U.S. patent application number 14/689625 was filed with the patent office on 2016-10-20 for composition and kits for pseudoplastic microgel matrices.
This patent application is currently assigned to ROCHAL INDUSTRIES, LLC. The applicant listed for this patent is ROCHAL INDUSTRIES, LLC. Invention is credited to Eunna Chung, Kelly Xiaoyu-Chen Leung, Katelyn Elizabeth Reilly, Ann Beal Salamone, Joseph Charles Salamone, Laura Jean Suggs.
Application Number | 20160303281 14/689625 |
Document ID | / |
Family ID | 57126813 |
Filed Date | 2016-10-20 |
United States Patent
Application |
20160303281 |
Kind Code |
A1 |
Salamone; Joseph Charles ;
et al. |
October 20, 2016 |
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.
Inventors: |
Salamone; Joseph Charles;
(San Antonio, TX) ; Salamone; Ann Beal; (San
Antonio, TX) ; Reilly; Katelyn Elizabeth; (San
Antonio, TX) ; Suggs; Laura Jean; (Austin, TX)
; Chung; Eunna; (Seoul, KR) ; Leung; Kelly
Xiaoyu-Chen; (San Antonio, TX) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ROCHAL INDUSTRIES, LLC |
San Antonio |
TX |
US |
|
|
Assignee: |
ROCHAL INDUSTRIES, LLC
San Antonio
TX
|
Family ID: |
57126813 |
Appl. No.: |
14/689625 |
Filed: |
April 17, 2015 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61L 27/227 20130101;
A61L 27/3834 20130101; A61L 27/52 20130101; A61L 27/26 20130101;
A61L 2400/06 20130101; A61L 27/3604 20130101; A61L 27/18 20130101;
A61L 27/225 20130101; A61L 27/38 20130101; A61L 27/22 20130101;
A61L 27/3633 20130101; A61L 2300/406 20130101; A61P 9/00 20180101;
A61L 27/24 20130101; A61L 27/54 20130101; A61L 27/26 20130101; C08L
71/02 20130101; A61L 27/26 20130101; C08L 89/00 20130101; A61L
27/18 20130101; C08L 71/02 20130101 |
International
Class: |
A61L 27/22 20060101
A61L027/22; A61L 27/36 20060101 A61L027/36; A61L 27/38 20060101
A61L027/38; A61L 27/52 20060101 A61L027/52; A61L 27/24 20060101
A61L027/24; A61L 27/54 20060101 A61L027/54 |
Claims
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
.alpha.-succinimidyloxyglutaryl-.omega.-succinimidyloxyglutaryloxypolyoxy-
ethylene (SG-PEG-SG),
.alpha.-aminopropyl-.omega.-aminopropoxypolyoxyethylene,
.alpha.-aminopropyl-.omega.-carboxypentyloxypolyoxyethylene,
.alpha.,.omega.-bis
{2-[(3-carboxy-1-oxopropyl)amino]ethyl}polyethylene glycol,
.alpha.-[3-(3-maleimido-1-oxopropyl)amino]propyl-.omega.-[3-(3-ma-
leimido-1-oxopropyl)amino]propoxypolyoxyethylene, pentaerythritol
tetra(aminopropyl)polyoxyethylene,
.alpha.-[3-(3-maleimido-1-oxopropyl)amino]propyl-.omega.-(succinimidyloxy-
carboxy)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
.alpha.-succinimidyloxyglutaryl-.omega.-succinimidyloxyglutarylo-
xypolyoxyethylene (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
.alpha.-succinimidyloxyglutaryl-.omega.-succinimidyloxyglutaryloxypolyoxy-
ethylene (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)
Description
FIELD OF THE INVENTION
[0001] This invention relates generally to crosslinked,
water-swellable microgel particles of proteins and protein-based
biological macromolecules that are pseudoplastic and flow in
aqueous media under shear and which can be coated, injected,
sprayed, painted, or implanted in tissues, organs, and wound void
spaces as well as surround tissue substitutes, where the microgel
particles aggregate as a monolithic microgel cluster in the absence
of shearing forces.
[0002] The microgel particles function as a viscoelastic matrix to
support cell growth, viability, and proliferation.
BACKGROUND
[0003] The field of hydrogels for use as biologically-compatible
macromolecules has been intensively investigated, commencing with
their commercial use as monolithic, cross-linked, soft contact lens
materials (U.S. Pat. No. 3,408,429). Because of their soft,
flexible nature, hydrogels are excellent materials for many
biomedical applications, including extended wear silicone-hydrogel
contact lenses, delivery of drugs and other pharmaceutically active
ingredients, development of new biomaterials, coating of
biomaterials, bioadhesives and sealants for cell encapsulation and
delivery, and cell culture substrates and scaffolds for tissue
regeneration.
[0004] Both natural and synthetic polymers can be used to create
scaffolds for tissue engineering applications. Natural polymers
often have inherent biocompatibility and can be biologically
active. Commonly used natural polymers that can be used as cell
scaffolds include collagen, hyaluronan, fibrin, fibrinogen, silk,
alginate, chitosan, dextran, and agarose, among others.
[0005] Synthetic polymer-based cell scaffolds allow control over
physical and chemical properties, such as stability, degradation
kinetics, physical properties, and mechanical strength. Many
synthetic polymers have been investigated for use as a matrix for
tissue engineering scaffolds, including poly(ethylene glycol)
(PEG), poly(lactic acid), poly(glycolic acid),
poly(lactic-co-glycolic acid), polycaprolactone,
poly(hydroxyalkanoate)s, and poly(vinyl alcohol). In particular,
PEG has been widely used as a scaffold material because of its
unique chemical and physical properties, such as solubility in
water and in organic solvents, nontoxicity, low protein adhesion
and nonimmunogenicity. In addition, PEG includes hydroxyl
end-groups that facilitate polymer modification, creating novel
structures and architectures with different chemical, physical, and
biological properties, particularly with biological moieties to
enhance their biological activity. Poly(ethylene glycol) has often
been used in combination with other polymers, to create scaffolds
for the purpose of regenerating a number of tissues, including
cartilage, bone, nerve, vasculature and muscle.
[0006] Compositions and methods for forming hydrogels in situ are
discussed in U.S. Pat. No. 6,818,018 through a combination of
physical and chemical crosslinking processes. The in situ formed
hydrogels may be applied in conjunction with bioactive molecules
that either are dissolved or dispersed within the hydrogels.
Methods of using such hydrogels as tissue coatings to prevent
postsurgical adhesion, as tissue augmentation or luminal occlusion
aids, as matrices for carrying cells, drugs or other bioactive
species, as tissue sealants or adhesives, and as medical device
coatings also are presented.
[0007] In U.S. Pat. Nos. 7,776,240 and 8,323,794, injectable
hydrogel microspheres are described by forming an emulsion where
hydrogel precursors are in a dispersed aqueous phase and the
hydrogel precursors are polymerized, yielding cross-linked
microspheres. Preferably, the hydrogel precursors are poly(ethylene
glycol) diacrylate and N-isopropylacylamide and the continuous
phase of the emulsion is an aqueous solution of dextran and a
dextran solubility reducer. Proteins, such as cytokines, can be
loaded into the microspheres.
[0008] U.S. Patent Application Publication Number 2004/0220296
discloses a gel formulation comprising poly(N-isopropylacrylamide),
which is also injectable in a liquid form. Solutions of this
polymer, copolymers or mixtures of the polymer with a second
polymer such as poly(ethylene glycol), polyvinylpyrrolidone or
poly(vinyl alcohol) are liquids at room temperature and solids at
body temperature. Methods are described of implanting a hydrogel
into a mammal by injecting the solution as a liquid at a
temperature below body temperature, which then undergoes thermal
phase transition to form a solid hydrogel in situ in the body as
the implant warms to body temperature.
[0009] In U.S. Patent Application Publication Number 2008/0268056 a
biocompatible substance is described that is useful for repairing a
vertebral compression fracture. The biocompatible substance can be
made from two or more biocompatible polymeric hydrogels via
physical crosslinking. The biocompatible substance thus made is
thermoresponsive and exhibits a lower critical solution
temperature. It undergoes volume and stage changes with temperature
in the range of 25.degree. C.-34.degree. C. The biocompatible
substance can be in a liquid injectable form at room temperature
and can gel within the human body at higher temperature. Other
thermoresponsive hydrogels for injection based upon synthetic
polymers are described in U.S. Patent Application Publication
Number 2009/0053276.
[0010] Processes for the preparation of injectable hyaluronan
hydrogels are reported in U.S. Patent Application Publication
Number 2005/0281880. The processes include crosslinking one or more
polymers and washing the subsequently formed gel, followed by
purification and homogenization by impeller stirring to produce an
injectable hydrogel. The swelling degree of the gels in phosphate
buffered saline can be about 4,000-5,000%. The gels can have
particle sizes on the order of 500 micrometers. The crosslinking
reaction can be carried out with a bi- or polyfunctional
crosslinking agent, such as an epoxide, aldehyde, polyaziridyl or
divinyl sulfone. The crosslinking agent can be 1,4-butanediol
diglycidyl ether. The process can be carried out at a pH of 11 or
higher at a temperature of 37-60.degree. C.
[0011] In U.S. Pat. No. 4,172,066, spheroidal microgels of a
water-swellable or water-swollen, crosslinked polymer consisting
essentially of water-soluble ethylenically unsaturated monomers are
described that are dispersed in water or other aqueous media and
are effective thickening agents which, when dispersed in an aqueous
medium, exhibit pseudoplastic rheology. Because of their uniform
small particle size, with water-swollen diameters generally within
the range from about 0.5 to about 200 micrometers, and their
ability to absorb substantial proportions of water, the microgels
are particularly suited for applications requiring thickening
agents, such as rapid sorption of aqueous fluids, e.g., sanitary
articles such as diapers, belt pads and the like, and for
applications wherein the swelling or partial plugging properties of
the polymer are particularly important, e.g., in the plugging of
porous formations or structures.
[0012] U.S. Pat. No. 8,038,721 discloses a soft, non-toxic tissue
filler that consists of spherically shaped solid particles having a
textured surface of a size range of between about 32 and 90
microns. The particles are suspended evenly in a gel as a carrier,
where the solid particles are preferably a non-ceramic cured
polymer such as poly(methyl methacrylate). The gel is a combination
of a cellulose polysaccharide, such as carboxymethylcellulose, and
an alcohol, such as poly(vinyl alcohol), dissolved in water or some
other solvent. The filler is used by injection in order to augment
a patient's soft tissue as well as to correct soft tissue
defects.
[0013] An injectable gel is described in U.S. Pat. No. 8,574,629,
comprising across-linked biopolymer-based matrix in which
previously cross-linked biopolymer particles have been
co-cross-linked with the matrix, where the biopolymer is selected
from the group consisting of sodium hyaluronate, chondroitin
sulfate, keratan, keratan sulfate, heparin, heparan sulfate,
cellulose and its derivatives, alginates, xanthan, carrageenan,
proteins, nucleic acids and mixtures thereof, and wherein the
crosslinking agent is butanediol diglycidyl ether.
[0014] U.S. Patent Application Publication Number 2012/0034271
discloses an injectable in situ disulfide-bond crosslinked
hydrogel, whose gelation process is completed in a syringe, wherein
the thiol groups are oxidized into disulfide bonds to form the
cross-linked hydrogel by oxygen dissolved in the crosslinking
active solution in the sealed injectable container, where the
in-situ crosslinked hydrogel is a derivative of polysaccharides,
proteins or synthetic macromolecules produced by one or more
chemical modifications.
[0015] A slowly polymerizing, biocompatible, biodegradable polymer
capable crosslinking to form a hydrogel that delivers isolated
cells into a patient to create an organ equivalent or tissue, such
as cartilage, is reported in U.S. Pat. No. 6,129,761. The polymer
is selected from the group consisting of modified hyaluronic acids,
synthetic modified alginates, polymers that are covalently
crosslinkable by a radical reaction and polymers that gel by
exposure to monovalent ions. The gels are reported to promote
engraftment and provide three-dimensional templates for new cell
growth. In one embodiment, cells are suspended in a polymer
solution and injected directly into a site in a patient, where the
polymer crosslinks to form a hydrogel matrix having cells dispersed
therein. In another embodiment, cells are suspended in a polymer
solution which is poured or injected into a mold having a desired
anatomical shape, then crosslinked to form a hydrogel matrix having
cells dispersed therein, which can be implanted into a patient.
[0016] A microporous injectable, soft elastic, fully resorbable
fibrin-based composition for use as a soft tissue lumen and void
filler is described in U.S. Patent Application Publication Number
2013/0209370. The composition combines a fibrinogen component, a
thrombin component, a plasticizer, and calcium-containing particles
having an average diameter between 0.01 .mu.m and 200 .mu.m. The
preparation of an injectable soft tissue void filler composition is
described by mixing the components together and/or homogenizing
said components.
[0017] In U.S. Pat. No. 6,290,729, thixotropic and pseudoplastic
polymers are described that exhibit shear thinning, whereby a
polymer becomes more fluent under shear, and reverts to a
high-viscosity or gelled form on cessation of shear. The '729
patent describes the formation of coatings that can be controlled
by introducing crosslinking agents, gelling agents or crosslinking
catalysts together with the fluent material, or thermoresponsive
behavior in the polymer, and then altering the conditions such that
crosslinking and/or gelling occurs in situ. A method for
controlling tissue repair or in-growth is described that includes
applying a polymeric material, at a site where tissue growth may
occur, wherein the polymeric material is applied in a first fluent
state and then converted in situ to a second non-fluent state.
[0018] Methods of preparing an extracellular matrix powder are
described in U.S. Patent Application Publication Number
2012/0264190. In the '190 Publication, the compositions can be used
for the formation of three-dimensional graft constructs for
implantation, injection, or the powdered or particulate
extracellular matrix material may be dispersed in a gel or ointment
for topical use to induce the repair of damaged or diseased tissues
in a host. The composition is a thermallyresponsive hydrogel that
is in a liquid form at room temperature and in gel form at a
temperature greater than room temperature or at normal body
temperature.
[0019] In U.S. Pat. No. 8,802,436, improved methods of
manufacturing bioactive gels from an extracellular matrix that
retain sufficient bioactivity to assist in tissue repair are
presented by particularizing the extracellular matrix to a particle
size in the range of about 1 .mu.m to about 1,000 .mu.m,
solubilizing the particularized powder in sodium hydroxide,
neutralizing the solubilized extracellular matrix with hydrochloric
acid, and, optionally, freezing or lyophilizing the frozen
extracellular matrix, and, optionally, reconstituting the
lyophilized gel in water or saline. The final consistency of all
gels is foam-like. Injection vehicles suitable for administering
particulate suspensions are described in
[0020] U.S. Pat. No. 7,582,311. Examples include polymer-based
formulations, associated pharmaceutical formulations, articles of
manufacture, and kits. The injection vehicles are reported to
include a pseudoplastic composition that improves injectability.
The injection vehicle comprises a flexible molecule, such as
hyaluronic acid or a derivative thereof, dissolved in a
physiological buffer, such as saline.
[0021] In U.S. Patent Application Publication Number 2004/0208938,
injectable compositions having improved injectability and methods
for the preparation of such injectable compositions are described.
In the '938 Publication, dry microparticles with an aqueous
injection vehicle form a suspension, and the suspension is then
mixed with a viscosity enhancing agent to increase the viscosity of
the fluid phase of the suspension to the desired level for improved
injectability.
[0022] U.S. Patent Application Publication Number 2010/0330184
describes injection vehicles suitable for administering particulate
suspensions, such as polymer-based formulations, as well as
associated pharmaceutical formulations, articles of manufacture,
and kits. The injection vehicles of the '184 Publication are
reportedly superior to conventional injection vehicles in that they
include a pseudoplastic flexible polymer composition, such as
solutions of hyaluronic acid, hyaluronic acid derivatives, and
combinations thereof, which improve injectability, facilitating
delivery of the desired dose.
[0023] An injectable polymer composition for use as a cell delivery
vehicle is discussed in U.S. Patent Application Publication Number
2013/0189230. The injectible polymer composition of the '230
Publication includes at least one thermal gelling polymer (methyl
cellulose), at least one anionic gelling polymer (hyaluronan), and
a water-based carrier, and is reported to be shear-thinning,
thixotropic and resorbable.
[0024] In U.S. Pat. No. 8,357,402, a flowable wound matrix material
is presented thats comprised of particles of a collagen matrix,
preferably a collagen/glycosaminoglycan (GAG) matrix, that, when
hydrated, reportedly can be effectively delivered to the wound
site. Reportedly, the flowable wound matrix can be effectively
delivered into wounds having varying depths and geometries and
provides a structural framework that serves as a scaffold for cell
ingrowth.
[0025] In U.S. Pat. No. 7,442,397, methods and materials related to
fibrin-based biomatrices are presented, where stem cells or
progenitor cells can be delivered to a diseased heart using a
fibrin-based biomatrix to assist or restore heart function. The
'397 Patent reportedly provides a method for repairing damaged
tissue in a mammal by introducing, on or into a tissue in need of
repair, a fibrinogen solution having stem or progenitor cells under
conditions such that a fibrin biomatrix including the cells forms
at or near the site of introduction. In one embodiment, the
fibrinogen solution is PEGylated. The conditions for forming the
fibrin biomatrix can include introducing a solution having a
fibrinogen-converting agent (e.g., a serine protease such as
thrombin) to the fibrinogen solution.
[0026] U.S. Pat. No. 5,733,563 reports hydrophilic water-swellable
gels consisting of a crosslinked mixture of a bifunctionalized
poly(ethylene oxide), activated with a suitable activating agent,
dissolved in an aqueous solution and an albumin-type protein. The
resulting hydrogels are based on the crosslinking of a protein,
namely albumin of various sources including, for example, bovine
serum albumin, with a bifunctional poly(alkene oxide),
preferentially poly(ethylene oxide), and most preferably
poly(ethylene glycol), or a mixture of bifunctional poly(alkene
oxide)s of various molecular masses. The mechanical properties of
the hydrogels can be improved by adding unreactive poly(ethylene
glycol) or other inert polymers of high molecular masses. The novel
hydrogels can be used for making contact lenses, controlled drug
release devices, immobilization matrices for enzymes or cells of
therapeutic interest, wound dressings and artificial skin.
[0027] A hydrogel system comprising polymer-conjugated albumin
molecules for controlled release delivery of therapeutic agents is
described in U.S. Patent Application Publication Number
2011/0238000. The polymer is a functionalized synthetic polymer,
preferably PEG-diacrylate. The polymer-conjugated albumin is
preferably mono-PEGylated albumin. The hydrogel system may comprise
a matrix to which the polymer-conjugated albumin molecules are
linked via a second functional group of the polymer. The matrix may
be formed from the same polymer of the polymer-albumin
conjugate.
[0028] U.S. Patent Application Publication Number 2010/0137510
reportedly discloses a polymer-protein precursor molecule that
comprises a thiolated protein and at east two synthetic polymers
covalently attached to thiol groups of the thiolated protein, each
of the at least two synthetic polymers having a functional group,
the functional group being selected capable of crosslinking with a
functional group of at least one other synthetic polymer being
covalently attached to at least one other polymer-protein precursor
molecule so as to form a scaffold. The functional group may be an
acrylate or vinyl sulfone, which are crosslinked by
photoinitiation. Soluble proteins of PEG-collagen, PEG-albumin, and
PEG-fibrinogen could be combined and photocrosslinked to form
hydrogel scaffolds.
[0029] U.S. Patent Application Publication Number 2003/0166867
discloses fibrin nanoparticles having a mean diameter of 200-2000
nm (0.2-2.0 .mu.m), and may be obtained by mixing an aqueous
solution comprising fibrinogen, thrombin and Factor XIII in an oil
emulsion at a temperature of 50-80.degree. C., without the addition
of an exogenous chemical crosslinking agent. Related U.S. Pat. No.
6,150,505 reports the preparation of fibrin microbeads of 50-200
.mu.m in diameter by reacting fibrinogen and thrombin, in heated
vegetable oil (70-80.degree. C.), in the presence of endogenous
factor XIII.
[0030] U.S. Pat. No. 8,858,925 reportedly discloses biodegradable
scaffolds composed of a naturally-occurring protein backbone of
fibrinogen crosslinked by a modification of the synthetic polymer
poly(ethylene glycol) that generates a PEGylated-fibrinogen
scaffold for use in treating disorders requiring tissue
regeneration. The '925 Patent uses a precursor molecule comprising
a fibrinogen protein that is denatured and retains an activity of
forming a scaffold and at least two poly(ethylene glycol) molecules
covalently connected to free thiol groups of the denatured
fibrinogen protein, where each of the at least two PEG molecules
comprises a functional group for crosslinking in order to form
hydrogel scaffolds. Crosslinking is performed by subjecting the
precursor molecules to a free-radical polymerization reaction,
preferably by photoinitiation of acrylated PEG's, which is done ex
vivo or in vivo.
BRIEF DESCRIPTION OF THE DRAWINGS
[0031] FIG. 1 shows images of the process for the preparation of
PEGylated fibrin or PEGylated fibrinogen microgel powders.
[0032] FIG. 2 is a graph showing the shear thinning behavior of a
10:1 molar ratio PEG-fibrin and 20:1 molar ratio PEG-fibrinogen
microgels. The cryomilled microgels were rehydrated at 300 mg/mL in
phosphate buffered saline. Values of slopes are as follows:
PEG-fibrinogen (-0.90) and PEG-fibrin (-0.98). Slopes of
approximately -1 indicate perfectly shear thinning materials.
[0033] FIG. 3 is a graph of modulus vs. frequency for 20:1
PEG-fibrinogen microgel particles rehydrated at 300 mg/mL in
phosphate buffered saline. Viscoelastic properties are recoverable
after repeated exposure to 1% strain. The slightly higher storage
and loss moduli for the repeated testing are an artifact of
microgel dehydration over time.
[0034] FIG. 4 is a graph comparing the storage (G') and loss (G'')
moduli of 10:1 PEG-fibrin and 20:1 PEG-fibrinogen microgels in a
plot of modulus vs. frequency. The microgels were rehydrated at a
concentration of 300 mg/mL in phosphate buffered saline.
[0035] FIG. 5 is a graph of viscosity vs. shear rate showing the
shear thinning behavior of 50:1 PEG-fibrinogen microgel particles
milled by mortar & pestle as well as cryomilling. The microgels
were rehydrated at 300 mg/mL in phosphate buffered saline.
[0036] FIG. 6 is a chart showing the proliferation of
adipose-derived stem cells (ADSC) on microgel particles of
PEG-fibrinogen (PEG-FGN) and PEG-fibrin over 1, 4 and 7 days.
[0037] FIG. 7 shows live cell staining images for human ADSCs on
10:1, 20:1, and 50:1 PEG-fibrinogen and PEG-fibrin microgels on day
7, which demonstrates that the ADSCs are viable on 20:1
PEG-fibrinogen and 50:1 PEG-fibrinogen microgels.
[0038] FIG. 8 shows images of the ex vivo subcutaneous injection
(rat) of dyed 20:1 PEG-FGN microgels. The microgel acts as a solid
after injection, retaining its shape and remaining at the site of
injection.
[0039] FIG. 9 is a graph of viscosity vs. shear rate showing the
shear thinning behavior of 20:1 PEG-FGN gels without and with PHMB
incorporated at rehydrated concentration of 1000 ppm PHMB. The
microgels were rehydrated in phosphate buffered saline at 300
mg/mL.
[0040] FIG. 10 is a graph of modulus vs. frequency showing the
storage and loss moduli of 20:1 PEG-FGN gels without and with PHMB
incorporated at rehydrated concentration (50 mg/mL) of 1000 ppm
PHMB. The microgels were rehydrated in phosphate buffered saline at
300 mg/mL.
[0041] FIG. 11 shows images of smooth muscle actin (SMA) staining
of 20:1 PEG-FGN microgels, which demonstrate angiogenesis at 7 and
14 days in the center of the microgels, alone and with
incorporation of ADSCs or morselized amnion.
[0042] FIG. 12 shows images of CM-dil labeled rat ADSCs, indicating
transplanted cell viability in 20:1 PEG-FGN microgels after 7 and
14 days. DAPI and smooth muscle actin (SMA) staining show cell
nuclei and microvascular formation.
SUMMARY
[0043] Water-insoluble but water-swellable and deformable
crosslinked PEGylated microgel particles of proteins, protein-based
macromolecules, or both are described. The proteins and
protein-based macromolecules can be selected from 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, on channel proteins, cell
adhesion proteins, neuropeptides, nucleoproteins, scleroproteins,
chromoproteins, conjugated proteins, protein-protein complexes,
protein-polysaccharide complexes, protein-lipid complexes,
protein-enzyme complexes, protein-polymer 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, which are pseudoplastic
and flow in aqueous media under shear and which can be injected or
made to flow, wherein the microgel particles can reform as a
cluster of microgel particles when shearing forces are removed.
[0044] The crosslinked PEGylated microgel particles can also be
utilized in their dried state and can be placed into or on a body
defect, wound, burn, or in, on, or surrounding a tissue substitute,
wherein the microgel particles can be hydrated by endogenous or
exogenous sources.
[0045] Of particular interest is the use of such protein-based
microgel particles as coated, injected, or implanted materials in
tissues, organs, wound void spaces, tissue substitutes, and skin
replacement products, where the hydrated microgel particles under
shear in aqueous media are pseudoplastic and can reform to a
cohesive microgel cluster when shear is removed. The microgel
particles can be applied in either their dried state as a powder or
as hydrated particles. The materials described herein can be
utilized in methods that include filling biological voids,
fissures, and soft tissue lumens, and providing a coating and
support for skin substitutes, where the microgel particles function
as a cell matrix.
[0046] Examples of voids include, but are not limited to, lesions,
fissures, fistulas and diverticula. These voids can be
physiological or the result of infection, surgery, cyst, tumor
removal, or traumatic injury or remodeling of soft tissue, such as
in skin and wound healing, plastic surgery, cosmetic surgery,
reconstructive surgery, coating/sealing of skin replacement
products, tendon repair, hernia repair, craniofacial surgery,
ophthalmic surgery, cervicofacial rhytidectomy, abdominoplasty,
breast augmentation, myocardium repair, cartilage repair, nerve
repair, spinal cord repair, liver tissue regeneration, bladder
repair, muscle repair, mastopexy, rheumatology, gynecomastia
reduction, body contouring, skin rejuvenation, skin resurfacing,
microsurgery, dermato-cosmetics for filling in wrinkles, masking
scars or enhancing lips, and the like.
[0047] Skin replacement utilizing tissue engineered skin
substitutes and spray-on cells is used to provide skin repair for
difficult-to-heal wounds, such as chronic wounds due to diabetes,
third-degree burns, and trauma wounds. Non-incorporation of the
skin replacement product and wound infection can be key
determinants for lack of success in wound healing using skin
replacement products. Skin replacement products often fill
approximately 60% of the wound void, leaving 40% of the wound area
without continuous contact for cell mobility as well as open areas
for desiccation/maceration and infection development. The microgel
particle-containing formulations described herein form a coating
that is conformable to surrounding tissues, filling wound void
space when applied as a powder and hydrated in situ or applied as
hydrated microgel particles, said hydrated microgel particles
enhance cell mobility between skin replacement products and the
wound bed, decreasing desiccation and maceration. When an
antimicrobial agent is added to the microgel particles, infection
can be reduced or eliminated, thereby improving healing
effectiveness, particularly for tunneling and undermining wounds,
burns, and skin replacement products that produce a higher take
rate in clinical outcome.
[0048] The microgel particles are derived from naturally-occurring
or genetically obtained proteins and protein-based biological
macromolecules selected from collagen, extracellular matrix,
albumin, plasma, fibrinogen, fibrin, thrombin, coagulation factors,
Von Willebrand Factor, hemoglobin, elastin, gelatin, keratin,
acellular dermis, amniotic membrane, adipose tissue, Matrigel,
cadaveric fascia, heart valves, blood vessels, skin, nerves,
skeletal muscle, mucin, aggrecan, actin, vitronectin, elastin,
perlecan, keratin, spectrin, placenta, liver, pancreas,
fibronectin, elastin, laminin, reticulin, chorion, umbilical cord,
small intestinal submucosa, large intestine, bladder acellular
matrix, stomach submucosa tissue, prostate, decorin, biglycan,
perlecan, lumican, fibromodulin, integrins, cadherins, agrin,
entactin, epiphycan, actin, pericardium, placenta, fetal tissue
from any mammalian organ, virus derivatives, combinations thereof,
and the like.
[0049] An injectable particle-based hydrated microgel is
advantageous in filling soft tissue defects. The microgel
particle-containing compositions can flow through an injection
needle by deformation of the microgel, if required by the inner
dimensions of the injection needle, and are shear thinning to allow
filling of tunneling and undermining wounds because the discrete
microgel particles can separate from a cluster of particles at rest
under a movable force thus lowering the viscosity, and because the
cluster of particles can reform when shear is removed, regenerating
the pseudopiastic matrix by the formation of a moldable,
conformable viscoelastic solid. The flowable characteristics of the
hydrated microgel particles, in conjunction with their resulting
viscoelastic solid-like behavior, enables the injectable,
pseudoplastic matrix to fill irregular wound surfaces or fill
tissue defects.
[0050] Preparation of derivatized proteins by PEGylation includes
conversion of the protein component to a more hydrophilic state
that is swellable but insoluble in aqueous media. The derivatized
protein functions as a hydrogel; however, crosslinking of the
derivatized proteins by multifunctional PEGylation can result in
monolithic gelation in aqueous media. The monolithic gel can then
be lyophilized and ground to a powder, which, upon hydration, forms
microgel particles. PEGylation of proteins and protein-based
macromolecules can occur through react on with available amino or
sulfhydryl groups, which are predominantly on the protein
component, PEGylating agents often comprise reactive functional
groups, including azide, carbonate, ester, aldehyde, acrylate,
carboxyl, carbodiimide, carbonylimidazole, dichlorotriazine, epoxy,
isocyanate, isothiocyanate, maleimide, nitrophenyl carbonate,
orthopyridyl disulfide, pyridinyloxycarbonyl, succinimidyl
carbonate, succinimidyl glutarate, succinimidyl methyl butanoate,
succinimidyl succinate, succinic acid, sulfhydryl, tresylate, vinyl
sulfone, and the like. Depending on the type of PEGylation agent
used for derivatizing the proteins and/or protein-based
macromolecules, other available functional groups, such as
carboxyl, guanidino, hydroxyl, imidazole, or tyrosine (phenolic)
groups can also be PEGylated. The molecular weight of the PEG
component and its molar ratio to the protein or protein-based
macromolecule to be PEGylated determines the degree of hydration to
be obtained, the degree of crosslinking, as well as the resulting
physical, mechanical, and biological properties of the PEGylated
composite. In general, the larger the PEG segment, the higher the
hydration (swelling) of the resulting gel, the stronger the gel,
the greater the stability of the protein component of the gel from
protease degradation, and the lower the biological activity of the
composite. The reduced biological activity of the protein component
of the composite may be related to steric hindrance caused by the
poly(ethylene glycol) units and by overall dilution in the
composition.
[0051] The mechanical properties of the microgels can be studied by
rheometric analysis. In rheometry, the storage modulus (G') and
loss modulus (G'') of weak biological materials, such as ocular
tissues (e.g., vitreous) and fat, are substantially lower than
related values for strong materials, such as muscle, tendon, and
cartilage, In this regard, values of G' range from 1 Pa to 1 MPa
and values of G'' range from 0.1 Pa to 1 MPa. In some embodiments
described herein, the storage modulus ranges from 10 Pa to 250,000
Pa, or from 50 Pa to 175,000 Pa, or from 100 Pa to 100,000 Pa. In
some embodiments, the loss modulus ranges from 5 Pa to 100,000 Pa,
or from 7,5 Pa to 50 Pa, or from 10 Pa to 10,000 Pa.
[0052] In some embodiments, the crosslinked, hydrated, PEGylated
microgel particles display pseudoplasticity under shear. This
pseudopiastic phenomenon is based on the water-insoluble, hydrated
particles themselves and not on soluble macromolecules.
Additionally, the rheological behavior of the hydrated microgel
particles described herein are surprisingly related to their
solid-like behavior, as demonstrated by loss tangent (tan delta)
values (G'IG') less than 1, as opposed to being more
fluid-like.
[0053] In some embodiments, the disclosure provides crosslinked,
water-swellable microgel particles of derivatized protein-based
biological macromolecules.
[0054] In some embodiments, the disclosure provides protein-based
biological macromolecules that are crosslinked by difunctional to
multifunctional PEGylation.
[0055] In some embodiments, the disclosure provides that the molar
ratio of PEGylating agent to protein ranges from 1:1 to 100:1.
[0056] In some embodiments, the disclosure provides for hydrated
microgel particles that are pseudoplastic under shear in aqueous
media.
[0057] In some embodiments, the disclosure provides for
compositions where, when shear is removed from the hydrated
microgel particles, a cluster of microgel particles reforms.
[0058] In some embodiments, the disclosure provides a dry microgel
powder and hydrated microgel particles.
[0059] In some embodiments, the disclosure provides for microgel
particles that can be coated, injected, sprayed, painted, or
implanted in or on tissues, organs, wound void spaces, tissue
substitutes, bandages, and medical devices.
[0060] In some embodiments, the disclosure provides methods to
treat wounds that have tunneling and undermining with the microgel
particles.
[0061] In some embodiments, the disclosure provides microgel
particles that are water-insoluble but water-swellable and
deformable under shear or compression.
[0062] In some embodiments, the protein portion of the microgel
particles is selected from 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,
protein-polymer 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.
[0063] In some embodiments, the protein portion of the microgel
particles is selected from fibrinogen, fibrin, albumin, plasma,
extracellular matrix and collagen.
[0064] In some embodiments, the microgel particles induce
angiogenesis in vivo.
[0065] In some embodiments, the microgel particles promote
transplanted cell survival in vivo.
[0066] In some embodiments, the protein PEGylating agent is
multifunctional.
[0067] In some embodiments, the protein PEGylating agent is
.alpha.-succinimidyloxyglutaryl-.omega.-succinimidyloxyglutaryloxypolyoxy-
ethylene (SG-PEG-SG).
[0068] In some embodiments, biologically active agents are
incorporated into the PEGylated protein microgel.
[0069] In some embodiments, the biologically active agents are
cells.
[0070] In some embodiments, the cells are of human origin and are
either autologous or allogeneic.
[0071] In some embodiments, the cells are stem cells of human
origin and are either autologous or allogeneic.
[0072] In some embodiments, it is a further object of the invention
that the biologically active agent is micronized decellularized
skin tissue.
[0073] In some embodiments, the biologically active agent is
micronized or morselized tissue, such as, but not limited to,
spinal cord, bladder, small intestinal submucosa, skin, dermis,
epidermis, fat, cartilage, placenta, extracellular matrix, tendon,
umbilical cord, cornea, heart, myocardium, liver, pancreas, and
muscle.
[0074] In some embodiments, the biologically active agent is
morselized amniotic tissue.
[0075] In some embodiments, the biologically active agent is minced
tissue.
[0076] In some embodiments, the biologically active agent is
micronized tissue.
[0077] In some embodiments, the biologically active agent is
granulated crosslinked bovine tendon collagen and/or
glycosaminoglycan.
[0078] In some embodiments, the biologically active agent is
amniotic fluid.
[0079] In some embodiments, the biologically active agent is
Wharton's jelly.
[0080] In some embodiments, the biologically active agent is
micronized skin tissue.
[0081] In some embodiments, the biologically active agent is a
growth factor.
[0082] In some embodiments, the biologically active agent has
antimicrobial properties.
[0083] In some embodiments, the antimicrobial agent is
poly(hexamethylene biguanide) and its salts.
[0084] In some embodiments, water-soluble polymers are added to the
PEGylated microgel particles.
[0085] In some embodiments, essential oils are added to the
PEGylated microgel particles.
[0086] In some embodiments, the crosslinked PEGylated microgel
particles can be applied as a powder, liquid, gel, paste, cream,
emulsion, or combinations thereof.
[0087] In some embodiments, the rheological storage modulus (G') is
greater than the loss modulus (G'') for the hydrated microgel
particles, with a loss tangent less than unity,
DETAILED DESCRIPTION
[0088] Compositions that include water-insoluble, microgel
particles selected from crosslinked, PEGylated proteins and
protein-based biological macromolecules that are pseudoplastic in
aqueous solution are provided. The solution of hydrated microgel
particles decreases in viscosity with applied shear and reforms
into a microgel cluster in the absence of shear. The molar ratio of
PEGylating agent to protein and protein-based biological
macromolecules is from 1:1 to 100:1. In some embodiments, the
concentration of microgel particles in the composition is from 10
mg/mL to 1,000 mg/mL, or from 25 mg/mL to 800 mg/mL, or from 50
mg/mL. to 750 mg/mL, or from 100 mg/mL to 500 mg/mL. The individual
and clustered hydrated microgel particles either alone or in a
mixture can have viscoelastic solid properties, storage modulus
greater than loss modulus, and loss tangent values less than 1.
[0089] Utilization of conventional monofunctional PEGylation for
the preparation of water-insoluble, water-swellable PEGylated
proteins is not generally preferred because soluble gels are often
obtained, which do not provide the desired mechanical properties.
Such soluble gels may provide a poor scaffold for added cells. In
some embodiments for the preparation of water-insoluble,
water-swellable PEGylated protein monolithic hydrogels described
herein. PEGylation by multifunctional reactive groups, on the
termini of a linear or branched, multi-armed poly(ethylene glycol)
(PEG) is used to produce a monolithic gel in aqueous media that can
be converted to insoluble microgel particles after drying and
converting to particles, such as by milling, eg., mortar and pestle
or cryomilling. In some embodiments, microgel particles can be
obtained directly during protein PEGylation preparation by rapid
stirring or shearing of the resulting gel. In some embodiments,
isolation of the monolithic gel is preferred because unreacted
components can be separated by extraction prior to microgel
particle formation.
[0090] In some embodiments, the PEGylation agents are selected from
.alpha.-aminopropyl-.omega.-aminopropoxypolyoxyethylene,
.alpha.-aminopropyl-.omega.-carboxypentyloxypolyoxyethylene,
.alpha.,
.omega.-bis{2-[(3-carboxy-1-oxopropyl)amino]ethyl}polyethylene
glycol,
.alpha.-[3-(3-maleimido-1-oxopropyl)amino]propyl-.omega.-[3-(3-maleimido--
1-oxopropyl)amino]propoxypolyoxyethylene, pentaerythritol
tetra(aminopropyl)polyoxyethylene,
.alpha.-[3-(3-maleimicio-1-oxopropyl)amino]propyl-.omega.-(succinimidylox-
ycarboxy)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-carboxypropy)-O'-[2-(3-mercaptopropionylamino)ethyl]polyethylene
glycol, O,O'-bis[2-(N-succinimidylsuccinylamino)ethyl]polyethylene
glycol, O,O'-bis(2-azidoethyl)polyethylene glyco, poy(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 the like, and combinations thereof. In some
embodiments, the PEGylation agent is
.alpha.-succinimidyloxyglutaryl-.omega.-succinimidyloxyglutaryloxypolyoxy-
ethylene (SG-PEG-SG), available from NOF America Corporation under
the mark SUNBRIGHT.RTM. DE-034GS, with a molecular weight of 3,400
Daltons (CAS Number: [154467-38-6]). SG-PEG-SG is believed to
crosslink proteins via elimination of N-hydroxysuccinimide of
protein amino groups, forming carbamate bridges between multiple
protein segments and the PEGylating agents. Thus, it is believed
that the PEGylating agent forms a bridge between fibrinogen
moieties.
[0091] In some embodiments, PEGylated fibrinogen and PEGylated
fibrin hydrated microgel particles provide a convenient delivery
format for utilization as a soft tissue filler, coating,
implantation, or injection in or on tissues, organs, and wound void
spaces as well as a conformable coating for tissue substitutes,
serving as degradable scaffolds to manage the wound environment and
promote angiogenesis during healing. Angiogenesis and
neovascularization are critical determinants of wound healing
outcomes as newly formed blood vessels participate in the healing
process, providing oxygen and nutrients to cells and growing
tissues. It is known that wound healing is facilitated by
fibrinopeptides, the degradation products of fibrinogen and fibrin,
which have a known role in enhancing angiogenesis and tissue
regrowth.
[0092] In contrast to a monolithic hydrogel, the individual
hydrated microgel particles have large pore sizes and large surface
areas for cell attachment. In addition to the unique flow
properties described herein, the flexibility and openness of the
microgel particle system enables cell mobility. In some embodiments
of the dried microgel particles, pore sizes range from 0.9 .mu.m to
40.7 .mu.m, while the length of the particles range from 10.9 .mu.m
to 1,347,7 .mu.m and a width of 2.2 .mu.m to 874,2 .mu.m. In some
embodiments of the dried microgel particles, pore sizes range from
0.9 .mu.m to 23.7 .mu.m, while the length of the particles range
from 5.3 .mu.m to 1,832.8 .mu.m and a width of 1.6 .mu.m to 894.2
.mu.m. In some embodiments, the dried microgel particles are
prepared by cryomilling or by mortar and pestle.
[0093] In some embodiments, when used as a cell scaffold, the dried
microgel is rapidly rehydrated by a solution containing cells. In
some embodiments, dry microgel powder is preloaded in a syringe,
and cells are drawn into the syringe in order to hydrate the
microgel powder before the cell-laden composition is injected into
a wound or defect. In other embodiments, dry microgel powder is
preloaded in a vial, then cells in solution are injected through
the septum into the vial in order to hydrate the microgels, before
the cell-laden system is drawn into a syringe and injected into
tissue, avoid, or a wound in need thereof. In some embodiments, the
hydrated pseudoplastic cell composition can be injected through a
syringe or cannula into tissue, a void, or a wound or applied by
coating for localized delivery. Upon application, the hydrated
microgel particles aggregate to form a microgel cluster by adherint
to each other and to the surrounding tissue. The microgel cluster
forms a cohesive hydrogel network that contains void spaces in and
on the microgel particles as well as between the microgel
particles. The shear thinning and rapid self-aggregating
characteristics of the microgel particles, in conjunction with
their ability to accommodate cells and other biological agents in
void spaces, coupled with their ability to fill the shape of the
cavity with an interface between the microgel and tissue, provides
a superior network for delivery of biological agents. As a result,
the water-insoluble, microgel particle compositions described
herein provide an excellent system for cell delivery.
[0094] PEGylated fibrinogen particles that have been lyophilized
and milled into a powder exhibit shear thinning, flowable behavior
when rehydrated. In some embodiments, the microgel particles adsorb
approximately 15 times their weight in saline solutions (93 wt %
saline), and are more hydrated than analogous PEGylated fibrin
particles, which adsorb approximately 7 times their weight in
saline solution (89 wt % saline) at the same degree of PEGylation
(mole ratio of 50:1, Table 1). Rather than forming a single solid
gel, these PEG-fibrinogen microgel particles interact with adjacent
PEG-fibrinogen microgel particles via hydrogen bonding to form a
"gel-like" solid with shear thinning properties.
[0095] Among the advantages of the microgel particles described
herein relative to a monolithic hydrogel prepared in situ in a host
are that the injected product can be pre-purified to eliminate
unreacted components; can be injected in hydrated microgel form to
a designated location based on the shear thinning properties, which
facilitates filling of soft tissue defects; can be stored at room
temperature in powder form; and can be mixed with cells during a
clinical procedure by drawing the cell solution into the syringe
with the microgel powder or otherwise contacting the cell solution
with the microgel powder. The porous network of the hydrated
microgel particles also enables cell mobility and improves cell
survival by nutrient exchange and host cell infiltration through
its pores and void spaces. This flexibility is in direct contrast
to a monolithic hydrogel, such as has been reported for
poly(ethylene glycol) (PEG) hydrogels as scaffolds, which can be
difficult for cells to infiltrate, due to the PEG hydrogel density
(U.S. Pat. No. 8,557,288).
[0096] In addition, in situ preparation of a monolithic gel often
utilizes photopolymerization of pendant acrylate groups on
PEGylated materials, or other vinyl-type monomers. While such a
procedure is cumbersome in a clinical environment, initiation of a
free radical polymerization in situ also generates heat, which can
be detrimental to surrounding tissue, or to added cells. Such a
process also presents the possibility of residual, unreacted
monomer remaining in the body, which may have cytotoxic
behavior.
[0097] In some embodiments, the microgel particles described herein
can be irregular in shape, or can have a defined shape, such as a
sphere or ellipse. In some embodiments, the microgel particles can
be microporous, mesoporous, or macroporous. Depending on the
embodiment, the microgel particles can therefore have an adjustable
size, an adjustable degree of hydration, a large surface area for
cell attachment or addition of a biological agent, and an interior
porous network for incorporation of other biomolecules. In
addition, the microgel particles are inherently biodegradable.
Furthermore, in contrast to a monolithic (bulk) hydrogel, because
of their smaller size, flexibility, deformation and compression
occur readily, the microgel particles are responsive to
environmental changes.
[0098] In some embodiments, the pseudoplastic microgel particle
compositions can include an aqueous solution with amniotic fluid,
morselized amniotic tissue, minced tissue, micronized tissue,
micronized decellularized tissue, plasma, blood, granulated
cross-linked bovine tendon collagen and glycosaminoglycans, cells
and stem cells in cell culture medium, synthetic or naturally
derived extracellular matrix components, including collagen,
glycosaminoglycans, fibrin, laminin, and fibronectin,
hydroxyapatite, 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, and bacterial polyesters, and combinations
thereof, and which can be injected into soft tissue, a void or a
wound.
[0099] In some embodiments, minced tissue is selected from 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. In some
embodiments, micronized tissues and micronized decellularized
tissues are selected from 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. The pseudoplastic microgel matrix can be used,
for example, for the treatment of diabetic foot ulcers with
tunneling and undermining, or in other wounds or surgical
procedures where a flowable viscoelastic matrix is desirable.
[0100] Fibrinogen is a water-soluble glycoprotein in vertebrates
that helps in the formation of blood clots. The fibrinogen molecule
is a water soluble, 340 kDa plasma glycoprotein that is converted
by thrombin into water-insoluble fibrin during blood clot
formation. Fibrinogen has a rod-like shape with dimensions of
approximately 9.times.47.5.times.6 nm with a negative net charge at
physiological pH (isoelectric point of pH 5.2). Fibrinogen is
available commercially from human plasma, bovine plasma, salmon
plasma, baboon plasma, murine plasma, mouse plasma, rat plasma,
canine plasma, and cat plasma. In some embodiments, human plasma is
preferred. In some embodiments Sigma-Aldrich (F3879) fibrinogen
from human plasma is utilized containing 50-70% protein, wherein
.gtoreq.80% of the protein is clottable. In some other embodiments,
fibrinogen may be utilized from autologous, allogeneic, or
xenogeneic sources. In still other embodiments, recombinant
fibrinogen may be utilized. In some embodiments, autologous human
fibrinogen is preferred.
[0101] Thrombin is a proteolytic enzyme that acts as a serine
protease in converting fibrinogen into insoluble strands of fibrin,
as well as, catalyzing many other coagulation-related reactions.
Thrombin is available from several types of plasma, such as human,
bovine, porcine, equine, rat, rabbit, and recombinant sources. In
some embodiments Sigma Aldrich (T7009) thrombin from human plasma,
.gtoreq.1,000 NIH units/mg protein, is preferred for the conversion
of PEGylated fibrinogen to PEGylated fibrin.
[0102] In some embodiments, the microgel composition is composed of
milled particles of fibrinogen or fibrin that are crosslinked with
di- to multi-functional PEGylating agents, creating water-insoluble
gel particles composed of various combinations of poly(ethylene
glycol) substituents between, predominantly, pendant amino groups
between the fibrinogen or fibrin protein chains. Each microgel
particle is a composite of a multitude of protein chains of
fibrinogen or fibrin, interspersed between shorter, but more
numerous, poly(ethylene glycol) chains. Formation of the microgel
particles occurs prior to application to tissue, organs, wounds,
and tissue substitutes, which avoids in situ instrumentation for
polymerization, provides the ability to remove contaminating
monomers and initiators prior to introduction to the body, and
avoid the generation of polymerization heat at the introduction
site. This combination helps facilitate success of injection.
[0103] PEGylation of protein substituents and/or protein-based
macromolecules can be done at molar ratios of 100:1, 75:1, 50:1,
35:1, 20:1, 15:1, 10:1, 7.5:1, 5:1, 2.5:1 and 1:1 of PEGylating
agent to protein component, or any range starting from or ending
with any of these molar ratios (e.g., 100:1 to 1:1, or 50:1 to
2.5:1, or 50:1 to 10:1, or 35:1 to 5:1). In some embodiments, molar
ratios of PEGylating agent to protein component are 10:1, 20:1,
35:1, and 50:1. In some embodiments, after lyophilizing the
monolithic gels, the freeze-dried polymers are powdered by mortar
and pestle or by cryomilling, and further separated by sieving,
giving median particle sizes of the dried microgel particles
ranging from 10 .mu.m to 1,000 .mu.m, or from 50 .mu.m to 500
.mu.m, or from 75 .mu.m to 250 .mu.m. In some embodiments, after
lyophilizing the monolithic gels, the freeze-dried polymers are
powdered by mortar and pestle or by cryomilling, giving median
particle size lengths of the dried particles ranging from 10 .mu.m
to 1,000 .mu.m, or from 50 .mu.m to 500 .mu.m, or from 75 .mu.m to
250 .mu.m.
[0104] In some embodiments, the microgel particles are hydrated
with a fluid. In some embodiments, the mobile phase of the fluid is
water, isotonic saline, balanced salt solution, buffer solution,
Ringer's solution, cell culture media, stem cell media, serum,
plasma, amniotic fluid, Wharton's jelly, nutrient broth, antiseptic
solutions, or a combination thereof. The degree of hydration of the
microgel particles is dependent upon at least the molar ratio of
PEGylating agent to derivatized protein, the degree of porosity,
and surface area of the resulting microgel particles, and the pH,
osmolality, and temperature of the mobile phase. In some
embodiments, where the fluid is an aqueous media, the aqueous media
can have a pH in the range 4.5-8.0, or 5.5 to 7.5. In some
embodiments the degree of hydration (swelling) of the microgel
particles can be at least 50 times, at least 40 times, at least 30
times, at least 20 times, at least 10 times, at least 5 times, or
at least 2 times the dry weight of the microgel particle, when
measured using saline solution.
[0105] In some embodiments, an antimicrobial agent is added to the
microgel particles to hinder development and proliferation of
microorganisms. In some embodiments, addition of an antimicrobial
agent helps reduce or eliminate microbial colonies and biofilm
formation. Because of the possibly of infection in voids, wounds,
and burns, the PEGylated protein microgel composition can include a
biological agent in an amount sufficient to hinder or eradicate
microorganisms.
[0106] Examples of biological agents include, but are not limited
to, antibiotics, antiseptics, anti-infective agents, antimicrobial
agents, antibacterial agents, antifungal agents, antiviral agents,
antiprotozoal agents, sporicidal agents, and antiparasitic agents.
In some embodiments, the biological agent is biodegradable,
non-cytotoxic to human and animal cells, or both biodegradable and
non-cytotoxic.
[0107] Examples of biocidal agents include, but are not limited to,
biguanides, such as poly(hexamethylene biguanide) (PHMB) and its
salts, a low molecular weight synthetic cationic biguanide polymer,
chlorhexidine and its salts, such as chlorhexidine digluconate, and
alexidine and its salts, such as alexidine dihydrochloride, where
the latter two are bis(biguanides), benzalkonium chloride,
benzethonium chloride, cetyltrimethylammonium bromide, glycerol
monolaurate, capryl glycol, gentamicin sulfate, iodine,
povidone-iodine, starch-iodine, neomycin sulfate, polymyxin B,
bacitracin, tetracyclines, clindamycin, gentamicin, nitrofurazone,
mafenide acetate, silver nanoparticles, silver sulfadiazine, silver
nitrate, terbinafine hydrochloride, miconazole nitrate,
ketoconazole, clotrimazole, itraconazole, metronidazole,
antimicrobial peptides, polyguaternium-1, polyquaternium-6,
polyquaternium-10, salts thereof, and combinations thereof.
[0108] In some embodiments, the antimicrobial biguanide is
poly(hexamethylene biguanide) hydrochloride (PHMB). PHMB can be
used because of its high biocidal activity against microorganisms,
combined with its biodegradation and low cytotoxicity. PHMB is
primarily active against Gram negative and Gram positive bacteria,
fungi, and viruses. In contrast to antibiotics, which are
considered regulated pharmaceutical drugs and to which bacterial
resistance can occur, such resistance does not occur with PHMB. As
used herein, an "antimicrobial agent" is a substance that kills
microorganisms or inhibits their growth or replication, while an
"anti-infective agent" is a substance that counteracts infection by
killing infectious agents, such as microorganisms, or preventing
them from spreading. Often, the two terms are used interchangeably.
As used herein, "PHMB" is considered an antimicrobial agent.
[0109] In some embodiments, the compositions containing hydrated or
rehydrated PEGylated protein microgel particles described herein
can include biocidal PHMB at a concentration ranging from 0.0001 wt
% (1 ppm) to 1 wt % (10,000 ppm), or ranging from 0.01 wt % (100
ppm) to 0.75 wt % (7,500 ppm), or ranging from 0.05 wt % (500 ppm)
to 0.5 wt % (5,000 ppm), or ranging from 0.1 wt % (1,000 ppm) to
0.25 wt % (2,500 ppm), based on the total weight of the
composition. In some embodiments, dry PEGyiated protein microgel
particle compositions described herein can include biocidal PHMB at
a concentration ranging from 0.002 wt % (20 ppm) to 25.0 wt %
(250,000 ppm), or ranging from 0.20 wt % to 15.0 wt % (150,000
ppm), or ranging from 1.0 wt % (10,000 ppm) to 10.0 wt % (100,000
ppm), or ranging from 2.0 wt % (20,000 ppm) to 4.0 wt % (40,000
ppm), based on the total weight of the composition.
[0110] In some embodiments, bis(biguanide)s, such as alexidine and
its salts and chlorhexidine and its salts, can be added to the
antimicrobial PEGylated protein microgel particle compositions in
concentrations from 0.001 wt % (10 ppm) to 4.0 wt % (40,000
ppm).
[0111] In some embodiments, surfactant-type antimicrobial agents,
such as benzethoni m chloride or benzalkonium chloride, can be
added to the antimicrobial PEGylated protein microgel particle
compositions in concentrations from 0.001 wt % (10 ppm) to 2.0 wt %
(20,000 ppm).
[0112] In some embodiments, lipophilic-type antimicrobial agents,
such as glycerol monolaurate or capryl glycol, can be added to the
antimicrobial PEGylated microgel particle protein compositions in
concentrations from 0.1 wt % (1,000 ppm) to 2.0 wt % (20,000
ppm).
[0113] In some embodiments, antimicrobial agents with reactive
functional groups, such as amino, imino, imidazoyl, sulfhydryl,
hydroxyl, phenolic, indolyl, guanidium, guanidinium, and carboxyl
groups, may be covalently incorporated into the PEGylated protein
microgel particle, forming a ternary composite of PEGylated
protein/antimicrobial agent. In some embodiments, covalently bound
ternary composites of PEGylated fibrinogen/PHMB microgel particles
are formed.
[0114] In some embodiments, aqueous PEGylated protein microgel
particle compositions can have an osmolality of 10-340 mOsm/kg. In
some embodiments where the PEGylated protein microgel particle
composition is an aqueous-based solution, gel, paste, emulsion, or
foam, a water-soluble polymer can be added to increase solution
viscosity and to prolong residence time on the surface of a tissue,
void, or wound, or subcutaneously in a void or wound. In some
embodiments, useful water-soluble polymers include, but are not
limited to, 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, carboxymethylquar, carboxymethylchitosan,
locust bean gum, carrageenan, xanthan gum, gellan gum, pullulan,
alginate, chondroitin sulfate, dextran, dextran sulfate. Aloe vera
gel, scleroglucan, schizophylian, 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(2-ethyl-2-oxazoline), 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, acrylamido- and
methacrylamido-trimethylammonium polymers, antimicrobial peptides,
and the like, and derivatives and combinations thereof.
[0115] In some embodiments, the preparation of PEGylated protein
microgel particle compositions in the form of viscous solutions,
gels, creams, pastes, emulsions, balms, and sprays, can be
facilitated by the inclusion of water-soluble polymer viscosity
builders in amounts ranging from about 0.01 to about 50.0 wt % from
0.1 to 45% wt, from 0.5 to 25 wt %, or from 1 .0 to 10.0 wt %.
[0116] In some embodiments, essential oils can be added to the
microgel particle compositions as fragrance or aromatic agents,
and/or as antimicrobial agents. Examples of essential oils useful
in the microgel particle compositions described herein include, but
are not limited to, thymol, menthol, sandalwood, camphor, cardamom,
cinnamon, jasmine, lavender, geranium, juniper, menthol, pine,
lemon, rose, eucalyptus, clove, orange, oregano, mint, linalool,
spearmint, peppermint, lemongrass, bergamot, citronella, cypress,
nutmeg, spruce, tea tree, wintergreen (methyl salicylate), vanilla,
and the like. In some embodiments, the essential oils can be
selected from thymol, sandalwood oil, wintergreen oil, eucalyptol,
pine oil, and combinations thereof. In some embodiments, essential
oils can be present in an amount ranging from 0% to 5 wt % based on
the weight of the PEGylated protein microgel particle composition.
In some embodiments, essential oils can be present in an amount of
at least 0.1 wt %, or at least 0.25 wt %, or at least 0.5 wt %,
based on the weight of the PEGylated protein microgel particle
composition.
[0117] In some embodiments, chlorophyllin, a water-soluble
semi-synthetic derivative of chlorophyll, may also be used to
control wound odor and to provide anti-inflammatory properties. In
some embodiments, chlorophyllin can be present in an amount ranging
from 0% to 5 wt % based on the weight of the PEGylated protein
microgel particle composition. In some embodiments, chlorophyllin
can be present in an amount of at least 0.1 wt %, or at least 0.25
wt %, or at least 0.5 wt % based on the weight of the PEGylated
protein microgel particle composition.
[0118] In some embodiments, the PEGylated protein microgel particle
composition can also include wetting agents, buffers, gelling
agents or emulsifiers. Other excipients could include various
water-based buffers ranging in pH from 5.0-7.5, surfactants,
silicones, polyether copolymers, vegetable and plant fats and oils,
hydrophilic and hydrophobic alcohols, vitamins, monoglycerides,
laurate esters, myristate esters, palmitate esters, and stearate
esters. In some embodiments, the PEGylated protein microgel
particle composition can be in a form including, but not limited
to, liquid, gel, paste, cream, emulsion, combinations thereof, and
the like. In some embodiments, the PEGylated protein microgel
particle composition is lyophilized to a dry powder. The
lyophilized PEGylated protein microgel particle composition may be
used in powder form, or the powder may be further processed (e.g.,
rehydrated) into solutions, suspensions, creams, lotions, gels,
pastes, emulsions, balms, sprays, foams, aerosols, films, or other
formulations.
[0119] In some embodiments, the PEGylated protein microgel
particles are in the form of nanoparticles, nanoshells, nanorods,
and combinations thereof. In some embodiments, the dehydrated or
lyophilized PEGylated protein microgel particles are in the form of
nanoparticles, nanoshells, nanorods, and combinations thereof.
[0120] As used herein, "aqueous media" refers to a spectrum of
water-based solutions including, but not limited to, homogeneous
solutions in water with solubilized components, cell media
solutions, buffer solutions, isotonic solutions, salt solutions,
emulsified solutions, surfactant solutions, amniotic fluids,
Wharton's jelly, serum, hydrophilic polymers, and viscous or gelled
homogeneous or emulsified solutions in water.
[0121] As used herein, "surfactant" has its standard meaning and
includes compounds that lower the surface tension (or interfacial
tension) between two liquids or between a liquid and a solid and
includes emulsifying agents, emulsifiers, detergents, wetting
agents, and surface-active agents.
[0122] As used herein, "hydrophilic" has its standard meaning and
includes compounds that have an affinity to water and can be ionic
or neutral or have polar groups in their structure that attract
water. For example, hydrophilic compounds can be miscible,
swellable, adsorbable, or soluble in water, with a stationary
contact angle with water of .ltoreq.590.degree. in water at room
temperature.
[0123] As used herein, "hydrophobic" refers to repelling water,
being insoluble or relatively insoluble in water, and lacking an
affinity for water with a stationary contact angle with water of
.gtoreq.90.degree. in water at room temperature. Hydrophobic
compounds with hydrophilic substituents, such as vicinal diols, may
form emulsions in water, with or without added surfactant, with the
hydrophilic substituent at the water interface and the hydrophobic
portion of the compound in the interior of the emulsion.
[0124] As used herein, "swellable" refers to materials that uptake,
absorb and/or adsorb fluids to their functional groups, surfaces,
pores, micropores, nanopores, holes, and interstitial networks.
[0125] As used herein, the term "PEGylation" pertains to modifying
a protein or protein-based macromolecule by covalently attaching
poly(ethylene glycol) (PEG) with reactive substituents to the
protein's available reactive functional groups, such as amino
groups or sulfhydryl groups, whereas "PEGylated" refers to a
protein having a PEG substituent attached thereto.
[0126] As used herein, "proteins" is intended to include
protein-based macromolecules and includes 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,
protein-enzyme complexes, protein-polymer 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.
[0127] As used herein, "derivatized proteins" are protein
components attached, bound, coordinated, or complexed with another
material, such as other proteins, polysaccharides,
oligosaccharides, glycosaminoglycans, lipids, phospholipids,
liposomes, synthetic polypeptides, DNA, RNA, synthetic polymers,
surfactants, metal atoms, nanoparticles, antimicrobial agents,
antibiotics, drugs, salts thereof, and the like.
[0128] As used herein, a "hydrogel" is an insoluble polymeric
network composed of normally water-soluble macromolecules that
exist in a crosslinked or pseudo-crosslinked state by covalent,
ionic, or physical interaction among macromolecular chains, where
the insoluble network adsorbs at least 10 % of its weight in water.
A hydrogel may contain one or more hydrophilic, polymeric
species.
[0129] As used herein, a "monolithic" hydrogel consists of a
single, large network of crosslinked, hydrophilic polymer or
polymers, which can be subdivided into smaller, crosslinked
microgel particles.
[0130] As used herein, a "microgel" is a gelatinous,
water-insoluble, hydrophilic particle ranging in length from 1
micrometer to 1,000 micrometers, with diameters of 1 micrometer to
1,000 micrometers or a dehydrated particle capable of exhibiting
those properties.
[0131] As used herein, "microgel particles" are mixed particles of
water-insoluble, water-swellable gel fragments that have varied
shapes, including spherical, elliptical, angular, regular
(organized) or irregular shapes, either hollow, microporous,
mesoporous, mac oporous, or with void spaces, or a combination
thereof, depending on the method of formation.
[0132] As used herein, the phrase "microporous" refers to materials
that have pore diameters less than 2 nm, "mesoporous" particles
have pore diameters between 2 and 50 nm, and "macroporous"
particles have pore diameters greater than 50 nm.
[0133] As used herein, "flowable" pertains to a volume of fluid or
gel that is capable of flowing through a passageway of any given
dimension, such as through a squeeze tube, pump, cannula, or
syringe.
[0134] As used herein, "injectable" describes the ability of a
solution, suspension, gel, emulsion, or microgel to pass through a
hypodermic needle or cannula.
[0135] As used herein, a "Newtonian fluid" exhibits a viscosity
that is independent of the shear conditions studied.
[0136] As used herein, a "non-Newtonian fluid" exhibits a viscosity
that is dependent upon the shear conditions studied.
[0137] As used herein, "pseudoplastic" pertains to a fluid
composition having a viscosity that decreases with increasing shear
rate, that is, shear thinning.
[0138] As used herein "shear rate" (also called shear strain rate)
is the rate of change of strain as a function of time.
[0139] As used herein, "loss modulus (G'')" is a measure of the
energy dissipated in a material under deformation (e.g., shear).
G'' is attributable to viscous flow, rather than elastic
deformation. Loss modulus is also known as viscous modulus.
[0140] As used herein, "storage modulus (G')" is a measure of the
energy stored in a material under deformation (e.g., shear). G' is
attributable to elastic deformation. Storage modulus is also known
as elastic modulus.
[0141] As used herein, "loss tangent" (also called tan delta, tan
.delta.) is the tangent of the phase angle (.delta.), which is the
ratio of the loss (viscous) modulus (G'') to the storage (elastic)
modulus (G'), that is, tan .delta.=G''/G'. The loss tangent is a
useful quantifier of the presence and extent of elasticity in a
fluid. Loss tangent values of less than unity indicate an
elastic-dominant (i.e., solid-like) behavior and values greater
than unity indicate a viscous-dominant (i.e., liquid-like)
behavior.
[0142] As used herein, "biologically active agents" has its
standard meaning and includes chemical or biological substances or
formulations that beneficially affect human or animal health and
well-being or is intended for use in the cure, mitigation,
treatment, prevention, or diagnosis of infection or disease, or is
destructive to or inhibits the growth of microorganisms.
[0143] As used herein, "antimicrobial agent" has its standard
meaning and include a substance that kills microorganisms or
inhibits their growth or replication, while an "anti-infective
agent" is defined as a substance that counteracts infection by
killing infectious agents, such as microorganisms, or preventing
them from spreading. Often, the two terms are used
interchangeably.
[0144] As used herein, "antibiotic" has its standard meaning and
includes those substances that were originally produced by a
microorganism or synthesized with active properties that can kill
or prevent the growth of another microorganism. The term antibiotic
is commonly used to refer to almost any prescribed drug that
attempts to eliminate infection.
[0145] As used herein, "excipient" has its standard meaning and
includes inert substances that form a vehicle, such as a liquid,
fluid, or gel, that solubilizes or disperses a PEGylated protein
microgel particle composition, which may include other added
ingredients.
[0146] As used herein, "viscoelasticity" is the property of
materials that exhibit both viscous and elastic characteristics
when undergoing deformation.
[0147] As used herein, "viscoelastic solids" are able to return to
their original shape when an applied shear load is removed, while
viscoelastic fluids do not.
[0148] As used herein, "soft tissue" has its standard meaning and
includes biological tissue that connects, supports, or surrounds
other structures and organs of the body, but does not include bone.
Examples of soft tissue include tendons, ligaments, fascia, skin,
fibrous tissues, fat, synovial membranes, muscles, nerves and blood
vessels.
[0149] As used herein, "nanoparticles" are particles between 1 and
500 nanometers in size and also include particles between 1 and 100
nanometers in size.
[0150] As used herein, "nanoshells" are spherical cores of a
particular compound surrounded by a shell or outer coating of
another, which are a few nanometers thick.
[0151] As used herein, "nanorods" are rod-shaped particles that
have a length at least twice a radius or width and are typically 1
to 100 nm in length.
[0152] As used herein, "microparticles" are particles of various
dimensions between 0.1 and 100 .mu.m in size.
[0153] As used herein, "microspheres" are spherical particles, with
diameters typically 1 .mu.m to 1,000 .mu.m. Microspheres are
sometimes referred to as microparticles.
[0154] In some embodiments, one or more observational or detectable
agents may be incorporated into the PEGylated protein microgel
particle composition to provide enhanced visualization or
facilitate proper placement. The agents may comprise, in other
embodiments, dyes, fluorescent substances, ultraviolet absorbers,
radioactive substances, pigments, or any combinations thereof.
[0155] In some embodiments, one or more biologically active agents
may be incorporated into the PEGylated protein microgel particle
composition to provide a medical benefit to a mammalian host,
Examples of biologically active agents that can be incorporated
into the PEGylated protein microgel particle composition include,
but are not limited to, cells, stem cells, amniotic tissue,
amniotic cells, growth factors, micronized decellularized skin
tissue, granulated crosslinked bovine tendon collagen and
glycosaminoglycans, antibiotics, antiseptics, anti-infective
agents, antimicrobial agents, antibacterial agents, antifungal
agents, antiviral agents, antiprotozoal 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-psoriasls
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, vasodila ors, 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,
sillca-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, bupivacalne, ketoprofen, opiolds,
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, and non-steroidal anti-inflammatory drugs or
similar materials, in either their salt form or their neutral form,
either being inherently hydrophilic or encapsulated within a
hydrophilic microparticle or nanoparticle. Such biologically active
agents could be in either of the (R)-, (R, S)-, or
(S)-configuration, or a combination thereof.
[0156] As used herein, "injection" has its standard meaning and
includes intradermal, subcutaneous, oral, intramuscular,
submucosal, subcutaneous, intranasal, vaginal, buccal, intrathecal,
epidural, intraparenchymal, ocular, subretinal, dental,
intratumoral, intracardiac, intra-articular, intravenous,
intracavernous, intraosseous, intraperitoneal, intra-abdominal,
intrafascial, intraogan, and intravitreal procedures.
[0157] As used herein, injections may be made into cavities/voids
created surgically, cavities/voids resulting from disease, and
cavities/voids resulting from injury.
[0158] As used herein, "cell culture" has its standard meaning and
includes the transfer of cells, tissues or organs from an animal or
plant and their subsequent placement into an environment conducive
to their survival and/or proliferation.
[0159] In some embodiments, the injectable PEGylated protein
microgel particle composition may include cells. Examples of cells
useful in the PEGylated protein microgel particle compositions
described herein include, but are not limited to, 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, platelets, and combinations thereof. In some
embodiments, the cells are eukaryotic or mammalian. In some
embodiments, the cells are of human origin. In some embodiments,
the cells may be autologous or allogeneic.
[0160] In some embodiments the injectable PEGylated protein
microgel particle composition may include 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,
hematopoletic 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 combinations thereof. In some
embodiments, the stem cells are mammalian. In some embodiments, the
stem cells are of human origin. In some embodiments, the stem cells
may be autologous or allogeneic.
[0161] In some embodiments, the injectable PEGylated protein
microgel particle compositions described herein may be used as a
scaffold matrix to deliver a therapeutically effective amount of
between 10,000 cells to about 1 billion cells.
[0162] In some embodiments, products derived from placental tissue
may be incorporated with the PEGylated protein microgel particle
composition for injection into a mammalian host. Placental tissues
are a source of collagen, elastin, fibronectin, and growth factors,
including platelet-derived growth factor (PDGF), vascular
endothelial growth factor (VEGF), epidermal growth factor (EGF),
fibroblast growth factor (FGF), and transforming growth factor beta
(TGF-.beta.), which can support tissue repair and regeneration. In
particular, amniotic tissue has anti-adhesive and antimicrobial
properties, and such tissue has been shown to support soft tissue
repair, reduce inflammation and minimize scar tissue formation,
which are significant benefits in the treatment of soft tissue
injuries.
[0163] Amniotic tissues have been described as being
immune-privileged in that an immune response in the human body
rarely occurs in response to the introduction of amniotic tissue.
In some embodiments a morselized, flowable tissue allograft derived
from amniotic tissues, available from BioD, LLC, commercialized as
BioDRestore.TM. Elemental Tissue Matrix, can be added to the
PEGylated protein microgel particle composition for a coating or
injection into soft tissue, or placement surrounding a tissue
substitute. For example, PEGylated fibrinogen microgel particle
compositions can include morselized, flowable tissue allograft
derived from amniotic tissues.
[0164] In some embodiments, the PEGylated protein microgel particle
composition may include growth factors. Examples of useful growth
factors include, but are not limited to, epidermal growth factor
(EGF), transforming growth factor beta (TGF-beta), fibroblast
growth factor (FGF), vascular endothelial growth factor (VEGF),
granulocyte macrophage colony stimulating factor (GM-CSF),
platelet-derived growth factor (PDGF), connective tissue growth
factor (CTGF), insulin-like growth factor (IGF), keratinocyte
growth factor (KGF), interleukin (IL) family, stromal cell derived
factor (SDF), heparin binding growth factor (HBGF), nerve growth
factor (NGF), brain-derived neurotrophic factor (BDNF), growth
differentiation factor (GDF), muscle morphogenic factor (MMF), and
tumor necrosis factor-alpha (TNF.alpha.),
[0165] In some embodiments, the multi-component PEGylated protein
microgel particle composition can be used as an injectable soft
tissue void filler and may also include any other component
suitable for augmenting, strengthening, supporting, repairing,
rebuilding, healing, occluding or filling a soft tissue.
[0166] The flow properties of the hydrated microgel particle
compositions can be determined by rheometry, wherein the storage
modulus (G') and loss modulus (G'') of biologically weak (soft)
materials, such as ocular tissues (vitreous) and fat, are
substantially lower than related values for strong materials, such
as muscle, tendon, and cartilage. In this regard, values of G'
range from 1 Pa to 1 MPa and values of G'' range from 0.1 Pa to 1
MPa, which include properties of the particular hydrated microgel
particle composition (e.g., individual microgel particles and
clusters of microgel particles, whether alone or with free liquid
or solid components). In some embodiments, the storage modulus for
the PEGylated protein microgel particle compositions described
herein ranges from 10 Pa to 250,000 Pa, from 50 Pa to 175,000 Pa,
and from 100 Pa to 100,000 Pa. In some embodiments, the loss
modulus ranges from 5 Pa to 100,000 Pa, from 7.5 Pa to 50 Pa, and
from 10 Pa to 10,000 Pa. The crosslinked, hydrated, PEGylated
microgel particles display pseudoplasticity under shear, with
viscosity decreasing with increasing shear rate. This pseudoplastic
phenomenon is based on the water-insoluble, hydrated particles
themselves and not on the fluid medium containing soluble
macromolecules. Additionally, the rheological behavior of the
microgel particle compositions, including the individual and
clustered microgel particles, described herein are surprisingly
related to their solid-like behavior, as opposed to being more
fluid-like, as demonstrated by loss tangent values (G''/G') less
than 1.
[0167] Also disclosed are uses of the compositions described herein
for treating a soft tissue lesion or injury. Any of the
compositions described herein can be used. In particular, the
compositions can be used for injection into soft tissue. The
injection can be 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. In some embodiments, the
water-insoluble, microgel particles of the compositions described
herein are in the form of a dry powder, and the use and or method
further comprises hydrating said dry powder prior to said injection
step. In such embodiments, after injection, the composition
promotes angiogenesis.
EXPERIMENTAL
[0168] The following materials and abbreviations are used in the
Experimental section: [0169] ADSC: Adipose derived stem cells,
primary human abdominoplasty cells. [0170] AHF: Cryoprecipitated
antihemophilic factor, South Texas Blood & Tissue Center, Lot
W1409114 212930. [0171] BSA: Bovine serum albumin, bioWORLD,
22070004-1, Lot V11121401. [0172] Calcium Chloride: Sigma C4901,
Lot 110M0105V. [0173] Carbopol: Lubrizol, Carbopol.RTM. Aqua SF-2,
Lot 0101014502. [0174] Collagen Type I: Corning Incorporated,
Product 354236, rat tail tendon, Lot 3298599. [0175] ECM:
Extracellular matrix, Engelbreth-Holm-Swarm murine sarcoma, Sigma
E1270, Lot 093M4006V. [0176] Gellan Gum: CP Kelco, Kelcogel CO-LA,
Lot 1E0566A. [0177] Guar Gum: Making Cosmetics, Lot 1092118.
[0178] Human Fibrinogen (FGN): Sigma F3879, Lots 061M7010,
071M7032V, SLBH0223V, SLBK3747V. [0179] Human Thrombin: Sigma
T7009, Lots 041M7007V, 011M7009V, SLBB4394V. [0180] Hydroxyethyl
Cellulose, Cationic: Dow Chemical Company, UCare.TM. Polymer
JR-30M, Lot XL2850GRXA. [0181] Hydroxypropylmethylcellulose:
Amerchol Methocel K15M, Lot WF15012N01. [0182] Morselized Amnion:
BioD, LLC, BioDRestore.TM., Tissue ID R0925131. [0183] NaOH: Sodium
hydroxide, Puritan Products 7705, Lot 011043. [0184] Needle: 18 G.
BD 305195, Lot 2089215. [0185] PBS: Phosphate Buffered Saline: pH
7.8-8.0 (without calcium and magnesium), INCELL ZSOL:F, Lots
A2014SEP10-01, Z2015JAN05-01; or Sigma-Aldrich D8537, Lots
RNBB9451, RNBC1143, RNBC8400. [0186] PHMB: Poly(hexamethylene
biguanide) Hydrochloride: Arch Chemicals, Cosmocil CQ.TM., Lots
9PL211280, 137261 or Lotioncrafter Biguanide 20, Lot
14RC169159-6380. [0187] Poly(ethylene glycol) Diacrylate: MW 575,
Sigma-Aldrich 437441, Lot MKBN7800V. [0188] Poly(vinyl alcohol):
DuPont, Elvanol.RTM., Lot 910113. [0189] PSG-(PEO).sub.4:
Pentaerythritol tetra(succinimidyloxyglutaryl) polyoxyethylene,
4arm (tetrafunctional), NOF America Corporation, Sunbright.RTM.
PTE-050GS (MW 5,000), Lot M8N526; [0190] PTE-100GS (MW 10,000), Lot
M9D105; and PTE200-GS (MW 20,000), Lot M119691. [0191] Pullulan:
Hayashibara, Lot 1G3021. [0192] SG-PEG-SG (PEG): NOF America
Corporation,
.alpha.-Succinimidyloxyglutaryl-.omega.-succinimidyloxyglutaryloxypolyoxy-
ethylene, (difunctional), NOF America Corporation; [0193]
Sunbright.RTM. DE-034GS (MW 3400), Lot M83541; DE-100GS (MW
10,000), Lot M107543; DE-200GS (MW 20,000), Lot M115700. [0194]
SG-PEG:
.alpha.-Succinimidyloxyglutaryl-.omega.-methoxypolyoxyethylene,
(monofunctional), NOF America Corporation, Sunbright.RTM. ME-050GS
(MW 5,000), Lot M10N587. [0195] Xanthan Gum: Bob's Red Mill, Lot
143454.
Protocols for Initial Hydrogel Formation:
Example 1
Poly(ethylene glycol)-Human Fibrinogen (PEG-FGN)
[0196] The preparation of various molar ratios of PEGylated
fibrinogen (PEG-fibrinogen, PEG-FGN) is illustrated by the
preparation of 20:1 molar PEG-FGN. Fibrinogen (FGN, MW 340 kDa) was
dissolved at 80 mg/mL in sterile pH 7.8-8.0 PBS (without calcium
and magnesium). The fibrinogen was dissolved at room temperature or
37.degree. C. SG-PEG-SG (MW 3.4 kDa) was dissolved at 16 mg/mL in
sterile pH 7.8-8.0 PBS (without calcium and magnesium) at room
temperature (.about.22.degree. C.). All lots of fibrinogen tested
performed similarly. The reactive difunctional PEG solution of
SG-PEG-SG was sterile filtered with a 0.20-0.22 .mu.m filter. The
SG-PEG-SG was then blended with FGN at 1:1 v/v (equivolume).
Gelation occurred within 5-30 minutes at room temperature or at
37.degree. C.
[0197] PEG-FGN was also prepared at molar ratios of 10:1, 35:1, or
50:1. It has been determined that lower levels of PEGylation
results in a higher concentration of fibrinogen incorporation (more
bioactive in the scaffold matrix), increases ease of manufacturing
(more PEG retains more water, making lyophilization more difficult,
and making milling in small particles very difficult), and
preferential adhesion and proliferation in vitro of adipose derived
stem cells (ADSC) on the 20:1 PEG-FGN scaffold compared to 35:1 and
50:1 PEG-FGN scaffolds.
[0198] PEG-FGN was also prepared at a molar ratio of 20:1 using the
following PEGylating agents:
[0199] PEG Diacrylate (difunctional);
[0200] SG-PEG-SG, Sunbright DE-034GS (MW 3400), DE-100GS (MW
10,000), DE-200GS (MW 20,000) (difunctional);
[0201] SG-PEG: Sunbright.RTM. ME-050GS (MW 5,000)
(monofunctional);
[0202] PSG-(PEO).sub.4, PTE-050GS (MW 5,000), Sunbright PTE-100GS
(MW 10,000), PTE200-GS (MW 20,000) (tetrafunctional);
where
[0203] DE:
NHS--OCO(CH.sub.2).sub.3COO--PEG-CO(CH.sub.2).sub.3COO--NHS;
[0204] ME: PEG-CO(CH.sub.2).sub.3COO--NHS;
[0205] PTE: PEG-(CO(CH.sub.2).sub.3COO--NHS).sub.4; and
[0206] NHS: N-Hydroxysuccinimide.
[0207] Increasing the molecular weight of the PEGylating agent
yielded a firmer gel. Difunctional PEG produced stiff gels, in
comparison to monofunctional PEG, which produced a fluid.
Increasing the functionality of the PEG produced stiffer gels at
the same molar ratios. PEG diacrylate did not form a gel under the
reaction conditions studied.
Example 2
PEG-FGN-PHMB
[0208] PEG-FGN hydrogels (20:1 molar) were prepared with the
addition of PHMB present in amounts ranging from 0 to 1000 ppm. The
preparation of PEG-FGN-PHMB composites is illustrated by the
incorporation of 100 ppm PHMB (concentration where the microgel is
rehydrated at 50 mg/mL), with other concentrations of PHMB being
prepared analogously.
[0209] PHMB was diluted in sterile pH 7.8-8.0 PBS without calcium
and magnesium. The calculated amount of the PHMB solution to be
added was based upon 0.01% w/w PHMB in the final rehydrated powder
(powdered product is rehydrated at 50 mg/mL). This requires
incorporation in the initial hydrogel of 48 .mu.L PHMB solution per
100 mL total gel volume. For a 100 mL batch, 0.048 mL (48 .mu.L)
PHMB (Cosmocil CQ) was added to 50 mL phosphate buffered saline.
The PEGylating agent was dissolved at 16 mg/mL in this dilute PHMB
solution. Gel formation was initiated by addition of 50 mL of 80
mg/mL fibrinogen solution.
Example 3
PEG-FGN-Polymers
[0210] PEG-FGN-water soluble polymer hydrogels (20:1 molar PEG to
FGN) were prepared with the incorporation of the following
water-soluble polymers: Carbopol at 27.6 mg/mL, pullulan at 27.6
mg/mL, guar gum at 20.7 mg/mL, hydroxyethyl cellulose at 3.4 mg/mL,
and collagen type I at 1.4 mg/mL. Carbopol is a crosslinked
synthetic polymer based upon acrylic acid; pullulan and guar gum
are natural polysaccharides; hydroxyethyl cellulose is a modified
polysaccharide; and collagen type I is a protein, and the most
abundant collagen of the human body. The water-soluble polymers
were added prior to PEGylation.
Example 4
PEG-Fibrin
[0211] PEG-fibrin gels were prepared at molar ratios of 10:1 to
50:1. Increasing PEGylation beyond 20:1 molar hindered the activity
of thrombin and the formation of fibrin gels, and longer reaction
times were needed. Thrombin was added to PEG-fibrinogen for
crosslinking of fibrinogen with thrombin concentrations of 2.5-12.5
U/mL gel.
[0212] The preparation of PEG-fibrin gels is illustrated for 10:1
molar PEG-fibrin with 2.5 U/mL thrombin. Fibrinogen (MW 340 kDa)
was dissolved at 80 mg/mL in sterile pH 7.8-8.0 phosphate buffered
saline (without calcium and magnesium). SG-PEG-SG (MW 3.4 kDa) was
dissolved at 8 mg/mL in sterile pH 7.8-8.0 PBS (without calcium and
magnesium) at room temperature. The reactive PEG solution was
sterile filtered with a 0.20-0.22 .mu.m filter. PEG was blended
with FGN at 1:1 v/v (equivolume) and reacted 5 min (PEGylated
fibrinogen solution). Thrombin (100 U/mL in deionized water) was
diluted to 5 U/mL in 40 mM calcium chloride. PEG-fibrin gels were
formed by mixing 1 part PEGylated fibrinogen solution with 1 part
PBS and 2 parts 5 U/mL thrombin in calcium chloride. Gelation
occurred within 5 minutes at room temperature or at 37.degree.
C.
[0213] PEG-fibrin was also prepared at molar ratios of 10:1, 35:1,
or 50:1 with thrombin concentrations of 2.5-12.5 U/mL gel according
to the above procedure. Previously, PEG-fibrin monolithic hydrogels
at molar ratios of PEG to fibrinogen, converted to fibrin via
thrombin, of 1:1, 2.5:1, 5:1, 7.5:1, 10:1, 15:1, 20:1, 100:1 have
been reported (Zhang et al., Tissue Engineering, 12(1), 9-19,
2006).
Example 5
PEG-Fibrin-Polymers
[0214] PEGylated fibrin (10:1) hydrogels were prepared with the
following water-soluble polymers incorporated at 2 mg/mL: xanthan
gum, gellan gum, guar gum, cationic hydroxyethylcellulose,
hydroxypropylmethylcellulose, and poly(vinyl alcohol). Xanthan gum,
gellan gum and guar gum are naturally-occurring polysaccharides,
cationic hydroxyethylcellulose and hydroxypropylmethylcellulose are
modified polysaccharides, and poly(vinyl alcohol) is a synthetic
vinyl-based polymer.
[0215] PEGylated fibrin (10:1) hydrogels were also prepared with
the following polymers incorporated: Carbopol at 20 mg/mL, pullulan
at 20 mg/mL, guar gum at 15 mg/mL, hydroxyethylcellulose at 2.5
mg/mL, and collagen type I at 1 mg/mL.
[0216] The hydrogels of PEG:Fibrin:polymers were prepared as
follows: Fibrinogen was PEGylated by blending the PEGylating agent
and fibrinogen at a molar ratio of 10:1. After PEGylating for 5
minutes, the water soluble polymer was added to the solution and
then thrombin was added to initiate crosslinking.
Example 6
PEG-Proteins
[0217] The following proteins were PEGylated at a molar ratio of
20:1: fibrinogen, bovine serum albumin, collagen type I,
Cryoprecipitated Antihemophilic Factor (AHF), and extracellular
matrix using a procedure analogous to that of Example 1. PEGylation
induced gel formation when the pH was increased to >7.0 with 1 M
NaOH. For in vivo applications and protein stability, pH ranges
from 6.6 to 8.0 can be used. The hydrated PEGylated gels were
converted to microgel particles by rapid mechanical stirring.
Pseudoplasticity of the resulting microgel particles was
demonstrated by loading the hydrated microgel particles into a
syringe and injecting the microgels with PBS through an 18 gauge
needle, wherein a cluster of microgels formed as shear was removed.
Alternately, the following proteins were PEGylated at a molar ratio
of 20:1: bovine serum albumin, collagen type I, and extracellular
matrix. The hydrogels were dried, rehydrated, and mechanically
blended to form microgels. Pseudoplasticity of the microgels was
demonstrated by loading the resulting hydrated microgel particles
into a syringe and injecting the microgel particles with PBS
through an 18 gauge needle, wherein a cluster of microgels formed
as shear was removed.
Protocols for Miorogel Particle Formation
[0218] The PEGylated fibrinogen- and fibrin-based hydrogels were
lyophilized 24-96 hours. Lyophilized gels were then milled using a
mortar and pestle or Spex SamplePrep 6870 CryoMill (Metuchen,
N.J.). Dry microgel powder could then be sieved to particle sizes
(<250 .mu.m, <150 .mu.m, <106 .mu.m, or <75 .mu.m)
using Tyler or Retsch stainless steel test sieves. The dried
microgel powder was rehydrated with PBS (with and without calcium
and magnesium), normal saline (0.9% sodium chloride), deionized
water, cell culture media (with and without cells), or morselized
amnion.
[0219] The process for microgel preparation is shown in FIG. 1
utilizing a crosslinked PEGylated fibrin with a PEG:fibrin molar
content of 10:1, wherein the initial monolithic gel (Gelled
PEG-fibrin) is lyophilized (Lyophilized PEG-fibrin), followed by
grinding to a powder by cryomilling (Milled PEG-fibrin), and
rehydrating to a microgel particles (Rehydrated PEG-fibrin). All
microgel powders were prepared by this procedure.
Swelling Behavior of PEG-Fibrinogen and PEG-Fibrin Microgels
[0220] In Table 1 are presented the swelling behavior of microgel
particles of 20:1 PEG-FGN and 10:1 PEG-fibrin. Swelling is
determined on the basis of water-uptake from a dried microgel
particle, that is, weight hydrated microgeliweight dry microgel. In
comparison to the 50:1 formulations of PEG-FGN and PEG-fibrin, the
fibrinogen formulation is more hydrophilic than the fibrin
formulation. This is perhaps related to the additional crosslinking
in PEG-fibrin because of the conversion of fibrinogen to fibrin by
thrombin.
[0221] When water-soluble polymers were incorporated into 10:1
PEG-fibrin and 20:1 PEG-fibrinogen microgel particles, water uptake
increased. Incorporation of these water-soluble hydrophilic
polymers increased hydrophilicity of the microgel particle network,
increasing the degree of swelling. Hydrophilic polymers examined
include PHMB, Carbopol, pullulan, guar gum, hydroxyethyl cellulose
(HEC), and type I collagen, PEG-fibrin gels contained Carbopol at
20 mg/mL, pullulan at 20 mg/mL, guar gum at 15 mg/mL,
hydroxyethylcellulose at 2.5 mg/mL, and collagen type I at 1 mg/mL.
PEG-FGN gels were prepared with Carbopol at 27.6 mg/mL, pullulan at
27.6 mg/mL, guar gum at 20.7 mg/mL, hydroxyethyl cellulose at 3.4
mg/mL, and collagen type I at 1.4 mg/mL.
TABLE-US-00001 TABLE 1 PEG-fibrin, PEG-fibrinogen microgel swelling
and % hydration Formulation Swelling % Hydration PEG-fibrin 10:1
6.1 .+-. 0.6 86 PEG-fibrin 20:1 7.3 .+-. 0.6 88 PEG-fibrin 50:1 7.9
.+-. 1.0 89 PEG-FGN 20:1 8.6 .+-. 1.1 90 PEG-FGN 35:1 10.2 .+-. 0.7
91 PEG-FGN 50:1 13.9 .+-. 1.4 93 PEG-FGN-PHMB 100 ppm 9.7 .+-. 0.4
91 PEG-fibrin-Carbopol 8.4 .+-. 1.8 89 PEG-fibrin-Pullulan 6.7 .+-.
0.1 87 PEG-fibrin-Guar Gum 8.4 .+-. 2.0 89 PEG-fibrin-HEC 11.4 .+-.
2.4 92 PEG-fibrin-Collagen 7.3 .+-. 0.5 88 PEG-FGN-Carbopol 11.8
.+-. 2.6 92 PEG-FGN-Pullulan 10.7 .+-. 2.8 91 PEG-FGN-Guar Gum 12.9
.+-. 3.5 93 PEG-FGN-HEC 11.4 .+-. 2.6 92 PEG-FGN-Collagen 9.1 .+-.
0.9 90
Rheological Behavior
[0222] It was determined that the hydrated microgel particles are
shear thinning, This unexpected property makes them particularly
useful for ease of fluid injection or placement at a specific site,
and allows the microgel particle composition to act as a
viscoelastic solid immediately after injection to maintain shape
and conform to native surrounding tissue. A rheometer (Anton-Paar
MCR 101 or Anton-Paar MCR 302, Ashland, Va.) was used to determine
the pseudoplastic behavior and viscoelastic properties of the
microgel particle compositions. Utilizing 20:1 molar ratio of
rehydrated PEG-fibrinogen and 10:1 rehydrated PEG-fibrin microgel
particles as examples made by cryomilling (or mortar and pestle),
the viscosity vs. shear rate shown in FIG. 2 demonstrates that the
crosslinked PEGylated protein microgel particle compositions
rapidly shear thin, as illustrated by the absolute values of slopes
of these plots, wherein the slope of PEG-fibrinogen is -0.90 and
that of PEG-fibrin is -0.98, where a slope of approximately -1
indicates a perfectly shear thinning material.
[0223] For 20:1 PEG-fibrinogen as an example, the mechanical
properties fully recover with no loss of viscoelasticity after
repeated exposure to 1% strain (FIG. 3).
[0224] Additionally, both PEG-fibrinogen and PEG-fibrin hydrated
microgel particle compositions act as viscoelastic solids with
storage module greater than loss moduli (FIG. 4, G'>G'', or loss
tangent <1) (Table 2).
TABLE-US-00002 TABLE 2 Loss Tangents for 10:1 PEG-fibrin,
PEG-fibrinogen, and PEG-FGN-PHMB at a frequency of 1 1/sec
Formulation Loss Tangent PEG-fibrin 10:1 0.18 PEG-FGN 20:1 0.29
PEG-FGN 35:1 0.22 PEG-FGN 50:1 0.19 PEG-FGN-PHMB 1000 ppm 0.21
[0225] The shear thinning behavior of 50:1 PEG-fibrinogen microgel
milled by mortar and pestle and by cryomilling is shown in FIG. 5
for a plot of viscosity vs. shear rate. Dried microgel particles
obtained by mortar and pestle were flatter and more flake-like,
whereas dried particles obtained by cryomilling were more
spherical. The microgels were rehydrated at 300 mg/mL in phosphate
buffered saline. The slopes of the plots are -0.86 for mortar and
pestle and -0.84 for cryomilling. The cryomilled particles were
somewhat more viscous, presumably due to decreased particle size
distribution and decreased average particle size.
Human Adipose-Derived Stem Cells
[0226] Human adipose-derived stem cells (ADSC) were isolated from
human abdominal fat and subcultured in MesenPRO RS.TM. Medium (Life
Technologies) with growth supplement and 1%
penicillin-streptomycin. Powdered samples of PEG-FGN and
PEG-fibrin, at 10:1, 20:1, and 50:1, (15 mg) were combined with 400
.mu.L of 2.5.times.10.sup.5 cells/mL suspension in cell culture
inserts (transparent PET membrane size=8.0 .mu.m, BD Biosciences)
on a 12 well plate (n=3). Another 600 .mu.L media was added to the
insert and another 1 mL outside, resulting in 2 mL total media
volume. The culture medium was exchanged daily and cell growth was
examined using CellTiter 96.RTM. Aqueous One Solution Cell
Proliferation Assay (MTS) (Promega) according to the manufacturer's
protocol. Afterwards, cells were stained with Calcein AM (4 mM)
live cell stain for 45 minutes and fixed with 10% formalin. The
macroscopic and fluorescent image of each sample was acquired using
a digital camera and confocal microscopy, respectively.
[0227] FIG. 6 shows that the stem cells significantly increased in
number on the microgels from day 1 to day 4. Cells continued to
proliferate on the 20:1 and 50:1 formulations from day 4 to day 7.
Live cell staining presented in FIG. 7 shows that the ADSCs are
viable on all the microgel particle compositions tested. The cells
appear to be forming networks on the 20:1 PEG-fibrinogen and 50:1
PEG-fibrinogen microgels.
[0228] Thus, a significant utilization of the microgel particle
compositions is their ability to reform as a duster of microgel
particles after injection or movement by fluid. In FIG. 8 is shown
20:1 PEGylated fibrinogen microgels injected subcutaneously ex vivo
in rats, wherein the microgel particles formed a cohesive solid and
maintained their shape at the injection site.
Antimicrobial Properties
[0229] In order to confer antimicrobial properties on the PEGylated
microgel particle compositions, PHMB was incorporated into the
PEGylation reaction with fibrinogen. FIG. 9 shows the
pseudoplasticity of the ternary complex of 20:1 PEG-FGN-PHIVIB at
1,000 ppm of PHMB, in comparison to 20:1 PEG-FGN. The values of
slopes are -1.01 for PEG-FGN and -1.06 for PEG-FGN-PHMB, with
slopes of approximately -1 indicating perfectly shear thinning
materials.
[0230] FIG. 10 shows a plot of modulus vs. frequency for the
storage and loss moduli of 20:1 PEG-FGN gels without and with PHMB.
The loss tangent for PEG-FGN was 0.25 while that of PEG-FGN-PHMB
was 0.21 at an angular frequency of 1 sec.sup.-1. The modulus data
illustrates that the addition of PHMB increased the solid-like
behavior of the PEG-FGN-PHMB complex in comparison to that of
PEG-FGN, indicating that PHMB was incorporated covalently into the
PEGylation reaction.
[0231] The covalent binding of PHMB into the PEGylated fibrinogen
complex, forming a covalently bonded PEG-FGN-PHMB ternary complex,
is supported in an antimicrobial study of the biocidal activity in
a zone-of-inhibition (ZOI) study of PHMB that was included in the
PEGylation of fibrinogen (covalently bound PHMB) and PHMB that was
added subsequently to the formation of PEG-fibrinogen (unbound
PHMB) against Methicillin resistant Staphylococcus aureus (MRSA,
ATCC #700787). The ZOI for PEG-fibrinogen with unbound PHMB
composite was <1.0 mm, while that of covalently bonded PHMB
ternary composite had no measurable ZOI, with inhibition only under
the microgel powder. Unbound cationic PHMB is expected to have a
significantly larger ZOI because of the mobility of free PHMB,
which may be ionically bound, not covalently bound, to anionic
sites on fibrinogen, while the covalently bonded PHMB requires the
microorganism to be in direct contact with the microgel
particle.
In Vivo Subcutaneous Injection
[0232] PEG-FGN (20:1 molar) was evaluated alone and in combination
with autologous stem cells (rat ADSCs) or BioDRestore.TM.
morselized amnion. Microgels sterilized with ethylene oxide were
injected into the rat dorsal subcutaneous region with a 21 gauge
needle. Five test groups were evaluated in duplicate: rat ADSCs,
BioDRestore.TM., PEG-FGN, PEG-FGN with rat ADSCs, and PEG-FGN with
BioDRestore.TM.. Results indicate that the microgels were easily
injected, formed a cohesive solid, and remained at the injection
site at days 7 and 14.
[0233] Hematoxylin & eosin (H&E) was used to stain the
fixed tissue sections on days 7 and 14 to visualize the cells,
collagen, and tissue remodeling. H&E staining showed minimal
inflammatory response with host cell remodeling of the microgels.
Remodeling of the microgels was even more evident on day 14,
especially in combination with ADSCs or BioDRestore.TM..
[0234] Smooth muscle actin (SMA) staining, an indicator for
pericytes, showed blood vessel infiltration in microgels by days 7
and 14. Blood vessel formation was enhanced by the incorporation of
allogeneic ADSCs or morselized amnion product (BioD) into the
microgel system, FIG. 11 demonstrates blood vessel infiltration in
the center of the microgels. With monolithic hydrogel systems, if
angiogenesis occurs, it rarely reaches the center of the
hydrogel.
[0235] In addition, cell viability was enhanced by injection with
the microgel particle system, which appears to shield the cells
from shear stress. PEG-FGN microgel compositions injected with
CM-dil labeled rat ADSCs (2.0.times.10.sup.5 cells/mL) were
retained at the injection site at days 7 and 14. FIG. 12
demonstrates stem cell viability in the microgels after 7 and 14
days in vivo. DAPI nuclear staining and SMA staining for
angiogenesis showed cells in the microgels were connected, spread
out, viable, and promoted angiogenesis, even after 14 days in
vivo.
[0236] While the above specification contains many specifics, these
should not be construed as limitations on the scope of the
invention, but rather as examples of preferred embodiments thereof.
Many other variations are possible. Accordingly, the scope of the
invention should be determined not by the embodiments illustrated,
but by the appended claims and their legal equivalents.
* * * * *