Composition and kits for pseudoplastic microgel matrices

Abstract

This invention relates generally to water-insoluble but water-swellable and deformable crosslinked PEGylated microgel particles of proteins and protein-based macromolecules that are pseudoplastic (shear thinning) and flow in aqueous media under shear and which can be injected or made to flow, wherein said microgel particles can reform as a duster of microgel particles when shearing forces are removed. The microgel particles function as a matrix to support cell growth, viability, and proliferation.

1 . A composition comprising:
 a plurality of water-insoluble, hydrogel microparticles comprising PEGylated gel microparticles selected from the group consisting of (1) crosslinked, PEGylated proteins, (2) crosslinked, PEGylated protein-based biological macromolecules, and (3) combinations thereof,   wherein said PEGylated hydrogel microparticles comprise a protein or protein-based biological macromolecule crosslinked with a PEGylating agent that is difunctional to multifunctional,   wherein, when said plurality of water-insoluble, hydrogel microparticles are hydrated, pseudoplastic.   
     
     
         2 . The composition according to  claim 1 , wherein a molar ratio of PEGylating agent to protein and/or protein-based biological macromolecules is from 1:1 to 100:1. 
     
     
         3 . The composition according to  claim 1 , wherein the hydrogel microparticles are hydrated. 
     
     
         4 . The composition according to  claim 1 , wherein the viscosity of the composition decreases with applied shear and microgel clusters form in the absence of shear,
 wherein said composition has viscoelastic solid properties, a storage modulus greater than loss modulus, and a loss tangent value less than 1.   
     
     
         5 . The composition according to  claim 1 , wherein said proteins and protein-based biological macromolecules are selected from the group consisting of extracellular matrices, glycoproteins, structural proteins, fibrous proteins, enzymes, proteoglycans, natural polypeptides, synthetic polypeptides, globular proteins, membrane proteins, plasma proteins, peptides, oligopeptides, antimicrobial peptides, peptide hormones, chaperones, metalloproteins, hemoproteins, coagulation proteins, immune system proteins, ion channel proteins, cell adhesion proteins, neuropeptides, nucleoproteins, scleroproteins, chromoproteins, conjugated proteins, protein-protein complexes, protein-polysaccharide complexes, protein-lipid complexes, motor proteins, mucoproteins, phosphoproteins, contractile proteins, transport proteins, signaling proteins, regulatory proteins, growth factors proteins, sensory proteins, defense proteins, storage proteins, receptor proteins, antibodies, recombinant proteins, fibrinogen, fibrin, thrombin, collagen, elastin, albumin, gelatin, keratin, laminin, and combinations thereof. 
     
     
         6 . The composition according to  claim 1 , wherein said protein PEGylating agent is selected from the group consisting of α-succinimidyloxyglutaryl-ω-succinimidyloxyglutaryloxypolyoxyethylene (SG-PEG-SG), α-aminopropyl-ω-aminopropoxypolyoxyethylene, α-aminopropyl-ω-carboxypentyloxypolyoxyethylene, α,ω-bis {2-[(3-carboxy-1-oxopropyl)amino]ethyl}polyethylene glycol, α-[3-(3-maleimido-1-oxopropyl)amino]propyl-ω-[3-(3-maleimido-1-oxopropyl)amino]propoxypolyoxyethylene, pentaerythritol tetra(aminopropyl)polyoxyethylene, α-[3-(3-maleimido-1-oxopropyl)amino]propyl-ω-(succinimidyloxycarboxy)polyoxyethylene, pentaerythritol tetra(succinimidyloxyglutaryl)polyoxyethylene, pentaerythritol tetra(mercaptoethyl)polyoxyethylene, hexaglycerol octa(succinimidyloxyglutaryl)polyoxyethylene, hexaglycerol octa(4-nitrophenoxycarbonyl)polyoxyethylene, 4-arm poly(ethylene glycol) tetraacrylate, 4-arm succinimidyloxyglutaryl)polyoxyethylene, bis(polyoxyethylene bis[imidazoyl carbonyl]), O-(3-carboxypropyl)-O′-[2-(3-mercaptopropionylamino)ethyl]polyethylene glycol, O,O′-bis[2-(N-succinimidylsuccinylamino)ethyl]polyethylene glycol, O,O′-bis(2-azidoethyl)polyethylene glycol, poly(ethylene glycol) diacrylate, poly(ethylene glycol) diglycidyl ether, poly(ethylene glycol) di(p-nitrophenyl carbonate), poly(ethylene glycol) di(vinyl sulfone), poly(ethylene glycol) di(proprionaldehyde), poly(ethylene glycol) di(benzotriazolyl carbonate), and combinations thereof. 
     
     
         7 . The composition according to  claim 1 , wherein said storage modulus values of the composition, the hydrated, water-insoluble, miefegel hydrogel microparticles, or both, are between 10 Pa to 250,000 Pa and said loss modulus values are between 5 Pa to 100,000 Pa. 
     
     
         8 . The composition according to  claim 1  further comprising an antibacterial agent, antifungal agent, antiprotozoal agent, antiviral agent, antibiotic, monoacyl glycerol, monoalkyl glycol, bis(biguanide) and its salts, poly(hexamethylene biguanide) and its salts, glycerol monolaurate, chlorhexidine, chlorhexidine digluconate, chlorhexidine diacetate, alexidine, alexidine hydrochloride, silver salts, benzalkonium chloride, benzethonium chloride, gentamicin sulfate, iodine, povidone-iodine, starch-iodine, neomycin sulfate, polymyxin B, bacitracin, tetracyclines, clindamycin, gentamicin, nitrofurazone, mafenide acetate, silver sulfadiazine, terbinafine hydrochloride, miconazole nitrate, ketoconazole, clotrimazole, itraconazole, metronidazole, antimicrobial peptides, polyquaternium-1, polyquaternium-6, polyquaternium-10, cationic guar, water-soluble derivatives of chitosan, salts thereof, and combinations thereof. 
     
     
         9 . The composition according to  claim 1 , further comprising water-soluble polymers at a concentration of from 0.01 weight % to 25 weight %, wherein the water-soluble polymers are selected from the group consisting of poly(ethylene glycol), poly(ethylene oxide), poly(vinyl alcohol) and copolymers, poly(N-vinylpyrrolidone) and copolymers, methylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, carboxymethylcellulose, guar gum, hydroxyethylguar, hydroxypropylguar, gelatin, albumin, hydroxypropylmethylguar, carboxymethylguar, carboxymethylchitosan, locust bean gum, carrageenan, xanthan gum, gellan gum, pullulan, dextran, dextran sulfate, Aloe vera gel, scleroglucan, schizophyllan, gum arabic, tamarind gum, poly(methyl vinyl ether), ethylene oxide-propylene oxide-ethylene oxide block copolymers, hyaluronan, chondroitin sulfate, keratan sulfate, dermatan sulfate, heparan sulfate, dextran, Carbomer and its salts, poly(acrylic acid) and its salts, poly(methacrylic acid) and its salts, poly(ethylene-co-acrylic acid), poly(vinyl methyl ether), poly(vinylphosphoric acid) salts, poly(vinylsulfonic acid) salts, sodium poly(2-acrylamido-2-methylpropanesulfonate), polyacrylamide(s), poly(N,N-dimethylacrylamide), poly(N-vinylacetamide), poly(N-vinylformamide), poly(2-hydroxyethyl methacrylate), poly(glyceryl methacrylate), poly(N-isopropylacrylamide) and poly(N-vinylcaprolactam), the latter two hydrated below their Lower Critical Solution Temperatures, polyquaternium-1, polyquaternium 6, polyquaternium-10, ionene polymers, cationic guar, pyridinium polymers, imidazolium polymers, diallyldimethylammonium polymers, poly(L-lysine), acryloyl-, methacryloyl-, and styryl-trimethylammonium polymers, acrylamide- and methacrylamido-trimethylammonium polymers, and antimicrobial peptides. 
     
     
         10 . The composition according to  claim 9 , wherein the composition is in the form of a dry or hydrated film comprising said plurality of water-insoluble, hydrogel microparticles. 
     
     
         11 . The composition according to  claim 1 , further comprising a biological component selected from the group consisting of cells; stem cells; morselized amniotic tissue;
 placental tissue; minced tissue, including minced skin tissue, muscle tissue, vascular tissue, nerve tissue, fat tissue, cartilage tissue, bone tissue, tendon tissue, bladder tissue, intestinal tissue, heart tissue, lung tissue, kidney tissue, liver tissue, pancreatic tissue, and vocal fold tissue; micronized tissue and micronized decellularized tissue, including skin tissue, muscle tissue, vascular tissue, nerve tissue, fat tissue, cartilage tissue, bone tissue, tendon tissue, bladder tissue, intestinal tissue, heart tissue, lung tissue, kidney tissue, liver tissue, pancreatic tissue, vocal fold tissue; synthetic or naturally derived extracellular matrix components, including collagen, glycosaminoglycans, fibrin, laminin, fibronectin; hydroxyapatite; granulated crosslinked bovine tendon collagen and glycosaminoglycans; honey; polysaccharides; biodegradable polymers, including polyglycolides, polylactides, poly(lactide-co-glycolide), polydioxanone, polycaprolactone, poly(trimethylene carbonate), poly(propylene fumarate), polyurethanes, poly(ester amide)s, poly(ortho ester)s, polyanhydrides, poly(amino acid)s, polyphosphazenes, bacterial polyesters; and combinations thereof.   
     
     
         12 . The composition according to  claim 11 , wherein the stem cells are selected from the group consisting of adult stem cells, embryonic stem cells, amniotic stem cells, induced pluripotent stem cells, fetal stem cells, tissue stem cells, adipose-derived stem cells, bone marrow stem cells, human umbilical cord blood stem cells, blood progenitor cells, mesenchymal stem cells, hematopoietic stem cells, epidermal stem cells, endothelial progenitor cells, epithelial stem cells, epiblast stem cells, cardiac stem cells, pancreatic stem cells, neural stem cells, limbal stem cells, perinatal stem cells, satellite cells, side population cells, multipotent stem cells, totipotent stem cells, unipotent stem cells, and mixtures thereof. 
     
     
         13 . The composition according to  claim 11 , wherein the cells are selected from the group consisting of fibroblasts, keratinocytes, neurons, glial cells, astrocytes, Schwann cells, dorsal root ganglia, adipocytes, endothelial cells, epithelial cells, chondrocytes, fibrochondrocytes, myocytes, cardiomyocytes, myoblasts, hepatocytes, tenocytes, intestinal epithelial cells, smooth muscle cells, stromal cells, neutrophils, lymphocytes, bone marrow cells, pericytes, platelets, and mixtures thereof. 
     
     
         14 . The composition according to  claim 1 , further comprising biologically active agents selected from the group consisting of nanoparticles, microparticles, antiseptics, anti-infective agents, antimicrobial agents, sporicidal agents, antiparasitic agents, peripheral neuropathy agents, neuropathic agents, chemotactic agents, analgesic agents, anti-inflammatory agents, anti-allergic agents, anti-hypertension agents, mitomycin-type antibiotics, polyene antifungal agents, antiperspirant agents, decongestants, anti-kinetosis agents, central nervous system agents, wound healing agents, anti-VEGF agents, anti-tumor agents, escharotic agents, anti-psoriasis agents, anti-diabetic agents, anti-arthritis agents, anti-itching agents, antipruritic agents, anesthetic agents, anti-malarial agents, dermatological agents, anti-arrhythmic agents, anticonvulsants, antiemetic agents, anti-rheumatoid agents, anti-androgenic agents, anthracyclines, anti-smoking agents, anti-acne agents, anticholinergic agents, anti-aging agents, antihistamines, anti-parasitic agents, hemostatic agents, vasoconstrictors, vasodilators, thrombogenic agents, anticlotting agents, cardiovascular agents, angina agents, erectile dysfunction agents, sex hormones, growth hormones, isoflavones, integrin binding sequence, biologically active ligands, cell attachment mediators, immunomodulators, tumor necrosis factor alpha, anti-cancer agents, antineoplastic agents, anti-depressant agents, antitussive agents, anti-neoplastic agents, narcotic antagonists, anti-hypercholesterolaemia agents, apoptosis-inducing agents, birth control agents, sunless tanning agents, emollients, alpha-hydroxyl acids, manuka honey, topical retinoids, hormones, tumor-specific antibodies, antisense oligonucleotides, small interfering RNA (siRNA), anti-VEGF RNA aptamer, nucleic acids, DNA, DNA fragments, DNA plasmids, Si-RNA, transfection agents, vitamins, essential oils, liposomes, silver nanoparticles, gold nanoparticles, drug-containing nanoparticles, albumin-based nanoparticles, chitosan-containing nanoparticles, polysaccharide-based nanoparticles, dendrimer nanoparticles, phospholipid nanoparticles, iron oxide nanoparticles, bismuth nanoparticles, gadolinium nanoparticles, metallic nanoparticles, ceramic nanoparticles, silica-based nanoparticles, virus-based nanoparticles, virus-like nanoparticles, antibiotic-containing nanoparticles, nitric oxide-containing nanoparticles, nanoshells, nanorods, polymeric micelles, silver salts, zinc salts, quantum dots nanoparticles, polymer-based microparticles, polymer-based microspheres, drug-containing microparticles, drug-containing microspheres, antibiotic-containing microparticles, antibiotic-containing microspheres, antimicrobial microparticles, antimicrobial microspheres, salicylic acid, benzoyl peroxide, 5-fluorouracil, nicotinic acid, nitroglycerin, clonidine, estradiol, testosterone, nicotine, motion sickness agents, scopolamine, fentanyl, diclofenac, buprenorphine, bupivacaine, ketoprofen, opioids, cannabinoids, enzymes, enzyme inhibitors, oligopeptides, cyclopeptides, polypeptides, proteins, prodrugs, protease inhibitors, cytokines, hyaluronic acid, chondroitin sulfate, dermatan sulfate, parasympatholytic agents, chelating agents, hair growth agents, lipids, glycolipids, glycoproteins, endocrine hormones, growth hormones, growth factors, differentiation factors, heat shock proteins, immunological response modifiers, saccharides, polysaccharides, insulin and insulin derivatives, steroids, corticosteroids, non-steroidal anti-inflammatory drugs, in either their salt form or their neutral form, and combinations thereof. 
     
     
         15 . The composition according to  claim 1 , wherein the water-insoluble, hydrogel microparticles are in the form of a dry powder. 
     
     
         16 . The composition according to  claim 1 , further comprising an aqueous media selected from water, isotonic saline, balanced salt solution, buffer solution, Ringer's solution, cell culture media, stem cell media, serum, blood plasma, amniotic fluid, Wharton's jelly, nutrient broth, antiseptic solutions, or combinations thereof. 
     
     
         17 . The composition according to  claim 16 , wherein the composition is in a form selected from the group consisting of solutions, suspensions, creams, lotions, gels, pastes, emulsions, balms, sprays, foams, aerosols, and other formulations thereof. 
     
     
         18 . The composition according to  claim 1 , where a protein that is PEGylated is selected from the group consisting of fibrinogen, fibrin, extracellular matrix, plasma proteins, and collagen; and the PEGylating agent is α-succinimidyloxyglutaryl-ω-succinimidyloxyglutaryloxypolyoxyethylene (SG-PEG-SG). 
     
     
         19 . The composition according to  claim 1 , where a protein that is PEGylated is selected from fibrinogen and fibrin and the microgel particles induce angiogenesis in a mammalian body. 
     
     
         20 . The composition according to  claim 1 , where a protein that is PEGylated is selected from fibrinogen and fibrin, the PEGylating agent is α-succinimidyloxyglutaryl-ω-succinimidyloxyglutaryloxypolyoxyethylene (SG-PEG-SG), and the composition comprises an active biological agent selected from poly(hexamethylene biguanide) and its salts. 
     
     
         21 . A method of preparing water-insoluble, microgel particles, comprising:
 providing a biological starting material comprising a protein, a protein-based biological macromolecule, or a combination of both;   reacting said biological starting material with a PEGylating agent;   PEGylating said biological starting material by crosslinking said biological starting material with said PEGylating agent to form difunctional to multifunctional PEGylation,   milling or shearing said PEGylated biological starting material under hydrated or dehydrated conditions to produce a plurality of microgel particles, wherein said microgel particles are pseudoplastic in aqueous solution,   wherein a solution of said hydrated microgel particles decreases in viscosity with applied shear and reforms a microgel cluster in the absence of shear,   wherein the molar ratio of PEGylating agent to biological starting material is from 1:1 to 100:1, and   wherein the hydrated microgel particles have viscoelastic solid properties, storage modulus greater than loss modulus, and loss tangent values less than 1.   
     
     
         22 . A method of treating a soft tissue lesion or injury, comprising:
 providing a composition according to  claim 1 , and   injecting the composition into soft tissue, wherein said injection is intradermal, subcutaneous, oral, intramuscular, submucosal, intranasal, vaginal, buccal, intrathecal, epidural, intraparenchymal, ocular, subretinal, dental, intratumoral, intracardiac, intra-articular, intravenous, intracavernous, intraosseous, intraperitoneal, intra-abdominal, intrafascial, intraogan, and intravitreal.   
     
     
         23 . The method of  claim 22 , wherein the water-insoluble, microgel particles of  claim 1  are in the form of a dry powder, and the method further comprises hydrating said dry powder prior to said injection step. 
     
     
         24 . The method of  claim 22 , wherein, after injection, the composition promotes angiogenesis. 
     
     
         25 - 29 . (canceled) 
     
     
         30 . The composition of  claim 1 , wherein, when said plurality of water-insoluble, hydrogel microparticles are hydrated, said composition has (i) viscoelastic solid properties, (ii) a storage modulus greater than loss modulus, (iii) a loss tangent value less than 1, or more than one of conditions (i), (ii), and (iii)