiments, the T cell is an autologous T cell or an allogeneic T cell. In a some embodiments, the T cell is a pan T cell or a T cell with a shared TCR clonotype. In a some embodiments, the T cell is selected from the group consisting of a naive T cell, a helper T cell, a cytotoxic T cell, a memory T cell, a regulatory T cell, an innate-like T cell, a natural killer T cell, a mucosal associated invariant T cell, a gamma delta T cell, and combinations thereof. In a some embodiments, the T cell is a genetically modified T cell, optionally wherein the genetically modified T cell is a T cell receptor (TCR) T cell or a chimeric antigen receptor (CAR) T cell.

As used herein, the term “T cell” refers to all types of immune cells expressing CD3 including, without limitation, T-helper cells (CD4+ cells), cytotoxic T-cells (CD8+ cells), T- regulatory cells (Treg), and gamma-delta T cells. As used herein, the term “cytotoxic cell” refer, without limitation, to cells capable of mediating cytotoxicity responses, such as CD8+ T cells, natural-killer (NK) cells, and neutrophils.

In some embodiments, provided herein is a surface-specific functionalization strategy that enables precise and efficient engineering of the concentrations and types of active agents, e.g., antibodies, presented on the surface of microgels and/or granular hydrogels, thus allowing for the expansion and phenotypic change of cells, such as T cells, in response to different presentation of cues. In some embodiments, the phenotypic change of cells, e.g., T cells, in response to different presentation of cues may be determined by using flow cytometry analysis.

In some embodiments, the compositions and methods provided herein can be used to produce a T cell characterized by a specific T cell phenotype. In some embodiments, the T cell may be characterized by an activation/inhibitory T cell phenotype, optionally wherein the activation/inhibitory T cell phenotype is based on a marker selected from the group consisting of PD1 , TIGIT, TIM3, LAG3, 0X40, CD39, CD25, CTLA4, and combinations thereof. In some embodiments, the T cell may be characterized by a memory T cell phenotype, optionally wherein the memory T cell phenotype is based on a marker selected from the group consisting of CD62L, CCR7, CD127, CD45RA, CD27, and combinations thereof. In some embodiments, the T cell may be characterized by a central memory-like phenotype, optionally wherein the central memory-like phenotype is based on a marker selected from the group consisting of CD44, CD62L, and combinations thereof. In some embodiments, the T cell may be characterized by a central memory-like (CD44+CD62L+) phenotype. In some embodiments, the T cell may be characterized by an effector-like phenotype, optionally wherein the effector-like phenotype is based on a marker selected from the group consisting of CD44, CD62L, and combinations thereof. In some embodiments, the T cell may be characterized by an effector-like phenotype (CD44+CD62L-). In some embodiments, the T cell may be characterized by a naive-like phenotype, optionally wherein the naive-like phenotype is based on a marker selected from the group consisting of CD44, CD62L, and combinations thereof. In some embodiments, the T cell may be characterized by a naive-like phenotype (CD44-CD62L+).

As used herein, the term “stem cell” generally includes pluripotent or multipotent stem cells. “Stem cells” includes, e.g., embryonic stem cells (ES); mesenchymal stem cells (MSG); induced-pluripotent stem cells (iPS); and committed progenitor cells (hematopoietic stem cells (HSC); bone marrow derived cells, neural progenitor cells, etc.).

As used herein, the term “T cell receptor” or “TCR” refers to a complex of membrane proteins that participate in the activation of T cells in response to the presentation of antigen. The TCR is responsible for recognizing antigens bound to major histocompatibility complex molecules. TCR is composed of a heterodimer of an alpha (a) and beta (P) chain, although in some cells the TCR consists of gamma and delta (y/b) chains. TCRs may exist in alpha/beta and gamma/delta forms, which are structurally similar but have distinct anatomical locations and functions. Each chain is composed of two extracellular domains, a variable and constant domain. In some embodiments, the TCR may be modified on any cell comprising a TCR, including, for example, a helper T cell, a cytotoxic T cell, a memory T cell, regulatory T cell, natural killer T cell, and gamma delta T cell. As used herein, the term “hematopoietic stem cells” or “HSC” refers to stem cells that can differentiate into the hematopoietic lineage and give rise to all blood cell types such as white blood cells and red blood cells, including myeloid {e.g., monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes/platelets, dendritic cells), and lymphoid lineages {e.g., T-cells, B-cells, K-cells). Stem cells are defined by their ability to form multiple cell types (multipotency) and their ability to selfrenew. Hematopoietic stem cells can be identified, for example by cell surface markers such as CD34-, CD133+, CD48-, CD150+, CD244-, cKit+, Scal+, and lack of lineage markers (negative for B220, CD3, CD4, CD8, Macl, Grl, and Teri I9, among others).

As used herein, the term “hematopoietic progenitor cells” or “HPC” encompasses pluripotent cells which are committed to the hematopoietic cell lineage, generally do not selfrenew, and are capable of differentiating into several cell types of the hematopoietic system, such as granulocytes, monocytes, erythrocytes, megakaryocytes, B-cells and T-cells, including, but not limited to, short term hematopoietic stem cells (ST-HSCs), multi-potent progenitor cells (MPPs), common myeloid progenitor cells (CMPs), granulocyte-monocyte progenitor cells (GMPs), megakaryocyte-erythrocyte progenitor cells (MEPs), and committed lymphoid progenitor cells (CLPs). The presence of hematopoietic progenitor cells can be determined functionally as colony forming unit cells (CFII-Cs) in complete methylcellulose assays, or phenotypically through the detection of cell surface markers e.g., CD45-, CD34+, Teri I9-, CD16/32, CD127, cKit, Seal) using assays known to those of skill in the art.

The term “reduced” or “reduce” or “decrease” as used herein generally means a decrease of at least 5%, for example a decrease by at least about 10%, or at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% decrease (i.e. substantially absent or below levels of detection), or any decrease between 5-100% as compared to a reference level, as that term is defined herein, and as determined by a method that achieves statistical significance (p <0.05).

The term “increased” or “increase” as used herein generally means an increase of at least 5%, for example an increase by at least about 10%, or at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase i.e., substantially above levels of detection), or any increase between 5-100% as compared to a reference level, as that term is defined herein, and as determined by a method that achieves statistical significance (p <0.05). In some embodiments, the methods described herein can result in a greater number of cells having a desired phenotype (e.g., T cell phenotype) localized in the scaffold material in vivo as compared to a reference, optionally, by at least about 5%, or, at least about 10%, or at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 95%, or at least about 99%, or up to and including a 100% increase, or any increase between about 5 and about 100%.

As used herein, the term “standard” or “reference” refers to a measured biological parameter including, but not limited to, the level {e.g., concentration) of a cell, e.g., an immune cell, in a known sample against which another sample is compared; alternatively, a standard can simply be a reference number that represents an amount of the measured biological parameter that defines a baseline for comparison. The reference number can be derived from either a sample taken from an individual, or a plurality of individuals or cells obtained therefrom. That is, the “standard” does not need to be a sample that is tested, but can be an accepted reference number or value. A series of standards can be developed that take into account an individual's status, e.g., with respect to age, gender, weight, height, ethnic background etc. A standard level can be obtained, for example, from a known sample from a different individual {e.g., not the individual being tested). A known sample can also be obtained by pooling samples from a plurality of individuals (or cells obtained therefrom) to produce a standard over an averaged population. Additionally, a standard can be synthesized such that a series of standards are used to quantify the biological parameter in an individual's sample. A sample from the individual to be tested can be obtained at an earlier time point (presumably prior to the onset of treatment) and serve as a standard or reference compared to a sample taken from the same individual after the onset of treatment. In such instances, the standard can provide a measure of the efficacy of treatment. Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 100 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,

42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,

66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,

90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100.

Throughout this disclosure, various aspects of the disclosure can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1 , 2, 2.7, 3, 4, 5, 5.3, and 6. As another example, a range such as 95-99% identity, includes something with 95%, 96%, 97%, 98% or 99% identity, and includes subranges such as 96- 99%, 96-98%, 96-97%, 97-99%, 97-98% and 98-99% identity. This applies regardless of the breadth of the range.

I. Compositions

In some aspects, provided herein are compositions comprising a hydrogel. In some embodiments, provided herein are hydrogels fabricated as microscale particles, known as microgels, with tailored size, morphology and mechanics, providing a highly tunable, modular and biocompatible system. In some embodiments, provided herein are granular hydrogels assembled from microgels.

Accordingly, some aspects of the present disclosure provide methods and compositions for fabricating and using microgels and/or granular hydrogels.

In one aspect, the present disclosure provides a microgel (e.g., granular hydrogel) platform that can present active agents, e.g., bioactive ligands, specifically on the surface to regulate cellular behaviors, such as T cell expansion and phenotypic change. In some embodiments, surface functionalization can be achieved by coating the microgel surface with oppositely charged polymers, resulting in a thin yet stable layer of functional polymers decorating the surface of microgels. In some embodiments, conjugation of activating antibodies and/or mitogenic cytokines, e.g., via chemo-selective chemistry, can allow modulation of the surface biochemical cues to cells, e.g., T cells precisely and efficiently. In some embodiments, the microgels modified with appropriate ligands can promote efficient polyclonal and antigen-specific T cell expansion. The present disclosure provides experimental data demonstrating, unexpectedly, that the concentration, ratio, and distribution of antibodies during T cell activation have profound effects on the resulting phenotype of primary mouse and human T cells. In addition, the experimental data demonstrated that stiffer and more elastic microgels promote the expansion and activation of the T cells.

In some aspects, provided herein is a surface-specific functionalization strategy that can provide a convenient and versatile means to modulate the surface biochemical properties of microgels (e.g., granular hydrogels), which can be used to manipulate the stimulation dose for personalized T cell therapies. The injectability of the microgels and granular hydrogels described herein, and stability of polymer coatings during injection can allow these materials to be delivered with minimally invasive procedures for in situ expansion of immune cells for cancer treatment, minimizing the risks of off target toxicities. The microgels and/or granular hydrogels described herein can also be used for T cell expansion and phenotypic regulation, as well as for the expansion and differentiation of a variety of cell types. As used herein the terms “microgel” and “core microgel” refer to a hydrogel fabricated as microscale particles, for example, a three-dimensional hydrogel particle that is about 0.001 pm to about 500 pm in diameter. The microgels may be formed of any suitable biomaterial, e.g., a non-degradable component and/or a degradable component. In some embodiments, a plurality of microgels can be assembled (e.g., jammed) to create a three dimensional scaffold {e.g., a granular hydrogel) that can serve, e.g., as a tissue substitute both in vitro and/or in vivo. To fabricate microgel assembly, microgels (e.g., core microgels) can first be generated via several techniques, such as batch emulsion, microfluidic emulsion, and as described herein. The microgels (e.g., core microgels) can then be assembled together under certain conditions. Various assembling techniques may be used for microgel assembly, including, e.g., chemical reaction, physical reaction, cell-cell interaction, and external driving force. In particular embodiments, microgel assembly into a granular hydrogel can be formed by microgel jamming, where microgels are packed into a limited space. In some embodiments, granular hydrogels can either be found in the jammed state or as free- floating, non-jammed particles in solution. Jammed microgels are dynamic structures that can be characterized by unique physical properties, such as self-assembly, shear-thinning, and self-healing. Unlike conventional crosslinked hydrogels, when microgels are packed closely together in a jammed state, the jammed microgels can appear like a solid, but when external forces are applied, the jammed microgels can display fluidic collective movement, i.e., shear-thinning behavior. In some embodiments, microgels can be used to construct heterogeneous and/or homogeneous scaffold structures. In some embodiments, the jammed microgels (e.g., granular hydrogel) may be characterized only by physical interactions between microgels, such as cohesive force, host-guest interaction, electrostatic interaction, and hydrogen bonding. In some embodiments, the jammed microgels (e.g., granular hydrogel) may be characterized by a secondary crosslinking. In some embodiments, the jammed microgels (e.g., granular hydrogel) are not characterized by a secondary crosslinking. In some embodiments, the jammed microgels (e.g., granular hydrogel) can be injected through a needle into a cavity, e.g., in a tissue of a subject. In some embodiments, the jammed microgels (e.g., granular hydrogel) can fill the space and can take the shape of the cavity. In some embodiment, the viscoelasticity of the microgels (e.g., granular hydrogel) can limit dispersion of microgels upon injection. In some embodiments, the microgels (e.g., granular hydrogel) can be characterized by self-healing properties that can enhance the stability of the microgels (e.g., granular hydrogel) for use in minimally invasive therapy.

The microgels may be of any shape, including, e.g., spheres, spheroids, ovals, ovoids, ellipsoids, discs, capsules, rectangles, polygons, toroids, cones, pyramids, rods, cylinders, and fibers, or any other suitable shape. In some embodiments, the microgel is spherical in form and is characterized by a diameter of about 5 pm to about 100 pm; about 5 m to about 10 pm; about 5 pm to about 25 pm; about 10 pm to about 20 pm; about 20 pm to about 30 pm; about 25 pm to about 50 pm; about 30 pm to about 40 pm; about 40 pm to about 50 pm; about 50 pm to about 60 pm; about 50 pm to about 75 pm; about 60 pm to about 70 pm; about 70 pm to about 80 pm; about 75 pm to about 100 pm; about 80 pm to about 90 pm; or about 90 pm to about 100 pm. In some embodiments, the microgel may comprise a diameter of about 1 pm, about 2 pm, about 3 pm, about 4 pm, about 5 pm, about 6 pm, about 7 pm, about 8 pm, about 9 pm, about 10 pm, about 11 pm, about 12 pm, about 13 pm, about 14 pm, about 15 pm, about 16 pm, about 17 pm, about 18 pm, about 19 pm, about 20 pm, about 21 pm, about 22 pm, about 23 pm, about 24 pm, about 25 pm, about 26 pm, about 27 pm, about 28 pm, about 29 pm, about 30 pm, about 31 pm, about 32 pm, about 33 pm, about 34 pm, about 35 pm, about 36 pm, about 37 pm, about 38 pm, about 39 pm, about 40 pm, about 41 pm, about 42 pm, about 43 pm, about 44 pm, about 45 pm, about 46 pm, about 47 pm, about 48 pm, about 49 pm, about 50 pm, about 51 pm, about 52 pm, about 53 pm, about 54 pm, about 55 pm, about 56 pm, about 57 pm, about 58 pm, about 59 pm, about 60 pm, about 61 pm, about 62 pm, about 63 pm, about 64 pm, about 65 pm, about 66 pm, about 67 pm, about 68 pm, about 69 pm, about 70 pm, about 71 pm, about 72 pm, about 73 pm, about 74 pm, about 75 pm, about 76 pm, about 77 pm, about 78 pm, about 79 pm, about 80 pm, about 81 pm, about 82 pm, about 83 pm, about 84 pm, about 85 pm, about 86 pm, about 87 pm, about 88 pm, about 89 pm, about 90 pm, about 91 pm, about 92 pm, about 93 pm, about 94 pm, about 95 pm, about 96 pm, about 97 pm, about 98 pm, about 99 pm, or about 100 pm.

In certain embodiments, the microgels may be configured to form a three- dimensional scaffold in situ upon administration to a subject. In some embodiments, a plurality of microgels can be assembled (e.g., jammed) in vitro or in vivo to create a three- dimensional scaffold {e.g., a granular hydrogel). Such three-dimensional scaffolds may comprise pores of a size that permit a eukaryotic cell, e.g., an immune cell, to traverse into or out of the scaffold. In some embodiments, the microgels (e.g., granular hydrogels) can comprise pores formed from void space in between microgels. The size, e.g., diameter, of the pores (e.g., void spaces) can be modulated based on, e.g., the diameter and size distribution of the microgels. The mean size of the microgels can also be modulated by the application of a polymer coating to the microgels. In some embodiments, the mean size of the microgels may be decreased after polymer coating. In some embodiments, the mean size of the microgels may be increased after polymer coating.

The pores (e.g., void spaces) may have a diameter of about 1 pm to about 1000 pm (e.g., about 5 pm, about 10 pm, about 15 pm, about 20 pm, about 25 pm, about 30 pm, about 35 pm, about 40 pm, about 45 pm, about 50 pm, about 55 pm, about 60 pm, about 65 pm, about 70 pm, about 75 pm, about 80 pm, about 85 pm, about 90 pm, about 95 pm, about 100 m, about 105 pm, about 110 pm, about 115 pm, about 120 pm, about 125 pm, about 130 pm, about 135 pm, about 140 pm, about 145 pm, about 150 pm, about 155 pm, about 160 pm, about 165 pm, about 170 pm, about 175 pm, about 180 pm, about 185 pm, about 190 pm, about 195 pm, about 200 pm, about 205 pm, about 210 pm, about 215 pm, about 220 pm, about 225 pm, about 230 pm, about 235 pm, about 240 pm, about 245 pm, about 250 pm, about 255 pm, about 260 pm, about 265 pm, about 270 pm, about 275 pm, about 280 pm, about 285 pm, about 290 pm, about 295 pm, about 300 pm, about 305 pm, about 310 pm, about 315 pm, about 320 pm, about 325 pm, about 330 pm, about 335 pm, about 340 pm, about 345 pm, about 350 pm, about 355 pm, about 360 pm, about 365 pm, about 370 pm, about 375 pm, about 380 pm, about 385 pm, about 390 pm, about 395 pm, about 400 pm, about 405 pm, about 410 pm, about 415 pm, about 420 pm, about 425 pm, about 430 pm, about 435 pm, about 440 pm, about 445 pm, about 450 pm, about 455 pm, about 460 pm, about 465 pm, about 470 pm, about 475 pm, about 480 pm, about 485 pm, about 490 pm, about 495 pm, about 500 pm, about 505 pm, about 510 pm, about 515 pm, about 520 pm, about 525 pm, about 530 pm, about 535 pm, about 540 pm, about 545 pm, about 550 pm, about 555 pm, about 560 pm, about 565 pm, about 570 pm, about 575 pm, about 580 pm, about 585 pm, about 590 pm, about 595 pm, about 600 pm, about 605 pm, about 610 pm, about 615 pm, about 620 pm, about 625 pm, about 630 pm, about 635 pm, about 640 pm, about 645 pm, about 650 pm, about 655 pm, about 660 pm, about 665 pm, about 670 pm, about 675 pm, about 680 pm, about 685 pm, about 690 pm, about 695 pm, about 700 pm, about 705 pm, about 710 pm, about 715 pm, about 720 pm, about 725 pm, about 730 pm, about 735 pm, about 740 pm, about 745 pm, about 750 pm, about 755 pm, about 760 pm, about 765 pm, about 770 pm, about 775 pm, about 780 pm, about 785 pm, about 790 pm, about 795 pm, about 800 pm, about 805 pm, about 810 pm, about 815 pm, about 820 pm, about 825 pm, about 830 pm, about 835 pm, about 840 pm, about 845 pm, about 850 pm, about 855 pm, about 860 pm, about 865 pm, about 870 pm, about 875 pm, about 880 pm, about 885 pm, about 890 pm, about 895 pm, about 900 pm, about 905 pm, about 910 pm, about 915 pm, about 920 pm, about 925 pm, about 930 pm, about 935 pm, about 940 pm, about 945 pm, about 950 pm, about 955 pm, about 960 pm, about 965 pm, about 970 pm, about 975 pm, about 980 pm, about 985 pm, about 990 pm, about 995 pm, or about 1000 pm in diameter). In certain embodiments, the pores may be formed by the complete or partial degradation of a component of the microgel, e.g., a degradable component. In certain embodiments, the pores may be formed by void space in between assembled microgels in a granular hydrogel. In some embodiments, microgels (e.g., granular hydrogels) may be characterized by a void fraction (e.g., the ratio of void volume to total volume) of about 15% to about 50% (e.g., about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about or 50%). In some embodiments, microgels (e.g., granular hydrogels) may be characterized by a porosity (e.g., void space %) of about 1% to about 20% (e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, or about 20%).

In some embodiments, microgels (e.g., granular hydrogels) may be characterized by a porosity (e.g., void space %) at the time of formation of about 1% to about 20% (e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, or about 20%), and/or at a time after formation (e.g., 3 days after formation) of about 1% to about 20% (e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, or about 20%). Such microgels (e.g., granular hydrogels) may be characterized by the presence or absence of a polymer coating, such as a polymer coating described herein. In some aspects, provided herein is a microgel, comprising: (i) a core microgel comprising a crosslinked polymer comprising a functional group selected from the group consisting of a crosslinked tetrazine (Tz) polymer, a crosslinked norbornene (Nb) polymer, and combinations thereof; (ii) a non-covalent polymer coating comprising a positively charged polymer applied to the surface of the core microgel; and (iii) a surface coating comprising a functionalized polymer applied to the surface of the coated core microgel to introduce a functional group for surface-specific conjugation of an active agent. In some embodiments, the core microgel is characterized by a degree of substitution (DS) of about 5 to about 15 functional groups per polymer chain. In some embodiments, the positively charged polymer comprises poly(D-lysine) (PDL). In some embodiments, the non-covalent polymer coating and the surface coating are independently characterized by a thickness of about 0.1 pm to about 2 pm. In some embodiments, the core microgel comprises a polymer selected from the group consisting of an alginate polymer, a hyaluronic acid (HA), a collagen polymer, a gelatin polymer, and combinations thereof. In some embodiments, the microgel is characterized by a diameter of about 25 pm to about 250 pm.

In one aspect, the present disclosure provides a microgel, comprising: (i) a non- degradable component; and/or (ii) a degradable component. The non-degradable component may comprise a first polymer and a second polymer, and the non-degradable component may comprise a third polymer. In some embodiments, the microgel may comprise both a non-degradable component and a degradable component.

The first polymer, the second polymer, and the third polymer may be independently selected from the group consisting of alginate, chitosan, polyethylene glycol (PEG), gelatin, hyaluronic acid, collagen, chondroitin, agarose, polyacrylamide, heparin, methacrylated alginate, derivatives thereof, and combinations thereof. In some embodiments, the first polymer and the second polymer are the same polymer. In some embodiments, the first polymer and the second polymer are independently an alginate, optionally wherein the first polymer and the second polymer independently comprise a modified alginate polymer, optionally wherein the first polymer and the second polymer independently comprise oxidized alginate, optionally wherein the first polymer and the second polymer are independently comprise methacrylate alginate, optionally wherein the first polymer and the second polymer independently comprise a click reagent.

In some embodiments, the first polymer and the second polymer independently comprise a modified polymer. In some embodiments, the first polymer and the second polymer independently comprise methacrylated alginate. In some embodiments, the first polymer and the second polymer independently comprise a click reagent. The click reagent may be selected from the group consisting of azide, dibenzocyclooctyne (DBCO), transcyclooctene, tetrazine (Tz), norbornene (Nb), and variants thereof. In some embodiments, the first polymer comprises a tetrazine (Tz) moiety. In some embodiments, the first polymer comprises tetrazine modified alginate (Alg-Tz). In some embodiments, the second polymer comprises a norbornene (Nb) moiety. In some embodiments, the second polymer comprises norbornene modified alginate (Alg-Nb). In some embodiments, the first polymer comprises tetrazine modified alginate (Alg-Tz) and the second polymer comprises norbornene modified alginate (Alg-Nb).

In some embodiments, the microgel may be about 1% to about 90% covalently crosslinked (e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, or about 90% covalently crosslinked).

In some embodiments, the microgel may independently comprises about 1% to about 100% of a crosslinked polymer comprising a functional group selected from the group consisting of a crosslinked tetrazine (Tz) polymer, a crosslinked norbornene (Nb) polymer, and combinations thereof (e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% of a crosslinked tetrazine (Tz) polymer, a crosslinked norbornene (Nb) polymer, and combinations thereof).

In some embodiments, the microgel may independently comprises about 1% to about 100% of a crosslinked tetrazine (Tz) polymer (e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% of a crosslinked tetrazine (Tz) polymer).

In some embodiments, the microgel may independently comprises about 1% to about 100% of a crosslinked norbornene (Nb) polymer (e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% of a crosslinked norbornene (Nb) polymer).

In some embodiments, the crosslinked tetrazine (Tz) polymer and/or the crosslinked norbornene (Nb) polymer may comprise at least one selected from the group consisting of an alginate polymer, a hyaluronic acid (HA) polymer, a collagen polymer, a gelatin polymer, a gelatin and alginate-type I collagen interpenetrating network, or a combination thereof.

In some embodiments, the microgel may independently comprises about 1% to about 100% Alg-Tz (e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% Alg-Tz).

In some embodiments, the microgel may comprise about 1% to about 100% Alg-Nb (e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% Alg-Nb).

In some embodiments, the microgel may comprise a ratio of norbornene (Nb)/tetrazine (Tz) of about 0.1 to about 10 (e.g., about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1 , about 1.1, about 1.2, about

1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2, about 2.1 , about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, about 3, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about

4.7, about 4.8, about 4.9, about 5, about 5.1 , about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6, about 6.1 , about 6.2, about 6.3, about

6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7, about 7.1 , about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8, about 8.1 , about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, about 9, about 9.1 , about 9.2, about 9.3, about 9.4, about 9.5, about 9.6, about 9.7, about

9.8, about 9.9, or about 10).

In some embodiments, the microgel may comprise a ratio of Alg-Tz: Alg-Nb of 1:1, 1 :3, or 3:1.

In some embodiments, the third polymer may comprise a modified polymer. In some embodiments, the third polymer may comprise an oxidized polymer. In some embodiments, the oxidized polymer is about 0.1% to about 99% oxidized (e.g., about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% oxidized). In some embodiments, the oxidized polymer is about 1% to about 10% oxidized, optionally wherein the oxidized polymer is about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, about 9%, about 9.5%, or about 10% oxidized. In some embodiments, the third polymer may comprise oxidized alginate. In some embodiments, the third polymer may degrade in vivo within about 1-day to about 30- days after administration to a subject.

In some embodiments, the microgel may comprise an active agent. In some embodiments, the active agent may be selected from the group consisting of a cell, a biological factor, a ligand (e.g., target peptides and/or proteins), a small molecule, and combinations thereof. In some embodiments, the active agent may be selected from the group consisting of an activating antibody, a mitogenic cytokine, and combinations thereof.

In some embodiments, the microgels enable encapsulation and release of bioactive factors in a controlled manner and exhibit mechanical properties similar to cells, which can enable the microgels to serve as aAPCs and/or APC mimetic scaffolds. Although bioactive ligands can be conjugated throughout the entire microgels, only those bioactive ligands presenting on the surface of the microgels can typically bind to T cell surface receptors to regulate T cell activation. Accordingly, the efficient and flexible conjugation to the microgel surface of target peptides or proteins that can bind to cell surface receptors, described herein, can be useful for T cell activation.

In some embodiments, the microgel may comprise an active agent (e.g., ligand) at a predefined density (e.g., ligand density) of about 0 to about 10 pg/cm² (e.g., about 0, about 0.025, about 0.05, about 0.1, about 0.2, about 0.4, about 0.6, about 0.8, about 1, about 1.2, about 1.4, about 1.6, about 1.8, about 2, about 2.2, about 2.4, about 2.6, about 2.8, about 3, about 3.2, about 3.4, about 3.6, about 3.8, about 4, about 4.2, about 4.4, about 4.6, about

4.8, about 5, about 5.2, about 5.4, about 5.6, about 5.8, about 6, about 6.2, about 6.4, about 6.6, about 6.8, about 7, about 7.2, about 7.4, about 7.6, about 7.8, about 8, about 8.2, about 8.4, about 8.6, about 8.8, about 9, about 9.2, about 9.4, about 9.6, about 9.8, or about 10 pg/cm²).

In some embodiments, the microgel may comprise an active agent (e.g., ligand) at a predefined density (e.g., ligand density) of about 3 pg/cm² to about 7 pg/cm² (e.g., about 3, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about

3.9, about 4, about 4.1 , about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5, about 5.1 , about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, or about 7 pg/cm²).

In some embodiments, the microgel may comprise an active agent (e.g., ligand) at a predefined density (e.g., ligand density) of about 0 to about 10 pg/cm² and a percentage of cytotoxicity of less than 20% (e.g., about 1% or less, about 2% or less, about 3% or less, about 4% or less, about 5% or less, about 6% or less, about 7% or less, about 8% or less, about 9% or less, about 10% or less, about 11% or less, about 12% or less, about 13% or less, about 14% or less, about 15% or less, about 16% or less, about 17% or less, about 18% or less, about 19% or less, or about 20% or less).

In some embodiments, the active agent may be present at between about 1 ng to about 1000 pg. In some embodiments, the active agent may be present at between about 1 ng to about 100 pg. In some embodiments, the active agent may be present at between about 1 pg to about 2 ng per microgel. In some embodiments, the active agent may be present at about 1 pg per microgel. In some embodiments, the active agent may comprise a growth factor. The growth factor may be selected from the group consisting of a BMP-2, a BMP-4, a BMP-6, a BMP-7, a BMP-12, a BMP-14, and a combination thereof. In some embodiments, the growth factor may comprise a BMP-2. In some embodiments, the growth factor may be present at between about 2 ng to about 500 ng per microgel.

In some embodiments, the active agent may comprise a differentiation factor. In some embodiments, the differentiation factor may be selected from the group consisting of a Delta-like 1 (DLL-1), a Delta-like 2 (DLL-2), a Delta-like 3 (DLL-3), a Delta-like 3 (DLL-3), a Delta-like 4 (DLL-4), a Jagged 1 , a Jagged 2, and a combination thereof. In some embodiments, the differentiation factor may comprise DLL-4. In some embodiments, the differentiation factor may be present at an amount at between about 1 ng to about 100 pg per microgel.

In some embodiments, the active agent is covalently and/or non-covalently attached to the microgel. The active agent may be covalently attached to the microgel utilizing click chemistry. For example, the active agent may be covalently linked to the scaffold utilizing N- hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) chemistry, NHS and dicyclohexylcarbodiimide (DCC) chemistry, avidin-biotin reaction, azide and dibenzocycloocytne chemistry, tetrazine and transcyclooctene chemistry, tetrazine and norbornene chemistry, or di-sulfide chemistry. In some embodiments, the active agent may be released from the microgel within about 1-day to about 30-days after administration to a subject. In some embodiments, the active agent is not covalently and/or non-covalently attached to the microgel. In some embodiments, the active agent is covalently and/or non-covalently attached to a polymer coating (e.g., a non-covalent polymer coating and/or a surface coating) of a microgel. Such polymer coatings may comprise one or more layers of a positively charged polymer and/or a negatively charged polymer. For example, the microgels may first be coated with a positively charged polymer such as, e.g., poly(D-lysine) (PDL), to form a first layer, and then be coated with a functionalized polymer such as, e.g., functionalized alginate, to form a second layer and to introduce functional groups on the surface of the coated microgels. In some embodiments, the thin and stable coating layer of functionalized polymer, e.g., functionalized alginate, can allow incorporation of sufficient ligands only on the surface to mediate biological functions without introducing functional groups throughout the entire microgel that are not available to cell surface receptors. In some embodiment, the surface ligand density can be efficiently and precisely engineered through multiple approaches during the surface functionalization process. For example, the surface ligand density can be tuned by varying the degree of substitution (DS) of functional groups coupled to the polymers, e.g., alginate polymers, used for coating. In some embodiments, the surface ligand density can be tuned by modulating the density of coated polymers. In some embodiments, varying the concentration of ligand-modified polymer solution used to create the second layer from 0.01 to 1 mg/mL can results in a 25-fold increase of polymer density without significant changes in thickness. In some embodiments, surface ligands can be engineered via post-functionalization using orthogonal click chemistries to conjugate the target molecules to the microgel surface. For example, microgels crosslinked via the norbornene-tetrazine strategy can be subsequently coated with azide-modified polymer (e.g., azide-modified alginate) to allow surface-specific conjugation of dibenzocyclooctyne (DBCO)-modified ligands through strain-promoted azide-alkyne cycloaddition (SPAAC) in a controlled manner.

The surface functionalization strategy is also applicable to a range of coating and core polymers. In some embodiments, hyaluronic acid (HA) can be uniformly coated on the surface of alginate microgels. In addition, core microgels made of HA, gelatin and alginate- type I collagen interpenetrating network can be fabricated using, e.g., microfluidic emulsion, and a uniform and thin layer of alginate can be coated on the surface of these microgels. In some embodiments, the surface-specific chemical modification achieved via surface coating allows efficient fabrication of microgels with different surface functionalities by leveraging different polymers and chemo-selective chemistries to modify pre-synthesized microgels.

II. Microgel Scaffolds & Granular Hydrogels

The composition of the present disclosure comprise a microgel and/or a granular hydrogel. The microgel and/or the granular hydrogel can comprise one or more biomaterials. In some embodiments, the one or more biomaterials can comprise a polymer.

Preferably, the biomaterial is a biocompatible material that is non-toxic and/or non- immunogenic. As used herein, the term "biocompatible material" refers to any material that does not induce a significant immune response or deleterious tissue reaction, e.g., toxic reaction or significant irritation, over time when implanted into or placed adjacent to a biological tissue of a subject. In some embodiments, the biomaterial comprises a biocompatible polymer.

The microgel and/or the granular hydrogel can comprise biomaterials {e.g., one or more polymers) that are non-biodegradable and/or biodegradable. In certain embodiments, the microgel, e.g., granular hydrogel, can comprise a non-biodegradable material, such as a non-biodegradable polymer. In certain embodiments, the microgel, e.g., granular hydrogel, can comprise a biodegradable material, such as a biodegradable polymer. The biodegradable material may be degraded by physical and/or chemical action, e.g., level of hydration, heat, oxidation, or ion exchange or by cellular action, e.g., elaboration of enzyme, peptides, or other compounds by nearby or resident cells. In certain embodiments, the microgel, e.g., granular hydrogel, can comprise both non-degradable and degradable materials e.g., both non-degradable and degradable polymers).

In some embodiments, the microgel, e.g., granular hydrogel, can degrade at a predetermined rate based on a physical parameter selected from the group consisting of temperature, pH, hydration status, and porosity, the cross-link density, type, and chemistry or the susceptibility of main chain linkages to degradation. Alternatively, the microgel, e.g., granular hydrogel, can degrade at a predetermined rate based on a ratio of chemical polymers. For example, a high molecular weight polymer comprised of solely lactide degrades over a period of years, e.g., 1-2 years, while a low molecular weight polymer comprised of a 50:50 mixture of lactide and glycolide degrades in a matter of weeks, e.g., 1, 2, 3, 4, 6, or 10 weeks. A calcium cross-linked gels composed of high molecular weight, high guluronic acid alginate degrade over several months (1 , 2, 4, 6, 8, 10, or 12 months) to years (1 , 2, or 5 years) in vivo, while a gel comprised of low molecular weight alginate, and/or alginate that has been partially oxidized, will degrade in a matter of weeks.

Exemplary biomaterials suitable for use as microgels, e.g., granular hydrogels, in the present disclosure include glycosaminoglycan, silk, fibrin, MATRIGEL®, poly-ethyleneglycol (PEG), polyhydroxy ethyl methacrylate, polyacrylamide, poly (N-vinyl pyrolidone), (PGA), poly lactic-co-glycolic acid (PLGA), poly e-carpolactone (PCL), polyethylene oxide, poly propylene fumarate (PPF), poly acrylic acid (PAA), polyhydroxybutyric acid, hydrolysed polyacrylonitrile, polymethacrylic acid, polyethylene amine, esters of alginic acid; pectinic acid; and alginate, fully or partially oxidized alginate, hyaluronic acid, carboxy methyl cellulose, heparin, heparin sulfate, chitosan, carboxymethyl chitosan, chitin, pullulan, gellan, xanthan, collagen, gelatin, carboxymethyl starch, carboxymethyl dextran, chondroitin sulfate, cationic guar, cationic starch, and combinations thereof. In certain embodiments, the biomaterial is selected from the group consisting of alginate, fully or partially oxidized alginate, and combinations thereof. In some embodiments, the core microgel comprises at least one selected from the group consisting of an alginate polymer, a hyaluronic acid (HA) polymer, a collagen polymer, a gelatin polymer, a gelatin and alginate-type I collagen interpenetrating network, or a combination thereof.

In some embodiments, the microgels, e.g., granular hydrogels, comprise biomaterials, such as polymers, that are modified. In some embodiments, the modified biomaterial polymer comprises an oxidized polymer. In some embodiments, the modified polymer comprises a reduced polymer. In some embodiments, the modified polymer comprises a polymer modified with a click reaction moiety. Exemplary click reaction moieties include, but are not limited to, an azide moiety, a dibenzocyclooctyne (DBCO) moiety, a transcyclooctene moiety, a tetrazine (Tz) moiety, a norbornene (Nb) moiety, and variants thereof. In some embodiments, the modified polymer comprises at least one selected from the group consisting of a modified alginate polymer, a modified hyaluronic acid (HA) polymer, a modified collagen polymer, a modified gelatin polymer, and combinations thereof.

In some embodiments, the microgel may comprise a polymer modified with norbornene (Nb) and/or tetrazine (Tz). In some embodiments, the microgel may comprise a ratio of norbornene (Nb)/tetrazine (Tz) of about 0.1 to about 10 {e.g., about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about

1.1 , about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2, about 2.1 , about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about

2.8, about 2.9, about 3, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4, about 4.1, about 4.2, about 4.3, about 4.4, about

4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5, about 5.1 , about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6, about 6.1 , about

6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about

7.9, about 8, about 8.1 , about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, about 9, about 9.1 , about 9.2, about 9.3, about 9.4, about 9.5, about

9.6, about 9.7, about 9.8, about 9.9, or about 10). In some embodiments, the polymer modified with norbornene (Nb) and/or tetrazine (Tz) comprises at least one selected from the group consisting of a norbornene (Nb) and/or tetrazine (Tz) alginate polymer, a norbornene (Nb) and/or tetrazine (Tz) hyaluronic acid (HA) polymer, a norbornene (Nb) and/or tetrazine (Tz) collagen polymer, a norbornene (Nb) and/or tetrazine (Tz) gelatin polymer, and combinations thereof.

The degree of modification, such as oxidation, can be varied from about 1% to about 100%. As used herein, the degree of modification means the molar percentage of the sites on the biomaterial that are modified with a functional group. For example, the degree of modification can be about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21 %, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%,

30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41 %, 42%, 43%, 44%, 45%,

46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%,

62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,

78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,

94%, 95%, 96%, 97%, 98%, 99%, or 100%. It is intended that values and ranges intermediate to the recited values are part of this disclosure.

The degree of substitution (DS) of a polymer is the (average) number of substituent groups attached per base unit (in the case of condensation polymers) or per monomeric unit (in the case of addition polymers). In the context of alginate, for example, the degree of substitution (DS) may be given as the ratio of substituted alginate residues to the total number of alginate residues in percent (mol/mol). In some embodiments, the degree of substitution of a polymer, e.g., an alginate polymer, can be about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21 %, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%,

39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%,

55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%,

71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,

87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

In some embodiments, a polymer may be modified to achieve an average degree of substitution (DS) of between about 5 to about 15 {e.g., about 1 , about 1.5, about 2, about

2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10, about 10.5, about 11 , about

11.5, about 12, about 12.5, about 13, about 13.5, about 14, about 14.5, or about 15) functional groups per polymer chain.

In some embodiments, a polymer may be modified with a click reaction moiety to achieve an average degree of substitution (DS) of between about 5 to about 15 e.g., about 1 , about 1.5, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10, about 10.5, about 11 , about 11.5, about 12, about 12.5, about 13, about 13.5, about 14, about 14.5, or about 15) click reaction moieties per polymer chain. In some embodiments, an alginate polymer may be modified with norbornene (Alg- Nb) or tetrazine (Alg-Tz), e.g., by carbodiimide coupling, to achieve an average degree of substitution (DS) of between about 5 to about 15 {e.g., about 1 , about 1.5, about 2, about

2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10, about 10.5, about 11, about

11.5, about 12, about 12.5, about 13, about 13.5, about 14, about 14.5, or about 15) functional groups e.g., Nb or Tz) per alginate chain.

Exemplary modified biomaterials, e.g., microgels, e.g., granular hydrogels, include, but not limited to, reduced-alginate, oxidized alginate, MA-alginate (methacrylated alginate), MA-gelatin (methacrylated gelatin), hyaluronic acid, norbornene modified alginate (Alg-Nb), or tetrazine modified alginate (Alg-Tz).

In some embodiments, the microgel may comprise an polymer, e.g., a modified polymer, at a weight percent (wt%) of about 1 wt%, about 1.5 wt%, about 2 wt%, about 2.5 wt%, about 3 wt%, about 3.5 wt%, about 4 wt%, about 4.5 wt%, about 5 wt%, about 5.5 wt%, about 6 wt%, about 6.5 wt%, about 7 wt%, about 7.5 wt%, about 8 wt%, about 8.5 wt%, about 9 wt%, about 9.5 wt%, or about 10 wt%.

In some embodiments, the microgel may comprise a norbornene (Nb) modified polymer and/or a tetrazine (Tz) modified polymer at a weight percent (wt%) of about 1 wt%, about 1.5 wt%, about 2 wt%, about 2.5 wt%, about 3 wt%, about 3.5 wt%, about 4 wt%, about 4.5 wt%, about 5 wt%, about 5.5 wt%, about 6 wt%, about 6.5 wt%, about 7 wt%, about 7.5 wt%, about 8 wt%, about 8.5 wt%, about 9 wt%, about 9.5 wt%, or about 10 wt%.

In some embodiments, the microgel may comprise a norbornene (Nb) and/or tetrazine (Tz) alginate polymer, a norbornene (Nb) and/or tetrazine (Tz) hyaluronic acid (HA) polymer, a norbornene (Nb) and/or tetrazine (Tz) collagen polymer, a norbornene (Nb) and/or tetrazine (Tz) gelatin polymer, and combinations thereof at a weight percent (wt%) of about 1 wt% to about 10 wt% (e.g., about 1 wt%, about 1.5 wt%, about 2 wt%, about 2.5 wt%, about 3 wt%, about 3.5 wt%, about 4 wt%, about 4.5 wt%, about 5 wt%, about 5.5 wt%, about 6 wt%, about 6.5 wt%, about 7 wt%, about 7.5 wt%, about 8 wt%, about 8.5 wt%, about 9 wt%, about 9.5 wt%, or about 10 wt%).

In some embodiments, the microgel may comprise at least one selected from the group consisting of an alginate polymer, a hyaluronic acid (HA) polymer, a collagen polymer, a gelatin polymer, and combinations thereof at a weight percent (wt%) of about 1 wt% to about 10 wt% (e.g., about 1 wt%, about 1.5 wt%, about 2 wt%, about 2.5 wt%, about 3 wt%, about 3.5 wt%, about 4 wt%, about 4.5 wt%, about 5 wt%, about 5.5 wt%, about 6 wt%, about 6.5 wt%, about 7 wt%, about 7.5 wt%, about 8 wt%, about 8.5 wt%, about 9 wt%, about 9.5 wt%, or about 10 wt%). In certain embodiments, one or more active agents (e.g., the growth factors, the differentiation factors, and the homing factors), disclosed herein, may be attached to or encapsulated in the microgels, e.g., granular hydrogels. In some embodiments, one or more active agents disclosed herein may be covalently or non-covalently linked or attached to the microgel, e.g., microgel scaffold. In various embodiments, one or more active agents disclosed herein may be incorporated on, into, or present within the structure or pores of, the scaffold composition. In some embodiments, the active agent is not covalently and/or non- covalently attached to the microgel.

In some embodiments, the active agent is covalently and/or non-covalently attached to a polymer coating (e.g., a surface coating a non-covalent polymer coating and/or a surface coating) of a microgel. Such polymer coatings may comprise one or more layers of a positively charged polymer and/or a negatively charged polymer. For example, the microgels may first be coated with a positively charged polymer such as, e.g., poly(D-lysine) (PDL), to form a first layer, and then be coated with a functionalized polymer such as, e.g., functionalized alginate, to form a second layer and to introduce functional groups on the surface of the coated microgels. In some embodiments, active agents, such as surface ligands, can be attached to the polymer coating using, e.g., orthogonal click chemistries, to conjugate the active agent to the microgel surface via the introduced functional group. For example, microgels crosslinked via the norbornene-tetrazine strategy can be subsequently coated with azide-modified polymer (e.g., azide-modified alginate) to allow surface-specific conjugation of dibenzocyclooctyne (DBCO)-modified ligands through strain-promoted azidealkyne cycloaddition (SPAAC) in a controlled manner.

The microgels, e.g., granular hydrogels, of the present disclosure may comprise an external surface. Alternatively, or in addition, the scaffolds may comprise an internal surface. External or internal surfaces of the microgels, e.g., granular hydrogels, of the present disclosure may be solid or porous. Pore size of the scaffolds can be less than about 10 nm, between about 100 nm-20 pm, or greater than about 20 pm, e.g., up to and including 1000 pm in diameter. For example, the pores may be nanoporous, microporous, or macroporous. For example, the diameter of nanopores is less than about 10 nm; the diameter of micropores is in the range of about 100 nm-20 pm; and, the diameter of macropores is greater than about 20 pm, e.g., greater than about 50 pm, e.g., greater than about 100 pm, e.g., greater than about 400 pm, e.g., greater than 600 pm or greater than 800 pm. In some embodiment the diameter of the pore is between about 50 pm and about 80 pm.

In some embodiments, the microgels, e.g., granular hydrogels, of the present disclosure may be organized in a variety of geometric shapes (e.g., spheres, discs, beads, pellets), niches, planar layers (e.g., thin sheets). For example, discs of about 0.1-200 millimeters in diameter, e.g., 5, 10, 20, 40, or 50 millimeters may be implanted subcutaneously. The disc may have a thickness of 0.1 to 10 millimeters, e.g., 1 , 2, or 5 millimeters. The discs are readily compressed or lyophilized for administration to a patient. An exemplary disc for subcutaneous administration has the following dimensions: 8 millimeters in diameter and 1 millimeter in thickness.

In some embodiments, the microgel and/or granular hydrogel scaffolds may comprise multiple components and/or compartments. In certain embodiments, a multiple compartment device is assembled in vivo by applying sequential layers of similarly or differentially doped gel or other scaffold material to the target site. For example, the device is formed by sequentially injecting the next, inner layer into the center of the previously injected material using a needle, thereby forming concentric spheroids. In certain embodiments, non-concentric compartments are formed by injecting material into different locations in a previously injected layer. A multi-headed injection device extrudes compartments in parallel and simultaneously. The layers are made of similar or different biomaterials differentially doped with pharmaceutical compositions. Alternatively, compartments self-organize based on their hydro-philic/phobic characteristics or on secondary interactions within each compartment. In certain embodiments, multicomponent scaffolds are optionally constructed in concentric layers each of which is characterized by different physical qualities such as the percentage of polymer, the percentage of crosslinking of polymer, chemical composition of the hydrogel, pore size, porosity, and pore architecture, stiffness, toughness, ductility, viscoelasticity, the growth factors, the differentiation factors, and/or homing factors incorporated therein and/or any other compositions incorporated therein.

Microgel Stiffness (e.g., Elastic Modulus)

Some aspects of the present disclosure provide microgels {e.g., granular hydrogels) characterized by a predefined stiffness, e.g., a predefined elastic modulus, and methods of producing such microgels e.g., granular hydrogels). The present disclosure provides experimental data demonstrating that the elastic moduli of the microgels {e.g., granular hydrogels) can be tuned, e.g., by varying the ratio between a polymer modified with norbornene (Nb) and a polymer modified with tetrazine (Tz). The present disclosure provides experimental data demonstrating, unexpectedly, that when microgels of different stiffness were cocultured with CD4+ and CD8+ T cells (CD4/CD8 = 1), increasing the elastic moduli of microgels from about 1 to about 3 kPa led to, e.g., an increase in T cell fold expansion and upregulation of the expression of CD25 and 0X40 activation markers.

In some embodiments, microgels with different stiffness can be synthesized by varying the ratio between a polymer modified with norbornene (Nb) and a polymer modified with tetrazine (Tz) at an overall polymer concentration of about 0.5 wt% to about 5 wt% (e.g., about 0.5 wt%, about 1 wt%, about 1.5 wt%, about 2 wt%, about 2.5 wt%, about 3 wt%, about 3.5 wt%, about 4 wt%, about 4.5 wt%, or about 5 wt%).

In some embodiments, the microgels (e.g., granular hydrogels) may be characterized by a predefined stiffness, e.g., a predefined elastic modulus, of about 0.5 kPa to about 10 kPa (e.g., about 0.5 kPa, about 1 kPa, about 1.5 kPa, about 2 kPa, about 2.5 kPa, about 3 kPa, about 3.5 kPa, about 4 kPa, about 4.5 kPa, about 5 kPa, about 5.5 kPa, about 6 kPa, about 6.5 kPa, about 7 kPa, about 7.5 kPa, about 8 kPa, about 8.5 kPa, about 9 kPa, about 9.5 kPa, or about 10 kPa).

In some embodiments, the microgels (e.g., granular hydrogels) may be characterized by a predefined stiffness, e.g., a predefined elastic modulus, of about 1 kPa to about 10 kPa; about 1 kPa to about 9 kPa; about 1 kPa to about 8 kPa; about 1 kPa to about 7 kPa; about 1 kPa to about 6 kPa; about 1 kPa to about 5 kPa; about 1 kPa to about 4 kPa; about 1 kPa to about 3 kPa; about 1 kPa to about 2 kPa; or about 5 kPa to about 10 kPa.

In some embodiments, the microgels (e.g., granular hydrogels) may be characterized by a predefined stiffness, e.g., a predefined elastic modulus, of about 1 kPa to about 5 kPa (e.g., about 1 kPa, about 1.5 kPa, about 2 kPa, about 2.5 kPa, about 3 kPa, about 3.5 kPa, about 4 kPa, about 4.5 kPa, or about 5 kPa) and a Nb/Tz ratio of about 0 to about 10 (e.g., about 0, about 0.1 , about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1 , about 1.5, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, or about 10).

In some embodiments, the microgels (e.g., granular hydrogels) may be characterized by a predefined stiffness, e.g., a predefined elastic modulus, of about 1 kPa to about 2.5 kPa (e.g., about 1 kPa, about 1.5 kPa, about 2 kPa, or about 2.5 kPa) and a Nb/Tz ratio of about 0.2 to about 0.5 (e.g., about 0.2, about 0.3, about 0.4, or about 0.5).

In some embodiments, the microgels (e.g., granular hydrogels) may be characterized by a predefined stiffness, e.g., a predefined elastic modulus, of greater than about 2.5 kPa (e.g., about 3 kPa to about 4 kPa) and a Nb/Tz ratio of about 0.6 to about 1.5 (e.g., about 0.6, about 0.7, about 0.8, about 0.9, about 1 , or about 1.5).

In some embodiments, the microgels (e.g., granular hydrogels) may be characterized by a predefined stiffness, e.g., a predefined elastic modulus, of less than about 2.5 kPa (e.g., about 0.1 kPa to about 2.5 kPa) and a Nb/Tz ratio of about 1.5 to about 7 (e.g., about 1.5, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, or about 7).

In some embodiments, the elastic modulus of the microgels can be measured using atomic force microscope (AFM). For example, nanoindentation tests may be conducted on a NanoWizard II AFM (JPK Instruments AG). In some embodiments, silicone cantilevers with a polystyrene tip, a force constant of 0.2 N m-¹, and a resonance frequency of 13 kHz werecan be used (NanoAndMore GmbH, Watsonville, CA, USA) for the AFM measurements. In some embodiments, the contact force can be set to 0.1 V, and the pulling range can be set from 1500 to 3000 nm. Force-distance curves in 20 x 20 pm area can be recorded and calculated to give the elastic modulus.

Elastic Microgels

Some aspects of the present disclosure provide elastic microgels and methods of producing such microgels. In some embodiments, the generation of elastic microgels comprises synthesizing covalently crosslinked microgels. In some embodiments, the synthesis of covalently crosslinked microgels comprises a dispersed phase containing about 1 wt% to about 5 wt% of a polymer described herein (e.g., about 1 wt% to about 5 wt% alginate, e.g., about 2 wt% alginate) prepared as a mixture of Tz and Nb modified polymer (e.g., alginate) dissolved separately at, e.g., 1-3 wt%, in DI water. In some embodiments, a mixture of fluorosurfactant (1 %) in fluorocarbon oil can be used as the continuous phase. In some embodiments, alginate-Tz and alginate-Nb solutions can be injected at 150 pL IT¹ and the continuous phase can be injected at 1000 pL IT¹. In some embodiments, the emulsion can then be collected in a tube and left at room temperature for 24 h to allow covalent crosslinking between alginate polymers. In some embodiments, after the reaction has been completed, the continuous phase can be removed, and 33% 1 H, 1 H, 2H, 2/7-perfluoro-1 -octanol in HFE can be added in excess at, e.g., a 1 :3 volume ratio, to the collected microgels to break the emulsion. In some embodiments, the microgels can be washed three times with beads buffer (e.g., 130 mM NaCI, 25 mM HEPES, 2 mM CaCh, pH 7.5), redispersed in beads buffer, and stored at 4 °C until further use.

Viscoelastic Microgels

Some aspects of the present disclosure provide viscoelastic microgels and methods of producing such microgels. In some embodiments, the generation of viscoelastic microgels comprises the synthesis of Ca²⁺ crosslinked polymer microgels (e.g., alginate microgels). In some embodiments, the synthesis of Ca²⁺ crosslinked polymer microgels (e.g., alginate microgels) comprises a dispersed phase comprising about 1 wt% to about 5 wt% of an unmodified polymer (e.g., about 1 wt% to about 5 wt% unmodified alginate, e.g., about 1 to about 2 wt% unmodified alginate) and about 50 mM CaEDTA for use in the batch emulsion technique, described herein. In some embodiments, a mixture of fluorosurfactant (1%) and acidic acid (0.05-0.2 v%) in fluorocarbon oil can be used as the continuous phase. In some embodiments, the alginate solution and the continuous phase can be injected at flow rates of 300 and 1000 pL h’¹, respectively. In some embodiments, the emulsion can then be collected and mixed with 50% 1/7,1/7,2/7,2/7-perfluoro-1 -octanol in HFE at, e.g., a 1 :1 volume ratio, to break the emulsion. In some embodiments, the microgels can be washed three times with beads buffer, redispersed in beads buffer, and stored at 4 °C until further use.

Polymer Coatings

Some aspects of the present disclosure provide microgels (e.g., granular hydrogels) comprising a polymer coating and methods of producing such microgels.

In some embodiments, the polymer coating comprises one or more layers. In some embodiments, the polymer coating comprises poly(D-lysine) (PDL). In some embodiments, the polymer coating comprises a functionalized polymer, e.g., functionalized alginate. In some embodiments, the microgels (e.g., granular hydrogels) comprises a polymer coating comprising poly(D-lysine) (PDL). In some embodiments, the microgels (e.g., granular hydrogels) comprises a polymer coating comprising a functionalized polymer, e.g., a functionalized alginate, such as alginate-Tz and/or alginate-Nb. In some embodiments, the microgels (e.g., granular hydrogels) comprises a polymer coating comprising poly(D-lysine) (PDL) and a polymer coating comprising a functionalized polymer, e.g., a functionalized alginate, such as alginate-Tz and/or alginate-Nb.

In some embodiments, the generation of microgels (e.g., granular hydrogels) comprising a polymer coating comprises concentrating microgels by centrifugation, e.g., at 300 ref for 3 min, and redispersing the concentrated microgels in a solution of poly(D-lysine) (PDL) (e.g., 50-150 kDa, 0.1 mg mL^-1 in beads buffer) at a concentration of, e.g., about 4 x 10⁵ microgels per mL. In some embodiments, the microgels can then immediately be concentrated by centrifugation, e.g., at 300 ref for 3 min, washed three times with beads buffer, redispersed in beads buffer at initial stock solution concentration, and stored at 4 °C until further use. In some embodiments, the microgels can be redispersed in a solution of functionalized polymer, e.g., functionalized alginate (e.g., about 0.01 mg/mL to about 1 mg/mL) in beads buffer at a concentration of, e.g., about 4 x 10⁵ microgels per mL, and collected by centrifugation, e.g., at 300 ref for 3 min. In some embodiments, microgels can be washed three times with beads buffer, redispersed in beads buffer at initial stock solution concentration, and stored at 4 °C until further use.

Coating Density

Some aspects of the present disclosure provide microgels (e.g., granular hydrogels) comprising a polymer coating characterized by a predefined coating density and methods of producing such microgels.

In some embodiments, the microgels (e.g., granular hydrogels) may comprise a polymer coating characterized by a predefined coating density of about 0 pg/cm² to about 5 pg/cm² (e.g., about 0.5 pg/cm², about 1 pg/cm², about 1.5 pg/cm², about 2 pg/cm², about 2.5 pg/cm², about 3 pg/cm², about 3.5 pg/cm², about 4 pg/cm², about 4.5 pg/cm², about 5 pg/cm², or about 5.5 pg/cm²).

In some embodiments, the microgels (e.g., granular hydrogels) may comprise a polymer coating characterized by a predefined coating density of about 0 pg/cm² to about 5 pg/cm² (e.g., about 0.5 pg/cm², about 1 pg/cm², about 1.5 pg/cm², about 2 pg/cm², about 2.5 pg/cm², about 3 pg/cm², about 3.5 pg/cm², about 4 pg/cm², about 4.5 pg/cm², about 5 pg/cm², or about 5.5 pg/cm²), wherein the polymer (e.g., alginate) concentration is about 0.01 mg/mL to about 2 mg/mL (e.g., about 0.01 mg/mL, about 0.1 mg/mL, about 0.2 mg/mL, about 0.3 mg/mL, about 0.4 mg/mL, about 0.5 mg/mL, about 0.6 mg/mL, about 0.7 mg/mL, about 0.8 mg/mL, about 0.9 mg/mL, about 1 mg/mL, about 1.1 mg/mL, about 1.2 mg/mL, about 1.3 mg/mL, about 1.4 mg/mL, about 1.5 mg/mL, about 1.6 mg/mL, about 1.7 mg/mL, about 1.8 mg/mL, about 1.9 mg/mL, or about 2.0 mg/mL).

In some embodiments, the microgels (e.g., granular hydrogels) may comprise a polymer coating characterized by a predefined thickness (e.g., distance from surface) of about 0 pm to about 3 pm (e.g., about 0.001 pm, 0.01 pm, about 0.1 pm, about 0.2 pm, about 0.3 pm, about 0.4 pm, about 0.5 pm, about 0.6 pm, about 0.7 pm, about 0.8 pm, about 0.9 pm, about 1 pm, about 1.1 pm, about 1.2 pm, about 1.3 pm, about 1.4 pm, about 1.5 pm, about 1.6 pm, about 1.7 pm, about 1.8 pm, about 1.9 pm, about 2 pm, about 2.1 pm, about 2.2 pm, about 2.3 pm, about 2.4 pm, about 2.5 pm, about 2.6 pm, about 2.7 pm, about 2.8 pm, about 2.9 pm, or about 3 pm).

In some embodiments, the amount of polymer coated on the microgels (e.g., granular hydrogels) can be determined by the difference between the amount of polymer used for coating and the remaining amount in the solution after coating. For example, the amount of alginate coated on the microgels can be determined by the difference between the amount of alginate used for coating and the remaining amount in the solution after coating. In some embodiments, alginate-rhodamine B can be used as a model polymer for coating to quantify the concentration of alginate in solutions. In some embodiments, microgels can be washed three times after coating and all the supernatants can be collected after each centrifugation. In some embodiments, alginate concentration in the original solution can be used for coating and all the supernatants can be quantified by fluorescent intensity at 586 nm (excitation wavelength 561 nm) based on a calibration curve. In some embodiments, the density of coating can be calculated by the amount of alginate-rhodamine B coated on the surface and the overall surface area of microgels.

Coating Stability Some aspects of the present disclosure provide microgels (e.g., granular hydrogels) comprising a polymer coating characterized by a predefined coating stability and methods of producing such microgels.

In some embodiments, the microgels (e.g., granular hydrogels) may comprise a polymer coating characterized by a predefined coating stability in which at least about 80% or more (e.g., about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) of the polymer coating remained after soaking in buffer and/or culture media for at least about 7 days or more.

In some embodiments, the microgels (e.g., granular hydrogels) may comprise a polymer coating characterized by a predefined coating stability in which at least about 80% or more (e.g., about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) of the polymer coating remained after soaking in buffer and/or culture media for at least about 3 weeks or more.

In some embodiments, the microgels (e.g., granular hydrogels) may comprise a polymer coating characterized by a predefined coating stability in which at least about 80% or more (e.g., about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) of the polymer coating remained after soaking in buffer and/or culture media for at least about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, or about 10 months or more.

In some embodiments, the microgels (e.g., granular hydrogels) may comprise a polymer coating characterized by a predefined coating stability in which at least about 80% or more (e.g., about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) of the polymer coating thickness remained after soaking in buffer and/or culture media for a predetermined period of time (e.g., about 1 day to about 7 days, about 1 week to about 4 weeks, or about 1 month to about 12 months or more). In some embodiments, the microgels (e.g., granular hydrogels) may comprise a polymer coating characterized by a thickness of about 0.5 pm to about 1.5 pm and at least about 80% or more (e.g., about 80%, about 85%, about 90%, about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) of the polymer coating thickness can remain after soaking in buffer and/or culture media for a predetermined period of time (e.g., about 1 day to about 7 days, about 1 week to about 4 weeks, or about 1 month to about 12 months or more). In some embodiments, the stability of the polymer coating can be determined by polymer dissolution in a surrounding buffer solution. In some embodiments, alginate- rhodamine B coated microgels can be soaked in beads buffer (e.g., 4 x 10⁵ microgels per mL) at room temperature. In some embodiments, the buffer solution can be collected and replaced by fresh beads buffer on, e.g., days 1 , 4, 7, 10, 14, and 21. In some embodiments, the concentration of released alginate-rhodamine B can be determined as described herein.

Microgel Jamming

Some aspects of the present disclosure provide microgels which when jammed together can assemble to form granular hydrogels, a type of injectable microporous scaffold, and methods of producing such microgels {e.g., granular hydrogels). The granular hydrogels can be characterized by a porous network.

In some embodiments, the generation of granular hydrogels comprises concentrating the microgels by centrifugation, e.g., at 300 ref for 3 min. In some embodiments, mixing complementary microgels collected separately together. In some embodiments, a pre-rinsed membrane (0.22 pm) can be folded into a cone shape and placed in a 1 .5 mL Eppendorf tube, and the pellet of microgels can then be loaded onto the membrane and centrifuged, e.g., at 50 ref for 20, 5, or 1 seconds to produced jammed microgels, e.g., granular hydrogels. The jammed microgels were can be retrieved from the membrane.

Porosity

Some aspects of the present disclosure provide microgels e.g., granular hydrogels) comprising a plurality of pores {e.g., void space between particles) characterized by a predefined porosity and methods of producing such microgels {e.g., granular hydrogels).

In some embodiments, the microgels {e.g., granular hydrogels) may be characterized by a predefined porosity (e.g., void space between particles) of about 5% to about 20% (e.g., about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, or about 20% or more).

In some embodiments, the microgels {e.g., granular hydrogels) may be characterized by a predefined porosity (e.g., void space between particles) of about 5% to about 20% (e.g., about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, or about 20% or more) and an elastic modulus of about 250 Pa to about 4000 Pa (e.g., about 300 Pa, about 400 Pa, about 500 Pa, about 600 Pa, about 700 Pa, about 800 Pa, about 900 Pa, about 1000 Pa, about 1100 Pa, about 1200 Pa, about 1300 Pa, about 1400 Pa, about 1500 Pa, about 1600 Pa, about 1700 Pa, about 1800 Pa, about 1900 Pa, about 2000 Pa, about 2100 Pa, about 2200 Pa, about 2300 Pa, about 2400 Pa, about 2500 Pa, about 2600 Pa, about 2700 Pa, about 2800 Pa, about 2900 Pa, about 3000 Pa, about 3100 Pa, about 3200 Pa, about 3300 Pa, about 3400 Pa, about 3500 Pa, about 3600 Pa, about 3700 Pa, about 3800 Pa, about 3900 Pa, about 4000 Pa, about 4100 Pa, about 4200 Pa, about 4300 Pa, about 4400 Pa, or about 4500 Pa).

In some embodiments, the microgels {e.g., granular hydrogels) may be characterized by a predefined porosity (e.g., void space between particles) of about 8% to about 12% (e.g., about 8%, about 9%, about 10%, about 11 %, or about 12%) and an elastic modulus of about 325 Pa to about 350 Pa (e.g., about 325 Pa, about 326 Pa, about 327 Pa, about 328 Pa, about 329 Pa, about 330 Pa, about 331 Pa, about 332 Pa, about 333 Pa, about 334 Pa, about 335 Pa, about 336 Pa, about 337 Pa, about 338 Pa, about 339 Pa, about 340 Pa, about 341 Pa, about 342 Pa, about 343 Pa, about 344 Pa, about 345 Pa, about 346 Pa, about 347 Pa, about 348 Pa, about 349 Pa, or about 350 Pa).

In some embodiments, the microgels {e.g., granular hydrogels) may be characterized by a predefined porosity (e.g., void space between particles) of about 8% to about 12% (e.g., about 8%, about 9%, about 10%, about 11 %, or about 12%) and an elastic modulus of about 1090 Pa to about 1120 Pa (e.g., about 1090 Pa, about 1091 Pa, about 1092 Pa, about 1093 Pa, about 1094 Pa, about 1095 Pa, about 1096 Pa, about 1097 Pa, about 1098 Pa, about 1099 Pa, about 1100 Pa, about 1101 Pa, about 1102 Pa, about 1103 Pa, about 1104 Pa, about 1105 Pa, about 1106 Pa, about 1107 Pa, about 1108 Pa, about 1109 Pa, about 1110 Pa, about 1111 Pa, about 1112 Pa, about 1113 Pa, about 1114 Pa, about 1115 Pa, about 1116 Pa, about 1117 Pa, about 1118 Pa, about 1119 Pa, about 1120 Pa, about 1121 Pa, about 1122 Pa, about 1123 Pa, about 1124 Pa, or about 1125 Pa).

In some embodiments, the microgels {e.g., granular hydrogels) may be characterized by a predefined porosity (e.g., void space between particles) of about 8% to about 12% (e.g., about 8%, about 9%, about 10%, about 11 %, or about 12%) and an elastic modulus of about 2230 Pa to about 2260 Pa (e.g., about 2230 Pa, about 2231 Pa, about 2232 Pa, about 2233 Pa, about 2234 Pa, about 2235 Pa, about 2236 Pa, about 2237 Pa, about 2238 Pa, about 2239 Pa, about 2240 Pa, about 2241 Pa, about 2242 Pa, about 2243 Pa, about 2244 Pa, about 2245 Pa, about 2246 Pa, about 2247 Pa, about 2248 Pa, about 2249 Pa, about 2250 Pa, about 2251 Pa, about 2252 Pa, about 2253 Pa, about 2254 Pa, about 2255 Pa, about 2256 Pa, about 2257 Pa, about 2258 Pa, about 2259 Pa, or about 2260 Pa).

In some embodiments, the microgels {e.g., granular hydrogels) may be characterized by a predefined porosity (e.g., void space between particles) of about 8% to about 12% (e.g., about 8%, about 9%, about 10%, about 11 %, or about 12%) and an elastic modulus of about 3390 Pa to about 3420 Pa (e.g., about 3390 Pa, about 3391 Pa, about 3392 Pa, about 3393 Pa, about 3394 Pa, about 3395 Pa, about 3396 Pa, about 3397 Pa, about 3398 Pa, about 3399 Pa, about 3400 Pa, about 3401 Pa, about 3402 Pa, about 3403 Pa, about 3404 Pa, about 3405 Pa, about 3406 Pa, about 3407 Pa, about 3408 Pa, about 3409 Pa, about 3410 Pa, about 3411 Pa, about 3412 Pa, about 3413 Pa, about 3414 Pa, about 3415 Pa, about 3416 Pa, about 3417 Pa, about 3418 Pa, about 3419 Pa, or about 3420 Pa).

Hydrogel and Cryogel Scaffolds

Some aspects of the present disclosure provide a microgel (e.g., granular hydrogel) scaffold comprising a polymer selected from the group consisting of an alginate polymer, a hyaluronic acid (HA), a collagen polymer, a gelatin polymer, and combinations thereof. In certain embodiments, the microgels (e.g., granular hydrogels) of the present disclosure comprise one or more hydrogels. A hydrogel is a polymer gel comprising a network of crosslinked polymer chains. A hydrogel is usually a composition comprising polymer chains that are hydrophilic. The network structure of hydrogels allows them to absorb significant amounts of water. Some hydrogels are highly stretchable and elastic; others are viscoelastic. Hydrogel are sometimes found as a colloidal gel in which water is the dispersion medium. In certain embodiments, hydrogels are highly absorbent (they can contain over 99% water (v/v)) natural or synthetic polymers that possess a degree of flexibility very similar to natural tissue, due to their significant water content. In certain embodiments, a hydrogel may have a property that, when an appropriate shear stress is applied, the deformable hydrogel is dramatically and reversibly compressed (up to 95% of its volume), resulting in injectable macroporous preformed scaffolds. Hydrogels have been used for therapeutic applications, e.g., as vehicles for in vivo delivery of therapeutic agents, such as small molecules, cells and biologies. Hydrogels are commonly produced from polysaccharides, such as alginates. The polysaccharides may be chemically manipulated to modulate their properties and properties of the resulting hydrogels.

The hydrogels of the present disclosure may be either porous or non-porous. Preferably the compositions of the disclosure are formed of porous hydrogels. For example, the hydrogels may be nanoporous wherein the diameter of the pores is less than about 10 nm; microporous wherein the diameter of the pores is preferably in the range of about 100 nm-20 pm; or macroporous wherein the diameter of the pores is greater than about 20 pm, more preferably greater than about 100 pm and even more preferably greater than about 400 pm. In certain embodiments, the hydrogel is macroporous with pores of about 50-80 pm in diameter. In certain embodiments, the hydrogel is macroporous with aligned pores of about 400-500 pm in diameter. Methods of preparing porous hydrogel products are known in the art. (See, e.g., U.S. Pat. No. 6,511 ,650, incorporated herein by reference).

The hydrogel may be constructed out of a number of different rigid, semi-rigid, flexible, gel, self-assembling, liquid crystalline, or fluid compositions such as peptide polymers, polysaccharides, synthetic polymers, hydrogel materials, ceramics (e.g., calcium phosphate or hydroxyapatite), proteins, glycoproteins, proteoglycans, metals and metal alloys. The compositions are assembled into hydrogels using methods known in the art, e.g., injection molding, lyophilization of preformed structures, printing, self-assembly, phase inversion, solvent casting, melt processing, gas foaming, fiber forming/processing, particulate leaching, microfluidics, or a combination thereof. The assembled devices are then implanted or administered, e.g., by injection, to the body of an individual to be treated.

The composition comprising a hydrogel may be assembled in vivo in several ways. The hydrogel is made from a gelling material, which is introduced into the body in its ungelled form where it gels in situ. Exemplary methods of delivering components of the composition to a site at which assembly occurs include injection through a needle or other extrusion tool, spraying, painting, or methods of deposit at a tissue site, e.g., delivery using an application device inserted through a cannula. In some embodiments, the ungelled or unformed hydrogel material is mixed with at least one pharmaceutical composition prior to introduction into the body or while it is introduced. The resultant in vivo/in situ assembled device, e.g., hydrogel, contains a mixture of the at least one pharmaceutical composition.

In situ assembly of the hydrogel may occur as a result of spontaneous association of polymers or from synergistically or chemically catalyzed polymerization. Synergistic or chemical catalysis is initiated by a number of endogenous factors or conditions at or near the assembly site, e.g., body temperature, ions or pH in the body, or by exogenous factors or conditions supplied by the operator to the assembly site, e.g., photons, heat, electrical, sound, or other radiation directed at the ungelled material after it has been introduced. The energy is directed at the hydrogel material by a radiation beam or through a heat or light conductor, such as a wire or fiber optic cable or an ultrasonic transducer. Alternatively, a shear-thinning material, such as an amphiphile, is used which re-cross links after the shear force exerted upon it, for example by its passage through a needle, has been relieved.

In certain embodiments, the microgels, e.g., microgel scaffolds, may be configured to form a three-dimensional scaffold in situ upon administration to a subject. Such three- dimensional scaffolds may comprise pores of a size that permit a eukaryotic cell, e.g., an immune cell, to traverse into or out of the scaffold. The pores may have a diameter of about 1 pm to about 1000 pm (e.g., about 5 pm, about 10 pm, about 15 pm, about 20 pm, about 25 pm, about 30 pm, about 35 pm, about 40 pm, about 45 pm, about 50 pm, about 55 pm, about 60 pm, about 65 pm, about 70 pm, about 75 pm, about 80 pm, about 85 pm, about 90 pm, about 95 pm, about 100 pm, about 105 pm, about 110 pm, about 115 pm, about 120 pm, about 125 pm, about 130 pm, about 135 pm, about 140 pm, about 145 pm, about 150 pm, about 155 pm, about 160 pm, about 165 pm, about 170 pm, about 175 pm, about 180 pm, about 185 pm, about 190 pm, about 195 pm, about 200 pm, about 205 pm, about 210 m, about 215 pm, about 220 pm, about 225 pm, about 230 pm, about 235 pm, about 240 pm, about 245 pm, about 250 pm, about 255 pm, about 260 pm, about 265 pm, about 270 pm, about 275 pm, about 280 pm, about 285 pm, about 290 pm, about 295 pm, about 300 pm, about 305 pm, about 310 pm, about 315 pm, about 320 pm, about 325 pm, about 330 pm, about 335 pm, about 340 pm, about 345 pm, about 350 pm, about 355 pm, about 360 pm, about 365 pm, about 370 pm, about 375 pm, about 380 pm, about 385 pm, about 390 pm, about 395 pm, about 400 pm, about 405 pm, about 410 pm, about 415 pm, about 420 pm, about 425 pm, about 430 pm, about 435 pm, about 440 pm, about 445 pm, about 450 pm, about 455 pm, about 460 pm, about 465 pm, about 470 pm, about 475 pm, about 480 pm, about 485 pm, about 490 pm, about 495 pm, about 500 pm, about 505 pm, about 510 pm, about 515 pm, about 520 pm, about 525 pm, about 530 pm, about 535 pm, about 540 pm, about 545 pm, about 550 pm, about 555 pm, about 560 pm, about 565 pm, about 570 pm, about 575 pm, about 580 pm, about 585 pm, about 590 pm, about 595 pm, about 600 pm, about 605 pm, about 610 pm, about 615 pm, about 620 pm, about 625 pm, about 630 pm, about 635 pm, about 640 pm, about 645 pm, about 650 pm, about 655 pm, about 660 pm, about 665 pm, about 670 pm, about 675 pm, about 680 pm, about 685 pm, about 690 pm, about 695 pm, about 700 pm, about 705 pm, about 710 pm, about 715 pm, about 720 pm, about 725 pm, about 730 pm, about 735 pm, about 740 pm, about 745 pm, about 750 pm, about 755 pm, about 760 pm, about 765 pm, about 770 pm, about 775 pm, about 780 pm, about 785 pm, about 790 pm, about 795 pm, about 800 pm, about 805 pm, about 810 pm, about 815 pm, about 820 pm, about 825 pm, about 830 pm, about 835 pm, about 840 pm, about 845 pm, about 850 pm, about 855 pm, about 860 pm, about 865 pm, about 870 pm, about 875 pm, about 880 pm, about 885 pm, about 890 pm, about 895 pm, about 900 pm, about 905 pm, about 910 pm, about 915 pm, about 920 pm, about 925 pm, about 930 pm, about 935 pm, about 940 pm, about 945 pm, about 950 pm, about 955 pm, about 960 pm, about 965 pm, about 970 pm, about 975 pm, about 980 pm, about 985 pm, about 990 pm, about 995 pm, or about 1000 pm in diameter). In certain embodiments, the pores may be formed by the complete or partial degradation of a component of the microgel, e.g., a degradable component.

In some embodiments, the hydrogel may be assembled ex vivo. In some embodiments, the hydrogel is injectable. For example, the hydrogels are created outside of the body as macroporous scaffolds. Upon injection into the body, the pores collapse causing the gel to become very small and allowing it to fit through a needle. See, e.g., WO 2012/149358; and Bencherif et al., 2012, Proc. Natl. Acad. Sci. USA 109.48:19590-5, the content of which are incorporated herein by reference).

Suitable hydrogels for both in vivo and ex vivo assembly of hydrogel devices are well known in the art and described, e.g., in Lee et al., 2001, Chem. Rev. 7:1869-1879. The peptide amphiphile approach to self-assembly assembly is described, e.g., in Hartgerink et al., 2002, Proc. Natl. Acad. Sci. USA 99:5133-5138. A method for reversible gellation following shear thinning is exemplified in Lee et al., 2003, Adv. Mat. 15:1828-1832.

In certain embodiments, exemplary hydrogels are comprised of materials that are compatible with attachment and/or encapsulation of materials including polymers, nanoparticles, active agents, polypeptides, and cells . Exemplary hydrogels are fabricated from alginate, polyethylene glycol (PEG), PEG-acrylate, agarose, hyaluronic acid, or synthetic protein (e.g., collagen or engineered proteins (/.e., self-assembly peptide-based hydrogels)). For example, a commercially available hydrogel includes BD™ PuraMatrix™. BD™ PuraMatrix™ Peptide Hydrogel is a synthetic matrix that is used to create defined three dimensional (3D) micro-environments for cell culture.

In some embodiments, the hydrogel is a biocompatible polymer matrix that is biodegradable in whole or in part. Examples of materials which can form hydrogels include alginates and alginate derivatives, polylactic acid, polyglycolic acid, poly(lactic-co-glycolic acid) (PLGA) polymers, gelatin, collagen, agarose, hyaluronic acid, hyaluronic acid derivative, natural and synthetic polysaccharides, polyamino acids such as polypeptides particularly poly(lysine), polyesters such as polyhydroxybutyrate and poly-epsilon- caprolactone, polyanhydrides; polyphosphazines, poly(vinyl alcohols), poly(alkylene oxides) particularly poly(ethylene oxides), poly(allylamines)(PAM), poly(acrylates), modified styrene polymers such as poly(4-aminomethylstyrene), pluronic polyols, polyoxamers, poly(uronic acids), poly(vinylpyrrolidone), and copolymers of the above, including graft copolymers. Synthetic polymers and naturally-occurring polymers such as, but not limited to, collagen, fibrin, hyaluronic acid, agarose, and laminin-rich gels may also be used. The term “derivative,” as used herein, refers to a compound that is derived from a similar compound by a chemical reaction. For example, oxidized alginate, which is derived from alginate through oxidization reaction, is a derivative of alginate,

The implantable composition can have virtually any regular or irregular shape including, but not limited to, spherical, spheroid, cubic, polyhedron, prism, cylinder, rod, disc, or other geometric shape. Accordingly, in some embodiments, the implant is of cylindrical form from about 0.5 to about 10 mm in diameter and from about 0.5 to about 10 cm in length. Preferably, its diameter is from about 1 to about 5 mm and its length from about 1 to about 5 cm.

In some embodiments, the compositions of the disclosure are of spherical form. When the composition is in a spherical form, its diameter can range, in some embodiments, from about 0.5 to about 50 mm in diameter. In some embodiments, a spherical implant’s diameter is from about 5 to about 30 mm. In an exemplary embodiment, the diameter is from about 10 to about 25 mm. In some embodiments, the microgel is spherical in form and is characterized by a diameter of about 10 pm to about 100 pm. In some embodiments, the microgel may comprise a diameter of about 10 pm, about 11 pm, about 12 pm, about 13 pm, about 14 pm, about 15 pm, about 16 pm, about 17 pm, about 18 pm, about 19 pm, about 20 pm, about 21 pm, about 22 pm, about 23 pm, about 24 pm, about 25 pm, about 26 pm, about 27 pm, about 28 pm, about 29 pm, about 30 pm, about 31 pm, about 32 pm, about 33 pm, about 34 pm, about 35 pm, about 36 pm, about 37 pm, about 38 pm, about 39 pm, about 40 pm, about 41 pm, about 42 pm, about 43 pm, about 44 pm, about 45 pm, about 46 pm, about 47 pm, about 48 pm, about 49 pm, about 50 pm, about 51 pm, about 52 pm, about 53 pm, about 54 pm, about 55 pm, about 56 pm, about 57 pm, about 58 pm, about 59 pm, about 60 pm, about 61 pm, about 62 pm, about 63 pm, about 64 pm, about 65 pm, about 66 pm, about 67 pm, about 68 pm, about 69 pm, about 70 pm, about 71 pm, about 72 pm, about 73 pm, about 74 pm, about 75 pm, about 76 pm, about 77 pm, about 78 pm, about 79 pm, about 80 pm, about 81 pm, about 82 pm, about 83 pm, about 84 pm, about 85 pm, about 86 pm, about 87 pm, about 88 pm, about 89 pm, about 90 pm, about 91 pm, about 92 pm, about 93 pm, about 94 pm, about 95 pm, about 96 pm, about 97 pm, about 98 pm, about 99 pm, or about 100 pm.

In certain embodiments, the microgel, e.g., microgel scaffold, comprises clickhydrogels and/or click-cryogels. A click hydrogel or cryogel is a gel in which cross-linking between hydrogel or cryogel polymers is facilitated by click reactions between the polymers. Each polymer may contain one of more functional groups useful in a click reaction. Given the high level of specificity of the functional group pairs in a click reaction, active compounds can be added to the preformed device prior to or contemporaneously with formation of the hydrogel device by click chemistry. Non-limiting examples of click reactions that may be used to form click-hydrogels include Copper I catalyzed azide-alkyne cycloaddition, strain- promoted assize-alkyne cycloaddition, thiol-ene photocoupling, Diels-Alder reactions, inverse electron demand Diels-Alder reactions, tetrazole-alkene photo-click reactions, oxime reactions, thiol-Michael addition, and aldehyde-hydrazide coupling. Non-limiting aspects of click hydrogels are described in Jiang et al., 2014, Biomaterials, 35:4969-4985, the entire content of which is incorporated herein by reference.

In various embodiments, a click alginate is utilized (see, e.g., PCT International Patent Application Publication No. WO 2015/154078 published October 8, 2015, hereby incorporated by reference in its entirety).

In some embodiments, the concentration of crosslinks e.g., noncovalent and/or covalent crosslinks) per hydrogel is at least about 10% (w/w), e.g., at least about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% (w/w). In some embodiments, the concentration of crosslinks per hydrogel is about 10% to about 100% (w/w), e.g., about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% (w/w). In some embodiments, the concentration of crosslinks per hydrogel is about 25% to about 50% (w/w). In some embodiments, the concentration of crosslinks per hydrogel is about 25% to about 75% (w/w). In some embodiments, the concentration of crosslinks per hydrogel is about 50% to about 75% (w/w). In some embodiments, the concentration of crosslinks per hydrogel is about 75% to about 100% (w/w).

In some embodiments, the click-hydrogel devices and scaffold materials include a hydrogel comprising a first polymer and a second polymer. The first polymer and the second polymer can be the same or different. In some embodiments, the first polymer and the second polymer are the same type of polymer. In some embodiments, the first polymer and/or the second polymer are independently selected from the group consisting of alginate, chitosan, polyethylene glycol (PEG), gelatin, hyaluronic acid, collagen, MATRIGEL®, chondroitin, agarose, polyacrylamide, and heparin. In some embodiments, the first polymer and the second polymer are the same polymer independently selected from the group consisting of alginate, chitosan, polyethylene glycol (PEG), gelatin, hyaluronic acid, collagen, MATRIGEL®, chondroitin, agarose, polyacrylamide, and heparin. In some embodiments, the hydrogel is an interpenetrating polymer network (IPN) hydrogel.

In some embodiments, polymers, e.g., alginate polymers, are modified with tetrazine or norbornene groups that can subsequently be covalently cross-linked to form click- crosslinked hydrogels, e.g., click alginate hydrogels. In some embodiments, the first polymer and the second polymer may be formulated for specific applications by controlling the molecular weight, degree of modification e.g., % oxidation and/or % crosslinking), rate of degradation, and method of scaffold formation.

Such scaffolds and scaffold materials, as well as methods for producing such scaffolds, are described in PCT International Patent Application Publication No. WO 2015/154078 published October 8, 2015, the entire content of which is incorporated herein by reference. For example, a click hydrogel may be prepared in a process: a) providing a first polymer comprising a first click reaction moiety and a second polymer comprising a second click reaction moiety. In non-limiting examples, the first click reaction moiety and the second click reaction moiety may be react with each other in a copper I catalyzed azidealkyne cycloaddition, strain-promoted assize-alkyne cycloaddition, thiol-ene photo coupling, a Diels-Alder reaction, an inverse electron demand Diels-Alder reaction, a tetrazole-alkene photo-click reaction, a oxime reaction, a thiol-Michael addition, or via aldehyde-hydrazide coupling. In an embodiment, the first click reaction moiety is a diene moiety and the second click reaction moiety is a dienophile moiety. In an embodiment, the first click reaction moiety is a tetrazine moiety and the second click reaction moiety is a norbornene moiety. As used herein, the terms "tetrazine" and "tetrazine moiety" include molecules that comprise 1 , 2,4,5- tetrazine substituted with suitable spacer for linking to the polymer {e.g., alkylamines like methylamine or pentylamine), and optionally further substituted with one or more substituents at any available position. Exemplary tetrazine moieties suitable for the compositions and methods of the disclosure are described in Karver et al. Bioconjugate Chem. 22(2011):2263-2270, and WO 2014/ 065860, both incorporated herein by reference). As used herein, the terms "norbornene" and "norbornene moieties" include but are not limited to norbornadiene and norbornene groups further comprising suitable spacer for linking to the polymer {e.g., alkylamines like methylamine or pentylamine), and optionally further substituted with one or more substituents at any available position. Such moieties include, for example, norbornene-S-methylamine and norbomadienemethylamine.

In certain embodiments, a hydrogel (e.g., cryogel) system can deliver one or more agent (e.g., a growth factor such as BMP-2, and/or a differentiation factor, such as a DLL-4, while creating a space for cells (e.g., stem cells such as hematopoietic stem cells (HSC) infiltration and trafficking). In some embodiments, the hydrogel system according to the present disclosure delivers BMP-2, which acts as a hematopoietic stem cell (HSC) and/or hematopoietic progenitor cell enhancement/recruitment factor, and DLL-4 as a differentiation factor, which facilitates T cell lineage specification of hematopoietic stem cell and/or hematopoietic progenitor cells.

In some embodiments, a cryogel composition, e.g., formed of MA-alginate, can function as a delivering platform by creating a local niche, such as a specific niche for enhancing T-lineage specification. In some embodiments, the cryogel creates a local niche in which the encounter of cells, such as recruited stem cells or progenitor cells, and various exemplary agent of the disclosure, such as the growth factor and/or differentiation factor can be controlled. In certain embodiments, the cells and the exemplary agents of the present disclosure are localized into a small volume, and the contacting of the cells and the agents can be quantitatively controlled in space and time.

In certain embodiments, the hydrogel (e.g., cryogel) can be engineered to coordinate the delivery of both growth factor and differentiation factor in space and time, potentially enhancing overall immune modulation performance by adjusting the differentiation and/or specification of recruited cells, such as hematopoietic stem cells or progenitor cells. In certain embodiments, the cells and growth factor/differentiation factor are localized into a small volume, and the delivery of factors in space and time can be quantitatively controlled. As the growth/differentiation factors are released locally, few systemic effects are anticipated, in contrast to systemically delivered agents, such as growth factors.

Examples of polymer compositions from which the cryogel or hydrogel is fabricated are described throughout the present disclosure, and include alginate, hyaluronic acid, gelatin, heparin, dextran, carob gum, PEG, PEG derivatives including PEG-co-PGA and PEG-peptide conjugates. The techniques can be applied to any biocompatible polymers, e.g., collagen, chitosan, carboxymethylcellulose, pullulan, polyvinyl alcohol (PVA), Poly(2- hydroxyethyl methacrylate) (PHEMA), Poly(N-isopropylacrylamide) (PNIPAAm), or Poly(acrylic acid) (PAAc). For example, in a particular embodiment, the composition comprises an alginate-based hydrogel/cryogel. In another example, the scaffold comprises a gelatin-based hydrogel/cryogel.

Cryogels are a class of materials with a highly porous interconnected structure that are produced using a cryotropic gelation (or cryogelation) technique. Cryogels also have a highly porous structure. Typically, active compounds are added to the cryogel device after the freeze formation of the pore/wall structure of the cryogel. Cryogels are characterized by high porosity, e.g., at least about 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95% pores with thin pore walls that are characterized by high density of polymer crosslinking. As used herein, the term “porosity” refers to the percentage of the volume of pores to the volume of the scaffold. It is intended that values and ranges intermediate to the recited values are part of this disclosure. The walls of cryogels are typically dense and highly cross-linked, enabling them to be compressed through a needle into a subject without permanent deformation or substantial structural damage.

In various embodiments, the pore walls comprise at least about 10, 15, 20, 25, 30, 35, or 40% (w/v) polymer. It is intended that values and ranges intermediate to the recited values are part of this disclosure. In other embodiments, the pore walls comprise about 10- 40% polymer. In some embodiments, a polymer concentration of about 0.5-4% (w/v) (before the cryogelation) is used, and the concentration increases substantially upon completion of cryogelation. Non-limiting aspects of cryogel gelation and the increase of polymer concentration after cryogelation are discussed in Beduer et al., 2015 Advanced Healthcare Materials 4.2: 301-312, the entire content of which is incorporated herein by reference.

In certain embodiments, cryogelation comprises a technique in which polymerizationcrosslinking reactions are conducted in quasi-frozen reaction solution. Non-limiting examples of cryogelation techniques are described in U.S. Patent Application Publication No. 20140227327, published August 14, 2014, the entire content of which is incorporated herein by reference. An advantage of cryogels compared to conventional macroporous hydrogels obtained by phase separation is their high reversible deformability. Cryogels may be extremely soft but can be deformed and reform their shape. In certain embodiments, cryogels can be very tough, can withstand high levels of deformations, such as elongation and torsion and can also be squeezed under mechanical force to drain out their solvent content. The improved deformability properties of alginate cryogels originate from the high crosslinking density of the unfrozen liquid channels of the reaction system.

In the cryogelation process, during freezing of the macromonomer (e.g., methacrylated alginate) solution, the macromonomers and initiator system (e.g., APS/TEMED) are expelled from the ice concentrate within the channels between the ice crystals, so that the reactions only take place in these unfrozen liquid channels. After polymerization and, after melting of ice, a porous material is produced whose microstructure is a negative replica of the ice formed. Ice crystals act as porogens. Desired pore size is achieved, in part, by altering the temperature of the cryogelation process. For example, the cryogelation process is typically carried out by quickly freezing the solution at -20 °C. Lowering the temperature to, e.g., -80° C , would result in more ice crystals and lead to smaller pores. In some embodiments, the cryogel is produced by cryo-polymerization of at least methacrylated (MA)-alginate and MA-PEG. In some embodiments, the cryogel is produced by cryo-polymerization of at least MA-alginate, the growth factor, the differentiation factor, and MA-PEG.

In some embodiments, the disclosure also features gelatin scaffolds, e.g., gelatin hydrogels such as gelatin cryogels, which are a cell-responsive platform for biomaterialbased therapy. Gelatin is a mixture of polypeptides that is derived from collagen by partial hydrolysis. These gelatin scaffolds have distinct advantages over other types of scaffolds and hydrogels/cryogels. For example, the gelatin scaffolds of the disclosure support attachment, proliferation, and survival of cells and are degraded by cells, e.g., by the action of enzymes such as matrix metalloproteinases (MMPs) (e.g., recombinant matrix metalloproteinase-2 and -9).

In certain embodiments, prefabricated gelatin cryogels rapidly reassume their approximately original shape ("shape memory") when injected subcutaneously into a subject (e.g., a mammal such as a human, dog, cat, pig, or horse) and elicit little or no harmful host immune response (e.g., immune rejection) following injection.

In some embodiments, the hydrogel (e.g., cryogel) comprises polymers that are modified, e.g., sites on the polymer molecule are modified with a methacrylic acid group (methacrylate (MA)) or an acrylic acid group (acrylate). Exemplary modified hydrogels/cryogels are MA- alginate (methacrylated alginate) or MA-gelatin. In the case of MA-alginate or MA-gelatin, 50% corresponds to the degree of methacrylation of alginate or gelatin. This means that every other repeat unit contains a methacrylated group. The degree of methacrylation can be varied from about 1% to about 100%. Preferably, the degree of methacrylation varies from about 1% to about 90%. In certain embodiments, polymers can also be modified with acrylated groups instead of methacrylated groups. The product would then be referred to as an acrylated-polymer. The degree of methacrylation (or acrylation) can be varied for most polymers. However, some polymers (e.g., PEG) maintain their water-solubility properties even at 100% chemical modification. After crosslinking, polymers normally reach near complete methacrylate group conversion indicating approximately 100% of cross-linking efficiency. As used herein, the term “cross-linking efficiency” refers to the percentage of macromonomers that are covalently linked. For example, the polymers in the hydrogel are 50-100% crosslinked (covalent bonds). The extent of crosslinking correlates with the durability of the hydrogel. Thus, a high level of crosslinking (90-100%) of the modified polymers is desirable.

For example, the highly crosslinked hydrogel/cryogel polymer composition is characterized by at least about 50% polymer crosslinking (e.g., about 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%; it is intended that values and ranges intermediate to the recited values are part of this disclosure). The high level of crosslinking confers mechanical robustness to the structure. Preferably, the percentage of crosslinking is less than about 100%. The composition is formed using a free radical polymerization process and a cryogelation process. For example, the cryogel is formed by cryopolymerization of methacrylated gelatin, methacrylated alginate, or methacrylated hyaluronic acid. In some embodiments, the cryogel comprises a methacrylated gelatin macro monomer or a methacrylated alginate macromonomer at concentration of about 1.5% (w/v) or less (e.g., about 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2% or less; it is intended that values and ranges intermediate to the recited values are part of this disclosure). In some embodiments, the methacrylated gelatin or alginate macromonomer concentration is about 1% (w/v).

In certain embodiments, the cryogel comprises at least about 75% (v/v) pores, e.g., about 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% (v/v) or more pores. It is intended that values and ranges intermediate to the recited values are part of this disclosure. In some embodiments, the pores are interconnected. Interconnectivity is important to the function of the hydrogel and/or cryogel, as without interconnectivity, water would become trapped within the gel. Interconnectivity of the pores permits passage of water (and other compositions such as cells and compounds) in and out of the structure. In certain embodiments, in a fully hydrated state, the hydrogel (e.g., cryogel) comprises at least about 90% water (volume of water I volume of the scaffold) (e.g., between about 90-99%, at least about 92%, 95%, 97%, 99%, or more). For example, at least about 90% (e.g., at least about 92%, 95%, 97%, 99%, or more) of the volume of the cryogel is made of liquid (e.g., water) contained in the pores. It is intended that values and ranges intermediate to the recited values are part of this disclosure. In certain embodiments, in a compressed or dehydrated hydrogel, up to about 50%, 60%, 70% of that water is absent, e.g., the cryogel comprises less than about 25% (e.g., about 20%, 15%, 10%, 5% or less) water.

In certain embodiments, the cryogels of the disclosure comprise pores large enough for a cell to travel through. For example, the cryogel contains pores of about 20-500 pm in diameter, e.g., about 20-30pm, about 30-150pm, about 50-500 pm, about 50-450 pm, about 100-400 pm, about 200-500 pm. In some embodiments, the hydrated pore size is about 1- 500 pm (e.g., about 10-400 pm, about 20-300 pm, about 50-250 pm). In certain embodiments, the cryogel contains pores about 50-80 pm in diameter.

In some embodiments, injectable hydrogels or cryogels are further functionalized by addition of a functional group selected from the group consisting of: amino, vinyl, aldehyde, thiol, silane, carboxyl, azide, or alkyne. Alternatively or in addition, the cryogel is further functionalized by the addition of a further cross-linker agent (e.g., multiple arms polymers, salts, aldehydes, etc.). The solvent can be aqueous, and in particular, acidic or alkaline. The aqueous solvent can comprise a water-miscible solvent (e.g., methanol, ethanol, DMF, DMSO, acetone, dioxane, etc).

For cryogels, the cryo-crosslinking may take place in a mold and the cryogels (which may be injected) can be degradable. The pore size can be controlled by the selection of the main solvent used, the incorporation of a porogen, the freezing temperature and rate applied, the crosslinking conditions (e.g. polymer concentration), and also the type and molecule weight of the polymer used. The shape of the cryogel may be dictated by a mold and can thus take on any shape desired by the fabricator, e.g., various sizes and shapes (disc, cylinders, squares, strings, etc.) are prepared by cryogenic polymerization.

Injectable cryogels can be prepared in the micrometer-scale to centimeter-scale. Exemplary volumes vary from a few hundred pm³ (e.g., about 100-500 pm³) to about 10 cm³. In certain embodiment, an exemplary scaffold composition is between about 100 pm³ to 100 mm³ in size. In various embodiments, the scaffold is between about 10 mm³ to about 100 mm³ in size. In certain embodiments, the scaffold is about 30 mm³ in size.

In some embodiments, the cryogels are hydrated, loaded with compounds and loaded into a syringe or other delivery apparatus. For example, the syringes are prefilled and refrigerated until use. In another example, the cryogel is dehydrated, e.g., lyophilized, optionally with a compound (such as a growth factor or differentiation factor) loaded in the gel and stored dry or refrigerated. Prior to administration, a cryogel-loaded syringe or apparatus may be contacted with a solution containing compounds to be delivered. For example, the barrel of the cryogel pre-loaded syringe is filled with a physiologically- compatible solution, e.g., phosphate-buffered saline (PBS). Alternatively, the cryogel may be administered to a desired anatomical site followed by administration of the physiologically- compatible solution, optionally containing other ingredients, e.g., a growth factor and/or a differentiation factor or together with one or more compounds disclosed herein. The cryogel is then rehydrated and regains its shape integrity in situ. In certain embodiments, the volume of PBS or other physiologic solution administered following cryogel placement is generally about 10 times the volume of the cryogel itself.

The cryogel also has the advantage that, upon compression, the cryogel composition maintains structural integrity and shape memory properties. For example, the cryogel is injectable through a hollow needle. For example, the cryogel returns to its approximately original geometry after traveling through a needle (e.g., a 16 gauge (G) needle, e.g., having a 1.65 mm inner diameter). Other exemplary needle sizes are 16-gauge, an 18-gauge, a 20- gauge, a 22- gauge, a 24-gauge, a 26-gauge, a 28-gauge, a 30-gauge, a 32-gauge, or a 34- gauge needle. Injectable cryogels have been designed to pass through a hollow structure, e.g., very fine needles, such as 18-30 G needles. In certain embodiments, the cryogel returns to its approximately original geometry after traveling through a needle in a short period of time, such as less than about 10 seconds, less than about 5 seconds, less than about 2 seconds, or less than about 1 second.

The cryogels may be injected to a subject using any suitable injection device. For example, the cryogels may be injected using syringe through a needle. A syringe may include a plunger, a needle, and a reservoir that comprises compositions of the present disclosure. The injectable cryogels may also be injected to a subject using a catheter, a cannula, or a stent.

The injectable cryogels may be molded to a desired shape, in the form of rods, square, disc, spheres, cubes, fibers, foams. In some cases, the cryogel is in the shape of a disc, cylinder, square, rectangle, or string. For example, the cryogel composition is between about 100 pm³ to 10 cm³ in size, e.g., between 10 mm³ to 100 mm³ in size. For example, the cryogel composition is between about 1 mm in diameter to about 50 mm in diameter (e.g., about 5 mm). Optionally, the thickness of the cryogel is between about 0.2 mm to about 50 mm (e.g., about 2 mm).

Three exemplary cryogel materials systems are described below. a) Methacrylated gelatin cryogel (CryoGe I MA) - An exemplary cryogel utilized methacrylated gelatin and the results are described in detail in U.S. Patent Application Publication No. 2014-0227327, published August 14, 2014, the entire contents of which are incorporated herein by reference. b) Methacrylated alginate cryogel (CryoMAAIginate) - An exemplary cryogel utilized methacrylated alginate and the results are described in detail in U.S. Patent Application Publication No. 2014-0227327, published August 14, 2014, the entire contents of which are incorporated herein by reference. c) Click Alginate cryogel with Laponite nanoplatelets (CryoClick) - The base material is click alginate (PCT International Patent Application Publication No. WO 2015/154078 published October 8, 2015, hereby incorporated by reference in its entirety). In some examples, the base material contains laponite (commercially available silicate clay used in many consumer products such as cosmetics). Laponite has a large surface area and highly negative charge density which allows it to adsorb positively charged moieties on a variety of proteins and other biologically active molecules by an electrostatic interaction, thereby allowing drug loading. When placed in an environment with a low concentration of drug, adsorbed drug releases from the laponite in a sustained manner. This system allows release of a more flexible array of various agents, e.g., growth factors, compared to the base material alone.

Various embodiments of the present subject matter include delivery vehicles comprising a pore-forming scaffold composition. For example, pores (such as macropores) are formed in situ within a hydrogel following hydrogel injection into a subject. Pores that are formed in situ via degradation of a sacrificial porogen hydrogel within the surrounding hydrogel (bulk hydrogel) facilitate recruitment and trafficking of cells, as well as the release of any composition or agent of the present disclosure, for example, a growth factor, such as BMP-2, a differentiation factor , or a homing factor, or any combination thereof. In some embodiments, the sacrificial porogen hydrogel, the bulk hydrogel, or both the sacrificial porogen hydrogel and the bulk hydrogel may comprise any composition or agent of the present disclosure, for example, a growth factor, a differentiation factor, and/or, a homing factor, or any combination thereof.

In various embodiments, the pore-forming composition becomes macroporous over time when resident in the body of a recipient animal such as a mammalian subject. For example, the pore-forming composition may comprise a sacrificial porogen hydrogel and a bulk hydrogel, wherein the sacrificial porogen hydrogel degrades at least about 10% faster (e.g., at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 50% faster) than the bulk hydrogel. It is intended that values and ranges intermediate to the recited values are part of this disclosure. The sacrificial porogen hydrogel may degrade leaving macropores in its place. In certain embodiments, the macropores are open interconnected macropores. In some embodiments, the sacrificial porogen hydrogel may degrade more rapidly than the bulk hydrogel, because the sacrificial porogen hydrogel (i) is more soluble in water (comprises a lower solubility index), (ii) is cross-linked to protease-mediated degradation motifs as described in U.S. Patent Application Publication No. 2005-0119762, published June 2, 2005 (incorporated herein by reference in its entirety), (iii) comprises a shorter polymer that degrades more quickly compared to that of a longer bulk hydrogel polymer, (iv) is modified to render it more hydrolytically degradable than the bulk hydrogel (e.g., by oxidation), and/or (v) is more enzymatically degradable compared to the bulk hydrogel.

In various embodiments, a scaffold is loaded (e.g., soaked with) with one or more active compounds after polymerization. In certain embodiments, device or scaffold polymer forming material is mixed with one or more active compounds before polymerization. In some embodiments, a device or scaffold polymer forming material is mixed with one or more active compounds before polymerization, and then is loaded with more of the same or one or more additional active compounds after polymerization.

In some embodiments, pore size or total pore volume of a composition or scaffold is selected to influence the release of compounds from the device or scaffold. Exemplary porosities (e.g., nanoporous, microporous, and macroporous scaffolds and devices) and total pore volumes (e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95% or more of the volume of the scaffold) are described herein. It is intended that values and ranges intermediate to the recited values are part of this disclosure. Increased pore size and total pore volume increases the amount of compounds that can be delivered into or near a tissue, such as bone marrow. In some embodiments, a pore size or total pore volume is selected to increase the speed at which active ingredients exit the composition or scaffold. In various embodiments, an active ingredient may be incorporated into the scaffold material of a hydrogel or cryogel, e.g., to achieve continuous release of the active ingredient from the scaffold or device over a longer period of time compared to active ingredient that may diffuse from a pore cavity.

Porosity influences recruitment of cells into devices and scaffolds, growth of cells embedded in devices and scaffolds, and/or the release of substances from devices and scaffolds. Pores may be, e.g., nanoporous, microporous, or macroporous. For example, the diameter of nanopores is less than about 10 nm. Micropores are in the range of about 100 nm to about 20 pm in diameter. Macropores are greater than about 20 pm (e.g., greater than about 100 pm or greater than about 400 pm). Exemplary macropore sizes include about 50 pm, about 100 pm, about 150 pm, about 200 pm, about 250 pm, about 300 pm, about 350 pm, about 400 pm, about 450 pm, about 500 pm, about 550 pm, and about 600 pm. Macropores are those of a size that permit a eukaryotic cell to traverse into or out of the composition. In one example, a macroporous composition has pores of about 400 pm to about 500 pm in diameter. In some embodiments, the pore diameter can be about 0.5 pm to about 10 pm (e.g., about 0.5 pm, about 1 pm, about 1.5 pm, about 2 pm, about 2.5 pm, about 3 pm, about 3.5 pm, about 4 pm, about 4.5 pm, about 5 pm, about 5.5 pm, about 6 pm, about 6.5 pm, about 7 pm, about 7.5 pm, about 8 pm, about 8.5 pm, about 9 pm, about 9.5 pm, or about 10 pm). The preferred pore size depends on the application. In various embodiments, the composition is manufactured in one stage in which one layer or compartment is made and infused or coated with one or more compounds. Exemplary bioactive compositions comprise polypeptides or polynucleotides. In certain embodiments, the composition is manufactured in two or more (3, 4, 5, 6, .... 10 or more) stages in which one layer or compartment is made and infused or coated with one or more compounds followed by the construction of second, third, fourth or more layers, which are in turn infused or coated with one or more compounds in sequence. In some embodiments, each layer or compartment is identical to the others or distinguished from one another by the number or mixture of bioactive compositions as well as distinct chemical, physical and biological properties. Polymers may be formulated for specific applications by controlling the molecular weight, rate of degradation, and method of scaffold formation. Coupling reactions can be used to covalently attach bioactive agent, such as the differentiation factor to the polymer backbone.

In some embodiments, one or more compounds is added to the scaffold compositions using a known method including surface absorption, physical immobilization, e.g., using a phase change to entrap the substance in the scaffold material. For example, a growth factor is mixed with the scaffold composition while it is in an aqueous or liquid phase, and after a change in environmental conditions (e.g., pH, temperature, ion concentration), the liquid gels or solidifies thereby entrapping the bioactive substance. In some embodiments, covalent coupling, e.g., using alkylating or acylating agents, is used to provide a stable, long term presentation of a compound on the scaffold in a defined conformation. Exemplary reagents for covalent coupling of such substances are provided in the table below.

Table 1 : Methods to Covalently Couple Peptides/Proteins to Polymers

DCC: dicyclohexylcarbodiimide

Alginate Scaffolds

In certain embodiments, the composition of the disclosure comprises an alginate hydrogel, e.g., an alginate microgel. Alginates are versatile polysaccharide based polymers that may be formulated for specific applications by controlling the molecular weight, rate of degradation and method of scaffold formation. Alginate polymers are comprised of two different monomeric units, (1 -4)-linked p-D-mannuronic acid (M units) and a L-guluronic acid (G units) monomers, which can vary in proportion and sequential distribution along the polymer chain. Alginate polymers are polyelectrolyte systems which have a strong affinity for divalent cations {e.g., Ca⁺², Mg⁺², Ba⁺²) and form stable hydrogels when exposed to these molecules. See Martinsen A., et al., 1989, Biotech. & Bioeng., 33: 79-89). For example, calcium cross-linked alginate hydrogels are useful for dental applications, wound dressings chondrocyte transplantation and as a matrix for other cell types. Without wishing to be bound by theory, it is believed that G units are preferentially crosslinked using calcium crosslinking, whereas click reaction based crosslinking is more indiscriminate with respect to G units or M units (/.e., both G and M units can be crosslinked by click chemistry). Alginate scaffolds and the methods for making them are known in the art. See, e.g., International Patent Application Publication No. WO 2017/075055 A1, published on May 4, 2017, the entire contents of which are incorporated herein by reference.

In some embodiments, the microgel may comprise an alginate polymer, e.g., a modified alginate polymer, at a weight percent (wt%) of about 1 wt%, about 1.5 wt%, about 2 wt%, about 2.5 wt%, about 3 wt%, about 3.5 wt%, about 4 wt%, about 4.5 wt%, about 5 wt%, about 5.5 wt%, about 6 wt%, about 6.5 wt%, about 7 wt%, about 7.5 wt%, about 8 wt%, about 8.5 wt%, about 9 wt%, about 9.5 wt%, or about 10 wt%. In some embodiments, the microgel may comprise a norbornene modified alginate (Alg-Nb) and/or a tetrazine modified alginate (Alg-Tz) at a weight percent (wt%) of about 1 wt%, about 1.5 wt%, about 2 wt%, about 2.5 wt%, about 3 wt%, about 3.5 wt%, about 4 wt%, about 4.5 wt%, about 5 wt%, about 5.5 wt%, about 6 wt%, about 6.5 wt%, about 7 wt%, about 7.5 wt%, about 8 wt%, about 8.5 wt%, about 9 wt%, about 9.5 wt%, or about 10 wt%.

The alginate polymers useful in the context of the present disclosure can have an average molecular weight from about 20 kDa to about 500 kDa, e.g., from about 20 kDa to about 40 kDa, from about 30 kDa to about 70 kDa, from about 50 kDa to about 150 kDa, from about 130 kDa to about 300 kDa, from about 230 kDa to about 400 kDa, from about 300 kDa to about 450 kDa, or from about 320 kDa to about 500 kDa. In one example, the alginate polymers useful in the present disclosure may have an average molecular weight of about 32 kDa. In another example, the alginate polymers useful in the present disclosure may have an average molecular weight of about 265 kDa. In some embodiments, the alginate polymer has a molecular weight of less than about 1000 kDa, e.g., less than about 900 KDa, less than about 800 kDa, less than about 700 kDa, less than about 600 kDa, less than about 500 kDa, less than about 400 kDa, less than about 300 kDa, less than about 200 kDa, less than about 100 kDa, less than about 50 kDa, less than about 40 kDa, less than about 30 kDa or less than about 25 kDa. In some embodiments, the alginate polymer has a molecular weight of about 1000 kDa, e.g., about 900 kDa, about 800 kDa, about 700 kDa, about 600 kDa, about 500 kDa, about 400 kDa, about 300 kDa, about 200 kDa, about 100 kDa, about 50 kDa, about 40 kDa, about 30 kDa or about 25 kDa. In one embodiment, the molecular weight of the alginate polymers is about 20 kDa.

Coupling reactions can be used to covalently attach bioactive agent, such as an atom, a chemical group, a nucleoside, a nucleotide, a nucleobase, a sugar, a nucleic acid, an amino acid, a peptide, a polypeptide, a protein, or a protein complex, to the polymer backbone.

The term “alginate,” used interchangeably with the term “alginate polymers,” includes unmodified alginate or modified alginate. Modified alginate includes, but not limited to, oxidized alginate (e.g., comprising one or more algoxalate monomer units), reduced alginate (e.g., comprising one or more algoxinol monomer units), MA-alginate (methacrylated alginate), hyaluronic acid, norbornene modified alginate (Alg-Nb), and/or tetrazine modified alginate (Alg-Tz).

In some embodiments, oxidized alginate comprises alginate comprising one or more aldehyde groups, or alginate comprising one or more carboxylate groups. In other embodiments, oxidized alginate comprises highly oxidized alginate, e.g., comprising one or more algoxalate units. Oxidized alginate may also comprise a relatively small number of aldehyde groups (e.g., less than 15%, e.g., 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% aldehyde groups or oxidation on a molar basis). It is intended that values and ranges intermediate to the recited values are part of this disclosure.

In some embodiments, an alginate polymer may be modified to achieve an average degree of substitution (DS) of between about 5 to about 15 {e.g., about 1, about 1.5, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10, about 10.5, about 11, about 11.5, about 12, about 12.5, about 13, about 13.5, about 14, about 14.5, or about 15) functional groups per alginate chain.

In some embodiments, an alginate polymer may be modified with a click reaction moiety to achieve an average degree of substitution (DS) of between about 5 to about 15 {e.g., about 1 , about 1.5, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10, about 10.5, about 11, about 11.5, about 12, about 12.5, about 13, about 13.5, about 14, about 14.5, or about 15) click reaction moieties per alginate chain.

In some embodiments, an alginate polymer may be modified with norbornene (Alg- Nb) or tetrazine (Alg-Tz), e.g., by carbodiimide coupling, to achieve an average degree of substitution (DS) of between about 5 to about 15 e.g., about 1 , about 1.5, about 2, about

11.5, about 12, about 12.5, about 13, about 13.5, about 14, about 14.5, or about 15) functional groups {e.g., Nb or Tz) per alginate chain.

In some embodiments, the microgel may comprise an alginate polymer modified with norbornene (Nb) and/or tetrazine (Tz). In some embodiments, the alginate microgel may comprise a ratio of norbornene (Nb)/tetrazine (Tz) of about 0.1 to about 10 {e.g., about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1 , about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about

2.7, about 2.8, about 2.9, about 3, about 3.1 , about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4, about 4.1 , about 4.2, about 4.3, about

4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5, about 5.1 , about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6, about 6.1 , about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7, about 7.1 , about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about

7.8, about 7.9, about 8, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, about 9, about 9.1, about 9.2, about 9.3, about 9.4, about

9.5, about 9.6, about 9.7, about 9.8, about 9.9, or about 10). In certain embodiments, alginate microgels may be fabricated using microfluidic emulsion, which can provide defined size and shape by controlled droplet formation. Alginate polymer may be first modified with norbornene (Alg-Nb) or tetrazine (Alg-Tz), e.g., by carbodiimide coupling, to achieve an average degree of substitution (DS) of about 13 or about 11.5 functional groups per alginate chain, respectively, as quantified, e.g., by proton nuclear magnetic resonance spectra. Stock solutions of Alg-Nb and Alg-Tz may then be mixed at a final concentration of 2 wt% in a microfluidic device and injected to form microdroplets by emulsion, which may then be crosslinked, e.g., overnight, to generate microgels with a diameter of about 77 ± 2 pm.

The term “alginate” or “alginate polymers” may also include alginate, e.g., unmodified alginate, oxidized alginate or reduced alginate, or methacrylated alginate or acrylated alginate. Alginate may also refer to any number of derivatives of alginic acid (e.g., calcium, sodium or potassium salts, or propylene glycol alginate ). See, e.g., WO1998012228A1, hereby incorporated by reference.

Hyaluronic Acid Scaffolds

In certain embodiments, the composition of the present disclosure comprises a hyaluronic acid hydrogel, e.g., a hyaluronic acid microgel. Hyaluronic acid (HA; conjugate base hyaluronate), is an anionic, nonsulfated glycosaminoglycan distributed widely throughout connective, epithelial, and neural tissues. One of the chief components of the extracellular matrix, hyaluronic acid contributes significantly to cell proliferation and migration. Natural hyaluronic acid is an important component of articular cartilage, muscular connective tissues, and skin.

Hyaluronic acid is a polymer of disaccharides, composed of D-glucuronic acid and N- acetyl-D-glucosamine, linked via alternating p-(1 — >4) and p-(1 — >3) glycosidic bonds. Hyaluronic acid can be 25,000 disaccharide repeats in length. Polymers of hyaluronic acid can range in size from 5,000 to 20,000,000 Da. Hyaluronic acid can also contain silicon.

Hyaluronic acid is energetically stable, in part because of the stereochemistry of its component disaccharides. Bulky groups on each sugar molecule are in sterically favored positions, whereas the smaller hydrogens assume the less-favorable axial positions.

Hyaluronic acid can be degraded by a family of enzymes called hyaluronidases, which are present in many mammals, e.g., a human. Hyaluronic acid can also be degraded via non-enzymatic reactions. These include acidic and alkaline hydrolysis, ultrasonic disintegration, thermal decomposition, and degradation by oxidants.

Due to its high biocompatibility and its common presence in the extracellular matrix of tissues, hyaluronic acid is used to form hydrogels, e.g., cryogels, as a biomaterial scaffold in tissue engineering research. Hyaluronic acid hydrogels are formed through crosslinking. Hyaluronic acid can form a hydrogel, e.g., cryogel, into a desired shape to deliver therapeutic molecules into a host. Hyaluronic acids, for use in the present compositions, can be crosslinked by attaching thiols, methacrylates, hexadecylamides, and tyramines. Hyaluronic acids can also be crosslinked directly with formaldehyde or with divinylsulfone.

The term “hyaluronic acid,” includes unmodified hyaluronic acid or modified hyaluronic acid. Modified hyaluronic acid includes, but is not limited to, oxidized hyaluronic acid and/or reduced hyaluronic acid. In some embodiments, the modified hyaluronic acid comprises a hyaluronic acid modified with a click reaction moiety. Exemplary click reaction moieties include, but are not limited to, an azide moiety, a dibenzocyclooctyne (DBCO) moiety, a transcyclooctene moiety, a tetrazine (Tz) moiety, a norbornene (Nb) moiety, and variants thereof.

The term “hyaluronic acid” or “hyaluronic acid polymers” may also include hyaluronic acid, e.g., unmodified hyaluronic acid, oxidized hyaluronic acid or reduced hyaluronic acid, or methacrylated hyaluronic acid or acrylated hyaluronic acid. Hyaluronic acid may also refer to any number of derivatives of hyaluronic acid.

Collagen Scaffolds

In certain embodiments, the composition of the present disclosure comprises a collagen hydrogel, e.g., a collagen microgel.

In some embodiments, the hydrogel comprises a modified collagen. The modified collagen can comprise a first click reagent, wherein the first click reagent can be capable of covalently crosslinking with a bioorthogonal crosslinker comprising a second click reagent.

In some embodiments, the collagen {e.g., modified collagen) is selected from the group consisting of a type I collagen e.g., COL1A1 and/or COL1A2), a type II collagen {e.g., COL2A1), a type III collagen {e.g., COL3A1), a type IV collagen {e.g., COL4A1, COL4A2, COL4A3, COL4A4, COL4A5, and/or COL4A6), a type V collagen {e.g., COL5A1 , COL5A2, and/or COL5A3), a type VI collagen {e.g., COL6A1, COL6A2, COL6A3, and/or COL6A5), a type VII collagen {e.g., COL7A1), a type VIII collagen {e.g., COL8A1 and/or COL8A2), a type IX collagen {e.g., COL9A1, COL9A2, and/or COL9A3), a type X collagen {e.g., COL10A1), a type XI collagen {e.g., COL11A1 and/or COL11A2), a type XII collagen {e.g., COL12A1), a type XIII collagen {e.g., COL13A1), a type XIV collagen {e.g., COL14A1), a type XV collagen {e.g., COL15A1), a type XVI collagen {e.g., COL16A1), a type XVII collagen {e.g., COL17A1), a type XVIII collagen {e.g., COL18A1), a type XIX collagen e.g., COL19A1), a type XX collagen e.g., COL20A1), a type XXI collagen e.g., COL21A1), a type XXII collagen e.g., COL22A1), a type XXIII collagen e.g., COL23A1), a type XXIV collagen e.g., COL24A1), a type XXV collagen {e.g., COL25A1), a type XXVI collagen {e.g., EMID2), a type XXVII collagen (e.g., COL27A1), a type XXVIII collagen (e.g., COL28A1), a type XXIX collagen (e.g., COL29A1), and combinations thereof.

In some embodiments, the collagen (e.g., modified collagen) is selected from the group consisting of a type I collagen, a type II collagen, a type III collagen, a type IV collagen, a type V collagen, a type VI collagen, a type VII collagen, a type VIII collagen, a type IX collagen, a type X collagen, a type XI collagen, a type XII collagen, a type XIII collagen, a type XIV collagen, a type XV collagen, a type XVI collagen, a type XVII collagen, a type XVIII collagen, a type XIX collagen, a type XX collagen, a type XXI collagen, a type XXII collagen, a type XXIII collagen, a type XXIV collagen, a type XXV collagen, a type XXVI collagen, a type XXVII collagen, a type XXVIII collagen, a type XXIX collagen, and combinations thereof.

In some embodiments, the collagen is selected from the group consisting of COL1A1 , COL1A2, COL2A1, COL3A1, COL4A1, COL4A2, COL4A3, COL4A4, COL4A5, COL4A6, COL5A1, COL5A2, COL5A3, COL6A1, COL6A2, COL6A3, COL6A5, COL7A1, COL8A1 , COL8A2, COL9A1, COL9A2, COL9A3, COL10A1 , COL11A1 , COL11A2, COL12A1, COL13A1, COL14A1 , COL15A1, COL16A1 , COL17A1 , COL18A1, COL19A1, COL20A1, COL21A1, COL22A1 , COL23A1, COL24A1 , COL25A1 , EMID2, COL27A1, COL28A1, COL29A1, and combinations thereof. In some embodiments, the collagen is selected from the group consisting of COL1A1, COL1A2, and combinations thereof. In some embodiments, the collagen comprises COL1A1.

In some embodiments, the collagen comprises a type I collagen {e.g., COL1A1 and/or COL1A2). In some embodiments, the collagen comprises a type II collagen e.g., COL2A1). In some embodiments, the collagen comprises a type III collagen {e.g., COL3A1). In some embodiments, the collagen comprises a type IV collagen {e.g., COL4A1 , COL4A2, COL4A3, COL4A4, COL4A5, and/or COL4A6). In some embodiments, the collagen comprises a type V collagen {e.g., COL5A1, COL5A2, and/or COL5A3). In some embodiments, the collagen comprises a type VI collagen {e.g., COL6A1 , COL6A2, COL6A3, and/or COL6A5). In some embodiments, the collagen comprises a type VII collagen {e.g., COL7A1). In some embodiments, the collagen comprises a type VIII collagen {e.g., COL8A1 and/or COL8A2). In some embodiments, the collagen comprises a type IX collagen {e.g., COL9A1 , COL9A2, and/or COL9A3). In some embodiments, the collagen comprises a type X collagen {e.g., COL10A1). In some embodiments, the collagen comprises a type XI collagen {e.g., COL11A1 and/or COL11A2). In some embodiments, the collagen comprises a type XII collagen {e.g., COL12A1). In some embodiments, the collagen comprises a type XIII collagen {e.g., COL13A1). In some embodiments, the collagen comprises a type XIV collagen {e.g., COL14A1). In some embodiments, the collagen comprises a type XV collagen {e.g., COL15A1). In some embodiments, the collagen comprises a type XVI collagen (e.g., COL16A1). In some embodiments, the collagen comprises a type XVII collagen e.g., COL17A1). In some embodiments, the collagen comprises a type XVIII collagen e.g., COL18A1). In some embodiments, the collagen comprises a type XIX collagen e.g., COL19A1). In some embodiments, the collagen comprises a type XX collagen e.g., COL20A1). In some embodiments, the collagen comprises a type XXI collagen {e.g., COL21A1). In some embodiments, the collagen comprises a type XXII collagen {e.g., COL22A1). In some embodiments, the collagen comprises a type XXIII collagen {e.g., COL23A1). In some embodiments, the collagen comprises a type XXIV collagen {e.g., COL24A1). In some embodiments, the collagen comprises a type XXV collagen {e.g., COL25A1). In some embodiments, the collagen comprises a type XXVI collagen {e.g., EMID2). In some embodiments, the collagen comprises a type XXVII collagen {e.g., COL27A1). In some embodiments, the collagen comprises a type XXVIII collagen {e.g., COL28A1). In some embodiments, the collagen comprises a type XXIX collagen {e.g., COL29A1).

In some embodiments, the collagen is a modified collagen comprising a click reagent.

In some embodiments, the collagen {e.g., modified collagen) is present in an amount of about 1 mg/mL to about 10 mg/mL {e.g., about 1 mg/mL, about 1.5 mg/mL, about 2 mg/mL, about 2.5 mg/mL, about 3 mg/mL, about 3.5 mg/mL, about 4 mg/mL, about 4.5 mg/mL, about 5 mg/mL, about 5.5 mg/mL, about 6 mg/mL, about 6.5 mg/mL, about 7 mg/mL, about 7.5 mg/mL, about 8 mg/mL, about 8.5 mg/mL, about 9 mg/mL, about 9.5 mg/mL, or about 10 mg/mL). In some embodiments, the collagen {e.g., modified collagen) is present in an amount of about 1 mg/mL to about 2 mg/mL. In some embodiments, the collagen {e.g., modified collagen) is present in an amount of about 1 mg/mL to about 3 mg/mL. In some embodiments, the collagen {e.g., modified collagen) is present in an amount of about 2 mg/mL to about 3 mg/mL. In some embodiments, the collagen {e.g., modified collagen) is present in an amount of about 2 mg/mL to about 4 mg/mL. In some embodiments, the collagen {e.g., modified collagen) is present in an amount of about 3 mg/mL to about 4 mg/mL. In some embodiments, the collagen {e.g., modified collagen) is present in an amount of about 3 mg/mL to about 5 mg/mL. In some embodiments, the collagen {e.g., modified collagen) is present in an amount of about 4 mg/mL to about 5 mg/mL. In some embodiments, the collagen {e.g., modified collagen) is present in an amount of about 4 mg/mL to about 6 mg/mL. In some embodiments, the collagen {e.g., modified collagen) is present in an amount of about 5 mg/mL to about 6 mg/mL. In some embodiments, the collagen {e.g., modified collagen) is present in an amount of about 5 mg/mL to about 7 mg/mL. In some embodiments, the collagen {e.g., modified collagen) is present in an amount of about 6 mg/mL to about 7 mg/mL. In some embodiments, the collagen {e.g., modified collagen) is present in an amount of about 6 mg/mL to about 8 mg/mL. In some embodiments, the collagen {e.g., modified collagen) is present in an amount of about 7 mg/mL to about 8 mg/mL. In some embodiments, the collagen {e.g., modified collagen) is present in an amount of about 7 mg/mL to about 9 mg/mL. In some embodiments, the collagen e.g., modified collagen) is present in an amount of about 8 mg/mL to about 9 mg/mL. In some embodiments, the collagen {e.g., modified collagen) is present in an amount of about 8 mg/mL to about 10 mg/mL. In some embodiments, the collagen {e.g., modified collagen) is present in an amount of about 9 mg/mL to about 10 mg/mL.

Gelatin Scaffolds

In certain embodiments, the composition of the present disclosure comprises a gelatin hydrogel, e.g., a gelatin microgel.

Gelatin is a heterogenous mixture of polypeptides that can be derived from collagen by partial hydrolysis. Collagen is an insoluble fibrous protein that occurs in vertebrates and is the main component of connective tissues and bones. For example, the collagen that can be used to make gelatin is isolated, e.g., from the connective tissues and bones of animals, e.g., from skin and bones. Gelatin is commercially available at a pharmaceutical grade. Exemplary types of gelatin include gelatin derived from porcine skin, beef skin, or bone. For example, gelatin can be derived by acid treatment of collagenous material (also called Type A gelatin) or alkali treatment of collagenous material (also called Type B gelatin). Other examples of gelatin include recombinant human gelatin and low endotoxin gelatin preparation from animal origin. In some embodiments, the hydrogel comprises a modified gelatin.

Porous and Pore-forming Scaffolds

The microgels, e.g., microgel scaffolds, of the present disclosure may be nonporous or porous. In certain embodiments, the microgels, e.g., microgel scaffolds, of the present disclosure are porous. Porosity of the scaffold composition influences migration of the cells through the device. Pores may be nanoporous, microporous, or macroporous. For example, the diameter of nanopores is less than about 10 nm. Micropores are in the range of about 100 nm to about 20 pm in diameter. Macropores are greater than about 20 pm {e.g., greater than about 100 pm or greater than about 400 pm) in diameter. Exemplary macropore sizes include about 50 pm, 100 pm, 150 pm, 200 pm, 250 pm, 300 pm, 350 pm, 400 pm, 450 pm, 500 pm, 550 pm, and 600 pm in diameter. It is intended that values and ranges intermediate to the recited values are part of this disclosure. Macropores are of a size that permits a eukaryotic cell to traverse into or out of the composition. In certain embodiments, a macroporous composition has pores of about 400 pm to 500 pm in diameter. The size of pores may be adjusted for different purpose. For example, for cell recruitment and cell release, the pore diameter may be greater than 50 pm. In certain embodiments, a macroporous composition has pores of about 50 pm - about 80 pm in diameter.

In some embodiments, the scaffolds contain pores before the administration into a subject. In some embodiments, the scaffolds comprise a pore-forming scaffold composition. Pore-forming scaffolds and the methods for making pore-forming scaffolds are known in the art. See, e.g., U.S. Patent Publication US2014/0079752A1 , the content of which is incorporated herein by reference. In certain embodiments, the pore-forming scaffolds are not initially porous, but become macroporous over time resident in the body of a recipient animal such as a mammalian subject. In certain embodiments, the pore-forming scaffolds are hydrogel scaffolds. The pore may be formed at different time, e.g., after about 12 hours, or 1, 3, 5, 7, or 10 days or more after administration, i.e., resident in the body of the subject.

In certain embodiments, the pore-forming scaffolds comprise a first hydrogel and a second hydrogel, wherein the first hydrogel degrades at least about 10% faster (e.g., at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50% faster, at least about 2 times faster, or at least about 5 times faster) than the second hydrogel. It is intended that values and ranges intermediate to the recited values are part of this disclosure. In certain embodiments, the first hydrogel comprises a porogen that degrades leaving a pore in its place. For example, the first hydrogel is a porogen and the resulting pore after degradation in situ is within 25% of the size of the initial porogen, e.g., within 20%, within 15%, or within 10% of the size of the initial porogen. Preferably, the resulting pore is within 5% of the size of the initial porogen. It is intended that values and ranges intermediate to the recited values are part of this disclosure. The first hydrogel may degrade faster than the second hydrogel due to the difference in their physical, chemical, and/or biological properties. In certain embodiments, the first hydrogel degrades more rapidly than the second hydrogel, because the first hydrogel is more soluble in water (comprises a lower solubility index). In certain embodiments, the first hydrogel degrades more rapidly because it is cross-linked to protease-mediated degradation motifs as described in U.S. Patent Publication US2005/0119762A1 , the content of which is incorporated herein by reference.

In certain embodiments, the molecular mass of the polymers used to form the first hydrogel composition (a porogen) is approximately 50 kilodaltons (kDa), and the molecular mass of the polymers used to form the second hydrogel composition (bulk) is approximately 250 kDa. A shorter polymer (e.g., that of a porogen) degrades more quickly compared to that of a longer polymer (e.g., that of the bulk composition). In certain embodiments, a composition is modified to render it more hydrolytically degradable by virtue of the presence of sugar groups (e.g., approximately 3-10% sugar of an alginate composition). In certain embodiments, the porogen hydrogel is chemically modified, such as oxidized, to render it more susceptible to degradation. In some embodiments, the porogen hydrogel is more enzymatically degradable compared to the bulk hydrogel. The composite (first and second hydrogel) composition is permeable to bodily fluids, e.g., containing an enzyme which is exposed to the composition and degrades the porogen hydrogel. In some embodiments, the second hydrogel is cross-linked around the first hydrogel, i.e., the porogens (first hydrogel) are completely physically entrapped in the bulk (second) hydrogel.

The click reagents disclosed herein can be provided in the bulk hydrogel or the porogen hydrogel. In exemplary embodiments, the click reagents, e.g., polymers or nanoparticles, are provided in the bulk hydrogel.

In certain embodiments, hydrogel micro-beads (“porogens”) are formed. Porogens are encapsulated into a “bulk” hydrogel that is either non-degradable or which degrades at a slower rate compared to the porogens. Immediately after hydrogel formation, or injection into the desired site in vivo, the composite material lacks pores. Subsequently, porogen degradation causes pores to form in situ. The size and distribution of pores are controlled during porogen formation, and mixing with the polymers which form the bulk hydrogel.

In some embodiments, the polymer utilized in the pore-forming scaffolds is naturally- occurring or synthetically made. In one example, both the porogens and bulk hydrogels are formed from alginate.

In certain embodiments, the alginate polymers suitable for porogen formation have a molecular weight from 5,000 to 500,000 Daltons. The polymers are optionally further modified (e.g., by oxidation with sodium periodate, (Bouhadir et al., 2001, Biotech. Prog. 17:945-950, hereby incorporated by reference), to facilitate rapid degradation. In certain embodiments, the polymers are crosslinked by extrusion through a nebulizer with co-axial airflow into a bath of divalent cation (for example, Ca²⁺ or Ba²⁺) to form hydrogel microbeads. Higher airflow rate leads to lower the porogen diameter.

In some embodiments, the porogen hydrogel microbeads contain oxidized alginate. For example, the porogen hydrogel can contain about 1-50% (w/v) oxidized alginate. In exemplary embodiments, the porogen hydrogel can contain about 1-10% oxidized alginate. In one embodiment, the porogen hydrogel contains about 7.5% oxidized alginate.

In certain embodiments, the concentration of divalent ions used to form porogens may vary from about 5 to about 500 mM, and the concentration of polymer from about 1 % to about 5% by weight/volume. However, any method which produces porogens that are significantly smaller than the bulk phase is suitable. Porogen chemistry can further be manipulated to produce porogens that interact with host proteins and/or cells, or inhibit interactions with host proteins and/or cells.

The alginate polymers suitable for formation of the bulk hydrogel have a molecular weight from about 5,000 to about 500,000 Da. The polymers may be further modified (for example, by oxidation with sodium periodate), to facilitate degradation, as long as the bulk hydrogel degrades more slowly than the porogen. The polymers may also be modified to present biological cues to control cell responses (e.g., integrin binding adhesion peptides such as RGD). Either the porogens or the bulk hydrogel may also encapsulate bioactive factors such as oligonucleotides, growth factors or drugs to further control cell responses. The concentration of divalent ions used to form the bulk hydrogel may vary from about 5 to about 500 mM, and the concentration of polymer from about 1% to about 5% by weight/volume. The elastic modulus of the bulk polymer is tailored for its purpose, e.g., to recruit stem cells or progenitor cells.

Methods relevant to generating the hydrogels described herein include the following. Bouhadir et al., 1999, Polymer, 40: 3575-84 (incorporated herein by reference in its entirety) describes the oxidation of alginate with sodium periodate, and characterizes the reaction. Bouhadir et al., 2001 , Biotechnol. Prog., 17: 945-50 (incorporated herein by reference in its entirety) describes oxidation of high molecular weight alginate to form alginate dialdehyde (alginate dialdehyde is high molecular weight (M_w) alginate in which a certain percent, e.g., 5%, of sugars in alginate are oxidized to form aldehydes), and application to make hydrogels degrade rapidly. Kong et al., 2002, Polymer, 43: 6239-46 (incorporated herein by reference in its entirety) describes the use of gamma-irradiation to reduce the weight-averaged molecular weight (M_w) of guluronic acid (GA) rich alginates without substantially reducing GA content (e.g., the gamma irradiation selectively attacks mannuronic acid, MA blocks of alginate). Alginate is comprised of GA blocks and MA blocks, and it is the GA blocks that give alginate its rigidity (elastic modulus). Kong et al., 2002, Polymer, 43: 6239-46 (incorporated herein by reference in its entirety) shows that binary combinations of high M_w, GA rich alginate with irradiated, low M_w, high GA alginate crosslinks with calcium to form rigid hydrogels, but which degrade more rapidly and also have lower solution viscosity than hydrogels made from the same overall weight concentration of only high M_w, GA rich alginate. Alsberg et al., 2003, J Dent Res, 82(11): 903-8 (incorporated herein by reference in its entirety) describes degradation profiles of hydrogels made from irradiated, low M_w, GA- rich alginate, with application in bone tissue engineering. Kong et al., 2004, Adv. Mater, 16(21): 1917-21 (incorporated herein by reference) describes control of hydrogel degradation profile by combining gamma irradiation procedure with oxidation reaction, and application to cartilage engineering. Techniques to control degradation of hydrogen biomaterials are well known in the art. For example, Lutolf MP et al., 2003, Nat Biotechnol., 21: 513-8 (incorporated herein by reference in its entirety) describes poly(ethylene glycol) based materials engineered to degrade via mammalian enzymes (MMPs). Bryant SJ et al., 2007, Biomaterials, 28(19): 2978-86 (US 7,192,693 B2; incorporated herein by reference in its entirety) describes a method to produce hydrogels with macro-scale pores. A pore template (e.g., polymethylmethacrylate beads) is encapsulated within a bulk hydrogel, and then acetone and methanol are used to extract the porogen while leaving the bulk hydrogel intact. Silva et al., 2008, Proc. Natl. Acad. Sci USA, 105(38): 14347-52 (incorporated herein by reference in its entirety; US 2008/0044900) describes deployment of endothelial progenitor cells from alginate sponges. The sponges are made by forming alginate hydrogels and then freeze- drying them (ice crystals form the pores). Ali et al., 2009, Nat Mater (incorporated herein by reference in its entirety) describes the use of porous scaffolds to recruit dendritic cells and program them to elicit anti-tumor responses. Huebsch et al., 2010, Nat Mater, 9: 518-26 (incorporated herein by reference in its entirety) describes the use of hydrogel elastic modulus to control the differentiation of encapsulated mesenchymal stem cells.

In some embodiments, the scaffold composition comprises open interconnected macropores. Alternatively or in addition, the scaffold composition comprises a pore-forming scaffold composition. In certain embodiments, the pore-forming scaffold composition may comprise a sacrificial porogen hydrogel and a bulk hydrogel, wherein the pore-forming scaffold composition lacks macropores. For example, the sacrificial porogen hydrogel may degrade at least 10% faster than the bulk hydrogel leaving macropores in its place following administration of said pore-forming scaffold into a subject. In some embodiments, the sacrificial porogen hydrogel is in the form of porogens that degrade to form said macropores. For example, the macropores may comprise pores having a diameter of, e.g., about 10-400 pm.

III. Active Agents

The compositions (e.g., microgels and/or granular hydrogels) of the present disclosure can comprise an active agent.

As used herein, the term “active agent” refers to an active ingredient that is intended for use in a particular application. In some embodiments, the term “active agent” refers to an agent that possesses therapeutic, prophylactic, or diagnostic properties in vivo, for example when administered to a human subject or an animal, including mammals and domestic animals.

Examples of active agents include, but are not limited to, amino acids, proteins, peptides, bioactive ligands, antibodies, growth factors, nucleic acids, vectors, sugars, antigens, vaccines, viruses, enzymes, cells, small molecules, drugs, and any combination thereof. In some embodiments, the active agent may be selected from the group consisting of a growth factor, a differentiation factor, a homing factor, a chemoattractant, an adjuvant, an antigen, and a combination thereof. In some embodiments, the active agent may comprise a bioactive ligand (e.g., a T cell ligand) capable of activating T cells and regulating their functions. These activating molecules may mediate direct, indirect, or semi-direct activation of a target population of T cells. In some embodiments, the T cell activating molecules mediate direct activation of T cells.

The term “T cell ligand” can refer to any natural or synthetic molecule (e.g., small molecule, protein, peptide, lipid, carbohydrate, and/or nucleic acid) that can bind to the T cell. Such ligands may be T-cell activating ligands or T-cell inhibiting ligands. In some embodiments, the T cell ligands can be attached and/or presented on the surface of the microgel, e.g., granular hydrogel.

In some embodiments, the T cell ligand can be an antibody molecule or antigenbinding fragment thereof. The term “antibody,” as used herein, broadly refers to any immunoglobulin (Ig) molecule comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains, or any functional fragment, mutant, variant, or derivation thereof, which retains the essential epitope binding features of an Ig molecule. Such mutant, variant, or derivative antibody formats are known in the art. Examples of antibodies and antigenbinding fragments thereof include, but are not limited to: single-chain Fvs (scFvs), Fab fragments, Fab’ fragments, F(ab’)2, disulfide-linked Fvs (sdFvs), Fvs, and fragments containing either a VL or a VH domain.

In some embodiments, the T cell ligand can be an anti-idiotype antibody or derivative thereof that binds to the antigen-binding domain of another antibody or a receptor molecule, such as a T cell receptor (TCR). In some embodiments, the one or more T-cell ligands can be selected from the group consisting of an anti-idiotype CD3 antibody (aCD3) or an antigen-binding fragment thereof; an anti-idiotype CD5 antibody (aCD5) or an antigenbinding fragment thereof; an anti-idiotype CD7 antibody (aCD7) or an antigen-binding fragment thereof; an anti-idiotype CD28 antibody (aCD28) or an antigen-binding fragment thereof; an anti-idiotype CD19 antibody (aCD19) or an antigen-binding fragment thereof; an anti-idiotype CD20 antibody (aCD20) or an antigen-binding fragment thereof; an anti-idiotype CD22 antibody (aCD22) or an antigen-binding fragment thereof; an anti-idiotype CD70 antibody (aCD70) or an antigen-binding fragment thereof; an anti-idiotype CD123 antibody (aCD123) or an antigen-binding fragment thereof; an anti-idiotype CS1 antibody (aCS1) or an antigen-binding fragment thereof; an anti-idiotype BCMA antibody (aBCMA) or an antigen-binding fragment thereof; an anti-idiotype SLAMF7 antibody (aSLAMF7) or an antigen-binding fragment thereof; an anti-idiotype Claudin-6 antibody (aClaudin-6) or an antigen-binding fragment thereof; an anti-idiotype NKG2D antibody (aNKG2D) or an antigenbinding fragment thereof; an anti-idiotype NKG2DL antibody (aNKG2DL) or an antigenbinding fragment thereof; an anti-idiotype GD2 antibody (aGD2) or an antigen-binding fragment thereof; an anti-idiotype Her2 antibody (aHer2) or an antigen-binding fragment thereof; and an anti-idiotype mesothelin antibody (aMSLN) or an antigen-binding fragment thereof; or a combination thereof.

In some embodiments, the T-cell ligand can comprise a T-cell antigen or derivative thereof that binds to a T cell receptor (TOR) of the T-cells in the subject. In some embodiments, the T cell ligand can be selected from the group consisting of a CD3 molecule or a fragment thereof; a CD5 molecule or a fragment thereof; a CD7 molecule or a fragment thereof; a CD28 molecule or a fragment thereof; a CD19 molecule or a fragment thereof; a CD20 molecule or a fragment thereof; a CD22 molecule or a fragment thereof; a CD70 molecule or a fragment thereof; a CD123 molecule or a fragment thereof; a CS1 molecule or a fragment thereof; a BCMA molecule or a fragment thereof; a SLAMF7 molecule or a fragment thereof; a Claudin-6 molecule or a fragment thereof; a NKG2D molecule or a fragment thereof; a NKG2DL molecule or a fragment thereof; a GD2 molecule or a fragment thereof; a Her2 molecule or a fragment thereof; and a mesothelin (MSLN) molecule or a fragment thereof; or a combination thereof.

In some embodiments, the T cell ligand can be selected from the group consisting of an anti-CD3 antibody or an antigen-binding fragment thereof, an anti-macrophage scavenger receptor (MSR1) antibody or an antigen-binding fragment thereof, an anti-T-cell receptor (TCR) antibody or an antigen-binding fragment thereof, an anti-CD2 antibody or an antigenbinding fragment thereof, an anti-CD47 antibody or an antigen-binding fragment thereof, a major histocompatibility complex (MHC) molecule loaded with an MHC peptide or a multimer thereof, and an MHC-immunoglobulin (Ig) conjugate or a multimer thereof, ICAM-1 , or a combination thereof.

In one embodiment, the target T-cells are activated in a CD3-dependent manner. It is generally believed that T cell activation requires a T cell receptor (TCR) to recognize its cognate peptide in the context of an MHC molecule. In addition, the association of CD3 with the TCR-peptide-MHC complex transmits the activation signal to intracellular signaling molecules to initiate a signaling cascade in the T cell. See, Ryan et al., Nature Reviews Immunology 10, 7, 2010. The CD3 receptor complex found on T-cells contains a CD3y chain, a CD35 chain, and two CD3E chains, which associate with TCR and the ^-chain (zetachain; CD247) to generate an activation signal in T cells. The TCR, ^-chain, and CD3 molecules together constitute the T cell receptor (TCR) complex. Binding of an activating molecule, e.g., an antibody, to one or more of the members of the TCR complex may activate the T-cell. In some embodiments, the active agent may comprise bioactive ligands capable of binding to a co-stimulatory antigen. The term “co-stimulatory molecule” refers to a group of immune cell surface receptor/ligands which engage between T cells and antigen presenting cells and generate a stimulatory signal in T cells which combines with the stimulatory signal (i.e. , “co-stimulation”) in T cells that results from T cell receptor (“TCR”) recognition of antigen on antigen presenting cells. In some embodiments, the active agent may comprise a T-cell costimulatory molecule which can bind to, e.g., CD28, 4.1 BB (CD137), 0X40 (CD134), CD27 (TNFRSF7), GITR (CD357), CD30 (TNFRSF8), HVEM (CD270), LT R (TNFRSF3), DR3 (TNFRSF25), ICOS (CD278), CD226 (DNAM1), CRTAM (CD355),TIM1 (HAVCR1 , KIM1), CD2 (LFA2, 0X34), SLAM (CD150, SLAMF1), 2B4 (CD244, SLAMF4), Ly108 (NTBA, CD352, SLAMF6), CD84 (SLAMF5), Ly9 (CD229, SLAMF3), CD279 (PD-1) and/or CRACC (CD319, BLAME).

In some embodiments, the T-cell activating antibody used in the compositions and methods of the disclosure comprises an anti-CD3 antibody. Exemplary anti-CD3 antibodies include, without limitation, muromonab (OKT3), otelixizumab (TRX4), teplizumab (hOKT3y1 (Ala-Ala)), visilizumab, an antibody recognizing 17-19 kD e-chain of CD3 within the CD3 antigen/T cell antigen receptor (TCR) complex (HIT3a), and an antibody recognizing a 20 kDa subunit of the TCR complex within CD3e (UCHT 1), or an antigen-binding fragment thereof. Other anti-CD3 antibodies, including, antigen-binding fragments thereof are known in the art.

In some embodiments, the active agent (e.g., antibody or an antigen binding fragment thereof) can be modified with a click reaction moiety. Exemplary click reaction moieties include, but are not limited to, an azide moiety, a dibenzocyclooctyne (DBCO) moiety, a transcyclooctene moiety, a tetrazine (Tz) moiety, a norbornene (Nb) moiety, and variants thereof. In some embodiments, the active agent (e.g., antibody or an antigenbinding fragment thereof) can be modified with an average of about 1 to about 10 (e.g., about 0.5, about 1 , about 1 .5, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, or about 10) click reaction moieties, e.g., dibenzocyclooctyne (DBCO) moieties, per active agent.

In some embodiments, the T-cell activating molecule can be an anti-CD3 antibody or an antigen-binding fragment thereof. In some embodiments, the anti-CD3 antibody or an antigen-binding fragment thereof can be modified with a click reaction moiety. In some embodiments, the anti-CD3 antibody or an antigen-binding fragment thereof can be modified with a dibenzocyclooctyne (DBCO) moiety.

In some embodiments, the T-cell activating molecule may include, for example, an anti-CD28 antibody or an antigen-binding fragment thereof. In some embodiments, the anti- CD28 antibody or an antigen-binding fragment thereof can be modified with a click reaction moiety. In some embodiments, the anti-CD28 antibody or an antigen-binding fragment thereof can be modified with a dibenzocyclooctyne (DBCO) moiety.

In some embodiments, the microgels {e.g., granular hydrogels) can be modified (e.g., surface modified) to comprise an antibody. In some embodiments, the microgels {e.g., granular hydrogels) can be modified (e.g., surface modified) to comprise an aCD3 and/or aCD28 antibodies modified with DBCO, e.g., by reducing the disulfide linkage using TCEPHCI (1 :30 molar ratio) and then reacting with DBCO-PEG12-maleimide (Conju Probe, 1 :60 molar ratio) at 4 °C overnight.

In some embodiments, the anti-CD3 antibody or an antigen-binding fragment thereof can be modified with an average of about 1 to about 10 (e.g., about 0.5, about 1, about 1.5, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about

6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, or about 10) click reaction moieties, e.g., dibenzocyclooctyne (DBCO) moieties, per antibody.

In some embodiments, the anti-CD28 antibody or an antigen-binding fragment thereof can be modified with an average of about 1 to about 10 (e.g., about 0.5, about 1 , about 1.5, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, or about 10) click reaction moieties, e.g., dibenzocyclooctyne (DBCO) moieties, per antibody.

4.8, about 5, about 5.2, about 5.4, about 5.6, about 5.8, about 6, about 6.2, about 6.4, about

6.6, about 6.8, about 7, about 7.2, about 7.4, about 7.6, about 7.8, about 8, about 8.2, about 8.4, about 8.6, about 8.8, about 9, about 9.2, about 9.4, about 9.6, about 9.8, or about 10 pg/cm²).

3.9, about 4, about 4.1 , about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5, about 5.1 , about 5.2, about 5.3, about 5.4, about 5.5, about

5.6, about 5.7, about 5.8, about 5.9, about 6, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, or about 7 pg/cm²). In some embodiments, the microgel may comprise an active agent (e.g., ligand) at a predefined density (e.g., ligand density) of about 0 to about 10 pg/cm² and a percentage of cytotoxicity of less than 20% (e.g., about 1% or less, about 2% or less, about 3% or less, about 4% or less, about 5% or less, about 6% or less, about 7% or less, about 8% or less, about 9% or less, about 10% or less, about 11% or less, about 12% or less, about 13% or less, about 14% or less, about 15% or less, about 16% or less, about 17% or less, about 18% or less, about 19% or less, or about 20% or less). In some embodiments, the active agent may be present at between about 1 ng to about 1000 pg. In some embodiments, the active agent may be present at between about 1 ng to about 100 pg. In some embodiments, the active agent may be present at between about 1 pg to about 2 ng per microgel. In some embodiments, the active agent may be present at about 1 pg per microgel. In some embodiments, the active agent may be present at between about 1 ng to about 500 ng. In some embodiments, the active agent may be present at between about 1 ng to about 100 ng e.g., about 1 ng, about 2 ng, about 3 ng, about 4 ng, about 5 ng, about 6 ng, about 7 ng, about 8 ng, about 9 ng, about 10 ng, about 11 ng, about 12 ng, about 13 ng, about 14 ng, about 15 ng, about 16 ng, about 17 ng, about 18 ng, about 19 ng, about 20 ng, about 21 ng, about 22 ng, about 23 ng, about 24 ng, about 25 ng, about 26 ng, about 27 ng, about 28 ng, about 29 ng, about 30 ng, about 31 ng, about 32 ng, about 33 ng, about 34 ng, about 35 ng, about 36 ng, about 37 ng, about 38 ng, about 39 ng, about 40 ng, about 41 ng, about 42 ng, about 43 ng, about 44 ng, about 45 ng, about 46 ng, about 47 ng, about 48 ng, about 49 ng, about 50 ng, about 51 ng, about 52 ng, about 53 ng, about 54 ng, about 55 ng, about 56 ng, about 57 ng, about 58 ng, about 59 ng, about 60 ng, about 61 ng, about 62 ng, about 63 ng, about 64 ng, about 65 ng, about 66 ng, about 67 ng, about 68 ng, about 69 ng, about 70 ng, about 71 ng, about 72 ng, about 73 ng, about 74 ng, about 75 ng, about 76 ng, about 77 ng, about 78 ng, about 79 ng, about 80 ng, about 81 ng, about 82 ng, about 83 ng, about 84 ng, about 85 ng, about 86 ng, about 87 ng, about 88 ng, about 89 ng, about 90 ng, about 91 ng, about 92 ng, about 93 ng, about 94 ng, about 95 ng, about 96 ng, about 97 ng, about 98 ng, about 99 ng, or about 100 ng).

In certain embodiments, the active agent can retain their bioactivity over an extended period of time. The term “bioactivity,” as used herein, refers to the beneficial or adverse effects of an active agent. The bioactivity of the active agent may be measured by any appropriate means. In some embodiments, the active agent retains their bioactivity for at least 10 days, 12 days, 14 days, 20 days, or 30 days or more after the incorporation into or onto the scaffold and/or a polymer coating.

Growth Factors The compositions (e.g., microgels and/or granular hydrogels) of the present disclosure can comprise a growth factor. The term “growth factor,” as used herein, refers to an agent that is capable of stimulating cellular growth, proliferation, healing, and/or cellular differentiation. In certain embodiments, growth factors are polypeptides. Growth factor polypeptides typically act as signaling molecules. In certain embodiments, the growth factor polypeptides are cytokines.

In certain embodiments, the growth factor can recruit a cell to the scaffold following the administration of the composition to a subject. The recruited cell may be autologous. For example, the recruited cell may be a stromal cell from the subject. In certain embodiments, the autologous cell may be a stem cell (e.g., umbilical cord stem cells) of the subject. The recruited cell may also be syngeneic, allogeneic or xenogeneic. As used herein, the term “syngeneic” refers to genetically identical, or sufficiently identical and immunologically compatible as to allow for transplantation. For example, syngeneic cells may include transplanted cells obtained from an identical twin. As used herein, the term “allogeneic” refers to cells that are genetically dissimilar, although from individuals of the same species. As used herein, the term “xenogeneic” refers to cells derived from a different species and therefore genetically different.

For example, the recruited cell may be a donor cell in a transplantation. In certain embodiments, the transplantation is a hematopoietic stem cell transplantation (HSCT). As used herein, HSCT refers to the transplantation of multipotent hematopoietic stem cells or hematopoietic progenitor cells, usually derived from bone marrow, peripheral blood, or umbilical cord blood.-HSCT may be autologous (the patient's own stem cells or progenitor cells are used), allogeneic (the stem cells or progenitor cells come from a donor), syngeneic (from an identical twin) or xenogenic (from different species).

The growth factors of the present disclosure may induce the formation of a tissue or organ within or around the administered composition. In certain embodiments, the tissue or organ is a bony tissue or hematopoietic tissue. The tissue formation may be restricted to the scaffold of the composition.

Methods of incorporating polypeptides (e.g., growth factor and/or differentiation polypeptides) are known in the art. See, US Patent Nos.: 8,728,456; 8,067,237; and 10,045,947; US Patent Publication No.: US20140079752; International Patent Publication No.: WO 2017/136837; International Patent Application Publication No.: WO 2020/131582; incorporated herein by reference in their entirety. The release of the growth factor polypeptides may be controlled. The methods of controlled release of polypeptides (e.g., growth factor polypeptides) are known in the art. See, US Patent Nos.: 8,728,456; 8,067,237; 10,045,946, incorporated by reference in their entirety. In certain embodiments, the growth factors (e.g., BMP-2) may be released over an extended period of time, such as 7-30 days or longer. The controlled release of the growth factors may affect the timing of the formation of the tissue or organ within the scaffold. In certain examples, the release of the growth factors is controlled with the goal of creating a functional, active bone nodule or tissue within one to two weeks after subcutaneous injection of the compositions of the present disclosure.

In certain embodiments, the growth factors retain their bioactivity over an extended period of time. The term “bioactivity,” as used herein, refers to the beneficial or adverse effects of an agent, such as a growth factor. The bioactivity of the growth factor may be measured by any appropriate means. For example, the bioactivity of BMP-2 may be measured by its capacity to induce the formation of bone nodule or tissue and/or recruit cells into the scaffold. In certain example, the growth factors retain their bioactivity for at least 10 days, 12 days, 14 days, 20 days, or 30 days after the incorporation of the growth factors into the scaffold.

Exemplary growth factors include, but are not limited to, bone morphogenetic proteins (BMP), epidermal growth factor (EGF), transforming growth factor beta (TGF-P), granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), nerve growth factor (NGF), neurotrophins, Platelet-derived growth factor (PDGF), erythropoietin (EPO), thrombopoietin (TPO), myostatin (GDF-8), growth differentiation factor-9 (GDF9), acidic fibroblast growth factor (aFGF or FGF-1), basic fibroblast growth factor (bFGF or FGF-2), epidermal growth factor (EGF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), and interleukins.

In some embodiments, the growth factor comprises a protein belonging to the transforming growth factor beta (TGF-P) superfamily. As used herein, TGF-p superfamily is a large group of structurally related cell regulatory proteins. TGF-p superfamily includes four major subfamilies: the TGF-p subfamily, the bone morphogenetic proteins and the growth differentiation factors, the activing and inhibin subfamilies, and a group encompassing various divergent members. Proteins from the TGF-p superfamily are active as homo- or heterodimer, the two chains being linked by a single disulfide bond. TGF-p superfamily proteins interact with a conserved family of cell surface serine/threonine-specific protein kinase receptors, and generate intracellular signals using a conserved family of proteins called SMADs. TGF-p superfamily proteins play important roles in the regulation of basic biological processes such as growth, development, tissue homeostasis and regulation of the immune system.

Exemplary TGF-p superfamily proteins include, but are not limited to, AMH, ARTN, BMP10, BMP15, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8A, BMP8B, GDF1 , GDF10, GDF11, GDF15, GDF2, GDF3, GDF3A, GDF5, GDF6, GDF7, GDF8, GDF9, GDNF, INHA, INHBA, INHBB, INHBC, INHBE, LEFTY1, LEFTY2, MSTN, NODAL, NRTN, PSPN, TGF-pi, TGF-P2, TGF-P3, and TGF-P4. In a particular embodiment, the growth factor is BMP2.

In certain embodiments, the growth factor comprises a bone morphogenetic protein (BMP). As used herein, a BMP is a protein belonging to a group of growth factors also known as cytokines and as metabologens. BMPs can induce the formation of bone and cartilage and constitute a group of important morphogenetic signals, orchestrating tissue architecture throughout the body. Absence or deficiency of BMP signaling may be an important factor in diseases or disorders.

In certain embodiments, the BMP is selected from a group consisting of a BMP-2, a BMP-4, a BMP-6, a BMP-7, a BMP-12, a BMP-14, and any combination thereof. In certain embodiments, the BMP is BMP-2. BMP-2 plays an important role in the development of bone and cartilage. BMP-2 can potently induce osteoblast differentiation in a variety of cell types.

In certain embodiments, the growth factor comprises a TGF-p subfamily protein. As used herein, TGF-p subfamily protein or TGF-p is a multifunctional cytokine that includes four different isoforms (TGF-pi, TGF-P2, TGF-P3, and TGF-P4). Activated TGF-p complexes with other factors to form a serine/threonine kinase complex that binds to TGF-p receptors, which is composed of both type 1 and type 2 receptor subunits. After the binding of TGF-p, the type 2 receptor kinase phosphorylates and activates the type 1 receptor kinase that activates a signaling cascade. This leads to the activation of different downstream substrates and regulatory proteins, inducing transcription of different target genes that function in differentiation, chemotaxis, proliferation, and activation of many immune cells.

In certain embodiments, the growth factor comprises a TGF-pi . TGF-pi plays a role in the induction from CD4+ T cells of both induced Tregs (iTregs), which have a regulatory function, and Th17 cells, which secrete pro-inflammatory cytokines. TGF-pi alone precipitates the expression of Foxp3 and Treg differentiation from activated T helper cells.

The growth factors, (e.g., BMP-2 or TGF-pi), may be isolated from endogenous sources or synthesized in vivo or in vitro. Endogenous growth factor polypeptides may be isolated from healthy human tissue. Synthetic growth factor polypeptides are synthesized in vivo following transfection or transformation of template DNA into a host organism or cell, e.g., a mammalian or human cell line. Alternatively, synthetic growth factor polypeptides are synthesized in vitro by cell free translation or other art-recognized methods Sambrook, J., Fritsch, E.F., and Maniatis, T., Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, NY, Vol. 1 , 2, 3 (1989), herein incorporated by reference.

In certain embodiments, growth factor (e.g., BMP-2 or TGF-pi) polypeptides may be recombinant. In some embodiments, growth factor polypeptides are humanized derivatives of mammalian growth factor polypeptides. Exemplary mammalian species from which growth factor polypeptides are derived include, but are not limited to, mouse, rat, hamster, guinea pig, ferret, cat, dog, monkey, or primate. In some embodiments, the growth factor is a recombinant human protein . In some embodiments, the growth factor is a recombinant murine (mouse) protein. In some embodiments, the growth factor is a humanized derivative of a recombinant mouse protein.

In certain embodiments, the growth factor polypeptides may be modified to increase protein stability in vivo. In certain embodiments, the growth factor polypeptides may be engineered to be more or less immunogenic. The terms “immunogenic” and “immunogenicity” refer to the ability of a particular substance, such as a protein, an antigen, or an epitope, to provoke an immune response in the body of a human and other animal.

In certain embodiments, the growth factors may be present at between about 0.001 nmol and about 1000 nmol per scaffold, or about 0.001 and about 100 nmol per scaffold, or about 0.001 nmol and about 1 nmol per scaffold.

In some embodiments, the growth factors may be present at between about 1 ng to 1000 micrograms per scaffold. For example, the growth factors may be present at an amount between about 1 pg and about 1000 pg, between about 1 pg and 500 pg, between about 1 pg and about 200 pg, between about 1 pg and about 100 pg, between about 1 pg and about 50 pg, or between about 1 pg and 10 pg.

In some embodiments, the growth factor may be present at between about 1 ng to about 1000 pg. In some embodiments, the growth factor may be present at between about 1 ng to about 100 pg. In some embodiments, the growth factor may be present at between about 1 pg to about 2 ng per microgel. In some embodiments, the growth factor may be present at about 1 pg per microgel. In some embodiments, the growth factor may be present at between about 1 ng to about 500 ng. In some embodiments, the growth factor may be present at between about 1 ng to about 100 ng {e.g., about 1 ng, about 2 ng, about 3 ng, about 4 ng, about 5 ng, about 6 ng, about 7 ng, about 8 ng, about 9 ng, about 10 ng, about 11 ng, about 12 ng, about 13 ng, about 14 ng, about 15 ng, about 16 ng, about 17 ng, about

18 ng, about 19 ng, about 20 ng, about 21 ng, about 22 ng, about 23 ng, about 24 ng, about

25 ng, about 26 ng, about 27 ng, about 28 ng, about 29 ng, about 30 ng, about 31 ng, about

32 ng, about 33 ng, about 34 ng, about 35 ng, about 36 ng, about 37 ng, about 38 ng, about

39 ng, about 40 ng, about 41 ng, about 42 ng, about 43 ng, about 44 ng, about 45 ng, about

46 ng, about 47 ng, about 48 ng, about 49 ng, about 50 ng, about 51 ng, about 52 ng, about

53 ng, about 54 ng, about 55 ng, about 56 ng, about 57 ng, about 58 ng, about 59 ng, about

60 ng, about 61 ng, about 62 ng, about 63 ng, about 64 ng, about 65 ng, about 66 ng, about

67 ng, about 68 ng, about 69 ng, about 70 ng, about 71 ng, about 72 ng, about 73 ng, about

74 ng, about 75 ng, about 76 ng, about 77 ng, about 78 ng, about 79 ng, about 80 ng, about 81 ng, about 82 ng, about 83 ng, about 84 ng, about 85 ng, about 86 ng, about 87 ng, about

88 ng, about 89 ng, about 90 ng, about 91 ng, about 92 ng, about 93 ng, about 94 ng, about

95 ng, about 96 ng, about 97 ng, about 98 ng, about 99 ng, or about 100 ng). In some embodiments, the growth factor may be present at greater than about 2 ng.

In certain embodiments, the composition of the present disclosure comprises nanogram quantities of growth factors (e.g., about 1 ng to about 1000 ng of BMP-2). For example, the growth factors may be present at an amount between about 5 ng and about 500 ng, between about 5 ng and about 250 ng, between about 5 ng and about 200 ng, between about 10 ng and about 200 ng, between about 25 ng and about 200 ng, between about 50 ng and 200 ng, between about 100 ng and 200 ng, and about 200 ng. Nanogram quantities of the growth factor are also released in a controlled manner. The nanogram quantities of the growth factors and/or the controlled release can contribute to reduced toxicity of the compositions and methods of the present disclosure as compared to other delivery system, which uses high dose of growth factors and has suboptimal release kinetics.

In various embodiments, the amount of growth factors present in a scaffold may vary according to the size of the scaffold. For example, the growth factor may be present at about 0.03 ng/mm³ (the ratio of the amount of growth factors in weight to the volume of the scaffold) to about 350 ng/mm³, such as between about 0.1 ng/mm³ and about 300 ng/mm³ , between about 0.5 ng/mm³ and about 250 ng/mm³, between about 1 ng/mm³ and about 200 ng/mm³, between about 2 ng/mm³ and about 150 ng/mm³, between about 3 ng/mm³ and about 100 ng/mm³, between about 4 ng/mm³ and about 50 ng/mm³, between about 5 ng/mm³ and 25 ng/mm³, between about 6 ng/mm³ and about 10 ng/mm³, or between about 6.5 ng/mm³ and about 7.0 ng/mm³.

In some embodiments, the amount of growth factors may be present at between about 300 ng/mm³ and about 350 pg/mm³, such as between about 400 ng/mm³ and between about 300 pg/mm³, between about 500 ng/mm³ and about 200 pg/mm³, between about 1 pg/mm³ and about 100 pg/mm³, between about 5 pg/mm³ and about 50 pg/mm³, between about 10 pg/mm³ and about 25 pg/mm³.

Differentiation Factors

The compositions (e.g., microgels and/or granular hydrogels) of the present disclosure can comprise a differentiation factor. As used herein, a differentiation factor is an agent that can induce the differentiation of a cell, for example, a recruited cell. In certain embodiments, the differentiation factor is a polypeptide. As used herein, “differentiation,” “cell differentiation,” “cellular differentiation,” or other similar terms refer to the process where a cell changes from one cell type to another. In certain embodiments, the cell changes to a more specialized type, e.g., from a stem cell or a progenitor cell to a T cell progenitor cell. Differentiation occurs numerous times during the development of a multicellular organism as it changes from a simple zygote to a complex system of tissues and cell types.

Differentiation continues in adulthood as adult stem cells divide and create fully differentiated daughter cells during tissue repair and during normal cell turnover. Differentiation may change a cell's size, shape, membrane potential, metabolic activity, and responsiveness to signals. These changes may be due to highly controlled modifications in gene expression.

Among dividing cells, there are multiple levels of cell potency, the cell's ability to differentiate into other cell types. A greater potency indicates a larger number of cell types that can be derived. A cell that can differentiate into all cell types, including the placental tissue, is known as totipotent. A cell that can differentiate into all cell types of the adult organism is known as pluripotent. In mammals, e.g., human being, a pluripotent cell may include embryonic stem cells and adult pluripotent cells. Induced pluripotent stem (iPS) cells may be created from fibroblasts by induced expression of certain transcription factors, e.g., Oct4, Sox2, c-Myc, and KIF4. A multipotent cell is one that can differentiate into multiple different, but closely related cell types. Oligopotent cells are more restricted than multipotent, but can still differentiate into a few closely related cell types. Finally, unipotent cells can differentiate into only one cell type, but are capable of self-renewal.

In certain embodiments, the differentiation factors of the present disclosure induce the differentiation of stem cells or progenitor cells into T-cell progenitor cells. As used herein, the term “T cell progenitor cell” refers to a progenitor cell that ultimately can differentiate to a T lymphocyte (T cell). The term “lymphocyte,” as used herein, refers to one of the subtypes of white blood cell in a vertebrate’s (e.g., human being) immune system. Lymphocytes include natural killer cells, T cells, and B cells. Lymphocytes originate from a common lymphoid progenitor during hematopoiesis, a process during which stem cells differentiate into several kinds of blood cells within the bone marrow, before differentiating into their distinct lymphocyte types.

In some embodiments, the T cell progenitor cell comprises a common lymphoid progenitor cell. The term “common lymphoid progenitor cell,” as used herein, refers to the earliest lymphoid progenitor cells, which give rise to lymphocytes including T-lineage cells, B-lineage cells, and natural killer (NK) cells. In various embodiment, the T cell progenitor cell comprises a T cell competent common lymphoid progenitor cell. The term “T cell competent common lymphoid progenitor cell,” as used herein, refers to a common lymphoid progenitor cell that differentiates into T-lineage progenitor cell. A T cell competent common lymphoid progenitor is usually characterized by lacking of biomarker Ly6D. The composition of the present disclosure can create an ectopic niche that mimics important features of bone marrow and induces the differentiation of stem cells or progenitor cells into T cell progenitor cells.

In certain embodiments, the lymphocytes comprise T cells. In some embodiments, the T cells are naive T cells. As used herein, a naive T cell is a T cell that has differentiated in bone marrow. Naive T cells may include CD4⁺ T cells, CD8⁺ T cells, and regulatory T cells (Treg).

In certain embodiments, the differentiation factors induce the differentiation of the recruited cells into T cell progenitor cells. In certain embodiments, the differentiation factors induce the differentiation of the recruited cells into T cell progenitor cells through the Notch signaling pathway. The Notch signaling pathway is a highly conserved cell signaling system present in many multicellular organisms. Mammals possess four different Notch receptors, referred to as Notchl , Notch2, Notch3, and Notch4. Notch signaling plays an important role in T cell lineage differentiation from common lymphoid progenitor cells. In certain embodiments, the differentiation factors bind to one or more Notch receptors and activates the Notch signaling pathway. In certain embodiments, the differentiation factor is selected from a group consisting of a Delta-like 1 (DLL-1), a Delta-like 2 (DLL-2), a Delta-like 3 (DLL- 3), a Delta-like 3 (DLL-3), a Delta-like 4 (DLL-4), a Jagged 1, a Jagged 2, and any combination thereof. In certain embodiments, the binding of the differentiation factor to one or more Notch receptors activates the Notch signaling pathway and induces T cell lineage differentiation.

In certain embodiments, the differentiation factor is a Delta-like 4 (DLL-4). DLL-4 is a protein that is a homolog of the Drosophila Delta protein. The Delta protein family includes Notch ligands that are characterized by a DSL domain, EGF repeats, and a transmembrane domain.

In certain embodiments, the differentiation factor polypeptides are isolated from endogenous sources or synthesized in vivo or in vitro. Endogenous differentiation factor polypeptides may be isolated from healthy human tissue. Synthetic differentiation factor polypeptides are synthesized in vivo following transfection or transformation of template DNA into a host organism or cell, e.g., a mammal or cultured human cell line. Alternatively, synthetic differentiation factor polypeptides are synthesized in vitro by cell free translation or other art-recognized methods Sambrook, J., Fritsch, E.F., and Maniatis, T., Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, NY, Vol. 1, 2, 3 (1989), herein incorporated by reference).

In certain embodiments, differentiation factor polypeptides may be recombinant. In some embodiments, the differentiation factor polypeptides are humanized derivatives of mammalian differentiation factor polypeptides. Exemplary mammalian species from which the differentiation factor polypeptides are derived include, but are not limited to, mouse, rat, hamster, guinea pig, ferret, cat, dog, monkey, or primate. In some embodiments, the differentiation factor is a recombinant human protein . In some embodiments, the differentiation factor is a recombinant murine (mouse) protein. In some embodiments, the differentiation factor is a humanized derivative of a recombinant mouse protein.

In certain embodiments, the differentiation factor polypeptides may be modified to achieve a desired activity, for example, to increase protein stability in vivo. In certain embodiments, the differentiation factor polypeptides may be engineered to be more or less immunogenic.

In certain embodiments, the differentiation factor (e.g., DLL-4) may be covalently linked to the scaffold of the present disclosure. For example, rather than being released from a scaffold material, a differentiation factor may be covalently bound to polymer backbone and retained within the composition that forms following implantation of the composition in the subject. By covalently binding or coupling a differentiation factor to the scaffold material, such differentiation factor will be retained within the scaffold that forms following administration of the composition to a subject, and thus will be available to promote the differentiation of stem cells or progenitor cells, as contemplated herein. In certain embodiments, the differentiation factors are conjugated to the scaffold material utilizing N- hydroxysuccinimide (NHS) and l-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) chemistry. Any methods of covalently binding or coupling differentiation factors known in the art may be used and are not limited. See "Bioconjugate Techniques Bioconjugate Techniques (Third Addition)", Greg T. Hermanson, Academic , Greg T. Hermanson, Academic Press, 2013 Press, 2013. In some embodiments, the differentiation factor may be covalently linked to the scaffold utilizing click chemistry. The methods of covalently binding or coupling differentiation factors include, but are not limited to, avidin-biotin reaction, azide and dibenzocycloocytne chemistry, tetrazine and transcyclooctene chemistry, tetrazine and norbornene chemistry, or di-sulfide bond.

In certain embodiments, the differentiation factors (e.g., DLL-4) of the present disclosure further comprise a tether (e.g., PEG, PEG2k) and a methacrylate group (MA). In certain embodiments, the differentiation factor is methacrylated DLL-4-PEG2k.

In certain embodiments, the covalent linking retains the differentiation factors within the scaffold to provide the differentiation signal to the recruited cells in the scaffold. For example, less than 1 % of the total differentiation factor is detected outside of the scaffold. The bioactivity of the differentiation factor may be retained for an extended period of time, such as at least three months after incorporation to the scaffold. The bioactivity of the differentiation factors may be measured by any appropriate methods, such as a colorimetric assay for DLL-4. In certain embodiments, the differentiation factors may be present at between about 0.01 nmol and 1000 nmol, about 0.1 nmol and 100 nmol, or about 1 nmol and 10 nmol per scaffold.

In some embodiments, the differentiation factors may be present at between about 1 ng and 1000 micrograms per scaffold. For example, the differentiation factor may be present at between about 10 ng and about 500 pg, between about 50 ng and about 250 pg, between about 100 ng and about 200 pg, between about 1 pg and about 100 pg, between about 1 pg and about 50 pg, between about 1 pg and about 25 pg, between about 1 pg and about 10 pg, between about 2 pg and about 10 pg, or about 6 pg.

In some embodiments, the differentiation factor may be present at between about 1 ng to about 1000 pg. In some embodiments, the differentiation factor may be present at between about 1 ng to about 100 pg. In some embodiments, the differentiation factor may be present at between about 1 pg to about 2 ng per microgel. In some embodiments, the differentiation factor may be present at about 1 pg per microgel. In some embodiments, the differentiation factor may be present at between about 1 ng to about 500 ng. In some embodiments, the differentiation factor may be present at between about 1 ng to about 100 ng {e.g., about 1 ng, about 2 ng, about 3 ng, about 4 ng, about 5 ng, about 6 ng, about 7 ng, about 8 ng, about 9 ng, about 10 ng, about 11 ng, about 12 ng, about 13 ng, about 14 ng, about 15 ng, about 16 ng, about 17 ng, about 18 ng, about 19 ng, about 20 ng, about 21 ng, about 22 ng, about 23 ng, about 24 ng, about 25 ng, about 26 ng, about 27 ng, about 28 ng, about 29 ng, about 30 ng, about 31 ng, about 32 ng, about 33 ng, about 34 ng, about 35 ng, about 36 ng, about 37 ng, about 38 ng, about 39 ng, about 40 ng, about 41 ng, about 42 ng, about 43 ng, about 44 ng, about 45 ng, about 46 ng, about 47 ng, about 48 ng, about 49 ng, about 50 ng, about 51 ng, about 52 ng, about 53 ng, about 54 ng, about 55 ng, about 56 ng, about 57 ng, about 58 ng, about 59 ng, about 60 ng, about 61 ng, about 62 ng, about 63 ng, about 64 ng, about 65 ng, about 66 ng, about 67 ng, about 68 ng, about 69 ng, about 70 ng, about 71 ng, about 72 ng, about 73 ng, about 74 ng, about 75 ng, about 76 ng, about 77 ng, about 78 ng, about 79 ng, about 80 ng, about 81 ng, about 82 ng, about 83 ng, about 84 ng, about 85 ng, about 86 ng, about 87 ng, about 88 ng, about 89 ng, about 90 ng, about 91 ng, about 92 ng, about 93 ng, about 94 ng, about 95 ng, about 96 ng, about 97 ng, about 98 ng, about 99 ng, or about 100 ng). In some embodiments, the differentiation factor may be present at greater than about 2 ng.

In various embodiments, the amount of differentiation factor present in a scaffold may vary according to the size of the scaffold. For example, the differentiation factor may be present at about 0.03 ng/mm³ (the ratio of the amount of differentiation factor in weight to the volume of the scaffold) to about 350 pg/mm³, such as between about 0.1 ng/mm³ and about 300 pg/mm³, between about 1 ng/mm³ and about 250 pg/mm³, between about 10 ng/mm³ and about 200 pg/mm³, between about 0.1 pg/mm³ and about 100 pg/mm³, between about 0.1 pg/mm³ and 50 about pg/mm³, or between about 0.1 pg/mm³ and about 20 pg/mm³, between about 0.1 pg/mm³ and about 10 pg/mm³, between about 0.1 pg/mm³ and about 5 pg/mm³, between about 0.1 pg/mm³ and about 1 pg/mm³, between about 0.1 pg/mm³ and 0.5 pg/mm³, or about 0.2 pg/mm³.

In certain embodiments, the DLL-4 may be present at about 6 pg per scaffold.

Homing Factors

The compositions (e.g., microgels and/or granular hydrogels) of the present disclosure can comprise a homing factor. As used herein, the term “homing factor” refers to an agent that is capable of inducing directed movement of a cell, e.g., a stem cell or a progenitor cell. In certain embodiments, the homing factors of the present disclosure are signaling proteins that can induce directed chemotaxis in nearby responsive cells. In various embodiments, the homing factors are cytokines and/or chemokines.

In certain embodiments, the inclusion of such homing factors in the compositions of the present disclosure promotes the homing of cells (e.g., transplanted stem cells and/or progenitor cells) to the scaffold composition administered to a subject. In certain aspects, such homing factors promote the infiltration of the cells (e.g., transplanted stem cells or progenitor cells) to the scaffold composition administered to the subject. In some embodiments, the homing factors comprise stromal cell derived factor (SDF-1). In certain embodiments, the homing factors are encapsulated in the material. In certain embodiments, the homing factors are released from the material over an extended period of time (e.g., about 7-30 days or longer, about 17-18 days).

In certain embodiments, the homing factors retain their bioactivity over an extended period of time. The bioactivity of the growth factor may be measured by any appropriate means. In certain example, the homing factors retain their bioactivity for at least 10 days, 12 days, 14 days, 20 days, or 30 days after the incorporation of the homing factors into the scaffold.

In some embodiments, the homing factors may be present at between about 0.01 nmol and 1000 nmol, about 0.1 nmol and 100 nmol, or about 1 nmol and 10 nmol per scaffold.

In some embodiments, the homing factors may be present at between about 1 ng and 1000 micrograms per scaffold. For example, the homing factor may be present at between about 10 ng and about 500 pg, between about 50 ng and about 250 pg, between about 100 ng and about 200 pg, between about 1 pg and about 100 pg, between about 1 pg and about 50 pg, between about 1 pg and about 25 pg, between about 1 pg and about 10 pg, between about 2 pg and about 10 pg, or about 6 pg. In some embodiments, the homing factor may be present at between about 1 ng to about 1000 pg. In some embodiments, the homing factor may be present at between about 1 ng to about 100 pg. In some embodiments, the homing factor may be present at between about 1 pg to about 2 ng per microgel. In some embodiments, the homing factor may be present at about 1 pg per microgel. In some embodiments, the homing factor may be present at between about 1 ng to about 500 ng. In some embodiments, the homing factor may be present at between about 1 ng to about 100 ng {e.g., about 1 ng, about 2 ng, about 3 ng, about 4 ng, about 5 ng, about 6 ng, about 7 ng, about 8 ng, about 9 ng, about 10 ng, about 11 ng, about 12 ng, about 13 ng, about 14 ng, about 15 ng, about 16 ng, about 17 ng, about 18 ng, about 19 ng, about 20 ng, about 21 ng, about 22 ng, about 23 ng, about 24 ng, about 25 ng, about 26 ng, about 27 ng, about 28 ng, about 29 ng, about 30 ng, about 31 ng, about 32 ng, about 33 ng, about 34 ng, about 35 ng, about 36 ng, about 37 ng, about 38 ng, about 39 ng, about 40 ng, about 41 ng, about 42 ng, about 43 ng, about 44 ng, about 45 ng, about 46 ng, about 47 ng, about 48 ng, about 49 ng, about 50 ng, about 51 ng, about 52 ng, about 53 ng, about 54 ng, about 55 ng, about 56 ng, about 57 ng, about 58 ng, about 59 ng, about 60 ng, about 61 ng, about 62 ng, about 63 ng, about 64 ng, about 65 ng, about 66 ng, about 67 ng, about 68 ng, about 69 ng, about 70 ng, about 71 ng, about 72 ng, about 73 ng, about 74 ng, about 75 ng, about 76 ng, about 77 ng, about 78 ng, about 79 ng, about 80 ng, about 81 ng, about 82 ng, about 83 ng, about 84 ng, about 85 ng, about 86 ng, about 87 ng, about 88 ng, about 89 ng, about 90 ng, about 91 ng, about 92 ng, about 93 ng, about 94 ng, about 95 ng, about 96 ng, about 97 ng, about 98 ng, about 99 ng, or about 100 ng). In some embodiments, the homing factor may be present at greater than about 2 ng.

Chemoattractants

The compositions (e.g., microgels and/or granular hydrogels) of the present disclosure can comprise a chemoattractant for cells. The term “chemoattractant,” as used herein, refers to any agent that attracts a motile cell, such as an immune cell. In certain embodiments, the chemoattractant for immune cells is a growth factor, a cytokine, and/or a chemokine.

In some embodiments, the chemoattractant is a growth factor. The compositions of the present disclosure can comprise a growth factor. The term “growth factor,” as used herein, refers to an agent that is capable of stimulating cellular growth, proliferation, healing, and/or cellular differentiation. In certain embodiments, growth factors are polypeptides. Growth factor polypeptides typically act as signaling molecules. In certain embodiments, the growth factor polypeptides are cytokines. Exemplary cytokines include, but are not limited to, interleukins, lymphokines, monokines, interferons, and colony stimulating factors.

Exemplary growth factors include, but are not limited to, transforming growth factor beta (TGF-P), granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), nerve growth factor (NGF), neurotrophins, Platelet- derived growth factor (PDGF), erythropoietin (EPO), thrombopoietin (TPO), myostatin (GDF- 8), growth differentiation factor-9 (GDF9), acidic fibroblast growth factor (aFGF or FGF-1), basic fibroblast growth factor (bFGF or FGF-2), epidermal growth factor (EGF), hepatocyte growth factor (HGF).

In some embodiments, the chemoattractant is a chemokine. Exemplary chemokines include, but are not limited to, CO chemokines, CXC chemokines, C chemokines, CX3C chemokines.

In some embodiments, the chemoattractant is a cytokine. Exemplary cytokines include, but are not limited to, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12 1L-15, 1L-17, 1 L-18, TNF-a, IFN-y, and IFN-a.

In some embodiments, the compositions of the present disclosure include a chemoattractant for immune cells. In some embodiments, the compositions of the present disclosure comprise a compound that attracts an immune cell to or into the device, wherein the immune cell comprises a macrophage, T-cell, B-cell, natural killer (NK) cell, or dendritic cell. Non-limiting examples of compounds useful for attracting an immune cell to or into the device comprises granulocyte-macrophage colony stimulating factor (GM-CSF), an FMS-like tyrosine kinase 3 ligand (Flt3L), chemokine (C-C motif) ligand 19 (CCL-19), chemokine (C-C motif) ligand 20 (CCL20), chemokine (C-C motif) ligand 21 (CCL-21), a N-formyl peptide, fractalkine, monocyte chemotactic protein- 1, and macrophage inflammatory protein-3 (MIP- 3a).

In certain embodiments, the chemoattractant for immune cells is Granulocytemacrophage colony-stimulating factor (GM-CSF). Granulocyte-macrophage colonystimulating factor (GM-CSF) is a protein secreted by macrophages, T cells, mast cells, endothelial cells and fibroblasts. Specifically, GM-CSF is a cytokine that functions as a white blood cell growth factor. GM-CSF stimulates stem cells to produce granulocytes and monocytes. Monocytes exit the blood stream, migrate into tissue, and subsequently mature into macrophages.

In some embodiments, the compositions of the present disclosure can comprise and release GM-CSF polypeptides to attract host DCs to the device. Contemplated GM-CSF polypeptides are isolated from endogenous sources or synthesized in vivo or in vitro. Endogenous GM-CSF polypeptides may be isolated from healthy human tissue. Synthetic GM-CSF polypeptides are synthesized in vivo following transfection or transformation of template DNA into a host organism or cell, e.g., a mammalian or human cell line. Alternatively, synthetic GM-CSF polypeptides are synthesized in vitro by polymerase chain reaction (PCR) or other art-recognized methods Sambrook, J., Fritsch, E.F., and Maniatis, T., Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, NY, Vol. 1 , 2, 3 (1989), herein incorporated by reference).

In certain embodiments, GM-CSF polypeptides may be recombinant. In some embodiments, GM-CSF polypeptides are humanized derivatives of mammalian GM-CSF polypeptides. Exemplary mammalian species from which GM-CSF polypeptides are derived include, but are not limited to, mouse, rat, hamster, guinea pig, ferret, cat, dog, monkey, or primate. In some embodiments, GM-CSF is a recombinant human protein (PeproTech, Catalog # 300-03). In some embodiments, GM-CSF is a recombinant murine (mouse) protein (PeproTech, Catalog #315-03). In some embodiments, GM-CSF is a humanized derivative of a recombinant mouse protein.

In certain embodiments, GM-CSF polypeptides may be modified to increase protein stability in vivo. In certain embodiments, GM-CSF polypeptides may be engineered to be more or less immunogenic. Endogenous mature human GM-CSF polypeptides are glycosylated, reportedly, at amino acid residues 23 (leucine), 27 (asparagine), and 39 (glutamic acid) (see US Patent No. 5,073,627). In certain embodiments, GM-CSF polypeptides of the present invention may be modified at one or more of these amino acid residues with respect to glycosylation state.

The chemoattractant for immune cells may recruit immune cells to the scaffolds of the present invention. Immune cells include cells of the immune system that are involved in immune response. Exemplary immune cells includes, but not limited to, T cells, B cells, leucocytes, lymphocytes, antigen presenting cells, dendritic cells, neutrophils, eosinophils, basophils, monocytes, macrophages, histiocytes, mast cells, and microglia.

In certain embodiments, the chemoattractant can recruit a cell to the scaffold following the administration of the composition to a subject. The recruited cell may be autologous. For example, the recruited cell may be an immune cell, such as a T cell, from the subject. The recruited cell may also be syngeneic, allogeneic, or xenogeneic. As used herein, the term “syngeneic” refers to genetically identical, or sufficiently identical and immunologically compatible as to allow for transplantation. For example, syngeneic cells may include transplanted cells obtained from an identical twin. As used herein, the term “allogeneic” refers to cells that are genetically dissimilar, although from individuals of the same species. As used herein, the term “xenogeneic” refers to cells derived from a different species and therefore genetically different.

For example, the recruited cell may be a donor cell in a transplantation. In certain embodiments, the transplantation is a hematopoietic stem cell transplantation (HSCT). As used herein, HSCT refers to the transplantation of multipotent hematopoietic stem cells or hematopoietic progenitor cells, usually derived from bone marrow, peripheral blood, or umbilical cord blood. HSCT may be autologous (the patient's own stem cells or progenitor cells are used), allogeneic (the stem cells or progenitor cells come from a donor), syngeneic (from an identical twin) or xenogenic (from different species).

Methods of incorporating polypeptides (e.g., growth factor polypeptides) are known in the art. See, US Patent Nos.: 8,728,456; 8,067,237; and 10,045,947; US Patent Publication No.: US20140079752; International Patent Publication No.: WO2017/136837; incorporated herein by reference in their entirety. The release of the growth factor polypeptides may be controlled. The methods of controlled release of polypeptides (e.g., growth factor polypeptides) are known in the art. See, US Patent Nos.: 8,728,456; 8,067,237; 10,045,946, incorporated by reference in their entirety. In certain embodiments, the growth factors may be released over an extended period of time, such as 7-30 days or longer.

Antigens

The compositions (e.g., microgels and/or granular hydrogels) of the present disclosure can comprise an antigen. The antigen can be a cancer antigen or a non-cancer antigen (e.g., a microbial antigen or a viral antigen). In one embodiment, the antigen is a polypeptide. In one embodiment, the polypeptide antigen comprises a stretch of at least 10 consecutive amino acids identical to a stretch of at least 10 consecutive amino acids of a cancer antigen, a microbial antigen, or a viral antigen. In some embodiments, the antigen is a cancer antigen. The device comprising a cancer antigen can be used to vaccinate and/or provide protective immunity to a subject to whom such a device was administered. In some embodiments, a cancer/tumor antigen is from a subject who is administered a device provided herein. In certain embodiments, a cancer/tumor antigen is from a different subject. In various embodiments, a cancer antigen is present in a cancer cell lysate. For example, the tumor cell lysate may comprise one or more lysed cells from a biopsy. In some embodiments, the cancer antigen is present on an attenuated live cancer cell. For example, the attenuated live cancer cell may be an irradiated cancer cell. Antigens may be used alone or in combination with GM-CSF, CpG-ODN sequences, or immunomodulators. Moreover, antigens can be provided simultaneously or sequentially with GM-CSF, CpG-ODN sequences, or immunomodulators.

One or more antigens may be selected based on an antigenic profile of a subject's cancer or of a pathogen. In certain embodiments, the device lacks a cancer antigen prior to administration to a subject. In some embodiments, the compositions of the present disclosure can comprise an immunoconjugate, wherein the immunoconjugate comprises an immunostimulatory compound covalently linked to an antigen. In various embodiments, the antigen comprises a cancer antigen, such as a central nervous system (CNS) cancer antigen, CNS germ cell tumor antigen, lung cancer antigen, leukemia antigen, acute myeloid leukemia antigen, multiple myeloma antigen, renal cancer antigen, malignant glioma antigen, medulloblastoma antigen, breast cancer antigen, prostate cancer antigen, Kaposi's sarcoma antigen, ovarian cancer antigen, adenocarcinoma antigen, or melanoma antigen. In some embodiments, treating the subject comprises reducing metastasis in the subject.

Exemplary cancer antigens encompassed by the compositions, methods, and devices of the present invention include, but are not limited to, tumor lysates extracted from biopsies, and irradiated tumor cells. Exemplary polypeptide cancer antigens include one or more of the following proteins, or fragments thereof: MAGE series of antigens (MAGE-1 is an example), MART-1/melana, tyrosinase, ganglioside, gp1OO, GD-2, O-acetylated GD-3, GM-2, MUG-1 , Sos1, Protein kinase C-binding protein, Reverse transcriptase protein, AKAP protein, VRK1, KIAA1735, T7-1, T11-3, T11-9, Homo Sapiens telomerase ferment (hTRT), Cytokeratin-19 (CYFRA21-1), SQUAMOUS CELL CARCINOMA ANTIGEN 1 (SCCA-1), (PROTEIN T4-A), SQUAMOUS CELL CARCINOMA ANTIGEN 2 (SCCA-2), Ovarian carcinoma antigen CA125 (1A1-3B) (KIAA0049), MUCIN 1 (TUMOR-ASSOCIATED MUCIN), (CARCINOMA-ASSOCIATED MUCIN), (POLYMORPHIC EPITHELIAL MUCIN), (PEM),(PEMT), (EPISIALIN), (TUMOR-ASSOCIATED EPITHELIAL MEMBRANE ANTIGEN), (EMA), (H23AG), (PEANUT-REACTIVE URINARY MUCIN), (PUM), (BREAST CARCINOMA- ASSOCIATED ANTIGEN DF3), CTCL tumor antigen se1-1 , CTCL tumor antigen se14-3, CTCL tumor antigen se20-4, CTCL tumor antigen se20-9, CTCL tumor antigen se33-1, CTCL tumor antigen se37-2, CTCL tumor antigen se57-1, CTCL tumor antigen se89-1, Prostate-specific membrane antigen, 5T4 oncofetal trophoblast glycoprotein, Orf73 Kaposi's sarcoma-associated herpesvirus, MAGE-C1 (cancer/testis antigen CT7), MAGE-B1 ANTIGEN (MAGE-XP ANTIGEN) (DAM10), MAGE-B2 ANTIGEN (DAM6), MAGE-2 ANTIGEN, MAGE-4a antigen, MAGE-4b antigen, Colon cancer antigen NY-CO-45, Lung cancer antigen NY-LU-12 variant A, Cancer associated surface antigen, Adenocarcinoma antigen ART1, Paraneoplastic associated brain-testis-cancer antigen (onconeuronal antigen MA2; paraneoplastic neuronal antigen), Neuro-oncological ventral antigen 2 (NOVA2), Hepatocellular carcinoma antigen gene 520, TUMOR-ASSOCIATED ANTIGEN 00-029, Tumor-associated antigen MAGE-X2, Synovial sarcoma, X breakpoint 2, Squamous cell carcinoma antigen recognized by T cell, Serologically defined colon cancer antigen 1 , Serologically defined breast cancer antigen NY-BR-15, Serologically defined breast cancer antigen NY-BR-16, Chromogranin A; parathyroid secretory protein 1, DUPAN- 2, CA 19-9, CA 72-4, CA 195, Carcinoembryonic antigen (CEA), Trp2, ovalbumin, M27, and M30. In embodiments, the antigen comprises a fragment of one or more of the following proteins. In exemplary embodiments, the fragment can comprise 10 or more consecutive amino acids identical in sequence to one or more of the foregoing proteins. In some embodiments, the fragment can comprise 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000 or more amino acids. In one embodiment, the fragment can comprise 10-500 amino acids.

In one embodiment, the antigen is a melanoma antigen. Exemplary melanoma antigens include, but are not limited to, tyrosinase, gp75 (tyrosinase related protein- 1 (TRP- 1 )), gp100 (Pmel17), Melan A/MART-1, TRP-2, MAGE family, BAGE family, GAGE family, NY-ESO-1, CDK4, p- catenin, mutated introns, N-acetylglucosaminyltransferase V gene product, MUM-1, p15, gangliosides (e.g., GM2, GD2, GM3, GD3), high molecular weight chondroitin sulfate proteoglycan, p97 melanotransferrin, and SEREX antigens (e.g., D-1, SSX-2) (Hodi FS, Clin Cancer Res, February 1 , 2006, 12: 673-678), or fragments thereof.

In certain embodiments, the antigen comprises a non-tumor antigen such as a microbial antigen. For example, the microbial antigen may comprise a bacterial antigen, a fungal antigen, an archaean antigen, or a protozoan antigen. In some embodiments, the microbial antigen is a viral antigen, e.g., an HIV antigen or influenza antigen. In some embodiments, the antigen is from a microbe such as a bacterium, virus, protozoan, archaean, or fungus. Various embodiments relate to vaccinating against or treating a bacterial, viral, or fungal infection. In various embodiments, a delivery vehicle comprising an antigen from a pathogen. For example, a pathogen includes but is not limited to a fungus, a bacterium (e.g., Staphylococcus species, Staphylococcus aureus, Streptococcus species, Streptococcus pyogenes, Pseudomonas aeruginosa, Burkholderia cenocepacia, Mycobacterium species, Mycobacterium tuberculosis, Mycobacterium avium, Salmonella species, Salmonella typhi, Salmonella typhimurium, Neisseria species, Brucella species, Bordetella species, Borrelia species, Campylobacter species, Chlamydia species, Chlamydophila species, Clostrium species, Clostrium botulinum, Clostridium difficile, Clostridium tetani, Helicobacter species, Helicobacter pylori, Mycoplasma pneumonia, Corynebacterium species, Neisseria gonorrhoeae, Neisseria meningitidis, Enterococcus species, Escherichia species, Escherichia coli, Listeria species, Francisella species, Vibrio species, Vibrio cholera, Legionella species, or Yersinia pestis), a virus (e.g., adenovirus, Epstein-Barr virus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Herpes simplex virus type 1 , 2, or 8, human immunodeficiency virus, influenza virus, measles, Mumps, human papillomavirus, poliovirus, rabies, respiratory syncytial virus, rubella virus, or varicella-zoster virus), a parasite or a protozoa (e.g., Entamoeba histolytica, Plasmodium, Giardia lamblia, Trypanosoma brucei, or a parasitic protozoa such as malaria-causing Plasmodium). In one embodiment, a pathogen antigen can be derived from a pathogen cell or particle described herein.

The compositions (e.g., microgels and/or granular hydrogels) of the present disclosure can comprise an adjuvant. The term “adjuvant,” as used herein, refers to compounds that can be added to, e.g., vaccines, to stimulate immune responses against antigens. Adjuvants may enhance the immunogenicity of highly purified or recombinant antigens. Adjuvants may reduce the amount of antigen or the number of immunizations needed to achieve protective immunity. For example, adjuvants may activate antibodysecreting B cells to produce a higher amount of antibodies. Alternatively, adjuvants can act as a depot for an antigen, present the antigen over a longer period of time, which could help maximize the immune response and provide a longer-lasting protection. Adjuvants may also be used to enhance the efficacy of a vaccine by helping to modify the immune response to particular types of immune system cells, for example, by activating T cells instead of antibody-secreting B cells depending on the purpose of the vaccine. Adjuvants are also used in the production of antibodies from immunized animals (Petrovskyl et al, 2002, Immunology and Cell Biology 82: 488-496).

Exemplary adjuvants include, but are not limited to, aluminium hydroxide, aluminum phosphate, calcium phosphate, Quil A, Quil A derived saponin QS-21 , or other types of saponins, Detox, ISCOMs, cell wall peptidoglycan or lipopolysaccharide of Gram-negative bacteria, trehalose dimycolate, bacterial nucleic acids such as DNA containing CpG motifs, FIA, Montanide, Adjuvant 65, Freund's complete adjuvant, Freund's incomplete adjuvant, Lipovant, interferon, granulocyte-macrophage colony stimulating factor (GM-CSF), AS03, AS04, IL-1 , IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, STING, Tolllike receptor (TLR) ligand, CD40L, ovalbumin (OVA), monophosphoryl lipid A (MPL), polyinosinic:polycytidylic acid (poly(l:C)), a combination of LPS (or MPLA) and OxPAPC, MF59, N-acetyl muramyl-L-alanyl-D-isoglutamine (MDP), poly (DL-lactide-coglycolide) microspheres, paraffin oil, squalene, virosome, gamma inulin, glucans, dextrans, lentinans, glucomannans and galactomannans, pathogen-associated molecular patterns (PAMPs), damage-associated molecular pattern molecules (DAMPs), antibodies against immune suppressive molecules (e.g., antibody or antagonist against transforming growth factor (TGF)-beta, A2aR antagonists), Freund’s complete adjuvant, Freund’s incomplete adjuvant, lipopolysaccharides (LPS), Fas ligand, Trail, lymphotactin, Mannan (M-FP), APG-2, Hsp70 and Hsp90.

IV. Methods of Making

The present disclosure provides methods of making microgels, e.g., granular hydrogels. Some aspects of the present disclosure provide a method of preparing a microgel, comprising: (i) providing a core microgel comprising a crosslinked polymer comprising a functional group selected from the group consisting of a crosslinked tetrazine (Tz) polymer, a crosslinked norbornene (Nb) polymer, and combinations thereof, optionally wherein the core microgel is characterized by a degree of substitution (DS) of about 5 to about 15 functional groups per polymer chain; (ii) applying a non-covalent polymer coating comprising a positively charged polymer to the surface of the core microgel, optionally wherein the positively charged polymer comprises poly(D-lysine) (PDL); (iii) applying a surface coating comprising a functionalized polymer to the surface of the coated core microgel to introduce a functional group for surface-specific conjugation of an active agent; and (iv) optionally conjugating an active agent comprising a complementary functional group conjugated to the functional group of the surface coating.

Some aspects of the present disclosure provide a method of preparing a method of preparing a granular hydrogel, comprising: (i) providing a composition comprising a plurality of microgels and a continuous aqueous phase; (ii) concentrating the microgels into a pellet via centrifugation; (iii) loading the pellet onto a membrane filter and removing the continuous aqueous phase or a portion thereof via centrifugation, thereby forming a granular hydrogel.

The microgels, e.g., granular hydrogels, of the present disclosure may be formed by a method comprising forming emulsions. Without wishing to be bound by theory, the size of the resulting microgels can be determined, at least in part, by the size of the emulsion. Thus, adjusting the size of the emulsion at the emulsion formation step can be used to tune the physicochemical properties e.g., size) of the microgels. Such adjusting can be achieved, for example, by controlling the flow rate and/or dimensions of the microfluidic chip. Formation of emulsion-templated microgels can also include a hydrophobic treatment, e.g., in which the walls of the microfluidic chip are contacted with a predetermined amount of a surfactant.

In one aspect, the present disclosure provides a method of preparing a microgel, comprising: (i) providing a microfluidics chip; (ii) providing an aqueous phase comprising a first polymer and a second polymer; (iii) providing a continuous oil phase comprising an oil and a surfactant; and (iv) contacting the aqueous phase with the continuous oil phase in the microfluidics chip to form an emulsion, thereby preparing the microgel. The microfluidics chip may comprise at least two aqueous inlets, at least one oil inlet, and at least one outlet. In some embodiments, the microfluidics chip may comprise at least one junction, wherein the junction permits the aqueous phase to contact the continuous oil phase to form an emulsion. Exemplary microfluidics chips are known in the art (see, e.g., PCT International Patent Application Publication No. WO 2015/069634, herein incorporated by reference in its entirety.

In various embodiments, the first polymer and the second polymer may be independently selected from the group consisting of a non-degradable polymer, a degradable polymer, and a combination thereof. In some embodiments, the first polymer and the second polymer may be independently selected from the group consisting of alginate, chitosan, polyethylene glycol (PEG), gelatin, hyaluronic acid, collagen, chondroitin, agarose, polyacrylamide, heparin, derivatives thereof, and combinations thereof. In some embodiments, the first polymer and the second polymer can be the same polymer. In some embodiments, the first polymer and the second polymer can be independently an alginate, optionally wherein the first polymer and the second polymer independently comprise a modified alginate polymer, optionally wherein the first polymer and the second polymer independently comprise oxidized alginate, optionally wherein the first polymer and the second polymer are independently comprise methacrylate alginate, optionally wherein the first polymer and the second polymer independently comprise a click reagent.

In some embodiments, the first polymer and the second polymer independently may comprise a modified polymer. For example, the first polymer and the second polymer may independently comprise an oxidized polymer. The oxidized polymer may be about 0.1% to about 99% oxidized (e.g., about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% oxidized). The first polymer and the second polymer may independently comprise oxidized alginate.

In some embodiments, the first polymer and the second polymer may independently comprise a click reagent. For example, the first polymer and the second polymer may independently comprise a click reagent selected from the group consisting of azide, dibenzocyclooctyne (DBCO), transcyclooctene, tetrazine (Tz), norbornene (Nb), and variants thereof. In some embodiments, the first polymer may comprise a tetrazine (Tz) moiety. In some embodiments, the first polymer may comprise tetrazine modified alginate (Alg-Tz). In some embodiments, the second polymer may comprise a norbornene (Nb) moiety. In some embodiments, the second polymer may comprise norbornene modified alginate (Alg-Nb).

In some embodiments, the first polymer and the second polymer are independently dissolved in deionized water. In some embodiments, the first polymer and the second polymer are independently provided at a concentration of about 0.1% (w/v) to about 10% (w/v). In some embodiments, the first polymer is provided at a concentration of about 0.5% (w/v) to about 1.5% (w/v). In some embodiments, the second polymer is provided at a concentration of about 1.5% (w/v) to about 2.5% (w/v).

In some embodiments, the oil comprises HFE7500. In some embodiments, the oil comprises mineral oil. In some embodiments, the oil comprises silicone.

In some embodiments, the surfactant is selected from the group consisting of an amphoteric surfactant, an anionic surfactant, a cationic surfactant, a nonionic surfactant, and a combination thereof. In some embodiments, the surfactant comprises a nonionic surfactant selected from the group consisting of Brij 93, SPAN 80, ABIL EM90, PGPR, and a combination thereof. In some embodiments, when mineral oil and/or silicone oil is included, the surfactant may be a nonionic surfactant such as Brij 93, SPAN 80, ABIL EM90, and/or PGPR.

In some embodiments, the surfactant comprises fluorosurfactant. In some embodiments, the continuous oil phase comprises about 0.5% (w/v) to about 2% (w/v) fluorosurfactant in HFE7500 solution, optionally wherein the continuous oil phase comprises about 1% (w/v) fluorosurfactant in HFE7500 solution.

In some embodiments, the methods described herein may further comprise injecting the Alg-Nb and the Alg-Tz into the microfluidics chip, optionally wherein the Alg-Nb and the Alg-Tz are injected at a rate of about 25 pl/hour to about 100 pl/hour, optionally wherein the Alg-Nb and the Alg-Tz are injected at a rate of about 50 pl/hour. In some embodiments, the methods described herein may further comprise injecting the continuous oil phase at a rate of about 175 pl/hour to about 500 pl/hour, optionally wherein the continuous oil phase is injected at a rate of about 200 pl/hour. In some embodiments, the methods described herein may further comprise allowing the Alg-Nb and the Alg-Tz solutions to form an emulsion when they encounter the continuous oil phase at a junction inside the microfluidics chip, thereby forming an emulsion-templated microgel. In some embodiments, the methods described herein may further comprise collecting the emulsion. In some embodiments, the methods described herein may further comprise maintaining the emulsion at room temperature for at least about 6 hours to about 24 hours to allow covalent crosslinking between the Alg-Nb and the Alg-Tz polymers. In some embodiments, the methods described herein may further comprise treating the emulsion with a demulsification and washing process, optionally wherein the treating comprises contacting the emulsion with an about 30-50% (v/v), optionally an about 40% (v/v), 1H,1 H,2H,2H-Perfluoro-1-octanol (PFO) solution, an about 0.1-1 % (v/v), optionally an about 0.5% (v/v) Tween 20 solution, and an about 0.5% (w/v) to about 1.5% (w/v), optionally an about 0.8% (w/v) sodium chloride (NaCI) solution, sequentially. In some embodiments, the methods described herein may further comprise isolating the microgel. In some embodiments, the methods described herein may further comprise dispersing the microgel in an aqueous solution, optionally wherein the aqueous solution comprises a saline solution, optionally wherein the aqueous solution comprises phosphate-buffered saline (PBS), optionally wherein the aqueous solution comprises calcium chloride (CaCh) and/or NaCI, optionally wherein the aqueous solution comprises about 2 mM CaCh and/or about 0.8% NaCI, optionally wherein the aqueous solution comprises DMEM media and 10% FBS. In some embodiments, the methods described herein may further comprise lyophilizing the microgel. In some embodiments, the methods described herein may further comprise storing the microgel at about 4°C.

In some embodiments, the methods described herein may further comprise coating the microgels (e.g., granular hydrogels) with a polymer coating. In some embodiments, the polymer coating comprises one or more layers. In some embodiments, the polymer coating comprises at least one selected from the group consisting of an alginate polymer, a hyaluronic acid (HA) polymer, a collagen polymer, a gelatin polymer, and combinations thereof. In some embodiments, the polymer coating comprises a modified polymer. In some embodiments, the modified polymer comprises a polymer modified with a click reaction moiety (e.g., a functionalized polymer). Exemplary click reaction moieties include, but are not limited to, an azide moiety, a dibenzocyclooctyne (DBCO) moiety, a transcyclooctene moiety, a tetrazine (Tz) moiety, a norbornene (Nb) moiety, and variants thereof. In some embodiments, the modified polymer comprises at least one selected from the group consisting of a modified alginate polymer, a modified hyaluronic acid (HA) polymer, a modified collagen polymer, a modified gelatin polymer, and combinations thereof.

In some embodiments, the polymer coating comprises a functionalized polymer, e.g., functionalized alginate. In some embodiments, the microgels (e.g., granular hydrogels) comprises a polymer coating comprising a functionalized polymer, e.g., a functionalized alginate, such as alginate-Tz and/or alginate-Nb. In some embodiments, the microgels (e.g., granular hydrogels) comprises a polymer coating comprising poly(D-lysine) (PDL). In some embodiments, the microgels (e.g., granular hydrogels) comprises a polymer coating comprising poly(D-lysine) (PDL) and a polymer coating comprising a functionalized polymer, e.g., a functionalized alginate, such as alginate-Tz and/or alginate-Nb.

In some embodiments, the methods described herein for the generation of microgels (e.g., granular hydrogels) comprising a polymer coating comprises concentrating microgels by centrifugation, e.g., at 300 ref for 3 min, and redispersing the concentrated microgels in a solution of poly(D-lysine) (PDL) (e.g., 50-150 kDa, 0.1 mg mL^-1 in beads buffer) at a concentration of, e.g., about 4 x 10⁵ microgels per mL. In some embodiments, the microgels can then immediately be concentrated by centrifugation, e.g., at 300 ref for 3 min, washed three times with beads buffer, redispersed in beads buffer at initial stock solution concentration, and stored at 4 °C until further use. In some embodiments, the microgels can be redispersed in a solution of functionalized polymer, e.g., functionalized alginate (e.g., about 0.01 mg/mL to about 1 mg/mL) in beads buffer at a concentration of, e.g., about 4 * 10⁵ microgels per mL, and collected by centrifugation, e.g., at 300 ref for 3 min. In some embodiments, microgels can be washed three times with beads buffer, redispersed in beads buffer at initial stock solution concentration, and stored at 4 °C until further use.

In some embodiments, the methods described herein may further comprise conjugating an active agent comprising a complementary functional group conjugated to the functional group of the surface coating. In some embodiments, the methods described herein may further comprise contacting the microgel with an active agent, optionally wherein the contacting occurs at about 4°C for about 1 hour to about 5 hours, optionally wherein the contacting occurs at about 4°C for about 3 hours.

V. Methods of Use

In one aspect, the present disclosure provides methods of modifying a cellular behavior selected from the group consisting of cell phenotype, morphology, spreading, proliferation, differentiation, activation, expansion, and combinations thereof. In certain embodiments of the present disclosure, the methods comprise contacting a population of cells with a microgel or a granular hydrogel of the present disclosure. In some embodiments of the present disclosure, the methods of modifying a cellular behavior selected from the group consisting of cell phenotype, morphology, spreading, proliferation, differentiation, activation, expansion, and combinations thereof comprise administering to the subject one or more compositions of the present disclosure.

In one aspect, the present disclosure provides methods of activating and expanding a population of cells, such as immune cells, e.g., T cells. In certain embodiments of the present disclosure, the methods comprise contacting the population of cells (e.g., immune cells, e.g., T cells) with a microgel or a granular hydrogel of the present disclosure. In some embodiments of the present disclosure, the methods of activating and expanding a population of cells (e.g., T cells) comprise administering to the subject one or more compositions of the present disclosure.

In one aspect, the present disclosure provides methods of promoting polyclonal and antigen-specific immune cell (e.g., T cell) expansion. In certain embodiments of the present disclosure, the methods comprise contacting the population of immune cells (e.g., T cells) with a microgel or a granular hydrogel of the present disclosure. In some embodiments of the present disclosure, the methods of promoting polyclonal and antigen-specific immune cell (e.g., T cell) expansion comprise administering to the subject one or more compositions of the present disclosure.

In one aspect, the present disclosure provides methods of enhancing antigen-specific enrichment of a subpopulation of immune cells, e.g., T cells. In certain embodiments of the present disclosure, the methods comprise contacting the population of immune cells, e.g., T cells, with a microgel or a granular hydrogel of the present disclosure. In some embodiments of the present disclosure, the methods of enhancing antigen-specific enrichment of a subpopulation of immune cells, e.g., T cells, comprise administering to the subject one or more compositions of the present disclosure.

In one aspect, the present disclosure provides methods of controlling T cell proliferation and T cell phenotype. In certain embodiments of the present disclosure, the methods comprise contacting the population of T cells with a microgel or a granular hydrogel of the present In some embodiments of the present disclosure, the methods of promoting polyclonal and antigen-specific immune cell (e.g., T cell) expansion comprise administering to the subject one or more compositions of the present disclosure.

In one aspect, the present disclosure provides methods of regulating the proliferation and differentiation of cells, such as stem cells, e.g., mesenchymal stem cells (MSCs). In certain embodiments of the present disclosure, the methods of regulating the proliferation and differentiation of cells (e.g., stem cells, e.g., MSCs) comprise contacting the population of cells with a microgel or a granular hydrogel of the present disclosure. In some embodiments of the present disclosure, the methods of regulating the proliferation and differentiation of cells (e.g., stem cells, e.g., MSCs) comprise administering to the subject one or more compositions of the present disclosure.

VI. Pharmaceutical Compositions

For administration to a subject, the microgels, e.g., granular hydrogels, described herein can be provided as pharmaceutically acceptable (e.g., sterile) compositions. These pharmaceutically acceptable compositions can be formulated together with one or more pharmaceutically acceptable carriers (additives) and/or diluents.

As described in detail below, the pharmaceutical compositions of the present disclosure can be specifically formulated for administration in solid or liquid form, including those adapted for the following: (1) parenteral administration, for example, by subcutaneous, intramuscular, intravenous (e.g., bolus or infusion) or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; (2) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), lozenges, dragees, capsules, pills, tablets (e.g., those targeted for buccal, sublingual, and/or systemic absorption), boluses, powders, granules, pastes for application to the tongue; (3) topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin; (4) intravaginally or intrarectally, for example, as a pessary, cream or foam; (5) sublingually; (6) ocularly; (7) transdermally; (8) transmucosally; or (9) nasally. Additionally, compounds can be implanted into a patient or injected using a drug delivery system. See, for example, Urquhart, et al., Ann. Rev. Pharmacol. Toxicol. 24: 199-236 (1984); Lewis, ed. “Controlled Release of Pesticides and Pharmaceuticals” (Plenum Press, New York, 1981); U.S. Pat. No. 3,773,919; and U.S. Pat. No. 35 3,270,960, content of all of which is herein incorporated by reference.

As used herein, the term “pharmaceutically acceptable” or “pharmacologically acceptable” refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. Moreover, for animal (e.g., human) administration, it will be understood that compositions should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards.

As used herein, the term “pharmaceutically acceptable carrier” means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids (23) serum component, such as serum albumin, HDL and LDL; (22) C2-C12 alcohols, such as ethanol; and (23) other non-toxic compatible substances employed in pharmaceutical formulations. Wetting agents, coloring agents, release agents, coating agents, disintegrating agents, binders, sweetening agents, flavoring agents, perfuming agents, protease inhibitors, plasticizers, emulsifiers, stabilizing agents, viscosity increasing agents, film forming agents, solubilizing agents, surfactants, preservative and antioxidants can also be present in the formulation. The terms such as “excipient”, “carrier”, “pharmaceutically acceptable carrier” or the like are used interchangeably herein.

VII. Kits

The disclosure includes various kits which comprise a microgel, e.g., granular hydrogel, of the disclosure.

Although exemplary kits are described below, the contents of other useful kits will be apparent to the skilled artisan in light of the present disclosure. Each of these kits is included within the disclosure.

In some embodiments, the kit further comprises an applicator useful for administering the microgel, e.g., granular hydrogel. The particular applicator included in the kit will depend on, e.g., the method used to administer the microgel, e.g., granular hydrogel, and such applicators are well-known in the art and may include, among other things, a pipette, a syringe, a dropper, a needle, and the like.

Moreover, in some embodiments the kit further comprises an instructional material which describe the use of the kit to perform the methods described herein. These instructions simply embody the disclosure provided herein.

In some embodiments, the kit includes a pharmaceutically-acceptable carrier. The composition is provided in an appropriate amount as set forth elsewhere herein. Further, the route of administration and the frequency of administration are as previously set forth elsewhere herein.

The kit may further encompass an additional agent comprising a wide plethora of molecules, such as, but not limited to, the active agents as set forth elsewhere herein. However, the skilled artisan armed with the teachings provided herein, would readily appreciate that the disclosure is in no way limited to these, or any other, combination of molecules. Rather, the combinations set forth herein are for illustrative purposes and they in no way limit the combinations encompassed by the present disclosure. EXAMPLES

Example 1 : Surface-Functionalized Microgels as Artificial Antigen-Presenting Cells to Regulate Expansion of T Cells

Artificial antigen-presenting cells (aAPCs) are currently used to manufacture T cells for adoptive therapy in cancer treatment, but a readily tunable and modular system could enable both rapid T cell expansion and control over T cell phenotype. The present example provides experimental data demonstrating that microgels with tailored surface biochemical properties can serve as aAPCs to mediate T cell activation and expansion. Surface functionalization of microgels was achieved via layer-by-layer coating using oppositely charged polymers, forming a thin but dense polymer layer on the surface. This facile and versatile approach is compatible with a variety of coating polymers and allows efficient and flexible surface-specific conjugation of defined peptides or proteins. The present example demonstrates that tethering appropriate stimulatory antibodies on the microgel surface efficiently activates T cells for polyclonal and antigen-specific expansion. The expansion, phenotype and functional outcome of primary mouse and human T cells can be regulated by modulating the concentration, ratio and distribution of stimulatory ligands presented on microgel surfaces as well as the stiffness and viscoelasticity of the microgels.

1. Introduction

Adoptive cell therapy (ACT) of T cells, in which isolated T cells are manipulated and expanded ex vivo before infusing into patients, has proven to be an effective treatment for certain cancers.^11-31 The activation and expansion of T cells involves signals for T-cell receptor (TCR) stimulation and co-stimulation together with growth factors such as interleukin 2 (IL-2) to stimulate isolated T cells ex vivo, which in the body are provided by antigen-presenting cells (APC).^[4-51 Technologies that allow rapid T cell expansion and tune T cell phenotype in a controlled manner provide powerful tools to generate functional therapeutic T cells.

Biomaterials have served as artificial antigen-presenting cells (aAPCs) by locally providing required stimulatory cues for T cell activation to mimic the endogenous T cell-APC interaction and improve the therapeutic efficacy of ACT.^16-71 Leveraging the flexible design in various material properties allows biomaterials to modulate T cell proliferation, function, and phenotype. Inorganic,^18-91 polymeric,^110-111 liposomal^112-131 and lipid-modified¹¹⁴¹ particles conjugated with stimulatory ligands for T-cell receptor (TCR) stimulation and co-stimulation have been explored for T cell activation, and provide various advantages owing to their preparation process and physical properties.¹⁶¹ The size,^{110 151} morphology,¹¹⁶¹ ligand composition^{19 171} and mobility^118-191 of aAPCs have profound effects on T cell expansion and phenotype. In addition to particle-based materials, APC mimetic scaffolds assembled from carbon nanotube bundles¹²⁰¹ or lipid-coated mesoporous silica rods^121-221 provide a 3D niche with large surface area for clustering of ligands and cells, resulting in efficient expansion of T cells. Extracellular matrix-mimetic hydrogels incorporating bioactive ligands are also capable of activating T cells and regulating their functions, ^123-251 in a manner dependent on the mechanics of the hydrogel.^125-271 Despite the development of aAPCs, those with flexibly tunable mechanical properties are still underexplored. And considering the importance of various biochemical and physical properties in T cell activation, a biomaterial system with multiple layers of tunability is of interest for research and T cell manufacturing.

Hydrogels can be fabricated as microscale particles, also known as microgels, with tailored size, morphology and mechanics, providing a highly tunable, modular and biocompatible system.¹²⁸¹ When jammed together, microgels can assemble to form granular hydrogels,^128-291 a type of injectable microporous scaffold that has been used in 3D bioprinting,¹³⁰¹ wound healing¹³¹¹ and tissue regeneration.^132-351 Microgels enable encapsulation and release of bioactive factors in a controlled manner¹³⁶¹ and exhibit mechanical properties similar to cells,^137-381 showing potential as aAPCs and APC mimetic scaffolds. However, bioactive ligands are generally conjugated throughout the entire microgels, while only those presenting on the surface can typically bind to T cell surface receptors to regulate T cell activation. Therefore, efficient and flexible conjugation to the microgel surface of target peptides or proteins that can bind to cell surface receptors is still challenging, thus limiting their application for T cell activation.

Described herein is the development of a powerful approach to fabricate microgels as aAPCs via surface functionalization of microgels using layer-by-layer coating. Sequentially adsorbing oppositely charged polymers formed a thin but dense layer on the surface with a high stability. This strategy is applicable to a variety of microgel polymers, coating polymers and allows versatile chemistry for further modification, thus providing a convenient means to modulate microgel surface properties independent of the mechanical properties. Efficient conjugation of stimulatory ligands specifically to the microgel surface promoted polyclonal and antigen-specific T cell expansion. It is further demonstrated that modulating the concentration, ratio and distribution of stimulatory ligands on microgel surfaces as well as the stiffness and viscoelasticity of microgels allows control over the expansion, function and phenotype of primary mouse and human T cells.

2. Results

2.1. Synthesis of Microgels and Granular Hydrogels Alginate microgels were fabricated using microfluidic emulsion, which provides defined size and shape by controlled droplet formation (FIG. 1 A). Alginate was first modified with norbornene (Alg-Nb) or tetrazine (Alg-Tz) by carbodiimide coupling to achieve an average degree of substitution (DS) of 13 or 11.5 functional groups per alginate chain respectively, as quantified by proton nuclear magnetic resonance spectra (FIG. 7, FIG. 8). Stock solutions of Alg-Nb and Alg-Tz were then mixed at a final concentration of 2 wt% in the microfluidic device and injected to form microdroplets by emulsion, which then crosslinked overnight to generate microgels with a diameter of 77 ± 2 pm (FIG. 1B). The elastic moduli of the microgels could be tuned by varying the ratio between Alg-Nb and Alg- Tz (FIG. 1C).

Alginate microgels can be jammed by centrifugation¹³⁰'³²³⁹¹ over a membrane to remove a portion of the continuous aqueous phase between particles to fabricate granular hydrogels (FIG. 1D). The resulting microporous structure was visualized by incorporating 2 MDa fluorescein (FITC)-labelled dextran (FIG. 9, FIG. 10). Varying the time for centrifugation modulated the porosity of the granular hydrogels in a reproducible manner, independent of the stiffness of the microgel building blocks (FIG. 1E, FIG. 11). The microporous structure allows cells to penetrate through granular hydrogels of 900 pm thick following cell seeding on the surface after 2 days (FIG. 12). As T cells can migrate at 10-15 pm/min,^[401 they likely had completely infiltrated the gels earlier than the 48 h observation point.

2.2 Surface Functionalization of Microgels

We next sought an efficient chemical strategy to functionalize the surface of microgels by non-covalent polymer coatings. Alginate microgels were first immersed in a solution of poly(D-lysine) (PDL) to deposit a layer of positively charged polymers and then immersed in a solution of functionalized alginate to coat the second layer and introduce functional groups on the surface (FIG. 2A-2B, Table 2). Both layers of polymers were uniformly and efficiently coated on the surface of microgels, and the thickness of both PDL layer and alginate coating was 0.74 ± 0.11 pm (FIG. 2C, FIG. 13). The diameter of the microgels slightly decreased to 72 ± 2 pm after coating (FIG. 14). This non-covalent coating showed high stability on microgel surfaces, as more than 90% of polymers remained when microgels were soaked in beads buffer (HEPES) over 3 weeks and in T cell culture media over 7 days (FIG. 2D-2E). Surprisingly, after soaking microgels in beads buffer at 4°C over 10 months, we still observed a thin alginate layer of 0.91 ± 0.22 pm, despite some polymer aggregation on the surface (FIG. 15).

Table 2 Zeta potential of microgels before and after polymer coating.

The thin and stable coating layer of alginate coating allows incorporation of sufficient ligands only on the surface to mediate biological functions without introducing functional groups throughout the entire microgel that are not available to cell surface receptors. The surface ligand density can be efficiently and precisely engineered through multiple approaches during the surface functionalization process. First, the surface ligand density can be tuned by varying the DS of functional groups coupled to the alginate polymers used for coating. When increasing the DS of FITC on alginate, for example, the fluorescent intensity of alginate-FITC on the microgel surface significantly increased while the coating density remained constant (FIG. 16). Second, the surface ligand density can also be easily tuned by modulating the density of coated polymers. Varying the concentration of ligand-modified alginate solution used to create the second layer from 0.01 to 1 mg/mL resulted in a 25-fold increase of polymer density without significant changes in thickness (FIG. 2F, FIG. 17). Third, surface ligands can be engineered via post-functionalization using orthogonal click chemistries to conjugate the target molecules to the microgel surface. For example, microgels crosslinked via the norbornene-tetrazine strategy can be subsequently coated with azide-modified alginate, allowing surface-specific conjugation of dibenzocyclooctyne (DBCO)-modified ligands through strain-promoted azide-alkyne cycloaddition (SPAAC) in a controlled manner (FIG. 2G).

The surface functionalization strategy is also applicable to a range of coating and core polymers. FITC-labelled hyaluronic acid (HA) can be uniformly coated on the surface of alginate microgels (FIG. 18). In addition, core microgels made of HA, gelatin and alginate- type I collagen interpenetrating network were fabricated using microfluidic emulsion, and a uniform and thin layer of fluorescent dye-labelled alginate was also observed on the surface of these microgels after coating, demonstrating the versatility of the approach (FIG. 2H). Overall, the surface-specific chemical modification achieved via surface coating allows efficient fabrication of microgels with different surface functionalities by leveraging different polymers and chemo-selective chemistries to modify pre-synthesized microgels.

We next examined the distribution of the coating polymer in the microgels. We synthesized Rhodamine B-labelled alginate microgels containing excess tetrazine (Nb/Tz = 1/2) and used sulfo-Cy5 labelled alginate as the coating polymer. FITC-TCO was allowed to react with residue tetrazines on the microgels after coating, to detect their availability in the microgel. As expected, the fluorescent signal from all three dyes overlapped at the outer shell of the microgels (FIG. 21-2 J) , indicating the diffusion of polymers vertical to the surface into the microgels instead of solely depositing a layer on the surface. This diffusion mechanism is further supported by the finding that polymer coatings had no significant impact on interparticle covalent crosslinking between microgels with complementary functional groups. Interparticle crosslinking between microgels with excess Tz (Nb/Tz = 1/2) and excess Nb (Nb/Tz = 2/1) in the jammed state resulted in a significant improvement of granular hydrogel stability compared to those crosslinked only by physical interactions (FIG.

19). Formation of interparticle covalent crosslinking remained efficient between microgels with polymer coatings, as these granular hydrogels were stable over 3 weeks when subsequently soaked in buffer and the porosity remained similar after 3 days (FIG. 19, FIG.

20), suggesting that the Nb and Tz functional moieties are exposed on surface rather than remaining underneath the polymer coatings. This phenomenon is likely attributed to the out- of-plane diffusion of charged polymers, a process commonly observed in layer-by-layer assembly approaches.^141-421 The PDL and alginate coating likely undergo some interdiffusion, although the high molecular weight and high charge density of PDL and alginate results in low chain mobility,¹⁴³¹ likely restricting their diffusion within a thin layer (< 1 pm). To test this possibility, low molecular weight PDL (1-5 kDa) was used for coating, and was found to diffuse throughout the entire microgel (FIG. 21).

2.3 Primary Mouse T Cell Activation

We next leveraged this surface-specific functionalization strategy to conjugate essential antibodies for T cell activation and expansion (FIG. 3A). T cell activation generally requires both T cell receptor stimulation and co-stimulation. Activating antibodies to the appropriate T cell surface receptors CD3 (aCD3) and CD28 (aCD28) were modified with DBCO groups by first reducing the disulfide linkages and then conjugating with maleimide- PEG12-DBCO via thiol-ene reaction. The amount of DBCO labeled on each antibody was approximately 4.5 on average, as measured by UV-vis spectroscopy (FIG. 3B, FIG. 22). In this site-specific modification, the reduction of disulfide linkages may reduce the stability and bioactivity of the antibodies. These activating antibodies were surface presented from microgels using the post-functionalization approach, by first coating alginate-azide on the surface of microgels and then reacting with DBCO-modified aCD3 and aCD28 (FIG. 3A).

Polyclonal T cell activation was evaluated by culture of CD4+ primary mouse T cells isolated from C57BL/6J mice with suspended microgels conjugated with aCD3 and aCD28 on the surface (surface specific) at an overall antibody density of 0.4 pg/cm², which is half of that on Dynabeads. By day 3, the proliferation rate significantly increased from 5% without antibodies to 90% in the presence of aCD3 and aCD28, suggesting antibodies are still functional. This number was slightly higher than commercial CD3/CD28 T-cell expansion beads (Dynabeads), which also showed robust proliferation (FIG. 3C-3D). Massive cell clusters were observed surrounding the microgels (FIG. 3E, surface-specific). When the same amount of TCO-modified aCD3 and aCD28 antibodies were directly conjugated to microgels via free tetrazines on the polymer used to fabricate the microgels (Nb/Tz = 1:2), only 13% of the CD4+ T cells were activated to proliferate (FIG. 3C-3E, entire microgel), which is attributed to a significantly reduced surface density of antibodies when conjugated throughout the entire microgel (FIG. 23).

We also investigated whether our approach is applicable to antigen-specific activation and expansion of primary mouse CD8+ T cells, which is relevant to cancer treatment. Microgels were first coated with biotin-modified alginate, which allows streptavidin to specifically bind to the surface due to high affinity between biotin and streptavidin (FIG. 3F, FIG. 24). A biotinylated H-2K(b) MHC class I monomer presenting SIINFEKL peptide and biotinylated aCD28 were attached to the surface for antigen-specific activation of OT-1 cells. When OT-1 cells (CD8+ T cells) were co-cultured with the surface-coated microgels for 3 days, a dramatic enhancement of proliferation rate was observed, compared to conditions without antibodies or with Dynabeads (FIG. 3G, FIG. 25, FIG. 26).

We next assessed the antigen-specific enrichment of a subpopulation of CD8+ T cells specific to SIINFEKL peptide. To introduce SHNFEKL-specific T cells, we mixed CD8+ T cells from wild type and OT-1 mice at a ratio of 200:1 and co-cultured with the MHC class I and aCD28-presenting microgels. By day 7, the frequency of SHNFEKL-specific CD8+ T cells increased from 0.7% to 87%, corresponding to a 190-fold expansion (FIG. 3H-3J). By contrast, only a slight increase in frequencies of the antigen-specific subpopulation was observed when cultured with Dynabeads, as expected. To address the possibility of antigenspecific expansion directly from primary T cell isolation, we also vaccinated wild type mice with vaccines presenting ovalbumin to induce antigen-specific T cells. The frequency of SHNFEKL-specific CD8+ T cells isolated from the spleen or lymph nodes 7 days after vaccination was 1.5% and 0.6%, respectively (FIG. 27, FIG. 28). When CD8+ T cells isolated from vaccinated mice were co-cultured with the MHC class I and aCD28-presenting microgels over 7 days, we also observed significant increases in the frequency of SHNFEKL- specific CD8+ T cells (FIG. FIG. 27, FIG. 28).

2.4 Tuning T cell proliferation and phenotype of primary mouse T cells by modulating surface biochemical properties

The surface-specific functionalization strategy enables precise and efficient engineering of the concentrations and types of antibodies presented on the surface, thus allowing us to explore the expansion and phenotypic change of T cells in response to different presentation of cues. Raising the concentration of aCD3 and aCD28 over 2 orders of magnitude, at a aCD3/aCD28 ratio of 1, led to an increase in T cell fold expansion when CD4+ and CD8+ T cells were co-cultured with microgels (CD4/CD8 = 1) (FIG. 4A, FIG. 29). CD4+ and CD8+ T cells that were expanded with higher ligand density upregulated the expression of CD25 and OX-40 activation markers (FIG. 30). Commercial Dynabeads and microgels presenting antibodies at a matched density of 0.79 pg/cm² exhibited similar expansion.¹²¹¹ The ultimate CD4/CD8 ratio was also dependent on the ligand density (FIG. 4B). Increasing the ligand concentration above a threshold of 0.1 pg/cm² promoted rapid and substantial CD8-biased skewing. Increasing ligand density also resulted in a reduction of CD44-CD62L+ T cells in both the CD4+ and CD8+ populations, suggesting fewer T cells associated with a naive-like phenotype but more T cells associated with central memory-like (CD44+CD62L+) and effector-like phenotypes (CD44+CD62L-) (FIG. 4C). The cytotoxicity function of expanded CD8+ T cells was also evaluated using an in vitro killing assay of B16- F10 target cells presenting ovalbumin by expanded OT-I T cells. Cytotoxic function initially increased with ligand density, and then saturated (FIG. 4D). When T cells were co-cultured with B16-F10 cells that don’t express ovalbumin, minimum killing was observed, as expected (FIG. 31).

The aCD3/aCD28 ratio was next altered. When increasing aCD3/aCD28 from 1:1 to 7:1 at an overall ligand density of 0.4 pg/cm², negligible difference was observed in fold expansion, CD4/CD8 ratio and differentiation status when CD4+ and CD8+ T cells were co- cultured with microgels (CD4/CD8 = 1) (FIG. 4E-4G, FIG. 32). However, decreasing the aCD3/aCD28 ratio affected the activation of T cells, leading to a reduction in fold expansion and an increasing population of T cells associated with a naive-like phenotype (CD44- CD62L+) in accordance with downregulation of CD25 and OX-40 (FIG. 33). This finding aligns with the finding that TCR stimulation is required before co-stimulation for optimal T cell activation.¹⁴⁴¹

To explore if spatial heterogeneity of signaling would impact T cell expansion and phenotype, we combined microgels conjugated with aCD3 and aCD28 (aCD3/ aCD28 ratio = 1) and blank microgels (without antibody conjugation) (FIG. 4H). The dose of antibodies in the mixed microgels conditions were tuned by varying the mixing ratios of microgels to match the dose in conditions of a single type of antibody-presenting microgel, giving the same average ligand density over the entire population of microgels. CD4+ and CD8+ T cells (CD4/CD8 = 1) cultured in mixed microgels demonstrated an increase in fold expansion and decrease in CD4/CD8 ratio, compared to cells cultured with a single type of microgel at the same dose of antibodies (FIG. 4I-4J). Heterogeneous ligand distribution also resulted in an upregulation of the expression of CD25 and OX-40 and a reduction in the population of naive-like T cells (FIG. 34, FIG. 35). Heterogeneous distribution of the ligands provides some local areas with high density of ligands for promoting stimulation, which likely leads to enhanced T cell activation and expansion. Altogether, these results indicate one can leverage the flexibility of the surface-specific functionalization strategy to modulate the concentration, ratio and distribution of activating antibodies to regulate T cell expansion and the resulting phenotypes.

IL-2, the third signal for productive T cell activation, is generally supplemented as soluble factors in the culture medium, but we hypothesized that DBCO-modified cytokines could also be immobilized on the microgel surface to regulate T cell activation. IL-2 was first functionalized with DBCO by reacting with DBCO-sulfo-NHS via aminolysis, and conjugated to coated microgels together with aCD3/aCD28 (FIG. 4K). After multiple washing steps to remove unreacted IL-2, minimum IL-2 release was detected. When cultured with CD4+ and CD8+ T cells (CD4/CD8 = 1), IL-2 immobilized on the surface of microgels supported T cell activation, with increasing IL-2 density resulting in greater fold expansion (FIG. 4L, FIG. 36, FIG. 37). Interestingly, the low density of immobilized IL-2 resulted in a CD4-biased expansion, while a high density promoted a substantial CD8-biased skewing (FIG. 4M). At low level of IL-2, an increase of FOXP3+CD25+ T cells was observed (FIG. 38). As regulatory T cells express a high affinity IL-2 receptor,¹⁴⁵¹ the low level of IL-2 likely enriched these cells, skewing the CD4/CD8 ratio.¹⁴⁶¹ Immobilizing IL-2 at a surface density of 2000 U/cm² led to similar activation of the T cells compared to supplementing IL-2 in the media, with minimum differences in T cell expansion and CD4/CD8 ratio (FIG. 4L-4M). Immobilizing cytokines on the surface of scaffolds can potentially provide a convenient means to locally regulate T cell activation in vivo and minimize non-targeted cytokine release to avoid unexpected side effects.^147-491 Our method allows precise engineering of IL-2 density on the surface to mediate the expansion and phenotypes of T cells, suggesting a strategy to specifically expand regulatory T cells when desired, and can also potentially capture subsequently administered DBCO-modified cytokines for presentation in a time-dependent manner.

To examine the impact of CD4+ cells on CD8+ cell expansion, we performed coculture studies or with CD8+ only when activated by microgels. The presence of CD4+ cells resulted in an enhanced expansion of CD8+ cells, an upregulation of activation markers, and a reduction in naive-like population (FIG. 39-41). However, no significant difference was observed in the expression of I FNy, TNFa and IL-2, suggesting a similar cytotoxic function (FIG. 42). By contrast, the presence of CD8+ cells showed an inverse impact on CD4+ population, leading to a reduction in expansion (FIG. 39). 2.5 Regulating T cell proliferation and phenotype of primary mouse T cells by modulating microgel mechanical properties

The surface-specific functionalization strategy also enables modulation of the mechanical properties of the microgels independent of their surface biochemical properties. Microgels with different stiffness were synthesized by varying the ratio between Alg-Nb and Alg-Tz at an overall alginate concentration of 2 wt% and functionalized with stimulatory antibodies on the surface. When microgels of different stiffness were co-cultured with CD4+ and CD8+ T cells (CD4/CD8 = 1), increasing the elastic moduli of microgels from 1kPa to 3 kPa led to an increase in T cell fold expansion and upregulation of the expression of CD25 and OX-40 activation markers (FIG. 5A, FIG. 43). Increasing the stiffness also resulted in a substantial CD8-biased skewing (FIG. 44). A reduction of CD44-CD62L+ T cells was observed in both the CD4+ and CD8+ populations, suggesting fewer T cells associated with naive-like phenotype (FIG. 5B).

The influence of the viscoelasticity of the microgels on T cell activation was next studied. Ionically crosslinked alginate gels exhibit viscoelastic features^150-521 and are used here as viscoelastic scaffolds for 3D cell culture. We synthesized microgels with calcium crosslinking, in place of covalent crosslinking. Alginate stock solution was first combined with calcium-ethylenediaminetetraacetic acid (EDTA) at neutral pH and then injected into the microfluidic device to mix with acid-containing oil. The binding affinity between EDTA and calcium decreased significantly from pH 7 to pH 4, thus releasing free calcium ions to crosslink the hydrogels when forming the microdroplets. The diameter of the microgels was 79 ± 2 pm and the elastic modulus was 3 kPa, similar to elastic microgels used for T cell activation (FIG. 45, FIG. 46).

Compared to elastic microgels with the same surface ligand density, viscoelastic microgels modified with aCD3/aCD28 showed a reduction in T cell expansion (FIG. 5C), similar to previously reported results in which T cells were activated in 3D collagen matrices of different viscoelasticity.¹²⁶¹ Viscoelastic microgels also led to the downregulation of the expression of CD25 and OX-40 activation markers and an increase of CD44-CD62L+ T cells, suggesting a less differentiated phenotype (FIG. 5D, FIG. 47). In addition, no significant difference in CD4/CD8 ratio was observed (FIG. 48).

To investigate if the size of the microgels affects T cell activation, we synthesized covalently crosslinked microgels with diameter of 18 ± 2 pm (FIG. 49), and functionalized with aCD3/aCD28 antibodies. While maintaining consistent surface density and dose of antibodies, decreasing microgel size resulted in a small increase in fold expansion and a reduction in CD4/CD8 ratio (FIG. 50, FIG. 51). Reduced microgel size also led to an upregulation of the expression of CD25 and OX-40 and a reduction in the population of naive-like T cells (FIG. 52, FIG. 53). We also examined the capability of our stategy for T cell activation in 2D culture. 96- well tissue culture plates were coated with PDL and alginate, and then functionalized with stimulatory antibodies at different densities. Similar to microgels, increasing ligand density on the plate resulted in an increase in T cell expansion (FIG. S48). However, a threshold of 0.2 ug/cm² was required for activation, which is significantly higher than found with 3D microgels, and the expansion plateaued at 0.8 ug/cm². Overall, the regime of ligand density that has tunable impact on T cell phenotypes was much narrower for coated plates compared to coated microgels (FIG. 54-57). More generally, compared to 2D plate culture, 3D culture provides higher achievable cell densities and more tunable properties, which is greatly preferable for T cell manufacturing.

2.6 Polyclonal expansion of primary human T cells

Finally, we investigated whether microgels could be used for polyclonal expansion of primary human T cells. Primary human T cells (mixture of CD4+ and CD8+) were isolated from blood samples from two healthy donors and cultured with microgels presenting human aCD3 and aCD28. Higher ligand densities initially led to an increase in fold expansion in cells from Donor #1, similar to the trend observed in mouse T cells, but expansion then diminished at ligand densities greater than 0.4 pg/cm² (FIG. 6A). The trend in fold expansion agreed with the expression of CD25 (FIG. 6B). The reduction of fold expansion at high ligand density is likely attributed to activation induced cell death (AICD), in which over activation results in apoptosis.¹⁵³¹ Donor #1 cells initially had a CD4/CD8 ratio of 1.9 (FIG. 6C), and when activated with hydrogels presenting a ligand density below 0.4 pg/cm², the resulting T cell population was biased towards the CD8+ population. Increasing the ligand density above 0.4 pg/cm² resulted in a more balanced CD4+ and CD8+ ratio. This phenomenon may be caused by the different sensitivities of CD4+ and CD8+ T cells to activating antibodies. The expanded T cells were also phenotypically different as the density of ligands presenting on hydrogels was varied. A high ligand density resulted in a reduction of CD45RA+CCR7+ T cells, suggesting a more differentiated phenotype in both CD4+ and CD8+ T cells (FIG. 6D). A similar trend was also observed in the expression of CD45RA and CD62L (FIG. 58). T cells exposed to high density of ligands also expressed more CD39+, suggesting a more effector-like phenotype (FIG. 6E). In addition to differentiation status, the expression of inhibitory markers was also affected by the surface ligand density (FIG. 59). PD-1 and Lag-3 expression among both CD4+ and CD8+ T cells first progressively increased with ligand density, suggesting a more exhausted phenotype. However, increasing the ligand density above 0.4 pg/cm² corresponded with a slightly lower level of PD-1 and Lag-3 expression. Expression of these inhibitory markers suggests that persistent stimulation of T cells at high does will lead to exhaustion over time, and ultimately T cell dysfunction. A potential strategy to balance proliferation and exhaustion is to limit the timeframe of stimulation using degradable materials.

To examine whether the T cell response would be consistent between different donors, we analyzed T cells from Donor #2 after activation with antibody-presenting hydrogels. Similar trends of expansion and phenotypic changes from cells of the two donors were observed (FIG. 60-66). However, a difference was found in activation potential and sensitivity between donors. For example, the proliferation for Donor #1 peaked at 0.4 pg/cm², while the largest expansion for Donor #2 occured at 1.6 pg/cm² (FIG. 6A, FIG. 60). Donor-to-donor variability due to different health conditions, genders and ages is an important factor affecting how T cells respond to stimulation.¹²²’^54-561 The facile functionalization strategy developed here for microgels could readily be used to provide patient-specific stimulation to accommodate individual differences in the donor cells.

3. Conclusion

Here we demonstrate a microgel platform that presents bioactive ligands specifically on the surface to regulate T cell expansion and phenotypic change. Surface functionalization was achieved by coating the microgel surface with oppositely charged polymers, resulting in a thin yet stable layer of functional polymers decorating the surface of microgels. Conjugation of activating antibodies and mitogenic cytokines via chemo-selective chemistry allows one to modulate the surface biochemical cues to T cells precisely and efficiently. Microgels modified with appropriate ligands promoted efficient polyclonal and antigenspecific T cell expansion. Our findings demonstrate that the concentration, ratio and distribution of antibodies during T-cell activation have profound effects on the resulting phenotype of primary mouse and human T cells. In addition, stiffer and more elastic microgels promote the expansion and activation of the T cells.

This surface-specific functionalization strategy provides a convenient and versatile means to modulate the surface biochemical properties of microgels, which could be exploited to manipulate the stimulation dose for personalized T cell therapies.¹²²¹ The ready injectability of microgels and granular hydrogels^{130-32 391} and stability of polymer coatings during injection (FIG. 67) potentially could also allow these materials to be delivered with minimally invasive procedures in the future for in situ expansion of immune cells for cancer treatment, minimizing the risks of off-target toxicities. Quantitative analysis of the stability of the coating will need to be further analyzed if future studies use needle injection of these microgels. Considering the flexibility of microgels in other aspects, including their physical properties and ligand mobility, these can also be leveraged to investigate the impact of other important material properties on T cell expansion and phenotypic regulation, and used for the expansion of a variety of cell types. 4. Experimental Section

Elastic Microgel Preparation. The synthesis of covalently crosslinked microgels was adapted from an existing protocol.¹⁵⁷¹ The dispersed phase containing 2 wt% alginate was prepared as a mixture of Tz and Nb modified alginate dissolved separately at 1-3 wt% in DI water. A mixture of fluorosurfactant (1%) in fluorocarbon oil was used as the continuous phase. Alginate-Tz and alginate-Nb solutions were injected at 150 pL/h and the continuous phase was injected at 1000 pL/h. The emulsion was then collected in a tube and left at room temperature for 24 h to allow covalent crosslinking between alginate polymers. After the reaction was complete, the continuous phase was removed, and 33% 1/7, 1/7, 2/7, 2/7- perfluoro-1 -octanol in HFE was added in excess at 1:3 volume ratio to the collected microgels to break the emulsion. Finally, microgels were washed three times with beads buffer (130 mM NaCI, 25 mM HEPES, 2 mM CaCh, pH 7.5), redispersed in beads buffer and stored at 4°C until further use.

Viscoelastic Microgel Preparation. The synthesis of Ca²⁺ crosslinked alginate microgels was adapted from an existing protocol.¹⁵⁸¹ The dispersed phase containing 1-2 wt% unmodified alginate and 50 mM CaEDTA was identically prepared as described above for batch emulsion technique. A mixture of fluorosurfactant (1%) and acidic acid (0.05-0.2 v%) in fluorocarbon oil was used as the continuous phase. Alginate solution and the continuous phase were injected at flow rates of 300 and 1000 pL/h, respectively. The emulsion was then collected and mixed with 50% 1/7,1/7,2/7,2/7-perfluoro-1-octanol in HFE at 1:1 volume ratio to break the emulsion. Microgels were washed three times with beads buffer, redispersed in beads buffer and stored at 4°C until further use.

Polymer Coating. Microgels were first concentrated by centrifugation at 300 ref for 3 min and redispersed in a solution of poly(D-lysine) (PDL) (50-150 kDa, 0.1 mg/mL in beads buffer) at a concentration of 4*10⁵ microgels/mL. Microgels were then immediately concentrated by centrifugation at 300 ref for 3 min, washed three times with beads buffer, redispersed in beads buffer at initial stock solution concentration, and stored at 4°C until further use. Microgels were redispersed in a solution of functionalized alginate (0.01 - 1 mg/mL) in beads buffer at a concentration of 4*10⁵ microgels/mL and collected by centrifugation at 300 ref for 3 min. Microgels were washed three times with beads buffer, redispersed in beads buffer at initial stock solution concentration, and stored at 4°C until further use.

Coating Density. The amount of alginate coated on the microgels was determined by the difference between the amount of alginate used for coating and the remaining amount in solution after coating. Alginate-Rhodamine B was used as a model polymer for coating to quantify the concentration of alginate in solutions. Microgels were washed three times after coating and all the supernatants were collected after each centrifugation. Alginate concentration in the original solution used for coating and all the supernatants were quantified by fluorescent intensity at 586 nm (excitation wavelength 561 nm) based on a calibration curve. The density of coating was calculated by the amount of alginate- Rhodamine B coated on the surface and overall surface area of microgels. Three independent experimental replicates were used for all experiments.

Coating Stability. The stability of polymer coating was determined by polymer dissolution in the surrounding buffer solution. Alginate-Rhodamine B coated microgels were soaked in beads buffer (4*10⁵ microgels/mL) at room temperature. All buffer solutions were collected and replaced by fresh beads buffer on Day 1 , 4, 7, 10, 14, 21. The concentration of released alginate-Rhodamine B was determined as described before. Three independent experimental replicates were used for all experiments.

Microgel Jamming. Microgels were first concentrated by centrifugation at 300 ref for 3 min, if applicable, mixed with the desired complementary microgels collected separately. A pre-rinsed membrane (0.22 pm) was folded into a cone shape and placed in a 1.5 mL eppendorf tube. The pellet was then loaded onto the membrane and centrifuged at 50 ref for 20, 5 or 1 s. The jammed microgels were retrieved from the membrane and placed between two glass slides with spacers until the assembly was completed.

Porosity. Characterization of porosity was adapted from a previously reported method based on the fluorescence of void space between particles.¹⁵⁹¹ Briefly, a labeling solution for the interstitial space was prepared by dissolving FITC-dextran (2 MDa) in beads buffer at 40 pg/mL. Microgels were dispersed in the FITC-dextran solution before jamming. Granular hydrogels were imaged using an inverted confocal microscope (LSM 700 Confocal Microscope) and post- processed with Imaged to analyze the pores. Thresholding was based on the Triangle or Huang algorithm to binarize the stacks and the size range in the Analyze Particles function was set to 5 pm² to infinity. The resulting %area was averaged over all stacks to obtain the porosity of the sample.

Atomic force microscopy (AFM). The elastic modulus of microgels were measured using AFM as previously described.¹⁶⁰¹ The nanoindentation tests were conducted on a NanoWizard II AFM (JPK Instruments AG). Silicone cantilevers with a polystyrene tip, a force constant of 0.2 N/m, and a resonance frequency of 13 kHz were used (NanoAndMore GmbH, Watsonville, CA, USA) for the measurements. The contact force was set to 0.1 V, and the pulling range was set from 1500 to 3000 nm. 4,096 force-distance curves in 20 x 20 pm area were recorded and calculated to give the elastic modulus.

Antibody modification. aCD3 or aCD28 antibodies were modified with DBCO by reducing the disulfide linkage using TCEP-HCI (1:30 molar ratio) and then reacting with DBCO-PEG12-maleimide (Conju Probe, 1:60 molar ratio) at 4°C overnight. The mixture was purified using desalting column (3kDa), washed 7 times with 1X PBS and stored at 4°C before further use. The degree of modification on antibodies was quantified via UV-vis spectroscopy using nanodrop. The absorbance of unmodified antibody and DBCO-PEG12- maleimide at 280 nm and 310 nm was measured and plotted versus the concentration to obtain the standard calibration curve and extinction coefficient. The concentration of DBCO conjugated to antibodies was quantified based on the calibration curve of DBCO-PEG12- maleimide absorption at 310 nm. The concentration of antibodies after modification was quantified based on the calibration curve of antibody absorption at 280 nm after subtracting the absorbance from DBCO. The number of DBCO per antibody = concentration of DBCO/concentration of antibody, indicating an average of 4.5 DBCO groups on each antibody.

Antibody conjugation. For polyclonal expansion, alginate microgels coated with alginate-azide were mixed with DBCO-modified aCD3 and aCD28 at 4°C overnight. Microgels were washed three times with beads buffer, soaked in T cell media at 4°C overnight and washed three times with T cell media to remove physically absorbed antibodies. The conjugation efficiency was quantified by measuring the amount of unreacted antibodies in the buffer and culture media using nanodrop during reaction and washing steps. For antigen-specific expansion, microgels coated with biotin-modified alginate were mixed with streptavidin at room temperature for 1h at a biotin/streptavidin ratio of 1. Then biotinylated H-2K(b) MHC class I monomer presenting SIINFEKL peptide and biotinylated aCD28 were added to react for 1 h at room temperature. Microgels were washed three times with beads buffer, soaked in T cell media at 4°C overnight and washed three times with T cell media to remove physically absorbed antibodies.

Primary mouse T cell isolation. C57BL/6J mice were used for polyclonal T cell expansion studies and C57BL/6-Tg(TcraTcrb)1100Mjb/J (OT-I) mice were used for antigenspecific T cell expansion studies and cytotoxic function analysis. Primary mouse T cells were obtained from the spleen and isolated using CD4+ or CD8a+ T cell isolation MACS kits (Miltenyi Biotec) to obtain CD4+ T cells or CD8+ T cells. Mouse T cells were cultured in T cell media (RPMI 1640 supplemented with 10% HI-FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, 50 pM beta-mercaptoethanol, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, 10 mM HEPES, and 1% penicillin-streptomycin) supplemented with 100 ll/rnl recombinant mouse IL-2. All procedures involving animals were done in compliance with National Institutes of Health and Institutional guidelines with approval of Harvard University’s Institutional Animal Care and Use Committee. Animals were purchased from The Jackson Laboratory, female and between 6 and 9 weeks old. Animals were maintained on 12 h light cycles and fed chow and water ad libitum. Vaccination against ovalbumin. Vaccines were prepared following protocols published previously. ^[61'⁶²¹ In brief, mesoporous silica rods (MSRs) were suspended in sterile DPBS at 50 mg/mL. 2mg of MSRs were incubated with 200pg ovalbumin (InvivoGen) and additional 2mg were incubated with 100ug CpG-ODN 1826 (5'-TCCATGACGTTCCTGA CGTT-3') (Integrated DNA Technologies, Chicago, IL). The suspensions were gently shaken at room temperature for 7h, flash-frozen, and lyophilized overnight. The following day, a separate 1mg aliquot of MSR suspension was mixed with 1 pg granulocyte-macrophage colony-stimulation factor (GM-CSF, Peprotech) and shaken for 1 h at 37°C. The three MSR suspensions were combined into one single 150pL shot by adding sterile DI water, and injected through an 18G needle into the left flank of two C57BL6/J mice (The Jackson Laboratory).

Primary mouse T cell isolation from lymph nodes. Vaccinated mice were euthanized 7 days post-vaccination. Their ipsilateral draining inguinal, axillary, and brachial lymph nodes as well as their contralateral lymph nodes were harvested into 4mL RPMI containing 10% FBS, 150 U/mL collagenase IV (Thermo Fisher Scientific Inc.), and 0.1 ug/mL DNAse (F. Hoffmann-La Roche AG). The lymph nodes were dissociated using a GentleMACS Tissue Dissociator (Miltenyi Biotec) and incubated at 37°C for 30 min under mild agitation. Subsequently, the tissues were strained through a 40pm strainer, washed with PBS twice, and processed to obtain lymphocytes. T cells were isolated using CD4+ or CD8a+ T cell isolation MACS kits (Miltenyi Biotec) to obtain CD4+ T cells or CD8+ T cells.

Primary human T cell isolation. Human healthy de-identified blood collars were obtained from Brigham’s and Woman’s Hospital, processed in a Ficoll gradient to obtain enriched peripheral blood mononuclear cells (PBMCs) and frozen prior to use. Primary human T cells were isolated from PBMCs using the human pan-T cell isolation kit (Miltenyi Biotec) to obtain a mixture of CD4+ and CD8+ T cells. Human T cells were cultured in T cell media (RPMI 1640 supplemented with 10% HI-FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, 50 pM beta-mercaptoethanol, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, 10 mM HEPES, and 1 % penicillin-streptomycin) supplemented with 30 U/ml recombinant human IL-2.

T Cell Proliferation Assay. The isolated CD4+ or CD8+ cells were pre-labelled with 5 pM CellTrace yellow (ThermoFisher Scientific) at 37°C for 15 minutes. After PBS washing, the CellTrace-labelled CD4+ cells were mixed with activation stimuli (i.e. , Dynabeads or microgels), and seeded at a density of 10⁵ cells/well. Microgels were seeded at a density of 10⁴/well in suspension. Commercial Dynabeads (ThermoFisher Scientific) were used according to the manufacturer-optimized protocol included with the kit at a cell/Dynabeads ratio of 1. After 3 d, the CellTrace yellow fluorescence was measured using an Aurora Spectral Analyzer (Cytek). Microscopic Images were taken using EVOS FL microscope. Polyclonal T cell Expansion Studies. A mixture of isolated primary mouse or human CD4+ and CD8+ T cells (CD4/CD8 ratio = 1) were mixed with activation stimuli and cultured for 3 d as described above. Media was added to maintain the cells below a density of 2.5x10⁶ cells/mL throughout the culture period. Fold expansion was calculated by dividing the number of cells at the respective time point by the number of cells seeded at the start of culture.

T Cell Phenotypic Analysis. T cell phenotype was evaluated by using flow cytometry (Cytek Aurora). Gates were set using fluorescence minus one (FMO) controls. Data was analyzed using FCS express flow cytometry software. Anti-mouse antibodies for flow cytometry were obtained from BioLegend and ThermoFisher: CD4-BV785 (RM4-5), CD8a- eFluor450 (53.6.7), CD62L-BV510 (MEL-14), CD44-FITC (IM7), PD-1-PE/Dazzle (RMP1- 30), Lag-3-PE (C9B7W), CD25-APC (PC61), OX-40-PE/Cy7 (OX-86), Live/Dead (Fixable blue dead stain). Anti-human antibodies for flow cytometry were obtained from BioLegend: CD4- PerCP (SK3), CD8- APC/Cyanine7 (SK1), CD45RA- PE/Cyanine7 (H1100), CD62L- BV510 (DREG-56), CCR7-FITC (G043H7), CD25- PE/Dazzle (M-A251), CD39-BV711 (A1), PD-1-BV421 (EH12.2H7), Lag-3-APC (7H2C65), CD127-PE (A019D5).

Cytotoxic Function. The B16-F10 murine melanoma cells and B16-F10 expressing ovalbumin were obtained from American Type Culture Collection (ATCC) and expanded subconfluently in growth medium consisting of 10% fetal bovine serum, 1% penicillin/streptomycin in high-glucose Dulbecco’s Modified Eagle media (DMEM). Cells were passaged at 80% confluency and used at passage 10 or lower for all experiments. In killing assays, B16-F10 expressing ovalbumin were incubated with 1 pg/mL Calcein AM (Invitrogen) for 30 min at 37 °C, and then pulsed with 2 pg/ml SIINFEKL peptide for 60 min at 37 °C. T cells and B16-F10 were mixed at a ratio of 10:1 and co-cultured for 4 h. Cells were pelleted and fluorescence intensity of supernatant samples were quantified using a plate reader. For B16-F10 without expressing ovalbumin, cells were incubated with Calcein AM and directly mixed with T cells for co-culture.

Statistical Analysis. All statistical analysis was performed in R studio (version 2022.7.0.548). All levels of significance for differences observed between groups were evaluated using ANOVA one-way, followed by Tukey Honest Significant Differences test. Standard deviation is illustrated by error bars, and significance levels are stated as follows: * P < 0.05, ** P < 0.01 , *** P < 0.001 , **** P < 0.0001.

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Additional Experimental Methods

1. Materials synthesis and preparation

Alginate and hyaluronic acid (HA) were functionalized with tetrazine (Tz) or norbornene (Nb) following a reported protocol.¹¹¹ Briefly, alginate or HA was dissolved in MES buffer solution (0.1 M MES, 0.3 M NaCI, pH 6.5) at a concentration of 0.5 wt%. N- Hydroxysuccinimide (NHS) (2.2 g per 1 g alginate, 5.28 g per 1 g HA) and 1-ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDC) (3.7 g per 1 g alginate, 8.88 g per 1 g HA) were added respectively. Then, (4-(1 ,2,4,5-tetrazin-3-yl)phenyl)methanamine hydrochloride (217 mg per 1 g alginate, 520.8 mg per 1 g HA) or 5-norbornene-2-methylamine (119.4 pL per 1 g alginate, 286.56 pL per 1 g HA) were dissolved in MES buffer and added to the solution. The mixture was stirred for 20 h at room temperature in the dark and then centrifuged at 4000 rpm for 30 min. The supernatant was filtered through a 0.22 pm pore size filter and dialyzed (MWCO 3.5 kDa) against decreasing salt concentrations for 3 d. The solution was further purified with activated charcoal (0.5 g per 1 g alginate) for 30 min, filtered through a 0.22 pm pore size filter and lyophilized.

Alginate was functionalized with Arg-Gly-Asp peptide (RGD), Lissamine rhodamine B ethylenediamine (RhoB), and sulfo-cyanine5 NHS ester (sulfo-Cy5) following an established protocol.¹²¹ Briefly, alginate was dissolved in MES buffer solution at a concentration of 1 wt%. Sulfo-NHS (68 mg per 1 g alginate) and EDC (121 mg per 1 g alginate) were added successively. RGD (76 mg per 1 g alginate), RhoB (36 mg per 1 g alginate), or sulfo-Cy5 (27 mg per 1 g alginate) were dissolved in MES buffer and added to the solution. The mixture was stirred for 20 h at room temperature in the dark before quenching the reaction with hydroxyl amine (18 mg per 1 g alginate). The solution was then dialyzed (MWCO 3.5 kDa) against decreasing salt concentrations for 3 d, purified with activated charcoal (0.5 g per 1 g alginate) for 30 min, filtered through a 0.22 pm pore size filter and lyophilized.

Proton nuclear magnetic resonance spectroscopy (¹H NMR, D2O, 400 MHz) was used to quantify the degree of substitution (DS) of norbornene (Nb) or tetrazine (Tz) functional groups on alginate following a previously reported method using potassium hydrogen phthalate (KHP) as the internal standard (peak at 7.50 ppm).^[3] Functionalized alginate samples were dissolved at 0.75-1.65 wt% in D2O solvent containing 3 mM of KHP. ¹H NMR peaks corresponding to KHP and functional groups were integrated and compared to determine the concentrations of Nb or Tz based on the known internal standard concentration. The DS values were calculated based on the molar ratio between the functional groups and the alginate.

2. Microgel preparation

HA microgels. The dispersed phase containing 2 wt% HA was prepared as a mixture of Tz and Nb modified HA dissolved separately at 1-3 wt% in DI water. A mixture of fluorosurfactant (1%) in fluorocarbon oil (HFE, 3M Novec 7500) was used as the continuous phase. HA-Tz and HA-Nb solutions were injected at 150 pL/h and the continuous phase was injected at 1000 pL/h. All solutions were injected through polyethylene tubing (0.381 mm I.D x 1.143 mm O.D., Scientific Commodities Inc., Lake Havasu City, AZ, USA) into the microfluidic device using Luer-Lok syringes (BD Syringe) and a Harvard Apparatus PHD 2000 Programmable syringe pump. The emulsion was then collected in a tube and left at room temperature for 24 h to allow covalent crosslinking between HA polymers. After the reaction was complete, the continuous phase was removed, and 33% 1/7, 1/7, 2/7, 2/7- perfluoro-1 -octanol (PFO) in HFE was added in excess at 1:3 volume ratio to the collected microgels to break the emulsion. Finally, microgels were washed three times with beads buffer (130 mM NaCI, 25 mM HEPES, 2 mM CaCh, pH 7.5), redispersed in beads buffer (2 mL per h of synthesis) to obtain a concentration of 4*10⁵ microgels/mL, and stored at 4°C until further use.

Gelatin microgels. The dispersed phase containing 10% gelatin was prepared by dissolving gelatin in DI water at 50°C. A mixture of fluorosurfactant (1%) in HFE was used as the continuous phase. The dispersed phase and continuous phase were injected into the microfluidic device at 40 °C at flow rates of 300 pL/h and 1000 pL/h, respectively. The emulsion was then collected in a tube on ice for gelation. After the synthesis was complete, the continuous phase was removed, and 33% PFO in HFE was added in excess at 1:3 volume ratio to the collected microgels to break the emulsion. Finally, microgels were washed three times with beads buffer at 4°C, redispersed in beads buffer (2 mL per h of synthesis) to obtain a concentration of 4*10⁵ microgels/mL, and stored at 4°C until further use.

Alginate-collagen interpenetrating network (IPN) microgels. The dispersed phase was prepared by mixing Tz and Nb modified alginate, type I collagen and 1M sodium hydroxide in situ at a final concentration of 2 wt% alginate and 2 mg/mL collagen. A mixture of fluorosurfactant (1%) in HFE was used as the continuous phase. The dispersed phase and continuous phase were injected into the microfluidic device at flow rates of 300 pL/h and 1000 pL/h, respectively. The emulsion was then collected in a tube and left at 37 °C for 24 h for crosslinking. After the synthesis was complete, the continuous phase was removed, and 33% PFO in HFE was added in excess at 1 :3 volume ratio to the collected microgels to break the emulsion. Finally, microgels were washed three times with beads buffer, redispersed in beads buffer (2 mL per h of synthesis) to obtain a concentration of 4*10⁵ microgels/mL, and stored at 4°C until further use.

The diameter and size distribution of microgels were determined by brightfield and fluorescent imaging using EVOS FL microscope and confocal microscope (Upright Zeiss LSM 710). The images were post-processed using Imaged software to calculate the average diameter of microgels and standard deviation.

3. Rheological characterization

Rheological properties of granular hydrogels were characterized using an oscillatory shear rheometer (DHR-3, TA Instruments, New castle, DE, USA). Measurements were performed at 25°C using an 8 mm parallel plate geometry. Time sweeps (1 Hz, 1% strain) were applied to determine the storage and loss moduli of jammed microgels. Shear-thinning test was performed by measuring viscosity (q) under a ramp flow mode as the shear rates increased from 0.1 to 100 s^-1.

Reference

[1] Y. Hu, A. S. Mao, R. M. Desai, H. Wang, D. A. Weitz, D. J. Mooney, Lab Chip 2017, 17, 2481-2490.

[2] A. S. Mao, J.-W. Shin, S. Utech, H. Wang, O. Uzun, W. Li, M. Cooper, Y. Hu, L. Zhang, D. A. Weitz, D. J. Mooney, Nat. Mater. 2017, 16, 236-243.

[3] B. Ozkale, J. Lou, E. Ozelgi, A. Elosegui-Artola, C. M. Tringides, A. S. Mao, M. S. Sakar, D. J. Mooney, Lab Chip 2022, 22, 1962-1970.

[4] E. Sideris, D. R. Griffin, Y. Ding, S. Li, W. M. Weaver, D. Di Carlo, T. Hsiai, T. Segura, ACS Biomater. Sci. Eng. 2016, 2, 2034-2041.

Additional Methods

Microgels were concentrated by centrifugation at 300 ref for 3 min and redispersed in a solution of 0.1 mg/mL poly(D-lysine) at a concentration of 4*10⁵ microgels/mL. Microgels were then immediately concentrated by centrifugation at 300 ref for 3 min and washed three times. Next microgels were redispersed in a solution of azide-functionalized alginate (1 mg/mL, DS20) at a concentration of 4*10⁵ microgels/mL and collected by centrifugation at 300 ref for 3 min. Microgels were washed three times and redispersed in beads buffer at initial stock solution concentration before further use. DLL4 was first modified with DBCO by mixing with DBCO-sulfo-N-hydroxysuccinimidyl ester (Sigma) (1 :3 molar ratio) in beads buffer at 4 °C overnight. DBCO-modified DLL4 (1 pg) was then mixed with azide-coated microgels (3*10⁴ microgels) at 4 °C for 24 h, washed three times to achieve DLL4-functionalized microgels.

Example 2: Surface-Specific Functionalization of Granular Hydrogels to Regulate Cell Behaviors

Granular hydrogels assembled from microgels are potentially useful for cell therapy and tissue regeneration, but a general approach to incorporate bioactive ligands on the gel surface has been lacking. The present experiment presents a strategy to modulate the surface properties of granular hydrogels by functionalizing via layer-by-layer coating. Sequentially depositing poly(D-lysine) and alginate on the microgel surface resulted in a thin, dense and stable layer. This strategy is compatible with a variety of coating polymers and allows versatile chemistry for further surface modification. Using this strategy, granular hydrogels presenting RGD peptides and growth factors on the surface promoted spreading, proliferation and differentiation of mesenchymal stem cells (MSCs). Moreover, the experimental data demonstrate that tethering stimulatory antibodies efficiently activates T cells for expansion.

1. Introduction

Cell therapies (e.g., stem cell and T cell transfer) are attracting increasing attention, and biomaterials often serve as 3D cell carriers and provide essential signaling cues to direct cell behaviors.^1-2 Bioactive ligands that bind to cell surface receptors and deliver biochemical and mechanical cues are typically presented on the surface of these biomaterials.³ For example, adhesive ligands, such as RGD peptides, mediate cell adhesion, spreading, migration, and prevent anoikis.⁴ Immobilized growth factors and antibodies on the surface can stimulate cell proliferation and differentiation.⁵'⁶ The void space and pore size in biomaterials also play important roles in regulating cell deployment and engagement with the host, as interconnected microporous structures enable cell migration and communication.⁷'⁹ Overall, porous scaffolds with high surface area presenting essential ligands provide a powerful means to direct cell behavior and deployment for cell therapy.

Granular hydrogels generated from jamming and assembly of microgels provide an attractive type of microporous scaffold.¹⁰'¹¹ They have been used in 3D bioprinting,¹²'¹³ wound healing¹⁴ and tissue regeneration.¹⁵'¹⁷ Granular hydrogels exhibit shear-thinning properties allowing for needle injection into the body, and provide void spaces that are sufficiently large for cell migration.¹⁴’¹⁸'²⁰ To introduce bioactive ligands, ligand-functionalized polymers are typically utilized to fabricate the microgels, but this requires a new synthesis for incorporation of different ligands.^{14 21}'²² To improve the fabrication efficiency, a postfunctionalization approach was developed to modify pre-synthesized microgels presenting chemo-selective groups with a variety of ligands via click chemistry.²³ However, this approach results in conjugation of target peptides or proteins throughout the entire microgels, while only those presenting on the surface can typically bind to cell surface receptors to regulate cell behavior.

Herein, we developed a powerful approach to achieve surface functionalization of microgels via layer-by-layer coating, forming a thin but dense layer on the surface which does not affect the jamming and inter-particle crosslinking between microgels in the fabrication of granular hydrogels. This strategy is applicable to a variety of microgel polymers, coating polymers and allows versatile chemistry for further modification. We further demonstrate that incorporating RGD peptides, growth factors, and antibodies on microgel surfaces allows control over mesenchymal stem cells (MSCs) and T cells.

2. Results

2.1 Synthesis of Microgels and Granular Hydrogels

Alginate microgels were fabricated using microfluidic emulsion, which provides defined size and shape by controlled droplet formation (FIG. 68A). Alginate was first modified with norbornene (Alg-Nb) or tetrazine (Alg-Tz) by carbodiimide coupling to achieve an average degree of substitution (DS) of 13 or 11.5 functional groups per alginate chain respectively, as quantified by proton nuclear magnetic resonance spectra. Stock solutions of Alg-Nb and Alg-Tz were then mixed at a final concentration of 2 wt% in the microfluidic device and injected to form microdroplets by emulsion, which then crosslinked overnight to generate microgels with a diameter of 77 ± 2 pm (FIG. 68B). The elastic moduli of the microgels could be tuned by varying the ratio between Alg-Nb and Alg-Tz (FIG. 68C).

To fabricate granular hydrogels, alginate microgels were jammed by centrifugation over a membrane to remove a portion of the continuous aqueous phase between particles (FIG. 68D). The resulting microporous structure was visualized by incorporating 2 MDa fluorescein (FITC)-labelled dextran (FIG. 68E). Varying the time for centrifugation modulated the porosity of the granular hydrogels in a reproducible manner (FIG. 68F). The porosity of the granular hydrogels was independent of the stiffness of the microgel building blocks (FIG. 68G). By contrast, the storage moduli of the granular hydrogels were proportional to the stiffness of microgels. 2.2 Surface Functionalization of Microgels

We next sought an efficient chemical strategy to functionalize the surface of microgels by non-covalent polymer coatings. Alginate microgels were first immersed in a solution of poly(D-lysine) (PDL) to deposit a layer of positively charged polymers and then immersed in a solution of functionalized alginate to coat the second layer and introduce functional groups on the surface (FIG. 69A-69B). Both layers of polymers were uniformly and efficiently coated on the surface of microgels, and the thickness of both PDL layer and alginate coating was 0.74 ± 0.11 pm (FIG. 69C). The diameter of the microgels slightly decreased to 72 ± 2 pm after coating. The thin coating layer allows efficient modification only on the surface without introducing functional groups throughout the entire microgel. The alginate polymers formed a dense layer on the surface of microgels (0.58 ± 0.18 pg/cm²), which allows incorporation of sufficient ligands to mediate biological functions. For example, when RGD-modified alginate (DS 20) was used for coating, the ligand density on the surface was 57.4 ng/cm², similar to the concentration in many biological studies.⁴²⁴ The concentration of functional moieties on microgel surfaces can be easily tuned by varying the DS of functional groups coupled to the alginate polymers used for coating. When increasing the DS of FITC on alginate, for example, the fluorescent intensity of alginate-FITC on the microgel surface significantly increased while the coating density remained constant. The surface density of coated polymers can also be modulated by varying the concentration of alginate solution used to create the second layer after PDL coating, without significant changes in thickness (FIG. 69D). This non-covalent coating was stable on microgel surfaces over at least 3 weeks (FIG. 69E).

The surface functionalization strategy is applicable to a range of coating and core polymers. FITC-labelled hyaluronic acid (HA) can be uniformly coated on the surface of alginate microgels. In addition, core microgels made of HA, gelatin and alginate-type Icollagen interpenetrating network were fabricated using microfluidic emulsion, and a uniform and thin layer of fluorescent dye-labelled alginate was also observed on the surface of these microgels after coating, demonstrating the versatility of the approach (FIG. 69F). A further advantage of the surface-specific chemical modification is that it allows different click chemistries to be used to fabricate the microgel as versus surface functionalization. For example, microgels crosslinked via the norbornene-tetrazine strategy can be subsequently coated with azide-modified alginate, allowing surface conjugation of dibenzocyclooctyne (DBCO)-modified agents through strain-promoted azide-alkyne cycloaddition (SPAAC), which is orthogonal to the norbornene-tetrazine crosslinking reaction. Overall, the surfacespecific chemical modification achieved via surface coating allows efficient fabrication of microgels with different surface functionalities by leveraging different polymers and chemo- selective chemistries to modify pre-synthesized microgels. We next examined whether direct chemical conjugation to microgels post-fabrication could provide surface functionalization similar to that achieved with the coating above. Here, click chemistry was leveraged to modify microgels with functional moieties by covalent conjugation of FITC labelled trans-cyclooctene (TCO-FITC) to Tz-presenting microgels (Nb/Tz = 1/2). While TCO-FITC was mixed with microgels for only 1 min, it was homogeneously conjugated throughout the microgels (FIG. 69G), indicating diffusion of the TCO-FITC was significantly faster than the TCO-tetrazine reaction, which is one of the most efficient click chemistries used for bioconjugation. This result highlights the challenge of surface-specific modification via chemical conjugation post microgel fabrication.

2.3 Influence of Polymer Coating on Interparticle Crosslinking

The ability of coated microgels to generate a stable granular hydrogel scaffold via interparticle crosslinking was next explored. Granular hydrogels formed without chemical or physical crosslinking between microgels were not stable under shear flow, and disassembled when changing the media after 24 h (FIG. 70A). To improve the stability, we formed granular hydrogels using a combination of two batches of microgels, with one fabricated with excess Tz (Nb/Tz = 1/2), and the other with excess Nb (Nb/Tz = 2/1). This resulted in stable granular hydrogels, as measured by less than 17% loss of mass over 3 weeks in subsequent incubation (FIG. 70A), suggesting formation of covalent linkages between the distinct microgels. Moreover, only a small increase in porosity was observed after soaking in media for 3 days, further demonstrating stable network microstructures (FIG. 70B). As expected, non-crosslinked granular hydrogels demonstrated rapid stress relaxation, while covalently crosslinked granular hydrogels demonstrated much slower stress relaxation. This is likely attributed to the confinement of microgels when covalently linked to adjacent microgels.

Strikingly, polymer coatings on the surface of these microgels didn’t affect their assembly. The annealing process used to form interparticle covalent crosslinking remained efficient between microgels with polymer coatings, as these granular hydrogels were stable over 3weeks when subsequently soaked in buffer (FIG. 70A) and the porosity remained similar after 3 days (FIG. 70B). The polymer coatings on the microgels in the granular hydrogels were also stable over 3 weeks in the jammed state. To further understand why microgel assembly into stable granular hydrogels was not inhibited by the polymer coating, we synthesized Rhodamine-labelled alginate microgels containing excess tetrazine (Nb/Tz = 1/2) and used sulfo-Cy5 labelled alginate as the coating polymer. FITC-TCO was allowed to react with residue tetrazines on the microgels after coating, to detect their availability in the microgel. Interestingly, the fluorescent signal from FITC colocalized with that of Rhodamine B and sulfo-Cy5 (FIG. 70C). Quantitative analysis further demonstrated that the highest intensity of fluorescent signal of all three dyes overlapped at the outer shell of the microgels, indicating that tetrazine groups are still exposed on the surface of microgels and accessible to form inter-particle crosslinks (FIG. 70D).

This phenomenon is likely attributed to both the minimum thickness of the polymer coatings and out-of-plane diffusion of charged polymers. The quantity of polymer deposited on the surface is anticipated to be low, as only two layers of polymers were coated on microgels; the resulting coating is expected to be less than 10 nm when formed on hard materials.²⁵ In addition, out of plane diffusion, representing diffusion of polymers vertical to the surface, is commonly observed in layer-by-layer assembly approaches.^26-27 The PDL and alginate coating likely undergo some interdiffusion, although the high molecular weight and high charge density of PDL and alginate results in low chain mobility,²⁸ likely restricting their diffusion within a thin layer (< 1 pm). To test this possibility, low molecular weight PDL (1-5 kDa) was used for coating, and was found to diffuse throughout the entire microgel.

2.4 Regulating Proliferation and Differentiation of Mesenchymal Stem Cells (MSCs)

We next explored the ability of granular hydrogels to provide biochemical signals to support MSCs by incorporating or coupling bioactive peptides or proteins in the polymer coatings. To promote cell adhesion, an Arg-Gly-Asp (RGD) peptide was introduced on the surface of microgels by coating the microgels with RGD functionalized alginate (Alg-RGD). The surface RGD concentration was varied by changing the DS of RGD on the coating alginate polymers. Cell penetration was observed through granular hydrogels following cell seeding both with and without the RGD functionalization. By contrast, cells seeded on nanoporous alginate hydrogels remained on the gel surface. Cells in non-RGD coated microgels remained rounded, while significant cell spreading was observed with RGD coated microgels (FIG. 71A). Quantitative analysis confirmed that increasing the amount of RGD coated on microgel surfaces resulted in increasing cell area and aspect ratio, as well as decreasing circularity (FIG. 71 B-71C). Cell proliferation was enhanced at higher RGD concentration, as indicated by nuclear staining of EdU (FIG. 71 D). The flexibility of granular hydrogels was leveraged by combining Alg-RGD coated microgels (labelled with Rhodamine B) and non-RGD coated microgels (coating labelled with sulfo-Cy5). MSCs were found to be preferentially in contact with RGD-presenting microgels, as compared to non-RGD coated microgels (FIG. 71 E). Quantitative analysis revealed that more than 85% of the cells were adherent to RGD-coated microgels, as compared to 25% adjacent to non-RGD-coated microgels (FIG. 71 F).

The ability of microgels to provide surface presentation of large proteins was also explored. Bone morphogenetic protein 2 (BMP-2) is a growth factor that induces osteogenic differentiation of MSCs. To achieve surface functionalization with BMP-2, alginate-azide was first coated on the surface of microgels, followed by reacting with DBCO-modified BMP-2 through SPAAC click chemistry. BMP-2 presented from the surface significantly enhanced osteogenic differentiation of MSCs, as indicated by alkaline phosphatase (ALP) staining (FIG. 71G-71H). To further examine the benefit of surface conjugation, alginate-azide coated microgels were also simply mixed with the same amount of unmodified BMP-2 to allow physical absorption of the growth factor. Physically trapped BMP-2 induced osteogenic differentiation, but ALP expression was reduced as compared to surface functionalized factor (FIG. 71G-71H).

2.5 T Cell Activation

Surface functionalization was next leveraged to present essential antibodies for T cell activation and expansion. T cell activation generally requires both T-cell receptor stimulation and T cell co-stimulation, and activating antibodies to the appropriate T cell surface receptors were surface presented from microgels by first coating alginate-tetrazine on the surface of microgels (Nb/Tz = 1:1), and then reacting with trans-cyclooctene-modified activating antibodies for CD3 (aCD3) and CD28 (aCD28). By day 3 of co-culture of jammed microgels and CD4 T cells, massive cell clusters were observed surrounding the microgels (FIG. 72A). The proliferation rate significantly increased from 5% without antibodies to 90% in the presence of aCD3 and aCD28, which was slightly higher than commercial CD3/CD28 T-cell expansion beads (Dynabeads) (FIG. 72B-72C). When the same amount of aCD3 and aCD28 antibodies were directly conjugated to microgels via free tetrazines on the polymer used to fabricate the microgels (Nb/Tz = 1:2), only 13% of the CD4+ T cells were activated to proliferate. Polyclonal expansion of a mixture of CD4+ and CD8+ cells was also examined using the surface functionalization strategy. Here, culture with the antibody-presenting granular hydrogels led to a 60-fold cell expansion of CD4+ T cells over 5 days, similar to the Dynabead control (FIG. 72D), but a significantly greater expansion of CD8+ cells was observed in antibody-presenting granular hydrogels compared to Dynabeads. Granular hydrogels promoted substantial CD8-biased skewing, as demonstrated by the CD4/CD8 ratio in the final cell population (FIG. 72E).

3. Conclusion

Here is demonstrate a surface functionalization method capable of incorporating bioactive ligands on the surface of porous granular hydrogels to regulate the behavior and functions of therapeutic cells. Surface functionalization was achieved by coating the microgel surface with oppositely charged polymers, resulting in a thin yet stable layer of functional polymers decorating the surface of microgels. This approach is generally applicable to a variety of coating and core polymers, and allows versatile chemo-selective chemistry for subsequent surface modification. When surface functionalized microgels were used to fabricate granular hydrogels, the polymer coatings had minimum impact on porosity and interparticle crosslinking. Microgels and granular hydrogels presenting specific peptides and proteins promoted the proliferation and differentiation of stem cells, and expansion of T cells. This strategy provides a convenient and versatile means to modulate the surface biochemical properties of porous scaffolds. Considering the ready injectability of granular hydrogels, these materials can potentially be delivered with minimally invasive procedures for in situ expansion of stem cells and immune cells. This strategy can also be applied to other types of porous scaffolds, such as cryogels, to control their surface properties.

4. Experimental Section

Microgel Preparation. The synthesis of covalently crosslinked microgels was adapted from an existing protocol.²⁹ The dispersed phase containing 2 wt% alginate was prepared as a mixture of Tz and Nb modified alginate dissolved separately at 1-3 wt% in DI water. A mixture of fluorosurfactant (1%) in fluorocarbon oil was used as the continuous phase. Alginate-Tz and alginate-Nb solutions were injected at 150 pL/h and the continuous phase was injected at 1000 pL/h. The emulsion was then collected in a tube and left at room temperature for 24 h to allow covalent crosslinking between alginate polymers. After the reaction was complete, the continuous phase was removed, and 33% 1/7, 1/7, 2/7, 2/7- perfluoro-1 -octanol in HFE was added in excess at 1 :3 volume ratio to the collected microgels to break the emulsion. Finally, microgels were washed three times with beads buffer (130 mM NaCI, 25 mM HEPES, 2 mM CaCh, pH 7.5), redispersed in beads buffer and stored at 4°C until further use.

Seeding MSCs on Hydrogels. Microgels coated with alginate-RGD (DS 2, 20) or unmodified alginate were assembled to form granular hydrogels with interparticle crosslinking using the method described above. Bulk hydrogels were prepared by mixing equal volumes of 1% alginate-Tz and 1% alginate-Nb and left at room temperature for 24 h for crosslinking. After trypsinized and re-suspended as a single cell suspension in DMEM, D1 MSCs were seeded on the surface of hydrogel at a concentration of 10⁴ cells/cm² and cultured for 6 d. Culture media was changed every 2 days.

Immunohistochemical Staining. Hydrogels containing cells were fixed with 4% paraformaldehyde in serum-free DMEM at 37 °C for 45 min and washed three times by calcium containing PBS (cPBS). The samples were permeabilized with 0.1% Triton-X, blocked with 3% v/v goat serum and stained with phalloidin (AF-488, Life Technologies) and DAPI (Invitrogen). Cell proliferation assay was performed using Click-IT Edll (Invitrogen) to assess the cell-cycle progression of cells. Samples were imaged using an inverted confocal microscope (LSM 700 Confocal Microscope) and post- processed with Imaged.

Differentiation Assay. Alginate microgels were coated with alginate-azide (DS 20), reacted with DBCO-labelled BMP-2 and assembled to form granular hydrogels with interparticle crosslinking using the method described above. MSCs were seeded at 5*10⁴ cells/cm², cultured for 7 days and then fixed with 4% paraformaldehyde. The gels were soaked in 30% sucrose at 4 °C overnight and then in a mix of 50% of a 30% sucrose in cPBS solution and 50% OCT (Tissue-Tek) for 4 h. The gels were then embedded in OCT, frozen, and sectioned with a thickness of 50 pm using a cryostat (Leica CM1950). The sectioned samples were equilibrated in alkaline buffer (100 mM Tris-HCI, 100 mM NaCI, 0.1% Tween-20, 50 mM MgCh pH 8.2 for 20 min and stained in 500 pg/mL naphthol AS-MX phosphate (Sigma) and 500 pg/mL Fast Blue BB Salt Hemi ZnCh salt (Sigma) in alkaline buffer for 60 min to probe alkaline phosphatase (ALP). The sectioned samples were then stained with DAPI and imaged using Zeiss AxioScan Microscope.

CD4+ T Cell Proliferation Assay. aCD3 or aCD28 antibodies were modified with TCO by reducing the disulfide linkage by TCEP-HCI and then reacting with TCO-PEG5- maleimide. To conjugate antibodies, alginate microgels (Nb/Tz = 1:1) coated with alginatetetrazine or alginate microgels presenting Tz (Nb/Tz = 1/2) were mixed with TCO modified antibodies at a concentration of 1 pg aCD3 and 1 pg aCD28 per 4*10⁵ microgels at 4°C overnight. Microgels were washed three times with beads buffer, soaked in beads buffer at 4°C overnight and washed three times to remove physically absorbed antibodies. The isolated CD4+ cells were prelabeled with 5 pM CellTrace yellow (ThermoFisher Scientific) at 37°C for 15 minutes. After PBS washing, the CellTrace -labeled CD4+ cells were mixed with activation stimuli (i.e., Dynabeads or microgels), and seeded at a density of 5x10⁵ cells/mL. Commercial Dynabeads (ThermoFisher Scientific) were used according to the manufacturer- optimized protocol included with the kit. After 3 days, the CellTrace yellow fluorescence was measured using an Aurora Spectral Analyzer (Cytek). Microscopic Images were taken using EVOS FL microscope.

Polyclonal T cell Expansion Studies. Isolated primary mouse pan T cells were mixed with activation stimuli and cultured for 5 days as described above. Media was added to maintain the cells below a density of 2.5x10⁶ cells/mL throughout the culture period. CD4+ and CD8+ were analyzed via flow cytometry using anti-mouse CD4 antibody (Brilliant Violet 785, RM4-5, BioLegend #100552) and anti-mouse CD8a antibody (APC/Fire™ 750, 53-6.7, BioLegend # 100766). Antibodies were used at the manufacturer-recommended dilution. Dead cells were stained and excluded from analysis using LIVE/DEAD™ Fixable Blue Dead Cell Stain Kit (ThermoFisher Scientific, # L23105). Gates were set based on fluorescence minus one (FMO) controls. Fold expansion was calculated by dividing the number of cells at the respective timepoint by the number of cells seeded at the start of culture.

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INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned herein are hereby incorporated by reference in their entirety as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control. EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the disclosure described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

CLAIMS What is claimed is:

1. A microgel, comprising

(i) a core microgel comprising a crosslinked polymer comprising a functional group selected from the group consisting of a crosslinked tetrazine (Tz) polymer, a crosslinked norbornene (Nb) polymer, and combinations thereof,

(ii) a non-covalent polymer coating comprising a positively charged polymer applied to the surface of the core microgel, and

(iii) a surface coating comprising a functionalized polymer applied to the surface of the coated core microgel to introduce a functional group for surface-specific conjugation of an active agent.

2. The microgel of claim 1 , wherein the core microgel is characterized by a degree of substitution (DS) of about 5 to about 15 functional groups per polymer chain.

3. The microgel of claim 1 or 2, wherein the positively charged polymer comprises poly(D-lysine) (PDL).

4. The microgel of any one of claims 1-3, wherein the core microgel comprises at least one polymer selected from the group consisting of an alginate polymer, a hyaluronic acid (HA) polymer, a collagen polymer, a gelatin polymer, and combinations thereof.

5. The microgel of any one of claims 1-4, wherein the core microgel comprises a gelatin and alginate-type I collagen interpenetrating network.

6. The microgel of any one of claims 1-5, wherein the functionalized polymer comprises a click reaction moiety selected from the group consisting of an azide moiety, a dibenzocyclooctyne (DBCO) moiety, a transcyclooctene moiety, a tetrazine (Tz) moiety, a norbornene (Nb) moiety, and variants thereof, optionally wherein the functionalized polymer comprises an azide-modified alginate polymer.

7. The microgel of any one of claims 1-6, wherein the non-covalent polymer coating and the surface coating are independently characterized by a thickness of about 0.1 pm to about

2 pm, optionally wherein the non-covalent polymer coating and the surface coating are characterized by a combined thickness of about 0.5 pm to about 1.5 pm.

8. The microgel of any one of claims 1-7, wherein the microgel is characterized by a diameter of about 25 pm to about 250 pm, optionally wherein the microgel is characterized by a diameter of about 50 pm to about 100 pm.

9. The microgel of any one of claims 1-8, wherein:

(i) the microgel is characterized by a porosity (e.g., void space %) of about 1% to about 20%;

(ii) the microgel is characterized by a zeta potential of about-19.62 mV when uncoated, the microgel is characterized by a zeta potential of about +10.32 when coated with poly(D- lysine) (PDL), and/or the microgel is characterized by a zeta potential of about -13.17 mV when coated with poly(D-lysine) (PDL) and an azide-modified alginate polymer; and/or

(iii) the microgel is characterized by an elastic modulus of about 0.5 kPa to about 10 kPa.

10. A microgel of any one of claims 1-9, further comprising an active agent comprising a complementary functional group conjugated to the functional group of the surface coating, optionally wherein the active agent comprises a click reaction moiety selected from the group consisting of an azide moiety, a dibenzocyclooctyne (DBCO) moiety, a transcyclooctene moiety, a tetrazine (Tz) moiety, a norbornene (Nb) moiety, and variants thereof, optionally wherein the active agent is modified with an average of about 1 to about 10 click reaction moieties per active agent.

11. The microgel of claim 10, wherein the surface coating comprises an azide-modified polymer to allow surface-specific conjugation of a dibenzocyclooctyne (DBCO)-modified active agent through strain-promoted azide-alkyne cycloaddition (SPAAC), optionally wherein the active agent is modified with an average of about 3 to about 6 dibenzocyclooctyne (DBCO) moieties per active agent.

12. The microgel of any one of claims 1-11 , wherein the active agent is selected from the group consisting of an antibody or an antigen binding fragment thereof, a peptide, a protein, and combinations thereof.

13. The microgel of any one of claims 1-12, wherein the active agent is capable of modifying a cellular behavior selected from the group consisting of cell phenotype, morphology, spreading, proliferation, differentiation, activation, expansion and combinations thereof.

14. The microgel of any one of claims 1-13, wherein the active agent is capable of binding to a T cell surface receptor to promote T cell activation and expansion.

15. The microgel of any one of claims 1-14, wherein the active agent is selected from the group consisting of a aCD3 antibody, a aCD28 antibody, and combinations thereof.

16. The microgel of any one of claims 1-15, wherein the active agent comprises a peptide, optionally wherein the active agent comprises a Arg-Gly-Asp peptide (RGD).

17. The microgel of any one of claims 1-16, wherein the active agent comprises an antigen.

18. The microgel of any one of claims 1-17, wherein the active agent comprises a major histocompatibility complex (MHC) class I molecule and/or a MHC class II molecule, optionally wherein the active agent comprises a MHC class I molecule and/or a MHC class II molecule presenting a peptide.

19. The microgel of any one of claims 1-18, wherein the microgel comprises an active agent at a predefined density (e.g., ligand density) of about 3 pg/cm² to about 10 pg/cm².

20. The microgel of any one of claims 1-19, wherein the surface coating layer of functionalized polymer allows incorporation of an active agent only on the surface to mediate biological functions without introducing functional groups throughout the entire microgel that are not available to cell surface receptors.

21. A composition comprising the microgel of any one of claims 1-20 and a continuous aqueous phase.

22. A granular hydrogel comprising the microgel of any one of claims 1-20.

23. A pharmaceutical composition comprising the microgel of any one of claims 1-20, or the granular hydrogel of claim 22, and a pharmaceutically acceptable carrier.

24. A method of preparing a microgel, comprising: (i) providing a core microgel comprising a crosslinked polymer comprising a functional group selected from the group consisting of a crosslinked tetrazine (Tz) polymer, a crosslinked norbornene (Nb) polymer, and combinations thereof, optionally wherein the core microgel is characterized by a degree of substitution (DS) of about 5 to about 15 functional groups per polymer chain;

(ii) applying a non-covalent polymer coating comprising a positively charged polymer to the surface of the core microgel, optionally wherein the positively charged polymer comprises poly(D-lysine) (PDL);

(iii) applying a surface coating comprising a functionalized polymer to the surface of the coated core microgel to introduce a functional group for surface-specific conjugation of an active agent; and

(iv) optionally conjugating an active agent comprising a complementary functional group conjugated to the functional group of the surface coating.

25. A method of preparing a granular hydrogel, comprising:

(i) providing a composition comprising a plurality of microgels and a continuous aqueous phase;

(ii) concentrating the microgels into a pellet via centrifugation;

(iii) loading the pellet onto a membrane filter and removing the continuous aqueous phase or a portion thereof via centrifugation, thereby forming a granular hydrogel.

26. A method of activating and expanding a population of T cells, comprising contacting the population of T cells with the microgel of any one of claims 1-20, or the granular hydrogel of claim 22.

27. A method of promoting polyclonal and antigen-specific T cell expansion, comprising contacting the population of T cells with the microgel of any one of claims 1-20, or the granular hydrogel of claim 22.

28. A method of enhancing antigen-specific enrichment of a subpopulation of T cells, comprising contacting the population of T cells with the microgel of any one of claims 1-20, or the granular hydrogel of claim 22.

29. A method of controlling T cell proliferation and T cell phenotype, comprising contacting the population of T cells with the microgel of any one of claims 1-20, or the granular hydrogel of claim 22.

30. A method of regulating the proliferation and differentiation of stem cells, optionally mesenchymal stem cells (MSCs), comprising contacting the population of T cells with the microgel of any one of claims 1-20, or the granular hydrogel of claim 22.