WO2025160219A1

WO2025160219A1 - Methods and compositions for treating reperfusion injury following myocardial infarction

Info

Publication number: WO2025160219A1
Application number: PCT/US2025/012671
Authority: WO
Inventors: Matthew Andrew BORRELLI; Steven R. Little; Heth TURNQUIST; Jason Alan Burdick
Original assignee: University of Colorado Boulder; University of Pittsburgh; University of Colorado System
Current assignee: University of Colorado Boulder; University of Pittsburgh; University of Colorado System
Priority date: 2024-01-23
Filing date: 2025-01-23
Publication date: 2025-07-31
Anticipated expiration: 2026-07-23

Abstract

The presently disclosed subject matter relates to a controlled release drug delivery system for cardiovascular applications. Specifically, the discloses subject matter involves utilizing injectable hydrogels coupled with degradable microparticles, formulated into compositions to provide a sustained release of therapeutic agents. The present disclosure further includes the application of these composition for use in methods of treating cardiovascular conditions.

Description

METHODS AND COMPOSITIONS FOR TREATING REPERFUSION INJURY FOLLOWING MYOCARDIAL INFARCTION

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims the benefit of priority to U.S. Provisional Patent Application No. 63/624,223, filed on January 23, 2024, the contents of which are incorporated herein by reference in their entirety.

1. FIELD OF INVENTION

The disclosed subject matter pertains to a controlled drug delivery system for treatment of reperfusion injury following ischemic myocardial infarction (MI). The controlled delivery system integrates a hydrogel coupled with microparticles to enable localized, targeted, and sustained release of therapeutic agents.

2. BACKGROUND

Myocardial infarction (MI - i.e. heart attack) is highly prevalent in America. Over 800,000 Mis occur each year, of which 20% are rehospitalizations due to subsequent MI. This is a significant burden on the health care system, amounting to annual hospital costs of $12.1 billion dollars (2013 data) [1], Treatments such as fibrinolytics and percutaneous coronary intervention (PCI) can reduce initial tissue damage following MI, yet 40% of Americans experiencing an MI will die or develop heart disease within 5 years [1], This is the result of dysregulated inflammation that advances damage to healthy tissue, called reperfusion injury, and an ineffective repair response working in tandem to perpetuate pathologic tissue remodeling [2], Existing treatments for MI do not address the inflammatory or repair responses in the infarcted heart. Thus, there is an unmet clinical need to limit inflammatory and accelerate repair processes following MI. In a healthy steady state, the body’s own cells can naturally control inflammation and mediate tissue repair and remodeling through a sophisticated, dynamic system that acts locally and specifically [3-5], The presently disclosed subject matter precisely targets this imperative by harnessing and amplifying the body’s innate systems for inflammation control and local repair at the MI site, thereby holding significant potential to prevent reperfusion injury and enhance cardiac repair.

The role of Tregs in modulating the local immune response during MI and their influence on tissue repair and remodeling is increasingly apparent. New advancements have led to the development of degradable, polymer microparticles designed to locally enrich regulatory T cells (Treg), a cell type known for its role in suppressing inflammation and promoting repair [5], These microparticles have shown the capability to attract endogenous Treg or induce Treg from infiltrating naive T cells, offering promise in enhancing Treg populations within specific areas. Studies have indicated that this enrichment leads to a local reduction in inflammatory cell populations and an increase in reparative cell populations. This progress suggests the potential for reducing inflammatory damage and enhancing tissue repair following MI. Although these controlled release systems have been administered locally via subdermal injection, adapting this technology to solid organs, such as the heart, requires further innovation.

3. SUMMARY

The present disclosure provides compositions and methods for treating ischemic myocardial infarction. The disclosed subject matter is based, in part, on the development of a controlled release drug delivery system specifically tailored for application within the cardiovascular system.

In one aspect, the present disclosure provides a hydrogel comprising a sustained release microparticle, wherein the sustained release microparticle comprises a therapeutic agent. In certain embodiments, the hydrogel is a shear-thinning hydrogel.

In certain embodiments, the shear-thinning hydrogel comprises hyaluronic acid macromers functionalized with adamantane (HA-AD), cyclodextrin (HA-CD), or a combination thereof. In certain embodiments, the hydrogel comprises HA-AD at a concentration from about 1% to about 30% wt/vol. In certain embodiments, the hydrogel comprises HA-AD at a concentration of about 4% wt/vol. In certain embodiments, the hydrogel comprises HA-CD at a concentration from about 1% to about 30% wt/vol. In certain embodiments, the hydrogel comprises HA-CD at a concentration of about 4% wt/vol.

In certain embodiments, the hydrogel comprises hyaluronic acid at a concentration of 4% wt/vol. In certain embodiments, the shear-thinning hydrogel exhibits a lower diffusivity after curing.

In certain embodiments, the hydrogel retains microparticles at a delivery site. In certain embodiments, the hydrogel is a thermoresponsive hydrogel. In certain embodiments, the thermoresponsive hydrogel comprises a PEG (Polethylene Glycol), NIPAAm (N- Isopropylacrylamide), or a combination thereof. In certain embodiments, the thermoresponsive hydrogel comprises about 100 mg of NIPAAm. In certain embodiments, the thermoresponsive hydrogel has a lower critical solution temperature below 37°C. In certain embodiments, the thermoresponsive hydrogel has a lower critical solution temperature below 20°C. In certain embodiments, the thermoresponsive hydrogel reversibly solidifies into a gel at a temperature above the lower critical solution temperature.

In certain embodiments, the microparticle is present in the hydrogel at a concentration from about 10 mg/mL up to about 100 mg/mL. In certain embodiments, the microparticle has a diameter up to about 100 pm. In certain embodiments, the microparticle has a diameter larger than about 1 pm.

In certain embodiments, the therapeutic agent comprises a Treg cell factor. In certain embodiments, the Treg cell factor comprises a regulatory T cell stimulatory factor. In certain embodiments, the Treg cell factor comprises a T cell chemoattractant factor. In certain embodiments, the Treg cell factor is selected from the group comprising CCL22, IL2, TGF-P, IL33, rapamycin, IL 13, amphiregulin, and a combination thereof.

In certain embodiments, the hydrogel further comprises a second sustained release microparticle, wherein the second sustained release microparticle comprises a second therapeutic agent. In certain embodiments, the second therapeutic agent comprises a Treg cell factor. In certain embodiments, the Treg cell factor is selected from the group comprising CCL22, IL2, TGF-P, IL33, rapamycin, IL13, amphiregulin, and a combination thereof. In certain embodiments, the hydrogel further comprises an antiproliferative agent, an immunosuppressant drug, a non-thrombogenic substance, an anti-adhesive substance, and a combination thereof.

In another aspect, the present disclosure provides a pharmaceutical composition comprising the hydrogel and a pharmaceutical carrier comprising at least one excipient component. In certain embodiments, the at least one excipient component comprises a buffering agent, an antioxidant, an alkali salt, a preservative, or a combination thereof.

In a further aspect, the present disclosure provides a method of treatment for ischemic myocardial infarction in a subject in need thereof, comprising administering an effective amount of the hydrogel or pharmaceutical composition comprising a hydrogel. In certain embodiments, the hydrogel or composition is administered into the affected tissue.

In certain embodiments, the hydrogel or composition is administered at an amount from about 0.01 pg to about 100 g per kg of body weight of the subject. In certain embodiments, the hydrogel or composition is administered once or more daily, weekly, monthly, or yearly. In certain embodiments, the hydrogel or composition is orally, transdermally, topically, pulmonary inhalation, or parenterally administered. In certain embodiments, the parenterally administered hydrogel or composition is injected into an infarct or infarct bordering region.

In one aspect, the present disclosure provides a composition comprising a hydrogel and a sustained release microparticle, wherein the sustained release microparticle comprises a first therapeutic agent.

In certain embodiments, the hydrogel is a thermoresponsive hydrogel. In certain embodiments, the thermoresponsive hydrogel comprises a PEG (Polyethylene Glycol) and NIPAAm (N-Isopropylacrylamide).

In certain embodiments, the hydrogel is a shear-thinning hydrogel. In certain embodiments, the shear-thinning hydrogel comprises hyaluronic acid macromers functionalized with adamantane (HA-AD) and cyclodextrin (HA-CD).

In certain embodiments, the hydrogel further comprises a second therapeutic agent.

In certain embodiments, the first therapeutic agent comprises a Treg cell factor. In certain embodiments, the Treg cell factor is a regulatory T cell stimulatory factor. In certain embodiments, the Treg cell factor is a T cell chemoattractant factor. In certain embodiments, the Treg cell factor is selected from the group consisting of CCL22, IL2, and TGF-P, and combinations thereof.

In another aspect, the present disclosure provides methods for treating ischemic myocardial infarction by administering a composition described herein.

In another aspect, the present disclosure provides methods for treating inflammation of a target tissue by administering a composition described herein. In certain embodiments, the composition is delivered to the target tissue in an effective amount effective to reduce myeloid cell infiltration, shorten myeloid cell infiltration duration time, alter infiltrating myeloid cell phenotype towards a pro-repair phenotype, and/or mitigate spreading of inflammation.

4. BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the present disclosure and its features and advantages, reference is now made to the following description, taken in conjunction with the accompanying drawings.

Figures 1A-1C show microparticles having a predicted release profile of a T cell chemoattractant factor. Figure 1 A shows an illustration demonstrating microparticle release of the chemoattractant factor CCL22. Figure IB shows a representative SEM image of a microparticle. Figure 1C shows a cumulative release curve of CCL22 from a poly(D,L-lactide- coglycolide) (PLGA) encapsulated polymer. Data represents mean ± stdev, N = 3 replicates.

Figures 2A-2C show the process of injecting microparticles encapsulated within a hydrogel. Figure 2A shows a representative fluorescent image displaying the hydrogel- encapsulated microparticles injected into agar. Figure 2B quantifies the microparticle distribution at different injection sites by measuring total fluorescence in agar, normalized to gel auto-fluorescence. Figure 2C shows a comparison between the total material injected and the amount remaining in the needle and syringe. Data shown represent mean ± stdev.

Figures 3A-3C show the coupling of the hydrogel with the microparticles improved microparticle retention in the heart. Figure 2A shows a schematic illustrating the injection of AF680 labeled dextran loaded microparticles in a healthy rat heart. Figure 2B shows the retention of microparticles in a healthy rat heart at day 14 post injection, measured by mean fluorescent intensity (MFI) and normalized to the region of interest (ROI). Figure 2C shows representative fluorescent images demonstrating microparticle distribution. Scale ranges are as follows: Saline+MP (1.46e6 - 1.16e7, 2e6); Gel+MP (3.81e6 - 6.45e7, 2e7); Vehicle Control (1.42e6 - 1.10e7, 0.2e6) (min - max, major increment). * p < 0.05.

Figures 4A and 4B show an ex vivo assessment of ventricle function following left ventricular injection of hydrogel-coupled microparticles. Figure 4A shows a representative image of the hydrogel-coupled microparticles injection into the left ventricle. Figure 4B shows an assessment of change of pressure and workload following injection.

Figures 5A and 5B show the effect of CCL22-containing microparticles (CCL22MP) on the migration of Tregs in a hind-limb transplantation model. Figure 5 A shows representative images of Tregs (MFI) colocalized with CCL22 (right limb) but not Blank (left limb) microparticles (MP) (red/black). Figure 5B shows the administration of CCL22MP in a mouse hind-limb transplantation model enhance local Treg populations out to POD 29 - 43.

Figures 6A-6C show the effect of CCL22MP treatment on inflammatory cell populations and expression of Treg-associated factors. Figure 6A shows that CCL22MP application led to a reduction in inflammatory cells. Figure 6B shows that Treg associated cytokines are increased in response to CCL22MP. Figure 6C shows an increase in genetic expression of Treg associated genes in response to CCL22MP.

Figures 7A-7E show the topical application of CCL22MPs in a murine model of MI. Figure 7A shows a schematic of a permanent ligature model of MI. Figure 7B shows the release kinetics of CCL22MP. Data represents mean ± stdev. Figure 7C shows a representative trichrome staining for CCL22 treated and blank treated mice with quantification of infarction size by area % (n = 3, * p < 0.05). Arrows denote significant ventricular wall thinning. Figure 7D shows a quantification of infarction size with and without CCL22MP treatment. Figure 7E shows an echocardiography assessment of left ventricular functional parameters (n = 3, * p < 0.05, ** p < 0.001).

Figures 8A and 8B show the application of hydrogel-coupled microparticles in a model of ischemic-reperfusion MI. Figure 8 A shows a schematic illustrating the ischemic-reperfusion model. Figure 8B shows an assessment injection of CCL22 containing hydrogel-coupled microparticles with respect to ejection fraction, cardiac output and systolic volume.

Figures 9A and 9B show IL-33 expands ST2⁺ Tregs that secrete IL-10 and IL-13. Figure 9A shows IL-10 levels and Figure 9B shows IL-13 levels in supernatants from sorted CD4+ T cell populations cultured with or without IL-33 (20 ng/mL). *P < 0.05; **P < 0.01, ****P < 0.0001.

Figures 10A-10C show Treg repair factors are critical for repair. Figure 10A shows a survival curve in a bleomycin induced lung injury model. Figure 10B shows the frequency of inflammatory monocytes population following bleomycin injury. Figure 10C shows fibroblast proliferation rates for an in vitro wound healing assay.

Figures 11A and 11B show an IL-33 releasing hydrogel. Figure 11A shows a representative image of IL-33 loaded hydrogel (IL-33Gel) in saline release media. Figure 11B shows the instantaneous release kinetics of IL-33 loaded hydrogel. Data represents mean ± stdev for N = 3 replicates.

Figures 12A-12D show TriMP (IL-2, TGFP, and rapamycin) treatment alters regulatory to effector cell populations and reduces inflammatory factors. Figure 12A shows the frequency of CD4+ FoxP3+ Tregs in total CD3+ population. *p < 0.05 B). Figure 12B shows the frequency of CD4+ IFNy+ Thl in total CD3+ population. **p < 0.01 C). Figure 12C shows the mRNA expression of inflammatory factors. Figure 12D shows the mRNA expression of Treg-associated factors. Rejecting samples (n = 17-20, POD = 33-45), Surviving samples (n = 7, POD > 300); *p < 0.05, **p < 0.01, ***p < 0.001.

5. DETAILED DESCRIPTION

Reperfusion injury following ischemic myocardial infarction (MI) is largely immune mediated. Briefly, ischemic MI causes cardiac cell necrosis and the release of inflammatory damage markers. When ischemia ends and the affected cardiac tissue is reperfused, infiltrating immune effector cells see the inflammatory markers and become activated causing damage to adjacent healthy/surviving cells. The adult cardiac cells are unable to proliferate, and thus the damage caused by the immune system exacerbates the severity of MI and increases the likelihood of a patient developing heart failure.

The disclosed subject matter pertains to compositions and methods for modulating the immune response post ischemic myocardial infarction (MI) to prevent or treat reperfusion injury. Particularly, the disclosed subject matter is directed to a controlled drug release system that combines injectable hydrogel technology with degradable, polymer microparticles for, adapted for local administration to the cardiovascular tissue, such as the heart.

For clarity, and not by way of limitation, the detailed description of the invention is divided into the following subsections:

5.1 Definitions;

5.2 T cells;

5.3 Compositions;

5.4 Methods;

5.5 Kits; and

5.6 Exemplary embodiments.

5, 1, Definitions

The terms used in this specification generally have their ordinary meanings in the art, within the context of this disclosure and in the specific context where each term is used. Certain terms are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner in describing the compositions and methods of the disclosure and how to make and use them.

As used herein, the use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification can mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”

As used herein, the terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The present disclosure also contemplates other embodiments “comprising,” “consisting of’, and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.

As used herein, the term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, z.e., the limitations of the measurement system. For example, “about” can mean within 3 or more than 3 standard deviations, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2- fold, of a value.

An “individual” or “subject” herein is a vertebrate, such as a human or non-human animal, for example, a mammal. Mammals include, but are not limited to, humans, non-human primates, farm animals, sport animals, rodents and pets. Non-limiting examples of non-human animal subjects include rodents such as mice, rats, hamsters, and guinea pigs; rabbits; dogs; cats; sheep; pigs; goats; cattle; horses; and non-human primates such as apes and monkeys.

As used herein, the term “in need thereof’ would be a subject known or suspected of having or being at risk of developing a disease or condition, e.g., coronary artery disease.

As used herein, the term “disease” refers to any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ.

As used herein, the term “at risk for” refers to a medical condition or set of medical conditions exhibited by a patient which can predispose the patient to a particular disease or affliction. For example, these conditions can result from influences that include, but are not limited to, behavioral, emotional, chemical, biochemical, or environmental influences.

As used herein, the term “drug” or “compound” refers to any pharmacologically active substance capable of being administered which achieves a desired effect. Drugs or compounds can be synthetic or naturally occurring, non-peptide, proteins or peptides, oligonucleotides or nucleotides, polysaccharides, or sugars.

As used herein, the terms “pharmaceutically” or “pharmacologically acceptable” refer to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or a human.

As used herein, the term, “pharmaceutically acceptable carrier” includes any and all solvents, or a dispersion medium including, but not limited to, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils, coatings, isotonic and absorption delaying agents, liposome, commercially available cleansers, and the like. Supplementary bioactive ingredients also can be incorporated into such carriers.

As used herein, the term “administered” or “administering” a drug, refers to any method of providing a compound or drug to a patient such that the compound or drug has its intended effect on the patient. For example, one method of administering is by an indirect mechanism using a medical device such as, but not limited to a catheter, spray gun, syringe etc. A second exemplary method of administering is by a direct mechanism such as, oral ingestion, transdermal patch, topical, inhalation, suppository etc.

As used herein, the terms “effective amount” or “therapeutically effective amount” refer to a quantity of a specified agent sufficient to achieve a desired effect in a subject being treated with that agent. Ideally, a therapeutically effective amount of an agent is an amount sufficient to inhibit or treat the disease or condition without causing a substantial cytotoxic effect in the subject. The therapeutically effective amount of an agent will be dependent on the subject being treated, the severity of the affliction, and the manner of administration of the therapeutic composition. In certain embodiments, an effective amount can be formulated and/or administered in a plurality of doses, for example, as part of a dosing regimen.

As used herein, the term “treatment” refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop, or administering a compound or composition to a subject who does not exhibit signs of a disease or exhibits only early signs for the purpose of decreasing the risk of developing a pathology or condition, or diminishing the severity of a pathology or condition. As used herein, the term “ameliorating,” with reference to a disease or pathological condition, refers to any observable beneficial effect of the treatment. The beneficial effect can be evidenced, for example, by a delayed onset of clinical symptoms of the disease in a susceptible subject, a reduction in severity of some or all clinical symptoms of the disease, a slower progression of the disease, an improvement in the overall health or well-being of the subject, or by other parameters well known in the art that are specific to the particular disease.

As used herein, “preventing” a disease or condition refers to prophylactic administering a composition to a subject who does not exhibit signs of a disease or exhibits only early signs for the purpose of decreasing the risk of developing a pathology or condition, or diminishing the severity of a pathology or condition.

As used herein, the terms “reduce,” “inhibit,” “diminish,” “suppress,” “decrease,” “prevent,” “eliminate,” or any variation of these terms includes any measurable decrease or complete inhibition to achieve a desired result. The terms “reduce,” “inhibit,” “diminish,” “suppress,” “decrease,” “prevent,” “eliminate,” and grammatical equivalents (including “lower,” “smaller,” etc.) when in reference to the expression of any symptom in an untreated subject relative to a treated subject, mean that the quantity and/or magnitude of the symptoms in the treated subject is lower than in the untreated subject by any amount that is recognized as clinically relevant by any medically trained personnel. In one embodiment, the quantity and/or magnitude of the symptoms in the treated subject is at least 10% lower than, at least 25% lower than, at least 50% lower than, at least 75% lower than, and/or at least 90% lower than the quantity and/or magnitude of the symptoms in the untreated subject.

As used herein, the term “target tissue” as used herein, refers to any bodily tissue that can be affected by a medical condition and/or disorder (e.g., an immunological disease) to which a population of regulatory T cells can be directed to by induction with a combination of T cell factors released from microparticles having pre-determined release profiles.

As used herein, the term “T cell” as used herein, refers to any of several lymphocytes (e.g., helper T cell or regulatory T cell) that differentiate in the thymus, possess highly specific cell-surface antigen receptors, and include some that control the initiation or suppression of cell-mediated and humoral immunity (as by the regulation of T cell maturation and proliferation) and others that lyse antigen-bearing cells — also referred to as a T lymphocyte.

As used herein, the term “T cell factor,” “T cell stimulating factor,” “regulatory T cell factor,” “T cell inducing factor,” or “T cell chemoattractant factor” refer to any biological agent (z.e., for example, a protein, hormone, compound, drug etc.) capable of interacting with Treg cells. Such factors can become encapsulated within a microparticle or hydrogel and undergo controlled release during the degradation of the microparticle or hydrogel.

As used herein, the term “microparticle” as used herein, refers to any microscopic carrier to which a compound or drug can be attached. Microparticles generally refer to the general categories comprising liposomes, nanoparticles, microspheres, nanospheres, microcapsules, and nanocapsules. In certain embodiments, microparticles contemplated by this present disclosure are capable of formulations having controlled release properties.

As used herein, the term “hydrogel” is intended to connote that meaning normally associated with that term, z.e., a three-dimensional hydrophilic polymeric network that are hydrophilic, in which water is the dispersion medium, and are capable of maintaining their structural integrity. Hydrogels are highly swollen (they can contain over 99.9% water) natural or synthetic polymers. Hydrogels also possess a degree of flexibility very similar to natural tissue, due to their significant water content. As used herein, the term “biocompatible” refers to any material does not elicit a substantial detrimental response in the host. There is always a concern, when a foreign object is introduced into a living body, that the object will induce an immune reaction, such as an inflammatory response that will have negative effects on the host. In the context of this invention, biocompatibility is evaluated according to the application for which it was designed: for example; a bandage is regarded a biocompatible with the skin, whereas an implanted medical device is regarded as biocompatible with the internal tissues of the body. In certain embodiments, biocompatible materials include, but are not limited to, biodegradable and biostable materials.

As used herein, the term “biodegradable” refers to any material that can be acted upon biochemically by living cells or organisms, or processes thereof, including water, and broken down into lower molecular weight products such that the molecular structure has been altered.

As used herein, the term “polymer” refers to any unit-based chain of molecules. For example, such molecules can include but are not limited to gelatin, collagen, cellulose esters, dextran sulfate, pentosan polysulfate, chitin, saccharides, albumin, synthetic polyvinyl pyrrolidone, polyethylene oxide, polypropylene oxide, block polymers of polyethylene oxide and polypropylene oxide, polyethylene glycol, acrylates, acrylamides, methacrylates including, but not limited to, 2-hydroxyethyl methacrylate, poly(ortho esters), cyanoacrylates, gelatin- resorcin-aldehyde type bioadhesives, polyacrylic acid and copolymers and block copolymers thereof.

As used herein, the term “PLGA” refers to mixtures of polymers or copolymers of lactic acid and glycolic acid. As used herein, lactide polymers are chemically equivalent to lactic acid polymer and glycolide polymers are chemically equivalent to glycolic acid polymers. In one embodiment, PLGA contemplates an alternating mixture of lactide and glycolide polymers, and is referred to as a poly(lactide-co-glycolide) polymer.

As used, herein, the term “controlled release” refers to the escape of any attached or encapsulated factor at a predetermined rate. For example, a controlled release of a factor can occur resulting from the predicable biodegradation of a polymer particle (z.e., for example, an artificial antigen presenting cell). The rate of biodegradation can be predetermined by altering the polymer composition and/or ratio’s comprising the particle. Consequently, the controlled release can be short term or the controlled release can be long term. In one embodiment, the short term release is between about 30 minutes and about 1 hour. In one embodiment, the short term release is between about 1 hour and about 3 hours. In one embodiment, the short term release is between about 3 hours and about 10 hours. In one embodiment, the short term release is between 10 hours-24 hours. In one embodiment, the long term release is between about 24 hours and about 36 hours. In one embodiment, the long term release is between about 3 days and about 7 days. In one embodiment, the long term release is between about 7 days and about 1 month. In one embodiment, the long term release is between about 1 month and about 6 months. In one embodiment, the long term release is between about 6 months and about 1 year. In one embodiment, the long term release is at least one year.

The term “sustained release” refers to a microparticle that provides for gradual release of a therapeutic agent over an extended period of time. In certain embodiments, sustained release results in constant blood levels of a therapeutic agent over an extended time period.

The term “delayed release” refers to a microparticle in which there is a time delay between administration of the microparticle and the release of the therapeutic agent. “Delayed release” can involve gradual release of a therapeutic agent over an extended period of time, and thus can be “sustained release.”

As used herein, “long-term release” refers to a microparticle capable of delivering therapeutic levels of the agent for at least about 7 days, at least about 15 days, at least about 30 days, or at least about 60 days.

As used herein, the term “tissue transplant” refers to any replacement of a tissue and/or organ within an individual with a similar tissue and/or organ from a different individual. In some cases, the individuals are from the same species. In other cases, the individuals are from different species.

As used herein, the term “immunological tolerance” refers to any modification of the immune system wherein specific antibodies may not be produced, but the immune system remains responsive to other antigens. For example, specific immune related cells including, but not limited to, Treg cells, osteoclasts, and/or osteoblasts can be stimulated to induce immunosuppression. Such “immunological tolerance” can also be capable of controlling autoimmune diseases including, but not limited to, arthritis, Type I diabetes. Such “immunological tolerance” can also be capable of controlling inflammatory diseases including, but not limited to, periodontal disease.

As used herein, the term “transplant rejection reaction” or “graft versus host disease” refers to any activation of the immune system subsequent to the implantation of an exogenous tissue and/or organ into a patient that can result in damage and/or destruction of the transplanted tissue. Generally, transplant rejections are believed to be an adaptive immune response via cellular immunity (i.e., for example, mediated by killer T cells inducing apoptosis of target cells) as well as humoral immunity (mediated by activated B cells secreting antibody molecules), though the action is joined by components of innate immune response (phagocytes and soluble immune proteins).

As used herein, the term “chemoattractant factor” refers to any compound and/or molecule that induces movement of chemotactic cells in the direction of its highest concentration. For example, a chemoattractant factor can include, but is not limited to, CCL22.

As used herein, the term “chemotactic cells” refers to any biological cell exhibiting chemotaxis, wherein the chemotactic cells direct their movements according to certain chemicals in their environment.

The term “nucleic acid molecule” and “nucleotide sequence,” as used herein, refers to a single or double-stranded covalently-linked sequence of nucleotides in which the 3’ and 5’ ends on each nucleotide are joined by phosphodi ester bonds. The nucleic acid molecule can include deoxyribonucleotide bases or ribonucleotide bases, and can be manufactured synthetically in vitro or isolated from natural sources.

The terms “polypeptide,” “peptide,” “amino acid sequence” and “protein,” used interchangeably herein, refer to a molecule formed from the linking of at least two amino acids. The link between one amino acid residue and the next is an amide bond and is sometimes referred to as a peptide bond. A polypeptide can be obtained by a suitable method known in the art, including isolation from natural sources, expression in a recombinant expression system, chemical synthesis or enzymatic synthesis. The terms can apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. In certain embodiments, a polypeptide can include a conservative amino acid substitution. In certain embodiments, conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid within the same group. For example, amino acids can be classified by charge: positively-charged amino acids include lysine, arginine, histidine, negatively-charged amino acids include aspartic acid, glutamic acid, neutral charge amino acids include alanine, asparagine, cysteine, glutamine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. Amino acids can also be classified by polarity: polar amino acids include arginine (basic polar), asparagine, aspartic acid (acidic polar), glutamic acid (acidic polar), glutamine, histidine (basic polar), lysine (basic polar), serine, threonine, and tyrosine; non-polar amino acids include alanine, cysteine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine. In certain embodiments, no more than one, no more than two, no more than three, no more than four, no more than five residues within a specified sequence are altered. Exemplary conservative amino acid substitutions are shown in Table 1 below.

Table 1.

As used herein, the percent homology between two amino acid sequences is equivalent to the percent identity between the two sequences. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (z.e., % homology = # of identical positions/total # of positions x 100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. The percent homology between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4: 11-17 (1988)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percent homology between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

As used herein, “a functional fragment” of a molecule or polypeptide includes a fragment of the molecule or polypeptide that retains at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% of the primary function of the molecule or polypeptide.

5,2, T Cells

Over the past two decades, regulatory T cells (Treg) have been identified as one of the central components of the mammalian immune system. Sakaguchi et cd., “Immunologic tolerance maintained by CD25+CD4+ regulatory T cells: their common role in controlling autoimmunity, tumor immunity, and transplantation tolerance” Immunol Rev. (2001) 182: 18- 32; Sakaguchi et al., “Regulatory T cells and immune tolerance” Cell (2008) 133:775-787; Campbell et al. , “Phenotypical and functional specialization of FOXP3+ regulatory T cells” Nat Rev Immunol (2011) 11 : 119-130; and Bour- Jordan et al., “Regulating the regulators: costimulatory signals control the homeostasis and function of regulatory T cells” (2009) 229:41-66. The most commonly described, widely studied, and possibly most abundant regulatory T cells in the body are those that express CD4, CD25, and/or FoxP3. These CD4+CD25+FoxP3+ cells (Treg) play important roles in suppressing the activity of self- reactive immune cells and in re-establishing homeostasis following infection. Sakaguchi etal., “Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alphachains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases” J Immunol. (1995) 155: 1151-1164; Hori et al., “Control of regulatory T cell development by the transcription factor Foxp3” Science (2003) 299: 1057-1061; Fontenot et al., “Foxp3 programs the development and function of CD4+CD25+ regulatory T cells” Nat Immunol. (2003) 4:330-336; and Khattri et al., “An essential role for Scurfin in CD4+CD25+ T regulatory cells” Nat Immunol. (2003) 4:337-342.

CD4+CD25+ regulatory T-cells are believed to express the CCR4 receptor which binds to the chemokine CCL22. CCL22 was reported to be involved in the migration of regulatory T-cells to tumor sites because tumors also actively secrete CCL22. Curiel et aL, Nat Med 10:942-949 (2004). This represents just one of the methods that tumors have for evading immune recognition. Evasion of an immune response provides for tumor survival given that many of its basic biological features such as genetic instability, invasive growth, and tissue disruption are inherently pro-inflammatory. Besides attracting regulatory T-cells to its vicinity, tumors are believed to also have the ability to influence biological antigen presenting cells (z.e., for example, biological dendritic cells) to down-regulate processing and presentation of tumor associated antigens, inhibit co-stimulatory expression (z.e., retain immature phenotype), and alter their cytokine secretion profile towards immune tolerance. Pardoll, D., Annual Review of Immunology 21 :807-839 (2003). It has been reported that tumors also secrete TGF-P, which can influence regulatory T-cell mediated tolerance. Chen et al., Proc Natl Acad Sci USA 102:419-424 (2005). Although it is not necessary to understand the mechanism of an invention, it is believed that these other mechanisms similar to those of tumor immune evasion and survival can also be useful in modulating immune responses.

Treg cells also express a unique transcription factor called FoxP3, which is thought to be required for the development of Treg suppressive capacity and is a recognized marker for Treg cells. Hori etal., Science 299: 1057-1061 (2003); and Fontenot etal., Nat Immunol 4:330- 336 (2003). Furthermore, it is believed that the extent of expression of this transcription factor can correlate with the suppressive capacity and activation state of a Treg cell. It has been shown that transfer of the gene for FoxP3 into cells which are CD4+CD25- confers regulatory capacity which is otherwise absent. Fontenot etal., Nat Immunol 4:330-336 (2003). Treg cells can also express high levels of CTLA-4 (i.e., for example, CD152) which can also be involved in their regulatory capacity as this molecule can bind to the B7 class of co-stimulatory molecules in place of CD28 thereby resulting in the production of transforming growth factor- P, or TGF-p. Perez et al., Immunity 6:411-417 (1997). TGF-P, along with engagement of the T-cell receptor, has been demonstrated to differentiate naive, peripheral CD4+CD25- T-cells into CD4+CD25+ cells with suppressive capacity, suggesting that this factor can be important for the in vivo generation and maintenance of Treg cells. Walker etal., J Clin Invest 112: 1437- 1443 (2003). Regulatory T-cells are also believed to express chemokine receptors including, but not limited to, CCR4 and CCR8, rendering them fully capable of migration (i.e., for example, by chemotaxis) to a site of inflammation or to the lymph nodes upon appropriate signaling (lellem et al., J Exp Med 194:847-853 (2001).

5,3, Hydrogel-Coupled Microparticles and Compositions Thereof The presently disclosed subject matter pertains to a composition to provide a sustained and controlled release of a therapeutic agent(s). In certain embodiments, the composition comprises one or more microparticle coupled with a hydrogel, encapsulating a therapeutic agent(s). The hydrogel’s adhesive properties enable targeting of microparticles to specific locations, establishing an effective local delivery system. The coupling of the hydrogel and microparticle(s) is particularly beneficial within the cardiovascular system, as the hydrogel overcomes typical microparticle displacement caused by blood flow. The hydrogel-coupled microparticles have increased retention ensuring a sustained presence of encapsulated therapeutic agents, significantly augmenting their efficacy in targeted tissues.

In certain embodiments, the hydrogel-coupled microparticle composition is created by mixing a base precursor for the hydrogel, cross linkers, and initiators, initiating their polymerization over a specified duration to create the hydrogel. Subsequently, the hydrogel undergoes a washing step to eliminate any surplus initiator or unreacted materials, remaining in a liquid state, typically as an aqueous solution at room temperature until it’s prepared for application. During the formation of the hydrogel, microparticles, loaded with a therapeutic agent, can be introduced either prior to, during, or after its polymerization. This integration yields a suspension of solid microparticles within the hydrogel, with the quantity of microparticles loaded being variable. In certain embodiments, the microparticles are homogeneously dispersed within the hydrogel. In certain embodiments, the microparticles are intentionally heterogeneously dispersed within the hydrogel, contributing to localized variations in drug release or specific targeted delivery mechanisms.

In certain embodiments, the composition comprises a single microparticle population comprising a sustained release of a single therapeutic agent. In certain embodiments, the composition comprises a plurality of microparticle populations wherein each of the microparticle populations comprises a different agent. In certain embodiments, the microparticle populations release the different agents with an independent and distinct release profile. In certain embodiments, the therapeutic agent can be encapsulated within the hydrogel, enabling versatile delivery options within the composition.

In certain embodiments, the therapeutic agent release is dependent on degradation of the polymer microparticles. As the polymer chains break up, the agent can diffuse out of the initial polymer microparticle matrix where it will eventually reach the hydrogel matrix. At that point, the hydrogel can partially slow down the release of the agent but diffusion through the hydrogel is significantly faster than degradation of the polymer. In certain embodiments, the therapeutic agent release can be linear or non-linear (single or multiple burst release). In certain embodiments, the agent can be released without a burst effect. For example, the sustained release can exhibit a substantially linear rate of release of the therapeutic agent. However, the release rate can change to either increase or decrease depending on the formulation of the polymer microparticle and/or hydrogel. The desired release rate and target drug concentration can vary depending on the particular therapeutic agent chosen for the drug delivery system, the cardiovascular condition being treated, and the subject’s health.

In certain embodiments, the present disclosure provides compositions for providing a controlled release formulation comprising one or more therapeutic agents. In certain embodiments the one or more therapeutic agents is a T cell factor. In certain embodiments, the T cell factor can include, but is not limited to, a T cell inducing factor, a T cell chemoattractant factor, and/or T cell stimulatory factor. In certain embodiments, the one or more T cell factors can include, but are not limited to, IL-2, TGF-P, CCL22, IL-33, and rapamycin.

In certain embodiments, the T cell factor can induce, attract, and/or stimulate regulatory T cells (iTreg). In certain embodiments, the T cell factor can induce, attract, and/or stimulate natural regulatory T cells (nTreg).

In certain embodiments, the present disclosure provides a composition comprising a controlled release formulation of one or more factors to induce a Treg phenotype (e.g., determined by the expression of canonical Treg markers and migratory surface markers). In certain embodiments, the composition comprises one or more Treg induction factors (e.g., IL- 2, TGF-P and Rapamycin).

In certain embodiments, the present disclosure provides compositions comprising a controlled release formulation of one or more factors to attract and/or stimulate natural regulatory T cells (nTreg). In certain embodiments, the composition comprises at least one natural regulatory T cell chemoattractant factor (c.g, CCL22). In certain embodiments, the composition comprises one or more natural regulatory T cell stimulatory factors (e.g., CCL22).

In certain embodiments, the present disclosure provides a composition comprising a controlled release formulation of one or more factors that induces nTreg proliferation. In certain embodiments, the composition comprises one or more nTreg proliferation inducing factors (e.g., IL-33). In certain embodiments, the composition comprises one or more factors that stimulates the secretion of reparative factors, including, but not limited to, IL- 13 and amphiregulin (AREG). In certain embodiments, the composition comprises one or more nTreg stimulatory factors (e.g., IL-33). Additionally or alternatively, the presently disclosed subject matter provides hydrogels comprising microparticles (e.g., disclosed in Section 5.3.1).

In certain embodiments, the hydrogel allows the microparticles to be retained in desired anatomic locations (e.g., myocardium or other cardiac tissues) in order to provide an effective local delivery system. In certain embodiments, the hydrogel comprising the microparticles overcomes typical microparticle displacement that can be caused by blood flow.

In certain embodiments, said hydrogels comprising microparticles can be prepared by using any of the methods disclosed herein. For example, and without any limitation, the hydrogels can be prepared using the methods disclosed in the Example below.

In certain embodiments, the microparticles comprised in said hydrogel can provide sustained release of a single therapeutic agent. In certain embodiments, the hydrogel comprises two or more microparticles, wherein each microparticle comprises a different therapeutic agent. For example, but without any limitation, the hydrogel can include a first microparticle comprising CCL22 and a second microparticle comprising IL33. In certain embodiments, the hydrogel comprises two or more microparticles, wherein each microparticle comprises a distinct release profile. For example, but without any limitation, the hydrogel can include a first microparticle comprising a sustained release of a first therapeutic agent and a second microparticle comprising a controlled release of a second therapeutic agent.

In certain embodiments, the therapeutic agent release is dependent on degradation of the polymer microparticles. In certain embodiments, the therapeutic agent release can be linear or non-linear (single or multiple burst release). In certain embodiments, the agent can be released without a burst effect. For example, the sustained release can exhibit a substantially linear rate of release of the therapeutic agent. In certain embodiments, the presently disclosed hydrogel can release picogram to nanogram quantities of one or more Treg induction factors over several hours, several days, or several weeks. Notably, said release rate can change to either increase or decrease depending on the formulation of the polymer microparticle and/or hydrogel.

In certain embodiments the one or more therapeutic agents is a T cell factor. In certain embodiments, the T cell factor can include, but is not limited to, a T cell inducing factor, a T cell chemoattractant factor, and/or T cell stimulatory factor. In certain embodiments, the one or more T cell factors can include, but are not limited to, IL-2, TGF-P, CCL22, IL-33, IL- 13, amphiregulin, and rapamycin. In certain embodiments, the T cell factor can induce, attract, and/or stimulate regulatory T cells (iTreg). In certain embodiments, the T cell factor can induce, attract, and/or stimulate natural regulatory T cells (nTreg). In certain embodiments, the hydrogel comprises a microparticle comprising a therapeutic agent. In certain embodiments, the hydrogel comprises a microparticle comprising CCL-22.

In certain embodiments, the hydrogel comprises a first microparticle comprising a first therapeutic agent, a second microparticle comprising a second therapeutic agent, and a third microparticle comprising a third therapeutic agent. In certain embodiments, the hydrogel comprises a first microparticle comprising IL-2, a second microparticle comprising TGF-P, and a third microparticle comprising rapamycin.

In certain embodiments, the hydrogel comprises a first microparticle comprising a first factor to induce nTreg proliferation and a second microparticle comprising a second factor to induce nTreg proliferation. In certain embodiments, the hydrogel comprises a first microparticle comprising IL-33 and a second microparticle comprising amphiregulin (AREG).

In certain embodiments, the present disclosure provides a composition that can be administered as a liquid, offering versatility in its application. While the composition is adaptable for administration to different tissues and organs, its primary suitability lies in specific locations where fluid movement can otherwise dislodge the microparticles, such as within the cardiovascular system. The composition seamlessly conforms to the cardiovascular system’s walls, ensuring comfortable placement while facilitating the gradual release of the loaded agent. The composition can be administered on a regimen wherein the interval between successive administrations is greater than at least one day. For example, there can be an interval of at least one day, at least one week, or at least one month between administrations of the composition. In certain embodiments, the composition can be used for sustained monthly delivery of medication as a replacement for the current clinical standard administration. In certain embodiments, the hydrogel component of the composition can be biodegradable so that there is no need to remove the gelled substance. This composition disclosed herein decreases the dosing frequency, thereby increasing the likelihood of patient compliance and recovery/prevention of worsening symptoms.

The composition disclosed herein can include an excipient component, such as effective amounts of buffering agents, and antioxidants to protect a drug (the therapeutic agent) from the effects of ionizing radiation during sterilization. Suitable water-soluble buffering agents include, without limitation, alkali and alkaline earth carbonates, phosphates, bicarbonates, citrates, borates, acetates, succinates and the like, such as sodium phosphate, citrate, borate, acetate, bicarbonate, carbonate and the like. These agents are advantageously present in amounts sufficient to maintain a pH of the system of between about 2 to about 9 and more preferably about 4 to about 8. Suitable water-soluble preservatives include sodium bisulfite, sodium bisulfate, sodium thiosulfate, ascorbate, benzalkonium chloride, chlorobutanol, thimerosal, phenylmercuric acetate, phenylmercuric borate, phenylmercuric nitrate, parabens, methylparaben, polyvinyl alcohol, benzyl alcohol, phenylethanol and the like and mixtures thereof.

In certain embodiment, additional components can be added to the composition to allow for easier visualization of the composition suspension such as sodium fluorescein or other fluorescent molecules such as FITC, rhodamine, or AlexaFluors or dyes such as titanium dioxide.

5.3.1. Controlled Release Microparticles

Microparticles refer to the general categories comprising liposomes, nanoparticles, microspheres, nanospheres, microcapsules, and nanocapsules. Preferably, some microparticles contemplated by the present disclosure comprise poly(lactide-co-glycolide), aliphatic polyesters including, but not limited to, poly-glycolic acid and poly-lactic acid, hyaluronic acid, modified polysaccharides, chitosan, cellulose, dextran, polyurethanes, polyacrylic acids, pseudo-poly(amino acids), polyhydroxybutrate-related copolymers, polyanhydrides, polymethylmethacrylate, polyethylene oxide), lecithin and phospholipids.

In certain embodiments, the microparticles can have a diameter of less than 1000 pm, e.g., from about 10 pm to about 200 pm. In certain embodiments, the microparticles can have a diameter of from about 10 pm to about 90 pm, from about 20 pm to about 80 pm, from about 60 pm to about 120 pm, from about 70 pm to about 120 pm, from about 80 pm to about 120 pm, from about 90 pm to about 120 pm, from about 100 pm to about 120 pm, from about 60 pm to about 130 pm, from about 70 pm to about 130 pm, from about 80 pm to about 130 pm, from about 90 pm to about 130 pm, from about 100 pm to about 130 pm, from about 110 pm to about 130 pm, from about 60 pm to about 140 pm, from about 70 pm to about 140 pm, from about 80 pm to about 140 pm, from about 90 pm to about 140 pm, from about 100 pm to about 140 pm, from about 110 pm to about 140 pm, from about 60 pm to about 150 pm, from about 70 pm to about 150 pm, from about 80 pm to about 150 pm, from about 90 pm to about 150 pm, from about 100 pm to about 150 pm, from about 110 pm to about 150 pm, or from about 120 pm to about 150 pm. In certain embodiments, the microparticles can have a diameter of from about 1 pm to about 30 pm, from about 2 pm to about 30 pm, from about 5 pm to about 30 pm, from about 7 pm to about 30 pm, from about 10 pm to about 30 pm, from about 12 pm to about 30 pm, from about 15 pm to about 30 pm, from about 20 pm to about 30 pm, from about 5 pm to about 20 pm, from about 8 pm to about 20 pm, from about 10 pm to about 20 pm, from about 12 pm to about 20 pm, from about 15 pm to about 20 pm, or from about 10 pm to about 15 pm. In certain embodiments, the microparticles can have a diameter of from about 10 pm to about 20 pm.

In certain embodiments, the microparticles can have a diameter of from about 10 nm to about 1000 nm, from about 50 nm to about 1000 nm, from about 100 nm to about 1000 nm, from about 150 nm to about 1000 nm, from about 200 nm to about 1000 nm, from about 300 nm to about 1000 nm, from about 400 nm to about 1000 nm, from about 500 nm to about 1000 nm, from about 600 nm to about 1000 nm, from about 700 nm to about 1000 nm, from about 800 nm to about 1000 nm, from about 100 nm to about 500 nm, from about 150 nm to about 500 nm, from about 200 nm to about 500 nm, from about 250 nm to about 500 nm, from about 300 nm to about 500 nm, from about 400 nm to about 500 nm, from about 500 nm to about 900 nm, from about 600 nm to about 900 nm, from about 700 nm to about 900 nm, from about 800 nm to about 900 nm, from about 100 nm to about 200 nm, from about 100 nm to about 300 nm, from about 100 nm to about 400 nm, from about 600 nm to about 800 nm, or from about 700 nm to about 800 nm. In certain embodiments, the microparticles can have a diameter of from about 10 nm to about 100 nm, from about 20 nm to about 100 nm, from about 50 nm to about 100 nm, from about 70 nm to about 100 nm, from about 110 nm to about 200 nm, from about 120 nm to about 100 nm, from about 150 nm to about 200 nm, from about 200 nm to about 300 nm, or from about 250 nm to about 300 nm.

In certain embodiments, the microparticle can include one or more lipids. In certain embodiments, the lipids can be neutral, anionic or cationic at physiological pH. In certain embodiments, the lipids can be sterols. For example, in certain embodiments, the lipid microparticle include cholesterol, phospholipids and sphingolipids. In certain embodiments, the microparticles comprise PEGylated derivatives of the neutral, anionic, and cationic lipids. The incorporation of PEGylated derivatives can improve the stability of the microparticles. Non-limiting examples of PEGylated lipids include distearoylphosphatidylethanlamine- polyethylene glycol (DSPE-PEG), stearyl-polyethylene glycol and cholesteryl-polyethylene glycol. In certain embodiments, the microparticle can include substituted or unsubstituted fatty acids. Non-limiting examples of saturated fatty acids include caproic acid, enanthic acid, caprylic acid, pelargonic acid, capric acid, undecanoic acid, lauric acid, tridecanoic acid, myristic acid, pentadecanoic acid, palmitic acid, margaric acid, stearic acid, nonadecanoic acid, arachidic acid, heneicosanoic acid, behenic acid, tricosanoic acid, lignoceric acid, pentacosanoic acid, cerotic acid, heptacosanoic acid, montanic acid, nonacosanoic acid, melissic acid, henatriacontanoic acid, lacceroic acid, psyllic acid, geddic acid, ceroplastic acid, hexatri acontanoic acid, and combinations thereof. Non-limiting examples of unsaturated fatty acids include hexadecatri enoic acid, alpha-linolenic acid, stearidonic acid, eicosatrienoic acid, eicosatetraenoic acid, eicosapentaenoic acid, heneicosapentaenoic acid, docosapentaenoic acid, docosahexaenoic acid, tetracosapentaenoic acid, tetracosahexaenoic acid, linoleic acid, gamma-linolenic acid, eicosadienoic acid, dihomo-gamma-linolenic acid, arachidonic acid, docosadienoic acid, adrenic acid, docosapentaenoic acid, tetracosatetraenoic acid, tetracosapentaenoic acid, oleic acid, eicosenoic acid, mead acid, erucic acid, nervonic acid, rumenic acid, a-calendic acid, P-calendic acid, jacaric acid, a-eleostearic acid, P-eleostearic acid, catalpic acid, punicic acid, rumelenic acid, a-parinaric acid, P-parinaric acid, bosseopentaenoic acid, pinolenic acid, podocarpic acid, palmitoleic acid, vaccenic acid, gadoleic acid, erucic acid, and combinations thereof.

In certain embodiments, the microparticles include polymers. In certain embodiments, the polymer can be amphiphilic, hydrophilic, or hydrophobic. In certain embodiments, the polymer can be biocompatible, e.g., the polymer does not induce an adverse and/or inflammatory response when administered to a subject. For example, without limitation, a polymer can be selected from polydioxanone (PDO), polyhydroxyalkanoate, polyhydroxybutyrate, poly(glycerol sebacate), polyglycolide (i.e., poly(glycolic) acid) (PGA), polylactide (i.e., poly(lactic) acid) (PLA), poly(lactic) acid-co-poly(glycolic) acid (PLGA), poly(lactide-co-glycolide) (PLG), polycaprolactone, copolymers, or derivatives including these and/or other polymers. In certain embodiments, the polymer includes PEG. In certain embodiments, the polymer includes poly(lactide-co-glycolide) (PLG).

In certain embodiments, the microparticles include cationic polymers. In certain embodiments, the cationic polymers can be branched or linear. Cationic polymers are able to condense and protect negatively charged molecules such as DNA or RNA. In certain embodiments, without limitation, the cationic polymers can be polyethylenimines, polyhistidyl polymers, chitosan, poly(amino ester glycol urethane), polylysines, or amino cyclodextrin derivatives. In certain embodiments, the microparticle comprises linear polyethylenimine. In certain embodiments, the microparticle comprises chitosan.

In certain embodiments, the microparticles include anionic polymers. In certain embodiments, the anionic polymers can be branched or linear. Anionic polymers are able to condense and protect positively charged molecules such as metals (e.g., Ca++) and positively charged proteins. In certain embodiments, without limitation, the anionic polymers can be polyacrylic acid cystamine conjugates and derivatives thereof, sodium carboxy methyl starch (CMS) and derivatives thereof, carboxy methyl guar gum (CMG) and derivatives thereof, carboxymethyl cellulose and derivatives thereof, or alginate and derivative thereof. In certain embodiments, the microparticle comprises alginate or a derivative thereof.

In certain embodiments, the microparticle can show organ tropism and can have an organ-specific distribution. In certain embodiments, the microparticles include molecules providing for organ tropism or organ-specific distribution. For example, but without any limitation, the surface of the microparticles can be functionalized to bind biological molecules (e.g., a ligand or an antibody) targeting a specific tissue (e.g., epithelial cells). The surface functionalization of microparticles can be based on the use of homo- or hetero-bifunctional cross linkers to the aim to add an organic functional group (e.g., R-NH2, R-COOH, etc.), useful to bind biological molecules (e.g., a ligand or an antibody). In certain embodiments, the functionalization of the surface of the microparticles can be achieved using non-covalent conjugation. In certain embodiments, the functionalization of the surface of the microparticles can be achieved using non-covalent conjugation. The covalent conjugation allows modifications at several levels using sequential functionalization and can be exploited to achieve structures with multiple functions. For example, without any limitation, the microparticle can include a PEG molecule synthesized with specific functional groups at the ends which can be used as homo-bifunctional or hetero-bifunctional linkers to perform a wide range of functionalization processes. In certain embodiments, the biological molecule is an antibody targeting an epithelial cell surface molecule. Non-limiting examples of epithelial cell surface molecules include A33, ACE/CD143, ALCAM/CD166, Aminopeptidase B/RNPEP, Aminopeptidase Inhibitors, Aminopeptidase N/CD13, Amnionless, B7-H2, B7-H3, CA125/MUC16, CA15-3/MUC-1, E-Cadherin, CDla, CDld, CDldl, CD46, CD74, CEACAM-l/CD66a, CEACAM-3/CD66d, CEACAM-4, CEACAM-5/CD66e, CEACAM- 6/CD66c, CEACAM-7, Collagen I, CTRP5/ClqTNF5, Cubilin, DDR1, DDR1/DDR2, beta- Defensin 2, beta-Defensin 3, alpha-Defensin 1, alpha-Defensin 5, Endorepellin/Perlecan, EpCAM/TROPl, Fas Ligand/TNFSF6, Gastrokine 1, HIN-1/SCGB3A1, Hyaluronan, IGSF4C/SynCAM4, Integrin alpha 4/CD49d, Integrin alpha 4 beta 1, Integrin alpha 4 beta 7/LPAM-l, JAM- A, JAM-B/VE-JAM, JAM-C, LI CAM, Laminin- 1, MFG-E8, MSPR/Ron, MUC-1, MUC-19, MUC-4, Nectin-1, Nectin-2/CDl 12, Nectin-3, Nectin-4, Nidogen- 1/Entactin, Occludin, PD-L1/B7-H1, PLET-1, P1GF, Prostasin/Prss8, SLURP2, TfR (Transferrin R), and UGRP1/SCGB3A2.

In certain embodiments, the microparticle can adhere to specific tissues. In certain embodiments, the microparticles can be biodegradable or non-biodegradable. In certain embodiments, the microparticle can be comprised in a pharmaceutical composition. In certain embodiments, the microparticles include a therapeutic agent.

Microspheres and microcapsules are useful due to their ability to maintain a generally uniform distribution, provide stable controlled compound release and are economical to produce and dispense. Microspheres are obtainable commercially (Prolease®, Alkerme’s: Cambridge, Mass.). For example, a freeze-dried medium comprising at least one therapeutic agent is homogenized in a suitable solvent and sprayed to manufacture microspheres in the range of about 20 to about 90 pm. Techniques are then followed that maintain sustained release integrity during phases of purification, encapsulation, and storage. Scott et cd.. Improving Protein Therapeutics With Sustained Release Formulations, Nature Biotechnology, Volume 16: 153-157 (1998).

Modification of the microsphere composition by the use of biodegradable polymers can provide an ability to control the rate of therapeutic agent release. Miller et al., Degradation Rates of Oral Resorbable Implants (Polylactates and Polyglycolates): Rate Modification and Changes in PLA/PGA Copolymer Ratios, J. Biomed. Mater. Res., Vol. 11:711-719 (1977).

Alternatively, a sustained or controlled release microsphere preparation is prepared using an in-water drying method, where an organic solvent solution of a biodegradable polymer metal salt is first prepared. Subsequently, a dissolved or dispersed medium of a therapeutic agent is added to the biodegradable polymer metal salt solution. The weight ratio of a therapeutic agent to the biodegradable polymer metal salt can for example be about 1 : 100000 to about 1 : 1, preferably about 1 :20000 to about 1 :500 and more preferably about 1 : 10000 to about 1 :500. Next, the organic solvent solution containing the biodegradable polymer metal salt and therapeutic agent is poured into an aqueous phase to prepare an oil/water emulsion. The solvent in the oil phase is then evaporated off to provide microspheres. Finally, these microspheres are then recovered, washed, and lyophilized. Thereafter, the microspheres can be heated under reduced pressure to remove the residual water and organic solvent.

Other methods useful in producing microspheres that are compatible with a biodegradable polymer metal salt and therapeutic agent mixture are: i) phase separation during a gradual addition of a coacervating agent; ii) an in-water drying method or phase separation method, where an antiflocculant is added to prevent particle agglomeration and iii) by a spraydrying method.

Microparticles can also comprise a gelatin, or other polymeric cation having a similar charge density to gelatin (z.e., poly-L-lysine) and is used as a complex to form a primary microparticle. A primary microparticle is produced as a mixture of the following composition: i) Gelatin (60 bloom, type A from porcine skin), ii) chondroitin 4-sulfate (0.005%-0.1%), iii) glutaraldehyde (25%, grade 1), and iv) l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC hydrochloride), and ultra-pure sucrose (Sigma Chemical Co., St. Louis, Mo.). The source of gelatin is not thought to be critical; it can be from bovine, porcine, human, or other animal source. Typically, the polymeric cation is between 19,000-30,000 daltons. Chondroitin sulfate is then added to the complex with sodium sulfate, or ethanol as a coacervation agent.

Controlled release microcapsules can be produced by using known encapsulation techniques such as centrifugal extrusion, pan coating and air suspension. Such microspheres and/or microcapsules can be engineered to achieve desired release rates. For example, Oliosphere® (Macromed) is a controlled release microsphere system. These particular microspheres are available in uniform sizes ranging between about 5 and about 500 pm and composed of biocompatible and biodegradable polymers. Specific polymer compositions of a microsphere can control the therapeutic agent release rate such that custom-designed microspheres are possible, including effective management of the burst effect. ProMaxx® (Epic Therapeutics, Inc.) is a protein-matrix delivery system. The system is aqueous in nature and is adaptable to standard pharmaceutical delivery models. In particular, ProMaxx® are bioerodible protein microspheres that deliver both small and macromolecular drugs, and can be customized regarding both microsphere size and desired release characteristics.

A microsphere or microparticle can comprise a pH sensitive encapsulation material that is stable at a pH less than the pH of the internal mesentery. The typical range in the internal mesentery is pH about 7.6 to pH about 7.2. Consequently, the microcapsules should be maintained at a pH of less than about 7. However, if pH variability is expected, the pH sensitive material can be selected based on the different pH criteria needed for the dissolution of the microcapsules. The encapsulated compound, therefore, will be selected for the pH environment in which dissolution is desired and stored in a pH preselected to maintain stability. Examples of pH sensitive material useful as encapsulants are Eudragit® L-100 or S-100 (Rohm GMBH), hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate succinate, polyvinyl acetate phthalate, cellulose acetate phthalate, and cellulose acetate trimellitate. In one embodiment, lipids comprise the inner coating of the microcapsules. In these compositions, these lipids can be, but are not limited to, partial esters of fatty acids and hexitiol anhydrides, and edible fats such as triglycerides. Lew C. W., Controlled-Release pH Sensitive Capsule And Adhesive System And Method. U.S. Pat. No. 5,364,634 (herein incorporated by reference). In certain embodiments, the microparticle comprises a biodegradable construct thereby providing a controlled release of encapsulated, incorporated and/or attached factors (z.e., for example, therapeutic agents, antibodies, cytokines, or chemokines). In one embodiment, the particle comprises a degradable polyester including, but not limited to, poly (lactic-co-glycolic) acid (PLGA). PLGA has been used in FDA-approved grafts, sutures, and/or drug delivery microparticulates such as Lupron Depot®. Degradable PLGA polymer microparticles are superior to conventional latex or polystyrene “artificial APCs” because PLGA confers biodegradability. Further, unlike latex and polystyrene polymer particles that only allow surface attachment of proteins, so PLGA polymer particles, allow encapsulation of cell factor (e.g., IL-2, TGF-P, CL22, IL-33, and rapamycin) through a double emulsion/solvent evaporation procedure. Odonnell et al., Advanced Drug Delivery Reviews 28:25-42 (1997). Further, a controlled release of soluble cell factors from PLGA polymers can be engineered to create an appropriate local concentration of these cell factors, which would be accompanied by cell-to-cell contact with immobilized molecules on the particle surface. Such immobilized molecules (z.e., for example, a monoclonal antibody) can be easily bound to the particle through a streptavidin-biotin linkage using several established chemical techniques. Further, the controlled release of encapsulated one such soluble protein factor for seventy days from microparticles has been demonstrated (z.e., for example, CCL22). See, Figure 1C. Scanning electron microscopy confirmed the porous nature of the microparticles responsible for the controlled release characteristics. See, Figure. IB.

In certain embodiments, the releasing is controlled by using different molecular weight PLGA or through other fabrication parameters including, but not limited to, drug distribution, occlusion radius, amorphicity/crytallinity of the polymer, excipients etc. Rothstein et al., J Materials Chem 18: 1873-1880 (2008). An empirical process determines the final amounts of factors to be encapsulated given that the appropriate quantity of these factors for optimal stimulation of regulatory T-cells in vivo is yet still unknown.

The factors can be encapsulated individually or in any combination. In certain embodiments, the factors have independent and differential release profiles. In other words, the release profile for each factor in the formulation is released with a custom-tailored predetermined kinetic and temporal pattern. Rothstein et al., “A simple model framework for the prediction of controlled release from bulk eroding polymer matrices” J Mater Chem (2008) 18: 1873-1880; and Rothstein et al., “A unified mathematical model for the prediction of controlled release from surface and bulk eroding polymer matrices” Biomaterials (2009) 30: 1657-1664. As is shown herein, the specific composition of a microparticle can be determined, in advance, that results in the differential release profiles of each component. Although it is not necessary to understand the mechanism of an invention, it is believed that the interaction of the differential release profiles from these microparticle populations result in Treg cell population induction, attraction, or stimulation.

In certain embodiments, microparticles are capable of presenting incorporated and/or attached factors (i.e., for example, therapeutic agents, antibodies, cytokines, or chemokines). For example, microparticles can comprise factors that can activate specific immune-related blood cells, including, but not limited to, T cells. Such biological agents can comprise a T cell chemoattractant factor, a T cell inducing factor, and/or a T cell stimulatory factor. The microparticles can further present a specific biomimetic surface pattern that results in a T cell response such that the microparticles represent artificial presenting cells.

In certain embodiments, the microparticles provide a controlled release formulation comprising one or more therapeutic agents. In certain embodiments, the one or more therapeutic agents is a T cell factor. In certain embodiments, the T cell factor can include, but is not limited to, a T cell inducing factor, a T cell chemoattractant factor, and/or T cell stimulatory factor. In certain embodiments, the one or more T cell factors can include, but is not limited to IL-2, TGF-P, CCL22, IL-33, IL-13, amphiregulin, and rapamycin.

In certain embodiments, the microparticles provide a controlled release formulation of one or more factor to induce a Treg phenotype (e.g., determined by the expression of canonical Treg markers and migratory surface markers). In certain embodiments, the composition comprises one or more Treg induction factor (e.g., IL-2, TGF-P, and Rapamycin).

In certain embodiments, the microparticles provide a controlled release formulation of one or more factors to attract and/or stimulate natural regulatory T cells (nTreg). In certain embodiments, the composition comprises at least one natural regulatory T cell chemoattractant factor (e.g., CCL22). In certain embodiments, the composition comprises one or more natural regulatory T cell stimulatory factors (e.g., CCL22).

In certain embodiments, the microparticles provide a controlled release formulation of one or more factor that induces nTreg proliferation. In certain embodiments, the composition comprises one or more nTreg proliferation inducing factor (e.g., IL-33). In certain embodiments, the composition comprises one or more factor that stimulates the secretion of reparative factors, including but not limited to IL- 13 and amphiregulin (AREG). In certain embodiments, the composition comprises one or more nTreg stimulatory factor (e.g., IL-33).

Additional information on the features and components of the microparticles disclosed herein can be found in International Patent Publication Nos. WO2011006029, WO2013112456, and WO2014022685, the content of each of which is incorporated by reference in their entireties.

5.3.2. Hydrogels

The presently disclosed subject matter involves coupling of microparticles with a hydrogel. In certain embodiments, the hydrogel envelops or forms connections with the microparticles, effectively serving as both an adhesive and a protective partition for the microparticles.

Additionally or alternatively, the presently disclosed subject matter relates to hydrogels comprising microparticles. In certain embodiments, the microparticles comprise a therapeutic agent (e.g., IL-2, TGF-P, CCL22, IL-33, IL-13, amphiregulin, and rapamycin).

The purpose of the hydrogel is to enhance microparticle retention, particularly in regions affected by blood flow, effectively preventing microparticle displacement and providing a sustained release of a therapeutic agent.

In certain embodiments, the hydrogel is a settable (curable), shear-thinning hydrogel, which can comprise non-covalent crosslinks (giving rise to the ability to deform and flow into liquids under shear-stress and recover back into hydrogels upon stress removal), as well as chemical moieties which provide for the ability to form chemical covalent crosslinks which can then stabilize the hydrogel network. In certain embodiments, this recovery from shear is complete within minutes or even seconds.

These materials can encapsulate therapeutic cargo ex vivo in consistent and controlled conditions, and can be delivered with minimally invasive techniques via-shear-induced flow through a catheter, so as to be surgically implantable with minimal risk of premature polymerization/catheter clogging, as the settable, shear-thinning hydrogel will thin and flow while stress is applied, and can rapidly recover at the target site when stress is removed. Upon delivery, the hydrogels can be further stabilized by a secondary cross-linking, and those hydrogels with sufficient high and robust dual cross-linking functionality have the potential to support and enhance biomedical applications.

Similarly, in the present context, the term shear thinning has a meaning normally associated with that term — z.e., an effect where a fluid’s viscosity (the measure of a fluid’s resistance to flow) decreases with an increasing rate of shear stress. As contemplated herein, such shear-thinning hydrogels are composed of two or more polymers or oligomers that are held together in unique structural relationships by forces other than those of full covalent bonds. Non-covalent bonding is critical in maintaining the three-dimensional structure of the hydrogels. There are four commonly mentioned types of non-covalent interactions: hydrogen bonds, ionic bonds, van der Waals forces, and hydrophobic interactions, each of which is employed in various embodiments of the shear-thinning hydrogels contemplated herein.

As described herein, a shear-thinning hydrogel is a hydrogel capable of self-assembling into a gelled network by interaction of its associated non-covalent linkages. When subjected to a mechanical shear (such as when forced to flow through a needle, catheter, or cannula), at least some of the non-covalent linkages within the hydrogel disassociate, leading to a disassembly of the gel network and a temporary thinning of the gel (lowering of the viscosity). Upon the removal of the mechanical shear force, the original gel re-assembles/recovers to a state (e.g., viscosity, stiffness, or diffusivity) the same as, or close to, its pre-shear state. The term “shearthinning hydrogels ” are used in the literatures to describe such gels where the recovery of the hydrogel after shear can be nearly instantaneous or be as long as hours. While the present disclosure contemplates hydrogels which are included within this broad context, particular independent embodiments include those characterized as “rapid healing ” or “rapid recovery ” hydrogels, where, upon the removal of the mechanical shear force, the original gel recovers within about 30 minutes, preferably within about 20, about 10, about 5, or about 1 minute, or within about 60, about 45, about 15, about 10, about 5, or about 1 second. The types of shearthinning hydrogels falling within this narrower category are summarized in Guvendiren, etal., “Shear-thinning hydrogels for biomedical applications, ” Soft Matter, 2012, 8, 260-272, which is incorporated by reference herein in its entirety for all purposes. All of the hydrogels described within this Guvendiren article, modified to incorporate the chemical moieties capable of participating in at least one chemical covalent cross-linking reaction, as could be accomplished by the skilled artisan, are considered separate embodiments of the present disclosure.

For the sake of absolute clarity, in specific embodiments, the settable shear-thinning hydrogel comprises a peptide-based hydrogel, a protein-based hydrogel, a blended polymer hydrogel, a colloidal hydrogel, or a guest-host-based hydrogel.

In certain embodiments, each settable, shear thinning hydrogel comprises a guest-host- based hydrogel, comprising a host-polymer and a guest-polymer, linked through a plurality of host-guest pairings of non-covalent bonding moieties (plurality here refers to number of crosslinks, not necessarily types of non-covalent crosslinks). A subset of these embodiments provides that the host-polymer comprises a first hydrophilic polymer comprising a plurality of a moieties having a hydrophobic cavity; and the guest-polymer comprises a second hydrophilic polymer comprising a plurality of hydrophobic anchoring moieties (again here, plurality here refers to number of crosslinks, not necessarily types of non-covalent crosslinks). More specific embodiments provide that the moieties capable of providing a hydrophobic cavity comprise a calixarene, a cucurbit[n]uril, or a cyclodextrin, in each case optionally substituted with one or more pendant alkyl, alkanol (e.g., hydroxypropanol), alcohol, alkoxy, aromatic, sugar moieties or vinyl groups. Embodiments described as comprising an optionally substituted cyclodextrin, include those wherein the cyclodextrin is an alpha, beta, or gamma-cyclodextrin, preferably an optionally substituted beta-cyclodextrin.

Another subset of embodiments wherein the settable, shear thinning hydrogel of the present disclosure comprises a guest-host-based hydrogel includes those wherein the hydrophobic anchoring moiety comprises a linear, branched, cyclic, or polycyclic C6-20 hydrocarbon, C6-20 aryl or alkylaryl, hetero or alkylaromatic hydrocarbon moieties. In certain embodiments, the hydrophobic anchoring moiety comprises an adamantane.

Certain other embodiments described as involving a guest-host strategy, include those wherein the host-guest pairing of moiety comprise an alpha-cyclodextrin/hexyl group pair, an alpha-cyclodextrin/polyethylene oxide group pair, a beta-cyclodextrin/adamantane group pair, a beta-cyclodextrin/cyclohexyl group pair, a beta-cyclodextrin/benzyl group pair, a gamma- cyclodextrin/cyclodecyl group pair, a cucurbit[6]uril/hexanediamine group pair, or a cucurbit[6]uril/spermine group pair.

Within those embodiments described by a guest-host relationship, the first and second polymers associated with the host-polymer and guest-polymer, respectively, can each comprise any of the polymers described below, but preferred embodiments are those wherein at least one of the first or second hydrophilic polymers comprises hyaluronic acid. In other preferred embodiments, both the first and second hydrophilic polymers both comprise hyaluronic acid.

In a specific, non-limiting example, the host-polymer moiety comprises a polymer comprising hyaluronic acid to which is attached a plurality of a beta-cyclodextrin moieties; and the guest-polymer comprises a polymer comprising hyaluronic acid to which is attached a plurality of adamantine groups; and the at least one set of chemical moieties capable of chemically, covalently cross-linking the hydrogel is an acrylic or methacrylate group. When the components are mixed, the hydrophobic adamantine becomes non-covalently bound inside of the hydrophobic beta-cyclodextrin cavity to yield physical cross-links and self-assembly to form a settable, shear-thinning hydrogel. Secondary covalent cross-linking of the material is obtainable by the photocatalytic, free-radical crosslinking of the acrylate groups. In certain embodiments, the host-polymer comprises a moiety having a hydrophilic cavity linked to a first hydrophilic polymer; and the guest-polymer comprises a hydrophilic anchoring moiety linked to a second hydrophilic polymer. Such a hydrophilic cavity can comprise a cryptand or crown ether.

In certain embodiments, the settable, shear thinning hydrogel of the present disclosure operates by a two-component Dock-and Lock (DnL) self-assembling hydrogelation mechanism, using bio-conjugate materials. Such a mechanism, and the associated class of shear thinning hydrogels, is described in H. D. Lu, M. B. Charati, I. L. Kim, J. A. Burdick, Injectable Shear-Thinning Hydrogels Engineered with a Self-Assembling Dock-and-Lock Mechanism, Biomaterials, 33:2145-2133, 2012, which is incorporated by reference herein for all purposes. All of the hydrogels described within this Lu article, modified to incorporate the chemical moieties capable of participating in at least one chemical covalent cross-linking reaction, as could be accomplished by the skilled artisan, are considered separate embodiments of the present disclosure. In certain of these embodiments, the hydrogel comprises a docking and dimerization domain (rDDD), comprising a dimer of RIM cAMP dependent PKA recombinant protein, linked together by a hydrophilic peptide spacer containing integrin binding domains. These hydrogels can also or alternatively comprise a locking anchoring domain (LOCK-AD), wherein the LOCK-AD comprises an A-kinase anchoring polypeptide modified with solubilizing amino acid sequences conjugated hydrophilic polymer backbone. These rDDD and LOCK-AD moieties can be linked by any of the hydrophilic polymers described below, but preferably comprise polyethylene glycol or hyaluronic acid. In preferred embodiments, the at least one set of chemical moieties being capable of participating in at least one chemical covalent cross-linking reaction in these DnL hydrogels comprise an acrylate or methacrylate group at the peptide N terminus or along the hydrophilic polymer backbone, said acrylate or methacrylate group capable of polymerizing with exposure to light.

These polymeric DnL conjugated materials can undergo triggered self-assembly via a molecular recognition based ‘Dock-and-Lock’ mechanism under constant physiological conditions. In particular embodiments, these settable, shear thinning hydrogels can be ‘stabilized’ by the radical polymerization of reactive methacrylates that are also included on the polymer.

As described above, various embodiments, provide settable (curable), shear-thinning hydrogels, each hydrogel comprising a hydrophilic polymer network, said hydrophilic polymer network comprising non-covalent crosslinks and at least one set of chemical moieties being capable of participating in at least one chemical covalent cross-linking reaction. In certain embodiments, the “at least one set of chemical moieties being capable of participating in at least one chemical covalent cross-linking reaction” are operable (or begin to chemical crosslink) spontaneously upon formation (mixing) of the shear-thinning hydrogel. The types of chemical moieties which can accomplish this “spontaneous” covalent crosslinking can be described in terms of the chemistries described below, but preferred embodiments are those where the reactants are chosen such that the kinetics of the covalent cross-linking are “slow” with respect to mixing and application to the intended site. That is, the term “slow” reflects that the chemical covalent crosslinking provides an observable effect on the properties of the gel only at times in excess of 30 minutes. In but one example, systems comprising a hydrophilic polymer modified with vinyl sulfone and another modified with a thiol can be used.

In comparison, separate embodiments provide those settable (curable), shear-thinning hydrogels, wherein the at least one chemical covalent cross-linking reaction is initiated by an internal or external (both relative to the hydrogel itself) trigger. In these embodiments, the shear-thinning hydrogels can be described as “selective settable” hydrogels, the term “selective ” referring to the fact that the user can select when and how to initiate the chemical covalent cross-linking reactions (beyond the act simple mixing).

The hydrophilic polymer network of the settable shear thinning hydrogels can also comprise more than one — z.e., at least two — sets of chemical moieties, each set being capable of independently participating in at least one chemical covalent cross-linking reaction. That is, in various aspects of the present disclosure, a given hydrogel can contain one, two, or more sets of chemical moieties capable of participating in a chemical covalent crosslinking reaction. In separate embodiments, these occur spontaneously or as triggered. Each covalent crosslinking reaction can occur by a similar mechanism (e.g., a condensation reaction), albeit with different chemical moieties, or by different mechanisms. In either case the reactions can be independently triggered (e.g., by different wavelengths of light or application of different stimuli), by an internal or external stimulus or stimuli, or operate at different rates (e.g., two condensation reactions can have different kinetics by virtue of different nucleophiles, electrophiles, steric hindrance, etc.).

For each mechanism, the chemical covalent crosslinking results in a covalently crosslinked hydrogel having a mechanical stability that is higher than the mechanical stability of the shear-thinning hydrogel before chemical cross-linking. In separate embodiments, this “higher” mechanical stability can be described in terms of improved resistance to bio-erosion — defined in terms of disassociation of the non-covalent linkages; z.e., improved resistance correlating with longer times necessary to realize degradation of the polymer network — or increased viscosity, stiffness or higher storage or loss modulus of the polymer network. Within each of these property classes, this higher stability reflects an improvement or increase in at least one physical property of at least about 10%, at least about 25%, at least about 50%, or at least about 100%, or at least about 2 times, at least about 5 times, or at least about 10 times relative to the corresponding property of the shear-thinning hydrogel.

In other embodiments, the chemical covalent crosslinking moieties are capable of, or actually, resulting in a covalently cross-linked hydrogel having a mechanical stability that is higher than the mechanical stability of the shear-thinning hydrogel before chemical crosslinking and/or the chemical covalent crosslinking reaction provides a covalently cross-linked hydrogel exhibiting reduced diffusivity of an entrained material relative to the diffusivity exhibited by the shear-thinning hydrogel before chemical cross-linking. As contemplated herein, the entrained material can include a pharmaceutically active drug or neutraceutical, a population of cells, a nanoparticle, quantum dot, or magnetic material. The diffusivity rate would be measured by standards means, for example by measuring the release of a macromolecule of known molecular weight (e.g., a dextran or bovine serum albumin) form a hydrogel into solution or by measuring the uptake of the same molecules into the hydrogel.

As described above, the settable, shear-thinning hydrogels comprise a hydrophilic polymer network, comprising hydrophilic polymers or copolymers containing hydrophilic polymer subunits. These polymers can comprise natural, synthetic, biocompatible, biodegradable, non-biodegradable, and/or biosorbable building blocks. Unless specifically restricted to one or more of these categories, the polymers can comprise materials from any one of these categories. For performance reasons, it can be desirable to incorporate biodegradable or porogenic materials into the design

In certain embodiments, settable, shear-thinning hydrogels, comprise a hydrophilic polymer network. In certain embodiment, the hydrophilic polymer network comprises non- covalent crosslinks and at least one set of chemical moieties participating in at least one chemical covalent cross-linking reaction.

Polymer is not intended to necessarily refer to a single polymer molecule; rather it is intended to connote a mixture of individual molecules, said mixture having a distribution of molecular weights, as is understood by those skilled in the art. The present disclosure is not limited to any particular molecule weight distribution, provided the distribution provides a mixture suitable for the purposes described herein. For example, a polymer comprising hyaluronic acid refers to a mixture of individual polymer molecules, each molecule comprising hyaluronic acid. As used herein, the term “synthetic polymer” refers to polymers that are not found in nature, even if the polymers are made from naturally occurring biomaterials. Examples include, but are not limited to, poly(amino acids), copoly(ether-esters), polyalkylenes oxalates, polyamides, tyrosine derived polycarbonates, poly(iminocarbonates), polyorthoesters, polyoxaesters, polyamidoesters, polyoxaesters containing amine groups, poly(anhydrides), polyphosphazenes, polysiloxanes, and combinations thereof. Suitable synthetic polymers for use according to the teachings of the present disclosure can also include biosynthetic polymers based on sequences found in collagen, elastin, thrombin, fibronectin, starches, poly(amino acid), polypropylene fumarate), gelatin, alginate, pectin, fibrin, oxidized cellulose, chitin, chitosan, tropoelastin, hyaluronic acid, polyethylene, polyethylene terephthalate, poly(tetrafhioroethylene), polycarbonate, polypropylene and poly(vinyl alcohol), ribonucleic acids, deoxyribonucleic acids, polypeptides, proteins, polysaccharides, polynucleotides and combinations thereof.

As used herein, the term “natural polymer” refers to polymers that are naturally occurring. Non-limiting examples of such polymers include collagen-based materials, chitosan, hyaluronic acid, and alginate.

As used herein, the phrase “biocompatible polymer” refers to any polymer (synthetic or natural) which when in contact with cells, tissues or body or physiological fluid of an organism does not induce adverse effects such as immunological reactions and/or rejections and the like. It will be appreciated that a biocompatible polymer can also be a biodegradable polymer.

As used herein, the phrase “biodegradable polymer” refers to a synthetic or natural polymer which can be degraded (z.e., broken down) in the physiological environment such as by enzymes, microbes, or proteins. Biodegradability depends on the availability of degradation substrates (z.e., biological materials or portion thereof which are part of the polymer), the presence of biodegrading materials (e.g., microorganisms, enzymes, proteins) and the availability of oxygen (for aerobic organisms, microorganisms, or portions thereof), carbon dioxide (for anaerobic organisms, microorganisms, or portions thereof) and/or other nutrients. Aliphatic polyesters, poly(amino acids), polyalkylene oxalates, polyamides, polyamido esters, poly(anhydrides), poly(beta-amino esters), polycarbonates, polyethers, polyorthoesters, polyphosphazenes, and combinations thereof are considered biodegradable. More specific examples of biodegradable polymers include, but are not limited to, collagen (e.g., Collagen I or IV), fibrin, hyaluronic acid, polylactic acid (PLA), polyglycolic acid (PGA), polycaprolactone (PCL), poly(Lactide-co-Glycolide) (PLGA), polydioxanone (PDO), trimethylene carbonate (TMC), polyethylene glycol (PEG), Collagen, PEG-DMA, alginate or alginic acid, chitosan polymers, or copolymers or mixtures thereof.

As used herein, the phrase “non-biodegradable polymer” refers to a synthetic or natural polymer which is not degraded (z.e., broken down) in the physiological environment. Examples of non-biodegradable polymers include, but are not limited to, carbon, nylon, silicon, silk, polyurethanes, polycarbonates, polyacrylonitriles, polyanilines, polyvinyl carbazoles, polyvinyl chlorides, polyvinyl fluorides, polyvinyl imidazoles, polyvinyl alcohols, polystyrenes and poly(vinyl phenols), aliphatic polyesters, polyacrylates, polymethacrylates, acyl-substituted cellulose acetates, non-biodegradable polyurethanes, polystyrenes, chlorosulphonated polyolefins, polyethylene oxides, polytetrafluoroethylenes, polydialkylsiloxanes, and shape-memory materials such as poly (styrene-block-butadiene), copolymers or mixtures thereof.

As used herein, the phrase “biosorbable” refers to those polymers which are absorbed within the host body, either through a biodegradation process, or by simple dissolution in aqueous or other body fluids. Water soluble polymers, such as polyethylene oxide) are included in this class of polymers.

As used herein, the term “co-polymer” as used herein, refers to a polymer of at least two chemically distinct monomers. Non-limiting examples of co-polymers which can be used within the hydrogels of the present disclosure can include, PLA-PEG, PEGT-PBT, PLA-PGA, PEG-PCL, and PCL-PLA. The use of copolymers or mixtures of polymers/copolymers provides a flexible means of providing the required blend of properties. In but one non-limiting example, functionalized poly(P-amino esters), which can be formed by the conjugate addition of primary or secondary amines with di-acrylates, can provide a range of materials exhibiting a wide array of advantageous properties for this purpose. Such materials are described, for example, in Anderson, et al., “A Combinatorial Library of Photocrosslinkable and Degradable Materials,” Adv. Materials, vol. 18 (19), 2006, this reference being incorporated by reference in its entirety.

In certain preferred embodiments, the settable shear-thinning hydrogels comprise an agarose, alginate, RGD-modified alginate, amylase, amylpectin, cellularose, chitosan, collagen, dextran, fibrin, gelatin, glycogen, heparin, hyaluronic acid, oligo(poly(ethylene glycol)fumarate), poly(s-caprolactone), poly(ethylene glycol), poly(acrylamide), poly(P- aminoester), poly (caprolactone), multi-arm polyethylene glycol, poly-hydroxyethyl acrylate, poly(hydroxyethyl methacrylate), poly(N-isopropylacrylamide), poly(glycolic acid), poly(lactic acid), poly(lactic acid-glycolic acid), oligo(poly(ethylene glycol)fumarate), poly(vinyl alcohol), or a poly(vinyl acid).

With the respect to the chemical moieties capable of chemical covalent crosslinking, the term “at least one set” refers to the fact that typically, but not necessarily, are the chemical moieties are different chemical groups which react together to form a cross-link; z.e., from this perspective, the “at least one set” can be envisioned as comprising a matched pair of chemical groups. For example, a set can comprise a carboxylic acid (or equivalent) and an amine or alcohol (or equivalent), together capable of forming an amide or ester cross-linked linkage. In another example, a set can comprise a thiol group and a vinyl group, together capable of forming a thiol ether on reaction with light. Another set can comprise a hydrazide and an aldehyde or ketone, capable of forming a hydrazone. Or a set can comprise simply a single radical polymerizable moiety, such as an acrylate or methacrylate.

In individual embodiments, each of the different chemical groups which can react together to form a covalent cross-link within the network can be attached to the same or a different polymer within the polymer network. In non-limiting examples relating to the guest- host-based hydrogels described above, for a given set of chemical cross-linkable moieties, (a) one chemical group can be attached to the first polymer while the associated “matching” chemical group is attached to the second polymer, or (b) both chemical groups can be attached to either the first or second polymer⁷ or (c) a combination of the (a) and (b). Where more than one set of chemical covalent cross-linkable moieties are present, each set can be independently arranged are described above.

These at least one set of chemical covalent cross-linkable moieties can be attached as a pendant to at least one polymer of the network, either directly to the polymer backbone or via a linking group. In some embodiments, this linking group can be biodegradable (e.g., under physiological conditions), such that after the hydrogel is cross-linked, the linking group can degrade with time, thereby reducing the physical strength of the original cross-linked performance or releasing any cargo contained within the cross-linked hydrogel.

In other embodiments, the chemical covalent cross-linkable moieties can be embedded within the polymer backbone of at least one polymer of the network. Olefin or epoxy moieties can be examples of this strategy.

In other embodiments, the settable, shear thinning hydrogel can comprise moieties capable of fluorescing or phosphorescing after exposure to light. Such moieties are known in the art, for example a Cy7.5 dye. Such a marker would be useful, for example, to measure degradation (or stability) performance of the hydrogel in use, or trigger-able upon exposure to a specific analyte in a sensor application.

In certain embodiments, where the chemical moieties capable of chemical covalent crosslinking are activated, or “triggered” by exposure to radiation, for example light of a specific wavelength or wavelengths (ie., the hydrogel can contain multiple such chemical sets, each triggerable by a different wavelength of light). In such case, the stimulus/stimuli can be light having a wavelength within the near infrared to ultraviolet range. See, e.g., Tan, et cd.. J. Biomed Mad. Res., vol. 87 (4), 2008, pp. 1034-1043, which is incorporated by reference in its entirety, for examples of chemical moieties triggerable by light. In those compositions wherein the chemical moieties are light activated, it would also be advantageous that the hydrogel further comprises a photo-initiator; for example, l-[4-(2-hydroxyethoxy)-phenyl]-2-hydroxy- 2-methyl-l -propane- 1 -one, available from Ciba Specialty Chemicals, Inc. as IRGACURE 2959. Other exemplary photo-initiators include 2,4,6 trimethylbenzoyldiphenyl phosphine oxide, 2 -hydroxy -2 -m ethyl- 1 -phenyl -propanone, oligo (2-hydroxy-2-methyl-l-(4-(l- methylvinyl)vinyl) propanone), and 2,4,6-trimethylbenzophonone.

In certain embodiments, the external stimulus to the chemical covalent crosslinking reaction can be radiation in the microwave range (z.e., in the range of about 1 MHz to about 10 GHz). In still other embodiments, the external stimulus can be a change in pH or temperature, a free radical initiator, or a combination thereof. Where the chemical covalent cross-linking reaction is a free radical polymerization, the hydrogel can further comprise a thermal radical initiator. Exemplary free radical initiators include azobisisobutyronitrile, dilauroyl peroxide lauroyl acid, dioctanoyl peroxide caprylic acid, didecanoyl peroxide n-decanoic acid, di-n- propionyl peroxide propionic acid, bi s(3, 5, 5 -trimethyl hexanoyl) 3, 5, 5 -trimethyl peroxide hexanoic acid, dibenzoyl peroxide benzoic acid, bis(2,4-di chlorobenzoyl) 2,4 di chlorobenzoic acid peroxide, bis(o-methybenzoyl) peroxide o-methyl benzoic acid, acetyl cyclohexane sulphonyl cyclohexane sulphonic peroxide acid, t-butylperoxypivalate pivalic acid, t-butyl peroxy-2-ethylhexanoate 2-ethyl caproic acid, t-butyl peroxy isobutyrate isobutyric acid, t- butyl peroxybenzoate benzoic acid, and mixtures thereof.

In certain embodiments, the thermoresponsive hydrogel has a lower critical solution temperature (LCST) below body temperature. The thermoresponsive hydrogel remains fluid below physiological temperature (e.g., 37°C. for humans) or at or below room temperature (e.g., 25°C.), solidify (into a hydrogel) at physiological temperature, and are biocompatible. For example, the thermoresponsive hydrogel can be a clear liquid at a temperature below 34°C which reversibly solidifies into a gelled composition at a temperature above 34°C. Generally, the LCST-based phase transition occurs upon warming in situ as a result of entropically-driven dehydration of polymer components, leading to polymer collapse. Various naturally derived and synthetic polymers exhibiting this behavior can be utilized.

In certain embodiments, the covalent crosslinking reaction is a condensation reaction, Michael addition, or a free radical polymerization reaction. In related embodiments, the at least one set of chemical moieties capable of participating in a covalent chemical crosslinking reaction comprises an acrylate, acrylamide, optionally protected alcohol, aldehyde, alkyne, optionally protected amine, anhydride, azide, carboxy, epoxy, ester, hydrazide, ketone, maleimide, methacrylate, styrenyl, optionally protected thiol, or vinyl or vinyl sulfone group. In still further related embodiments, the product of the chemical covalent cross-linking reaction is an ester, ether, amide, hydrozone, polyacrylate, polymethacrylate, thioamide, thioester, thioether, or urethane. This skilled artisan would appreciate how to modify the desired polymer to attached or incorporate, the chemical covalent cross-linkable moiety.

In certain embodiments, the hydrogel is a shear-thinning hydrogel as described above. In certain embodiments, the shear-thinning hydrogel comprises hyaluronic acid. In certain embodiments, the shear-thinning hydrogel has a total hyaluronic acid content from about 1% to about 30% wt/vol, from about 1% to about 28% wt/vol, from about 1% to about 25% wt/vol, from about 1% to about 22% wt/vol, from about 1% to about 20% wt/vol, from about 1% to about 18% wt/vol, from about 1% to about 15% wt/vol, from about 1% to about 12% wt/vol, from about 1% to about 10% wt/vol, from about 1% to about 8% wt/vol, from about 1% to about 5% wt/vol, from about 2% to about 30% wt/vol, from about 2% to about 28% wt/vol, from about 2% to about 25% wt/vol, from about 2% to about 22% wt/vol, from about 2% to about 20% wt/vol, from about 2% to about 18% wt/vol, from about 2% to about 15% wt/vol, from about 2% to about 12% wt/vol, from about 2% to about 10% wt/vol, from about 2% to about 8% wt/vol, from about 2% to about 5% wt/vol, from about 3% to about 30% wt/vol, from about 3% to about 28% wt/vol, from about 3% to about 25% wt/vol, from about 3% to about 22% wt/vol, from about 3% to about 20% wt/vol, from about 3% to about 18% wt/vol, from about 3% to about 15% wt/vol, from about 3% to about 12% wt/vol, from about 3% to about 10% wt/vol, from about 3% to about 8% wt/vol, from about 3% to about 5% wt/vol, from about 3.2% to about 5% wt/vol, from about 3.4% to about 5% wt/vol, from about 3.5% to about 5% wt/vol, from about 3.6% to about 5% wt/vol, from about 3.7% to about 5% wt/vol, from about 3.8% to about 5% wt/vol, from about 3.9% to about 5% wt/vol, from about 3% to about 4.9% wt/vol, from about 3% to about 4.8% wt/vol, from about 3% to about 4.5% wt/vol, from about 3% to about 4.4% wt/vol, from about 3% to about 4.3% wt/vol, from about 3% to about 4.2% wt/vol, from about 3% to about 4.1% wt/vol, from about 3.2% to about 4.5% wt/vol, from about 3.4% to about 4.5% wt/vol, from about 3.4.5% to about 4.5% wt/vol, from about 3.6% to about 4.5% wt/vol, from about 3.7% to about 4.5% wt/vol, from about 3.8% to about 4.5% wt/vol, from about 3.9% to about 4.5% wt/vol, from about 3.2% to about 4.1% wt/vol, from about 3.4% to about 4.1% wt/vol, from about 3.4.1% to about 4.1% wt/vol, from about 3.6% to about 4.1% wt/vol, from about 3.7% to about 4.1% wt/vol, from about 3.8% to about 4.1% wt/vol, or from about 3.9% to about 4.1% wt/vol. In certain embodiments, the shear-thinning hydrogel has a total hyaluronic acid content from about 1% to about 10% wt/vol, from about 1.5% to about 10% wt/vol, from about 2% to about 10% wt/vol, from about 2.5% to about 10% wt/vol, from about 3% to about 10% wt/vol, from about 3.5% to about 10% wt/vol, from about 1% to about 9% wt/vol, from about 1.5% to about 9% wt/vol, from about 2% to about 9% wt/vol, from about 2.5% to about 9% wt/vol, from about 3% to about 9% wt/vol, from about 3.5% to about 9% wt/vol, from about 1% to about 8% wt/vol, from about 1.5% to about 8% wt/vol, from about 2% to about 8% wt/vol, from about 2.5% to about 8% wt/vol, from about 3% to about 8% wt/vol, from about 3.5% to about 8% wt/vol, from about 1% to about 7% wt/vol, from about 1.5% to about 7% wt/vol, from about 2% to about 7% wt/vol, from about 2.5% to about 7% wt/vol, from about 3% to about 7% wt/vol, from about 3.5% to about 7% wt/vol, from about 1% to about 6% wt/vol, from about 1.5% to about 6% wt/vol, from about 2% to about 6% wt/vol, from about 2.5% to about 6% wt/vol, from about 3% to about 6% wt/vol, from about 3.5% to about 6% wt/vol, from about 1% to about 5% wt/vol, from about 1.5% to about 5% wt/vol, from about 2% to about 5% wt/vol, from about 2.5% to about 5% wt/vol, from about 3% to about 5% wt/vol, or from about 3.5% to about 5% wt/vol. In certain embodiments, the shear-thinning hydrogel has a total hyaluronic acid content of about 4% wt/vol.

In certain embodiments, the shear-thinning hydrogel comprises hyaluronic acid macromers functionalized with adamantane (HA-AD), cyclodextrin (HA-CD), or a combination thereof.

In certain embodiments, the shear-thinning hydrogel comprises hyaluronic acid macromers functionalized with adamantane (HA-AD). In certain embodiments, the HA-AD is present in the shear-thinning hydrogel at a concentration from about 1% to about 30% wt/vol, from about 1% to about 28% wt/vol, from about 1% to about 25% wt/vol, from about 1% to about 22% wt/vol, from about 1% to about 20% wt/vol, from about 1% to about 18% wt/vol, from about 1% to about 15% wt/vol, from about 1% to about 12% wt/vol, from about 1% to about 10% wt/vol, from about 1% to about 8% wt/vol, from about 1% to about 5% wt/vol, from about 2% to about 30% wt/vol, from about 2% to about 28% wt/vol, from about 2% to about 25% wt/vol, from about 2% to about 22% wt/vol, from about 2% to about 20% wt/vol, from about 2% to about 18% wt/vol, from about 2% to about 15% wt/vol, from about 2% to about 12% wt/vol, from about 2% to about 10% wt/vol, from about 2% to about 8% wt/vol, from about 2% to about 5% wt/vol, from about 3% to about 30% wt/vol, from about 3% to about 28% wt/vol, from about 3% to about 25% wt/vol, from about 3% to about 22% wt/vol, from about 3% to about 20% wt/vol, from about 3% to about 18% wt/vol, from about 3% to about 15% wt/vol, from about 3% to about 12% wt/vol, from about 3% to about 10% wt/vol, from about 3% to about 8% wt/vol, from about 3% to about 5% wt/vol, from about 3.2% to about 5% wt/vol, from about 3.4% to about 5% wt/vol, from about 3.5% to about 5% wt/vol, from about 3.6% to about 5% wt/vol, from about 3.7% to about 5% wt/vol, from about 3.8% to about 5% wt/vol, from about 3.9% to about 5% wt/vol, from about 3% to about 4.9% wt/vol, from about 3% to about 4.8% wt/vol, from about 3% to about 4.5% wt/vol, from about 3% to about 4.4% wt/vol, from about 3% to about 4.3% wt/vol, from about 3% to about 4.2% wt/vol, from about 3% to about 4.1% wt/vol, from about 3.2% to about 4.5% wt/vol, from about 3.4% to about 4.5% wt/vol, from about 3.4.5% to about 4.5% wt/vol, from about 3.6% to about 4.5% wt/vol, from about 3.7% to about 4.5% wt/vol, from about 3.8% to about 4.5% wt/vol, from about 3.9% to about 4.5% wt/vol, from about 3.2% to about 4.1% wt/vol, from about 3.4% to about 4.1% wt/vol, from about 3.4.1% to about 4.1% wt/vol, from about 3.6% to about 4.1% wt/vol, from about 3.7% to about 4.1% wt/vol, from about 3.8% to about 4.1% wt/vol, or from about 3.9% to about 4.1% wt/vol. In certain embodiments, the HA- AD is present in the shearthinning hydrogel at a concentration of about 4% wt/vol.

In certain embodiments, the HA-AD has a degree of functionalization between about 1% and about 40%, between about 2.5% and about 40%, between about 5% and about 40%, between about 7.5% and about 40%, between about 10% and about 40%, between about 12.5% and about 40%, between about 15% and about 40%, between about 17.5% and about 40%, between about 19% and about 40%, between about 1% and about 35%, between about 2.5% and about 35%, between about 5% and about 35%, between about 7.5% and about 35%, between about 10% and about 35%, between about 12.5% and about 35%, between about 15% and about 35%, between about 17.5% and about 35%, between about 19% and about 35%, between about 1% and about 30%, between about 2.5% and about 30%, between about 5% and about 30%, between about 7.5% and about 30%, between about 10% and about 30%, between about 12.5% and about 30%, between about 15% and about 30%, between about 17.5% and about 30%, between about 19% and about 30%, between about 1% and about 27%, between about 2.5% and about 27%, between about 5% and about 27%, between about 7.5% and about 27%, between about 10% and about 27%, between about 12.5% and about 27%, between about 15% and about 27%, between about 17.5% and about 27%, between about 19% and about 27%, between about 1% and about 26%, between about 2.5% and about 26%, between about 5% and about 26%, between about 7.5% and about 26%, between about 10% and about 26%, between about 12.5% and about 26%, between about 15% and about 26%, between about 17.5% and about 26%, between about 19% and about 26%, between about 1% and about 25%, between about 2.5% and about 25%, between about 5% and about 25%, between about 7.5% and about 25%, between about 10% and about 25%, between about 12.5% and about 25%, between about 15% and about 25%, between about 17.5% and about 25%, between about 19% and about 25%, between about 1% and about 24%, between about 2.5% and about 24%, between about 5% and about 24%, between about 7.5% and about 24%, between about 10% and about 24%, between about 12.5% and about 24%, between about 15% and about 24%, between about 17.5% and about 24%, between about 19% and about 24%, between about 1% and about 23%, between about 2.5% and about 23%, between about 5% and about 23%, between about 7.5% and about 23%, between about 10% and about 23%, between about 12.5% and about 23%, between about 15% and about 23%, between about 17.5% and about 23%, between about 19% and about 23%, between about 1% and about 22%, between about 2.5% and about 22%, between about 5% and about 22%, between about 7.5% and about 22%, between about 10% and about 22%, between about 12.5% and about 22%, between about 15% and about 22%, between about 17.5% and about 22%, between about 19% and about 22%, between about 1% and about 21%, between about 2.5% and about 21%, between about 5% and about 21%, between about 7.5% and about 21%, between about 10% and about 21%, between about 12.5% and about 21%, between about 15% and about 21%, between about 17.5% and about 21%, between about 19% and about 21%, between about 1% and about 19% wt/vol, between about 2.5% and about 19% wt/vol, between about 5% and about 19% wt/vol, between about 7.5% and about 19% wt/vol, between about 10% and about 19%, between about 12.5% and about 19% wt/vol, between about 15% and about 19% wt/vol, or between about 17.5% and about 19% wt/vol. As used herein, said HA- AD degree of functionalization is calculated by dividing the number of AD groups added to the HA polymer by the total number of available binding sites. In certain embodiments, the HA- AD has a degree of functionalization of about 18%. In certain embodiments, the shear-thinning hydrogel comprises hyaluronic acid macromers functionalized with cyclodextrin (HA-CD). In certain embodiments, the HA-CD is present in the shear-thinning hydrogel at a concentration from about 1% to about 30% wt/vol, from about 1% to about 28% wt/vol, from about 1% to about 25% wt/vol, from about 1% to about 22% wt/vol, from about 1% to about 20% wt/vol, from about 1% to about 18% wt/vol, from about 1% to about 15% wt/vol, from about 1% to about 12% wt/vol, from about 1% to about 10% wt/vol, from about 1% to about 8% wt/vol, from about 1% to about 5% wt/vol, from about 2% to about 30% wt/vol, from about 2% to about 28% wt/vol, from about 2% to about 25% wt/vol, from about 2% to about 22% wt/vol, from about 2% to about 20% wt/vol, from about 2% to about 18% wt/vol, from about 2% to about 15% wt/vol, from about 2% to about 12% wt/vol, from about 2% to about 10% wt/vol, from about 2% to about 8% wt/vol, from about 2% to about 5% wt/vol, from about 3% to about 30% wt/vol, from about 3% to about 28% wt/vol, from about 3% to about 25% wt/vol, from about 3% to about 22% wt/vol, from about 3% to about 20% wt/vol, from about 3% to about 18% wt/vol, from about 3% to about 15% wt/vol, from about 3% to about 12% wt/vol, from about 3% to about 10% wt/vol, from about 3% to about 8% wt/vol, from about 3% to about 5% wt/vol, from about 3.2% to about 5% wt/vol, from about 3.4% to about 5% wt/vol, from about 3.5% to about 5% wt/vol, from about 3.6% to about 5% wt/vol, from about 3.7% to about 5% wt/vol, from about 3.8% to about 5% wt/vol, from about 3.9% to about 5% wt/vol, from about 3% to about 4.9% wt/vol, from about 3% to about 4.8% wt/vol, from about 3% to about 4.5% wt/vol, from about 3% to about 4.4% wt/vol, from about 3% to about 4.3% wt/vol, from about 3% to about 4.2% wt/vol, from about 3% to about 4.1% wt/vol, from about 3.2% to about 4.5% wt/vol, from about 3.4% to about 4.5% wt/vol, from about 3.4.5% to about 4.5% wt/vol, from about 3.6% to about 4.5% wt/vol, from about 3.7% to about 4.5% wt/vol, from about 3.8% to about 4.5% wt/vol, from about 3.9% to about 4.5% wt/vol, from about 3.2% to about 4.1% wt/vol, from about 3.4% to about 4.1% wt/vol, from about 3.4.1% to about 4.1% wt/vol, from about 3.6% to about 4.1% wt/vol, from about 3.7% to about 4.1% wt/vol, from about 3.8% to about 4.1% wt/vol, or from about 3.9% to about 4.1% wt/vol. In certain embodiments, the HA-CD is present in the shearthinning hydrogel at a concentration of about 4% wt/vol.

In certain embodiments, the HA-CD has a degree of functionalization between about 1% and about 40%, 2.5% and about 40%, between about 5% and about 40%, between about 7.5% and about 40%, between about 10% and about 40%, between about 12.5% and about 40%, between about 15% and about 40%, between about 17.5% and about 40%, between about 19% and about 40%, between about 1% and about 35%, between about 2.5% and about 35%, between about 5% and about 35%, between about 7.5% and about 35%, between about 10% and about 35%, between about 12.5% and about 35%, between about 15% and about 35%, between about 17.5% and about 35%, between about 19% and about 35%, between about 1% and about 30%, between about 2.5% and about 30%, between about 5% and about 30%, between about 7.5% and about 30%, between about 10% and about 30%, between about 12.5% and about 30%, between about 15% and about 30%, between about 17.5% and about 30%, between about 19% and about 30%, between about 1% and about 27%, between about 2.5% and about 27%, between about 5% and about 27%, between about 7.5% and about 27%, between about 10% and about 27%, between about 12.5% and about 27%, between about 15% and about 27%, between about 17.5% and about 27%, between about 19% and about 27%, between about 1% and about 26%, between about 2.5% and about 26%, between about 5% and about 26%, between about 7.5% and about 26%, between about 10% and about 26%, between about 12.5% and about 26%, between about 15% and about 26%, between about 17.5% and about 26%, between about 19% and about 26%, between about 1% and about 25%, between about 2.5% and about 25%, between about 5% and about 25%, between about 7.5% and about 25%, between about 10% and about 25%, between about 12.5% and about 25%, between about 15% and about 25%, between about 17.5% and about 25%, between about 19% and about 25%, between about 1% and about 24%, between about 2.5% and about 24%, between about 5% and about 24%, between about 7.5% and about 24%, between about 10% and about 24%, between about 12.5% and about 24%, between about 15% and about 24%, between about 17.5% and about 24%, between about 19% and about 24%, between about 1% and about 23%, between about 2.5% and about 23%, between about 5% and about 23%, between about 7.5% and about 23%, between about 10% and about 23%, between about 12.5% and about 23%, between about 15% and about 23%, between about 17.5% and about 23%, between about 19% and about 23%, between about 1% and about 22%, between about 2.5% and about 22%, between about 5% and about 22%, between about 7.5% and about 22%, between about 10% and about 22%, between about 12.5% and about 22%, between about 15% and about 22%, between about 17.5% and about 22%, between about 19% and about 22%, between about 1% and about 21%, between about 2.5% and about 21%, between about 5% and about 21%, between about 7.5% and about 21%, between about 10% and about 21%, between about 12.5% and about 21%, between about 15% and about 21%, between about 17.5% and about 21%, or between about 19% and about 21%. As used herein, said HA-CD degree of functionalization is calculated by dividing the number of CD groups added to the HA polymer by the total number of available binding sites. In certain embodiments, the HA-CD has a degree of functionalization of about 18%. In certain embodiments, the shear-thinning hydrogel comprises hyaluronic acid macromers functionalized with adamantane (HA-AD) and hyaluronic acid macromers functionalized with cyclodextrin (HA-CD). In certain embodiments, the HA-AD and the HA- CD are present in a ratio from about 1:100 to about 1:1, from about 1:90 to about 1:1, from about 1 :80 to about 1:1, from about 1 :70 to about 1:1, from about 1 :60 to about 1:1, from about 1 :50 to about 1:1, from about 1 :40 to about 1:1, from about 1 :30 to about 1:1, from about 1 :20 to about 1:1, from about 1 : 10 to about 1:1, from about 1 :9 to about 1:1, from about 1 :8 to about 1:1, from about 1 : 7 to about 1:1, from about 1 : 6 to about 1:1, from about 1 : 5 to about 1:1, from about 1:4 to about 1:1, from about 1:3 to about 1:1, from about 1:2 to about 1:1, from about 100: 1 to about 1:1, from about 90: 1 to about 1:1, from about 80: 1 to about 1:1, from about 70: 1 to about 1:1, from about 60:1 to about 1:1, from about 50:1 to about 1:1, from about 40:1 to about 1:1, from about 30: 1 to about 1:1, from about 20: 1 to about 1:1, from about 10: 1 to about 1:1, from about 9 : 1 to about 1:1, from about 8 : 1 to about 1:1, from about 7 : 1 to about 1:1, from about 6:1 to about 1:1, from about 5:1 to about 1:1, from about 4:1 to about 1:1, from about 3:1 to about 1:1, or from about 1:1 to about 1:1. In certain embodiments, the HA-AD and the HA-CD are present in a ratio of 1 : 1.

Other embodiments describe these additional materials as comprising biofactors, therapeutic agents, particles (e.g., microparticles, nanoparticles, quantum dots, or magnetic materials), or cells.

In one set of embodiments, these additional materials comprise at least one therapeutic compound or agent, capable of modifying cellular activity. Similarly, agents that act to increase cell attachment, cell spreading, cell proliferation, cell differentiation and/or cell migration in the scaffold can also be incorporated into the hydrogels. Such agents can be biological agents such as amino acids, peptides, polypeptides, proteins, DNA, RNA, lipids and/or proteoglycans. These agents can also include growth factors, cytokines, proteases, and protease substrates.

In one embodiment, at least one therapeutic agent comprises interleukin (IL)-33. IL-33 is a member of the IL-1 superfamily of cytokines, a determination based in part on the molecules (P-trefoil structure, a conserved structure type described in other IL-1 cytokines, including IL-a, IL-ip, IL-IRa and IL-18. In this structure, the 12 P-strands of the (P-trefoil are arranged in three pseudorepeats of four (P-strand units, of which the first and last (P -strands are antiparallel staves in a six-stranded (P -barrel, while the second and third P -strands of each repeat form a P -hairpin sitting atop the P -barrel. IL-33 binds to a high-affinity receptor family member ST2. IL-33 induces helper T cells, mast cells, eosinophils, and basophils to produce type 2 cytokines. Exemplary amino acid sequences for human IL-33 are provided in GENBANK® Accession Nos. NP_001186569.1, NP_001186570.1, NP_001300973.1, NP_001300974.1, and NP_001300975.1, all incorporated herein by reference. ST2, also known as IL1RL1, is member of the interleukin 1 receptor family. ST2 is also a member of the Toll-like receptor superfamily based on the function of its intracellular TIR domain, but its extracellular region is composed of immunoglobulin domains. The ST2 protein has two isoforms and is directly implicated in the progression of cardiac disease: a soluble form (referred to as soluble ST2 or sST2) and a membrane-bound receptor form (referred to as the ST2 receptor or ST2L). When the myocardium is stretched, the ST2 gene is upregulated, increasing the concentration of circulating soluble ST2. The ligand for ST2 is IL-33. Binding of IL-33 to the ST2 receptor, in response to cardiac disease or injury, such as an ischemic event, elicits a cardioprotective effect resulting in preserved cardiac function. This cardioprotective IL-33 signal is counterbalanced by the level of soluble ST2, which binds IL-33 and makes it unavailable to the ST2 receptor for cardioprotective signaling. As a result, the heart is subjected to greater stress in the presence of high levels of soluble ST2.

Additionally, and/or alternatively, the hydrogels of the present disclosure can comprise an antiproliferative agent, an immunosuppressant drug, a non-thrombogenic substance, an antiadhesive substance, and a combination thereof.

Additionally, and/or alternatively, the hydrogels of the present disclosure can comprise cells. The cells which can be used according to the teachings of the present disclosure can comprise non-autologous cells or non-autologous cells (e.g., allogeneic cells or xenogeneic cells), such as from human cadavers, human donors or xenogeneic (e.g., porcine or bovine) donors. The cells can comprise a heterogeneous population of cells or a homogeneous population of cells. Such cells can be for example, stem cells, progenitor cells, or differentiated cells. Stem cells can include adipose derived stem cells, embryonic stem cells, bone marrow stem cells, cord blood stem cells, mesenchymal stem cells, adult stem cells, and pluripotent or induced pluripotent stem cells. Mesenchymal stem cells are preferred. Furthermore, such cells can be live or non-viable and/or of autologous origin or non-autologous origin, such as postpartum-derived cells (as described in U.S. application Ser. Nos. 10/887,012 and 10/887,446). Typically, the cells are selected according to the tissue being generated.

In additional to the settable, shear thinning hydrogels (i.e., which exists before the covalent crosslinking reach on(s) has occurred or is complete), individual embodiments of the present disclosure can include those hydrogel compositions, based on the previous descriptions, which have undergone at least one of the covalent cross-linking reactions, either partially or completely. This includes embodiments where any number of the at least one set of the chemical moieties capable of covalent crosslinking of settable, shear thinning hydrogel has reacted, either partially or entirely.

In certain embodiments, the cured hydrogels exhibit a higher stability or lower diffusivity than the pre-cured (z.e., settable, shear thinning) hydrogel. In several of these embodiments, the cured, covalently cross-linked hydrogel exhibits a mechanical stability that is higher than the mechanical stability of the (pre-cured) shear-thinning hydrogel (z.e., before covalent crosslinking). In separate embodiments, this “higher” mechanical stability can be described in terms of improved resistance to bio-erosion — defined in terms of disassociation of the non-covalent linkages; z.e., improved resistance correlating with longer times necessary to realize degradation of the polymer network — or increased viscosity, stiffness or higher storage or loss modulus of the polymer network. Within each of these property classes, this higher stability reflects an improvement or increase in at least one physical property of at least about 10%, at least about 25%, at least about 50%, or at least about 100%, or at least about 2 times, at least about 5 times, or at least about 10 times, relative to the corresponding property of the shear-thinning hydrogel.

Further, the settable, shear-thinning hydrogels and associated cured hydrogels can be applied in-vivo and/or ex-vivo. Various embodiments provide that the settable or cured hydrogels are adapted to be medically acceptable for use in a mammal, including those where the mammal is a human. Such embodiments include those where the materials are at least biocompatible, and preferably approved by the United States Food and Drug Administration in the United States (or a corresponding regulatory agency in other countries).

The described hydrogels enhanced controlled drug release from microparticles, bolstering in vivo microparticle retention, and precisely regulating encapsulated drug release profiles and pharmacokinetics.

5,4, Methods of Treatment

In one aspect, the present disclosure provides methods for treating ischemia or ischemia-reperfusion injury in a subject in need thereof. In certain embodiments, the methods encompass the administration of a therapeutic agent via a controlled release delivery system. In certain embodiments, the controlled release delivery system comprises a hydrogel-coupled microparticle, offering a targeted and regulated approach to alleviate reperfusion injury.

In certain embodiments, the present disclosure provides for methods for treating, preventing, or reducing conditions associated with ischemia or ischemia-reperfusion injury, for instance, immune and/or inflammatory responses subsequent to infarction. In certain embodiments, the methods are adept at addressing immune and/or inflammation responses by targeting myeloid cell dynamics and tissue damage. In certain embodiment, the methods can reduce the total myeloid cell infiltrate, shortened duration of myeloid cell infiltrate, and/or alter the phenotype of infiltrating myeloid cells towards a pro-repair phenotype. In a certain embodiment, the methods can reduce infarct spreading, i.e., inflammatory damage seen following MI after reperfusion of the tissue.

As used herein, the term “ischemia or “ischemic” refers to a restriction in blood supply, resulting in an absolute or relative deficiency of oxygen reaching tissues, cells, or entire organs. This insufficient blood supply leads to tissue oxygen deprivation (anoxia), which can cause tissue dysfunction, necrosis (cell death), apoptosis (programmed cell death), and autophagy (cellular self-degradation). Insufficient oxygenation due to reduced blood supply can result in severe consequences for cellular health and function. Acute ischemia’s immediate aftermath is cell death (necrocytosis). When ischemic myocardial cells die, it can lead to cardiac exhaustion, potentially causing low blood pressure or inadequate blood supply to other organs, known as cardiogenic shock. Moreover, necrocytosis alters the electrical activity of myocardial cells, often resulting in life-threatening irregular heart rhythms like tachycardia, atrial fibrillation, or bundle branch blocks, which can cause sudden cardiac death. Additionally, the death of papillary muscles can impede heart valve function, leading to blood backflow and compromised pumping capacity. Myocardial cell death might also lead to the formation and rupture of aneurysms in the heart muscle. Early and effective intervention is crucial to mitigate these serious outcomes associated with acute ischemia. In cases of “chronic ischemia,” the gradual progression of blood supply deficiency occurs due to factors like atherosclerosis (where lipid plaque obstructs arterial pathways) or conditions causing decreased blood pressure, such as septicemia or cardiac failure. This leads to a slow and localized development of insufficient blood supply over time.

The term “reperfusion” refers to the restoration of blood supply to ischemic tissue, which had experienced reduced blood flow due to various reasons such as atherosclerosis, arterial constriction, or surgical procedures involving clamping. This renewed blood flow aims to revive the previously deprived tissue. However, the resumption of blood supply can be spontaneous or influenced by therapeutic interventions. While restoring blood flow is crucial for treating conditions like myocardial infarction or other ischemic events, excessive restriction or delays can exacerbate tissue damage and increase the size of the affected area. However, the restoration of blood flow itself can lead to reperfusion injury, causing further damage to the ischemic tissue. This phenomenon underscores the delicate balance in managing the reperfusion process to avoid additional harm to the already compromised tissue. In certain embodiments, the ischemia can be associated with or caused by acute myocardial infarction, elective angioplasty, coronary artery bypass graft, surgery involving cardiac bypass or organ or tissue transplantation, e.g., cardiac transplantation, stroke, head trauma, drowning, sepsis, cardiac arrest, drowning or shock, atherosclerosis, hypertension, cocaine-induced heart disease, smoking-induced heart disease, heart failure, pulmonary hypertension, hemorrhage, capillary leak syndrome (such as child and adult respiratory distress syndrome), multi-organ system failure, a state of low colloid oncotic pressure (such as starvation, anorexia nervosa, or hepatic failure with decreased production of serum proteins), anaphylaxis, hypothermia, cold injury, e.g., due to hypothermic perfusion, frostbite, hepatorenal syndrome, delirium tremens, a crush injury, mesenteric insufficiency, peripheral vascular disease, claudication, burn, electrocution, excessive drug-induced vasodilation, excessive drug-induced vasoconstriction, tissue rejection after transplantation, graft versus host disease, radiation exposure, e.g., during fluoroscopy or radiographic imaging, or exposure to high energy, e.g., exposure to laser light. Excessive drug-induced vasodilation can be caused by, for instance, nitroprusside, hy dralazone, dyazoxide, a calcium channel blocker, or a general anesthetic. Excessive drug-induced vasoconstriction can be caused by, for instance, neosynephrine, isoproterenol, dopamine, dobutamine, or cocaine.

In certain embodiments, the injury resulting from ischemia is the result of a myocardial ischemia. An injury resulting from a myocardial ischemia can result from, for example, a myocardial infarction (e.g., an acute myocardial infarction) in a subject.

In certain embodiments, the injury resulting from ischemia is an ischemia-reperfusion injury. As used herein, the term “ischemia-reperfusion injury” refers to an injury resulting from the restoration of blood flow to an area of a tissue or organ that had previously experienced deficient blood flow due to an ischemic event. Oxidative stresses associated with reperfusion can cause damage to the affected tissues or organs. Ischemia-reperfusion injury is characterized biochemically by a depletion of oxygen during an ischemic event followed by reoxygenation and the concomitant generation of reactive oxygen species during reperfusion.

In certain embodiments, the ischemia-reperfusion injury can be caused, for example, by a natural event (e.g, restoration of blood flow following a myocardial infarction), a trauma, or by one or more surgical procedures or other therapeutic interventions that restore blood flow to a tissue or organ that has been subjected to a diminished supply of blood. Such surgical procedures include, for example, coronary artery bypass graft surgery, coronary angioplasty, organ transplant surgery and the like (e.g, cardiopulmonary bypass surgery). In a particular embodiment, the compounds and methods of the present disclosure are useful for treating perioperative cardiac damage caused by an ischemia or ischemia-reperfusion injury.

In certain embodiments, damaged tissue caused by ischemia or cardiac infarct can be treated by the administration of the hydrogel and/or pharmaceutical composition, wherein the administration can be topical , pulmonary , oral, or parenteral. Local administration can also include intramyocardial injection and/or intraarticular injection into synovial spaces of the body (i.e., for example, into articulating bone joints and/or cartilage). Parenteral administration includes, but is not limited to, intramyocardial, intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or i ntraventri cul ar, admi ni str ati on .

5,5, Pharmaceutical Compositions

The present disclosure further provides pharmaceutical formulations and/or compositions. In certain embodiments, the pharmaceutical formulations and/or compositions can comprise a compound-loaded microparticle as described herein. Additionally or alternatively, the present disclosure further provides pharmaceutical formulations or compositions comprising the presently disclosed hydrogel (e.g., hydrogel comprising microparticles).

In certain embodiments, the pharmaceutical formulation and/or composition is a compound-loaded microparticle population. In certain embodiments, the compound-loaded microparticle population comprises a solid shape. In one embodiment, the solid shape of the compound-loaded microparticle population can include, but is not limited to, transdermal patches, ointments, lotions, creams, gels, suppositories, and powders. In one embodiment, the solid shape of the compound-loaded microparticle population can include, but is not limited to, powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets and/or tablets.

The pharmaceutical formulations and/or compositions of the present disclosure can be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated.

In certain embodiments, the administration of the formulation and/or composition can be topical (including, but not limited to, ophthalmic and to mucous membranes including vaginal and rectal delivery), pulmonary (e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), oral or parenteral. Local administration can also be topical, but includes as well, intraarticular injection into synovial spaces of the body (i.e., for example, into articulating bone joints and/or cartilage). Parenteral administration includes, but is not limited to, intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration.

Pharmaceutical compositions and formulations for topical administration can include, but are not limited to, transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like can be necessary or desirable.

Compositions and formulations for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets or tablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders can be desirable.

Compositions and formulations for parenteral, intrathecal or intraventricular administration can include sterile aqueous solutions that can also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.

Pharmaceutical compositions of the present disclosure include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions can be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids.

The pharmaceutical formulations of the present disclosure, which can conveniently be presented in unit dosage form, can be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

The compositions of the present disclosure can be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present disclosure can also, be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions can further contain substances that increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension can also contain stabilizers.

Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. The administering physician can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages can vary depending on the relative potency of individual drugs and can generally be estimated based on EC50s found to be effective in in vitro and in vivo animal models or based on the examples described herein. In general, dosage is from 0.01 pg to 100 g per kg of body weight, and can be given once or more daily, weekly, monthly or yearly. The treating physician can estimate repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues. Following successful treatment, it can be desirable to have the subject undergo maintenance therapy to prevent the recurrence of the disease state, wherein the compound is administered in maintenance doses, ranging from about 0.01 pg to about 100 g per kg of body weight, from once or more daily to once every 20 years, for example, such dosing can be weekly, monthly or yearly as necessary for maintenance therapy.

In certain embodiments, the pharmaceutical compositions can be formulated and used as foams. Pharmaceutical foams include formulations such as, but not limited to, emulsions, microemulsions, creams, jellies and liposomes. While basically similar in nature, these formulations vary in the components and the consistency of the final product.

The compositions of the present disclosure can additionally contain other adjunct components conventionally found in pharmaceutical compositions. Thus, for example, the compositions can contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or can contain additional materials useful in physically formulating various dosage forms of the compositions of the present disclosure, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present disclosure. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.

5,6, Kits

In another embodiment, the present disclosure contemplates kits comprising a composition disclosed herein and/or for practicing any one of the above-listed methods. In certain embodiments the kit can include a composition comprising a hydrogel component. In certain embodiments the kit can include a composition comprising one or more controlled released microparticle population. In certain embodiments the kit can include a composition comprising one or more therapeutic agent.

In certain embodiments, the present disclosure provides kits comprising a hydrogel disclosed herein (e.g., a hydrogel comprising microparticles) and/or for practicing any one of the above-described methods.

In certain embodiments, the therapeutic agent comprises a T cell factor, including but not limited to an T cell inducing factor, T cell chemoattractant factor, or T cell stimulatory factor. In certain embodiments, the T cell factor can induce, attract, or stimulate regulatory T cells (iTreg). In certain embodiments, the T cells factor can induce, attract, or stimulate natural regulatory T cells (nTreg).

In certain embodiments, the kit provides a controlled release formulation of at least one factor to induce a Treg phenotype (e.g., determined by the expression of canonical Treg markers and migratory surface markers). In certain embodiments, the kit comprises a controlled release formulation of least one Treg induction factor (e.g., IL-2, TGF-P and rapamycin).

In certain embodiments, the kit provides a controlled release formulation of at least one factor to attract or stimulate natural regulatory T cells (nTreg). In certain embodiments, the kit comprises a controlled release formulation of at least one factor to stimulate natural regulatory T cells (nTreg). In certain embodiments, the kit comprises a controlled release formulation of at least one natural regulatory T cell chemoattractant factor (e.g., CCL22). In certain embodiments, the kit comprises a controlled release formulation at least one natural regulatory T cell stimulatory factor (e.g., CCL22).

In certain embodiment, the kit a controlled release formulation of at least one factor that induces nTreg proliferation. In certain embodiment, the kit comprises a controlled release formulation of at least one factor that stimulates the secretion of reparative factors, including but not limited to IL-13 and amphiregulin (AREG) (e.g., IL-33). In certain embodiments, the kit provides comprises a controlled release formulation of at least one nTreg proliferation inducing factor (e.g., IL-33). In certain embodiments, the comprises a controlled release formulation of at least one nTreg stimulating factor (e.g., IL-33).

The kit can include a pharmaceutically acceptable excipient and/or a delivery vehicle. The reagents can be provided suspended in the excipient and/or delivery vehicle or may be provided as a separate component which can be later combined with the excipient and/or delivery vehicle. The kit can contain additional therapeutics to be co-administered with the composition(s).

The kits can also optionally include appropriate systems (e.g., opaque containers) or stabilizers (e.g., antioxidants) to prevent degradation of the reagents by light or other adverse conditions.

The kits can optionally include instructional materials containing directions (i.e., protocols) providing for the use of the reagents in the performance of the methods described herein. In particular the disease can include any one or more of the disorders described herein. While the instructional materials typically comprise written or printed materials, they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this invention. Such media include, but are not limited to, electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. Such media can include addresses to internet sites that provide such instructional materials.

In certain embodiments, the present disclosure contemplates a kit comprising a hydrogel-coupled microparticle composition(s) to provide an ‘off-the-shelf therapeutic for creating a local immunosuppressive environment and increasing the presence of Treg cells at sites of inflammation. In one embodiment, the kits can be used to treat various medical conditions including, but not limited to, ischemia or ischemia-reperfusion injury. Although it is not necessary to understand the mechanism of the present disclosure, it is believed that hydrogel-coupled microparticle composition, including a therapeutic agent(s), can be administered to treat diseases and disorders while the systemic immune system integrity such that the immune system can continue to fight infections and inhibit malignancies.

5,7 , Exemplary Embodiments

Al . In certain non-limiting embodiments, the present disclosure relates to a hydrogel comprising a sustained release microparticle, wherein the sustained release microparticle comprises a therapeutic agent.

A2. The foregoing hydrogel of Al, wherein the hydrogel is a shear-thinning hydrogel.

A3. The foregoing hydrogel of A2, wherein the shear-thinning hydrogel comprises hyaluronic acid macromers functionalized with adamantane (HA-AD), cyclodextrin (HA-CD), or a combination thereof.

A4. The forgoing hydrogel of A2 or A3, wherein the hydrogel comprises HA-AD at a concentration from about 1% to about 30% wt/vol. A5. The foregoing hydrogel of A4, wherein the hydrogel comprises HA- AD at a concentration of about 4% wt/vol.

A6. The foregoing hydrogel of A2-A5, wherein the hydrogel comprises HA-CD at a concentration from about 1% to about 30% wt/vol.

A7. The foregoing hydrogel of A6, wherein the hydrogel comprises HA-CD at a concentration of about 4% wt/vol.

A8. The foregoing hydrogel of and one of A1-A7, wherein the hydrogel comprises hyaluronic acid at a concentration of 4% wt/vol.

A9. The foregoing hydrogel of any one of A2-A8, wherein the shear-thinning hydrogel exhibits a lower diffusivity after curing.

A10. The foregoing hydrogel of any one of A2-A9, wherein the hydrogel retains microparticles at a delivery site.

Al l. The foregoing hydrogel of Al, wherein the hydrogel is a thermoresponsive hydrogel.

Al 2. The foregoing hydrogel of Al l, wherein the thermoresponsive hydrogel comprises a PEG (Polethylene Glycol), NIPAAm (N-Isopropylacrylamide), or a combination thereof.

Al 3. The foregoing hydrogel of Al 1 or A 12, wherein the thermoresponsive hydrogel comprises about 100 mg of NIPAAm.

Al 4. The foregoing hydrogel of any one of Al 1 -Al 3, wherein the thermoresponsive hydrogel has a lower critical solution temperature below 37 °C.

Al 5. The foregoing hydrogel of any one of Al 1 -Al 4, wherein the thermoresponsive hydrogel has a lower critical solution temperature below 20 °C.

Al 6. The foregoing hydrogel of any one of Al 1 -Al 5, wherein the thermoresponsive hydrogel reversibly solidifies into a gel at a temperature above the lower critical solution temperature.

Al 7. The foregoing hydrogel of any one of Al -Al 6, wherein the microparticle is present in the hydrogel at a concentration from about 10 mg/mL up to about 100 mg/mL.

A18. The foregoing hydrogel of any one of A1-A17, wherein the microparticle has a diameter up to about 100 pm.

Al 9. The foregoing hydrogel of any one of Al -Al 8, wherein the microparticle has a diameter larger than about 1 pm. A20. The foregoing hydrogel of any one of Al -Al 9, wherein the therapeutic agent comprises a Treg cell factor.

A21. The foregoing hydrogel of A20, wherein the Treg cell factor comprises a regulatory T cell stimulatory factor.

A22. The foregoing hydrogel of A20 or A21, wherein the Treg cell factor comprises a T cell chemoattractant factor.

A23. The foregoing hydrogel of any one of A20-A22, wherein the Treg cell factor is selected from the group comprising CCL22, IL2, TGF-P, IL33, rapamycin, IL13, amphiregulin, and a combination thereof.

A24. The foregoing hydrogel of any one of A1-A23, further comprising a second sustained release microparticle, wherein the second sustained release microparticle comprises a second therapeutic agent.

A25. The foregoing hydrogel of A24, wherein the second therapeutic agent comprises a Treg cell factor.

A26. The foregoing hydrogel of A25, wherein the Treg cell factor is selected from the group comprising CCL22, IL2, TGF-P, IL33, rapamycin, IL13, amphiregulin, and a combination thereof.

A27. The foregoing hydrogel of any one of A1-A26, further comprising an antiproliferative agent, an immunosuppressant drug, a non-thrombogenic substance, an antiadhesive substance, and a combination thereof.

A28. A pharmaceutical composition comprising the foregoing hydrogel of any one of A1-A27 and a pharmaceutical carrier comprising at least one excipient component.

A29. The foregoing pharmaceutical composition of A28, wherein the at least one excipient component comprises a buffering agent, an antioxidant, an alkali salt, a preservative, or a combination thereof.

A30. A method of treatment for ischemic myocardial infarction in a subject in need thereof, comprising administering an effective amount of the hydrogel of any one of A1-A27 or the composition of A28 or A29

A31. The foregoing method of A30, wherein the hydrogel or composition is administered into the affected tissue.

A32. The foregoing method of A30 or A31, wherein the hydrogel or composition is administered at an amount from about 0.01 pg to about 100 g per kg of body weight of the subject. A33. The foregoing method of any one of A30- A32, wherein the hydrogel or composition is administered once or more daily, weekly, monthly, or yearly.

A34. The foregoing method of any one of A30- A33, wherein the hydrogel or composition is orally, transdermally, topically, pulmonary inhalation, or parenterally administered.

A35. The foregoing method of A34, wherein the parenterally administered hydrogel or composition is injected into an infarct or infarct bordering region.

Bl. A composition comprising: a) a hydrogel; and b) a sustained release microparticle, wherein the sustained release microparticles comprises a first therapeutic agent.

B2. The foregoing composition of Bl, wherein the hydrogel is a thermoresponsive hydrogel.

B3. The foregoing composition of B2, wherein the thermoresponsive hydrogel comprises a PEG (Polyethylene Glycol) and NIPAAm (N-Isopropyl acrylamide).

B4. The foregoing composition of Bl, wherein the hydrogel is a shear-thinning hydrogel.

B5. The foregoing composition of B4, wherein the shear-thinning hydrogel comprises hyaluronic acid macromers functionalized with adamantane (HA-AD) and cyclodextrin (HA-CD).

B6. The foregoing composition of any one of B1-B5, wherein the hydrogel further comprises a second therapeutic agent.

B7. The foregoing composition of B6, wherein the first therapeutic agent comprises a Treg cell factor.

B8. The foregoing composition of B7, wherein the Treg cell factor comprises a regulatory T cell stimulatory factor.

B9. The foregoing composition of B7, wherein the Treg cell factor comprises a T cell chemoattractant factor.

BIO. The foregoing composition of B7, wherein the Treg cell factor is selected from the group consisting of CCL22, IL2, and TGF-P, and combinations thereof.

Cl. A method for treating ischemic myocardial infarction in a subject, wherein the method comprises administering to the subject a composition comprising: a) a hydrogel; and b) a sustained release microparticle, wherein the sustained release microparticle comprises a first therapeutic agent.

DI. A method for treating inflammation of a target tissue in a subj ect in need thereof, wherein the method comprises administering to the subject a composition comprising: a) a hydrogel; and b) a first sustained release microparticle, wherein the first sustained release microparticle comprises a first therapeutic agent.

D2. The foregoing method of DI, wherein the hydrogel is a thermoresponsive hydrogel.

D3. The foregoing method of C 1 or D 1 , wherein the hydrogel is a thermoresponsive hydrogel.

D4. The foregoing method of D3, wherein the hydrogel comprises a PEG (Polyethylene Glycol) and NIPAAm (N-Isopropylacrylamide).

D5. The foregoing method of Cl or DI, wherein the hydrogel is a shear-thinning hydrogel.

D6. The foregoing method of D5, wherein the shear-thinning hydrogel comprises hyaluronic acid macromers functionalized with adamantane (HA-AD) and cyclodextrin (HA- CD).

D7. The foregoing method of any one of Cl, D1-D6, wherein the hydrogel further comprises a second therapeutic agent.

D8. The foregoing method of Cl or DI, wherein the first therapeutic agent comprises a Treg cell factor.

D9. The foregoing method of D8, wherein the soluble Treg cell factor comprises a regulatory T cell stimulatory factor.

DIO. The foregoing method of D8, wherein the soluble Treg cell factor comprises a T cell chemoattractant factor.

Dl l. The foregoing method of D8, wherein the soluble Treg cell factor is selected from the group consisting of CCL22, IL2, and TGF- P, and combinations thereof.

6. EXAMPLES

The presently disclosed subject matter will be better understood by reference to the following Example, which are provided as exemplary of the presently disclosed subject matter, and not by way of limitation.

6.1. Introduction Significant research has focused on understanding the immune response following myocardial infarction (MI) [16, 24-27] and subsequent heart failure [27, 28], This has led to researchers endeavoring to modulate the immune response through delivery of specific immunokines, cytokines with immunologic effect, such as IL- 10 [29-31], or delivery of various immunomodulatory nucleic acids [32, 33], While these approaches have led to improvement in outcomes following MI in small animal models, single factor delivery in highly complex cellular systems has not been adequately robust to capture all intended benefits. Particularly, interactions between the immune system and repair processes, orchestrated by living cells, often requires several soluble factors, cell-cell contact, and specific timing to effectively initiate and direct cells in the local microenvironment [34-36], Accordingly, the present disclosure provides an approach to amplify the body’s own regulatory strategies through local enrichment of the body’s own natural regulatory T cells (nTreg) in terms of both presence and activity in the infarct microenvironment to minimize immune-mediated damage and enhance repair. nTregs secrete multiple factors that suppress inflammatory responses while enhancing local repair with appropriate spatial and temporal context. In short, nTregs represent an existing, endogenous target whose sophisticated regulatory activity can be amplified through appropriate stimuli at the site of desired action. With the appropriate dose and kinetics of delivery, the present disclosure provides an approach that has significant potential to address the inflammatory response in a way that is dynamic and context-rich through natural cellular regulation. In contrast, previous approaches have lacked this specificity [37], have been ineffective [27], and can even increase the risk of subsequent cardiac events or complications [38],

Degradable microparticle (MP) formulations can be used to enhance the presence of the body’s own regulatory cells at the application site. The Treg-recruiting MP take advantage of the enriched CXCR4 chemokine receptor expressed on Tregs via C-C motif ligand 22 (CCL22), which is a chemokine upregulated by tumors causing the attraction of nTregs to tumors [39], This Treg-recruiting approach (CCL22MP) relied upon sustained release technology to maintain a gradient of CCL22 from the site of application (Figures 1A-1C and 2A-2C. Previous work suggests that CCL22MP attracts nTregs from neighboring tissues or the vasculature including the lymphatic and circulatory systems [40], This technology has successfully applied to increase local nTreg concentrations in several disease models characterized by an unwanted or disproportionate immune response that does not effectively resolve (e.g., transplant [41], dry-eye disease [42], and periodontitis [43]). The MP delivery approach effectively attracts nTregs to the site of application for a sustained period causing immunosuppression, tolerance, and immune polarization.

In the context of MI, limited studies have indicated that Tregs [34, 44] and their secreted factors [45-47] are critical to support immune resolution and healing. However, these prior studies examined the benefits of Tregs through their ablation, and not through their enhancement. Separately, there is evidence that enhancing the number of mobilized Treg through administration of a bolus dose of CXCR4 antagonist is beneficial [48], Notably, as a result of the bolus nature of the dose, this approach was transient and did not directly target the infarct. The present disclosure investigated the localized controlled release of CCL22, a chemokine known for its affinity to Treg cells expressing elevated levels of CCR4. This approach finely modulated regulatory responses within the infarcted myocardium at the specific site of application. Accordingly, an approach to local, sustained attraction of Tregs directly to the infarcted myocardium, provides a new strategy in the context of MI. The present disclosure further elucidated the effects generated by an augmented Treg population on cardiac repair and remodeling processes.

6.2. Methods

Microparticle fabrication. Microparticles containing Alexafluor680-labeled Dextran (10k MW, 125 pg) or rmCCL22 (5 pg) were made via double emulsion-evaporation technique.

Hydrogel fabrication. Thermogels were prepared via aqueous free radical polymerization. Briefly, the selected 100 pL PEG (MW 200 da) was added to 100 mg of NIPAAm with 2 ml of a 0.1 mg-mE¹ ammonium persulfate aqueous solution. This mixture was vortexed until the solution was homogeneously mixed. To this solution, 5 pl of tetramethylethylenediamine was added and then refrigerated overnight. The resulting polymer was washed 5 times using DI water at 45 °C. Thermogels were formulated to have an LCST of 34-35°C. ydr were formed from guest-host reactions between Hyaluronic acid macromers that have been functionalized with adamantane (HA- AD) and cyclodextrin (HA-CD) (20 & 18% functionalization, respectively). HA-AD and HA-CD were resuspended (4% wt./vol total HA) in PBS with MPs at the specified concentration and mixed in a 1 mL syringe.

Microparticle release and degradation assays. 10 mg of MPs were added to microcentrifuge tubes and re-suspended in 1 mL of release media (1% wt./vol Bovine Serum Albumin in PBS). The samples were incubated at 37°C on an end-to-end rotator and at specified time intervals, samples were collected and replaced with release media. Released rmCCL22 was quantified using DuoSet ELISA kits (R&D Systems). Data shown is mean ± SD. Animals. Animals were bred at the University of Pittsburgh and shipped to Envigo’s surgical facility in Indianapolis, IN or purchased and shipped directly to Envigo. For myocardial infarction procedures, B6 WT mice (age 8-12 weeks) were purchased from Jackson Labs. MI was induced via ligation of the left aortic descending artery permanently (Thermogel Experiments) or transiently (30 mins) (Injectable Hydrogel Experiments). Wistar rats (aged 10- 12 weeks) were purchased from Jackson Labs for performing MP retention studies. Briefly, the rats were intubated, the heart was exposed, and HA gels, containing 20 mg MP/mL, were injected (100 uL) into the hearts (healthy) of Wistar rats (aged 8 - 10 wks). MI induction and iHEART treatment occurred at Envigo’s surgical facility and animals were housed on-site until study terminal end-points were reached. Hearts were explanted by Envigo and same-day shipped to the University of Pittsburgh for downstream processing.

MI Induction and treatment. The left aortic descending (LAD) artery ligature MI model is representative of clinical conditions [108-110], LAD artery ligation was performed by ventilating the mouse, opening the chest, and tying a suture around the LAD artery to occlude blood flow [111]. Immediately following MI induction, iHEART formulations were injected into the infarct and border region as three (3) 30 pl injections (90 pL Total), similar to prior hydrogel studies [85, 88, 112],

Ex vivo heart injection. Hearts were excised, flushed with PBS + heparin, attached to a Langendorff apparatus, and perfused on working heart mode to evaluate the effect of hydrogel injection on cardiac function.

Assessment of functional outcomes. Animals were evaluated at baseline (prior to MI induction), immediately after MI induction, POD 7, and POD 28 for cardiac function with echocardiography (Fuji/Visualsonics Vevo 3100 ultrasound scanner). Specifically, changes in cardiac function, including end systolic pressure-volume relationship, ejection fraction, cardiac output, +/- dP/dt, stroke volumes, end-diastolic volumes, and systolic volumes were evaluated using routinely performed techniques [88, 113],

Echocardiography. Transthoracic echocardiography was performed using a VisualSonics Vevo 3100, high frequency ultrasound machine with the MS 400 transducer (VisualSonics Inc). The mice were anesthetized with isoflurane at 3% induction. During the echocardiogram, isoflurane was reduced to 1.5% to maintain a heart rate greater than 400 bpm. A short axis image of the left ventricle (SAX) was obtained, and an M-mode image was taken at mid ventricular, with evidence of the papillary muscles. A parasternal long axis of the left ventricle (PSLAX) was also acquired. Analysis was then completed with VevoLab Software provided by VisualSonics Inc. An average of at least 3 cardiac cycles was analyzed in all measurements. The SAX M-Mode image was used to obtain heart rate, ventricular wall thickness, ventricular cavity size, ejection fraction, fractional shortening, and cardiac output. To acquire the volume of the left ventricular cavity in PSLAX, the left cavity area was measured in systole and diastole.

Flow cytometric quantification of immune populations. Explanted hearts were enzymatically digested and CD45+ (leukocyte marker [114]) enriched cells were quantified via flow cytometry. Briefly, CD45+ cells were stained for phenotypic markers of Neutrophils (CDl lb, CD16, Ly6G), monocytes (CDl lb, CD68, Ly6C), macrophages (CD11B, CD11C, CD206, iNOS, F4-80, LYVE-1), and T-lymphocytes (CD3, CD4, CD8, CD25, FoxP3, RORyt, T-bet, Gata-3, ST2, AREG, and IL-13). Additionally, flow cytometry was used to assess iHEART’s ability to induce Treg from conventional CD4+ T cells or stabilize infiltrating nTreg. Treg and CD4+ T cell fate mapping reporter mice systems show a color change corresponding to Foxp3 Gene activation [115, 116], The reporter mice system is discussed in greater detail with respect to Figure 15. T-lymphocyte associated markers were also employed to quantify population shifts.

Tissue remodeling quantification by histology. On POD 28, H&E, quantitative immunohistochemical(IHC), and immunofluorescent (IF) staining were performed on explanted hearts. Transverse tissue cryosections (10 pm thickness) were prepared from the apex to the base of the left ventricle (LV) and sections near the mid-papillary level to above the apex will be stained and used to visualize and quantify morphological features of LV remodeling [88, 117], Evaluation of cell and vessel invasion, density, and deposited matrix will be done on regions of interest (RO I) where the infarct, border-zone, and iHEART are detectable. For each metric quantified, values for a single biological replicate will be obtained by averaging at least 3 ROIs within each animal. Trichrome staining will be used to quantify the size (% area of LV) of collagenous scar tissue and its interaction with iHEART, as well as collagen fiber density, thickness, and fiber orientation within the tissue [118], Macrophages will be stained for pan marker: CD68+, pro inflammatory: iNOS+ CD86+ [119], and pro-healing: CD206+, Arginase 1 [120], and T-lymphocytes for markers: CD3+, and FoxP3+ [88], For rare leukocyte populations that are not typically cardiac resident (Tregs, CD4+ T cells), Hearts are harvested and sectioned for IF staining during more immunologically timepoints (POD 3-7). POD 28 staining did not always show the target populations, due to scar tissue deposited by activated fibroblasts (myofibroblasts). The presence of the fibroblast cells around iHEART was evaluate for their density and phenotype with alpha-smooth muscle actin (a-SMA) staining. CD31+ staining was used to identify blood capillaries, which are known to sprout as an early reparative response to MI, and vessel maturation was evaluated using vessel size and a-SMA staining [98^ 110], Blood vessel density was determined by counting double positive CD31+ a-SMA+ vessels in the LV and ratioing to total LV area.

Statistics. Group size and power analysis were determined for studies involving the mouse MI model, injecting hydrogels into small rodent models, cell populations quantified by flow cytometry. Experiments were designed based on a -20% standard deviation, which is seen as a conservative estimate. A relative standard deviation of -20% is typically used for cardiac outcomes (e.g., ejection fraction). Based on a 20% estimated difference between groups, a statistical power (1-13) of 80% at the significance level of a = 5% was obtained with a sample size of n = 16 biological repeats per group. In recognition of multiple comparisons, animal loss during surgery and for study design consistency, n = 20 animals per group were used for post-MI outcomes. For flow cytometry and other immune cell characterization, experiments were design for 14% standard deviation, 20% difference between groups, significance level of a = 5%, and a statistical power (1-13) of 80% rendering n = 8 biological replicates per group. In recognition of multiple comparisons, animal loss during surgery, and for study design consistency, n = 10 animals per group were used. Quantitative outcomes from in vivo cardiac function measurements and ex vivo histological analysis were tested for normal distribution, and differences between groups will be assessed by one-way ANOVA (normal distribution) or Kruskal -Wallis test (non-normal distribution) followed by Sidak’s multiple comparisons test. A probability of less than 5% (p < 0.05) of no difference between groups was considered to be significant.

6.3. Results

6.3.1. Enhanced microparticle retention.

Previous work has shown that the direct injection of microparticles into the heart tissue resulted in poor retention due to coronary blood flow perfusing the tissue [49], which reduced potential efficacy. Consequently, microparticles are seldom used for cardiac delivery of bioactive molecules [50], In previous studies with CCL22MP were shown to have significant retention of injected payload, administered via subdermal injection [51], Thus, a change of approach to facilitate cardiac retention of microparticles was necessary to expand their use as a delivery system in cardiac applications. Injectable hydrogels (water-swollen polymer networks) have gained attention because they have controllable biophysical and biochemical properties, can be designed to be injectable, can deliver biologies (e.g., cells, cell-derived products, cytokines, or growth factors) locally, and have good retention in solid organs [52-55], However, release of biologies often exhibits burst release due to the presence of highly connected pores in hydrogels [56], requiring highly engineered hydrogels or modified biologic molecules [54, 57] to achieve controlled delivery for a sustained period.

The present disclosure demonstrated the application of an innovative platform known as Injectable Hydrogel to Enhance or Attract Regulatory T cells (iHeart). This present disclosure demonstrated the development of the iHeart platform, integrating microparticle and injectable hydrogel technologies to enhance retention within solid organs and enable controlled release of therapeutic agents with a unique spatiotemporal presentation (involving sustained release microparticles and a bursting hydrogel). Previous efforts by others have explored similar strategies, seeking to improve the retention of nanocarriers like micelles and coacervates to achieve multifactor delivery in cardiac applications [29, 58],

An evaluation of the retention of MPs using iHEART in rat hearts was performed (Figures 3A-3C). Fluorescently labeled dextran was encapsulated into PLGA MP [40], Fluorescent MP were then suspended in hydrogel (Gel+MP) or saline (Saline+MP) at a concentration of 20 mg MP/mL and injected (100 uL) into the hearts (healthy) of Wistar rats (aged 8 - 10 wks). Blank MP were suspended in hydrogel (20 mg MP/mL) and injected as a vehicle control. Fourteen (14) days after injection, animals were sacrificed, and hearts were explanted for fluorescent imaging. The results revealed a significant improvement in the retention of microparticles when suspended in hydrogel vs. saline, as measured by mean fluorescent intensity (MFI) normalized to the region of interest (ROI). The ROI was defined as the entire heart to minimize selection bias. Qualitatively, improved retention at the injection site resulted in a high fluorescent intensity peak followed by an ellipsoidal radiance of fluorescence due to diffusion of encapsulated dextran from the MP in the Gel+MP group. The Gel+MP experimental group performed better than Saline+MP in nearly all replicates, although there a greater degree of variance. The fluorescence was quantified in the loaded syringe, injected gels, and in the post-injection needle & syringe (Figures 2A-2C) to establish the source of variance. To do so, multiple (3) injections (per syringe) of Gel+MP into agar gel at different concentrations (20 & 100 mg/mL) were performed for fluorescent MP. The data demonstrated that the mixing of microparticles in the gel is relatively homogenous, but there is variation in the injection payload due to losses in the needle and syringe walls. Payload loss is more pronounced at higher concentrations due to increased potential for the needle to plug. Thus, although the Gel+MP experimental group performed well, the approach can be improved by implementing engineering controls (Needle and syringe geometry) and operability improvements (single syringe for injection, planned needle changeout, increased needle diameter, implement max concentration of 20 mg MP/mL). Additionally, left ventricle iHEART injection in an ex vivo model of ventricle function showed no signs of negative effects to cardiac function (Figures 4A and 4B).

6,3.2. iHEART-mediated attraction and enhancement of nTreg to the infarcted heart altered local immune populations, influenced repair, and affected heart function.

The present disclosure concerns a delivery system composed of an injectable, Hydrogel loaded with degradable polymeric MP to Enhance or Attract Regulatory T cells, herein referred to as iHEART, to treat MI. The present disclosure demonstrates that by enhancing local Treg populations in the infarcted myocardium will improve inflammation resolution, tissue repair and remodeling, and functional outcomes following MI. The iHEART technology was utilized to attract endogenous, thymic-derived Tregs (nTreg) to the infarcted myocardium. The present disclosure elucidated how local enhancement of distinct Treg populations shaped the infarct microenvironment.

This present disclosure is based on evidence that strongly suggests that both nTregs and iTregs provide significant benefit following inflammatory damage through immune suppression [41-43, 68, 69] and enhancement of repair processes [43, 84], The present disclosure shows that CCL22MP administration to the heart produces a positive impact on heart function, which was associated with reduced infarction size.

The iHEART platform was used to delivered biologic molecules to the infarcted myocardium to enhance local populations of natural regulatory T cells (nTreg) leading to a reduction in inflammatory damage and infarct size while improving heart function through repair. In particular, the present disclosure assessed migration kinetics of Tregs in response to CCL22MP and quantified the effects of Treg attraction via CCL22MP.

CCL22MP can attract Treg to the site of administration. Previous work has characterized migration kinetics of adoptively transferred nTregs to the site of CCL22MP administration [40], In the present disclosure, fluorescently labeled MP (red/black) (CCL22 Loaded and Blank) were suspended in saline and injected into the hindlimbs of mice. Next, nTregs modified to express the luciferase gene (MFI) were injected via Intraperitoneally (IP). Blank MP (left limb) did not show co-localization with nTregs, whereas CCL22MP (right limb) showed colocalization 4 days following injection (see Figure 5 A). Subsequent experimentation performed in a hind-limb transplantation model showed that mice receiving CCL22MP displayed significantly enhanced endogenous nTreg populations in the skin of the grafts at postoperative days 29 - 43 (Figure 5B) [94], These data indicate CCL22MP can attract nTregs to the site of application resulting in increased nTreg frequency for several weeks, which are impactful on inflammatory and even immune rejection outcomes. 7r' eg attracting MP (CCL22MP) was shown to prevent and/or reverse inflammatory disease progression. The ability of CCL22MP administration to attract nTregs to the site of application and the resulting nTreg’s ability to ameliorate symptoms of disease has been characterized in several inflammatory diseases. Specifically, application of this technology in a mouse model of periodontal disease significantly reduced key indicators of disease [43], In these studies, it was found that CCL22MP treatment significantly reduced inflammatory cell populations (Figure 6A) and enhanced tissue mRNA expression and cytokine levels of nTreg associated factors (Figures 6B and 6C). Similar results have been demonstrated in murine models of Transplant [41] and Dry-eye disease [42], These data strongly suggest that local administration of CCL22MP in models of inflammatory disease significantly enhances nTreg presence leading to diminished inflammation.

CCL22 MP treatment improved heart function at POD 7 and 28. To determine if the effect of CCL22MP treatment following MI, studies were performed in a mouse LAD ligature model of MI (Figure 7A). The present disclosure showed that CCL22MP was suspended in a thermoresponsive gel and the gel and MP solution was then applied, topically, to the infarcted heart, ensuring a controlled and gradual release of CCL2MP in the targeted area (Figure 7B). A significant reduction in mean infarction size of -10% was observed between CCL22MP and BlankMP groups (Figure 7C and 7D). Significant improvements in ejection fraction (POD 7 & 28), systolic volume (POD 7 & 28), stroke volume (POD 28), and diastolic volume (POD 28) were observed in the CCL22MP group compared to the Blank MP group (Figure 7E). These data indicated that CCL22MP treatment following MI can improve heart function by reducing tissue damage. The thermoresponsive hydrogel and MP system was not observed in POD 28 histology suggesting suboptimal retention, thus, the iHEART system, which is demonstrated to be retained in the heart, can achieve greater effects. Similarly, iHEART application in a model of ischemia-reperfusion MI showed a trend toward improvement in cardiac function (POD7) (Figures 8A and 8B).

6.3.3. IL-33 expands ST2⁺ Treg subset and stimulates the production of reparative factors.

IL-33, a typically sequestered cytokine that is released after cellular injury and stress [75], triggers natural nTreg proliferation and stimulates the secretion of reparative factors such as IL-13 and amphiregulin (AREG) [74, 76, 77], Given the upregulation of IL-33 in the infarcted heart [78-80], it is thought that limited local natural regulatory T cell (nTreg) concentration, rather than IL-33, contributes to suboptimal injury resolution and repair following myocardial infarction (MI). However, IL-33 supplementation beyond what naturally occurs, can provide further benefit. Indeed, studies have shown that systemic IL-33 treatment can be protective following MI [80, 81], IL-33 has also been shown to be a critical factor in skeletal muscle injury for tissue retention of nTregs [78], However, there are reports that indicate systemic IL-33 supplementation in injury and disease has potential to cause inflammatory skewing [82] and perpetuate ineffective repair [83], These methods of supplementation did not recapitulate biorelevant presentation because they were not performed locally or with kinetics akin to natural presentation in response to injury (short-term, transient). Thus, biorelevant short-term supplementation, locally, supplied by encapsulation can reduce the potential for negative effects of IL-33 supplementation to occur.

To understand the effects that IL-33 has on nTreg, a transgenic knockout (KO) mouse lacking IL-33 (IL-33-/- ) was utilized. ST2 expressing (ST2+ ) nTreg were separated from the ST2- nTreg. As a control, canonical ST2+ Th2 CD4+ T cells were also assessed. Co-culture of ST2+ nTreg with recombinant IL-33 significantly elevated levels of IL-10 (Figure 9A) and IL- 13 (Figure 9B) in culture supernatants. To investigate what role Treg-secreted IL- 13 plays in injury and repair, a transgenic mouse with nTreg lacking IL-13 (Foxp3Cre xil4/il 13flox ; IL- 13 KO) was generated to evaluate the injury and repair response in a bleomycin lung injury model. Reduced survival (Figure 10 A) and increased inflammatory cell populations (Figure 10B) in IL-13 KO mice in comparison to controls were observed. To investigate what role nTreg-secreted amphiregulin (AREG) plays in injury and repair, mice with nTreg lacking AREG (Foxp3Cre xAregfl/fl; AREG KO) were generated. nTregs isolated from AREG KO and co-cultured with recombinant IL-33 and fibroblasts isolated from transgenic mice lacking ST2 (St2-/- ; ST2 KO). WT, but not AREG KO Treg, increased ST2 KO fibroblast proliferation in the presence or IL-33 (Figure 10C). Together, these data indicated that IL- 33 signaling through the ST2 receptor on nTreg can upregulate reparative factors (IL-13, AREG) that are implicated in effective healing following injury [74, 77, 84],

The present disclosure demonstrated IL-33 -releasing injectable hydrogel (IL-33Gel) (Figures 11A-11C) to facilitate short-term, local supplementation of this factor. The iHEART system can be used to administer various other factors, including CCL22MP, with and without IL- 33Gel, or can even utilize TriMP alone.

In this fashion, the present disclosure tested the controlled release technology can be applied to (i) augment local Treg concentration, (ii) promote nTreg-mediated repair following MI, (iii) stabilize nTreg phenotype in the inflammatory MI local environment, and (iv) limit inflammation with increased local density of iTreg. No study has demonstrated an approach that modulates the nTreg or iTreg activity locally to treat MI. The present disclosure demonstrated a first sustained-release formulation of IL-33, which has broad potential to enhance tissue repair diseases.

6.3.4. Enhancing Treg Stability and Immunomodulation with TriMP

Although nTreg recruitment has demonstrated effectiveness in a number of other models of inflammation and even transplantation tolerance [40-43], nTregs are a relatively rare population (-0.5% of total immune cells in the periphery [59, 60]) which can impose a limit to efficacy. Also, it has been previously demonstrated that it is possible for nTregs to differentiate into inflammatory Thl/Thl7 differentiated CD4+ cells under certain circumstances, minimizing or abrogating immunosuppressive action [6, 61, 62], Indeed, this has been documented to occur naturally following MI in the short term [63] and several weeks after MI [23], Thus, it is important to develop therapies that can stabilize nTreg phenotype while promoting beneficial effector functions. Certain soluble factors can be delivered to modulate Treg phenotype and function. One such factor of interest, TGFB, is known to enhance nTreg proliferation and immunosuppression [3, 64], A previously developed microparticle formulation (TriMP) has been designed to deliver TGFb, IL-2, and rapamycin [65], aimed to inhibit IL-2 signaling and thereby impede the expansion of effector CD4+ T cells (Teff). TriMP induced Tregs (iTreg) from infiltrating CD4+ T cells facilitating significant increases in local Treg population. The application of TriMP has been shown to alter the ratio of Treg to Teff cells reversing or preventing signs of disease in animal models of arthritis [66], allergic dermatitis [67], dry-eye disease [68], and transplantation [69],

Tri factors maintain iTreg stability. During the development of TriMP, a series of experiments were conducted to characterize iTreg induced by Tri Factor administration [104], To determine if induced Treg (iTreg) were stable, Treg were induced from conventional CD4+ T Cells (Tconv) using the individual Tri Factors, TGFB1 or Rapamycin. The induced populations were restimulated via TCR activation (CD3/CD28 costimulatory Dynal beads) with IL-2 (without Tri factors) or with Tri Factors (IL-2, TGFB1, rapamycin) in the with or without activated Teff cells. Significant reductions in Foxp3+ cell populations occurred following culture with Teff cells in groups lacking Tri factors as compared to those receiving Tri factors. This indicates that Tri factor supplementation is required throughout inflammatory signaling to prevent loss of iTregs. Most importantly, this demonstrates that Tri factors can induce Treg and prevent their polarization toward inflammatory cell populations.

TriMP enhances Treg immunosuppressive ability. Previous studies with TriMP, concerned how treatment with TriMP alters Tregs. To this end, in vitro experimentation with Tconv and nTreg isolated from spleens was performed [65], Tconv cells were stained with CFSE and co cultured with nTregs or Tregs treated with TriMP (iTreg). CFSE signal strength was measured using flow cytometry, in which decreased CFSE readings (left skew of the histogram) occurred if cells become activated and proliferate. The TriMP treated Treg group exhibited greater suppression (smaller proliferating cell numbers) as compared to the nTreg group for all dilutions trialed . In subsequent work, an in vivo assessment of TriMP’ s ability to induce immunosuppressive Treg was conducted. Using an allergic dermatitis model, TriMP or vehicle control (Blank MP) was administered 2 days before sensitization. 4 days after sensitization, skin draining lymph nodes (DLNs) were harvested and analyzed using flow cytometry [67], TriMP -treated iTregs isolated from the DLN exhibited increases in several inhibitory ligands that facilitate immunosuppression (CTLA-4, GITR, LAP) relative to nTregs . These data indicated TriMP induced iTreg have enhanced immunosuppressive capacity relative to nTreg.

TriMP increases regulatory T cell populations and associated markers while reducing inflammatory markers in inflammatory diseases. To investigate how TriMP alters the local environment, TriMP was administered in a hindlimb transplant model. The immunologic activity in the transplanted limbs was assessed on POD 33 - 45 (rejecting grafts) and POD >300 (surviving grafts receiving TriMP). In the DLNs, there were increased populations of Tregs (Figure 12A) and greatly reduced Thl associated, inflammatory CD4+ T cells (Figure 12B). The population changes corresponded to reductions in mRNA expression of inflammatory markers, TNF and IL-17a (Figure 12C). mRNA expression of Treg-associated factors, IL-10 and TGFB1, were also significantly upregulated in response to TriMP treatment (Figure 112D). The paradoxical decrease in Foxp3 expression in the surviving grafts (TriMP treated) is due to reduced inflammation causing reduced total numbers of Treg that are more functional giving rise to the increased IL-10 and TGFB1 expression. IFN-y is typically considered to be an inflammatory factor, but this factor helps direct alloantigen-specific Treg survival and function in transplant models [105, 106], Similar population frequency and cytokine changes have been observed following TriMP treatment in several other murine models of inflammatory disorders (dry-eye disease [68], arthritis [66], and allergic dermatitis [67]). Thus, these data strongly suggest that TriMP administration alters the ratio of Treg:Teff leading to diminished inflammatory markers.

6.4. Discussion

Cardiovascular disease remains the leading cause of death, worldwide. The etiology of cardiovascular disease is multifaceted, but acute or chronic ischemia, presenting as myocardial infarction (MI), is a precipitating event. MI leads to rapid progression of cardiovascular disease due to early tissue injury and later pathologic tissue remodeling [2], Over 9.5 million deaths annually can be attributed directly to MI (2016 data) [1], and patients surviving their first MI have a 20% likelihood of being re-hospitalized due to refractory MI or a related cardiac event [1], Surviving patients experience a significant reduction in their health and quality of life following MI due to ischemic heart disease, in which 33% of patients progress to total heart failure within a few years [7], Together, the health care costs associated with ischemic heart disease and MI hospitalizations account for $134.5 billion each year [1], With nearly 153.5 million people living with ischemic heart disease, worldwide [1], there is a great need to provide better solutions to a disease that significantly taxes health care systems, debilitates affected individuals, and results in significant loss of life. Ischemic Injury.

Ischemic injury. During ischemic injury, blood flow is blocked or reduced creating a local hypoxic environment that causes cardiomyocytes (CMs) to necrose. CM necrosis releases typically sequestered self-materials containing damage associated molecular patterns (DAMPs) that trigger and amplify local inflammation and tissue damage. For poorly understood reasons, the heart does not naturally replace lost tissue with functional CMs, causing any damage inflicted to be permanent [8], Delays in reestablishment of blood flow (reperfusion) results in larger infarcts [9, 10], In turn, larger infarcts translate to reduced cardiac function, drastically increase the likelihood of developing heart failure, and significantly reduce the quality of life of a patient. Consequently, vasodilators [11], thrombolytics [12], and other treatments aiming to re-establish blood flow [13] have become the clinical standard for treating MI patients. However, reperfusion can paradoxically exacerbate damage to the heart, referred to as reperfusion injury (RI), accounting for nearly half of the final infarct size [10], These existing treatments do little to address subsequent RI. RI can be caused by several factors but inflammation has been identified as one of the greatest contributors [10], A number of new strategies to treat RI have been explored, but have failed to demonstrate meaningful clinical benefits, as succinctly reviewed by Yellon et al [10], A key challenge is that multiple cellular processes become dysregulated because of RI requiring a multifaceted approach to impart a tangible benefit [14, 15], Unlike previous approaches to address RI, the immune system has natural and sophisticated regulatory mechanisms capable of addressing dysregulated cellular processes.

The immune response to MI. The development of inflammation seen in MI is a multistage process. DAMPs released by necrotic cells cause numerous immune cells to infiltrate the infarct. These first responders cause uncontrolled inflammatory signaling and other effector actions (e.g., NETosis, oxidative species, granzymes) damaging remaining healthy tissue, a phenomenon referred to as infarct spreading [10, 16], After several days, this immune response shifts toward a pro-wound healing response via the adaptive immune system in which FoxP3+ CD4+ Regulatory T cells (Tregs) play a vital role in the transition [17, 18], The magnitude of the initial immune response and the way that it ultimately resolves appears to significantly shape the repair and remodeling [19], Yet, inflammation often does not adequately resolve due to a variety of poorly understood reasons including on-going stimulation of immune effector cells, ineffective apoptosis of effector cells, or persistent cellular debris from apoptotic and necrotic bodies [20-22], Indeed, following infarction there are persistent environmental cues that appear to perpetuate inflammatory phenotypes of local immune cells [16, 23], Directing the immune response following MI with appropriate context to resolve inflammation, enhance repair, and minimize damage holds the potential to significantly reduce RI and the repair and scar formation processes.

The present disclosure provides a first-of-its-kind approach that combines new regulatory T cell-attracting or -inducing microparticles (MP) with an injectable hydrogel to facilitate solid organ delivery to the heart. This approach is directed to increasing the population of Tregs in the infarct and enhancing their ability to resolve inflammation and/or promote repair following MI leading to improved outcomes (e.g., reduced infarction size, improved scar formation, greater vascularization of tissue, among others). To date, no other studies have attempted to directly manipulate regulatory T cells to treat MI, and thus the present disclosure stands to significantly advance the field of cardiac tissue repair. Utilizing this technology can guide the body’s own cellular systems to prevent or reduce infarct spreading and it can enhance the switch toward a wound healing response translating to a smaller, more stable infarct.

* * *

Although the presently disclosed subject matter and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the present disclosure. Moreover, the scope of the present application is not intended to be limited to the particular embodiments of the process, machine, manufacture, and compositions of matter, means, methods and steps described in the specification. As one of ordinary skill in the art will readily appreciate from the present disclosure of the presently disclosed subject matter, processes, machines, manufacture, compositions of matter, means, methods, or steps, presently existing or later to be developed that perform substantially the same function or achieve substantially the same result as the corresponding embodiments described herein can be utilized according to the presently disclosed subject matter. Accordingly, the appended claims are intended to include within their scope such processes, machines, manufacture, compositions of matter, means, methods, or steps. Various patents, patent applications, publications, product descriptions, protocols, and sequence accession numbers are cited throughout this application, this present disclosures of which are incorporated herein by reference in their entireties for all purposes.

6.5. References

[1] E.J. Benjamin, P. Muntner, A. Alonso, M.S. Bittencourt, C.W. Callaway, A.P. Carson, A.M. Chamberlain, A.R. Chang, S. Cheng, S.R. Das, F.N. Delling, L. Djousse, M.S.V. Elkind, J.F. Ferguson, M. Fornage, L.C. Jordan, S.S. Khan, B.M. Kissela, K.L. Knutson, T.W. Kwan, D.T. Lackland, T.T. Lewis, J.H. Lichtman, C.T. Longenecker, M.S. Loop, P.L. Lutsey, S.S. Martin, K. Matsushita, A.E. Moran, M.E. Mussolino, M. O’Flaherty, A. Pandey, A.M. Perak, W.D. Rosamond, G.A. Roth, U.K. A. Sampson, G.M. Satou, E.B. Schroeder, S.H. Shah, N.L. Spartano, A. Stokes, D.L. Tirschwell, C.W. Tsao, M.P. Turakhia, L.B. VanWagner, J.T. Wilkins, S.S. Wong, S.S. Virani, E. American Heart Association Council on, C. Prevention Statistics, S. Stroke Statistics, Heart Disease and Stroke Statistics-2019 Update: A Report From the American Heart Association, Circulation, 139 (2019) e56-e528.

[2] M.A. Konstam, D.G. Kramer, A.R. Patel, M.S. Maron, J.E. Udelson, Left ventricular remodeling in heart failure: current concepts in clinical significance and assessment, JACC Cardiovasc Imaging, 4 (2011) 98-108.

[3] M.A. Sugimoto, L.P. Sousa, V. Pinho, M. Perretti, M.M. Teixeira, Resolution of Inflammation: What Controls Its Onset?, Front Immunol, 7 (2016) 160.

[4] D.A. Cronkite, T.M. Strutt, The Regulation of Inflammation by Innate and Adaptive Lymphocytes, J Immunol Res, 2018 (2018) 1467538.

[5] J. Li, J. Tan, M.M. Martino, K.O. Lui, Regulatory T-Cells: Potential Regulator of Tissue Repair and Regeneration, Front Immunol, 9 (2018) 585.

[6] S. Piconese, V. Barnaba, Stability of Regulatory T Cells Undermined or Endorsed by Different Type-1 Cytokines, Adv Exp Med Biol, 850 (2015) 17-30.

[7] T.J. Cahill, R.K. Kharbanda, Heart failure after myocardial infarction in the era of primary percutaneous coronary intervention: Mechanisms, incidence and identification of patients at risk, World J Cardiol, 9 (2017) 407-415.

[8] D.C. Zebrowski, S. Vergarajauregui, C.C. Wu, T. Piatkowski, R. Becker, M. Leone, S. Hirth, F. Ricciardi, N. Falk, A. Giessl, S. Just, T. Braun, G. Weidinger, F.B. Engel, Developmental alterations in centrosome integrity contribute to the postmitoticstate of mammalian cardiomyocytes, Elife, 4 (2015).

[9] E.T. Hasche, C. Fernandes, S.B. Freedman, R.W. Jeremy, Relation Between Ischemia Time, Infarct Size, and LeftVentricular Function in Humans, Circulation, 92 (1995) 710-719.

[10] D.M. Yellon, D.J. Hausenloy, Myocardial reperfusion injury, N Engl J Med, 357 (2007) 1121-1135.

[11] K. Chatterjee, W.W. Parmley, Vasodilator therapy for acute myocardial infarction and chronic congestive heart failure, Journal of the American College of Cardiology, 1 (1983) 133- 153.

[12] J. Hirsh, G. Guyatt, G.W. Albers, R. Harrington, H.J. Schunemann, Antithrombotic and thrombolytic therapy: American College of Chest Physicians Evidence-Based Clinical Practice Guidelines (8th Edition), Chest, 133 (2008) 110S-112S.

[13] F. Van De Werf, D.S. Bairn, Reperfusion for ST-segment elevation myocardial infarction: an overview of current treatment options, Circulation, 105 (2002) 2813-2816.

[14] J.S. Burchfield, M. Xie, J.A. Hill, Pathological ventricular remodeling: mechanisms: part 1 of 2, Circulation, 128 (2013) 388-400.

[15] S. Hernandez-Resendiz, K. Chinda, S.B. Ong, H. Cabrera-Fuentes, C. Zazueta, D.J. Hausenloy, The Role of Redox Dysregulation in the Inflammatory Response to Acute Myocardial Ischaemia-reperfusion Injury - Adding Fuel to the Fire, CurrMed Chem, 25 (2018) 1275-1293.

[16] G. Courties, M.A. Moskowitz, M. Nahrendorf, The innate immune system after ischemic injury: lessons to be learned from the heart and brain, JAMA Neurol, 71 (2014) 233-236.

[17] F.K. Swirski, M. Nahrendorf, Leukocyte behavior in atherosclerosis, myocardial infarction, and heart failure, Science, 339 (2013) 161-166.

[18] J. Weirather, U.D. Hofmann, N. Beyersdorf, G.C. Ramos, B. Vogel, A. Frey, G. Ertl, T. Kerkau, S. Frantz, Foxp3+ CD4+ T cells improve healing after myocardial infarction by modulating monocyte/macrophage differentiation, Circ Res, 115 (2014) 55-67.

[19] J.E. Park, A. Barbul, Understanding the role of immune regulation in wound healing, The American Journal of Surgery, 187 (2004) S11-S16.

[20] V. Kain, S.D. Prabhu, G.V. Halade, Inflammation revisited: inflammation versus resolution of inflammation following myocardial infarction, Basic Res Cardiol, 109 (2014) 444.

[21] P. Panizzi, F.K. Swirski, J.L. Figueiredo, P. Waterman, D.E. Sosnovik, E. Aikawa, P. Libby, M. Pittet, R. Weissleder, M. Nahrendorf, Impaired infarct healing in atherosclerotic mice with Ly-6C(hi) monocytosis, J Am Coll Cardiol, 55 (2010) 1629- 1638. [22] T. Lawrence, D.W. Gilroy, Chronic inflammation: a failure of resolution?, Int J Exp Pathol, 88 (2007) 85- 94.

[23] S.S. Bansal, M.A. Ismahil, M. Goel, G. Zhou, G. Rokosh, T. Hamid, S.D. Prabhu, Dysfunctional and Proinflammatory Regulatory T-Lymphocytes Are Essential for Adverse Cardiac Remodeling in Ischemic Cardiomyopathy, Circulation, 139 (2019) 206-221.

[24] L. Timmers, G. Pasterkamp, V.C. de Hoog, F. Arslan, Y. Appelman, D.P. de Kleijn, The innate immune response in reperfused myocardium, Cardiovasc Res, 94 (2012) 276-283.

[25] R. Gentek, G. Hoeffel, The Innate Immune Response in Myocardial Infarction, Repair, and Regeneration, in: S. Sattler, T. Kennedy -Lydon (Eds.) The Immunology of Cardiovascular Homeostasis and Pathology, Springer International Publishing, Cham, 2017, pp. 251-272.

[26] S.L. Lai, R. Marin-Juez, D.Y.R. Stainier, Immune responses in cardiac repair and regeneration: a comparative point of view, Cell Mol Life Sci, 76 (2019) 1365-1380.

[27] M. Panahi, A. Papanikolaou, A. Torabi, J.G. Zhang, H. Khan, A. Vazir, M.G. Hasham, J.G.F. Cleland, N.A. Rosenthal, S.E. Harding, S. Sattler, Immunomodulatory interventions in myocardial infarction and heart failure: a systematic review of clinical trials and meta-analysis of IL-1 inhibition, Cardiovasc Res, 114 (2018) 1445-1461.

[28] E. De Angelis, M. Pecoraro, M.R. Rusciano, M. Ciccarelli, A. Popolo, Cross-Talk between Neurohormonal Pathways and the Immune System in Heart Failure: A Review of the Literature, Int J Mol Sci, 20 (2019).

[29] A. J. Rocker, D.J. Lee, R. Shandas, D. Park, Injectable Polymeric Delivery System for Spatiotemporal and Sequential Release of Therapeutic Proteins To Promote Therapeutic Angiogenesis and Reduce Inflammation, ACS Biomaterials Science & Engineering, 6 (2020) 1217-1227.

[30] X. Meng, J. Li, M. Yu, J. Yang, M. Zheng, J. Zhang, C. Sun, H. Liang, L. Liu, Transplantation of mesenchymal stem cells overexpressing IL10 attenuates cardiac impairments in rats with myocardial infarction, J Cell Physiol, 233 (2018) 587- 595.

[31] M. Jung, Y. Ma, R.P. Iyer, K.Y. DeLeon-Pennell, A. Yabluchanskiy, M.R. Garrett, M.L. Lindsey, IL- 10 improves cardiac remodeling after myocardial infarction by stimulating M2 macrophage polarization and fibroblast activation, Basic Res Cardiol, 112 (2017) 33.

[32] B. Saleh, H.K. Dhaliwal, R. Portillo-Lara, E. Shirzaei Sani, R. Abdi, M.M. Amiji, N. Annabi, Local Immunomodulation Using an Adhesive Hydrogel Loaded with miRNA-Laden Nanoparticles Promotes Wound Healing, Small, 15 (2019) e!902232. [33] Z. Wei, S. Qiao, J. Zhao, Y. Liu, Q. Li, Z. Wei, Q. Dai, L. Kang, B. Xu, miRNA-181a over-expression in mesenchymal stem cell-derived exosomes influenced inflammatory response after myocardial ischemia-reperfusion injury, Life Sci, 232 (2019) 116632.

[34] A. Saxena, M. Dobaczewski, V. Rai, Z. Haque, W. Chen, N. Li, N.G. Frangogiannis, Regulatory T cells are recruited in the infarcted mouse myocardium and may modulate fibroblast phenotype and function, Am J Physiol Heart Circ Physiol, 307 (2014) H1233-1242.

[35] J. Barthes, H. Ozcelik, M. Hindie, A. Ndreu-Halili, A. Hasan, N.E. Vrana, Cell microenvironment engineering and monitoring for tissue engineering and regenerative medicine: the recent advances, Biomed Res Int, 2014 (2014) 921905.

[36] S. Van Linthout, K. Miteva, C. Tschope, Crosstalk between fibroblasts and inflammatory cells, Cardiovasc Res, 102 (2014) 258-269.

[37] P.T. O’Gara, F.G. Kushner, D.D. Ascheim, D.E. Casey, Jr., M.K. Chung, J. A. de Lemos, S.M. Ettinger, J.C. Fang, F.M. Fesmire, B.A. Franklin, C.B. Granger, H.M. Krumholz, J.A. Linderbaum, D.A. Morrow, L.K. Newby, J.P. Omato, N. Ou, M. J. Radford, J.E. Tamis-Holland, J.E. Tommaso, C.M. Tracy, Y.J. Woo, D.X. Zhao, C.A.T. Force, 2013 ACCF/AHA guideline for the management of ST-elevation myocardial infarction: executive summary: a report of the American College of Cardiology Foundation/American Heart Association Task Force on Practice Guidelines, Circulation, 127 (2013) 529-555.

[38] G.H. Gislason, S. Jacobsen, J.N. Rasmussen, S. Rasmussen, P. Buch, J. Friberg, T.K. Schramm, S.Z. Abildstrom, L. Kober, M. Madsen, C. Torp-Pedersen, Risk of death or reinfarction associated with the use of selective cyclooxygenase-2 inhibitors and nonselective nonsteroidal antiinflammatory drugs after acute myocardial infarction, Circulation, 113 (2006) 2906-2913.

[39] A.W. Mailloux, M.R. Young, NK-dependent increases in CCL22 secretion selectively recruits regulatory T cells to the tumor microenvironment, J Immunol, 182 (2009) 2753-2765.

[40] S. Jhunjhunwala, G. Raimondi, A.J. Glowacki, S.J. Hall, D. Maskarinec, S.H. Thome, A.W. Thomson, S.R. Little, Bioinspired controlled release of CCL22 recruits regulatory T cells in vivo, Adv Mater, 24 (2012) 4735-4738. [41] J.D. Fisher, W. Zhang, S.C. Balmert, A.M. Aral, A.P. Acharya, Y. Kulahci, J. Li, H.R. Tumquist, A.W. Thomson, M.G. Solari, V.S. Gorantla, S.R. Little, In situ recruitment of regulatory T cells promotes donor-specific tolerance in vascularized composite allotransplantation, Science Advances, 6 (2020).

[42] M.L. Ratay, A.J. Glowacki, S.C. Balmert, A.P. Acharya, J. Polat, L.P. Andrews, M.V. Fedorchak, J.S. Schuman, D.A.A. Vignali, S.R. Little, Treg-recruiting microspheres prevent inflammation in a murine model of dry eye disease, J Control Release, 258 (2017) 208-217. [43] A.J. Glowacki, S. Yoshizawa, S. Jhunjhunwala, A.E. Vieira, G.P. Garlet, C. Sfeir, S.R. Little, Prevention of inflammationmediated bone loss in murine and canine periodontal disease via recruitment of regulatory lymphocytes, ProcNatl Acad Sci U S A, 110 (2013) 18525-18530.

[44] T.T. Tang, J. Yuan, Z.F. Zhu, W.C. Zhang, H. Xiao, N. Xia, X.X. Yan, S.F. Nie, J. Liu, S.F. Zhou, J. J. Li, R. Yao, M.Y. Liao, X. Tu, Y.H. Liao, X. Cheng, Regulatory T cells ameliorate cardiac remodeling after myocardial infarction, Basic Res Cardiol, 107 (2012) 232.

[45] P. Krishnamurthy, J. Rajasingh, E. Lambers, G. Qin, D.W. Losordo, R. Kishore, IL- 10 inhibits inflammation and attenuates left ventricular remodeling after myocardial infarction via activation of STAT3 and suppression of HuR, Circ Res, 104 (2009) e9-18.

[46] U. Hofmann, S. Knorr, B. Vogel, J. Weirather, A. Frey, G. Ertl, S. Frantz, Interleukin- 13 deficiency aggravates healing and remodeling in male mice after experimental myocardial infarction, Circ Heart Fail, 7 (2014) 822-830.

[47] M. Koeppen, J.W. Lee, S.W. Seo, K.S. Brodsky, S. Kreth, I.V. Yang, P.M. Buttrick, T. Eckle, H.K. Eltzschig, Hypoxiainducible factor 2-alpha-dependent induction of amphiregulin dampens myocardial ischemia-reperfusion injury, Nat Commun, 9 (2018) 816.

[48] Y. Wang, K. Dembowsky, E. Chevalier, P. Stuve, M. Korf-Klingebiel, M. Lochner, L.C. Napp, H. Frank, E. Brinkmann, A. Kanwischer, J. Bauersachs, M. Gyongyosi, T. Sparwasser, K.C. Wollert, C-X-C Motif Chemokine Receptor 4 Blockade Promotes Tissue Repair After Myocardial Infarction by Enhancing Regulatory T Cell Mobilization and Immune-Regulatory Function, Circulation, 139 (2019) 1798-1812.

[49] C.J. Teng, J. Luo, R.C. Chiu, D. Shum-Tim, Massive mechanical loss of microspheres with direct intramyocardial injection in the beating heart: implications for cellular cardiomyoplasty, J Thorac Cardiovasc Surg, 132 (2006) 628-632.

[50] M.A. Borrelli, H.R. Turnquist, S.R. Little, Biologies and their delivery systems: Trends in myocardial infarction, Adv Drug Deliv Rev, 173 (2021) 181-215.

[51] T. Yokochi, Y. Inoue, J. Yokoo, Y. Kimura, N. Kato, Retention of bacterial lipopolysaccharide at the site of subcutaneous injection, Infect Immun, 57 (1989) 1786-1791.

[52] E. Tous, B. Purcell, J.L. Ifkovits, J. A. Burdick, Injectable acellular hydrogels for cardiac repair, J Cardiovasc Transl Res, 4 (2011) 528-542.

[53] K. Huang, E.W. Ozpinar, T. Su, J. Tang, D. Shen, L. Qiao, S. Hu, Z. Li, H. Liang, K. Mathews, V. Scharf, D.O. Freytes, K Cheng, An off-the-shelf artificial cardiac patch improves cardiac repair after myocardial infarction in rats and pigs, Sci Transl Med, 12 (2020).

[54] A.N. Steele, M.J. Paulsen, H. Wang, L.M. Stapleton, H.J. Lucian, A. Eskandari, C.E. Hironaka, J.M. Farry, S.W. Baker, A.D. Thakore, K.J. Jaatinen, Y. Tada, M.J. Hollander, K.M. Williams, A. J. Seymour, K.P. Totherow, A.C. Yu, J.R. Cochran, E.A. Appel, Y.J. Woo, Multiphase catheter-injectable hydrogel enables dual-stage protein-engineered cytokine release to mitigate adverse left ventricular remodeling following myocardial infarction in a small animal model and a large animal model, Cytokine, 127 (2020) 154974.

[55] J. A. Burdick, R.L. Mauck, S. Gerecht, To Serve and Protect: Hydrogels to Improve Stem Cell-Based Therapies, Cell Stem Cell, 18 (2016) 13-15.

[56] J. Li, D.J. Mooney, Designing hydrogels for controlled drug delivery, Nat Rev Mater, 1 (2016).

[57] A.J. Rufaihah, N.A. Johari, S.R. Vaibavi, M. Plotkin, D.T. Di Thien, T. Kofidis, D. Seliktar, Dual delivery of VEGF and ANG-1 in ischemic hearts using an injectable hydrogel, Acta Biomater, 48 (2017) 58-67.

[58] H.K. Awada, N.R. Johnson, Y. Wang, Sequential delivery of angiogenic growth factors improves revascularization and heart function after myocardial infarction, J Control Release, 207 (2015) 7-17.

[59] N. Yu, X. Li, W. Song, D. Li, D. Yu, X. Zeng, M. Li, X. Leng, X. Li, CD4(+)CD25 (+)CD127 (low/-) T cells: a more specific Treg population in human peripheral blood, Inflammation, 35 (2012) 1773-1780.

[60] L. Li, Q.G. Chao, L.Z. Ping, C. Xue, Z.Y. Xia, D. Qian, H. Shi-ang, The prevalence of FOXP3+ regulatory T-cells in peripheral blood of patients with NSCLC, Cancer Biother Radiopharm, 24 (2009) 357-367.

[61] A. Jager, V.K. Kuchroo, Effector and regulatory T-cell subsets in autoimmunity and tissue inflammation, Scand J Immunol, 72 (2010) 173-184. [62] D.V. Sawant, D.A. Vignali, Once a Treg, always a Treg?, Immunol Rev, 259 (2014) 173-191.

[63] R. Sharir, J. Semo, S. Shimoni, T. Ben-Mordechai, N. Landa-Rouben, S. Maysel- Auslender, A. Shaish, M. Entin-Meer, G. Keren, J. George, Experimental myocardial infarction induces altered regulatory T cell hemostasis, and adoptive transfer attenuates subsequent remodeling, PLoS One, 9 (2014) el 13653.

[64] W. Chen, W. Jin, N. Hardegen, K.J. Lei, L. Li, N. Marinos, G. McGrady, S.M. Wahl, Conversion of peripheral CD4+CD25- naive T cells to CD4+CD25+ regulatory T cells by TGF-beta induction of transcription factor Foxp3, J Exp Med, 198 (2003) 1875-1886.

[65] S. Jhunjhunwala, S.C. Balmert, G. Raimondi, E. Dons, E.E. Nichols, A.W. Thomson, S.R. Little, Controlled release formulations of IL-2, TGF-betal and rapamycin for the induction of regulatory T cells, J Control Release, 159 (2012) 78-84. [66] E.J. Bassin, A.R. Buckley, J.D. Piganelli, S.R. Little, TRI microparticles prevent inflammatory arthritis in a collageninduced arthritis model, PLoS One, 15 (2020) e0239396.

[67] S.C. Balmert, C. Donahue, J.R. Vu, G. Erdos, L.D. Falo, Jr., S.R. Little, In vivo induction of regulatory T cells promotes allergen tolerance and suppresses allergic contact dermatitis, J Control Release, 261 (2017) 223-233.

[68] M.L. Ratay, S.C. Balmert, A.P. Acharya, A.C. Greene, T. Meyyappan, S.R. Little, TRI Microspheres prevent key signs of dry eye disease in a murine, inflammatory model, Sci Rep, 7 (2017) 17527.

[69] J.D. Fisher, S.C. Balmert, W. Zhang, R. Schweizer, J.T. Schnider, C. Komatsu, L. Dong, V.E. Erbas, J.V. Unadkat, A.M. Aral, A.P. Acharya, Y. Kulahci, H.R. Tumquist, A.W. Thomson, M.G. Solari, V.S. Gorantla, S.R. Little, Treg-inducing microparticles promote donor-specific tolerance in experimental vascularized composite allotransplantation, Proc Natl Acad Sci U S A, 116 (2019) 25784-25789.

[70] D.K. Reichenbach, V. Schwarze, B.M. Matta, V. Tkachev, E. Lieberknecht, Q. Liu, B.H. Koehn, D. Pfeifer, P.A. Taylor, G. Prinz, H. Dierbach, N. Shekel, Y. Beck, M. Wamcke, T. Junt, A. Schmitt-Graeff, S. Nakae, M. Folio, T. Wertheimer, L. Schwab, J. Devlin, S.C. Watkins, J. Duyster, J.L. Ferrara, H.R. Tumquist, R. Zeiser, B.R. Blazar, The IL-33/ST2 axis augments effector T-cell responses during acute GVHD, Blood, 125 (2015) 3183-3192.

[71] B.M. Matta, J.M. Lott, L.R. Mathews, Q. Liu, B.R. Rosborough, B.R. Blazar, H.R. Tumquist, IL-33 is an unconventional Alarmin that stimulates IL-2 secretion by dendritic cells to selectively expand IL-33R/ST2+ regulatory T cells, J Immunol, 193 (2014) 4010-4020.

[72] T. Li, Z. Zhang, J.G. Bartolacci, G.K. Dwyer, Q. Liu, L.R. Mathews, M. Velayutham, A.S. Roessing, Y.C. Lee, H. Dai, S. Shiva, M.H. Oberbamscheidt, J.L. Dziki, S.J. Mullet, S.G. Wendell, J.D. Wilkinson, S.A. Webber, M. Wood-Trageser, S.C. Watkins, A. J. Demetris, G.S. Hussey, S.F. Badylak, H.R. Tumquist, Graft IL-33 regulates infiltrating macrophages to protect against chronic rejection, J Clin Invest, 130 (2020) 5397-5412.

[73] H.R. Tumquist, Z. Zhao, B.R. Rosborough, Q. Liu, A. Castellaneta, K. Isse, Z. Wang, M. Lang, D.B. Stolz, X.X. Zheng, A.J. Demetris, F.Y. Liew, K.J. Wood, A.W. Thomson, IL-33 expands suppressive CD1 lb+ Gr-l(int) and regulatory T cells, including ST2L+ Foxp3+ cells, and mediates regulatory T cell-dependent promotion of cardiac allograft survival, J Immunol, 187 (2011) 4598-4610.

[74] Q. Liu, G.K. Dwyer, Y. Zhao, H. Li, L.R. Mathews, A.B. Chakka, U.R. Chandran, J. A. Demetris, J.F. Alcorn, K.M. Robinson, L.A. Ortiz, B.R. Pitt, A.W. Thomson, M.H. Fan, T.R. Billiar, H.R. Turnquist, IL-33 -mediated IL-13 secretion by ST2+ Tregs controls inflammation after lung injury, JCI Insight, 4 (2019).

[75] C. Cayrol, J.P. Girard, Interleukin-33 (IL-33): A nuclear cytokine from the IL-1 family, Immunol Rev, 281 (2018) 154-168.

[76] J. Siede, A. Frohlich, A. Datsi, A.N. Hegazy, D.V. Varga, V. Holecska, H. Saito, S. Nakae, M. Lohning, IL-33 Receptor-Expressing Regulatory T Cells Are Highly Activated, Th2 Biased and Suppress CD4 T Cell Proliferation through IL-10 and TGFbeta Release, PLoS One, 11 (2016) e0161507.

[77] D. Burzyn, W. Kuswanto, D. Kolodin, J.L. Shadrach, M. Cerletti, Y. Jang, E. Sefik, T.G. Tan, A. J. Wagers, C. Benoist, D. Mathis, A special population of regulatory T cells potentiates muscle repair, Cell, 155 (2013) 1282-1295.

[78] W. Kuswanto, D. Burzyn, M. Panduro, K.K. Wang, Y.C. Jang, A.J. Wagers, C. Benoist, D. Mathis, Poor Repair of Skeletal Muscle in Aging Mice Reflects a Defect in Local, Interleukin-33 -Dependent Accumulation of Regulatory T Cells, Immunity, 44 (2016) 355-367.

[79] J. Schmitz, A. Owyang, E. Oldham, Y. Song, E. Murphy, T.K. McClanahan, G. Zurawski, M. Moshrefi, J. Qin, X. Li, D.M. Gorman, J.F. Bazan, R.A. Kastelein, IL-33, an interleukin- 1- like cytokine that signals via the IL-1 receptor-related protein

ST2 and induces T helper type 2-associated cytokines, Immunity, 23 (2005) 479-490. [80] J. Sanchez-Mas, A. Lax, C. Asensio-Lopez Mdel, M.J. Fernandez-Del Palacio, L. Caballero, G. Santarelli, J.L. Januzzi, D.A. Pascual-Figal, Modulation of IL-33/ST2 system in postinfarction heart failure: correlation with cardiac remodelling markers, Eur J Clin Invest, 44 (2014) 643- 651.

[81] P.T. Veeraveedu, S. Sanada, K. Okuda, H.Y. Fu, T. Matsuzaki, R. Araki, M. Yamato, K. Yasuda, Y. Sakata, T. Yoshimoto, T. Minamino, Ablation of IL-33 gene exacerbate myocardial remodeling in mice with heart failure induced by mechanical stress, Biochem Pharmacol, 138 (2017) 73-80.

[82] A. Gabryelska, P. Kuna, A. Antczak, P. Bialasiewicz, M. Panek, IL-33 Mediated Inflammation in Chronic Respiratory Diseases-Understanding the Role of the Member of IL-1 Superfamily, Front Immunol, 10 (2019) 692.

[83] R. Ghali, N.J. Habeichi, A. Kaplan, C. Tannous, E. Abidi, A. Bekdash, R. Farhat, H. Itani, A. Jurjus, G.W. Booz, Z. Mallat, F.A. Zouein, IL-33 induces type-2-cytokine phenotype but exacerbates cardiac remodeling post-myocardial infarction with eosinophil recruitment, worsened systolic dysfunction, and ventricular wall rupture, Clin Sci (Lond), 134 (2020) 1191- 1218. [84] N. Arpaia, J.A. Green, B. Moltedo, A. Arvey, S. Hemmers, S. Yuan, P.M. Treuting, A.Y. Rudensky, A Distinct Function of Regulatory T Cells in Tissue Protection, Cell, 162 (2015) 1078-1089.

[85] L.L. Wang, Y. Liu, J. J. Chung, T. Wang, A.C. Gaffey, M. Lu, C.A. Cavanaugh, S. Zhou, R. Kanade, P. Atluri, E.E. Morrisey, J.A. Burdick, Local and sustained miRNA delivery from an injectable hydrogel promotes cardiomyocyte proliferation and functional regeneration after ischemic injury, Nat Biomed Eng, 1 (2017) 983-992.

[86] M.H. Chen, L.L. Wang, J. J. Chung, Y.H. Kim, P. Atluri, J.A. Burdick, Methods To Assess Shear-Thinning Hydrogels for Application As Injectable Biomaterials, ACS Biomater Sci Eng, 3 (2017) 3146-3160.

[87] C.W. Chen, L.L. Wang, S. Zaman, J. Gordon, M.F. Arisi, C.M. Venkataraman, J. J. Chung, G. Hung, A.C. Gaffey, L.A. Spruce, H. Fazelinia, R.C. Gorman, S.H. Seeholzer, J.A. Burdick, P. Atluri, Sustained release of endothelial progenitor cell-derived extracellular vesicles from shear-thinning hydrogels improves angiogenesis and promotes function after myocardial infarction, Cardiovasc Res, 114 (2018) 1029-1040.

[88] M.H. Chen, J. J. Chung, J.E. Mealy, S. Zaman, E.C. Li, M.F. Arisi, P. Atluri, J.A. Burdick, Injectable Supramolecular Hydrogel/Microgel Composites for Therapeutic Delivery, Macromol Biosci, 19 (2019) el 800248.

[89] S.R. Eckhouse, B.P. Purcell, J.R. McGarvey, D. Lobb, C.B. Logdon, H. Doviak, J.W. O’Neill, J.A. Shuman, C.P. Novack, K.N. Zellars, S. Pettaway, R.A. Black, A. Khakoo, T. Lee, R. Mukheijee, J.H. Gorman, R.C. Gorman, J.A. Burdick, F.G. Spinale, Local hydrogel release of recombinant TIMP-3 attenuates adverse left ventricular remodeling after experimental myocardial infarction, Sci Transl Med, 6 (2014) 223ra221.

[90] B.P. Purcell, D. Lobb, M.B. Charati, S.M. Dorsey, R.J. Wade, K.N. Zellars, H. Doviak, S. Pettaway, C.B. Logdon, J.A. Shuman, P.D. Freels, J.H. Gorman, 3rd, R.C. Gorman, F.G. Spinale, J.A. Burdick, Injectable and bioresponsive hydrogels for on-demand matrix metalloproteinase inhibition, Nat Mater, 13 (2014) 653-661.

[91] B.P. Purcell, S.C. Barlow, P.E. Perreault, L. Freeburg, H. Doviak, J. Jacobs, A. Hoenes, K.N. Zellars, A.Y. Khakoo, T. Lee, J.A. Burdick, F.G. Spinale, Delivery of a matrix metalloproteinase-responsive hydrogel releasing TIMP-3 after myocardial infarction: effects on left ventricular remodeling, Am J Physiol Heart Circ Physiol, 315 (2018) H814-H825.

[92] H. Dai, P. Lan, D. Zhao, K. Abou-Daya, W. Liu, W. Chen, A. J. Friday, A.L. Williams, T. Sun, J. Chen, W. Chen, S. Mortin-Toth, J.S. Danska, C. Wiebe, P. Nickerson, T. Li, L.R. Mathews, H.R. Tumquist, M.L. Nicotra, S. Gingras, E. Takayama, H. Kubagawa, M.J. Shlomchik, M.H. Oberbamscheidt, X.C. Li, F.G. Lakkis, PIRs mediate innate myeloid cell memory to nonself MHC molecules, Science, 368 (2020) 1122-1127.

[93] A.W. Thomson, H.R. Tumquist, A.F. Zahorchak, G. Raimondi, Tolerogenic dendritic cell- regulatory T-cell interaction and the promotion of transplant tolerance, Transplantation, 87 (2009) S86-90.

[94] J.D. Fisher, W. Zhang, S.C. Balmert, A.M. Aral, A.P. Acharya, Y. Kulahci, J. Li, H.R. Turnquist, A.W. Thomson, M.G. Solari, V.S. Gorantla, S.R. Little, In situ recruitment of regulatory T cells promotes donor-specific tolerance in vascularized composite allotransplantation, Sci Adv, 6 (2020) eaax8429.

[95] G.K. Dwyer, L.M. D’Cruz, H.R. Tumquist, Emerging Functions of IL-33 in Homeostasis and Immunity, Annu Rev Immunol, 40 (2022) 15-43.

[96] M.C. Fishbein, D. Maclean, P.R. Maroko, Experimental myocardial infarction in the rat: qualitative and quantitative changes during pathologic evolution, Am J Pathol, 90 (1978) 57- 70.

[97] J. Takagawa, Y. Zhang, M.L. Wong, R.E. Sievers, N.K. Kapasi, Y. Wang, Y. Yeghiazarians, R.J. Lee, W. Grossman, M.L. Springer, Myocardial infarct size measurement in the mouse chronic infarction model: comparison of area- and length-based approaches, J Appl Physiol (1985), 102 (2007) 2104-2111.

[98] G. Ren, L.H. Michael, M.L. Entman, N.G. Frangogiannis, Morphological characteristics of the microvasculature in healing myocardial infarcts, J Histochem Cytochem, 50 (2002) 71- 79.

[99] U. Amit, D. Kain, A. Wagner, A. Sahu, Y. Nevo-Caspi, N. Gonen, N. Molotski, T. Konfino, N. Landa, N. Naftali-Shani, G. Blum, E. Merquiol, D. Karo-Atar, Y. Kanfi, G. Paret, A. Munitz, H.Y. Cohen, E. Ruppin, S. Hannenhalli, J. Leor, New Role for Interleukin- 13 Receptor alphal in Myocardial Homeostasis and Heart Failure, J Am Heart Assoc, 6 (2017).

[100] T. Hong, S. Li, X. Guo, Y. Wei, J. Zhang, X. Su, M. Zhou, H. Jin, Q. Miao, L. Shen, M. Zhu, B. He, IL- 13 Derived Type 2 Innate Lymphocytes Ameliorates Cardiomyocyte Apoptosis Through STAT3 Signaling Pathway, Front Cell Dev Biol, 9 (2021) 742662.

[101] C. Berasain, M.A. Avila, Amphiregulin, Semin Cell Dev Biol, 28 (2014) 31-41.

[102] E. Ubil, J. Duan, I.C. Pillai, M. Rosa-Garrido, Y. Wu, F. Bargiacchi, Y. Lu, S. Stanbouly, J. Huang, M. Rojas, T.M. Vondriska, E. Stefani, A. Deb, Mesenchymal-endothelial transition contributes to cardiac neovascularization, Nature, 514 (2014) 585-590.

[103] S.A. Bageghni, K.E. Hemmings, N. Zava, C.P. Denton, K.E. Porter, J.F.X. Ainscough, M.J. Drinkhill, N.A. Turner, Cardiac fibroblast-specific p38alpha MAP kinase promotes cardiac hypertrophy via a putative paracrine interleukin-6 signaling mechanism, FASEB J, 32 (2018) 4941-4954.

[104] S. Jhunjhunwala, L.C. Chen, E.E. Nichols, A.W. Thomson, G. Raimondi, S.R. Little, All-trans retinoic acid and rapamycin synergize with transforming growth factor-betal to induce regulatory T cells but confer different migratory capacities, J Leukoc Biol, 94 (2013) 981-989.

[105] M. Nomura, S.J. Hodgkinson, G.T. Tran, N.D. Verma, C. Robinson, K.M. Plain, R. Boyd, B.M. Hall, Cytokines affecting CD4(+)T regulatory cells in transplant tolerance. II. Interferon gamma (IFN-gamma) promotes survival of alloantigenspecific CD4(+)T regulatory cells, Transpl Immunol, 42 (2017) 24-33.

[106] M. Hill, P. Thebault, M. Segovia, C. Louvet, G. Beriou, G. Tilly, E. Merieau, I. Anegon, E. Chiffoleau, M.C. Cuturi, Cell therapy with autologous tolerogenic dendritic cells induces allograft tolerance through interferon-gamma and epsteinbarr virus-induced gene 3, Am J Transplant, 11 (2011) 2036-2045.

[107] M. Karim, C.I. Kingsley, A.R. Bushell, B.S. Sawitzki, K.J. Wood, Alloantigen-induced CD25+CD4+ regulatory T cells can develop in vivo from CD25-CD4+ precursors in a thymusindependent process, J Immunol, 172 (2004) 923-928.

[108] A. Frank, M. Bonney, S. Bonney, L. Weitzel, M. Koeppen, T. Eckle, Myocardial ischemia reperfusion injury: from basic science to clinical bedside, Semin Cardiothorac Vase Anesth, 16 (2012) 123-132.

[109] B. Ibanez, S. James, S. Agewall, M.J. Antunes, C. Bucciarelli-Ducci, H. Bueno, A.L.P. Caforio, F. Crea, J. A. Goudevenos, S. Halvorsen, G. Hindricks, A. Kastrati, M.J. Lenzen, E. Prescott, M. Roffi, M. Valgimigli, C. Varenhorst, P. Vranckx, P. Widimsky, E.S.C.S.D. Group, 2017 ESC Guidelines for the management of acute myocardial infarction in patients presenting with ST-segment elevation: The Task Force for the management of acute myocardial infarction in patients presenting with ST-segment elevation of the European Society of Cardiology (ESC), Eur Heart J, 39 (2018) 119-177.

[110] J. Yu, K.L. Christman, E. Chin, R.E. Sievers, M. Saeed, R.J. Lee, Restoration of left ventricular geometry and improvement of left ventricular function in a rodent model of chronic ischemic cardiomyopathy, J Thorac Cardiovasc Surg, 137 (2009) 180-187.

[111] M.V. Kolk, D. Meyberg, T. Deuse, K.R. Tang-Quan, R.C. Robbins, H. Reichenspurner, S. Schrepfer, LAD-ligation: a murine model of myocardial infarction, J Vis Exp, (2009). [112] J.E. Mealy, J. J. Chung, H.H. Jeong, D. Issadore, D. Lee, P. Atluri, J. A. Burdick, Injectable Granular Hydrogels with Multifunctional Properties for Biomedical Applications, Adv Mater, 30 (2018) el705912.

[113] J. J. Chung, J. Han, L.L. Wang, M.F. Arisi, S. Zaman, J. Gordon, E. Li, S.T. Kim, Z. Tran, C.W. Chen, A.C. Gaffey, J.A. Burdick, P. Atluri, Delayed delivery of endothelial progenitor cell-derived extracellular vesicles via shear thinning gel improves postinfarct hemodynamics, J Thorac Cardiovasc Surg, 159 (2020) 1825-1835 el822.

[114] J.G. Altin, E.K. Sloan, The role of CD45 and CD45 -associated molecules in T cell activation, Immunol Cell Biol, 75 (1997) 430-445.

[115] X. Zhou, S.L. Bailey-Bucktrout, L.T. Jeker, C. Penaranda, M. Martinez-Llordella, M. Ashby, M. Nakayama, W. Rosenthal, J.A. Bluestone, Instability of the transcription factor Foxp3 leads to the generation of pathogenic memory T cells in vivo, Nat Immunol, 10 (2009) 1000-1007.

[116] P.D. Bittner-Eddy, L.A. Fischer, M. Costalonga, Cre-loxP Reporter Mouse Reveals Stochastic Activity of the Foxp3 Promoter, Front Immunol, 10 (2019) 2228.

[117] L.L. Wang, J. J. Chung, E.C. Li, S. Uman, P. Atluri, J.A. Burdick, Injectable and protease- degradable hydrogel for siRNA sequestration and triggered delivery to the heart, J Control Release, 285 (2018) 152-161. [118] K.A. Wegner, A. Keikhosravi, K.W. Eliceiri, C.M. Vezina, Fluorescence of Picrosirius Red Multiplexed With Immunohistochemistry for the Quantitative Assessment of Collagen in Tissue Sections, J Histochem Cytochem, 65 (2017) 479-490.

[119] S.B. Weisser, K.W. McLarren, E. Kuroda, L.M. Sly, Generation and characterization of murine alternatively activated macrophages, Methods Mol Biol, 946 (2013) 225-239.

[120] N. Makita, Y. Hizukuri, K. Yamashiro, M. Murakawa, Y. Hayashi, IL- 10 enhances the phenotype of M2 macrophages induced by IL-4 and confers the ability to increase eosinophil migration, Int Immunol, 27 (2015) 131-141.

[121] M. Wang, Y. Wang, A. Abarbanell, J. Tan, B. Weil, J. Herrmann, D.R. Meldrum, Both endogenous and exogenous testosterone decrease myocardial STAT3 activation and SOCS3 expression after acute ischemia and reperfusion, Surgery, 146 (2009) 138-144.

[122] R.D. Patten, I. Pourati, M.J. Aronovitz, A. Alsheikh-Ali, S. Eder, T. Force, M.E. Mendelsohn, R.H. Karas, 17 Betaestradiol differentially affects left ventricular and cardiomyocyte hypertrophy following myocardial infarction and pressure overload, J Card Fail, 14 (2008) 245-253. [123] M.A. Cavasin, Z. Tao, S. Menon, X.P. Yang, Gender differences in cardiac function during early remodeling after acute myocardial infarction in mice, Life Sci, 75 (2004) 2181- 2192.

[124] Q. Chen, R. Williams, C.L. Healy, C.D. Wright, S.C. Wu, T.D. O’Connell, An association between gene expression and better survival in female mice following myocardial infarction, J Mol Cell Cardiol, 49 (2010) 801-811.

[125] S.J. Robertson, P. Lemire, H. Maughan, A. Goethel, W. Turpin, L. Bedrani, D.S. Guttman, K. Croitoru, S.E. Girardin, D.J. Philpott, Comparison of Co-housing and Littermate Methods for Microbiota Standardization in Mouse Models, Cell Rep, 27 (2019) 1910-1919 el912.

Claims

WHAT IS CLAIMED IS:

1. A hydrogel comprising a sustained release microparticle, wherein the sustained release microparticle comprises a therapeutic agent.

2. The hydrogel of claim 1, wherein the hydrogel is a shear-thinning hydrogel.

3. The hydrogel of claim 2, wherein the shear-thinning hydrogel comprises hyaluronic acid macromers functionalized with adamantane (HA-AD), cyclodextrin (HA-CD), or a combination thereof.

4. The hydrogel of claim 3, wherein the hydrogel comprises HA-AD at a concentration from about 1% to about 30% wt/vol.

5. The hydrogel of claim 4, wherein the hydrogel comprises HA-AD at a concentration of about 4% wt/vol.

6. The hydrogel of claim 3, wherein the hydrogel comprises HA-CD at a concentration from about 1% to about 30% wt/vol.

7. The hydrogel of claim 6, wherein the hydrogel comprises HA-CD at a concentration of about 4% wt/vol.

8. The hydrogel of claim 1, wherein the hydrogel comprises hyaluronic acid at a concentration of 4% wt/vol.

9. The hydrogel of claim 2, wherein the shear-thinning hydrogel exhibits a lower diffusivity after curing.

10. The hydrogel of claim 9, wherein the hydrogel retains microparticles at a delivery site.

11. The hydrogel of claim 1, wherein the hydrogel is a thermoresponsive hydrogel.

12. The hydrogel of claim 11, wherein the thermoresponsive hydrogel comprises a PEG (Polethylene Glycol), NIPAAm (N-Isopropylacrylamide), or a combination thereof.

13. The hydrogel of claim 12, wherein the thermoresponsive hydrogel comprises about 100 mg of NIPAAm.

14. The hydrogel of claim 11, wherein the thermoresponsive hydrogel has a lower critical solution temperature below 37°C.

15. The hydrogel of claim 14, wherein the thermoresponsive hydrogel has a lower critical solution temperature below 20°C.

16. The hydrogel of claim 15, wherein the thermoresponsive hydrogel reversibly solidifies into a gel at a temperature above the lower critical solution temperature.

17. The hydrogel of claim 1, wherein the microparticle is present in the hydrogel at a concentration from about 10 mg/mL up to about 100 mg/mL.

18. The hydrogel claim 1, wherein the microparticle has a diameter up to about 100 pm.

19. The hydrogel of claim 18, wherein the microparticle has a diameter larger than about 1 pm.

20. The hydrogel of claim 1, wherein the therapeutic agent comprises a Treg cell factor.

21. The hydrogel of claim 20, wherein the Treg cell factor comprises a regulatory T cell stimulatory factor.

22. The hydrogel of claim 20, wherein the Treg cell factor comprises a T cell chemoattractant factor.

23. The hydrogel of claim 20, wherein the Treg cell factor is selected from the group comprising CCL22, IL2, TGF-P, IL33, rapamycin, IL13, amphiregulin, and a combination thereof.

24. The hydrogel of claim 1, further comprising a second sustained release microparticle, wherein the second sustained release microparticle comprises a second therapeutic agent.

25. The hydrogel of claim 24, wherein the second therapeutic agent comprises a Treg cell factor.

26. The hydrogel of claim 25, wherein the Treg cell factor is selected from the group comprising CCL22, IL2, TGF-P, IL33, rapamycin, IL13, amphiregulin, and a combination thereof.

27. The hydrogel of claim 1, further comprising an antiproliferative agent, an immunosuppressant drug, a non-thrombogenic substance, an anti-adhesive substance, and a combination thereof.

28. A pharmaceutical composition comprising the hydrogel of any one of claims 1-27 and a pharmaceutical carrier comprising at least one excipient component.

29. The pharmaceutical composition of claim 28, wherein the at least one excipient component comprises a buffering agent, an antioxidant, an alkali salt, a preservative, or a combination thereof.

30. A method of treatment for ischemic myocardial infarction in a subject in need thereof, comprising administering an effective amount of the hydrogel of any one of claims 1- 27 or the composition of claim 28 or 29.

31. The method of claim 30, wherein the hydrogel or composition is administered into the affected tissue.

32. The method of claim 31, wherein the hydrogel or composition is administered at an amount from about 0.01 pg to about 100 g per kg of body weight of the subject.

33. The method of claim 32, wherein the hydrogel or composition is administered once or more daily, weekly, monthly, or yearly.

34. The method of claim 33, wherein the hydrogel or composition is orally, transdermally, topically, pulmonary inhalation, or parenterally administered.

35. The method of claim 34, wherein the parenterally administered hydrogel or composition is injected into an infarct or infarct bordering region.

36. A composition comprising:

(a) a hydrogel; and

(b) a sustained release microparticle, wherein the sustained release microparticle comprises a first therapeutic agent.

37. The composition of claim 36, wherein the hydrogel is a thermoresponsive hydrogel.

38. The composition of claim 37, wherein the thermoresponsive hydrogel comprises a PEG (Polyethylene Glycol) and NIPAAm (N-Isopropylacrylamide).

39. The composition of claim 36, wherein the hydrogel is a shear-thinning hydrogel.

40. The composition of claim 39, wherein the shear-thinning hydrogel comprises hyaluronic acid macromers functionalized with adamantane (HA-AD) and cyclodextrin (HA-CD).

41. The composition of claim 36, wherein the hydrogel further comprises a second therapeutic agent.

42. The composition of claim 36, wherein the first therapeutic agent comprises a Treg cell factor.

43. The composition of claim 42, wherein the Treg cell factor comprises a regulatory T cell stimulatory factor.

44. The composition of claim 42, wherein the Treg cell factor comprises a T cell chemoattractant factor.

45. The composition of claim 42, wherein the Treg cell factor is selected from the group consisting of CCL22, IL2, and TGF-P, and combinations thereof.

46. A method for treating ischemic myocardial infarction in a subject, wherein the method comprises administering to the subject a composition comprising:

(a) a hydrogel; and

47. A method for treating inflammation of a target tissue in a subject in need thereof, wherein the method comprises administering to the subject a composition comprising: (a) a hydrogel; and

(b) a first sustained release microparticle, wherein the first sustained release microparticle comprises a first therapeutic agent.

48. The method of claim 47, wherein the composition is delivered to the target tissue in an amount effective to:

(a) reduce myeloid cell infiltration;

(b) shorten myeloid cell infiltration duration time;

(c) alter infiltrating myeloid cell phenotype towards a pro-repair phenotype; or

(d) mitigate spreading of inflammation.

49. The method of claim 46 or 47, wherein the hydrogel is a thermoresponsive hydrogel.

50. The method of claim 49, wherein the thermoresponsive hydrogel comprises a PEG (Polyethylene Glycol) and NIPAAm (N-Isopropylacrylamide).

51. The method of claim 46 or 47, wherein the hydrogel is a shear-thinning hydrogel.

52. The method of claim 51, wherein the shear-thinning hydrogel comprises hyaluronic acid macromers functionalized with adamantane (HA-AD) and cyclodextrin (HA-CD).

53. The method of claim 52, wherein the hydrogel further comprises a second therapeutic agent.

54. The method of claim 46 or 47, wherein the first therapeutic agent comprises a Treg cell factor.

55. The method of claim 54, wherein the soluble Treg cell factor comprises a regulatory T cell stimulatory factor.

56. The method of claim 54, wherein the soluble Treg cell factor comprises a T cell chemoattractant factor.

57. The method of claim 54, wherein the soluble Treg cell factor is selected from the group consisting of CCL22, IL2, and TGF- P, and combinations thereof.