[go: up one dir, main page]

WO2025091007A1 - Photo-responsive therapeutic encapsulation compositions and devices - Google Patents

Photo-responsive therapeutic encapsulation compositions and devices Download PDF

Info

Publication number
WO2025091007A1
WO2025091007A1 PCT/US2024/053211 US2024053211W WO2025091007A1 WO 2025091007 A1 WO2025091007 A1 WO 2025091007A1 US 2024053211 W US2024053211 W US 2024053211W WO 2025091007 A1 WO2025091007 A1 WO 2025091007A1
Authority
WO
WIPO (PCT)
Prior art keywords
alginate
molecular weight
average molecular
methacrylation
efficiency
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2024/053211
Other languages
French (fr)
Inventor
Omid Veiseh
Martha FOWLER
Shalini Pandey
Alvaro Moreno LOZANO
Julian Krause
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
William Marsh Rice University
Original Assignee
William Marsh Rice University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by William Marsh Rice University filed Critical William Marsh Rice University
Publication of WO2025091007A1 publication Critical patent/WO2025091007A1/en
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/738—Cross-linked polysaccharides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin

Definitions

  • the present disclosure relates to the fields of biology, medicine, bioengineering and medical devices. More particular, it relates to the development and use of hydrogels designed to deliver therapeutic agents to a subject.
  • the hydrogels comprise an alginate methacrylate polymer.
  • Encapsulation within semi-permeable hydrogels represents a local immuno-i solation strategy for many therapies without the need for systemic immunosuppression (Chang, 1964; Lim and Sun, 1980).
  • the hydrogel sphere may facilitate the diffusion of nutrients necessary for cell function while excluding immune cells that would reject the foreign cells.
  • Alginate spheres are one of the most widely investigated cell encapsulation materials because this anionic polysaccharide forms a hydrogel in the presence of divalent cations under cell friendly conditions. This natural co-polymer can then exhibit differential physical properties depending on the ratio and sequential arrangement of mannuronic and guluronic acid residues, the molecular weight, the concentration and the divalent cations used to form the gels (Strand et al., 2017).
  • alginate with a high guluronic-block content has a higher binding affinity for barium ions and will form a tighter, more stable network compared with the same alginate type crosslinked with calcium (Haug and Smidsrod, 1970).
  • electrostatic complexation of a positively charged polymer to the negatively charged alginate surface can provide an outer layer to reduce sphere porosity and increase sphere stability (Kollmer el al., 2015).
  • a final outer alginate layer or chemical modification of the polycation used for coating has been investigated to reduce the positive surface charge density of the sphere (Kollmer et al., 2015; Mooranian etal., 2016; Kleinberger etal., 2016).
  • Many alginate sphere formulations are produced with variations in the alginate concentration, the crosslinking ion or the inclusion or exclusion of a polycation layer.
  • composition comprising:
  • hydrogel system wherein said system comprises at least one of A, B or C, wherein:
  • A is a photo-responsive alginate, wherein the molecular weight is less than 95 kDa and wherein the methacrylation efficiency is from about 15% to about 95%;
  • B is a photo-responsive alginate, wherein the molecular weight is from about 55 kDa to about 240 kDa and wherein the methacrylation efficiency is from about 1% to about 65%; and C is a photo-responsive alginate, wherein the molecular weight is from about 180 kDa to about 320 kDa and wherein the methacrylation efficiency from about 1% to about 25%; and
  • the hydrogel system comprises a monomer of the formula: wherein n and m are at least I .
  • n is 1-5. In some embodiments, m is 1-5. In some aspects, n and m are the same.
  • the present disclosure provides a drug delivery device comprising:
  • hydrogel system wherein said system comprises at least one of A, B or C, wherein:
  • A is a photo-responsive alginate, wherein the molecular weight is less than 95 kDa and wherein the methacrylation efficiency is from about 15% to about 95%;
  • B is a photo-responsive alginate, wherein the molecular weight is from about 55 kDa to about 240 kDa and wherein the methacrylation efficiency is from about 1% to about 65%, and
  • C is a photo-responsive alginate, wherein the molecular weight is from about 180 kDa to about 320 kDa and wherein the methacrylation efficiency from about 1% to about 25%, and
  • the methacryl ati on efficiency of A is about 20%, about 46%, about 60% or about 90%.
  • the methacrylation efficiency of B is about 5%, about 10%, about 20%, about 46% or about 60%.
  • the methacrylation efficiency of C is about 5%, about 10% or about 20%.
  • A is combined with B at a ratio of about 1:0 to about 0: 1; A is combined with C at a ratio of about 1 :0 to about 0: 1; or B is combined with C at a ratio of about 1 :0 to about 0:1.
  • A is combined with Bat a ratio of about 7:3 to about 3:7, A is combined with C at a ratio of about 7:3 to about 3:7; or B is combined with C at a ratio of about 7:3 to about 3:7.
  • A is combined with B at a ratio of about 7:3; / ⁇ is combined with Cat a ratio of about 7:3; or Bis combined with Cat a ratio of about 7:3.
  • A is combined with B at a ratio of about 6:4; A is combined with C at a ratio of about 6:4; or Bis combined with Cat a ratio of about 6:4.
  • A is combined with Bat a ratio of about 5:5: A is combined with Cat a ratio of about 5:5; or Bis combined with Cat a ratio of about 5:5.
  • A is combined with Bat a ratio of about 4:6; A is combined with Cat a ratio of about 4:6; or Bis combined with Cat a ratio of about 4:6.
  • A is combined with B at a ratio of about 3:7, A is combined with C at a ratio of about 3:7; or Bis combined with Cat a ratio of about 3:7.
  • the therapeutic agent comprises an engineered cell or protein, a secreted protein, a hormone, a cytokine, an antibody, an enzyme or a peptide. In certain embodiments, the therapeutic agent comprises an engineered cell
  • the present disclosure provides a method of treating a disease or disorder in a patient in need thereof comprising administering to the patient a device or composition described herein.
  • FIG. 1. Alginate methacrylate synthesis
  • FIG. 2 Alginate methacrylate NMR
  • FIG. 3. AIM A formulation gelation assay
  • FIG. 4. A1MA formulation hydrogel shape retention
  • FIG. 5 Absolute weight change values of AIM A formulations in solution after two weeks
  • FIG. 6 ARPE-19 cell viability of A1MA base formulations
  • FIG. 7 Mechanical strength of AIM A formulations
  • FIG. 8 Molding platform for A1MA formulation of varying shapes and sizes [00028] FIG. 9. PDMS molding device
  • FIG. 10 Programmable RPE-VEGF-C cell therapy enables stable and local production with inducible safety switch for termination.
  • a Overall schematic of engineered RPE-VEGF-C cells with inducible small molecule for cell death
  • b VEGF-C production of VEGF-C engineered (RPE-VEGF-C) and non-engineered cell lines over 24 hours in culture
  • c VEGF-C production over time in RPE-VEGFC cell lines in culture
  • d schematic of in vivo experiments of RPE-VEGF-C with and without engineered kill switch
  • e fluorescence imaging of cell viability
  • f in vivo VEGF-C production over 24 hours
  • g RPE-VEGF-C viability and production with engineered kill switch at 10 pM, IpM, lOOnM, lOnM, InM, O.lnM, O.OlnM, and OnM of small molecule activator
  • h fluorescence imaging of cell viability
  • FIG. 11 Development of A1MA library and high-throughput screen to enable leading materials for RPE-VEGFC cell therapy, a, screening strategy for A1MA library, b, shape retention of ionically crosslinked alginate with barium chloride, and A1MA at different methacrylation efficiencies upon UV crosslinking, c, gelation assay with Rhodamine B loaded A1MA formulation samples, d, mechanical strength of A1MA library formulations, e, AUC of weight alteration, f, VEGF-c diffusion in A1MA gels after 24 hours.
  • compositions of matter comprising photo-responsive library platforms that can be fabricated into devices for management of diseases.
  • the compositions and/or devices described herein are hydrogels.
  • the hydrogels are alginate methacrylate formulations that are crosslinkable, mechanically stable, permeable and support cell viability.
  • the present disclosure describes a library of alginate methacrylate (A1MA) synthesized by reacting sodium alginates of different molecular weights (UP VLVG with molecular weight ⁇ 75 kDa, SLG-20 with molecular weight 75 - 220 kDa and SLG-100 with molecular weight 200-300 kDa) with 2-aminoethyl methacrylate (AEMA) and having different methacrylate efficiency.
  • A1MA formulations may utilize the nomenclature described in Table 1 below.
  • the Al MA formulations may be combined at ratios of 1 :0, 7:3, 6:4, 5:5, 4:6, 3:7 or 0: 1. in preferred embodiments, the AIMA formulations may be combined at the ratios described by Table 2. These and other aspects of the disclosure are described in detail below. [00035] Table 2. Preferred AIMA Formulations [00036] DEFINITIONS
  • Heal refers to the partial or complete restoration of a cell or tissue containing a wound, e.g., a wound described herein.
  • Alginate is a collective term used to refer to linear polysaccharides formed from -D- mannuronate and a-L-guluronate in any MIG ratio, as well as salts and derivatives thereof.
  • alginate encompasses any polymer having the structure shown by Formula I, as well as salts thereof
  • Hydrogel refers to a substance formed when an organic polymer (natural or synthetic) is cross-linked via covalent, ionic, or hydrogen bonds to create a three-dimensional open-lattice structure which entraps water molecules to form a gel.
  • Biocompatible hydrogel refers to a polymer forms a gel which is not toxic to living cells and allows sufficient diffusion of oxygen and nutrients to the entrapped therapeutic agent to maintain viability.
  • subject refers to an individual bearing a wound and/or the recipient of a wound healing device described herein.
  • the subject may include a human (i.e., a. male or female of any age group, e.g..
  • a pediatric subject e.g., infant, child, adolescent
  • adult subject e.g., young adult, middle-aged adult, or senior adult
  • non-human animals for example, mammals (e.g., primates (e.g., cynomolgus monkeys, rhesus monkeys): commercially relevant mammals such as cattle, pigs, horses, sheep, goats, cats, and/or dogs) and birds (e.g., commercially relevant birds such as chickens, ducks, geese, and/or turkeys).
  • the animal is a mammal.
  • the animal may be a male or female and at any stage of development.
  • a non-human animal may be a transgenic animal.
  • treatment refers to reversing, alleviating, delaying the onset of or inhibiting the progress of one or more of a symptom, manifestation, or underlying cause of, e.g., a wound, e.g., as described herein. Treating may entail administering or applying the wound healing device described herein. In an embodiment, treating comprises reducing, reversing, alleviating, delaying the onset of, or inhibiting the worsening of a wound in a subject. In some embodiments, “treatment,” “treat,” and “treating” require that signs or symptoms of the disease, disorder, or condition have developed or have been observed. In other embodiments, treatment may be administered in the absence of signs or symptoms of the disease or condition, e.g., in preventive treatment. In some embodiments, treatment comprises prevention and in other embodiments it does not.
  • administering are used interchangeably to refer to causing a subject to receive a treatment.
  • administering a therapeutic agent to the subject means moving the particle inside the body of the subject.
  • active agent, active pharmaceutical ingredient, pharmacologically active agent, and drug are used interchangeably herein to refer to a bioactive material or compound which, when administered to an organism (human or animal) induces a desired pharmacologic and/or physiologic effect by local and/or systemic action.
  • determining As used herein, the terms “determining,” “measuring,” “assessing,” and “assaying” are used interchangeably and include both quantitative and qualitative determinations.
  • acrylation and “methaciylation” refer to the addition of an acrylate or methacrylate group respectively.
  • the term “methaciylation efficiency” refers to the number of methacrylate groups added to a monomer or polymer described herein. Methacryl ation efficiency is represented as a percentage of the polymer that has been substituted with a methacrylate unit.
  • a “therapeutically effective amount”, a “therapeutically effective dose” or “therapeutic dose” is an amount sufficient to effect desired clinical results (e.g., achieve therapeutic efficacy, achieve a desired therapeutic response).
  • a therapeutically effective dose can be administered in one or more administrations.
  • a therapeutically effective dose of a compositions is an amount that is sufficient, when administered to the individual, to palliate, ameliorate, stabilize, reverse, prevent, slow or delay the progression of a disease state (e.g., neurodegenerative disease, etc ) present in the subject.
  • a "pharmaceutically acceptable excipient,” “pharmaceutically acceptable diluent,” “pharmaceutically acceptable carrier,” and “pharmaceutically acceptable adjuvant” means an excipient, diluent, carrier, and adj uvant that are useful in preparing a pharmaceutical composition that are generally safe, non-toxic and neither biologically nor otherwise undesirable, and include an excipient, diluent, carrier, and adjuvant that are acceptable for veterinary' use as well as human pharmaceutical use.
  • “A pharmaceutically acceptable excipient, diluent, carrier and adjuvant” as used in the specification and claims includes both one and more than one such excipient, diluent, carrier, and adjuvant.
  • a "pharmaceutical composition” is meant to encompass a composition suitable for administration to a subject, such as a mammal, especially a human.
  • a “pharmaceutical composition” is sterile, and preferably free of contaminants that are capable of eliciting an undesirable response within the subject (e.g, the compound(s) in the pharmaceutical composition is pharmaceutical grade).
  • Pharmaceutical compositions can be designed for administration to subjects or patients in need thereof via a number of different routes of administration including intravenous, buccal, rectal, parenteral, intraperitoneal, intradermal, intratracheal, intramuscular, subcutaneous, and the like.
  • co-administration and “in combination with” include the administration of two or more therapeutic agents either simultaneously, concurrently or sequentially.
  • the agents are present in the cell or in the subject's body at the same time or exert their biological or therapeutic effect at the same time.
  • the therapeutic agents are in the same composition or unit dosage form. In other embodiments, the therapeutic agents are in separate compositions or unit dosage forms.
  • a first agent can be administered prior to (e.g., minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, I hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of a second therapeutic agent.
  • the compounds disclosed herein may also comprise one or more isotopic substitutions.
  • H may be in any isotopic form, including ! H, 2 H (Dor deuterium), and 3 H (Tor tritium);
  • C may be in any isotopic form, including 12 C, 1J C, and ,4 C;
  • 0 may be in any isotopic form, including 16 0 and 18 0, and the like.
  • the compositi ons/devices of the present disclosure comprising a therapeutic agent have an inner housing chamber with which the therapeutic agents are housed. This in turn is surrounded by a biocompatible supporting structure/latice or are disposed in a capsule formed from a biocompatible material.
  • a biocompatible supporting structure/latice or are disposed in a capsule formed from a biocompatible material.
  • the inventors contemplate the use of alginates to create protective coatings for therapeutic agents contained in the devices and capsules according to the present disclosure.
  • the alginate is SLG20.
  • the SLG20 is about 0. 1%- 3% SLG20.
  • Modified alginate polymers can be of any desired molecular weight.
  • the weight average molecular weight of the alginates is preferably between 1 ,000 and 1 ,000,000 Daltons, more preferably between 10,000 and 500,000 Daltons as determined by gel permeation chromatography.
  • Modified alginate polymers can contain any ratio of covalently modified monomers. In some embodiments, greater than 2.5%, 5%, 7.5%, 10%, 12%, 14%, 15%, 16%, 18%, 20%, 22%, 24%, 25%, 26%, 28%, 30%, 32.5%, 35%, 37.5%, 40%, 45%, 50%, 55%, or 60% of the monomers in the modified alginate polymer are covaleritiy modified monomers. Greater than 10%, greater than 20%, or greater than 30% of the monomers in the modified alginate polymer are covalently modified monomers.
  • Modified alginate polymers can be produced incorporating covalently modified monomers possessing a range of different hydrogen bonding potentials, hydrophobicities/hydrophilicilies, and charge states.
  • the inclusion of covalently modified monomers into an alginate polymer alters the physiochemical properties of alginate polymer. Accordingly, the physiochemical properties of alginates can be tuned for desired applications by the selective incorporation of covalently modified monomers.
  • the glass transition temperature (Tg) can be varied by the incorporation of covalently modified monomers.
  • the modified alginate polymer powder possess a Tg, as measured by differential scanning calorimetry (DSC), of greater than 50°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, 90°C, 95°C, 100°c, 105°C, 110°c, 115°C, 120°c, 125°C, 130°C, 135°C, 140°C, 145°C, 150°C, 160°C, 175°C, 190°C, or200°c.
  • DSC differential scanning calorimetry
  • the hydrophobicity/hydrophilicity of alginates can be varied by the incorporation of hydrophobic and/or hydrophilic covalently modified monomers.
  • the modified alginate polymer contains one or more hydrophobic covalently modified monomers.
  • the relative hydrophobicity/hydrophilicity of modified alginates can be quantitatively assessed by measuring the contact angle of a water droplet on a film of the modified alginate polymer using a goniometer.
  • the modified alginate has a contact angle of less than 90° (i.e. it is hydrophilic).
  • the modified alginate has a contact angle of more than 90° (i.e., it is hydrophobic).
  • the modified alginate has a contact angle of more than 95°, 100°, 105°, 110°, 115°, or 120°.
  • the modified alginate polymer can be ionically crosslinked by a polyvalent cation such as Ca ⁇ +, Sr ⁇ +, or Ba ⁇ + to form hydrogels.
  • a polyvalent cation such as Ca ⁇ +, Sr ⁇ +, or Ba ⁇ + to form hydrogels.
  • the modified alginate polymer forms hydrogels such that the fluorescence intensity measured using the high throughput hydrogel formation assay described herein is greater than 10,000, 15,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, or 55,000.
  • the modified alginate polymer forms hydrogels such that the fluorescence intensity measured using the high throughput hydrogel formation assay described herein is greater than 15,000. In particular embodiments, the modified alginate polymer forms hydrogels such that the fluorescence intensity measured using the high throughput hydrogel formation assay described herein is between 15,000 and 55,000, preferably between 20,000 and 55,000, more preferably between 25,000 and 55,000.
  • the porosity and surface area of modified alginates can be measured using BET analysis. Prior to BET analysis, solvent and volatile impurities are removed by prolonged heating of the modified alginate gel under vacuum. Subsequently, the hydrogel samples are cooled under vacuum, for example by liquid nitrogen, and analyzed by measuring the volume of gas (typically N2, Kr, CO2, or Ar gas) adsorbed to the hydrogel at specific pressures. Analysis of the physisorption of the gas at variable pressures is used to characterize the total surface area and porosity of gels formed by the modified alginate polymers. A particular method of determining hydrogel porosity is BET analysis
  • the modified alginate forms a hydrogel with sufficient porosity to permit nutrients, waste, and the hormones and/or proteins secreted from encapsulated cells to diffuse freely into and out of the capsules, while simultaneously preventing the incursion of immune cells into the gel matrix.
  • the porosity of the hydrogel formed by the modified alginate polymer is increased by 5%, 10%, 15%, or 20% relative to the porosity of a hydrogel formed from the unmodified alginate polymer.
  • the porosity of the hydrogel formed by the modified alginate polymer is decreased by 5%, 10%, 15%, or 20% relative to the porosity of a hydrogel formed from the unmodified alginate polymer,
  • the modified alginate is biocompatible.
  • the biocompatibility of modified alginates can be quantitatively determined using a fluorescence-based in vivo biocompatibility assay.
  • the modified alginate polymer is biocompatible such that the fluorescence response normalized to unmodified alginate measured using the in vivo biocompatibility assay described herein is less than 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%.
  • the modified alginate polymer induces a lower foreign body response than unmodified alginate. This is indicated by fluorescence response normalized to unmodified alginate of less than 100%.
  • the modified alginate polymer is biocompatible such that the fluorescence response normalized to unmodified alginate measured using the in vivo biocompatibility assay described herein is less than 75%, more preferably less than 65%, and most preferably less than 50%.
  • the modified alginates can be chemically modified as described herein to any desired density of modifications.
  • the density of modifications is the average number of modifications (that is, attached compounds) per a given weight volume, or area of the surface of a capsule or product that includes the modified alginate.
  • a density at or above a threshold density can provide a beneficial effect, such as lower foreign body response.
  • a high density is not required.
  • the chemical modifications signal to, indicate to, or are identified by, one or more immune system or other body components to result in a beneficial effect, such as a lower foreign body response.
  • a lower density of modifications can be effective for this purpose.
  • Useful densities include densities of at least, of less than, of about, or of 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19,20,25,30,35,40,45,50,55,60,65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 550, 600, 650, 700, 750, 800, 850, 900, and 1000 modifications per square um, per ug, or per cubic gm. All ranges defined by any pair of these densities are also specifically contemplated and disclosed.
  • the density of the modifications on a surface, surfaces, or portions of a surface(s) of a capsule or product that, when the product is administered to (e.g., implanted in the body of) a subject, would be in contact with fluid(s), cell(s), tissue(s), other component(s), or a combination thereof of the subject's body is greater than the density of the modifications on other surfaces of the product.
  • Density can also be expressed in terms of the concentration of the surface modifications as measured by X-ray photoelectron spectroscopy (XPS).
  • XPS is a surfacesensitive quantitative spectroscopic technique that measures the elemental composition at the parts per thousand range of the elements that exist within a material .
  • XPS spectra are obtained by irradiating a material with a beam of X-rays while simultaneously measuring the kinetic energy and number of electrons that escape from the top O to 10 nm of the materia! being analyzed.
  • the percentage of the elements that come from the surface modifications can be calculated. This can be accomplished by, for example, taking the percentage of nitrogen (and/or other elements in the surface modifications) in the total elemental signal measured. Nitrogen is a useful indicator for the surface modification because many substrated and materials forming the capsule or product contain little nitrogen.
  • the percent of the element(s) used to indicate the surface modifications can be stated as the percent surface modifications. Also for convenience, the percent surface modifications can be referred to as the concentration of surface modifications.
  • I Jseful percent surface modifications include concentrations of about, less than or at O. l , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, and 100 percent surface modifications. All ranges defined by any pair of these concentrations are also specifically contemplated and disclosed.
  • the alginates may be used to form a capsule.
  • Capsules are particles having a mean diameter of about 150 pm to about 5 cm.
  • the disclosed capsules can be formed of cross-linked hydrogel.
  • the capsules for example, can be formed solely of cross-linked hydrogel, can have a cross-linked hydrogel core that is surrounded by one or more polymeric shells, can have one or more cross-linked hydrogel layers, can have a cross- linked hydrogel coating, or a combination thereof
  • the capsule may have any shape suitable for, for example, cell encapsulation.
  • the capsule may contain one or more cells dispersed in the cross-linked hydrogel, thereby "encapsulating" the cells.
  • Particular capsules are formed of or include one or more of the disclosed modified alginates.
  • Capsules can have a mean diameter of about 150 pm to about 8 mm. Capsules can have any mean diameter from about 150 pm to about 5 cm. In particular, the capsules have a mean diameter that is greater than 1 mm, more particularly 1.5 mm or greater. In some embodiments, the capsules can be as large as about 8 mm in diameter. E ?
  • the capsule can be in a size range of about 1 mm to 8 mm, I ram to 6 mm, 1 mm to 5 mm, 1 mm to 4 mm, 1 mm to 3 mm, 1 mm to 2 mm, 1 mm to 1.5 mm, 1 .5 mm to 8 mm, 1 .5 mm to 6 mm, 1 .5 mm to 5 mm, 1.5 ram to 4 mm, 1.5 mm to 3 mm, or 1.5 mm to 2 mm.
  • T 'he rate of molecules entering the capsule necessary for cell viability and the rate of therapeutic products and waste material exiting the capsule membrane can be selected by modulating capsule permeability.
  • Capsule permeability can also be modified to limit entry of immune cells, antibodies, and cytokines into the capsule.
  • known methods of forming hydrogel capsules can produce capsules the permeability of which limit entry of immune cells, antibodies, and cytokines into the capsule. Since different cell types have different metabolic requirements, the permeability of the membrane can be optimized based on the cell type encapsulated in the hydrogel. The diameter of the capsules is an important factor that influences both the immune response towards the cell capsules as well as the mass transport across the capsule membrane.
  • the unmodified alginate typically has a weight average molecular weight of about 50,000 Daltons to about 500,000 Daltons; however, unmodified alginates having molecular weights outside this range can also be used.
  • the average molecular weight is less than 95,000 Daltons, preferably less than 75,000 Daltons.
  • the average molecular weight is from about 50,000 to about 250,000 Daltons, preferably from about 75,000 Daltons to about 220,000 Daltons.
  • the average molecular weight is from about 175,000 to about 325,000 Daltons, preferably from about 200,000 Daltons to about 300,000 Daltons.
  • one or more additional hydrogel-fonning polymers are used in combination with unmodified alginate or in place of unmodified alginate.
  • Such polymers are known in the art. Examples include, but are not limited to, PEG, chitosan, dextran, hyaluronic acid, silk, fibrin, polyfvinyl alcohol) and polyfhydroxyl ethyl methacrylate).
  • the particles prepared from a mixture of modified alginate and unmodified alginate produced more homogenous microparticle populations in terms of shape and size as evaluated by scanning electron microscopy (SEM).
  • the hydrogel capsules can have any suitable shape.
  • Useful shapes include spheres, sphere-like shapes, spheroids, spheroid-like shapes, ellipsoids, ellipsoid-like shapes, stadiumoids, stadiumoid-like shapes, disks, disk-like shapes, cylinders, cylinder-like shapes, rods, rod-like shapes, cubes, cube-like shapes, cuboids, cuboid-like shapes, toruses, torus-like shapes, and flat and curved surfaces.
  • Products, devices, and surfaces that have been or will be coated can have any of these shapes or any shape suitable for the product or device.
  • Spheres, spheroids, and ellipsoids are shapes with curved surfaces that can be defined by rotation of circles, ellipses, or a combination around each of the three perpendicular axes, a, b, and c.
  • the three axes are the same length.
  • oblate spheroids also referred to as oblate 15 ellipsoids of rotation
  • the length of the axes are a ;::: b > c.
  • prolate spheroids also referred to as prolate ellipsoids of rotation
  • the length of the axes are a> b > c.
  • Stadium oi ds are rotational shapes of stadiums. Cylinders are rotational shapes of rectangles rotated on the long axis. Disks are squashed cylinders where the diameter is greater than the height. Rods are elongated cylinders where the long axis is ten or more times the diameter.
  • Sphere-like shape refers to an object having a surface that roughly forms a sphere, spheroid, ellipsoid, stadiumoid, cylinder, rod, cube, cuboid, or torus, respectively.
  • a sphere-like shape, spheroid-like shape, ellipsoid-like shape, stadiumoid-like shape, cylinder-like shape, rod- like shape, cube-like shape, cuboid-like shape, and torus-like shape can have waves and undulations.
  • a sphere-like shape is an ellipsoid (for its averaged surface) with seraiprincipal axes within 10% of each other.
  • the diameter of a sphere or sphere-like shape is the average diameter, such as the average of the semi-principal axes.
  • a spheroid-like shape is an ellipsoid (for its averaged surface) with semi-principal axes within 100% of each other.
  • the diameter of a spheroid or spheroid-like shape is the average diameter, such as the average of the semi-principal axes.
  • an ellipsoid-like shape is an ellipsoid (for its averaged surface) with semi-principal axes within 100% of each other.
  • the diameter of an ellipsoid or ellipsoid-like shape is the average diameter, such as the average of the semiprincipal axes.
  • a stadiumoid-like shape is a stadiumoid (for its averaged surface) with semi-principal axes of the ends within 20% of each other.
  • the diameter of a stadiumoid or stadiumoid-like shape is the average diameter, such as the average of the semi -principal axes.
  • the size of a stadiumoid or stadiumoid-like shape can be given as the average of the long axis.
  • a cylinder-like shape is a cylinder (for its averaged surface) with semi- principal axes within 20% of each other.
  • the diameter of a cylinder or cylinderlike shape is the average diameter, such as the average of the semi-principal axes.
  • the size of a cylinder or cylinder-like shape can be given as the average of the long axis.
  • a rod-like shape is a rod (for its averaged surface) with semi- principal axes within 10% of each other.
  • the di ameter of a rod or rod-like shape is the average diameter, such as the average of the semi-principal axes.
  • the size of a rod or rod-like shape can be given as the average of the long axis.
  • a cubelike shape is a cube (for its averaged surface) with sides within 10% of each other
  • the diameter of a cube or cube-like shape is the average side length.
  • a cuboid-like shape is a cuboid (for its averaged surface) with matching sides within 10% of each other.
  • the diameter of a cuboid or cuboid-like shape is the average side length.
  • a torus-like shape is a torus (for its averaged surface) with semiprincipal axes within 10% of each other.
  • the diameter of a torus or toms-like shape is the average diameter, such as the average of the semi -principal axes.
  • the size of a toms or torus-like shape can be given as the diameter across the ring.
  • “Flat side” refers to a contiguous area of more than 5% of a surface that has a curvature of 0.
  • “Sharp angle” refers to a location on a surface across which the tangent to the surface changes by more than 10% over a distance of 2% or less of the circumference of the surface. Edges, comers, grooves, and ridges in a surface are all forms of sharp angles.
  • Particular capsules can be made of biocompatible materials, have a diameter of at least 1 mm and less than 10 mm, has a spheroid-like shape, and have one or more of the additional characteristics: surface pores of the capsules greater than 0 nm and less than 10 um; surface of the capsules neutral or hydrophilic; curvature of the surface of the capsules at least 0.2 and is not greater than 2 on all points of the surface; and surface of the capsules lacking flat sides, sharp angles, grooves, or ridges.
  • the capsules elicit less of a fibrotic reaction after implantation than the same capsules lacking one or more of these characteristics that, are present on the capsules.
  • the capsules are provided as a preparation and at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the capsules in the preparation have a shape characteristic described herein, e.g., have a spheroidlike shape, or have a curvature of the surface of at least 0.2 to 2.0 on all points of the surface.
  • the hydrogel capsules have a mean diameter that is greater than 1 mm, particularly 1.5 mm or greater.
  • the hydrogel capsules can be as large as 8 ram in diameter.
  • the hydrogel capsules is in a size range of 1 mm to 8 mm, 1 mm to 6 mm, 1 mm to 5 mm, 1 mm to 4 mm, 1 mm to 3 mm, 1 mm to 2 mm,
  • the capsule has a mean diameter or size between 1 mm to 8 mm. In some embodiments, the capsule has a mean diameter or size between 1 mm to 4 mm. In some embodiments, the capsule has a mean diameter or size between 1 mm to 2 mm. In some embodiments, the capsules are provided as a preparation and at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the hydrogel capsules in the preparation have a diameter in a size range described herein
  • the methacrylation efficiency of the UPVLVG alginate is less than about 100%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is less than about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, or about 25%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is less than about 95%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is less than about 90%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is less than about 85%.
  • the methacrylation efficiency of the UPVLVG alginate is less than about 80%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is less than about 75%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is less than about 70%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is less than about 65%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is less than about 60%, In embodiments, the methacrylation efficiency of the UPVLVG alginate is less than about 55%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is less than about 50%.
  • the methacrylation efficiency of the UPVLVG alginate is less than about 45%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is less than about 40%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is less than about 35%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is less than about 30%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is less than about 25%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is 20%.
  • the methacrylation efficiency of the UPVLVG alginate is 46% In embodiments, the methacrylation efficiency of the UPVLVG alginate is 60%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is 90%.
  • the methacrylation efficiency of the UPVLVG alginate is between about 10% and about 100%. In embodiments, the methacrylation efficiency of the UPVL VG alginate is between about 12% and about 98%, about 14% and about 96%, about 16% and about 94%, about 18% and about 92%, or about 20% and about 90%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is between about 10% and about 100%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is between about 12% and about 98%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is between about 14% and about 96%.
  • the methacrylation efficiency of the UPVL V G alginate i s between about 16% and about 94%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is between about 18% and about 92%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is between about 20% and about 90%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is 20%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is 46%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is 60%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is 90%.
  • the methacrylation efficiency of the UPVLVG alginate is greater than about 5%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is greater than about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80% or about 85%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is greater than about 5%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is greater than about 10%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is greater than about 15%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is greater than about 20%
  • the methacrylation efficiency of the UPVLVG alginate is greater than about 25%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is greater than about 30%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is greater than about 35%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is greater than about 40%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is greater than about 45%.
  • the methacrylation efficiency of the UPVLVG alginate is greater than about 50%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is greater than about 55%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is greater than about 60%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is greater than about 65%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is greater than about 70%.
  • the methacrylation efficiency of the UPVLVG alginate is greater than about 75%.
  • the methaciylation efficiency of the UPVLVG alginate is greater than about 80%.
  • the methaciylation efficiency of the UPVLVG alginate is greater than about 85%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is 20%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is 46%. In embodiments, the methaciylation efficiency of the UPVLVG alginate is 60%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is 90%.
  • the methacrylation efficiency of the SLG20 alginate is less than about 70%. In embodiments, the methacrylation efficiency of the SI..G20 alginate is less than about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15% or about 10%. In embodiments, the methaciylation efficiency of the SLG20 alginate is less than about 70%. In embodiments, the methacrylation efficiency of the SLG20 alginate less than about 65%. In embodiments, the methacrvlation efficiency of the SLG20 alginate is less than about 60%. In embodiments, the methacrylation efficiency of the SLG20 alginate is less than about 55%.
  • the methacrylation efficiency of the SLG20 alginate is less than about 50%. In embodiments, the methacrylation efficiency of the SLG20 alginate is less than about 45%. In embodiments, the methacrylation efficiency of the SLG20 alginate less than about 40%. In embodiments, the methaciylation efficiency of the SI..G20 alginate is less than about 35%. In embodiments, the methacrylation efficiency of the SLG20 alginate is less than about 30%. In embodiments, the methacrylation efficiency of the SLG20 alginate is less than about 25%. In embodiments, the methacrylation efficiency of the SLG20 alginate less than about 20%.
  • the methacrylation efficiency of the SLG20 alginate is less than about 15%. In embodiments, the methacrylation efficiency of the SI..G20 alginate is less than about 10%. In embodiments, the methacrylation efficiency of the SLG20 alginate 5%. In embodiments, the methacrylation efficiency of the SLG20 alginate is 10%. In embodiments, the methacrylation efficiency of the SLG20 alginate is 20%. In embodiments, the methacrylation efficiency of the SLG20 alginate is 46%. In embodiments, the methacrylation efficiency of the SLG20 alginate is 60%.
  • the methacrylation efficiency of the SLG20 alginate is between about 1% and about 80%. In embodiments, the methacrylation efficiency of the SLG20 alginate is between about 2% and about 75%, about 3% and about 70%, about 4% and about 65% or about 5% and about 60%. In embodiments, the methacrylation efficiency of the SLG20 alginate is between about 1% and about 80%. In embodiments, the methacrylation efficiency of the SLG20 alginate is between about 2% and about 75%. In embodiments, the methacrylation efficiency of the SLG20 alginate is between about 3% and about 70%.
  • the methaciylation efficiency of the SLG20 alginate is between about 4% and about 65%. In embodiments, the methacrylation efficiency of the SLG20 alginate is between about 5% and about 60%. In embodiments, the methacrylation efficiency of the SLG20 alginate is 5%. In embodiments, the methacrylation efficiency of the SLG20 alginate is 10%. In embodiments, the methacrylation efficiency of the SLG20 alginate is 20%. In embodiments, the methacrylation efficiency of the SLG20 alginate is 46%. In embodiments, the methacrylation efficiency of the SLG20 alginate is 60%.
  • the methacrylation efficiency of the SLG20 alginate is greater than about 1%. In embodiments, the methacrylation efficiency of the SLG20 alginate is greater than about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, or about 55%. In embodiments, the methacrylation efficiency of the SLG20 alginate is greater than about 1%. In embodiments, the methacrylation efficiency of the SLG20 alginate is greater than about 5%. In embodiments, the methacrylation efficiency of the SLG20 alginate is greater than about 10%. In embodiments, the methacrylation efficiency of the SLG20 alginate is greater than about 15%.
  • the methacrylation efficiency of the SLG20 alginate is greater than about 20%. In embodiments, the methacrylation efficiency of the SLG20 alginate is greater than about 25%. In embodiments, the methacrylation efficiency of the SLG20 alginate is greater than about 30%. In embodiments, the methacrylation efficiency of the SLG20 alginate is greater than about 35%. In embodiments, the methacrylation efficiency of the SLG20 alginate is greater than about 40%. In embodiments, the methacrylation efficiency of the SLG20 alginate is greater than about 45%. In embodiments, the methacrylation efficiency of the SLG20 alginate is greater than about 50%.
  • the methacrylation efficiency of the SLG20 alginate is greater than about 55%. In embodiments, the methacrylation efficiency of the SLG20 alginate is 5%. In embodiments, the methaciylation efficiency of the SLG20 alginate is 10%. In embodiments, the methacrylation efficiency of the SLG20 alginate is 20%. In embodiments, the methacrylation efficiency of the SLG20 alginate is 46%. In embodiments, the methacrylation efficiency of the SLG20 alginate is 60%.
  • the methacrylation efficiency of the SLG 100 alginate is less than about 40%. In embodiments, the methacrylation efficiency of the SLG 100 alginate is less than about 35%, about 30%, about 25%, about 20%, about 15%, or about 10%. In embodiments, the methacrylation efficiency of the SLG100 alginate is less than about 40%. In embodiments, the methaciylation efficiency of the SLG100 alginate is less than about 35%. In embodiments, the methacrylation efficiency of the SLG100 alginate is less than about 30%. In embodiments, the methaciylation efficiency of the SLG 100 alginate is less than about 25%. In embodiments, the methacrylation efficiency of the SLG100 alginate is less than about 20%.
  • the methacrylation efficiency of the SLG100 alginate is less than about 15%. In embodiments, the methacrylation efficiency of the SLG100 alginate is less than about 10%. In embodiments, the methacrylation efficiency of the SLG100 alginate is 5%. In embodiments, the methacrylation efficiency of the SLG100 alginate is 10% In embodiments, the methacrylation efficiency of the SLG100 alginate is 20%.
  • the methacrylation efficiency of the SL.G100 alginate is between about 1% and about 40%. In embodiments, the methacrylation efficiency of the SLG100 alginate is between about 2% and about 35%, about 3% and about 30%, about 4% and about 25% or about 5% and about 20%. In embodiments, the methacrylation efficiency of the SLG100 alginate is between about 1% and about 40%. In embodiments, the methacrylation efficiency of the SLG100 alginate is between about 2% and about 35%. In embodiments, the methacrylation efficiency of the SLG100 alginate is between about 3% and about 30%. In embodiments, the methacrylation efficiency of the SLG100 alginate is between about 4% and about 25%.
  • the methacrylation efficiency of the SL.G1O0 alginate is between about 5% and about 20%. In embodiments, the methacrylation efficiency of the SLG100 alginate is 5%. In embodiments, the methacrylation efficiency of the SLG100 alginate is 10%. In embodiments, the methacrylation efficiency of the SLG100 alginate is 20%.
  • the methacrylation efficiency of the SLG100 alginate is greater than about 1 %. In embodiments, the methacrylation efficiency of the SLG100 alginate is greater than about 1%, about 2%, about 3%, about 4%, about 5%, about 10% or about 15%. In embodiments, the methacrylation efficiency of the SLG100 alginate is greater than about 1%. In embodiments, the methacrylation efficiency of the SLG1O0 alginate is greater than about 2%, In embodiments, the methacrylation efficiency of the SLG100 alginate is greater than about 3%. In embodiments, the methacrylation efficiency of the SLG100 alginate is greater than about 4%.
  • the methacrylation efficiency of the SLG100 alginate is greater than about 5%. In embodiments, the methacrylation efficiency of the SLG100 alginate is greater than about 10%. In embodiments, the methacrylation efficiency of the SLG100 alginate is greater than about 15%. In embodiments, the methacrylation efficiency of the SLG100 alginate is 5%. In embodiments, the methacrylation efficiency of the SLG100 alginate is 10%.
  • the average molecular weight of the alginate is less than about 95,000 Da In embodiments, the average molecular weight of the alginate is less than about 65,000 Da, about 55,000 Da, about 45,000 Da, about 35,000 Da, about 25,000 Da, about 14,000 Da, about 12,000 Da, about 10,000 Da, about 9,000 Da, about 8,000 Da, about 7,000 Da, about 6,000 Da, about 5,000 Da, about 4,000 Da, about 3,000 Da or about 2,000 Da. In embodiments, the average molecular weight of the alginate is less than about 65,000 Da. In embodiments, the average molecular weight of the alginate is less than about 55,000 Da. In embodiments, the average molecular weight of the alginate is less than about 45,000 Da.
  • the average molecular weight of the alginate is less than about 35,000 Da. In embodiments, the average molecular weight of the alginate is less than about 25,000 Da. In embodiments, the average molecular weight of the alginate is less than about 14,000 Da. In embodiments, the average molecular weight of the alginate is less than about 12,000 Da. In embodiments, the average molecular weight of the alginate is less than about 10,000 Da. In embodiments, the average molecular weight of the alginate is less than about 9,000 Da. In embodiments, the average molecular weight of the alginate is less than about 8,000 Da In embodiments, the average molecular weight of the alginate is less than about 7,000 Da.
  • the average molecular weight of the alginate is less than about 6,000 Da. In embodiments, the average molecular weight of the alginate is less than about 5,000 Da. In embodiments, the average molecular weight of the alginate is less than about 4,000 Da. In embodiments, the average molecular weight of the alginate is less than about 3,000 Da. In embodiments, the average molecular weight of the alginate is less than about 2,000 Da.
  • the average molecular weight of the alginate is between about 500 Da and 50,000 Da. In embodiments, the average molecular weight of the alginate is between about 500 Da and 40,000 Da., about 500 Da and 30,000 Da, about 500 Da and 20,000 Da, about 500 Da and 18,000 Da, about 500 Da and 16,000 Da, about 500 Da and 14,000 Da, about 500 Da and 12,000 Da, about 500 Da and 10,000 Da, about 500 Da and 9,000 Da, about 500 Da and 8,000 Da, about 500 Da and 7,000 Da, about 500 Da and 6,000 Da, about 500 Da and 5,000 Da, about 500 Da and 4,000 Da, about 500 Da and 3,000 Da, about 500 Da and 2,000 Da, or about 500 Da and 1,000 Da.
  • the average molecular weight of the alginate is between about 500 Da and 40,000 Da. In embodiments, the average molecular weight of the alginate is between about 500 Da and 30,000 Da. In embodiments, the average molecular weight of the alginate is between about 500 Da and 20.000 Da. In embodiments, the average molecular weight of the alginate is between about 500 Da and 18,000 Da. In embodiments, the average molecular weight of the alginate is between about 500 Da and 16,000 Da. In embodiments, the average molecular weight of the alginate is between about 500 Da and 14,000 Da. In embodiments, the average molecular weight of the alginate is between about 500 Da and 12,000 Da.
  • the average molecular weight of the alginate is between about 500 Da and 10,000 Da. In embodiments, the average molecular weight of the alginate is between about 500 Da and 9,000 Da. In embodiments, the average molecular weight of the al ginate is between about 500 Da and 8,000 Da. In embodiments, the average molecular weight of the alginate is between about 500 Da and 7,000 Da. In embodiments, the average molecular weight of the alginate is between about 500 Da and 6,000 Da. In embodiments, the average molecular weight of the alginate is between about 500 Da and 5,000 Da.
  • the average molecular weight of the alginate is between about 500 Da and 4,000 Da. In embodiments, the average molecular weight of the alginate is between about 500 Da and 3,000 Da. In embodiments, the average molecular weight of the alginate is between about 500 Da and 2,000 Da. In embodiments, the average molecular weight of the alginate is between about 500 Da and 1,000 Da.
  • the average molecular weight of the alginate is between about 1 ,000 Da and 50,000 Da. In embodiments, the average molecular weight of the alginate is between about 1,000 Da and 40,000 Da, about 1,000 Da and 30,000 Da, about 1,000 Da and 20,000 Da, about 1,000 Da and 18,000 Da, about 1,000 Da and 16,000 Da, about 1 ,000 Da and 14,000 Da, about 1,000 Da and 12,000 Da, about 1,000 Da and 10,000 Da, about 1,000 Da and 9,000 Da, about 1,000 Da and 8,000 Da, about 1,000 Da and 7,000 Da, about 1,000 Da and 6,000 Da, about 1 6,000 Da and 5,000 Da, about 1 ,000 Da and 4,000 Da, about 1 ,000 Da and 3,000 Da. or about 1 ,000 Da and 2,000 Da.
  • the average molecular weight of the alginate is between about 1,000 Da and 40,000 Da. In embodiments, the average molecular weight of the alginate is between about 1,000 Da and 30,000 Da. In embodiments, the average molecular weight of the alginate is between about 1,000 Da and 20,000 Da. In embodiments, the average molecular weight of the alginate is between about 1,000 Da and 18,000 Da. In embodiments, the average molecular weight of the alginate is between about 1,000 Da and 16,000 Da. In embodiments, the average molecular weight of the alginate is between about 1,000 Da and 14,000 Da. In embodiments, the average molecular weight of the alginate is between about 1,000 Da and 12,000 Da.
  • the average molecular weight of the alginate is between about 1 ,000 Da and 10,000 Da. In embodiments, the average molecular weight of the alginate is between about 1000 Da and 9,000 Da. In embodiments, the average molecular weight of the alginate is between about 1,000 Da and 8,000 Da. In embodiments, the average molecular weight of the alginate is between about 1,000 Da and 7,000 Da. In embodiments, the average molecular weight of the alginate is between about 1,000 Da and 6,000 Da. In embodiments, the average molecular weight of the alginate is between about 1 ,000 Da and 5,000 Da. In embodiments, the average molecular weight, of the alginate is between about 1 ,000 Da and 4,000 Da. In embodiments, the average molecular weight of the alginate is between about 1,000 Da and 3,000 Da. In embodiments, the average molecular weight of the alginate is between about 1,000 Da and 2,000 Da.
  • the average molecular weight of the alginate is between about
  • the average molecular weight of the alginate is between about 2,000 Da and 40,000 Da, about 2,000 Da and 30,000 Da, about 2,000 Da and 20,000 Da, about 2,000 Da and 18,000 Da, about 2,000 Da and 16,000 Da, about 2,000 Da and 14,000 Da, about 2,000 Da and 12,000 Da, about 2,000 Da and 10,000 Da, about 2,000 Da and 9,000 Da, about 2,000 Da and 8,000 Da, about 2,000 Da and 7,000 Da, about 2,000 Da and 6,000 Da, about 2,000 Da and 5,000 Da, about 2,000 Da and 4,000 Da, or about 2,000 Da and 3,000 Da. In embodiments, the average molecular weight of the alginate is between about 2,000 Da and 40,000 Da.
  • the average molecular weight of the alginate is between about 2,000 Da and 30,000 Da In embodiments, the average molecular weight of the alginate is between about 2,000 Da and 20,000 Da. In embodiments, the average molecular weight of the alginate is between about 2,000 Da and 18,000 Da. In embodi ments, the average molecular weight of the alginate is between about 2,000 Da and 16,000 Da. In embodiments, the average molecular weight of the alginate is between about 2,000 Da and 14,000 Da. In embodiments, the average molecular weight of the alginate is between about 2,000 Da and 12,000 Da. In embodiments, the average molecular weight of the alginate is between about 2,000 Da and 10,000 Da.
  • the average molecular weight of the alginate is between about 2,000 Da and 9,000 Da. In embodiments, the average molecular weight of the alginate is between about 2,000 Da and 8,000 Da. In embodiments, the average molecular weight of the alginate is between about 2,000 Da and 7,000 Da. In embodiments, the average molecular weight of the alginate is between about 2,000 Da and 6,000 Da. In embodiments, the average molecular weight of the alginate is between about 2,000 Da and 5,000 Da. In embodiments, the average molecular weight of the alginate is between about 2,000 Da and 4,000 Da. In embodiments, the average molecular weight of the alginate is between about 2,000 Da and 3,000 Da.
  • the average molecular weight of the alginate is between about 5,000 Da and 50,000 Da. In embodiments, the average molecular weight of the alginate is between about 5,000 Da and 40,000 Da, about 5,000 Da and 30,000 Da, about 5,000 Da and 20,000 Da, about 5,000 Da and 18,000 Da, about 5,000 Da and 16,000 Da, about 5,000 Da and 14,000 Da, about 5,000 Da and 12,000 Da, about 5,000 Da and 10,000 Da, about 5,000 Da and 9,000 Da, about 5,000 Da and 8,000 Da, about 5,000 Da and 7,000 Da, or about 5,000 Da and 6,000 Da. In embodiments, the average molecular weight of the alginate is between about 5,000 Da and 40,000 Da.
  • the average molecular weight of the alginate is between about 5,000 Da and 30,000 Da. In embodiments, the average molecular weight of the alginate is between about 5,000 Da and 20,000 Da. In embodiments, the average molecular weight of the alginate is between about 5,000 Da and 18,000 Da. In embodiments, the average molecular weight of the alginate is between about 5,000 Da and 16,000 Da. In embodiments, the average molecular weight of the alginate is between about 5,000 Da and 14,000 Da. In embodiments, the average molecular weight of the alginate is between about 5,000 Da and 12,000 Da. In embodiments, the average molecular weight of the alginate is between about 5,000 Da and 10,000 Da.
  • the average molecular weight of the alginate is between about 5,000 Da and 9,000 Da. In embodiments, the average molecular weight of the alginate is between about 5,000 Da and 8,000 Da. In embodiments, the average molecular weight of the alginate is between about 5,000 Da and 7,000 Da. In embodiments, the average molecular weight of the alginate is between about 5,000 Da and 6,000 Da.
  • the average molecular weight of the alginate is between about
  • the average molecular weight of the alginate is between about 10,000 Da and 40,000 Da, about 10,000 Da and 30,000 Da, about 10,000 Da and 20,000 Da, about 10,000 Da and 18,000 Da, about 10,000 Da and 16,000 Da, about 10,000 Da and 14,000 Da, or about 10,000 Da and 12,000 Da. In embodiments, the average molecular weight of the alginate is between about 10,000 Da and 40,000 Da. In embodiments, the average molecular weight of the alginate is between about 10,000 Da and 30,000 Da. In embodiments, the average molecular weight of the alginate is between about 10,000 Da and 20,000 Da. In embodiments, the average molecular weight of the alginate is between about 10,000 Da and 18,000 Da.
  • the average molecular weight of the alginate is between about 10,000 Da and 16,000 Da. In embodiments, the average molecular weight of the alginate is between about 10,000 Da and 14,000 Da. In embodiments, the average molecular weight of the alginate is between about 10,000 Da and 12,000 Da.
  • the average molecular weight of the alginate is greater than about 500 Da. In embodiments, the average molecular weight of the alginate i s greater than about 1,000 Da, about 2,000 Da, about 3,000 Da, about 4,000 Da, about 5,000 Da, about 6,000 Da, about 7,000 Da, about 8,000 Da, about 9,000 Da, about 10,000 Da, about 12,000 Da, about 14,000 Da, about 16,000 Da, about 18,000 Da, about 20,000 Da, about 30,000 Da, about 40,000 Da or about 50,000 Da In embodiments, the average molecular weight of the alginate is greater than about 1,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 2,000 Da.
  • the average molecular weight of the alginate is greater than about 3,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 4,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 5,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 6,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 7,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 8,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 9,000 Da, In embodiments, the average molecular weight of the alginate is greater than about 10,000 Da.
  • the average molecular weight of the alginate is greater than about 12,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 14,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 16,000 Da. In embodiments, the average molecular tveight of the alginate is greater than about 18,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 20,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 30,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 40,000 Da.
  • the average molecular weight of the alginate is less than about 260,000 Da. In embodiments, the average molecular weight of the alginate is less than about 250,000 Da, about 240,000 Da, about 230,000 Da, about 220,000 Da, about 210,000 Da, about
  • the average molecular weight of the alginate is less than about 260,000 Da. In embodiments, the average molecular weight of the alginate is less than about 250,000 Da. In embodiments, the average molecular weight of the alginate is less than about 240,000 Da. In embodiments, the average molecular weight of the alginate is less than about 260,000 Da. In embodiments, the average molecular weight of the alginate is less than about 250,000 Da. In embodiments, the average molecular weight of the alginate is less than about 240,000 Da. In embodiments, the average molecular weight of the alginate is less than about 230,000 Da.
  • the average molecular weight of the alginate is less than about 220,000 Da. In embodiments, the average molecular weight of the alginate is less than about 210,000 Da. In embodiments, the average molecular weight of the alginate is less than about 200,000 Da. In embodiments, the average molecular weight of the alginate is less than about 190,000 Da. In embodiments, the average molecular weight of the alginate is less than about 180,000 Da. In embodiments, the average molecular weight of the alginate is less than about 170,000 Da. In embodiments, the average molecular weight of the alginate is less than about 160,000 Da. In embodiments, the average molecular weight of the alginate is less than about 150,000 Da.
  • the average molecular weight of the alginate is less than about 140,000 Da. In embodiments, the average molecular weight of the alginate is less than about 130,000 Da. In embodiments, the average molecular weight of the alginate is less than about 120,000 Da. In embodiments, the average molecular weight of the alginate is less than about 110,000 Da. In embodiments, the average molecular weight of the alginate is less than about 100,000 Da. In embodiments, the average molecular weight of the alginate is less than about 90,000 Da. In embodiments, the average molecular weight of the alginate is less than about 80,000 Da. In embodiments, the average molecular weight of the alginate is less than about 70,000 Da. In embodiments, the average molecular weight of the alginate is less than about 60,000 Da. In embodiments, the average molecular weight of the alginate is less than about
  • the average molecular weight of the alginate is between about 50,000 Da and about 260,000 Da. tn embodiments, the average molecular weight of the alginate is between about 75,000 Da and 260,000 Da, about 75,000 Da and 250,000 Da, about 75,000 Da and 240,000 Da, about 75,000 Da and 230,000 Da, about 75,000 Da and 220,000 Da, about 75,000 Da and 210,000 Da, about 75,000 Da and 200,000 Da, about 75,000 Da and 190,000 Da, about 75,000 Da and 180,000 Da, about 75,000 Da and 170,000 Da, about 75,000 Da and 160,000 Da, about 75,000 Da and 150,000 Da, about 75,000 Da and 140,000 Da, about 75,000 Da and 130,000 Da, about 75,000 Da and 120,000 Da, about 75,000 Da and 110,000 Da, or about 75,000 Da and 100,000 Da.
  • the average molecular weight of the alginate is between about 75,000 Da and 260,000 Da. In embodiments, the average molecular weight of the alginate is between about 75,000 Da and 250,000 Da In embodiments, the average molecular weight of the alginate is between about 75,000 Da and 240,000 Da. In embodiments, the average molecular weight of the alginate is between about 75,000 Da and 230,000 Da. In embodiments, the average molecular weight of the alginate is between about 75,000 Da and 220,000 Da. In embodiments, the average molecular weight of the alginate is between about 75,000 Da and 210,000 Da. In embodiments, the average molecular weight of the alginate is between about 75,000 Da and 200,000 Da.
  • the average molecular weight of the alginate is between about 75,000 Da and 190,000 Da. In embodiments, the average molecular weight of the alginate is between about 75,000 Da and 180,000 Da. In embodiments, the average molecular weight of the alginate is between about 75,000 Da and 170,000 Da. In embodiments, the average molecular weight of the alginate is between about 75,000 Da and 160,000 Da. In embodiments, the average molecular weight of the alginate is between about 75,000 Da and 150,000 Da. In embodiments, the average molecular weight of the alginate is between about 75,000 Da and 140,000 Da. In embodiments, the average molecular weight of the alginate is between about 75,000 Da and 130,000 Da.
  • the average molecular weight of the alginate is between about 75,000 Da and 120,000 Da. In embodiments, the average molecular weight of the alginate is between about 75,000 Da and 110,000 Da. In embodiments, the average molecular weight of the alginate is between about 75,000 Da and 100,000 Da.
  • the average molecular weight of the alginate is greater than about 50,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 60,000 Da, about 70,000 Da, about 80,000 Da, about 90,000 Da, about 100,000 Da, about 110,000 Da, about 120,000 Da, about 130,000 Da, about 140,000 Da, about 150,000 Da, about 160,000 Da, about 170,000 Da, about 180,000 Da, about 190,000 Da, about 200,000 Da, about 210,000 Da, about 220,000 Da or about 230,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 50,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 60,000 Da.
  • the average molecular weight of the alginate is greater than about 70,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 80,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 90,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 100,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 110,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 120,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 130,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 140,000 Da.
  • the average molecular weight of the alginate is greater than about 150,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 160,000 Da. In embodiments, the average molecular weight of the alginate is greater than about. 170,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 180,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 190,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 200,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 210,000 Da.
  • the average molecular weight of the alginate is less than about 350,000 Da. In embodiments, the average molecular weight of the alginate is less than about 300,000 Da, about 290,000 Da, about 280,000 Da, about 270,000 Da, about 260,000 Da, about 250,000 Da, about 240,000 Da, about 230,000 Da, about 220,000 Da, about 210,000 Da, about 200,000 Da, about 190,000 Da, or about 180,000 Da. In embodiments, the average molecular weight of the alginate is less than about 350,000 Da. In embodiments, the average molecular weight of the alginate is less than about 300,000 Da. In embodiments, the average molecular weight of the alginate is less than about 290,000 Da.
  • the average molecular weight of the alginate is less than about 280,000 Da. In embodiments, the average molecular weight of the alginate is less than about 270,000 Da. In embodiments, the average molecular weight of the alginate is less than about 260,000 Da. In embodiments, the average molecular weight of the alginate is less than about 250,000 Da. In embodiments, the average molecular weight of the alginate is less than about 240,000 Da. In embodiments, the average molecular weight of the alginate is less than about 230,000 Da. In embodiments, the average molecular weight of the alginate is less than about 220,000 Da. In embodiments, the average molecular weight of the alginate is less than about 210,000 Da.
  • the average molecular weight of the alginate is less than about 200,000 Da. In embodiments, the average molecular weight of the alginate is less than about 190,000 Da. In embodiments, the average molecular weight of the alginate is less than about 180,000 Da.
  • the average molecular weight of the alginate is between about 180,000 Da and 350,000 Da. In embodiments, the average molecular weight of the alginate is between about 180,000 Da and 350,000 Da, about 180,000 Da and 300,000 Da, about 180,000 Da and 290,000 Da, about 180,000 Da and 280,000 Da, about 180,000 Da and 270,000 Da, about 180,000 Da and 260,000 Da, about 180,000 Da and 250,000 Da, about 180,000 Da and 240,000 Da, about 180,000 Da and 230,000 Da, about 180,000 Da and 220,000 Da, about 180,000 Da and 210,000 Da, about 180,000 Da and 200,000 Da, or about 180,000 Da and 190,000 Da.
  • the average molecular weight of the alginate is between about 180,000 Da and 350,000 Da. In embodiments, the average molecular weight of the alginate is between about 180,000 Da and 300,000 Da. In embodiments, the average molecular weight of the alginate is between about 180,000 Da and 290,000 Da. In embodiments, the average molecular weight of the alginate is between about 180,000 Da and 280,000 Da. In embodiments, the average molecular weight of the alginate is between about 180,000 Da and 270,000 Da. In embodiments, the average molecular weight of the alginate is between about 180,000 Da and 260,000 Da. In embodiments, the average molecular weight of the alginate is between about 180,000 Da and 250,000 Da.
  • the average molecular weight of the alginate is between about 180,000 Da and 240,000 Da. In embodiments, the average molecular weight of the alginate is between about 180,000 Da and 230,000 Da. In embodiments, the average molecular weight of the alginate is between about 180,000 Da and 220,000 Da. In embodiments, the average molecular weight of the alginate is between about 180,000 Da and 210,000 Da. In embodiments, the average molecular weight of the alginate is between about 180,000 Da and 200,000 Da. In embodiments, the average molecular weight of the alginate is between about 180,000 Da and 190,000 Da
  • the average molecular weight of the alginate is greater than about 180,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 190,000 Da, about 200,000 Da, about 210,000 Da, about 220,000 Da, about 230,000 Da, about 240,000 Da, about 250,000 Da, about 260,000 Da, about 270,000 Da, about 280,000 Da, about 290,000 Da, about 300,000 Da, about 310,000 Da, about 320,000 Da, or about 325,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 180,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 190,000 Da.
  • the average molecular weight of the alginate is greater than about 200,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 210,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 220,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 230,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 240,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 250,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 260,000 Da.
  • the average molecular weight of the alginate is greater than about 270,000 Da In embodiments, the average molecular weight of the alginate is greater than about 280,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 290,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 300,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 310,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 320,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 325,000 Da.
  • the hydrogel is photo-responsive, wherein the photo- responsive material comprises a. monomer of Formula (I) represented by the following structural formula:
  • n and m are each independently an integer from 1 to 100.
  • the hydrogel is a pH-responsive material, wherein the hydrogel forms upon a change in pH.
  • the flowable material is a photo-responsive material, wherein the hydrogel forms, ceases forming, or is capable of forming, upon the introduction, removal or change in the intensity of light of a particular wavelength or a range of wavelengths, e.g., visible, UV-A, UV-B, Infrared, X-Ray, inter alia.
  • the hydrogel is a moisture-responsive material, wherein the hydrogel forms, ceases forming, or is capable of forming, upon a change in the moisture content of the material or adjacent environment.
  • the polymeric components undergo self-assembly to form the resulting hydrogel in the presence of water.
  • Materials for assembling hydrogels may also include any provided in Hoffman, A.S. Adv Drug Deliv Rev 2012, 64, 18-23, such as agarose, alginate (e.g., the calcium or barium salt of alginic acid), alginate-g-(alginate), carboxymethyl chitin, carrageenan, chitosan, chondroitin sulfate, collagen-acrylate, dextran, dextran sulfate, fibrin, gelatin, hyaluronic acid (HA), hyaluronic acid/glycidyl methacrylate, HA-g-NIPAAM, PAAM, P(AN-co-allyl sulfonate), P(biscarboxyl-phenoxy-phosphazene), pectin, PEG optionally with cyclodextrins (CDs), PEG-g- P(AAM-co-Vamine), PEG-bis(PLA-acrylate), PEG-PCL-
  • the hydrogel is a chemically responsive material, wherein the hydrogel forms, ceases forming, or is capable of forming, upon the introduction or removal of a chemical stimulus.
  • the hydrogel ceases forming upon the completion or initiation of a chemical stimulus, e.g., a chemical stimulus provided in Zhang Y S. el al. Science. 2017, 356 (6337), eaaS627.
  • a chemical stimulus provided in Zhang Y S. el al. Science. 2017, 356 (6337), eaaS627.
  • the hydrogel can include a plurality of natural polymer macromers cross- linked with a plurality of crosslinks that are degradable after administration to a subject in vivo.
  • T lie number or percentage of cross-links linking the macromers can be varied to control the mechanical properties, swelling ratios, and degradation profiles of the hydrodgels. Degradation of the crosslinks in vivo allows the hydrogel to more readily biodegrade and be used for in vivo applications. Additionally, as discussed below the photocrosslinked hydrogel can be used as a substrate for the incorporation and/or attachment of various agents and/or cells.
  • the photocrosslinked hydrogel can be injectable and/or implantable, and can be in the form of a membrane, sponge, gel, solid scaffold, spun fiber, woven or unwoven mesh, nanoparticle, microparticle, or any other desirable configuration.
  • the photocrosslinked hydrogel can include at least on cross-link that can be hydrolyzed to allow" degradation of the hydrogel in vivo.
  • the cross-link can include ester, amide, acetal, and/or ketal groups or linkages that can be readily hydrolyzed in vivo to promote degradation of the hydrogel.
  • the hydrolyzable cross-link can include at least one hydrolyzable acrylate (e.g.. methacrylate) cross-link.
  • the hydrolyzable acrylate cross-link can include at least one hydrolyzable ester and/or hydrolysable amide linkage.
  • hydrolytic degradation of the hydrolyzable acrylate crosslink can create space for cell growth and deposition of a new extracellular matrix to replace the photocrosslinked hydrogel in vivo.
  • the photocrosslinked hydrogel can be modified or configured to differentially and/or controllably release at least one bioactive agent by forming at least one concentration gradient within the hydrogel.
  • the hydrogel can have multiple gradients in the same hydrogel, and the gradients can run in the same or opposite directions.
  • the gradients can be comprised of different components, such as different photoalginates having different molecular weights or acrylation (e.g., methacryl ati on) percentages, acrylated cell adhesion ligands, bioactive factors, cells, etc.
  • the photocrosslinked biodegradable hydrogel can be formed into a particular shape or form to facilitate release of one or more bi oactive agents according to a gradient release profile.
  • one or more materials or agents can be added to the photocrosslinked biodegradable hydrogel to facilitate differential and/or controlled release of one or more bioactive agents according to a gradient release profile.
  • the term “cell” can refer to any progenitor cell, such as totipotent stem cells, pluripotent stem cells, and multipotent stem cells, as well as any of their lineage descendant cells, including more differentiated cells.
  • progenitor cell such as totipotent stem cells, pluripotent stem cells, and multipotent stem cells, as well as any of their lineage descendant cells, including more differentiated cells.
  • stem cell and “progenitor cell” are used inter changeably herein.
  • the cells can derive from embryonic, fetal, or adult tissues.
  • progenitor cells can include totipotent stem cells, multipotent stem cells, mesenchymal stem cells (MSCs), hematopoietic stem cells, neuronal stem cells, hematopoietic stem cells, pancreatic stem cells, cardiac stem cells, embryonic stem cells, embryonic germ ceils, neural crest stem cells, kidney stem ceils, hepatic stem cells, lung stem cells, hemangioblast cells, and endothelial progenitor cells.
  • Additional exemplary progenitor cells can include de-differentiated chondrogenic cells, chondrogenic cells, cord blood stem cells, multi-potent adult progenitor cells, myogenic cells, osteogenic cells, tendogenic cells, ligamentogenic cells, adipogenic cells, and dermatogenic cells.
  • Devices or compositions described herein may contain a cell, for example, an engineered cell.
  • a cell may be derived from any mammalian organ or tissue, including the brain, nerves, ganglia, spine, eye, heart, liver, kidney, lung, spleen, bone, thymus, lymphatic system, skin, muscle, pancreas, stomach, intestine, blood, ovary, uterus, or testes.
  • a cell may be derived from a donor (e.g., an allogeneic cell), derived from a subject (e.g, an autologous cell), or from another species (e.g, a xenogeneic cell).
  • a cell can be grown in cell culture, or prepared from an established cell culture line, or derived from a donor (e.g., a living donor or a cadaver).
  • a cell is genetically engineered.
  • a cell is not genetically engineered.
  • a cell may include a stem cell, such as a reprogrammed stem cell, or an induced pluripotent cell.
  • Exemplary cells include mesenchymal stem cells (MSCs), fibroblasts (e.g., primary fibroblasts).
  • HEK cells e.g., HEK293T
  • Jurkat cells HeLa cells
  • retinal pigment epithelial (RPE) cells HUVEC cells
  • NIH3T3 cells CHO-K1 cells
  • COS-1 cells COS-7 cells
  • PC-3 cells HCT 116 cells
  • A549MCF-7 cells HuH-7 cells
  • U-2 OS cells HepG2 cells
  • Neuro-2a cells and SF9 cells.
  • a cell included in a device or composition described herein may produce or secrete a therapeutic agent.
  • a cell included in a device or composition described herein may produce or secrete a single type of therapeutic agent or a plurality of therapeutic agents.
  • a device or composition described herein may comprise a cell that is transduced or transfected with a nucleic acid (e.g, a vector) comprising an expression sequence of a therapeutic agent.
  • a. cell may be transduced or transfected with a lentivirus.
  • a nucleic acid introduced into a cell may be incorporated into a nucleic acid delivery system, such as a plasmid, or may be delivered directly.
  • a nucleic acid introduced into a cell may include a region to enhance expression of the therapeutic agent and/or to direct targeting or secretion, for example, a promoter sequence, an activator sequence, or a cell -signaling peptide, or a cell export peptide.
  • Exemplary promoters include EF-la, CMV, Ube, hPGK, VMD2, and CAG.
  • Exemplary activators include the TETI catalytic domain, P300 core, VPR, rTETR, Cas9 (e.g., from A pyogenes or A aureus), and Cpfl (e.g., from L. bacterium).
  • a device or composition described herei n may comprise a cell or a plurality of cells.
  • the concentration and total cell number may be varied depending on a number of factors, such as cell type, implantation location, and expected lifetime of the devices or compositions.
  • the total number of cells included in a device or composition described herein is greater than about 2, 4, 6, 8, 10, 20, 30, 40, 50, 75, 100,200,250, 500, 750, 1000, 1500, 2000, 5000, 10000, or more.
  • the total number of cells included in a device or composition described herein is greater than about 1.0 x 10 2 , 1.0 x 10 3 , 1 .0 x 10 4 .
  • the total number of cells included in a device or composition described herein is less than about than about 10000, 5000, 2500, 2000, 1500, 1000, 750, 500, 250, 200, 100, 75, 50, 40, 30, 20, 10, 8, 6, 4, 2, or less.
  • the total number of cells included in a device or composition described herein is less than about 1.0 x 10 10 , 1.0 x 10 9 , 1.0 x 10 s , 1.0 x 10 7 , 1.0 x lO 6 , 1.0 x 10 5 , 1.0 x 10 4 , 1.0 x 10 3 , 1.0 x 10 2 , or less.
  • a plurality of cells is present as an aggregate. In an embodiment, a plurality of cells is present as a cell dispersion.
  • cell viability, cell density, or cell expression level may be assessed.
  • cell viability, cell density, and cell expression level may be determined using standard techniques, such as cell microscopy, fluorescence microscopy, histology, or biochemical assay.
  • a device or composition described herein may contain a therapeutic agent, for example, produced or secreted by a cell.
  • a therapeutic agent may include a nucleic acid (e.g., an RNA, aDNA, or an oligonucleotide), a protein (e.g., an antibody, enzyme, cytokine, hormone, receptor), a lipid, a small molecule, a metabolic agent, an oligosaccharide, a peptide, an amino acid, an antigen.
  • a device or composition described herein comprises a cell or a plurality of cells that are genetically engineered to produce or secrete a therapeutic agent.
  • a device or composition described herein comprises a cell producing or secreting a protein.
  • the protein may be of any size, e.g., greater than about 100 Da, 200 Da, 250 Da, 500 Da, 750 Da, 1 KDa, 1.5 kDa, 2 kDa, 2.5 kDa, 3 kDa, 4 kDa, 5 kDa, 6 kDa, 7 kDa, 8 kDa, 9 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, 55 kDa, 60 kDa, 65 kDa, 70 kDa, 75 kDa, 80 kDa, 85 kDa, 90 kDa, 95 kDa, 100 kDa, 125 kDa, 150 kDa, 200
  • the protein is composed of a single subunit or multiple subunits (e.g., a dimer, trimer, tetramer, etc.).
  • a protein produced or secreted by a cell may be modified, for example, by glycosylation, methylation, or other known natural or synthetic protein modification
  • a protein may be produced or secreted as a pre-protein or in an inactive form and may require further modification to convert it into an active form.
  • Proteins produced or secreted by a cell may be include antibodies or antibody fragments, for example, an Fe region or variable region of an antibody.
  • Exemplary antibodies include anti-PD-1, anti-PD-LI, anti-CTLA4, anti-TNFa, and anti-VEGF antibodies.
  • An antibody may be monoclonal or polyclonal.
  • Other exemplar,' proteins include a lipoprotein, an adhesion protein, blood clotting factor (e.g., Factor VII, Factor VIII, Factor IX, GCG, or VWF), hemoglobin, enzymes, proenkephalin, a growth factor (e.g., EGF, IGF-1, VEGF alpha, HGF, TGFbeta, bFGF), or a cytokine,
  • a protein produced or secreted by a cell may include a hormone.
  • hormones include growth hormone, growth hormone releasing hormone, prolactin, lutenizing hormone (LH), anti-diuretic hormone (ADH), oxytocin, thyroid stimulating hormone (TSH), thyrotropin-release hormone (TRH), adrenocorticotropic hormone (ACTH), follicle- stimulating hormone (FSH), thyroxine, calcitonin, parathyroid hormone, aldosterone, cortisol, epinephrine, glucagon, insulin, estrogen, progesterone, and testosterone.
  • a protein produced or secreted by a cell may include a cytokine.
  • a cytokine maybe a pro- inflammatory cytokine or an anti-inflammatory' cytokine.
  • Example of cytokines include IL-1, IL- la, IL-1 , IL-IRA, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL- 12, IL- 12a.
  • IL- 12b IL-13, IL- 14, IL- 16, IL- 17, G-CSF, GM-CSF, IL-20, IFN-a, IFN- , IFN-y, CD 154, LT- , CD70, CD 153, CD178, TRAIL, TNF-a, TNF- , SCF, M-CSF, MSP, 4-1BBL, LIE, OSM, and others.
  • a cytokine may include any cytokine described in M.J. Cameron and D.J. Kelvin, Cytokines, Chemokines, and Their Receptors (2013), Austin Biosciences, which is incorporated herein by reference in its entirety.
  • a device or composition described herein may comprise a cell expressing a single type of therapeutic agent, e.g., a single protein or nucleic acid, or may express more than one type of therapeutic agent, e.g., a plurality of proteins or nucleic acids.
  • a device or composition described herein comprises a cell expressing two types of therapeutic agents (e.g., two types of proteins or nucleic acids).
  • a device or composition described herein comprises a cell expressing three types of therapeutic agents (e.g., three types of proteins or nucleic acids).
  • a device or composition described herein comprises a cell expressing four types of therapeutic agents (e.g., four types of proteins or nucleic acids)
  • a device or composition described herein comprises a cell expressing a single type of nucleic acid (e.g., DNA or RNA) or may express more than one type of nucleic acid, e.g., a plurality of nucleic acid (e.g., DNA or RNA).
  • a device or composition described herein comprises a cell expressing two types of nucleic acids (e.g., DNA or RNA).
  • an devices and compositions comprises a cell expressing three types of nucleic acids (e.g., DNA or RNA).
  • a device or composition described herein comprises a cell expressing four types of nucleic acids (e.g., DNA or RNA).
  • a device or composition described herein comprises a cell expressing a single type of protein, or may express more than one type of protein, e.g., a plurality of proteins.
  • a device or composition described herein comprises a cell expressing two types of proteins.
  • a device or composition described herein comprises a cell expressing three types of proteins.
  • a device or composition described herein comprises a cell expressing four types of proteins.
  • a device or composition described herein comprises a cell expressing a single type of enzyme, or may express more than one type of enzyme, e.g., a plurality of enzymes.
  • a device or composition described herein comprises a cell expressing two types of enzymes.
  • a device or composition described herein comprises a cell expressing three types of enzymes.
  • a device or composition described herein comprises a cell expressing four types of enzymes.
  • a device or composition described herein comprises a cell expressing a single type of antibody or antibody fragment or may express more than one type of antibody or antibody fragment, e.g., a plurality of antibodies or antibody fragments.
  • an devices and compositions comprises a cell expressing two types of antibodies or antibody fragments.
  • a device or composition described herein comprises a cell expressing three types of antibodies or antibody fragments.
  • a device or composition described herein comprises a cell expressing four types of antibodies or antibody fragments.
  • a device or composition described herein comprises a cell expressing a single type of hormone, or may express more than one type of hormone, e.g., a plurality of hormones
  • a device or composition described herein comprises a cell expressing two types of hormones.
  • a device or composition described herein comprises a cell expressing three types of hormones.
  • a device or composition described herein comprises a cell expressing four types of hormones.
  • a device or composition described herein comprises a cell expressing a single type of enzyme, or may express more than one type of enzyme, e.g., a plurality of enzymes.
  • a device or composition described herein comprises a cell expressing two types of enzymes. In an embodiment, a device or composition described herein comprises a cell expressing three types of enzymes. In an embodiment, a device or composition described herein comprises a cell expressing four types of enzymes.
  • a device or composition described herein comprises a cell expressing a single type of cytokine or may express more than one type of cytokine, e.g., a plurality of cytokines. In an embodiment, a device or composition described herein comprises a cell expressing two types of cytokines. In an embodiment, a device or composition described herein comprises a cell expressing three types of cytokines. In an embodiment, a device or composition described herein comprises a cell expressing four types of cytokines. [000155] Diseases
  • a device or composition comprising a hydrogel and a therapeutic agent as described herein can be used to treat a variety of diseases in a mammalian subject.
  • the disease is a brain disease, a cancer, diabetes, melanoma, solid tumor, Alzheimer's disea.se, Parkinson’s disease, stroke, epilepsy, depression, pain relief, or local anesthesia.
  • the cancer can be a cancer of the bladder, blood, bone, bone marrow, breast, colon, esophagus, gastrointestinal, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus.
  • the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma, basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary’ transitional cell carcinoma; adenocarcinoma, gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma, adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant, branchiolo-alveolar adenocarcinoma; papillary' adenocarcinoma; chromophobe carcinoma
  • an alginate methacrylate and a therapeutic agent as described herein are included in a pharmaceutical composition.
  • Pharmaceutical compositions of the present invention comprise an effective amount of one or more compounds of the present disclosure, e.g., a mechanoluminescent nanoparticle or mechanoluminescent liposome, or additional agent dissolved or dispersed in a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate.
  • compositions that contains a hydrogel as described herein or additional active ingredient will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington: The Science and Practice of Pharmacy, 21 s! Ed., Lippincott Williams and Wilkins, 2005, incorporated herein by reference. Moreover, for animal (e.g., human) administration, it will be understood that preparations should typically meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards.
  • compositions may comprise, for example, a compound, composition and/or device provided by WO 2022/266086 A2, which is incorporated herein by reference.
  • the pharmaceutical compositions and/or devices described herein may be formulated in conjunction with a compound, composition and/or device provided by WO 2022/266086 A2.
  • the pharmaceutical compositions and/or devices described herein may be formulated in conjunction with a triazole-containing polymer, such as a triazole-containing alginate.
  • the triazole-containing polymer may comprise a compound according to any one of the following formulae:
  • m and n result in a number of repeating units with a molecular weight from about 50,0000 Daltons to about 500,000 Daltons.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington - 23rd Edition, October 2020, incorporated herein by reference). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the pharmaceutical compositions is contemplated.
  • the hydrogel may comprise different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form, and whether it need to be sterile for such routes of administration as injection.
  • the present invention can be administered intravenously, intradermally, transdermally, intrathecally, intraarterially, intraperitoneally, intranasally, intravaginally, intrarectally, topically, intramuscularly, subcutaneously, mucosally, orally, topically, locally, inhalation (e.g., aerosol inhalation), via injection, infusion, continuous infusion, or localized perfusion bathing target cells directly.
  • compositions may comprise, for example, at least about 0 1% of an active compound.
  • the active compound may comprise between about 2% to about 75% of the weight of the unit, or between about 25% to about 60%, for example, and any range derivable therein.
  • the amount of active compound(s) in each therapeutically useful composition may be prepared is such a way that a suitable dosage will be obtained in any given unit dose of the compound.
  • a dose may also comprise from about 1 mi crogram/kg/body weight, about 5 mi crogram/kg/b ody weight, about 10 microgram/kg/body weight, about 50 m i crogram/kg/b ody weight, about 100 microgram/kg/body weight, about 200 microgram/kg/body weight, about 350 microgram/kg/body weight, about 500 microgram/kg/body weight, about 1 milligram/kg/body weight, about 5 milligram/kg/body weight, about 10 milligram/kg/body weight, about 50 milligram/kg/body weight, about 100 milligram/kg/body weight, about 200 milligram/kg/body weight, about 350 milligram/kg/body weight, about 500 milligram/kg/body weight, to about 1000 mg/kg/body weight or more per administration, and any range derivable therein.
  • a range of about 5 mg/kg/body weight to about 100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500 milligram/kg/body weight, etc. can be administered, based on the numbers described above.
  • a composition of the present invention may be administered via a parenteral route.
  • parenteral includes routes that bypass the alimentary tract.
  • the pharmaceutical compositions disclosed herein may be administered for example, but not limited to intravenously, intradermally, intramuscularly, intraarterially, intrathecally, subcutaneous, or intraperitoneally U.S. Pat. Nos. 6,7537,514, 6,613,308, 5,466,468, 5,543, 158; 5,641,515; and 5,399,363 (each specifically incorporated herein by reference in its entirety).
  • Solutions of the active compounds as free base or pharmacologically acceptable salts may be prepared in water suitably mixed with a surfactant, such as hydroxypropyl cellulose.
  • Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (U S. Patent 5,466,468, specifically incorporated herein by reference in its entirety).
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (i.e., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils.
  • a coating such as lecithin
  • surfactants for example
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • F or parenteral administration in an aqueous solution for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration.
  • sterile aqueous media that can be employed will be known to those of skill in the art in light of the present disclosure
  • one dosage may be dissolved in isotonic NaCl solution and either added hypoderm ocly sis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences” 15th Edition, pages 1035- 1038 and 1570-1580).
  • Some variation in dosage will necessarily occur depending on the condition of the subject being treated.
  • the person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
  • Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed byfiltered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • a powdered composition is combined with a liquid carrier such as, e.g., water or a saline solution, with or without a stabilizing agent.
  • a liquid carrier such as, e.g., water or a saline solution
  • the hydrogel can be stored with or without a drug or therapeutic agent, e.g., in a solution aqueous 5% glucose (w/v).
  • the hydrogel comprising a drug or therapeutic agent is dissolved in an aqueous 5% glucose (w/v) prior to administration by injection.
  • a variety of dosages of therapeutic agents may be administered to a mammalian subject.
  • the therapeutic agents may be administered to the subject in an amount of about 5- 10 tng/kg, 0.5, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, or 15 mg/kg, or any range derivable therein.
  • a variety of drugs and therapeutic agents can be included with or encapsulated in a hydrogel as described herein.
  • the drug may be a small molecule, a biological or protein therapeutic, or enzyme.
  • the drug is an anesthetic, a volatile anesthetic, an anticancer drug or chemotherapeutic, an anti-inflammatory' drug (e.g., NSAID etc.), an analgesic drug or a painkiller.
  • the drug or therapeutic agent, and hydrogel may be comprised in a pharmaceutical formulation that includes an excipient.
  • compositions or devices described herein could be used similarly in conjunction with other standard treatments.
  • composition or device described herein may be combined with any clinically acceptable treatment article.
  • the composition or device described herein and the other clinically acceptable treatment article are used concurrently.
  • the composition or device described herein is used before the clinically acceptable treatment article.
  • the composition or device described herein is used after the clinically acceptable treatment article.
  • the composition or device described herein may be combined with any clinically acceptable treatment modality.
  • compositions comprise a prophylactically or therapeutically effective amount of a bioactive agent, and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
  • carrier refers to a diluent, excipient, or vehicle with which the therapeutic is administered.
  • Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like.
  • Water is a particular carrier when the pharmaceutical composition is administered intravenously.
  • Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly forinjectable solutions.
  • Other suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
  • compositions can also contain minor amounts of weting or emul sifying agents, or pH buffering agents.
  • These compositions can take the form of soluti ons, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like.
  • Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical agents are described in "Remington's Pharmaceutical Sciences.”
  • Such compositions will contain a prophylactically or therapeutically effective amount of the agent, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient.
  • the formulation should suit the mode of administration, which can be oral, intravenous, intraarterial, intrabuccal, intranasal, nebulized, bronchial inhalation, intra-rectal, vaginal, topical or delivered by mechanical ventilation.
  • Pharmaceutically acceptable salts include the acid salts and those which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like.
  • inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like.
  • Saks formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropyl amine, tri methylamine, 2-etbylamino ethanol, histidine, procaine, and the like.
  • a library of alginate methacrylate (A1MA) was synthesized by reacting sodium alginates of different molecular weights (UPVLVG with molecular weigh t ⁇ 75 kDa, SLG-20 with molecular weight 75 - 220 kDa and SLG-100 with molecular weight 200-300 kDa) with 2- aminoethyl methacrylate (AEMA) for 24 hours (FIG. 1 and Table 3).
  • the product was dialyzed against DI water for 3 days, filtered through 70 micron pore sized filters and freeze dried. Alginates were stored at -20 degree Celsius until further use. Alginate methacrylate was then characterized using the 1 H NMR. The characteristic peaks for the methylene protons in the region 5.5 - 6.2 ppm confirmed the methacrylation (FIG. 2). All successfully synthesized were combined at ratios of 1:0, 7:3, 6:4, 5:5, 4:6, and 3:7 with the 1 :0 formulation being 100% the material or in other words the base formulation.
  • Example 2 Hydrogel Forming Ability and Shape Retention
  • A1MA formulations were added into 96 well plates at 100 uL with 1 p.L of 1 w/v% Rhodamine Bin DMSO and then placed under UV light at 4 mW/cm"2 for 15 minutes. The plates were incubated for 10 minutes and then washed three times with DI water. Fluorescence measurements were taken (540 nm excitation, 580 nm emission) followed by gross imaging to access shape retention of the hydrogels within the 96 well plate.
  • the negative control for the study was DI water and our positive control was a previously published alginate composed from 1.4 w't/v% SL20 ionically crosslinked in barium chloride solution for 30 minutes.
  • ARPE-19 cells were seeded into black 96 well plates at 10k cells/90uL and given 24 hours to adhere and form monolayers.
  • A1MA formulations were added into 96 well at 20 pL per well along with 60 pL of cell culture media. Alamar blue was then added into the wells at 10 pL per well and plates were incubator at 37 °C for 3 hours. Fluorescnce readings using TECAN plate reader (570 nm for measurement wavelength and 600 nra for reference wavelength).
  • ARPE-19 ells in phenol red-free media alone were used as the positive control.
  • Example 3 Mechanical Testing
  • A1MA formulations were added into 250 pl PDMS molds and placed under UV exposure at 4mW/cm"2 for 15 minutes. These Al MA gels were retrieved from the PDMS mold and used for compression testing. Compression testing may be performed by applying constant crosshead speed 10 pmis. Compression testing may be performed until fracture or until half the height of the hydrogel has been compressed. Stress vs. strain and modulus may be collected from the compression testing. Modulus may be obtained from the slope of stress vs. strain, limited to the first 15% of strain.
  • Example 4 Generation and screening of alginate methacrylate (A1MA) formulation library yields stable hydrogels for RPE-VEGFC cell factory encapsulation
  • A1MA alginate methacrylates
  • VLVG very low viscosity
  • LVG-20 low viscosity
  • AEMA 2- aminoethyl methacrylate
  • Modulus was measured from the slope of stress vs. strain, limited to the fracture point.
  • VLVG-M46 and LV20-M20 leading A1MA hydrogel formulations
  • RPE-19 cells were engineered to secrete VEGF-C growth factor via a PiggyBac vector system with Lipofectamine 3000 (ThermoFisher, &L3000001) to transfect DNA inside RPE cells.
  • Alginate methacrylate (A1MA) was synthesized by reacting sodium alginates of different molecular weights (UP VLVG with molecular weight ⁇ 75 kDa, SLG-20 with molecular weight 75 kDa - 220 kDa and SLG-100 with molecular weight 200 kDa-300 kDa) with 2- aminoethyl methacrylate (AEMA) for 24 h.
  • AEMA 2- aminoethyl methacrylate
  • the product was dialyzed against DI water for 3 days, filtered through 70 micron pore sized filters and freeze dried. Alginates were stored at -20 degree Celsius until further use. Alginate methacrylate was then characterized using the 1H NMR. The characteristic peaks for the methylene protons in the region 5.5 - 6.2 ppm confirmed the methacrylation.
  • A1MA formulations are added into 96 well plates at lOOuL with luL of lw/v% Rhodamine B in DMSO and then placed under UV light at 4mW/cm A 2 for 15minutes. The plates are incubated for 10 minutes and then washed three times with DI water. Fluorescence measurements were taken (540nm excitation, 580nm emission) followed by gross imaging to access shape retention of the hydrogels within the 96 well plate. The negative control for the study was DI water and our positive control was a previously published alginate composed from 1.4wt/v% SL20 ionically crosslinked in barium chloride solution for 30 minutes. Gelation was determined by the fluorescence intensity averaging above 15,000 AUG,
  • A1MA formulations were added into 96 well plates at lOOuL per well and placed under UV exposure at 4mW/cm A 2 for 15 minutes. These A1MA gels were retrieved from the 96 well plate and used for compression testing. Compression testing will be performed by applying constant crosshead speed 1.0 cm/min and 10N. Compression test will be performed until fracture. Stress vs strain and modulus will be collected from the compression testing. Modulus will be obtained from the slope of stress vs strain, limited to the first 2% of strain.
  • ARPE-19 cells were seeded into black 96 well plates at 10k cells/90uL and given 24 hours to adhere and form monolayers.
  • A1MA formulations were added into 96 well at 20uL per well along with 60uL of cell culture media. Alamar blue was then added into the wells at lOuL per well and plates were incubator at 37C for 3 hours. Fluorescence readings using TECAN plate reader (570 nm for measurement wavelength and 600 nm for reference wavelength).
  • ARPE-19 ells in phenol red-free media alone were used as the positive control.
  • compositions and devices disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and devices of this disclosure have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and devices and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the disclosure. More specifically, it wall be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the disclosure as defined by the appended claims.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Molecular Biology (AREA)
  • Medicinal Preparation (AREA)

Abstract

The present disclosure describes a device and/or composition comprising a hydrogel system, such as an alginate methacrylate hydrogel system, and a therapeutic agent. The device and or composition may contain one or more therapeutic agents, such as an engineered cell or protein, a secreted protein, a hormone, a cytokine, an antibody, an enzyme or a peptide Also described herein is a method of treating a disease or disorder using a device and/or composition comprising a hydrogel system and a therapeutic agent.

Description

PHOTO-RESPONSIVE THERAPEUTIC ENCAPSULATION
COMPOSITIONS AND DEVICES
[0001] This application is an International Application which claims priority from U.S, provisional patent application no. 63/593,813, filed on October 27, 2023, the entire contents of which are incorporated herein by reference.
[0002] All patents, patent applications and publications cited herein are hereby incorporated by reference in their entirety'. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.
[0003] This patent disclosure contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure as it appears in the U.S. Patent and Trademark Office patent file or records, but otherwise reserves any and all copyright rights.
BACKGROUND OF THE INVENTION
[0004] Field of the Invention
[0005] The present disclosure relates to the fields of biology, medicine, bioengineering and medical devices. More particular, it relates to the development and use of hydrogels designed to deliver therapeutic agents to a subject. In particular, the hydrogels comprise an alginate methacrylate polymer.
[0006] Description of Related Art
[0007] Encapsulation within semi-permeable hydrogels represents a local immuno-i solation strategy for many therapies without the need for systemic immunosuppression (Chang, 1964; Lim and Sun, 1980). The hydrogel sphere may facilitate the diffusion of nutrients necessary for cell function while excluding immune cells that would reject the foreign cells. Alginate spheres are one of the most widely investigated cell encapsulation materials because this anionic polysaccharide forms a hydrogel in the presence of divalent cations under cell friendly conditions. This natural co-polymer can then exhibit differential physical properties depending on the ratio and sequential arrangement of mannuronic and guluronic acid residues, the molecular weight, the concentration and the divalent cations used to form the gels (Strand et al., 2017). For instance, alginate with a high guluronic-block content has a higher binding affinity for barium ions and will form a tighter, more stable network compared with the same alginate type crosslinked with calcium (Haug and Smidsrod, 1970).
[0008] Additionally, electrostatic complexation of a positively charged polymer to the negatively charged alginate surface can provide an outer layer to reduce sphere porosity and increase sphere stability (Kollmer el al., 2015). A final outer alginate layer or chemical modification of the polycation used for coating has been investigated to reduce the positive surface charge density of the sphere (Kollmer et al., 2015; Mooranian etal., 2016; Kleinberger etal., 2016). Many alginate sphere formulations are produced with variations in the alginate concentration, the crosslinking ion or the inclusion or exclusion of a polycation layer.
[0009] However, one critical drawback of ionically cross-linked alginate gels is the limited long- term stability in physiological conditions, because these gels can be dissolved due to release of divalent ions into the surrounding media due to exchange reactions with monovalent cations. In addition, the calcium ions released from the gel may promote hemostasis, while the gel serves as a matrix for aggregation of platelets and erythrocytes (Lee and Mooney, 2012; Suzuki et al., 1998) Finally, limited control over the rate of biodegradation and brittleness remains an issue. Thus, there exists a need for mechanically stable alginate methacrylate hydrogels that are crosslinkable, permeable and support cell viability.
SUMMARY OF THE INVENTION
[00010] In some aspects, the present disclosure provides a composition comprising:
(i) a hydrogel system, wherein said system comprises at least one of A, B or C, wherein:
A is a photo-responsive alginate, wherein the molecular weight is less than 95 kDa and wherein the methacrylation efficiency is from about 15% to about 95%;
B is a photo-responsive alginate, wherein the molecular weight is from about 55 kDa to about 240 kDa and wherein the methacrylation efficiency is from about 1% to about 65%; and C is a photo-responsive alginate, wherein the molecular weight is from about 180 kDa to about 320 kDa and wherein the methacrylation efficiency from about 1% to about 25%; and
(11) a therapeutic agent.
[00011] In some embodiments, the hydrogel system comprises a monomer of the formula:
Figure imgf000004_0001
wherein n and m are at least I .
[00012] In some embodiments, n is 1-5. In some embodiments, m is 1-5. In some aspects, n and m are the same.
[00013] In some aspects, the present disclosure provides a drug delivery device comprising:
(i) a hydrogel system, wherein said system comprises at least one of A, B or C, wherein:
A is a photo-responsive alginate, wherein the molecular weight is less than 95 kDa and wherein the methacrylation efficiency is from about 15% to about 95%;
B is a photo-responsive alginate, wherein the molecular weight is from about 55 kDa to about 240 kDa and wherein the methacrylation efficiency is from about 1% to about 65%, and
C is a photo-responsive alginate, wherein the molecular weight is from about 180 kDa to about 320 kDa and wherein the methacrylation efficiency from about 1% to about 25%, and
(ii) a therapeutic agent. [00014] In some aspects, the methacryl ati on efficiency of A is about 20%, about 46%, about 60% or about 90%. In some embodiments, the methacrylation efficiency of B is about 5%, about 10%, about 20%, about 46% or about 60%. In some embodiments, the methacrylation efficiency of C is about 5%, about 10% or about 20%.
[00015] In some embodiments, A is combined with B at a ratio of about 1:0 to about 0: 1; A is combined with C at a ratio of about 1 :0 to about 0: 1; or B is combined with C at a ratio of about 1 :0 to about 0:1. In some embodiments, A is combined with Bat a ratio of about 7:3 to about 3:7, A is combined with C at a ratio of about 7:3 to about 3:7; or B is combined with C at a ratio of about 7:3 to about 3:7. In some embodiments, A is combined with B at a ratio of about 7:3; /\ is combined with Cat a ratio of about 7:3; or Bis combined with Cat a ratio of about 7:3. In some embodiments, A is combined with B at a ratio of about 6:4; A is combined with C at a ratio of about 6:4; or Bis combined with Cat a ratio of about 6:4. In some embodiments, A is combined with Bat a ratio of about 5:5: A is combined with Cat a ratio of about 5:5; or Bis combined with Cat a ratio of about 5:5. In some embodiments, A is combined with Bat a ratio of about 4:6; A is combined with Cat a ratio of about 4:6; or Bis combined with Cat a ratio of about 4:6. In some embodiments, A is combined with B at a ratio of about 3:7, A is combined with C at a ratio of about 3:7; or Bis combined with Cat a ratio of about 3:7. In some embodiments, Bis combined with Cat a ratio of about 7:3.
[00016] In some embodiments, the therapeutic agent comprises an engineered cell or protein, a secreted protein, a hormone, a cytokine, an antibody, an enzyme or a peptide. In certain embodiments, the therapeutic agent comprises an engineered cell
[00017] In some aspects, the present disclosure provides a method of treating a disease or disorder in a patient in need thereof comprising administering to the patient a device or composition described herein.
[00018] Other objects, features, and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. BRIEF DESCRIP TION OF THE DRAWINGS
[00019] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
[00020] FIG. 1. .Alginate methacrylate synthesis
[00021] FIG. 2. Alginate methacrylate NMR
[00022] FIG. 3. AIM: A formulation gelation assay
[00023] FIG. 4. A1MA formulation hydrogel shape retention
[00024] FIG. 5. Absolute weight change values of AIM A formulations in solution after two weeks
[00025] FIG. 6. ARPE-19 cell viability of A1MA base formulations
[00026] FIG. 7. Mechanical strength of AIM A formulations
[00027] FIG. 8. Molding platform for A1MA formulation of varying shapes and sizes [00028] FIG. 9. PDMS molding device
[00029] FIG. 10 Programmable RPE-VEGF-C cell therapy enables stable and local production with inducible safety switch for termination. a, Overall schematic of engineered RPE-VEGF-C cells with inducible small molecule for cell death, b, VEGF-C production of VEGF-C engineered (RPE-VEGF-C) and non-engineered cell lines over 24 hours in culture, c, VEGF-C production over time in RPE-VEGFC cell lines in culture, d, schematic of in vivo experiments of RPE-VEGF-C with and without engineered kill switch, e, fluorescence imaging of cell viability, f, in vivo VEGF-C production over 24 hours, g, RPE-VEGF-C viability and production with engineered kill switch at 10 pM, IpM, lOOnM, lOnM, InM, O.lnM, O.OlnM, and OnM of small molecule activator, h, fluorescence imaging of cell viability, i, cell viability with and without engineered kill switch, j, rVEGF-C concentration with and without engineered kill switch.
[00030] FIG. 11 Development of A1MA library and high-throughput screen to enable leading materials for RPE-VEGFC cell therapy, a, screening strategy for A1MA library, b, shape retention of ionically crosslinked alginate with barium chloride, and A1MA at different methacrylation efficiencies upon UV crosslinking, c, gelation assay with Rhodamine B loaded A1MA formulation samples, d, mechanical strength of A1MA library formulations, e, AUC of weight alteration, f, VEGF-c diffusion in A1MA gels after 24 hours.
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[00031] Described herein are compositions of matter comprising photo-responsive library platforms that can be fabricated into devices for management of diseases. In some embodiments, the compositions and/or devices described herein are hydrogels. In some embodiments, the hydrogels are alginate methacrylate formulations that are crosslinkable, mechanically stable, permeable and support cell viability.
[00032] In certain aspects, the present disclosure describes a library of alginate methacrylate (A1MA) synthesized by reacting sodium alginates of different molecular weights (UP VLVG with molecular weight < 75 kDa, SLG-20 with molecular weight 75 - 220 kDa and SLG-100 with molecular weight 200-300 kDa) with 2-aminoethyl methacrylate (AEMA) and having different methacrylate efficiency. In some aspects, the A1MA formulations may utilize the nomenclature described in Table 1 below.
[00033] Table 1. Proposed Nomenclature for AIM A Formulations at 2wt/v% (VLV = very low viscosity; LV = low viscosity; M = methacrylation)
Figure imgf000007_0001
[00034] In some aspects the Al MA formulations may be combined at ratios of 1 :0, 7:3, 6:4, 5:5, 4:6, 3:7 or 0: 1. in preferred embodiments, the AIMA formulations may be combined at the ratios described by Table 2. These and other aspects of the disclosure are described in detail below. [00035] Table 2. Preferred AIMA Formulations
Figure imgf000008_0001
Figure imgf000009_0001
Figure imgf000010_0001
[00036] DEFINITIONS
[00037] The use of the word "a" or "an" when used in conjunction with the term
"comprising" in the claims and/or the specification may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one." The word "about" means plus or minus 5% of the stated number. "Heal" as used herein refers to the partial or complete restoration of a cell or tissue containing a wound, e.g., a wound described herein.
[00038] "Alginate" is a collective term used to refer to linear polysaccharides formed from -D- mannuronate and a-L-guluronate in any MIG ratio, as well as salts and derivatives thereof. The term "alginate", as used herein, encompasses any polymer having the structure shown by Formula I, as well as salts thereof
[00039] "Hydrogel" refers to a substance formed when an organic polymer (natural or synthetic) is cross-linked via covalent, ionic, or hydrogen bonds to create a three-dimensional open-lattice structure which entraps water molecules to form a gel. Biocompatible hydrogel refers to a polymer forms a gel which is not toxic to living cells and allows sufficient diffusion of oxygen and nutrients to the entrapped therapeutic agent to maintain viability.
[00040] When a range of values is listed, it is intended to encompass each value and sub-range within the range. For example, "n is 1 -5" is intended to encompass, n = 1 , n ~ 2, n = 3, n = 4 and n= 5.
[00041] The terms "subject" or "patient," as used herein, refer to an individual bearing a wound and/or the recipient of a wound healing device described herein. The subject may include a human (i.e., a. male or female of any age group, e.g.. a pediatric subject (e.g., infant, child, adolescent) or adult subject (e.g., young adult, middle-aged adult, or senior adult)) and/or other non-human animals, for example, mammals (e.g., primates (e.g., cynomolgus monkeys, rhesus monkeys): commercially relevant mammals such as cattle, pigs, horses, sheep, goats, cats, and/or dogs) and birds (e.g., commercially relevant birds such as chickens, ducks, geese, and/or turkeys). In certain embodiments, the animal is a mammal. The animal may be a male or female and at any stage of development. A non-human animal may be a transgenic animal.
[00042] As used herein, the terms "treatment," "treat," and "treating" refer to reversing, alleviating, delaying the onset of or inhibiting the progress of one or more of a symptom, manifestation, or underlying cause of, e.g., a wound, e.g., as described herein. Treating may entail administering or applying the wound healing device described herein. In an embodiment, treating comprises reducing, reversing, alleviating, delaying the onset of, or inhibiting the worsening of a wound in a subject. In some embodiments, "treatment," "treat," and "treating" require that signs or symptoms of the disease, disorder, or condition have developed or have been observed. In other embodiments, treatment may be administered in the absence of signs or symptoms of the disease or condition, e.g., in preventive treatment. In some embodiments, treatment comprises prevention and in other embodiments it does not.
[00043] T he terms "applying" and "administering" are used interchangeably to refer to causing a subject to receive a treatment. For example, administering a therapeutic agent to the subject means moving the particle inside the body of the subject. The terms active agent, active pharmaceutical ingredient, pharmacologically active agent, and drug are used interchangeably herein to refer to a bioactive material or compound which, when administered to an organism (human or animal) induces a desired pharmacologic and/or physiologic effect by local and/or systemic action.
[00044] As used herein, the terms "determining," "measuring," "assessing," and "assaying" are used interchangeably and include both quantitative and qualitative determinations.
[00045] As used herein, the terms "acrylation" and "methaciylation" refer to the addition of an acrylate or methacrylate group respectively. The term "methaciylation efficiency" refers to the number of methacrylate groups added to a monomer or polymer described herein. Methacryl ation efficiency is represented as a percentage of the polymer that has been substituted with a methacrylate unit.
[00046] A "therapeutically effective amount", a "therapeutically effective dose" or "therapeutic dose" is an amount sufficient to effect desired clinical results (e.g., achieve therapeutic efficacy, achieve a desired therapeutic response). A therapeutically effective dose can be administered in one or more administrations. For purposes of this disclosure, a therapeutically effective dose of a compositions is an amount that is sufficient, when administered to the individual, to palliate, ameliorate, stabilize, reverse, prevent, slow or delay the progression of a disease state (e.g., neurodegenerative disease, etc ) present in the subject.
[00047] The chemical structures and formulae set forth herein are constructed according to the standard rules of chemical valency known in the chemical and biological arts. Also, all publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety.
[00048] .A "pharmaceutically acceptable excipient," "pharmaceutically acceptable diluent," "pharmaceutically acceptable carrier," and "pharmaceutically acceptable adjuvant" means an excipient, diluent, carrier, and adj uvant that are useful in preparing a pharmaceutical composition that are generally safe, non-toxic and neither biologically nor otherwise undesirable, and include an excipient, diluent, carrier, and adjuvant that are acceptable for veterinary' use as well as human pharmaceutical use. "A pharmaceutically acceptable excipient, diluent, carrier and adjuvant" as used in the specification and claims includes both one and more than one such excipient, diluent, carrier, and adjuvant.
[00049] As used herein, a "pharmaceutical composition" is meant to encompass a composition suitable for administration to a subject, such as a mammal, especially a human. In general a "pharmaceutical composition" is sterile, and preferably free of contaminants that are capable of eliciting an undesirable response within the subject (e.g, the compound(s) in the pharmaceutical composition is pharmaceutical grade). Pharmaceutical compositions can be designed for administration to subjects or patients in need thereof via a number of different routes of administration including intravenous, buccal, rectal, parenteral, intraperitoneal, intradermal, intratracheal, intramuscular, subcutaneous, and the like.
[00050] The terms "co-administration" and "in combination with" include the administration of two or more therapeutic agents either simultaneously, concurrently or sequentially. In one embodiment, the agents are present in the cell or in the subject's body at the same time or exert their biological or therapeutic effect at the same time. In one embodiment, the therapeutic agents are in the same composition or unit dosage form. In other embodiments, the therapeutic agents are in separate compositions or unit dosage forms. In certain embodiments, a first agent can be administered prior to (e.g., minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, I hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of a second therapeutic agent.
[00051] The compounds disclosed herein may also comprise one or more isotopic substitutions. For example, H may be in any isotopic form, including !H, 2H (Dor deuterium), and 3H (Tor tritium); C may be in any isotopic form, including 12C, 1JC, and ,4C; 0 may be in any isotopic form, including 160 and 180, and the like.
[00052] Polymer," as used herein, refers to a molecule comprising one or more repeating units, or monomers. Polymers may comprise only one species of repeating unit, i.e., a " homopolymer" as used herein, or may comprise multiple species of repeating units, i.e., a "heteropolymer." For example, polyethylene oxide is a homopolymer of ethylene oxide monomers.
[00053] Devices and Compositions
[00054] The compositi ons/devices of the present disclosure comprising a therapeutic agent have an inner housing chamber with which the therapeutic agents are housed. This in turn is surrounded by a biocompatible supporting structure/latice or are disposed in a capsule formed from a biocompatible material. The following discussion of materials and methods for production relate to these compositions/ devices.
[00055] The inventors contemplate the use of alginates to create protective coatings for therapeutic agents contained in the devices and capsules according to the present disclosure. In some embodiments, the alginate is SLG20. In some embodiments, the SLG20 is about 0. 1%- 3% SLG20.
[00056] One embodiment provides for the use of modified alginate polymers in the construction of therapeutic agent-containing devices. Modified alginate polymers can be of any desired molecular weight. The weight average molecular weight of the alginates is preferably between 1 ,000 and 1 ,000,000 Daltons, more preferably between 10,000 and 500,000 Daltons as determined by gel permeation chromatography.
[00057] Modified alginate polymers can contain any ratio of covalently modified monomers. In some embodiments, greater than 2.5%, 5%, 7.5%, 10%, 12%, 14%, 15%, 16%, 18%, 20%, 22%, 24%, 25%, 26%, 28%, 30%, 32.5%, 35%, 37.5%, 40%, 45%, 50%, 55%, or 60% of the monomers in the modified alginate polymer are covaleritiy modified monomers. Greater than 10%, greater than 20%, or greater than 30% of the monomers in the modified alginate polymer are covalently modified monomers.
[00058] Modified alginate polymers can be produced incorporating covalently modified monomers possessing a range of different hydrogen bonding potentials, hydrophobicities/hydrophilicilies, and charge states. The inclusion of covalently modified monomers into an alginate polymer alters the physiochemical properties of alginate polymer. Accordingly, the physiochemical properties of alginates can be tuned for desired applications by the selective incorporation of covalently modified monomers.
[00059] For example, the glass transition temperature (Tg), can be varied by the incorporation of covalently modified monomers. In some embodiments, the modified alginate polymer powder possess a Tg, as measured by differential scanning calorimetry (DSC), of greater than 50°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, 90°C, 95°C, 100°c, 105°C, 110°c, 115°C, 120°c, 125°C, 130°C, 135°C, 140°C, 145°C, 150°C, 160°C, 175°C, 190°C, or200°c. [00060] The hydrophobicity/hydrophilicity of alginates can be varied by the incorporation of hydrophobic and/or hydrophilic covalently modified monomers. In particular embodiments, the modified alginate polymer contains one or more hydrophobic covalently modified monomers. The relative hydrophobicity/hydrophilicity of modified alginates can be quantitatively assessed by measuring the contact angle of a water droplet on a film of the modified alginate polymer using a goniometer. In some embodiments, the modified alginate has a contact angle of less than 90° (i.e. it is hydrophilic). In particular embodiments, the modified alginate has a contact angle of more than 90° (i.e., it is hydrophobic). In some embodiments, the modified alginate has a contact angle of more than 95°, 100°, 105°, 110°, 115°, or 120°.
[00061] In embodiments used for cell encapsulation, the modified alginate polymer can be ionically crosslinked by a polyvalent cation such as Ca^+, Sr^+, or Ba^+ to form hydrogels. [00062] In some embodiments, the modified alginate polymer forms hydrogels such that the fluorescence intensity measured using the high throughput hydrogel formation assay described herein is greater than 10,000, 15,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, or 55,000.
[00063] In particular embodiments, the modified alginate polymer forms hydrogels such that the fluorescence intensity measured using the high throughput hydrogel formation assay described herein is greater than 15,000. In particular embodiments, the modified alginate polymer forms hydrogels such that the fluorescence intensity measured using the high throughput hydrogel formation assay described herein is between 15,000 and 55,000, preferably between 20,000 and 55,000, more preferably between 25,000 and 55,000.
[00064] The porosity and surface area of modified alginates can be measured using BET analysis. Prior to BET analysis, solvent and volatile impurities are removed by prolonged heating of the modified alginate gel under vacuum. Subsequently, the hydrogel samples are cooled under vacuum, for example by liquid nitrogen, and analyzed by measuring the volume of gas (typically N2, Kr, CO2, or Ar gas) adsorbed to the hydrogel at specific pressures. Analysis of the physisorption of the gas at variable pressures is used to characterize the total surface area and porosity of gels formed by the modified alginate polymers. A particular method of determining hydrogel porosity is BET analysis
[00065] In particular embodiments, the modified alginate forms a hydrogel with sufficient porosity to permit nutrients, waste, and the hormones and/or proteins secreted from encapsulated cells to diffuse freely into and out of the capsules, while simultaneously preventing the incursion of immune cells into the gel matrix. In some embodiments, the porosity of the hydrogel formed by the modified alginate polymer is increased by 5%, 10%, 15%, or 20% relative to the porosity of a hydrogel formed from the unmodified alginate polymer. In alternative embodiments, the porosity of the hydrogel formed by the modified alginate polymer is decreased by 5%, 10%, 15%, or 20% relative to the porosity of a hydrogel formed from the unmodified alginate polymer,
[00066] In particular embodiments used for cell encapsulation, the modified alginate is biocompatible. The biocompatibility of modified alginates can be quantitatively determined using a fluorescence-based in vivo biocompatibility assay.
[00067] In some embodiments, the modified alginate polymer is biocompatible such that the fluorescence response normalized to unmodified alginate measured using the in vivo biocompatibility assay described herein is less than 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%. In particular embodiments, the modified alginate polymer induces a lower foreign body response than unmodified alginate. This is indicated by fluorescence response normalized to unmodified alginate of less than 100%. In some embodiments, the modified alginate polymer is biocompatible such that the fluorescence response normalized to unmodified alginate measured using the in vivo biocompatibility assay described herein is less than 75%, more preferably less than 65%, and most preferably less than 50%.
[00068] The modified alginates can be chemically modified as described herein to any desired density of modifications. The density of modifications is the average number of modifications (that is, attached compounds) per a given weight volume, or area of the surface of a capsule or product that includes the modified alginate. Generally, a density at or above a threshold density can provide a beneficial effect, such as lower foreign body response. In some embodiments, a high density is not required. Without being bound to any particular theory of operation, it is believed that the chemical modifications signal to, indicate to, or are identified by, one or more immune system or other body components to result in a beneficial effect, such as a lower foreign body response. In some embodiments, a lower density of modifications can be effective for this purpose.
[00069] Useful densities include densities of at least, of less than, of about, or of 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19,20,25,30,35,40,45,50,55,60,65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 550, 600, 650, 700, 750, 800, 850, 900, and 1000 modifications per square um, per ug, or per cubic gm. All ranges defined by any pair of these densities are also specifically contemplated and disclosed.
[00070] In some embodiments, the density of the modifications on a surface, surfaces, or portions of a surface(s) of a capsule or product that, when the product is administered to (e.g., implanted in the body of) a subject, would be in contact with fluid(s), cell(s), tissue(s), other component(s), or a combination thereof of the subject's body is greater than the density of the modifications on other surfaces of the product.
[00071] Density can also be expressed in terms of the concentration of the surface modifications as measured by X-ray photoelectron spectroscopy (XPS). XPS is a surfacesensitive quantitative spectroscopic technique that measures the elemental composition at the parts per thousand range of the elements that exist within a material .
[00072] XPS spectra are obtained by irradiating a material with a beam of X-rays while simultaneously measuring the kinetic energy and number of electrons that escape from the top O to 10 nm of the materia! being analyzed. By measuring all elements present on the surface, the percentage of the elements that come from the surface modifications can be calculated. This can be accomplished by, for example, taking the percentage of nitrogen (and/or other elements in the surface modifications) in the total elemental signal measured. Nitrogen is a useful indicator for the surface modification because many substrated and materials forming the capsule or product contain little nitrogen. For convenience, the percent of the element(s) used to indicate the surface modifications can be stated as the percent surface modifications. Also for convenience, the percent surface modifications can be referred to as the concentration of surface modifications.
[00073] I Jseful percent surface modifications include concentrations of about, less than or at O. l , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, and 100 percent surface modifications. All ranges defined by any pair of these concentrations are also specifically contemplated and disclosed.
[00074] In one aspect, the alginates may be used to form a capsule. Capsules are particles having a mean diameter of about 150 pm to about 5 cm. The disclosed capsules can be formed of cross-linked hydrogel. Other than the encapsulated material, the capsules, for example, can be formed solely of cross-linked hydrogel, can have a cross-linked hydrogel core that is surrounded by one or more polymeric shells, can have one or more cross-linked hydrogel layers, can have a cross- linked hydrogel coating, or a combination thereof The capsule may have any shape suitable for, for example, cell encapsulation. The capsule may contain one or more cells dispersed in the cross-linked hydrogel, thereby "encapsulating" the cells. Particular capsules are formed of or include one or more of the disclosed modified alginates.
[00075] Capsules can have a mean diameter of about 150 pm to about 8 mm. Capsules can have any mean diameter from about 150 pm to about 5 cm. In particular, the capsules have a mean diameter that is greater than 1 mm, more particularly 1.5 mm or greater. In some embodiments, the capsules can be as large as about 8 mm in diameter. E?or example, the capsule can be in a size range of about 1 mm to 8 mm, I ram to 6 mm, 1 mm to 5 mm, 1 mm to 4 mm, 1 mm to 3 mm, 1 mm to 2 mm, 1 mm to 1.5 mm, 1 .5 mm to 8 mm, 1 .5 mm to 6 mm, 1 .5 mm to 5 mm, 1.5 ram to 4 mm, 1.5 mm to 3 mm, or 1.5 mm to 2 mm. [00076] T 'he rate of molecules entering the capsule necessary for cell viability and the rate of therapeutic products and waste material exiting the capsule membrane can be selected by modulating capsule permeability. Capsule permeability can also be modified to limit entry of immune cells, antibodies, and cytokines into the capsule. Generally, as shown by the examples, known methods of forming hydrogel capsules can produce capsules the permeability of which limit entry of immune cells, antibodies, and cytokines into the capsule. Since different cell types have different metabolic requirements, the permeability of the membrane can be optimized based on the cell type encapsulated in the hydrogel. The diameter of the capsules is an important factor that influences both the immune response towards the cell capsules as well as the mass transport across the capsule membrane.
[00077] The unmodified alginate typically has a weight average molecular weight of about 50,000 Daltons to about 500,000 Daltons; however, unmodified alginates having molecular weights outside this range can also be used. In some embodiments, the average molecular weight is less than 95,000 Daltons, preferably less than 75,000 Daltons. In some embodiments, the average molecular weight is from about 50,000 to about 250,000 Daltons, preferably from about 75,000 Daltons to about 220,000 Daltons. In some embodiments, the average molecular weight is from about 175,000 to about 325,000 Daltons, preferably from about 200,000 Daltons to about 300,000 Daltons.
[00078] In other embodiments, one or more additional hydrogel-fonning polymers are used in combination with unmodified alginate or in place of unmodified alginate. Such polymers are known in the art. Examples include, but are not limited to, PEG, chitosan, dextran, hyaluronic acid, silk, fibrin, polyfvinyl alcohol) and polyfhydroxyl ethyl methacrylate).
[00079] The particles prepared from a mixture of modified alginate and unmodified alginate produced more homogenous microparticle populations in terms of shape and size as evaluated by scanning electron microscopy (SEM).
[00080] In some embodiments, the hydrogel capsules can have any suitable shape. Useful shapes include spheres, sphere-like shapes, spheroids, spheroid-like shapes, ellipsoids, ellipsoid-like shapes, stadiumoids, stadiumoid-like shapes, disks, disk-like shapes, cylinders, cylinder-like shapes, rods, rod-like shapes, cubes, cube-like shapes, cuboids, cuboid-like shapes, toruses, torus-like shapes, and flat and curved surfaces. Products, devices, and surfaces that have been or will be coated can have any of these shapes or any shape suitable for the product or device.
[00081] Spheres, spheroids, and ellipsoids are shapes with curved surfaces that can be defined by rotation of circles, ellipses, or a combination around each of the three perpendicular axes, a, b, and c. For a sphere, the three axes are the same length. For oblate spheroids (also referred to as oblate 15 ellipsoids of rotation), the length of the axes are a;::: b > c. For prolate spheroids (also referred to as prolate ellipsoids of rotation ), the length of the axes are a= b < c. For tri-axial ellipsoids (also referred to as scalene ellipsoids), the length of the axes are a> b > c. Stadium oi ds are rotational shapes of stadiums. Cylinders are rotational shapes of rectangles rotated on the long axis. Disks are squashed cylinders where the diameter is greater than the height. Rods are elongated cylinders where the long axis is ten or more times the diameter.
[00082] "Sphere-like shape," "spheroid-like shape," "ellipsoid-like shape," "stadiumoid- like shape," "cylinder-like shape," "rod-like shape," "cube-like shape," "cuboid-like shape," and "torus- like shape" refers to an object having a surface that roughly forms a sphere, spheroid, ellipsoid, stadiumoid, cylinder, rod, cube, cuboid, or torus, respectively. Beyond a perfect or classical form of the shape, a sphere-like shape, spheroid-like shape, ellipsoid-like shape, stadiumoid-like shape, cylinder-like shape, rod- like shape, cube-like shape, cuboid-like shape, and torus-like shape can have waves and undulations.
[00083] Generally, a sphere-like shape is an ellipsoid (for its averaged surface) with seraiprincipal axes within 10% of each other. The diameter of a sphere or sphere-like shape is the average diameter, such as the average of the semi-principal axes. Generally, a spheroid-like shape is an ellipsoid (for its averaged surface) with semi-principal axes within 100% of each other. The diameter of a spheroid or spheroid-like shape is the average diameter, such as the average of the semi-principal axes. Generally, an ellipsoid-like shape is an ellipsoid (for its averaged surface) with semi-principal axes within 100% of each other. The diameter of an ellipsoid or ellipsoid-like shape is the average diameter, such as the average of the semiprincipal axes. Generally, a stadiumoid-like shape is a stadiumoid (for its averaged surface) with semi-principal axes of the ends within 20% of each other. The diameter of a stadiumoid or stadiumoid-like shape is the average diameter, such as the average of the semi -principal axes. Alternatively, the size of a stadiumoid or stadiumoid-like shape can be given as the average of the long axis. Generally, a cylinder-like shape is a cylinder (for its averaged surface) with semi- principal axes within 20% of each other. The diameter of a cylinder or cylinderlike shape is the average diameter, such as the average of the semi-principal axes.
[00084] Alternatively, the size of a cylinder or cylinder-like shape can be given as the average of the long axis. Generally, a rod-like shape is a rod (for its averaged surface) with semi- principal axes within 10% of each other. The di ameter of a rod or rod-like shape is the average diameter, such as the average of the semi-principal axes. Alternatively, the size of a rod or rod-like shape can be given as the average of the long axis. Generally, a cubelike shape is a cube (for its averaged surface) with sides within 10% of each other The diameter of a cube or cube-like shape is the average side length. Generally, a cuboid-like shape is a cuboid (for its averaged surface) with matching sides within 10% of each other. The diameter of a cuboid or cuboid-like shape is the average side length.
[00085] Generally, a torus-like shape is a torus (for its averaged surface) with semiprincipal axes within 10% of each other. The diameter of a torus or toms-like shape is the average diameter, such as the average of the semi -principal axes. Alternatively, the size of a toms or torus-like shape can be given as the diameter across the ring.
[00086] “Flat side" refers to a contiguous area of more than 5% of a surface that has a curvature of 0. "Sharp angle" refers to a location on a surface across which the tangent to the surface changes by more than 10% over a distance of 2% or less of the circumference of the surface. Edges, comers, grooves, and ridges in a surface are all forms of sharp angles.
[00087] Particular capsules can be made of biocompatible materials, have a diameter of at least 1 mm and less than 10 mm, has a spheroid-like shape, and have one or more of the additional characteristics: surface pores of the capsules greater than 0 nm and less than 10 um; surface of the capsules neutral or hydrophilic; curvature of the surface of the capsules at least 0.2 and is not greater than 2 on all points of the surface; and surface of the capsules lacking flat sides, sharp angles, grooves, or ridges. Generally, the capsules elicit less of a fibrotic reaction after implantation than the same capsules lacking one or more of these characteristics that, are present on the capsules.
[00088] In some embodiments, the capsules are provided as a preparation and at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the capsules in the preparation have a shape characteristic described herein, e.g., have a spheroidlike shape, or have a curvature of the surface of at least 0.2 to 2.0 on all points of the surface. [00089] In some embodiments, the hydrogel capsules have a mean diameter that is greater than 1 mm, particularly 1.5 mm or greater. In some embodiments, the hydrogel capsules can be as large as 8 ram in diameter. For example, the hydrogel capsules is in a size range of 1 mm to 8 mm, 1 mm to 6 mm, 1 mm to 5 mm, 1 mm to 4 mm, 1 mm to 3 mm, 1 mm to 2 mm,
1 mm to 1.5 mm, 1.5 mm to 8 mm, 1.5 mm to 6 mm, 1.5 mm to 5 mm, 1.5 mm to 4 mm, 1.5 mm to 3 mm, 1.5 mm to 2 mm, 2 mm to 8 mm, 2 mm to 7 mm, 2 mm to 6 mm, 2 mm to 5 mm,
2 mm to 4 mm, 2 mm to 3 mm, 2.5 mm to 8 mm, 2.5 mm to 7 mm, 2.5 mm to 6 ram, 2.5 mm to 5 mra, 2.5 mm to 4 mm, 2.5 mm to 3 mm, 3 mm to 8 ram, 3 mm to 7 mra, 3 mm to 6 mm,
3 mm to 5 mm, 3 mm to 4 mm, 3.5 mm to 8 mm, 3.5 mm to 7 mm, 3.5 mm to 6 mm, 3.5 mm to 5 mm, 3 5 mm to 4 mm, 4 mm to 8 mm, 4 mm to 7 mm, 4 mm to 6 mm, 4 mm to 5 mm, 4.5 mm to 8 mm, 4.5 mm to 7 mm, 4.5 mm to 6 mm, 4.5 mm to 5 mm, 5 mm to 8 mm, 5 mm to 7 mra, 5 rara to 6 mm, 5.5 mm to 8 mm, 5.5 mm to 7 mm, 5.5 mm to 6 mm, 6 mm to 8 mm, 6 mm to 7 mm, 6.5 mm to 8 mm, 6.5 mm to 7 mm, 7 mm to 8 mm, or 7.5 mm to 8 mm. In some embodiments, the capsule has a mean diameter or size between 1 mm to 8 mm. In some embodiments, the capsule has a mean diameter or size between 1 mm to 4 mm. In some embodiments, the capsule has a mean diameter or size between 1 mm to 2 mm. In some embodiments, the capsules are provided as a preparation and at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the hydrogel capsules in the preparation have a diameter in a size range described herein
[00090] In embodiments, the methacrylation efficiency of the UPVLVG alginate is less than about 100%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is less than about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, or about 25%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is less than about 95%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is less than about 90%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is less than about 85%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is less than about 80%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is less than about 75%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is less than about 70%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is less than about 65%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is less than about 60%, In embodiments, the methacrylation efficiency of the UPVLVG alginate is less than about 55%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is less than about 50%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is less than about 45%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is less than about 40%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is less than about 35%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is less than about 30%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is less than about 25%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is 20%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is 46% In embodiments, the methacrylation efficiency of the UPVLVG alginate is 60%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is 90%.
[00091] In embodiments, the methacrylation efficiency of the UPVLVG alginate is between about 10% and about 100%. In embodiments, the methacrylation efficiency of the UPVL VG alginate is between about 12% and about 98%, about 14% and about 96%, about 16% and about 94%, about 18% and about 92%, or about 20% and about 90%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is between about 10% and about 100%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is between about 12% and about 98%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is between about 14% and about 96%. In embodiments, the methacrylation efficiency of the UPVL V G alginate i s between about 16% and about 94%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is between about 18% and about 92%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is between about 20% and about 90%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is 20%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is 46%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is 60%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is 90%.
[00092] In embodiments, the methacrylation efficiency of the UPVLVG alginate is greater than about 5%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is greater than about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80% or about 85%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is greater than about 5%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is greater than about 10%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is greater than about 15%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is greater than about 20%
[00093] In embodiments, the methacrylation efficiency of the UPVLVG alginate is greater than about 25%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is greater than about 30%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is greater than about 35%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is greater than about 40%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is greater than about 45%.
[00094] In embodiments, the methacrylation efficiency of the UPVLVG alginate is greater than about 50%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is greater than about 55%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is greater than about 60%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is greater than about 65%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is greater than about 70%.
[00095] In embodiments, the methacrylation efficiency of the UPVLVG alginate is greater than about 75%.
[00096] In embodiments, the methaciylation efficiency of the UPVLVG alginate is greater than about 80%.
[00097] In embodiments, the methaciylation efficiency of the UPVLVG alginate is greater than about 85%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is 20%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is 46%. In embodiments, the methaciylation efficiency of the UPVLVG alginate is 60%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is 90%.
[00098] In embodiments, the methacrylation efficiency of the SLG20 alginate is less than about 70%. In embodiments, the methacrylation efficiency of the SI..G20 alginate is less than about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15% or about 10%. In embodiments, the methaciylation efficiency of the SLG20 alginate is less than about 70%. In embodiments, the methacrylation efficiency of the SLG20 alginate less than about 65%. In embodiments, the methacrvlation efficiency of the SLG20 alginate is less than about 60%. In embodiments, the methacrylation efficiency of the SLG20 alginate is less than about 55%. In embodiments, the methacrylation efficiency of the SLG20 alginate is less than about 50%. In embodiments, the methacrylation efficiency of the SLG20 alginate is less than about 45%. In embodiments, the methacrylation efficiency of the SLG20 alginate less than about 40%. In embodiments, the methaciylation efficiency of the SI..G20 alginate is less than about 35%. In embodiments, the methacrylation efficiency of the SLG20 alginate is less than about 30%. In embodiments, the methacrylation efficiency of the SLG20 alginate is less than about 25%. In embodiments, the methacrylation efficiency of the SLG20 alginate less than about 20%. In embodiments, the methacrylation efficiency of the SLG20 alginate is less than about 15%. In embodiments, the methacrylation efficiency of the SI..G20 alginate is less than about 10%. In embodiments, the methacrylation efficiency of the SLG20 alginate 5%. In embodiments, the methacrylation efficiency of the SLG20 alginate is 10%. In embodiments, the methacrylation efficiency of the SLG20 alginate is 20%. In embodiments, the methacrylation efficiency of the SLG20 alginate is 46%. In embodiments, the methacrylation efficiency of the SLG20 alginate is 60%.
[00099] In embodiments, the methacrylation efficiency of the SLG20 alginate is between about 1% and about 80%. In embodiments, the methacrylation efficiency of the SLG20 alginate is between about 2% and about 75%, about 3% and about 70%, about 4% and about 65% or about 5% and about 60%. In embodiments, the methacrylation efficiency of the SLG20 alginate is between about 1% and about 80%. In embodiments, the methacrylation efficiency of the SLG20 alginate is between about 2% and about 75%. In embodiments, the methacrylation efficiency of the SLG20 alginate is between about 3% and about 70%. In embodiments, the methaciylation efficiency of the SLG20 alginate is between about 4% and about 65%. In embodiments, the methacrylation efficiency of the SLG20 alginate is between about 5% and about 60%. In embodiments, the methacrylation efficiency of the SLG20 alginate is 5%. In embodiments, the methacrylation efficiency of the SLG20 alginate is 10%. In embodiments, the methacrylation efficiency of the SLG20 alginate is 20%. In embodiments, the methacrylation efficiency of the SLG20 alginate is 46%. In embodiments, the methacrylation efficiency of the SLG20 alginate is 60%.
[000100] In embodiments, the methacrylation efficiency of the SLG20 alginate is greater than about 1%. In embodiments, the methacrylation efficiency of the SLG20 alginate is greater than about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, or about 55%. In embodiments, the methacrylation efficiency of the SLG20 alginate is greater than about 1%. In embodiments, the methacrylation efficiency of the SLG20 alginate is greater than about 5%. In embodiments, the methacrylation efficiency of the SLG20 alginate is greater than about 10%. In embodiments, the methacrylation efficiency of the SLG20 alginate is greater than about 15%. In embodiments, the methacrylation efficiency of the SLG20 alginate is greater than about 20%. In embodiments, the methacrylation efficiency of the SLG20 alginate is greater than about 25%. In embodiments, the methacrylation efficiency of the SLG20 alginate is greater than about 30%. In embodiments, the methacrylation efficiency of the SLG20 alginate is greater than about 35%. In embodiments, the methacrylation efficiency of the SLG20 alginate is greater than about 40%. In embodiments, the methacrylation efficiency of the SLG20 alginate is greater than about 45%. In embodiments, the methacrylation efficiency of the SLG20 alginate is greater than about 50%. In embodiments, the methacrylation efficiency of the SLG20 alginate is greater than about 55%. In embodiments, the methacrylation efficiency of the SLG20 alginate is 5%. In embodiments, the methaciylation efficiency of the SLG20 alginate is 10%. In embodiments, the methacrylation efficiency of the SLG20 alginate is 20%. In embodiments, the methacrylation efficiency of the SLG20 alginate is 46%. In embodiments, the methacrylation efficiency of the SLG20 alginate is 60%.
[000101] In embodiments, the methacrylation efficiency of the SLG 100 alginate is less than about 40%. In embodiments, the methacrylation efficiency of the SLG 100 alginate is less than about 35%, about 30%, about 25%, about 20%, about 15%, or about 10%. In embodiments, the methacrylation efficiency of the SLG100 alginate is less than about 40%. In embodiments, the methaciylation efficiency of the SLG100 alginate is less than about 35%. In embodiments, the methacrylation efficiency of the SLG100 alginate is less than about 30%. In embodiments, the methaciylation efficiency of the SLG 100 alginate is less than about 25%. In embodiments, the methacrylation efficiency of the SLG100 alginate is less than about 20%. In embodiments, the methacrylation efficiency of the SLG100 alginate is less than about 15%. In embodiments, the methacrylation efficiency of the SLG100 alginate is less than about 10%. In embodiments, the methacrylation efficiency of the SLG100 alginate is 5%. In embodiments, the methacrylation efficiency of the SLG100 alginate is 10% In embodiments, the methacrylation efficiency of the SLG100 alginate is 20%.
[000102] In embodiments, the methacrylation efficiency of the SL.G100 alginate is between about 1% and about 40%. In embodiments, the methacrylation efficiency of the SLG100 alginate is between about 2% and about 35%, about 3% and about 30%, about 4% and about 25% or about 5% and about 20%. In embodiments, the methacrylation efficiency of the SLG100 alginate is between about 1% and about 40%. In embodiments, the methacrylation efficiency of the SLG100 alginate is between about 2% and about 35%. In embodiments, the methacrylation efficiency of the SLG100 alginate is between about 3% and about 30%. In embodiments, the methacrylation efficiency of the SLG100 alginate is between about 4% and about 25%. In embodiments, the methacrylation efficiency of the SL.G1O0 alginate is between about 5% and about 20%. In embodiments, the methacrylation efficiency of the SLG100 alginate is 5%. In embodiments, the methacrylation efficiency of the SLG100 alginate is 10%. In embodiments, the methacrylation efficiency of the SLG100 alginate is 20%.
[000103] In embodiments, the methacrylation efficiency of the SLG100 alginate is greater than about 1 %. In embodiments, the methacrylation efficiency of the SLG100 alginate is greater than about 1%, about 2%, about 3%, about 4%, about 5%, about 10% or about 15%. In embodiments, the methacrylation efficiency of the SLG100 alginate is greater than about 1%. In embodiments, the methacrylation efficiency of the SLG1O0 alginate is greater than about 2%, In embodiments, the methacrylation efficiency of the SLG100 alginate is greater than about 3%. In embodiments, the methacrylation efficiency of the SLG100 alginate is greater than about 4%. In embodiments, the methacrylation efficiency of the SLG100 alginate is greater than about 5%. In embodiments, the methacrylation efficiency of the SLG100 alginate is greater than about 10%. In embodiments, the methacrylation efficiency of the SLG100 alginate is greater than about 15%. In embodiments, the methacrylation efficiency of the SLG100 alginate is 5%. In embodiments, the methacrylation efficiency of the SLG100 alginate is 10%.
[000104] In embodiments, the average molecular weight of the alginate is less than about 95,000 Da In embodiments, the average molecular weight of the alginate is less than about 65,000 Da, about 55,000 Da, about 45,000 Da, about 35,000 Da, about 25,000 Da, about 14,000 Da, about 12,000 Da, about 10,000 Da, about 9,000 Da, about 8,000 Da, about 7,000 Da, about 6,000 Da, about 5,000 Da, about 4,000 Da, about 3,000 Da or about 2,000 Da. In embodiments, the average molecular weight of the alginate is less than about 65,000 Da. In embodiments, the average molecular weight of the alginate is less than about 55,000 Da. In embodiments, the average molecular weight of the alginate is less than about 45,000 Da. In embodiments, the average molecular weight of the alginate is less than about 35,000 Da. In embodiments, the average molecular weight of the alginate is less than about 25,000 Da. In embodiments, the average molecular weight of the alginate is less than about 14,000 Da. In embodiments, the average molecular weight of the alginate is less than about 12,000 Da. In embodiments, the average molecular weight of the alginate is less than about 10,000 Da. In embodiments, the average molecular weight of the alginate is less than about 9,000 Da. In embodiments, the average molecular weight of the alginate is less than about 8,000 Da In embodiments, the average molecular weight of the alginate is less than about 7,000 Da. In embodiments, the average molecular weight of the alginate is less than about 6,000 Da. In embodiments, the average molecular weight of the alginate is less than about 5,000 Da. In embodiments, the average molecular weight of the alginate is less than about 4,000 Da. In embodiments, the average molecular weight of the alginate is less than about 3,000 Da. In embodiments, the average molecular weight of the alginate is less than about 2,000 Da.
[000105] In embodiments, the average molecular weight of the alginate is between about 500 Da and 50,000 Da. In embodiments, the average molecular weight of the alginate is between about 500 Da and 40,000 Da., about 500 Da and 30,000 Da, about 500 Da and 20,000 Da, about 500 Da and 18,000 Da, about 500 Da and 16,000 Da, about 500 Da and 14,000 Da, about 500 Da and 12,000 Da, about 500 Da and 10,000 Da, about 500 Da and 9,000 Da, about 500 Da and 8,000 Da, about 500 Da and 7,000 Da, about 500 Da and 6,000 Da, about 500 Da and 5,000 Da, about 500 Da and 4,000 Da, about 500 Da and 3,000 Da, about 500 Da and 2,000 Da, or about 500 Da and 1,000 Da. In embodiments, the average molecular weight of the alginate is between about 500 Da and 40,000 Da. In embodiments, the average molecular weight of the alginate is between about 500 Da and 30,000 Da. In embodiments, the average molecular weight of the alginate is between about 500 Da and 20.000 Da. In embodiments, the average molecular weight of the alginate is between about 500 Da and 18,000 Da. In embodiments, the average molecular weight of the alginate is between about 500 Da and 16,000 Da. In embodiments, the average molecular weight of the alginate is between about 500 Da and 14,000 Da. In embodiments, the average molecular weight of the alginate is between about 500 Da and 12,000 Da. In embodiments, the average molecular weight of the alginate is between about 500 Da and 10,000 Da. In embodiments, the average molecular weight of the alginate is between about 500 Da and 9,000 Da. In embodiments, the average molecular weight of the al ginate is between about 500 Da and 8,000 Da. In embodiments, the average molecular weight of the alginate is between about 500 Da and 7,000 Da. In embodiments, the average molecular weight of the alginate is between about 500 Da and 6,000 Da. In embodiments, the average molecular weight of the alginate is between about 500 Da and 5,000 Da.
[000106] In embodiments, the average molecular weight of the alginate is between about 500 Da and 4,000 Da. In embodiments, the average molecular weight of the alginate is between about 500 Da and 3,000 Da. In embodiments, the average molecular weight of the alginate is between about 500 Da and 2,000 Da. In embodiments, the average molecular weight of the alginate is between about 500 Da and 1,000 Da.
[000107] In embodiments, the average molecular weight of the alginate is between about 1 ,000 Da and 50,000 Da. In embodiments, the average molecular weight of the alginate is between about 1,000 Da and 40,000 Da, about 1,000 Da and 30,000 Da, about 1,000 Da and 20,000 Da, about 1,000 Da and 18,000 Da, about 1,000 Da and 16,000 Da, about 1 ,000 Da and 14,000 Da, about 1,000 Da and 12,000 Da, about 1,000 Da and 10,000 Da, about 1,000 Da and 9,000 Da, about 1,000 Da and 8,000 Da, about 1,000 Da and 7,000 Da, about 1,000 Da and 6,000 Da, about 1 ,000 Da and 5,000 Da, about 1 ,000 Da and 4,000 Da, about 1 ,000 Da and 3,000 Da. or about 1 ,000 Da and 2,000 Da. In embodiments, the average molecular weight of the alginate is between about 1,000 Da and 40,000 Da. In embodiments, the average molecular weight of the alginate is between about 1,000 Da and 30,000 Da. In embodiments, the average molecular weight of the alginate is between about 1,000 Da and 20,000 Da. In embodiments, the average molecular weight of the alginate is between about 1,000 Da and 18,000 Da. In embodiments, the average molecular weight of the alginate is between about 1,000 Da and 16,000 Da. In embodiments, the average molecular weight of the alginate is between about 1,000 Da and 14,000 Da. In embodiments, the average molecular weight of the alginate is between about 1,000 Da and 12,000 Da. In embodiments, the average molecular weight of the alginate is between about 1 ,000 Da and 10,000 Da. In embodiments, the average molecular weight of the alginate is between about 1000 Da and 9,000 Da. In embodiments, the average molecular weight of the alginate is between about 1,000 Da and 8,000 Da. In embodiments, the average molecular weight of the alginate is between about 1,000 Da and 7,000 Da. In embodiments, the average molecular weight of the alginate is between about 1,000 Da and 6,000 Da. In embodiments, the average molecular weight of the alginate is between about 1 ,000 Da and 5,000 Da. In embodiments, the average molecular weight, of the alginate is between about 1 ,000 Da and 4,000 Da. In embodiments, the average molecular weight of the alginate is between about 1,000 Da and 3,000 Da. In embodiments, the average molecular weight of the alginate is between about 1,000 Da and 2,000 Da.
[000108] In embodiments, the average molecular weight of the alginate is between about
2,000 Da and 50,000 Da In embodiments, the average molecular weight of the alginate is between about 2,000 Da and 40,000 Da, about 2,000 Da and 30,000 Da, about 2,000 Da and 20,000 Da, about 2,000 Da and 18,000 Da, about 2,000 Da and 16,000 Da, about 2,000 Da and 14,000 Da, about 2,000 Da and 12,000 Da, about 2,000 Da and 10,000 Da, about 2,000 Da and 9,000 Da, about 2,000 Da and 8,000 Da, about 2,000 Da and 7,000 Da, about 2,000 Da and 6,000 Da, about 2,000 Da and 5,000 Da, about 2,000 Da and 4,000 Da, or about 2,000 Da and 3,000 Da. In embodiments, the average molecular weight of the alginate is between about 2,000 Da and 40,000 Da. In embodiments, the average molecular weight of the alginate is between about 2,000 Da and 30,000 Da In embodiments, the average molecular weight of the alginate is between about 2,000 Da and 20,000 Da. In embodiments, the average molecular weight of the alginate is between about 2,000 Da and 18,000 Da. In embodi ments, the average molecular weight of the alginate is between about 2,000 Da and 16,000 Da. In embodiments, the average molecular weight of the alginate is between about 2,000 Da and 14,000 Da. In embodiments, the average molecular weight of the alginate is between about 2,000 Da and 12,000 Da. In embodiments, the average molecular weight of the alginate is between about 2,000 Da and 10,000 Da. In embodiments, the average molecular weight of the alginate is between about 2,000 Da and 9,000 Da. In embodiments, the average molecular weight of the alginate is between about 2,000 Da and 8,000 Da. In embodiments, the average molecular weight of the alginate is between about 2,000 Da and 7,000 Da. In embodiments, the average molecular weight of the alginate is between about 2,000 Da and 6,000 Da. In embodiments, the average molecular weight of the alginate is between about 2,000 Da and 5,000 Da. In embodiments, the average molecular weight of the alginate is between about 2,000 Da and 4,000 Da. In embodiments, the average molecular weight of the alginate is between about 2,000 Da and 3,000 Da.
[000109] In embodiments, the average molecular weight of the alginate is between about 5,000 Da and 50,000 Da. In embodiments, the average molecular weight of the alginate is between about 5,000 Da and 40,000 Da, about 5,000 Da and 30,000 Da, about 5,000 Da and 20,000 Da, about 5,000 Da and 18,000 Da, about 5,000 Da and 16,000 Da, about 5,000 Da and 14,000 Da, about 5,000 Da and 12,000 Da, about 5,000 Da and 10,000 Da, about 5,000 Da and 9,000 Da, about 5,000 Da and 8,000 Da, about 5,000 Da and 7,000 Da, or about 5,000 Da and 6,000 Da. In embodiments, the average molecular weight of the alginate is between about 5,000 Da and 40,000 Da. In embodiments, the average molecular weight of the alginate is between about 5,000 Da and 30,000 Da. In embodiments, the average molecular weight of the alginate is between about 5,000 Da and 20,000 Da. In embodiments, the average molecular weight of the alginate is between about 5,000 Da and 18,000 Da. In embodiments, the average molecular weight of the alginate is between about 5,000 Da and 16,000 Da. In embodiments, the average molecular weight of the alginate is between about 5,000 Da and 14,000 Da. In embodiments, the average molecular weight of the alginate is between about 5,000 Da and 12,000 Da. In embodiments, the average molecular weight of the alginate is between about 5,000 Da and 10,000 Da. In embodiments, the average molecular weight of the alginate is between about 5,000 Da and 9,000 Da. In embodiments, the average molecular weight of the alginate is between about 5,000 Da and 8,000 Da. In embodiments, the average molecular weight of the alginate is between about 5,000 Da and 7,000 Da. In embodiments, the average molecular weight of the alginate is between about 5,000 Da and 6,000 Da.
[000110] In embodiments, the average molecular weight of the alginate is between about
10,000 Da and 50,000 Da. In embodiments, the average molecular weight of the alginate is between about 10,000 Da and 40,000 Da, about 10,000 Da and 30,000 Da, about 10,000 Da and 20,000 Da, about 10,000 Da and 18,000 Da, about 10,000 Da and 16,000 Da, about 10,000 Da and 14,000 Da, or about 10,000 Da and 12,000 Da. In embodiments, the average molecular weight of the alginate is between about 10,000 Da and 40,000 Da. In embodiments, the average molecular weight of the alginate is between about 10,000 Da and 30,000 Da. In embodiments, the average molecular weight of the alginate is between about 10,000 Da and 20,000 Da. In embodiments, the average molecular weight of the alginate is between about 10,000 Da and 18,000 Da. In embodiments, the average molecular weight of the alginate is between about 10,000 Da and 16,000 Da. In embodiments, the average molecular weight of the alginate is between about 10,000 Da and 14,000 Da. In embodiments, the average molecular weight of the alginate is between about 10,000 Da and 12,000 Da.
[000111] In embodiments, the average molecular weight of the alginate is greater than about 500 Da. In embodiments, the average molecular weight of the alginate i s greater than about 1,000 Da, about 2,000 Da, about 3,000 Da, about 4,000 Da, about 5,000 Da, about 6,000 Da, about 7,000 Da, about 8,000 Da, about 9,000 Da, about 10,000 Da, about 12,000 Da, about 14,000 Da, about 16,000 Da, about 18,000 Da, about 20,000 Da, about 30,000 Da, about 40,000 Da or about 50,000 Da In embodiments, the average molecular weight of the alginate is greater than about 1,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 2,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 3,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 4,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 5,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 6,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 7,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 8,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 9,000 Da, In embodiments, the average molecular weight of the alginate is greater than about 10,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 12,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 14,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 16,000 Da. In embodiments, the average molecular tveight of the alginate is greater than about 18,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 20,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 30,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 40,000 Da.
[000112] In embodiments, the average molecular weight of the alginate is less than about 260,000 Da. In embodiments, the average molecular weight of the alginate is less than about 250,000 Da, about 240,000 Da, about 230,000 Da, about 220,000 Da, about 210,000 Da, about
200,000 Da, about 190,000 Da, about 180,000 Da, about 170,000 Da, about 160,000 Da, about
150,000 Da, about 140,000 Da, about 130,000 Da, about 120,000 Da, about 110,000 Da, about
100,000 Da, about 90,000 Da, about 80,000 Da, about 70,000 Da, about 60,000 Da, about 50,000
Da, about 40,000 Da, or about 30,000 Da. In embodiments, the average molecular weight of the alginate is less than about 260,000 Da. In embodiments, the average molecular weight of the alginate is less than about 250,000 Da. In embodiments, the average molecular weight of the alginate is less than about 240,000 Da. In embodiments, the average molecular weight of the alginate is less than about 260,000 Da. In embodiments, the average molecular weight of the alginate is less than about 250,000 Da. In embodiments, the average molecular weight of the alginate is less than about 240,000 Da. In embodiments, the average molecular weight of the alginate is less than about 230,000 Da. In embodiments, the average molecular weight of the alginate is less than about 220,000 Da. In embodiments, the average molecular weight of the alginate is less than about 210,000 Da. In embodiments, the average molecular weight of the alginate is less than about 200,000 Da. In embodiments, the average molecular weight of the alginate is less than about 190,000 Da. In embodiments, the average molecular weight of the alginate is less than about 180,000 Da. In embodiments, the average molecular weight of the alginate is less than about 170,000 Da. In embodiments, the average molecular weight of the alginate is less than about 160,000 Da. In embodiments, the average molecular weight of the alginate is less than about 150,000 Da. In embodiments, the average molecular weight of the alginate is less than about 140,000 Da. In embodiments, the average molecular weight of the alginate is less than about 130,000 Da. In embodiments, the average molecular weight of the alginate is less than about 120,000 Da. In embodiments, the average molecular weight of the alginate is less than about 110,000 Da. In embodiments, the average molecular weight of the alginate is less than about 100,000 Da. In embodiments, the average molecular weight of the alginate is less than about 90,000 Da. In embodiments, the average molecular weight of the alginate is less than about 80,000 Da. In embodiments, the average molecular weight of the alginate is less than about 70,000 Da. In embodiments, the average molecular weight of the alginate is less than about 60,000 Da. In embodiments, the average molecular weight of the alginate is less than about
50,000 Da.
[000113] In embodiments, the average molecular weight of the alginate is between about 50,000 Da and about 260,000 Da. tn embodiments, the average molecular weight of the alginate is between about 75,000 Da and 260,000 Da, about 75,000 Da and 250,000 Da, about 75,000 Da and 240,000 Da, about 75,000 Da and 230,000 Da, about 75,000 Da and 220,000 Da, about 75,000 Da and 210,000 Da, about 75,000 Da and 200,000 Da, about 75,000 Da and 190,000 Da, about 75,000 Da and 180,000 Da, about 75,000 Da and 170,000 Da, about 75,000 Da and 160,000 Da, about 75,000 Da and 150,000 Da, about 75,000 Da and 140,000 Da, about 75,000 Da and 130,000 Da, about 75,000 Da and 120,000 Da, about 75,000 Da and 110,000 Da, or about 75,000 Da and 100,000 Da. In embodiments, the average molecular weight of the alginate is between about 75,000 Da and 260,000 Da. In embodiments, the average molecular weight of the alginate is between about 75,000 Da and 250,000 Da In embodiments, the average molecular weight of the alginate is between about 75,000 Da and 240,000 Da. In embodiments, the average molecular weight of the alginate is between about 75,000 Da and 230,000 Da. In embodiments, the average molecular weight of the alginate is between about 75,000 Da and 220,000 Da. In embodiments, the average molecular weight of the alginate is between about 75,000 Da and 210,000 Da. In embodiments, the average molecular weight of the alginate is between about 75,000 Da and 200,000 Da. In embodiments, the average molecular weight of the alginate is between about 75,000 Da and 190,000 Da. In embodiments, the average molecular weight of the alginate is between about 75,000 Da and 180,000 Da. In embodiments, the average molecular weight of the alginate is between about 75,000 Da and 170,000 Da. In embodiments, the average molecular weight of the alginate is between about 75,000 Da and 160,000 Da. In embodiments, the average molecular weight of the alginate is between about 75,000 Da and 150,000 Da. In embodiments, the average molecular weight of the alginate is between about 75,000 Da and 140,000 Da. In embodiments, the average molecular weight of the alginate is between about 75,000 Da and 130,000 Da. In embodiments, the average molecular weight of the alginate is between about 75,000 Da and 120,000 Da. In embodiments, the average molecular weight of the alginate is between about 75,000 Da and 110,000 Da. In embodiments, the average molecular weight of the alginate is between about 75,000 Da and 100,000 Da.
[000114] In embodiments, the average molecular weight of the alginate is greater than about 50,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 60,000 Da, about 70,000 Da, about 80,000 Da, about 90,000 Da, about 100,000 Da, about 110,000 Da, about 120,000 Da, about 130,000 Da, about 140,000 Da, about 150,000 Da, about 160,000 Da, about 170,000 Da, about 180,000 Da, about 190,000 Da, about 200,000 Da, about 210,000 Da, about 220,000 Da or about 230,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 50,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 60,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 70,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 80,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 90,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 100,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 110,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 120,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 130,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 140,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 150,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 160,000 Da. In embodiments, the average molecular weight of the alginate is greater than about. 170,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 180,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 190,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 200,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 210,000 Da.
[000115] bi embodiments, the average molecular weight of the alginate is less than about 350,000 Da. In embodiments, the average molecular weight of the alginate is less than about 300,000 Da, about 290,000 Da, about 280,000 Da, about 270,000 Da, about 260,000 Da, about 250,000 Da, about 240,000 Da, about 230,000 Da, about 220,000 Da, about 210,000 Da, about 200,000 Da, about 190,000 Da, or about 180,000 Da. In embodiments, the average molecular weight of the alginate is less than about 350,000 Da. In embodiments, the average molecular weight of the alginate is less than about 300,000 Da. In embodiments, the average molecular weight of the alginate is less than about 290,000 Da. In embodiments, the average molecular weight of the alginate is less than about 280,000 Da. In embodiments, the average molecular weight of the alginate is less than about 270,000 Da. In embodiments, the average molecular weight of the alginate is less than about 260,000 Da. In embodiments, the average molecular weight of the alginate is less than about 250,000 Da. In embodiments, the average molecular weight of the alginate is less than about 240,000 Da. In embodiments, the average molecular weight of the alginate is less than about 230,000 Da. In embodiments, the average molecular weight of the alginate is less than about 220,000 Da. In embodiments, the average molecular weight of the alginate is less than about 210,000 Da. In embodiments, the average molecular weight of the alginate is less than about 200,000 Da. In embodiments, the average molecular weight of the alginate is less than about 190,000 Da. In embodiments, the average molecular weight of the alginate is less than about 180,000 Da.
[000116] In embodiments, the average molecular weight of the alginate is between about 180,000 Da and 350,000 Da. In embodiments, the average molecular weight of the alginate is between about 180,000 Da and 350,000 Da, about 180,000 Da and 300,000 Da, about 180,000 Da and 290,000 Da, about 180,000 Da and 280,000 Da, about 180,000 Da and 270,000 Da, about 180,000 Da and 260,000 Da, about 180,000 Da and 250,000 Da, about 180,000 Da and 240,000 Da, about 180,000 Da and 230,000 Da, about 180,000 Da and 220,000 Da, about 180,000 Da and 210,000 Da, about 180,000 Da and 200,000 Da, or about 180,000 Da and 190,000 Da. In embodiments, the average molecular weight of the alginate is between about 180,000 Da and 350,000 Da. In embodiments, the average molecular weight of the alginate is between about 180,000 Da and 300,000 Da. In embodiments, the average molecular weight of the alginate is between about 180,000 Da and 290,000 Da. In embodiments, the average molecular weight of the alginate is between about 180,000 Da and 280,000 Da. In embodiments, the average molecular weight of the alginate is between about 180,000 Da and 270,000 Da. In embodiments, the average molecular weight of the alginate is between about 180,000 Da and 260,000 Da. In embodiments, the average molecular weight of the alginate is between about 180,000 Da and 250,000 Da. In embodiments, the average molecular weight of the alginate is between about 180,000 Da and 240,000 Da. In embodiments, the average molecular weight of the alginate is between about 180,000 Da and 230,000 Da. In embodiments, the average molecular weight of the alginate is between about 180,000 Da and 220,000 Da. In embodiments, the average molecular weight of the alginate is between about 180,000 Da and 210,000 Da. In embodiments, the average molecular weight of the alginate is between about 180,000 Da and 200,000 Da. In embodiments, the average molecular weight of the alginate is between about 180,000 Da and 190,000 Da
[000117] In embodiments, the average molecular weight of the alginate is greater than about 180,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 190,000 Da, about 200,000 Da, about 210,000 Da, about 220,000 Da, about 230,000 Da, about 240,000 Da, about 250,000 Da, about 260,000 Da, about 270,000 Da, about 280,000 Da, about 290,000 Da, about 300,000 Da, about 310,000 Da, about 320,000 Da, or about 325,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 180,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 190,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 200,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 210,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 220,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 230,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 240,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 250,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 260,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 270,000 Da In embodiments, the average molecular weight of the alginate is greater than about 280,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 290,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 300,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 310,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 320,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 325,000 Da.
[000118] In an embodiment, the hydrogel is photo-responsive, wherein the photo- responsive material comprises a. monomer of Formula (I) represented by the following structural formula:
Figure imgf000038_0001
or a pharmaceutically acceptable salt, stereoisomer, mixture of stereoisomers, or a tautomer thereof, wherein n and m are each independently an integer from 1 to 100. In some embodiments,
[000119] In an aspect, the hydrogel is a pH-responsive material, wherein the hydrogel forms upon a change in pH. In embodiments, the hydrogel forms, oris capable of flowing, when the pH is acidic, e.g., less than pH 7. In embodiments, the hydrogel forms, or is capable of forming, when the pH is basic, e.g., greater than pH 7. In embodiments, the hydrogel forms, or is capable of forming, when the pH is neutral, e.g., about pH 7. In embodiments, the hydrogel forms, or is capable of forming, when the pH is above or below the acid dissociation (Ka) or the base dissociation constant (Kb) of a moiety of the pH-responsive material.
[000120] In an aspect, the flowable material is a photo-responsive material, wherein the hydrogel forms, ceases forming, or is capable of forming, upon the introduction, removal or change in the intensity of light of a particular wavelength or a range of wavelengths, e.g., visible, UV-A, UV-B, Infrared, X-Ray, inter alia. In embodiments, the hydrogel forms, ceases forming, or is capable of forming, when the introduction of light of a particular wavelength or a range of wavelengths of a sufficient intensity triggers photopolymerization, optionally with a photo initiator.
[000121] In an aspect, the hydrogel is a moisture-responsive material, wherein the hydrogel forms, ceases forming, or is capable of forming, upon a change in the moisture content of the material or adjacent environment. In embodiments, the polymeric components undergo self-assembly to form the resulting hydrogel in the presence of water.
[000122] Materials for assembling hydrogels may also include any provided in Hoffman, A.S. Adv Drug Deliv Rev 2012, 64, 18-23, such as agarose, alginate (e.g., the calcium or barium salt of alginic acid), alginate-g-(alginate), carboxymethyl chitin, carrageenan, chitosan, chondroitin sulfate, collagen-acrylate, dextran, dextran sulfate, fibrin, gelatin, hyaluronic acid (HA), hyaluronic acid/glycidyl methacrylate, HA-g-NIPAAM, PAAM, P(AN-co-allyl sulfonate), P(biscarboxyl-phenoxy-phosphazene), pectin, PEG optionally with cyclodextrins (CDs), PEG-g- P(AAM-co-Vamine), PEG-bis(PLA-acrylate), PEG-PCL-PEG, , PEG-PLA-PEG, PEG-PLGA-PEG, P(PF-co-EG) optionally with acrylate groups, P(GEMA- sulfate), P(HEMA/Matrigel®), PHB, PLA-PEG-PLA, Pluronics, Pluronics and bioactive glass, P(MMA-co-HEMA), P(NIPAAM-co-AAC), P(NIPAAM-co-EMA,) PNVP, poly(D-lysine), poly (L-ly sine), poly(L- lysine)/polyacrylic acid pair, P(PEG/PBO terephthalate), P(PEG-co-peptides), PPO-PEO), P(PLGA-co-serine), pullulan, and PVAC/PVA.
[000123] In an aspect, the hydrogel is a chemically responsive material, wherein the hydrogel forms, ceases forming, or is capable of forming, upon the introduction or removal of a chemical stimulus.
[000124] In embodiments, the hydrogel ceases forming upon the completion or initiation of a chemical stimulus, e.g., a chemical stimulus provided in Zhang Y S. el al. Science. 2017, 356 (6337), eaaS627.
[000125] In some aspects, the hydrogel can include a plurality of natural polymer macromers cross- linked with a plurality of crosslinks that are degradable after administration to a subject in vivo.
[000126] T lie number or percentage of cross-links linking the macromers can be varied to control the mechanical properties, swelling ratios, and degradation profiles of the hydrodgels. Degradation of the crosslinks in vivo allows the hydrogel to more readily biodegrade and be used for in vivo applications. Additionally, as discussed below the photocrosslinked hydrogel can be used as a substrate for the incorporation and/or attachment of various agents and/or cells. The photocrosslinked hydrogel can be injectable and/or implantable, and can be in the form of a membrane, sponge, gel, solid scaffold, spun fiber, woven or unwoven mesh, nanoparticle, microparticle, or any other desirable configuration. [000127] In an aspect of the invention, the photocrosslinked hydrogel can include at least on cross-link that can be hydrolyzed to allow" degradation of the hydrogel in vivo. In one embodiment, the cross-link can include ester, amide, acetal, and/or ketal groups or linkages that can be readily hydrolyzed in vivo to promote degradation of the hydrogel. In one example, the hydrolyzable cross-link can include at least one hydrolyzable acrylate (e.g.. methacrylate) cross-link. The hydrolyzable acrylate cross-link can include at least one hydrolyzable ester and/or hydrolysable amide linkage. As explained further below, hydrolytic degradation of the hydrolyzable acrylate crosslink can create space for cell growth and deposition of a new extracellular matrix to replace the photocrosslinked hydrogel in vivo.
[000128] In one aspect of the present invention, the photocrosslinked hydrogel can be formed into alginate microbeads or microspheres capable of carrying and differentially and/or control lablv releasing at least one bioactive agent.
[000129] In some aspects of the present invention, the photocrosslinked hydrogel can be modified or configured to differentially and/or controllably release at least one bioactive agent by forming at least one concentration gradient within the hydrogel. The hydrogel can have multiple gradients in the same hydrogel, and the gradients can run in the same or opposite directions. The gradients can be comprised of different components, such as different photoalginates having different molecular weights or acrylation (e.g., methacryl ati on) percentages, acrylated cell adhesion ligands, bioactive factors, cells, etc. As discussed below, for example, the photocrosslinked biodegradable hydrogel can be formed into a particular shape or form to facilitate release of one or more bi oactive agents according to a gradient release profile. In some aspects, one or more materials or agents can be added to the photocrosslinked biodegradable hydrogel to facilitate differential and/or controlled release of one or more bioactive agents according to a gradient release profile.
[000130] Therapeutic Agents
[000131] A. Cells
[000132] In some aspects, the term "cell" can refer to any progenitor cell, such as totipotent stem cells, pluripotent stem cells, and multipotent stem cells, as well as any of their lineage descendant cells, including more differentiated cells. The terms "stem cell" and "progenitor cell" are used inter changeably herein. The cells can derive from embryonic, fetal, or adult tissues. Examples of progenitor cells can include totipotent stem cells, multipotent stem cells, mesenchymal stem cells (MSCs), hematopoietic stem cells, neuronal stem cells, hematopoietic stem cells, pancreatic stem cells, cardiac stem cells, embryonic stem cells, embryonic germ ceils, neural crest stem cells, kidney stem ceils, hepatic stem cells, lung stem cells, hemangioblast cells, and endothelial progenitor cells. Additional exemplary progenitor cells can include de-differentiated chondrogenic cells, chondrogenic cells, cord blood stem cells, multi-potent adult progenitor cells, myogenic cells, osteogenic cells, tendogenic cells, ligamentogenic cells, adipogenic cells, and dermatogenic cells.
[000133] Devices or compositions described herein may contain a cell, for example, an engineered cell. A cell may be derived from any mammalian organ or tissue, including the brain, nerves, ganglia, spine, eye, heart, liver, kidney, lung, spleen, bone, thymus, lymphatic system, skin, muscle, pancreas, stomach, intestine, blood, ovary, uterus, or testes.
[000134] A cell may be derived from a donor (e.g., an allogeneic cell), derived from a subject (e.g, an autologous cell), or from another species (e.g, a xenogeneic cell). In an embodiment, a cell can be grown in cell culture, or prepared from an established cell culture line, or derived from a donor (e.g., a living donor or a cadaver). In an embodiment, a cell is genetically engineered. In another embodiment, a cell is not genetically engineered. A cell may include a stem cell, such as a reprogrammed stem cell, or an induced pluripotent cell. Exemplary cells include mesenchymal stem cells (MSCs), fibroblasts (e.g., primary fibroblasts). HEK cells (e.g., HEK293T), Jurkat cells, HeLa cells, retinal pigment epithelial (RPE) cells, HUVEC cells, NIH3T3 cells, CHO-K1 cells, COS-1 cells, COS-7 cells, PC-3 cells, HCT 116 cells, A549MCF-7 cells, HuH-7 cells, U-2 OS cells, HepG2 cells, Neuro-2a cells, and SF9 cells.
[000135] A cell included in a device or composition described herein may produce or secrete a therapeutic agent. In an embodiment, a cell included in a device or composition described herein may produce or secrete a single type of therapeutic agent or a plurality of therapeutic agents. In an embodiment, a device or composition described herein may comprise a cell that is transduced or transfected with a nucleic acid (e.g, a vector) comprising an expression sequence of a therapeutic agent. For example, a. cell may be transduced or transfected with a lentivirus. A nucleic acid introduced into a cell (e.g., by transduction or transfection) may be incorporated into a nucleic acid delivery system, such as a plasmid, or may be delivered directly. In an embodiment, a nucleic acid introduced into a cell (e.g., as part of a plasmid) may include a region to enhance expression of the therapeutic agent and/or to direct targeting or secretion, for example, a promoter sequence, an activator sequence, or a cell -signaling peptide, or a cell export peptide. Exemplary promoters include EF-la, CMV, Ube, hPGK, VMD2, and CAG. Exemplary activators include the TETI catalytic domain, P300 core, VPR, rTETR, Cas9 (e.g., from A pyogenes or A aureus), and Cpfl (e.g., from L. bacterium).
[000136] A device or composition described herei n may comprise a cell or a plurality of cells. In the case of a plurality of cells, the concentration and total cell number may be varied depending on a number of factors, such as cell type, implantation location, and expected lifetime of the devices or compositions. In an embodiment, the total number of cells included in a device or composition described herein is greater than about 2, 4, 6, 8, 10, 20, 30, 40, 50, 75, 100,200,250, 500, 750, 1000, 1500, 2000, 5000, 10000, or more. In an embodiment, the total number of cells included in a device or composition described herein is greater than about 1.0 x 102, 1.0 x 103, 1 .0 x 104. 1.0 x 105, 1 .0 x 10 ', 1.0 x 107, 1.0 x 10s, 1.0 x 109, l O x 10i0, or more. In an embodiment, the total number of cells included in a device or composition described herein is less than about than about 10000, 5000, 2500, 2000, 1500, 1000, 750, 500, 250, 200, 100, 75, 50, 40, 30, 20, 10, 8, 6, 4, 2, or less. In an embodiment, the total number of cells included in a device or composition described herein is less than about 1.0 x 1010, 1.0 x 109, 1.0 x 10s, 1.0 x 107, 1.0 x lO6, 1.0 x 105, 1.0 x 104, 1.0 x 103, 1.0 x 102, or less. In an embodiment, a plurality of cells is present as an aggregate. In an embodiment, a plurality of cells is present as a cell dispersion.
[000137] Specific features of a cell contained within a device or composition described herein may be determined, e.g., prior to and/or after incorporation into a device or composition described herein. For example, cell viability, cell density, or cell expression level may be assessed. In an embodiment, cell viability, cell density, and cell expression level may be determined using standard techniques, such as cell microscopy, fluorescence microscopy, histology, or biochemical assay.
[000138] B. Bioactive Agents
[000139] A device or composition described herein may contain a therapeutic agent, for example, produced or secreted by a cell. A therapeutic agent may include a nucleic acid (e.g., an RNA, aDNA, or an oligonucleotide), a protein (e.g., an antibody, enzyme, cytokine, hormone, receptor), a lipid, a small molecule, a metabolic agent, an oligosaccharide, a peptide, an amino acid, an antigen. In an embodiment, a device or composition described herein comprises a cell or a plurality of cells that are genetically engineered to produce or secrete a therapeutic agent.
[000140] hi an embodiment, a device or composition described herein comprises a cell producing or secreting a protein. The protein may be of any size, e.g., greater than about 100 Da, 200 Da, 250 Da, 500 Da, 750 Da, 1 KDa, 1.5 kDa, 2 kDa, 2.5 kDa, 3 kDa, 4 kDa, 5 kDa, 6 kDa, 7 kDa, 8 kDa, 9 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, 55 kDa, 60 kDa, 65 kDa, 70 kDa, 75 kDa, 80 kDa, 85 kDa, 90 kDa, 95 kDa, 100 kDa, 125 kDa, 150 kDa, 200 kDa, 200 kDa, 250 kDa, 300 kDa, 400 kDa, 500 kDa, 600 kDa, 700 kDa, 800 Da, 900 kDa, or more. In an embodiment, the protein is composed of a single subunit or multiple subunits (e.g., a dimer, trimer, tetramer, etc.). A protein produced or secreted by a cell may be modified, for example, by glycosylation, methylation, or other known natural or synthetic protein modification
[000141] A protein may be produced or secreted as a pre-protein or in an inactive form and may require further modification to convert it into an active form.
[000142] Proteins produced or secreted by a cell may be include antibodies or antibody fragments, for example, an Fe region or variable region of an antibody. Exemplary antibodies include anti-PD-1, anti-PD-LI, anti-CTLA4, anti-TNFa, and anti-VEGF antibodies. An antibody may be monoclonal or polyclonal. Other exemplar,' proteins include a lipoprotein, an adhesion protein, blood clotting factor (e.g., Factor VII, Factor VIII, Factor IX, GCG, or VWF), hemoglobin, enzymes, proenkephalin, a growth factor (e.g., EGF, IGF-1, VEGF alpha, HGF, TGFbeta, bFGF), or a cytokine,
[000143] A protein produced or secreted by a cell may include a hormone. Exemplary hormones include growth hormone, growth hormone releasing hormone, prolactin, lutenizing hormone (LH), anti-diuretic hormone (ADH), oxytocin, thyroid stimulating hormone (TSH), thyrotropin-release hormone (TRH), adrenocorticotropic hormone (ACTH), follicle- stimulating hormone (FSH), thyroxine, calcitonin, parathyroid hormone, aldosterone, cortisol, epinephrine, glucagon, insulin, estrogen, progesterone, and testosterone.
[000144] A protein produced or secreted by a cell may include a cytokine. A cytokine maybe a pro- inflammatory cytokine or an anti-inflammatory' cytokine. Example of cytokines include IL-1, IL- la, IL-1 , IL-IRA, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL- 12, IL- 12a. IL- 12b, IL-13, IL- 14, IL- 16, IL- 17, G-CSF, GM-CSF, IL-20, IFN-a, IFN- , IFN-y, CD 154, LT- , CD70, CD 153, CD178, TRAIL, TNF-a, TNF- , SCF, M-CSF, MSP, 4-1BBL, LIE, OSM, and others.
[000145] For example, a cytokine may include any cytokine described in M.J. Cameron and D.J. Kelvin, Cytokines, Chemokines, and Their Receptors (2013), Landes Biosciences, which is incorporated herein by reference in its entirety.
[000146] A device or composition described herein may comprise a cell expressing a single type of therapeutic agent, e.g., a single protein or nucleic acid, or may express more than one type of therapeutic agent, e.g., a plurality of proteins or nucleic acids. In an embodiment, a device or composition described herein comprises a cell expressing two types of therapeutic agents (e.g., two types of proteins or nucleic acids). In an embodiment, a device or composition described herein comprises a cell expressing three types of therapeutic agents (e.g., three types of proteins or nucleic acids). In an embodiment, a device or composition described herein comprises a cell expressing four types of therapeutic agents (e.g., four types of proteins or nucleic acids)
[000147] In an embodiment, a device or composition described herein comprises a cell expressing a single type of nucleic acid (e.g., DNA or RNA) or may express more than one type of nucleic acid, e.g., a plurality of nucleic acid (e.g., DNA or RNA). In an embodiment, a device or composition described herein comprises a cell expressing two types of nucleic acids (e.g., DNA or RNA). In an embodiment, an devices and compositions comprises a cell expressing three types of nucleic acids (e.g., DNA or RNA). In an embodiment, a device or composition described herein comprises a cell expressing four types of nucleic acids (e.g., DNA or RNA).
[000148] In an embodiment, a device or composition described herein comprises a cell expressing a single type of protein, or may express more than one type of protein, e.g., a plurality of proteins. In an embodiment, a device or composition described herein comprises a cell expressing two types of proteins. In an embodiment, a device or composition described herein comprises a cell expressing three types of proteins. In an embodiment, a device or composition described herein comprises a cell expressing four types of proteins.
[000149] In an embodiment, a device or composition described herein comprises a cell expressing a single type of enzyme, or may express more than one type of enzyme, e.g., a plurality of enzymes. In an embodiment, a device or composition described herein comprises a cell expressing two types of enzymes. In an embodiment, a device or composition described herein comprises a cell expressing three types of enzymes. In an embodiment, a device or composition described herein comprises a cell expressing four types of enzymes.
[000150] In an embodiment, a device or composition described herein comprises a cell expressing a single type of antibody or antibody fragment or may express more than one type of antibody or antibody fragment, e.g., a plurality of antibodies or antibody fragments. In an embodiment, an devices and compositions comprises a cell expressing two types of antibodies or antibody fragments. In an embodiment, a device or composition described herein comprises a cell expressing three types of antibodies or antibody fragments. In an embodiment, a device or composition described herein comprises a cell expressing four types of antibodies or antibody fragments.
[000151] In an embodiment, a device or composition described herein comprises a cell expressing a single type of hormone, or may express more than one type of hormone, e.g., a plurality of hormones In an embodiment, a device or composition described herein comprises a cell expressing two types of hormones. In an embodiment, a device or composition described herein comprises a cell expressing three types of hormones. In an embodiment, a device or composition described herein comprises a cell expressing four types of hormones.
[000152] In an embodiment, a device or composition described herein comprises a cell expressing a single type of enzyme, or may express more than one type of enzyme, e.g., a plurality of enzymes.
[000153] In an embodiment, a device or composition described herein comprises a cell expressing two types of enzymes. In an embodiment, a device or composition described herein comprises a cell expressing three types of enzymes. In an embodiment, a device or composition described herein comprises a cell expressing four types of enzymes.
[000154] In an embodiment, a device or composition described herein comprises a cell expressing a single type of cytokine or may express more than one type of cytokine, e.g., a plurality of cytokines. In an embodiment, a device or composition described herein comprises a cell expressing two types of cytokines. In an embodiment, a device or composition described herein comprises a cell expressing three types of cytokines. In an embodiment, a device or composition described herein comprises a cell expressing four types of cytokines. [000155] Diseases
[000156] A device or composition comprising a hydrogel and a therapeutic agent as described herein can be used to treat a variety of diseases in a mammalian subject. In some embodiments, the disease is a brain disease, a cancer, diabetes, melanoma, solid tumor, Alzheimer's disea.se, Parkinson’s disease, stroke, epilepsy, depression, pain relief, or local anesthesia.
[000157] The cancer can be a cancer of the bladder, blood, bone, bone marrow, breast, colon, esophagus, gastrointestinal, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus. In addition, the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma, basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary’ transitional cell carcinoma; adenocarcinoma, gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma, adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant, branchiolo-alveolar adenocarcinoma; papillary' adenocarcinoma; chromophobe carcinoma; acidophil carcinoma, oxyphilic adenocarcinoma, basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma, endometroid carcinoma: skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma, mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary' carcinoma; lobular carcinoma, inflammatory carcinoma; paget's disease, mammary, acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w/squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; androblastoma, malignant; sertoli cell carcinoma; leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malig melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma, mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma, retinoblastoma; olfactory' neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; hodgkin’s disease; hodgkin’s; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-hodgkin's lymphomas; malignant histiocytosis, multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease, leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia, eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.
[000158] Pharmaceutical Compositions
[000159] In some embodiments, an alginate methacrylate and a therapeutic agent as described herein are included in a pharmaceutical composition. Pharmaceutical compositions of the present invention comprise an effective amount of one or more compounds of the present disclosure, e.g., a mechanoluminescent nanoparticle or mechanoluminescent liposome, or additional agent dissolved or dispersed in a pharmaceutically acceptable carrier. The phrases "pharmaceutical or pharmacologically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate. The preparation of a pharmaceutical composition that contains a hydrogel as described herein or additional active ingredient will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington: The Science and Practice of Pharmacy, 21s! Ed., Lippincott Williams and Wilkins, 2005, incorporated herein by reference. Moreover, for animal (e.g., human) administration, it will be understood that preparations should typically meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards.
[000160] In some embodiments, pharmaceutical compositions may comprise, for example, a compound, composition and/or device provided by WO 2022/266086 A2, which is incorporated herein by reference. In some embodiments, the pharmaceutical compositions and/or devices described herein may be formulated in conjunction with a compound, composition and/or device provided by WO 2022/266086 A2. In some embodiments, the pharmaceutical compositions and/or devices described herein may be formulated in conjunction with a triazole-containing polymer, such as a triazole-containing alginate. In some embodiments, the triazole-containing polymer may comprise a compound according to any one of the following formulae:
Figure imgf000048_0001
Figure imgf000049_0001
Figure imgf000050_0001
wherein:
Figure imgf000051_0001
m and n result in a number of repeating units with a molecular weight from about 50,0000 Daltons to about 500,000 Daltons.
[000161] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington - 23rd Edition, October 2020, incorporated herein by reference). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the pharmaceutical compositions is contemplated.
[000162] The hydrogel may comprise different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form, and whether it need to be sterile for such routes of administration as injection. The present invention can be administered intravenously, intradermally, transdermally, intrathecally, intraarterially, intraperitoneally, intranasally, intravaginally, intrarectally, topically, intramuscularly, subcutaneously, mucosally, orally, topically, locally, inhalation (e.g., aerosol inhalation), via injection, infusion, continuous infusion, or localized perfusion bathing target cells directly.
[000163] In some embodiments, pharmaceutical compositions may comprise, for example, at least about 0 1% of an active compound. In other embodiments, the active compound may comprise between about 2% to about 75% of the weight of the unit, or between about 25% to about 60%, for example, and any range derivable therein. Naturally, the amount of active compound(s) in each therapeutically useful composition may be prepared is such a way that a suitable dosage will be obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelflife, as wrell as other pharmacological considerations will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable.
[000164] In other non-limiting examples, a dose may also comprise from about 1 mi crogram/kg/body weight, about 5 mi crogram/kg/b ody weight, about 10 microgram/kg/body weight, about 50 m i crogram/kg/b ody weight, about 100 microgram/kg/body weight, about 200 microgram/kg/body weight, about 350 microgram/kg/body weight, about 500 microgram/kg/body weight, about 1 milligram/kg/body weight, about 5 milligram/kg/body weight, about 10 milligram/kg/body weight, about 50 milligram/kg/body weight, about 100 milligram/kg/body weight, about 200 milligram/kg/body weight, about 350 milligram/kg/body weight, about 500 milligram/kg/body weight, to about 1000 mg/kg/body weight or more per administration, and any range derivable therein. In non-limiting examples of a derivable range from the numbers listed herein, a range of about 5 mg/kg/body weight to about 100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500 milligram/kg/body weight, etc., can be administered, based on the numbers described above.
[000165] In further embodiments, a composition of the present invention may be administered via a parenteral route. As used herein, the term "parenteral" includes routes that bypass the alimentary tract. Specifically, the pharmaceutical compositions disclosed herein may be administered for example, but not limited to intravenously, intradermally, intramuscularly, intraarterially, intrathecally, subcutaneous, or intraperitoneally U.S. Pat. Nos. 6,7537,514, 6,613,308, 5,466,468, 5,543, 158; 5,641,515; and 5,399,363 (each specifically incorporated herein by reference in its entirety).
[000166] Solutions of the active compounds as free base or pharmacologically acceptable salts may be prepared in water suitably mixed with a surfactant, such as hydroxypropyl cellulose. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (U S. Patent 5,466,468, specifically incorporated herein by reference in its entirety). In all cases the form must be sterile and must be fluid to the extent that easy injectability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (i.e., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin. [000167] F or parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this connection, sterile aqueous media that can be employed will be known to those of skill in the art in light of the present disclosure For example, one dosage may be dissolved in isotonic NaCl solution and either added hypoderm ocly sis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035- 1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologies standards Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. A powdered composition is combined with a liquid carrier such as, e.g., water or a saline solution, with or without a stabilizing agent. In some preferred embodiments, the hydrogel can be stored with or without a drug or therapeutic agent, e.g., in a solution aqueous 5% glucose (w/v). In some embodiments, the hydrogel comprising a drug or therapeutic agent is dissolved in an aqueous 5% glucose (w/v) prior to administration by injection.
[000168] A variety of dosages of therapeutic agents may be administered to a mammalian subject. For example, the therapeutic agents may be administered to the subject in an amount of about 5- 10 tng/kg, 0.5, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, or 15 mg/kg, or any range derivable therein.
[000169] A variety of drugs and therapeutic agents can be included with or encapsulated in a hydrogel as described herein. The drug may be a small molecule, a biological or protein therapeutic, or enzyme. In some embodiments, the drug is an anesthetic, a volatile anesthetic, an anticancer drug or chemotherapeutic, an anti-inflammatory' drug (e.g., NSAID etc.), an analgesic drug or a painkiller. The drug or therapeutic agent, and hydrogel may be comprised in a pharmaceutical formulation that includes an excipient.
[000170] Combination Therapy
[000171] In the context of the present disclosure, it also is contemplated that the compositions or devices described herein could be used similarly in conjunction with other standard treatments.
[000172] To effect an improved or enhanced therapy using the therapeutic agent of the present disclosure and another agent to therapy, one would generally contact a cell with the therapeutic at the same time or with one modality preceding the other such that, if applied separately, one would generally ensure that a significant period of time did not expire between the time of each treatment such that the therapeutic and other would still be able to exert an advantageously combined effect on the cell. In such instances, it is contemplated that one would contact the cell with both modalities within about 12-24 hours of each other and, more preferably, within about 6-12 hours of each other, with a delay time of only about 12 hours being most preferred. In some situations, it may be desirable to extend the time period for treatment significantly, however, where several days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the respective administrations.
[000173] It also is conceivable that more than one administration of either the therapeutic or the other agent will be desired. Various combinations may be employed, where the therapeutic according to the present disclosure is "A" and the other therapy is "B", as exemplified below'
A/B/A B/A/B B/B/A A/A/B B/A/A A/B/B B/B/B/A B/B/A/B
A/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B B/B/B/A
A/A/A/B B/A/A/A A/B/A/A A/A/B/A A/B/B/B B/A/B/B B/B/A/B
Other combinations are contemplated.
[000174] The composition or device described herein may be combined with any clinically acceptable treatment article. In some embodiments, the composition or device described herein and the other clinically acceptable treatment article are used concurrently. In some embodiments, the composition or device described herein is used before the clinically acceptable treatment article. In some embodiments, the composition or device described herein is used after the clinically acceptable treatment article. In some embodiments, the composition or device described herein may be combined with any clinically acceptable treatment modality.
[000175] Formulation and Administration
[000176] The present disclosure provides pharmaceutical compositions. Such compositions comprise a prophylactically or therapeutically effective amount of a bioactive agent, and a pharmaceutically acceptable carrier. In a specific embodiment, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a particular carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly forinjectable solutions. Other suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
[000177] The composition, if desired, can also contain minor amounts of weting or emul sifying agents, or pH buffering agents. These compositions can take the form of soluti ons, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical agents are described in "Remington's Pharmaceutical Sciences." Such compositions will contain a prophylactically or therapeutically effective amount of the agent, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration, which can be oral, intravenous, intraarterial, intrabuccal, intranasal, nebulized, bronchial inhalation, intra-rectal, vaginal, topical or delivered by mechanical ventilation.
[000178] Pharmaceutically acceptable salts include the acid salts and those which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Saks formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropyl amine, tri methylamine, 2-etbylamino ethanol, histidine, procaine, and the like.
[000179] EXAMPLES
[000180] The following examples are included to demonstrate preferred embodiments. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventor to function well in the practice of embodiments, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure.
[000181] Example 1: Synthesis of Alginate Methacrylate
[000182] The example below sets forth the fabrication of a hydrogel system for use in the encapsulation of a therapeutic agent, such as engineered cells and proteins, secreted proteins, hormones, cytokines, antibodies, enzymes, peptides, and other bioactive agents. A library of alginate methacrylate (A1MA) was synthesized by reacting sodium alginates of different molecular weights (UPVLVG with molecular weigh t< 75 kDa, SLG-20 with molecular weight 75 - 220 kDa and SLG-100 with molecular weight 200-300 kDa) with 2- aminoethyl methacrylate (AEMA) for 24 hours (FIG. 1 and Table 3). The product was dialyzed against DI water for 3 days, filtered through 70 micron pore sized filters and freeze dried. Alginates were stored at -20 degree Celsius until further use. Alginate methacrylate was then characterized using the 1H NMR. The characteristic peaks for the methylene protons in the region 5.5 - 6.2 ppm confirmed the methacrylation (FIG. 2). All successfully synthesized were combined at ratios of 1:0, 7:3, 6:4, 5:5, 4:6, and 3:7 with the 1 :0 formulation being 100% the material or in other words the base formulation.
[000183] Table 3. AIM A Formulations
Figure imgf000057_0001
Figure imgf000058_0001
Figure imgf000059_0001
Figure imgf000060_0001
Figure imgf000061_0001
Figure imgf000062_0001
Figure imgf000063_0001
Figure imgf000064_0001
Figure imgf000065_0001
Figure imgf000066_0001
[000184] Example 2: Hydrogel Forming Ability and Shape Retention
[000185] A1MA formulations were added into 96 well plates at 100 uL with 1 p.L of 1 w/v% Rhodamine Bin DMSO and then placed under UV light at 4 mW/cm"2 for 15 minutes. The plates were incubated for 10 minutes and then washed three times with DI water. Fluorescence measurements were taken (540 nm excitation, 580 nm emission) followed by gross imaging to access shape retention of the hydrogels within the 96 well plate. The negative control for the study was DI water and our positive control was a previously published alginate composed from 1.4 w't/v% SL20 ionically crosslinked in barium chloride solution for 30 minutes. Gelation was determined by the fluorescence intensity averaging above 15,000 AUG (FIG. 3). Hydrogel shape retention was evaluated as a measure of the ability of formulations to retain shape after gelation (FIG . 4). The weight changes of these formulations are monitored for the formulations that possess the least change in weight over time (FIG. 5).
[000186] ARPE-19 cells were seeded into black 96 well plates at 10k cells/90uL and given 24 hours to adhere and form monolayers. A1MA formulations were added into 96 well at 20 pL per well along with 60 pL of cell culture media. Alamar blue was then added into the wells at 10 pL per well and plates were incubator at 37 °C for 3 hours. Fluorescnce readings using TECAN plate reader (570 nm for measurement wavelength and 600 nra for reference wavelength). ARPE-19 ells in phenol red-free media alone were used as the positive control. [000187] Example 3: Mechanical Testing
[000188] A1MA formulations were added into 250 pl PDMS molds and placed under UV exposure at 4mW/cm"2 for 15 minutes. These Al MA gels were retrieved from the PDMS mold and used for compression testing. Compression testing may be performed by applying constant crosshead speed 10 pmis. Compression testing may be performed until fracture or until half the height of the hydrogel has been compressed. Stress vs. strain and modulus may be collected from the compression testing. Modulus may be obtained from the slope of stress vs. strain, limited to the first 15% of strain.
[000189] Example 4: Generation and screening of alginate methacrylate (A1MA) formulation library yields stable hydrogels for RPE-VEGFC cell factory encapsulation
[000190] The incorporation of methacrylate groups in the alginate backbone enables them to rapidly crosslink in response to UV light and enhance their strength and stability. We synthesized alginate methacrylates (A1MA) in-house by reacting sodium alginates of very low viscosity (VLVG) with molecular weight < 75 kDa and low viscosity (LVG-20) also known as SLG20 with molecular weight 75 kDa - 220 kDa, at methacrylation efficiencies between 20-46% with 2- aminoethyl methacrylate (AEMA). We previously characterized A1MA using the 1H NMR. The characteristic peaks for the methylene protons in the region 5.5 - 6.2 ppm confirmed the methacrylation.
[000191] We examined the ability of A1MA to undergo gelation and form hydrogels that could retain the shape of their mold upon exposure to UV light. Gelation and hydrogel shape retention were evaluated for the ability of the A1MA formulation to take the shape of a 96-well plate. The hydrogels that retained the shape of mold were further screened for mechanical strength. To test for mechanical strength, VLVG and SLG20 A1MA were added into 96 well plates at 1 OOuL per well and placed under UV exposure at 4mW/cmA2 for 15 minutes. These A1MA gels were retrieved from the 96-well plate and used for compression testing. Compression testing was performed by applying a constant crosshead linear rate of 10 um/s. Compression testing was performed until hydrogel fracture. Modulus was measured from the slope of stress vs. strain, limited to the fracture point. We were able to identify leading A1MA hydrogel formulations (VLVG-M46 and LV20-M20) as novel strong alginates that will help to maintain the stability of transplanted cells and therapeutics.
[000192] Methods
[000193] Engineering and culture of RPE-VEGFC cellular therapy with safety switch [000194] RPE-19 cells were engineered to secrete VEGF-C growth factor via a PiggyBac vector system with Lipofectamine 3000 (ThermoFisher, &L3000001) to transfect DNA inside RPE cells.
[000195] Alginate methacrylate (A1MA) synthesis and library generation
[000196] Alginate methacrylate (A1MA) was synthesized by reacting sodium alginates of different molecular weights (UP VLVG with molecular weight < 75 kDa, SLG-20 with molecular weight 75 kDa - 220 kDa and SLG-100 with molecular weight 200 kDa-300 kDa) with 2- aminoethyl methacrylate (AEMA) for 24 h. The product was dialyzed against DI water for 3 days, filtered through 70 micron pore sized filters and freeze dried. Alginates were stored at -20 degree Celsius until further use. Alginate methacrylate was then characterized using the 1H NMR. The characteristic peaks for the methylene protons in the region 5.5 - 6.2 ppm confirmed the methacrylation.
Table 4
Figure imgf000068_0001
Figure imgf000069_0001
ME: Methacrylation efficiency, M: Methacrylation
[00202] Rhodamine B gelation assay in vitro
[00203] A1MA formulations are added into 96 well plates at lOOuL with luL of lw/v% Rhodamine B in DMSO and then placed under UV light at 4mW/cmA2 for 15minutes. The plates are incubated for 10 minutes and then washed three times with DI water. Fluorescence measurements were taken (540nm excitation, 580nm emission) followed by gross imaging to access shape retention of the hydrogels within the 96 well plate. The negative control for the study was DI water and our positive control was a previously published alginate composed from 1.4wt/v% SL20 ionically crosslinked in barium chloride solution for 30 minutes. Gelation was determined by the fluorescence intensity averaging above 15,000 AUG,
[00204] Mechanical strength
[00205] A1MA formulations were added into 96 well plates at lOOuL per well and placed under UV exposure at 4mW/cmA2 for 15 minutes. These A1MA gels were retrieved from the 96 well plate and used for compression testing. Compression testing will be performed by applying constant crosshead speed 1.0 cm/min and 10N. Compression test will be performed until fracture. Stress vs strain and modulus will be collected from the compression testing. Modulus will be obtained from the slope of stress vs strain, limited to the first 2% of strain.
[00206] RPE cell viability encapsulated within A1MA
[00207] ARPE-19 cells were seeded into black 96 well plates at 10k cells/90uL and given 24 hours to adhere and form monolayers. A1MA formulations were added into 96 well at 20uL per well along with 60uL of cell culture media. Alamar blue was then added into the wells at lOuL per well and plates were incubator at 37C for 3 hours. Fluorescence readings using TECAN plate reader (570 nm for measurement wavelength and 600 nm for reference wavelength). ARPE-19 ells in phenol red-free media alone were used as the positive control.
[00208] Table 5. Complete A1MA formulation library.
Figure imgf000070_0001
Figure imgf000071_0001
[000197] All of the compositions and devices disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and devices of this disclosure have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and devices and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the disclosure. More specifically, it wall be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the disclosure as defined by the appended claims.
[000198] References
[000199] T he following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference:
WO 2022/266086 A2
Chang, Science, 146, 524-525 (1964). Haug and Smidsrod, Acta Chem Scand, 24, 843 -& (1970).
Kleinberger el al., J Biomater Sci Polym Ed, 27, 351-369 (2016).
Kollmer el al., Tissue Eng Part B Rev, (2015).
Lee and Mooney, Prag Polym Set., 37(1): 106-126, 2012.
Lim and Sun, Science, 210, 908-910 (1980).
Mooranian etal., Artif Cells Nanomed Biotechnol, 44, 194-200 (2016).
Strand etal., Stem Cells TranslMed, 6, 1053-1058 (2017).
Suzuki et al., J Biomed Mater Res. 39:317-322, 1998.

Claims

WHAT IS CLAIMED IS:
1. A composition comprising:
(i) a hydrogel system, wherein said system comprises at least one of A, B or C, wherein:
A is a photo-responsive alginate, wherein the molecular weight is less than 95 kDa and wherein the rnethacryla.ti on efficiency is from about 15% to about 95%;
B is a photo-responsive alginate, wherein the molecular weight is from about 55 kDa to about 240 kDa and wherein the methacrylation efficiency is from about 1% to about 65%; and
C is a photo-responsive alginate, wherein the molecular weight is from about 180 kDa to about 320 kDa and wherein the methacrylation efficiency from about 1% to about 25%; and
(ii) a therapeutic agent. The composition of claim 1, wherein the hydrogel system comprises a monomer of the formula:
Figure imgf000073_0001
wherein n and m are at least 1.
3 The composition according to claim 2 wherein n is 1-5. The composition according to claim 2 wherein m is 1-5.
5 The composition according to any one of claims 2-4, wherein n and mare the same.
6 A drug delivery' device comprising:
(i) a hydrogel system, wherein said system comprises at least one of A, B or C, wherein:
A is a photo-responsive alginate, wherein the molecular weight is less than 95 kDa and wherein the methacrylation efficiency is from about 15% to about 95%;
B is a photo-responsive alginate, wherein the molecular weight is from about 55 kDa to about 240 kDa and wherein the methacrylation efficiency is from about 1% to about 65%; and
C is a photo-responsive alginate, wherein the molecular weight is from about 180 kDa to about 320 kDa and wherein the methacrylation efficiency from about 1% to about 25%; and
(ii) a therapeutic agent The composition or device according to any one of claims 1-6, wherein the methacrylation efficiency of A is about 20%, about 46%, about 60% or about 90%. The composition or device according to any one of claims 1-6, wherein the methacrylation efficiency of Bis about 5%, about 10%, about 20%, about 46% or about 60%. The composition or device according to any one of claims 1-6, wherein the methacrylation efficiency of C is about 5%, about 10% or about 20%. The composition or device according to any one of claims 1-9, wherein: i. A is combined with B at a ratio of about 1 :0 to about 0: 1; ii. A is combined with C at a ratio of about 1 :0 to about 0: 1 ; or iii. B is combined with C at a ratio of about 1 :0 to about 0: 1. The composition or device according to any one of claims 1-10, wherein: i. A is combined with B at a ratio of about 7:3 to about 3:7; ii. A is combined with Cat a ratio of about 7:3 to about 3:7; or iii. B is combined with Cat a ratio of about 7:3 to about 3:7. The composition or device according to any one of claims 1-11, wherein: i. A is combined with Bat a ratio of about 7:3; ii. A is combined with Cat a ratio of about 7:3; or iii. B is combined with Cat a ratio of about 7:3. The composition or device according to any one of claims 1-11, wherein: i. A is combined with B at a ratio of about 6:4; ii. A is combined with C at a ratio of about 6:4; or iii. B is combined with C at a ratio of about 6:4. The composition or device according to any one of claims 1-11, wherein: i. A is combined with B at a ratio of about 5:5; ii. A is combined with C at a ratio of about 5:5; or iii. B is combined with Cat a ratio of about 5:5.
15. The composition or device according to any one of claims 1-11, wherein: i. A is combined with B at a ratio of about 4:6; ii. A is combined with C at a ratio of about 4:6; or iii. Bis combined with Cat a ratio of about 4:6.
16. The composition or device according to any one of claims 1-11, wherein: i. A is combined with Bat a ratio of about 3:7; ii. A is combined with Cat a ratio of about 3 :7; or iii. B is combined with Cat a ratio of about 3:7.
17. The composition or device according to any one of claims 1-1 1, wherein B is combined with C at a ratio of about 7:3.
18. The composition or devi ce according to any one of claims 1-17, wherein the therapeutic agent comprises an engineered cell or protein, a secreted protein, a hormone, a cytokine, an antibody, an enzyme or a peptide.
19. The drug delivery device according to claim 18, wherein the therapeutic agent comprises an engineered cell.
20. A method of treating a disease or disorder in a patient in need thereof, comprising administering to the patient a device or composition according to any one of claims 1-19.
PCT/US2024/053211 2023-10-27 2024-10-28 Photo-responsive therapeutic encapsulation compositions and devices Pending WO2025091007A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202363593813P 2023-10-27 2023-10-27
US63/593,813 2023-10-27

Publications (1)

Publication Number Publication Date
WO2025091007A1 true WO2025091007A1 (en) 2025-05-01

Family

ID=95516465

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2024/053211 Pending WO2025091007A1 (en) 2023-10-27 2024-10-28 Photo-responsive therapeutic encapsulation compositions and devices

Country Status (1)

Country Link
WO (1) WO2025091007A1 (en)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9642914B2 (en) * 2008-12-30 2017-05-09 Case Western Reserve University Photocrosslinked biodegradable hydrogel
US20190076373A1 (en) * 2011-04-28 2019-03-14 President And Fellows Of Harvard College Injectable preformed macroscopic 3-dimensional scaffolds for minimally invasive administration
US20200129660A1 (en) * 2013-03-07 2020-04-30 Case Western Reserve University Bioadhesive hydrogels

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9642914B2 (en) * 2008-12-30 2017-05-09 Case Western Reserve University Photocrosslinked biodegradable hydrogel
US20190076373A1 (en) * 2011-04-28 2019-03-14 President And Fellows Of Harvard College Injectable preformed macroscopic 3-dimensional scaffolds for minimally invasive administration
US20200129660A1 (en) * 2013-03-07 2020-04-30 Case Western Reserve University Bioadhesive hydrogels

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
FRANCIS LYDIA, GRECO KARIN V, BOCCACCINI ALDO R, ROETHER JUDITH J, ENGLISH NICHOLAS R, HUANG HONGLEI, PLOEG R, ANSARI TAHERA: "Development of a novel hybrid bioactive hydrogel for future clinical applications", JOURNAL OF BIOMATERIALS APPLICATIONS., TECHNOMIC, LANCASTER, PA., US, vol. 33, no. 3, 1 September 2018 (2018-09-01), US , pages 447 - 465, XP093313097, ISSN: 0885-3282, DOI: 10.1177/0885328218794163 *
JEON ET AL.: "Photocrosslinked alginate hydrogels with tunable biodegradation rates and mechanical properties", BIOMATERIALS, vol. 30, May 2009 (2009-05-01), pages 2724 - 2734, XP025970939, DOI: 10.1016/j.biomaterials.2009.01.034 *

Similar Documents

Publication Publication Date Title
Wang et al. Microencapsulation using natural polysaccharides for drug delivery and cell implantation
Sanjanwala et al. Polysaccharide-based hydrogels for drug delivery and wound management: a review
Aebischer et al. Macroencapsulation of dopamine-secreting cells by coextrusion with an organic polymer solution
Chen et al. Scaffold-mediated delivery for non-viral mRNA vaccines
Bruggeman et al. Temporally controlled growth factor delivery from a self-assembling peptide hydrogel and electrospun nanofibre composite scaffold
Jiang et al. Brain microenvironment responsive and pro‐angiogenic extracellular vesicle‐hydrogel for promoting neurobehavioral recovery in type 2 diabetic mice after stroke
Meng et al. Targeting delivery of miR-146a via IMTP modified milk exosomes exerted cardioprotective effects by inhibiting NF-κB signaling pathway after myocardial ischemia-reperfusion injury
US20140017304A1 (en) Method for encapsulated therapeutic products and uses thereof
JP2017536127A (en) Encapsulated stem cells for treating inflammatory diseases
Tian et al. Preparation and characterization of galactosylated alginate–chitosan oligomer microcapsule for hepatocytes microencapsulation
CN109925516A (en) A kind of composite hydrogel and preparation method thereof loading excretion body
Gattás-Asfura et al. Promoting dendrimer self-assembly enhances covalent layer-by-layer encapsulation of pancreatic islets
Yang et al. Initial IL-10 production dominates the therapy of mesenchymal stem cell scaffold in spinal cord injury
AU2018327318A1 (en) Adaptation of Hollow-Fiber-based cell culture technology for the manufacturing of Neo-Islets or exosomes from stem cells
CN101601862A (en) Application of Porous Starch and Its Derivatives as Drug Carriers
Baruch et al. Alginate-PLL cell encapsulation system Co-entrapping PLGA-microspheres for the continuous release of anti-inflammatory drugs
Feng et al. Mannose-modified erythrocyte membrane-encapsulated chitovanic nanoparticles as a DNA vaccine carrier against reticuloendothelial tissue hyperplasia virus
DeVolder et al. Three dimensionally flocculated proangiogenic microgels for neovascularization
WO2025091007A1 (en) Photo-responsive therapeutic encapsulation compositions and devices
CN102731775A (en) Poly spermine cations, construction method thereof, and preparation method of nano-grade particles
CN107693505B (en) A kind of oil-water dual-soluble ROS-sensitive nanoparticles and preparation method thereof
CN113521309A (en) Application of human hepatocyte growth factor gene in the treatment of eczema and its microneedling device
CN115990134A (en) Injectable hydrogel/nanogel drug-loaded slow-release system and preparation method and application thereof
KR102464966B1 (en) INJECTABLE γ-PGA-BASED HYDROGEL AND METHOD FOR PREPARING THE SAME
Dhayani et al. An Amphiphilic Double‐Brush Polymer Hydrogel for Sustained Release of Small Molecules and Biologics: Insulin Delivering‐Hydrogel to Control Hyperglycemia

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 24883492

Country of ref document: EP

Kind code of ref document: A1