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US20050176620A1 - Crosslinked compounds and methods of making and using thereof - Google Patents

Crosslinked compounds and methods of making and using thereof Download PDF

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US20050176620A1
US20050176620A1 US10/519,173 US51917305A US2005176620A1 US 20050176620 A1 US20050176620 A1 US 20050176620A1 US 51917305 A US51917305 A US 51917305A US 2005176620 A1 US2005176620 A1 US 2005176620A1
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group
compound
residue
protein
polysaccharide
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Glenn Prestwich
Xiao Shu
Yi Luo
Kelly Kirker
Yanchun Liu
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University of Utah Research Foundation Inc
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Priority to US12/234,445 priority patent/US8859523B2/en
Priority to US12/244,135 priority patent/US7928069B2/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/61—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups

Definitions

  • a physiologically compatible macromolecular scaffold capable of being produced in a straightforward manner is needed before they will be useful as therapeutic aids. Described herein are compounds and methods that are capable of coupling two or more molecules, such as macromolecules, under mild conditions.
  • crosslinked compounds Described herein are crosslinked compounds. Also described herein are methods of making and using crosslinked compounds.
  • FIG. 1 shows the reaction scheme for producing HA-thiolated derivatives.
  • FIG. 2 shows (a) absorption at 242 nm as a function of pH for HA-DTPH and HA-DTBH solution and (b) logarithmic plot of log[(A max ⁇ A i )/A i ] vs. pH.
  • the pK a values correspond to the intercept with the abscissa.
  • FIG. 3 shows the swelling of HA-DTPH and HA-DTBH films in PBS at pH 7.4.
  • the open circles and triangles are the films coupled via oxidation with 0.3% H 2 O 2 after air oxidation, and the closed circles and triangles are films coupled by air oxidation only.
  • FIG. 5 shows the release of blue dextran from HA-DTPH in PBS containing different concentrations of DTT at pH 7.4.
  • FIG. 6 shows fibroblast proliferation in HA-DTPH hydrogel after in vitro culture of 0, 1, 2, and 3 days.
  • FIG. 7 shows the synthesis of thiolated HA and gelatin.
  • FIG. 8 shows the effect of salt concentration on polyelectrolyte complex formation in mixed HA-gelatin solutions.
  • HA-DTPH and gelatin-DTPH, both 3.0% (w/v) were dissolved in 0.02 M PBS, the pH was adjusted to 7.4 (3.0% w/v), and the solutions were then mixed at different ratios.
  • the absorption was determined at 15 min (open circles, no added salt) and 2 h (open diamonds, 1.0% NaCl) after the preparation of solution in 0.5-cm spectrophotometer cell.
  • FIG. 9 shows the determination of disulfide density in HA-DTPH gelatin-DTPH hydrogel films.
  • the hydrogel films were prepared with 3.0% (w/v) polymer in 0.02 M PBS (pH 7.4) with 1.0% (w/v) NaCl.
  • FIG. 11 shows the enzymatic degradation of mixed HA-gelatin films.
  • Panel A HA-gelatin, 20:80.
  • Panel B HA-gelatin, 40:60.
  • the hydrogel films were prepared with 3.0% (w/v) polymer in 0.02 M PBS (pH 7.4) with 1.0% (w/v) NaCl.
  • FIG. 12 shows the cell attachment and spreading of fibroblasts of HA-gelatin films. Fluorescent microscopic images of adherent and spread Balb/c 3T3 fibroblast on the surface of HA-gelatin hydrogel films after 24 h of in vitro culture. The cells were initially seeded at 25,000 cells/cm2 and were stained with F-DA. Panel a: 100% HA film; Panel b: HA-gelatin, 80:20; Panel c: HA-gelatin, 40:60 film; and Panel d: 100% gelatin-DTPH film. Original magnification: Panels a, b, c, and d at ⁇ 100.
  • FIG. 13 shows the proliferation of Balb/c 3T3 fibroblast on the surface of HA-gelatin hydrogel film.
  • Tissue culture polystyrene (PS) was used as control, and the relative cell density on tissue culture polystyrene after one day of in vitro culture was defined as 1.0.
  • the inset shows the proliferation ratio (PR) as a function of percent gelatin (% G) in the hydrogel.
  • FIG. 14 shows structures of ⁇ , ⁇ -unsaturated esters and amides of poly(ethylene glycol) crosslinked with thiolated HA and thiolated gelatin.
  • FIG. 15 shows the conjugate addition between PEGDA, PEGDM, PEGDAA, PEGDMA and cysteine.
  • FIG. 16 shows the conjugate addition of HA-DTPH, HA-DTBH and PEG-acrylate.
  • FIG. 17 shows the digestion of HA-DTPH-PEGDA with HAse.
  • FIG. 19 shows the proliferation of T31 fibroblasts in HA-DTPH-PEGDA gel.
  • FIG. 20 shows the gross view of explants of HA-DTPH-PEGDA seeded with T3 1 fibroblasts after subcutaneous implantation in vivo in nude mice.
  • FIG. 21 shows the histological examination of the explants after incubation in nude mice for 2 weeks (Panel A), 4 weeks (Panel B), and 8 weeks (Panel C), immunohistochemistry (fibronectin). Original magnification ⁇ 200.
  • FIG. 22 shows the synthesis of HA-DTPH-MMC.
  • FIG. 23 shows the synthesis of HA-DTPH-PEGDA-MMC.
  • FIGS. 24 a and 24 b show the results of in vitro MMC release results.
  • “Optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.
  • the phrase “optionally substituted lower alkyl” means that the lower alkyl group can or can not be substituted and that the description includes both unsubstituted lower alkyl and lower alkyl where there is substitution.
  • Ranges may be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, another aspect includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.
  • X and Y are present at a weight ratio of 2:5, and are present in such ratio regardless of whether additional components are contained in the compound.
  • a weight percent of a component is based on the total weight of the formulation or composition in which the component is included.
  • a residue of a chemical species refers to the moiety that is the resulting product of the chemical species in a particular reaction scheme or subsequent formulation or chemical product, regardless of whether the moiety is actually obtained from the chemical species.
  • a polysaccharide that contains at least one —COOH group can be represented by the formula Y—COOH, where Y is the remainder (i.e., residue) of the polysaccharide molecule.
  • alkyl group as used herein is a branched or unbranched saturated hydrocarbon group of 1 to 24 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, pentyl, hexyl, heptyl, octyl, decyl, tetradecyl, hexadecyl, eicosyl, tetracosyl and the like.
  • a “lower alkyl” group is an alkyl group containing from one to six carbon atoms.
  • polyalkylene group as used herein is a group having two or more CH 2 groups linked to one another.
  • the polyalkylene group can be represented by the formula —(CH 2 ) n —, where n is an integer of from 2 to 25.
  • polyether group as used herein is a group having the formula —[(CHR) n O] m —, where R is hydrogen or a lower alkyl group, n is an integer of from 1 to 20, and m is an integer of from 1 to 100.
  • examples of polyether groups include, polyethylene oxide, polypropylene oxide, and polybutylene oxide.
  • polythioether group as used herein is a group having the formula —[(CHR) n S] m —, where R is hydrogen or a lower alkyl group, n is an integer of from 1 to 20, and m is an integer of from 1 to 100.
  • polyimino group as used herein is a group having the formula —[(CHR) n NR] m —, where each R is, independently, hydrogen or a lower alkyl group, n is an integer of from 1 to 20, and m is an integer of from 1 to 100.
  • polyester group as used herein is a group that is produced by the reaction between a compound having at least two carboxylic acid groups with a compound having at least two hydroxyl groups.
  • polyamide group as used herein is a group that is produced by the reaction between a compound having at least two carboxylic acid groups with a compound having at least two unsubstituted or monosubstituted amino groups.
  • aryl group as used herein is any carbon-based aromatic group including, but not limited to, benzene, naphthalene, etc.
  • aromatic also includes “heteroaryl group,” which is defined as an aromatic group that has at least one heteroatom incorporated within the ring of the aromatic group. Examples of heteroatoms include, but are not limited to, nitrogen, oxygen, sulfur, and phosphorus.
  • the aryl group can be substituted or unsubstituted.
  • the aryl group can be substituted with one or more groups including, but not limited to, alkyl, alkynyl, alkenyl, aryl, halide, nitro, amino, ester, ketone, aldehyde, hydroxy, carboxylic acid, or alkoxy.
  • FIG. 1 depicts one aspect of the method described above for producing a first thiolated compound having the formula III, where Y is hyaluronan.
  • the first step involves reacting a macromolecule having the formula Y—COOH with the dibydrazide/disulfide compound having the formula A.
  • the reaction is performed in the presence of a condensing agent.
  • a condensing agent is any compound that facilitates the reaction between the dihydrazide group of compound A and the COOH group on the macromolecule.
  • the condensing agent is a carbodiimide, including, but not limited to, 1-ethyl-3-[3-(dimethylamino)propyl]-carbodiimide (EDCI). As depicted in FIG.
  • a mixture of products (B and C) are produced after the first step.
  • the disulfide bond in compounds B and C is cleaved with a reducing agent.
  • the reducing agent is dithiothreitol. Cleavage of the disulfide bonds in compounds B and C produces the first tholated compound having the formula III.
  • the macromolecule is any compound having at least one group that can react with a hydrazide compound.
  • the macromolecule has at least one —COOH group or the salt or ester thereof.
  • the macromolecule is an oligonucleotide, a nucleic acid or a metabolically stabilized analogue thereof, a polypeptide, a lipid, a glycoprotein, or a glycolipid.
  • the macromolecule is a polysaccharide, a protein, or a synthetic polymer.
  • the macromolecule can be a pharmaceutically-acceptable compound.
  • the pharmaceutically-acceptable compounds can include substances capable of preventing an infection systemnically in the biological system or locally at the defect site, as for example, anti-inflammatory agents such as, but not limited to, pilocarpine, hydrocortisone, prednisolone, cortisone, diclofenac sodium, indomethacin, 6 ⁇ -methyl-prednisolone, corticosterone, dexamethasone, prednisone, and the like; antibacterial agents including, but not limited to, penicillin, cephalosporins, bacitracin, tetracycline, doxycycline, gentamycin, chloroquine, vidarabine, and the like; analgesic agents including, but not limited to, salicylic acid, acetaminophen, ibuprofen, naproxen, piroxicam, flurbiprofen, morphine, and the like
  • the phannaceutically-acceptable compound can be a substance or metabolic precursor which is capable of promoting growth and survival of cells and tissues or augmenting the functioning of cells is useful, as for example, a nerve growth promoting substance such as a ganglioside, a nerve growth factor, and the like; a hard or soft tissue growth promoting agent such as fibronectin (FN), human growth honnone (HGH), a colony stimulating factor, bone morphogenic protein, platelet-derived growth factor (PDGF), insulin-derived growth factor (IGF-I, IGF-II), transforming growth factor-alpha (TGF-alpha), transforming growth factor-beta (TGF-beta), epiderrnal growth factor (EGF), fibroblast growth factor (FGF), interleukin-1 (IL-1), vascular endothelial growth factor (VEGF) and keratinocyte growth factor (KGF), dried bone material, and the like; and antineoplastic agents such as methotrexate, 5-fluorour
  • the pharmaceutically-acceptable compound can include hormones such as progesterone, testosterone, and follicle stimulating hormone (FSH) (birth control, fertility-enhancement), insulin, and the like; antihistamines such as diphenhydramine, and the like; cardiovascular agents such as papaverine, streptokinase and the like; anti-ulcer agents such as isopropamide iodide, and the like; bronchodilators such as metaproternal sulfate, aminophylline, and the like; vasodilators such as theophylline, niacin, minoxidil, and the like; central nervous system agents such as tranquilizer, B-adrenergic blocking agent, dopamine, and the like; antipsychotic agents such as risperidone, narcotic antagonists such as naltrexone, naloxone, buprenorphine; and other like substances. All compounds are available from Sigma Chemical Co. (Milwaukee, Wis, testosterone,
  • Polysaccharides useful in the methods described herein have at least one group, such as a carboxylic acid group or the salt or ester thereof, that can react with a dihydrazide.
  • the polysaccharide is a glycosaminoglycan (GAG).
  • GAG is one molecule with many alternating subunits. For example, HA is (GlcNAc-GlcUA-)x. Other GAGs are sulfated at different sugars.
  • GAGs are represented by the formula A-B-A-B-A-B, where A is a uronic acid and B is an aminosugar that is either O— or N-sulfated, where the A and B units can be heterogeneous with respect to epimeric content or sulfation. Any natural or synthetic polymer containing uronic acid can be used.
  • Y in formula III is a sulfated-GAG.
  • GAGs there are many different types of GAGs, having commonly understood structures, which, for example, are within the disclosed compositions, such as chondroitin sulfate, dermatan, heparan, heparin, dermatan sulfate, and heparan sulfate. Any GAG known in the art can be used in any of the methods described herein. Glycosaminoglycans can be purchased from Sigma, and many other biochemical suppliers. Alginic acid, pectin, and carboxymethylcellulose are among other carboxylic acid containing polysaccharides useful in the methods described herein.
  • the polysaccharide Y in formula III is hyaluronan (HA).
  • HA is a non-sulfated GAG.
  • Hyaluronan is a well known, naturally occurring, water soluble polysaccharide composed of two alternatively linked sugars, D-glucuronic acid and N-acetylglucosamine. The polymer is hydrophilic and highly viscous in aqueous solution at relatively low solute concentrations. It often occurs naturally as the sodium salt, sodium hyaluronate. Methods of preparing commercially available hyaluronan and salts thereof are well known.
  • Hyaluronan can be purchased from Seikagaku Company, Clear Solutions Biotech, Inc., Pharmacia Inc., Sigma Inc., and many other suppliers. For high molecular weight hyaluronan it is often in the range of 100 to 10,000 disaccharide units.
  • the lower limit of the molecular weight of the hyaluronan is from 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, or 100,000
  • the upper limit is 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, or 1,000,000, where any of the lower limits can be combined with any of the upper limits.
  • Y in formula III is not hyaluronan.
  • Y in formula m can also be a synthetic polymer.
  • the synthetic polymer has at least one carboxylic acid group or the salt or ester thereof, which is capable of reacting with a hydrazide.
  • the synthetic polymer residue in formula III comprises the synthetic polymer comprises glucuronic acid, polyacrylic acid, polyaspratic acid, polytartaric acid, polyglutamic acid, or polyfumaric acid.
  • Y in formula III is a protein.
  • Proteins useful in the methods described herein include, but are not limited to, an extracellular matrix protein, a chemically-modified extracellular matrix protein, or a partially hydrolyzed derivative of an extracellular matrix protein.
  • the proteins may be naturally occurring or recombinant polypeptides possessing a cell interactive domain.
  • the protein can also be mixtures of proteins, where one or more of the proteins are modified. Specific examples of proteins include, but are not limited to, collagen, elastin, decorin, laminin, or fibronectin.
  • L in formula III is a polyalkylene group. In another aspect, L in formula III is a C 1 to C 20 polyalkylene group. In another aspect, L in formula I is CH 2 CH 2 or CH 2 CH 2 CH 2 . In another aspect, Y is a residue of a polysaccharide or protein and L is CH 2 CH 2 or CH 2 CH 2 CH 2 .
  • the second thiolated compound is any compound having at least one thiol group.
  • the first and second thiolated compounds can be the same or different compounds.
  • the second thiolated compound can be any macromolecule described above.
  • the second thiolated compound is a polysaccharide having at least one SH group. Any of the polysaccharides described above can be used as the second thiolated compound.
  • the second thiolated compound comprises a sulfated-glycosaminoglycan.
  • the second thiolated compound includes chondroitin sulfate, dermatan, heparan, heparin, dermatan sulfate, heparan sulfate, alginic acid, pectin, carboxymethylcellulose, or hyaluronan having at least one SH gpup.
  • the second thiolated compound has the formula II wherein
  • the macromolecule residue Z can be any of the macromolecules described above.
  • the second thiolated compound can be a protein having at least one thiol group.
  • the protein comprises an extracellular matrix protein or a chemically-modified extracellular matrix protein.
  • the protein comprises collagen, elastin, decorin, laminin, or fibronectin
  • L in formula II is a polyalkylene group. In another aspect, L in formula II is a C 1 to C 20 polyalkylene group. In another aspect, L in formula II is CH 2 CH 2 or CH 2 CH 2 CH 2 . In one aspect, Z is a residue of hyaluronan and L in formula II is CH 2 CH 2 or CH 2 CH 2 CH 2 . In a further aspect, Z is a residue of gelatin and L in formula II is CH 2 CH 2 or CH 2 CH 2 CH 2 .
  • the reaction between the first and second thiolated compounds is performed in the presence of an oxidant.
  • the reaction between the first and second thiolated compounds can be conducted in the presence of any gas that contains oxygen.
  • the oxidant is air.
  • This aspect also contemplates the addition of a second oxidant to expedite the reaction.
  • the reaction can be performed under an inert atmosphere (i.e., oxygen free), and an oxidant is added to the reaction.
  • oxidants useful in this method include, but are not limited to, molecular iodine, hydrogen peroxide, alkyl hydroperoxides, peroxy acids, dialkyl sulfoxides, high valent metals such as Co +3 and Ce +4 , metal oxides of manganese, lead, and chromium, and halogen transfer agents.
  • the oxidants disclosed in Capozzi, G.; Modena, G. In The Chemistry of the Thiol Group Part II ; Patai, S., Ed.; Wiley: New York, 1974; pp 785-839, which is incorporated by reference in its entirety, are useful in the methods described herein.
  • the reaction between the first and second thiolated compounds can be conducted in a buffer solution that is slightly basic.
  • the amount of the first thiolated compound relative the amount of the second thiolated compound can vary.
  • the volume ratio of the first thiolated compound to the second thiolated compound is from 99:1, 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, 10:90, or 1:99.
  • the first and second thiolated compound react in air and are allowed to dry at room temperature.
  • the dried material can be exposed to a second oxidant, such as hydrogen peroxide.
  • the resultant compound can then be rinsed with water to remove any unreacted first and/or second thiolated compound and any unused oxidant.
  • One advantage of preparing coupled compound via the oxidative coupling methodology described herein is that crosslinking can occur in an aqueous media under physiologically benign conditions without the necessity of additional crosslink-ing reagents.
  • fragment refers to the entire molecule itself or a portion or segment of a larger molecule.
  • Y in formula VI may be high molecular weight polysaccharide that is crosslinked by disulfide linkage with another polysaccharide, synthetic polymer, or thiolated polymer to produce the coupled compound.
  • the coupled compound may have multiple disulfide linkages.
  • the compound has at a minimum one unit depicted in formula VI, which represents at least one disulfide linkage as the result of at least one first thiolated compound that reacted with at least one second thiolated compound via oxidation.
  • the macromolecule (Y) and thiolated compound (G) can be any of the macromolecules described above.
  • Y in formula VI is a polysaccharide, a protein, or a synthetic polymer.
  • the fragment comprises the formula VIII wherein
  • Y in formula VIII is a residue of any of the glycosaminoglycans described above including, but not limited to, chondroitin sulfate, dermatan, heparan, heparin, dermatan sulfate, heparan sulfate, alginic acid, pectin, or carboxymethylcellulose.
  • L in formula VIII is CH 2 CH 2 or CH 2 CH 2 CH 2 .
  • G is a residue of any of the polysaccharides described above, including a glycosaminoglycan such as chondroitin sulfate, dermatan, heparan, heparin, dermatan sulfate, heparan sulfate, alginic acid, pectin, carboxymethylcellulose, or hyaluronan.
  • a glycosaminoglycan such as chondroitin sulfate, dermatan, heparan, heparin, dermatan sulfate, heparan sulfate, alginic acid, pectin, carboxymethylcellulose, or hyaluronan.
  • described herein is a method for coupling two or more compounds by reacting a first thiolated macromolecule having at least one SH group with at least one compound having at one thiol-reactive electrophilic functional group.
  • the compound has at least two-thiol reactive functional groups.
  • any of the macromolecules described above can be used in this aspect.
  • Two or more different macromolecules can be used in this method.
  • a second thiolated macromolecule can be used in combination with the first thiolated macromolecule.
  • the first and second thiolated macromolecule can be the same or different compounds.
  • the macromolecule is a polysaccharide.
  • the polysaccharide is a sulfated-glycosaminoglycan including, but not limited to, chondroitin sulfate, dermatan, heparan, heparin, dermatan sulfate, heparan sulfate, alginic acid, pectin, or carboxymethylcellulose.
  • the polysaccharide is hyaluronan.
  • the polysaccharide has the formula III described above.
  • Y is a residue of hyaluronan and L is CH 2 CH 2 or CH 2 CH 2 CH 2 .
  • the macromolecule is a compound having the formula III, wherein Y is a protein. Any of the proteins described above can be used in this aspect.
  • the protein is collagen, elastin, decorin, laminin, or fibronectin.
  • a compound having at least one thiol-reactive electrophilic group is also used in this aspect of the method.
  • the term “thiol-reactive electrophilic group” as used herein is any group that is susceptible to nucleophilic attack by the lone-pair electrons on the sulfur atom of the thiol group or by the thiolate anion.
  • Examples of thiol-reactive electrophilic groups include groups that have good leaving groups.
  • an alkyl group having a halide or alkoxy group attached to it or an ⁇ -halocarbonyl group are examples of thiol-reactive electrophilic groups.
  • the thiol-reactive electrophilic group is an electron-deficient vinyl group.
  • an electron-deficient vinyl group is a group having a carbon-carbon double bond and an electron-withdrawing group attached to one of the carbon atoms.
  • An electron-deficient vinyl group is depicted in the formula C ⁇ ⁇ C ⁇ X, where X is the electron-withdrawing group.
  • the electron-withdrawing group is attached to C ⁇ , the other carbon atom of the vinyl group (C ⁇ ) is more susceptible to nucleophilic attack by the thiol group. This type of addition to an activated carbon-carbon double bond is referred to as a Michael addition.
  • Examples of electron-withdrawing groups include, but are not limited to, a nitro group, a cyano group, an ester group, an aldehyde group, a keto group, a sulfone group, or an amide group.
  • Examples of compounds possessing thiol-reactive electrphilic groups include, but are not limited to, maleimides, vinyl sulfones. acrylonitriles, ⁇ -methylene esters, quinone methides, acryloyl esters or amides, or ⁇ -halo esters or amides.
  • the thiol-reactive compound has two electron-deficient vinyl groups, wherein the two electron-deficient vinyl groups are the same.
  • the thiol-reactive compound is a diacrylate, a dimethacrylate, a diacrylamide, a dimethacrylamide, or a combination thereof.
  • the thiol-reactive compound has the formula V wherein
  • R 3 and R 4 are hydrogen, U and V are oxygen, and M is a polyether group. In another aspect, R 3 and R 4 are hydrogen, U and V are NH, and M is a polyether group. In a further aspect, R 3 and R 4 are methyl, U and V are oxygen, and M is a polyether group. In another aspect, R 3 and R 4 are methyl, U and V are NH, and M is a polyether group.
  • the thiol-reactive compound is any of pharmaceutically-acceptable compounds described above containing at least one thiol-reactive electrophilic group.
  • FIG. 22 depicts one embodiment of this aspect. Mitomycin C (MMC) is converted to the corresponding acrylate (MMC-acrylate). MMC-acrylate is then coupled with the hydrazide-modified hyaluronan thiol compound HA-DTPH to produce HA-DT H-MMC.
  • HA-DT H-MMC contains one or more free thiols groups, which then can couple with PEGDA to produce HA-DTPH-PEGDA-MMC, which is depicted in FIG. 23 .
  • the first thiolated macromolecule has the formula III described above, wherein Y is a residue of polysaccharide, and L is CH 2 CH 2 or CH 2 CH 2 CH 2 , and the thiol-reactive compound has the formula V described above, wherein R 3 and R 4 are, independently, hydrogen or lower alkyl; U and V are, independently, O or NR 5 , wherein R 5 is, independently, hydrogen or lower alkyl; and M is a polyether group.
  • Y is a residue of hyaluronan
  • the reaction further comprises reacting gelatin having at least one thiol group with the compound having the formula V
  • the polysaccharide includes a first polysaccharide and second polysaccharide having the formula I, wherein in the first polysaccharide, Y is a residue of a first sulfated-glycosaminoglycan, and in the second polysaccharide, Y is a residue of a second sulfated-glycosaminoglycan, wherein the first and second sulfated-glycosaminoglycans are the same or different;
  • the polysaccharide includes a first polysaccharide and second polysaccharide having the formula I, wherein in the first polysaccharide, Y is a residue of hyaluronan, and in the second polysaccharide, Y is a residue of a sulf
  • described herein is a method for coupling a compound by reacting a first thiolated macromolecule having at least one thiol-reactive electrophilic functional group with at least one compound having at least two thiol groups.
  • the first thiolated macromolecule having at least one thiol-reactive electrophilic functional group and the thiolated compound have the formula I wherein
  • examples of compounds having at least two thiol groups include, but are not limited to, propane-1,3-dithiol, polyethylene glycol- ⁇ , ⁇ -dithiol, para, ortho, or meta-bisbenzyl thiol, dithiothreitol, a peptide containing two or more cysteine residues, or dendrimeric thiols.
  • the fragment has the formula IV wherein
  • Y in formula IV has the formula IX wherein
  • Z in formula IV has the formula X wherein
  • the reaction between the thiol reactive compound and thiol compound is generally conducted at a pH of from 7 to 12, 7.5 to 11, 7.5 to 10, or 7.5 to 9.5, or a pH of 8.
  • the solvent used can be water (alone) or an aqueous containing organic solvent.
  • a base such as a primary, secondary, or tertiary amine can used.
  • an excess of thiol compound is used relative to the thiol-reactive compound in order to ensure that all of the thiol-reactive compound is consumed during the reaction.
  • the thiol compound can react with itself or another thiol compound via oxidative addition to form a disulfide linkage in addition to reacting with the thiol-reactive compound.
  • a protein having at least one hydrazide-reactive group is reacted with a compound having at least one hydrazide group.
  • a protein having at least one hydrazide group is reacted with a compound having at least one hydrazide-reactive group.
  • the hydrazide-reactive group can be a —COOH group (or the salt or ester thereof), an aldehyde group, or a ketone group.
  • the coupled protein has the formula XI wherein
  • the protein residue can be any protein that has at least one hydrazide-reactive group or at least one hydrazide group.
  • the protein can be an extracellular matrix protein, a partially hydrolyzed extracellular matrix protein, or a chernically-modifled extracellular matrix protein.
  • the protein is collagen, elastin, decorin, laminin, or fibronectin.
  • E in formula XI is a reporter group.
  • reporter groups include, but are not limited to, a fluorescent tag, a radiolabel, a targeting moiety, a lipid, a peptide, a radionuclide chelator with a radionuclide, a spin-label, a PEG camouflage, a glass surface, a plastic surface, or a combination thereof.
  • hydrazide-modified fluorescent groups include, but are not limited to, BODIPY-hydrazide, fluorescein hydrazide, or NBD-hexanoyl -hydrazide.
  • hydrazide-modified radiolabels include, but are not limited to, 125I-tyrosine-hydrazide, 3H-acetyl-hydrazide, or 14 C-acetyl-hydrazide.
  • hydrazide-modified targeting moieties include, but are not limited to, 6-aminohexanoylhydrazide (Z) of integrin targeting peptide, such as ZYRGDS, Z-tat decapeptide for cell penetration, Z-GFLG for lysosome targeting, HA oligosaccharide hydrazide for CD-44 cancer targeting, or N-Ac glucosamine derivative for liver targeting.
  • hydrazide-modified lipids include, but are not limited to, hydrazide of 2′-succinate of Taxol or 2′succinate of a glucocorticosteroids, alkanoic or perfluoroalkanoate hydrazides, phosphatidylserine hydrazide, or cholic acid hydrazide.
  • hydrazide-modified radionuclides include, but are not limited to, the reaction product between DTPA anhydride and hydrazine to produce the corresponding hydrazide, coupling the hydrazide to a protein, then adding a nuclide such as In-111, Tc-99m, or Y-90.
  • spin labels include, but are not limited to, proxyl or doxyl groups.
  • glass surfaces include, but are not limited to, glass silanized with an epoxy or activated ester or a thiol-reactive electrophilic functional group, beads, or coverslips.
  • plastics include, but are not limited to, plasma-etched polypropylene, chemically-modified polystyrene with hydrazide, or any other plastic material.
  • E is a crosslinkable thiol reactive-electrophilic groups such, but not limited to, acrylic hydrazide or methacrylic hydrazide.
  • kits including (1) a compound having at least one hydrazide group; (2) a condensing agent; (3) a buffer reagent; and (4) a purification column.
  • the compound can be any compound having at least one hydrazide group and at least one of the reporter groups described above.
  • Use of the kit generally involves admixing components (1)-(3) together with a protein having at least one hydrazide-reactive group. Components (1)-(3) and the protein can be added in any order. After the protein and the compound having at least one hyrazide group have reacted with one another to produce the coupled protein, the coupled protein is then purified by passing the admixture containing the coupled protein through a purification column. Purification columns and techniques for using the same are known in the art.
  • any of the compounds produced by the methods described above can include at least one pharmaceutically-acceptable compound.
  • the resulting pharmaceutical composition can provide a system for sustained, continuous delivery of drugs and other biologically-active agents to tissues adjacent to or distant from the application site.
  • the biologically-active agent is capable of providing a local or systemic biological, physiological or therapeutic effect in the biological system to which it is applied.
  • the agent can act to control infection or inflammation, enhance cell growth and tissue regeneration, control tumor growth, act as an analgesic, promote anti-cell attachment, and enhance bone growth, among other functions.
  • any of the compounds described herein can contain combinations of two or more pharmaceutically-acceptable compounds.
  • the pharmaceutically-acceptable compounds can include substances capable of preventing an infection systemically in the biological system or locally at the defect site, as for example, anti-inflammatory agents such as, but not limited to, pilocarpine, hydrocortisone, prednisolone, cortisone, diclofenac sodium, indomethacin, 6 ⁇ -methyl-prednisolone, corticosterone, dexamethasone, prednisone, and the like; antibacterial agents including, but not limited to, penicillin, cephalosporins, bacitracin, tetracycline, doxycycline, gentamycin, chloroquine, vidarabine, and the like; analgesic agents including, but not limited to, salicylic acid, acetaminophen, ibuprofen, naproxen, piroxicam, flurbiprofen, morphine, and the like; local anesthetics including, but not limited to, cocaine, lidoca
  • a substance or metabolic precursor which is capable of promoting growth and survival of cells and tissues or augmenting the functioning of cells is useful, as for example, a nerve growth promoting substance such as a ganglioside, a nerve growth factor, and the like; a hard or soft tissue growth promoting agent such as fibronectin (FN), human growth hormone (HGH), a colony stimulating factor, bone morphogenic protein, platelet-derived growth factor (PDGF), insulin-derived growth factor (IGF-I, IGF-II), transforming growth factor-alpha (TGF-alpha), transforming growth factor-beta (TGF-beta), epidermal growth factor (EGF), fibroblast growth factor (FGF), interleukin-1 (IL-1), vascular endothelial growth factor (VEGF) and keratinocyte growth factor (KGF), dried bone material, and the like; and antineoplastic agents such as methotrexate, 5-fluorouracil, adriamycin, vinblastine, cisp
  • hormones such as progesterone, testosterone, and follicle stimulating hormone (FSH) (birth control, fertility-enhancement), insulin, and the like; antihistamines such as diphenhydramine, and the like; cardiovascular agents such as papaverine, streptokinase and the like; anti-ulcer agents such as isopropamide iodide, and the like; bronchodilators such as metaproternal sulfate; aminophylline, and the like; vasodilators such as theophylline, niacin, minoxidil, and the like; central nervous system agents such as tranquilizer, B-adrenergic blocking agent, dopamine, and the like; antipsychotic agents such as risperidone, narcotic antagonists such as naltrexone, naloxone, buprenorphine; and other like substances. All compounds are available from Sigrna Chemical Co. (Milwaukee, Wis.).
  • compositions can be prepared using techniques known in the art.
  • the composition is prepared by admixing a compound described herein with a pharmaceutically-acceptable compound.
  • admixing is defined as mixing the two components together so that there is no chemical reaction or physical interaction.
  • admixing also includes the chemical reaction or physical interaction between the compound and the pharmaceutically-acceptable compound. Covalent bonding to reactive therapeutic drugs, e.g., those having reactive carboxyl groups, can be undertaken on the compound.
  • carboxylate-containing chemicals such as anti-inflammatory drugs ibuprofen or hydrocortisone-hemisuccinate can be converted to the corresponding N-hydroxysuccinimide (NHS) active esters and can further react with the NH 2 group of the dihydrazide-modified polysaccharide.
  • NHS N-hydroxysuccinimide
  • electrostatic or hydrophobic interactions can facilitate retention of a pharmaceutically-acceptable compound in a modified: polysaccharide.
  • the hydrazido group can non-covalently interact, e.g., with carboxylic acid-containing steroids and their analogs, and anti-inflammatory drugs such as Ibuprofen (2-(4-iso-butylphenyl) propionic acid).
  • the protonated hydrazido group can form salts with a wide variety of anionic materials such as proteins, heparin or dermatan sulfates, oligonucleotides, phosphate esters, and the like.
  • the actual preferred amounts of active compound in a specified case will vary according to the specific compound being utilized, the particular compositions formulated, the mode of application, and the particular situs and subject being treated. Dosages for a given host can be determined using conventional considerations, e.g. by customary comparison of the differential activities of the subject compounds and of a known agent, e.g., by means of an appropriate conventional pharmacological protocol. Physicians and formulators, skilled in the art of determining doses of pharmaceutical compounds, will have no problems determining dose according to standard recommendations (Physicians Desk Reference, Barnhart Publishing (1999).
  • compositions described herein can be formulated in any excipient the biological system or entity can tolerate.
  • excipients include, but are not limited to, water, saline, Ringer's solution, dextrose solution, Hank's solution, and other aqueous physiologically balanced salt solutions.
  • Nonaqueous vehicles such as fixed oils, vegetable oils such as olive oil and sesame oil, triglycerides, propylene glycol, polyethylene glycol, and injectable organic esters such as ethyl oleate can also be used.
  • Other useful formulations include suspensions containing viscosity enhancing agents, such as sodium carboxymethylcellulose, sorbitol, or dextran.
  • Excipients can also contain minor amounts of additives, such as substances that enhance isotonicity and chemical stability.
  • buffers include phosphate buffer, bicarbonate buffer and Tris buffer, while examples of preservatives include thimerosol, cresols, formalin and benzyl alcohol.
  • Pharmaceutical carriers are known to those skilled in the art. These most typically would be standard carriers for administration to humans, including solutions such as sterile water, saline, and buffered solutions at physiological pH.
  • Molecules intended for pharmaceutical delivery can be formulated in a pharmaceutical composition.
  • Pharmaceutical compositions can include carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the molecule of choice.
  • Pharmaceutical compositions can also include one or more active ingredients such as antimicrobial agents, antiinflammatory agents, anesthetics, and the like.
  • the pharmaceutical composition can be administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated. Administration can be topically (including ophthalmically, vaginally, rectally, intranasally).
  • Preparations for administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.
  • non-aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles if needed for collateral use of the disclosed compositions and methods, include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
  • Intravenous vehicles if needed for collateral use of the disclosed compositions and methods, include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like.
  • Preservatives and other additives can also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.
  • Formulations for topical administration can include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders.
  • Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like can be necessary or desirable.
  • Dosing is dependent on severity and responsiveness of the condition to, be treated, but will normally be one or more doses per day, with course of treatment lasting from several days to several months or until one of ordinary skill in the art determines the delivery should cease. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates.
  • any of the compounds and pharmaceutical compositions can include living cells.
  • living cells include, but are not limited to, fibroblasts, hepatocytes, chondrocytes, stem cells, bone marrow, muscle cells, cardiac myocytes, neuronal cells, or pancreatic islet cells.
  • compositions described herein can be used for a variety of uses related to drug delivery, small molecule delivery, wound healing, burn injury healing, and tissue regeneration.
  • the disclosed compositions are useful for situations which benefit from a hydrated, pericellular environment in which assembly of other matrix components, presentation of growth and differentiation factors, cell migration, or tissue regeneration are desirable.
  • the compounds and pharmaceutical compositions described herein can be placed directly in or on any biological system without purification as it is composed of biocompatible materials.
  • sites the compounds can be placed include, but not limited to, soft tissue such as muscle or fat; hard tissue such as bone or cartilage: areas of tissue regeneration; a void space such as periodontal pocket; surgical incision or other formed pocket or cavity; a natural cavity such as the oral, vaginal, rectal or nasal cavities, the cul-de-sac of the eye, and the like; the peritoneal cavity and organs contained within, and other sites into or onto which the compounds can be placed including a skin surface defect such as a cut, scrape or burn area.
  • the present compounds can be biodegradeable and naturally occurring enzymes will act to degrade them over time.
  • Components of the compound can be “bioabsorbable” in that the components of the compound will be broken down and absorbed within the biological system, for example, by a cell, tissue and the like. Additionally, the compounds, especially compounds that have not been rehydrated, can be applied to a biological system to absorb fluid from an area of interest.
  • the compounds described herein can be used as a carrier for a wide variety of releasable biologically active substances having curative or therapeutic value for human or non-human animals. Many of these substances which can be carried by the compound are discussed above. Included among biologically active materials which are suitable for incorporation into the gels of the invention are therapeutic drugs, e.g., anti-inflammatory agents, anti-pyretic agents, steroidal and non-steroidal drugs for anti-inflammatory use, hormones, growth factors, contraceptive agents, antivirals, antibacterials, antifungals, analgesics, hypnotics, sedatives, tranquilizers, anti-convulsants, muscle relaxants, local anesthetics, antispasmodics, antiulcer drugs, peptidic agonists, sympathiomimetic agents, cardiovascular agents, antitumor agents, oligonucleotides and their analogues and so forth.
  • a biologically active substance is added in pharmaceutically active amounts.
  • the compounds and compositions described herein can be used for the delivery of living cells to a subject. Any of the living cells described above can be used in the aspect.
  • the compounds and compositions can be used for the delivery of growth factors and molecules related to growth factors.
  • the growth factors can be a nerve growth promoting substance such as a ganglioside, a nerve growth factor, and the like; a hard or soft tissue growth promoting agent such as fibronectin (FN), humran growth hormone (HGH), a colony stimulating factor, bone morphogenic protein, platelet-derived growth factor (PDGF), insulin derived growth factor (IGF-I, IGF-II), transforming growth factor-alpha (TGF-alpha), transforming growth factor-beta (TGF-beta), epidermal growth factor (EGF), fibroblast growth factor (FGF), interleukin-1 (IL-1).
  • Preferred growth factors are bFGF and TGF- ⁇ .
  • VEGF vascular endothelial growth factor
  • KGF keratinocyte growth factor
  • anti-inflammatories bearing carboxyl groups such as ibuprofen, naproxen, ketoprofen and indomethacin
  • Other biologically active substances are peptides, which are naturally occurring, non-naturally occurring or synthetic polypeptides or their isosteres, such as small peptide hormones or hormone analogues and protease inhibitors.
  • Spermicides, antibacterials, antivirals, antifungals and antiproliferatives such as fluorodeoxyuracil and adriamycin can also be used. These substances are all known in the art. Compounds are available from Sigma Chemical Company (St. Louis, Mo.).
  • therapeutic drugs as used herein is intended to include those defined in the Federal Food, Drug and Cosmetic Act.
  • USP United States Pharmacopeia
  • NF National Formulary
  • the pharmaceutically acceptable compound is pilocarpine, hydrocortisone, prednisolone, cortisone, diclofenac sodium, indomethacin, 6 ⁇ -methyl-prednisolone, corticosterone, dexamethasone and prednisone.
  • delivery of a pharmaceutically-acceptable compound is for a medical purpose selected from the group of delivery of contraceptive agents, treating postsurgical adhesions, promoting skin growth, preventing scarring, dressing wounds, conducting viscosurgery, conducting viscosupplementation, engineering tissue.
  • the rate of drug delivery depends on the hydrophobicity of the molecule being released. Hydrophobic molecules, such as dexamethazone and prednisone are released slowly from the compound as it swells in an aqueous environment, while hydrophilic molecules, such as pilocarpine, hydrocortisone, prednisolone, cortisone, diclofenac sodium, indomethacin, 6 ⁇ -methyl-prednisolone and corticosterone, are released quickly.
  • hydrophilic molecules such as pilocarpine, hydrocortisone, prednisolone, cortisone, diclofenac sodium, indomethacin, 6 ⁇ -methyl-prednisolone and corticosterone
  • the delivery of molecules or reagents related to angiogenesis and vascularization are achieved.
  • agents such as VEGF, that stimulate microvascularization.
  • methods for the delivery of agents that can inhibit angiogenesis and vascularization such as those compounds and reagents useful for this purpose disclosed in but not limited to U.S. Pat. No. 6,174,861 for “Methods of inhibiting angiogenesis via increasing in vivo concentrations of endostatin protein;” U.S. Pat. No. 6,086,865 for “Methods of treating angiogenesis-induced diseases and pharmaceutical compositions thereof;” U.S. Pat. No.
  • Described herein are methods for improving wound healing in a subject in need of such improvement by contacting any of the compounds or pharmaceutical compositions described herein with a wound of a subject in need of wound healing improvement. Also provided are methods to deliver at least one pharmaceutically-acceptable compound to a patient in need of such delivery by contacting any of the compounds or pharmaceutical compositions described herein with at least one tissue capable of receiving said pharmaceutically-acceptable compound.
  • compositions can be used for treating a wide variety of tissue defects in an animal, for example, a tissue with a void such as a periodontal pocket, a shallow or deep cutaneous wound, a surgical incision, a bone or cartilage defect, and the like.
  • the compounds described herein can be in the form of a hydrogel film.
  • the hydrogel film can be applied to a defect in bone tissue such as a fracture in an arm or leg bone, a defect in a tooth, a cartilage defect in the joint, ear, nose, or throat, and the like.
  • the hydrogel film composed of the compound described herein can also function as a barrier system for guided tissue regeneration by providing a surface on or through which the cells can grow. To enhance regeneration of a hard tissue such as bone tissue, it is preferred that the hydrogel film provides support for new cell growth that will replace the matrix as it becomes gradually absorbed or eroded by body fluids.
  • the hydrogel film composed of a compound described herein can be delivered onto cells, tissues, and/or organs, for example, by injection, spraying, squirting, brushing, painting, coating, and the like. Delivery can also be via a cannula, catheter, syringe with or without a needle, pressure applicator, pump, and the like.
  • the compound can be applied onto a tissue in the form of a film, for example, to provide a film dressing on the surface of the tissue, and/or to adhere to a tissue to another tissue or hydrogel film, among other applications.
  • the compounds described herein are administered via injection.
  • injectable hydrogels are preferred for three main reasons.
  • an injectable hydrogel could be formed into any desired shape at the site of injury. Because the initial hydrogels can be sols or moldable putties, the systems can be positioned in complex shapes and then subsequently crosslinked to conform to the required dimensions.
  • the hydrogel would adhere to the tissue during gel formation, and the resulting mechanical interlocking arising from surface microroughness would strengthen the tissue-hydrogel interface.
  • introduction of an in situ-crosslinkable hydrogel could be accomplished using needle or by laparoscopic methods, thereby minimizing the invasiveness of the surgical technique.
  • the compounds described herein can be used to treat periodontal disease, gingival tissue overlying the root of the tooth can be excised to form an envelope or pocket, and the composition delivered into the pocket and against the exposed root.
  • the compounds can also be delivered to a tooth defect by making an incision through the gingival tissue to expose the root, and then applying the material through the incision onto the root surface by placing, brushing, squirting, or other means.
  • the compounds described herein can be in the form of a hydrogel film that can be placed on top of the desired area.
  • the hydrogel film is malleable and can be manipulated to conform to the contours of the tissue defect.
  • the compounds described herein can be applied to an implantable device such as a suture, claps, prosthesis, catheter, metal screw, bone plate, pin, a bandage such as gauze, and the like, to enhance the compatibility and/or performance or function of an implantable device with a body tissue in an implant site.
  • the compounds can be used to coat the implantable device.
  • the compounds could be used to coat the rough surface of an implantable device to enhance the compatibility of the device by providing a biocompatable smooth surface which reduces the occurrence of abrasions from the contact of rough edges with the adjacent tissue.
  • the compounds can also be used to enhance the performance or function of an implantable device.
  • the hydrogel film when the compound is a hydrogel film, can be applied to a gauze bandage to enhance its compatibility or adhesion with the tissue to which it is applied.
  • the hydrogel film can also be applied around a device such as a catheter or colostomy that is inserted through an incision into the body to help secure the catheter/colosotomy in place and/or to fill the void between the device and tissue and form a tight seal to reduce bacterial infection and loss of body fluid.
  • compositions and methods can be applied to a subject in need of tissue regeneration.
  • cells can be incorporated into the compounds described herein for implantation.
  • the subject is a mammal.
  • Preferred mammals to which the compositions and methods apply are mice, rats, cows or cattle, horses, sheep, goats, cats, dogs, and primates, including apes, chimpanzees, orangatangs, and humans.
  • the compounds and compositions described herein can be applied to birds.
  • the disclosed methods and compositions When being used in areas related to tissue regeneration such as wound or burn healing, it is not necessary that the disclosed methods and compositions eliminate the need for one or more related accepted therapies. It is understood that any decrease in the length of time for recovery or increase in the quality of the recovery obtained by the recipient of the disclosed compositions or methods has obtained some benefit. It is also understood that some of the disclosed compositions and methods can be used to prevent or reduce fibrotic adhesions occurring as a result of wound closure as a result of trauma, such surgery. It is also understood that collateral affects provided by the disclosed compositions and compounds are desirable but not required, such as improved bacterial resistance or reduced pain etc.
  • compositions and methods can be easily compared to the specific examples and embodiments disclosed herein, including the non-polysaccharide based reagents discussed in the Examples. By performing such a comparison, the relative efficacy of each particular embodiment can be easily determined.
  • Particularly preferred assays for the various uses are those assays which are disclosed in the Examples herein, and it is understood that these assays, while not necessarily limiting, can be performed with any of the compositions and methods disclosed herein.
  • reaction conditions e.g., component concentrations, desired solvents, solvent mixtures, temperatures, pressures and other reaction ranges and conditions that can be used to optimize the product purity and yield obtained from the described process. Only reasonable and routine experimentation will be required to optimize such process conditions.
  • Fermentation-derived hyaluronan (HA, sodium salt, M w 1.5 MDa) was obtained from Clear Solutions Biotech, Inc. (Stony Brook, N.Y.).
  • 1-Ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDCI), 3,3′-dithiobis(propanoic acid), 4,4-dithiobis(butanoic acid), and poly(ethylene glycol) acrylate (M w 375), and hydrazine hydrate were from Aldrich Chemical Co. (Milwaukee, Wis.).
  • Dulbecco's phosphate buffered saline (PBS), bovine testicular hyaluronidase (HAse, 330 U/mg) and blue dextran (M w 200,000) was from Sigma Chemical Co. (St. Louis, Mo.).
  • Dithiothreitol (DTT) was from Diagnostic Chemicals Limited (Oxford, Conn.).
  • 5,5′-Dithio-bis(2-nitrobenzoic acid) (DTNB) was from Acros (Houston, Tex.).
  • Poly(ethylene glycol)-diacrylate (PEGDA), poly(ethylene glycol)-dimethacrylate (PEGDM), poly(ethylene glycol)-diacrylamide (PEGDAA) and poly(ethylene glycol)-dimethacrylamide (PEGDMA) were synthesized from poly(ethylene glycol) or poly(ethylene glycol) diamine (Mw 3,400, Shearwater Polymers) as described in Elbert D L and Hubbell J A. “Conjugate addition reactions combined with free-radical crosslinking for the design of materials for tissue engineering,” Biomacromolecules 2001;2:430441, which is incorporated by reference in its entirety. Gelatin from bovine skin (Types B and A, gel strength approx.
  • Dulbecco's phosphate buffered saline PBS
  • cystein bovine testicular hyaluronidase
  • HAse bovine testicular hyaluronidase
  • bacterial collagenase from Clostriditim histolyticum EC 3.4.24.3, 388 U/mg
  • 3-(4,5-dimethylthiazol-2-yl)-2,5, diphenyl tetrazolium bromide (MTT) were obtained from Sigma Chemical Co. (St. Louis, Mo.).
  • 5,5′-Dithio-bis(2-nitrobenzoic acid) DINB was purchased from Acros (Houston, Tex.).
  • GPC Gel permeation chromatography analysis was performed using the following system: Waters 515 HPLC pump, Waters 410 differential refractometer, WatersTM 486 tunable absorbance detector, Ultrahydrogel 250 or 1000 columns (7.8 mm i.d. ⁇ 130 cm) (Milford, Mass.)
  • the system was calibrated with standard HA samples provided by Dr. U. Wik (Phanmacia, Uppsala, Sweden). Fluorescence images of viable cells were recorded using a Nikon Eclipse TE300 with epi-fluorescence capabilities.
  • Cell proliferation was determined using a biochemical assay (Cell-Titer 96 Proliferation Kit, Promega, Madison, Wis.), MTT assay, or MTS assay at 550 nm, which was recorded on an OPTI Mx microplate reader (Molecular Devices, Sunnyvale, Calif.).
  • the diesters were hydrazinolyzed with hydrazine hydrate to form the corresponding dihydrazides.
  • DTP Very crudesse, K. P.; Marecak, D. M.; Marecek, J. F.; Prestwich, G. D. Bioconjugate Chem.
  • LMW low molecular weight
  • High molecular weight HA (1.5 MDa) (20 g) was dissolved in 2.0 L distilled water, and the solution pH was adjusted to ca. 0.5 by the addition of concentrated HCl. The degradation was carried out at 37° C., 130 rpm stirring for 24 h. After that, the pH of the solution was adjusted to 7.0 by the addition of 1.0 N NaOH before transfer to dialysis tubing (M w cut-off 3,500) and dialyzed against water for four days. The solution was then centrifuged, and the supernatant was lyophilized to give 15 g LMW HA (M w 246 kDa, Mn 120 kDa, polydispersity index 1.97).
  • Thiolated HA derivatives with different loadings were prepared following a general protocol ( FIG. 1 ).
  • LMW HA (20 g, 50 mmol) was dissolved in 2.0 L of water, 23.8 g of DTP or 26.6 g of DTB (100 mmol) was added while stirring.
  • the pH of the reaction mixture was adjusted to 4.75 by the addition of 1.0 N HCl.
  • 19.2 g of EDCI (100 mmol) in solid form was added.
  • the pH of the reaction mixture was maintained at 4.75 with aliquots of 1.0 N HCl.
  • the reaction was stopped by addition of 1.0 N NaOH, raising the pH of the reaction mixture to 7.0.
  • 100 g DTF ca.
  • the purity of thiolated HA was measured by GPC and 1 H NMR, and the degree of substitution (SD) was determined by 1 H NMR.
  • the free thiols on the side chain of HA-DTPH and HA-DTBH were determined by a modified Ellman method (Butterworth, P. H. W.; Baum, H.; Porter, J. W. Arch. Biochem. Biophlys. 1967, 118, 716-723). SD (%) and thiol content (%) were defined as the number of DTP (or DTB) residues and thiols per 100 disaccharide units, respectively.
  • HA-DTBH M w 165 kDa, M, 63 kDa, polydispersity index 2.62, SD 72%) and HA-DTPH (M w 136 kDa, M n 61 kDa, polydispersity index 2.23, SD 58%).
  • the structures of HA-DTPH and HA-DTBH were confirmed by 1 H NMR spectroscopy in D 2 O.
  • pK a of thiols in HA-DTPH and HA-DTBH was determined spectrophotometlically based on the UV absorption of thiolates as proposed by Benesch and Benesch (Benesch, R.; Benesch, R. E. Proc. Nat. Acad. Sci. USA 1958, 44, 848-853).
  • Solutions of HA-DTPH and HA-DTBH (ca. 5 mg) were dissolved in 100 mL 0.001 N HCl containing 0.1 N NaCl (stable ionic strength). Freshly-prepared solutions were immediately measured in the UV region with a scan from 190 to 300 nm.
  • the pK a values were determined spectrophotometrically based on the UV absorption of thiolates (Benesch). With increasing pH, the absorption of solutions increased abruptly—especially at the pH near the pK a of thiols ( FIG. 2 a ). According to the procedure reported by Lutolf and co-workers (Lutolf, M. P.; Tirelli, N.; Ceiritelli, S.; Cavalli, L.; Hubbell, J. A. Bioconjugate Chem. 2001, 12, 1051-1056) the intercept with the abscissa in a graphical representation of log[(A max ⁇ A i )/A i ] vs.
  • the solution (flow)-gel (no flow) transition was determined by a flow test utilizing a test tube inverting method reported by Jeong et al. (Jeong, B.; Bae, Y. H.; Kim, S. W. Macromol. 1999, 32, 7064-7069).
  • HA-DTBH and HA-DTPH were dissolved in PBS to give 3.0% (w/v) solutions under N 2 protection.
  • the solution pH was adjusted to 5.0, 6.0, 7.0, 8.0 and 9.0 by 1.0 N NaOH.
  • Freshly-prepared solutions (1.0 mL) with different pH were immediately injected into glass tubes (0.8 cm in diameter, 7.5 cm in length). After exposure to air at room temperature for 15 or 30 min, the test tube was inverted. If no fluidity was visually observed in 1 min, we concluded that that a gel had formed.
  • Preparation of disulfide-crosslinked HA films HA-DTBH and HA-DTPH were dissolved in PBS to give 3.0% (w/v) solutions and the solution pH was adjusted to 7.4 by the addition of 1.0 N NaOH.
  • 0.15% (w/v) blue dextran (M w 200,000) was included as a model drug.
  • 25 mL of the solution was poured into a 9-cm petri dish and allowed to dry at room temperature. After ca. three days, a film ready. As required, the film was further oxidized by immersion in 0.3% H 2 O 2 for 1 h. The film was then rinsed with distilled water, cut into 6-mm diameter discs, and dried at room temperature for one day and then at 1 mm Hg for one week, to give films with 0.1 mm thickness. Swelling determination.
  • Discs of HA-DTBH and HA-DTPH film (6 mm in diameter) were weighed (W 0 ), immersed in glass vials containing 10 mL PBS (pH 7.4), and placed in a shaking incubator at 37° C., at 300 rpm. At predetermined time intervals, the wet films were weighed (W 1 ) immediately after the removal of the surface water by blotting between two pieces of filter paper. Swelling ratio (R) was defined as W t /W 0 .
  • the swelling of HA-DTPH and HA-DTBH films in PBS was in accordance with the disulfide content in the films as shown in FIG. 3 .
  • the air oxidized films swelled significantly because of low degree of crosslinking, with a swelling ratio at 5.5 h of 16.2 for HA-DTBH film and 9.5 for HA-DTPH.
  • These ratios are similar to PEG-dialdehyde crosslinked HA adipic dihydrazide hydrogels used for drug release (Luo, Y., Kirker, K. R.; Prestwich, G. D. J. Controlled Rel. 2000, 69, 169-184) and wound repair (Kirker, K.
  • Disuifide content determination Discs of HA-DTBH and HA-DTPH film were degraded by acid hydrolysis (0.1 N HCl). The total sulfur content (disulfide plus thiol) was measured using 2-nitro-5-thiosulphobenzoate (NTSB) according to Thannhauser et al. (Thannhauser, T. W.; Konishi, Y.; Scheraga, H. A. Meth. Enzymol. 1987, 143, 115-119). In addition, the free thiol content was measured by the Ellman method (Ellman, G. L. Arch. Biochem. Biophys. 1958, 74, 443-450). Disulfide content was calculated from the difference between total sulfur content and free thiol content.
  • FIG. 4 shows the oxidation with dilute H 2 O 2 increased the number of disulfide linkages.
  • the disulfide content in HA-DTPH film increased from 0.175 to 0.212 mmol/g after the oxidation of H 2 O 2 .
  • HA-DTBH film fewer disulfide linkages were formed due to air oxidation because the thiol was less reactive (the value was 0.125 mmol/g); however, this could be increased significantly to 0.25 mmol/g by oxidation with H 2 O 2 .
  • no additional thiol groups are detected within both the HA-DTPH and the HA-DTBH films, and only ca.
  • the hydrogel films were incubated in PBS that contained different concentrations of DTT at pH 7.4 (data not shown). Even with DTT concentration as low as 10 mM, films generated from both air and H 2 O 2 oxidation swelled significantly and dissolved gradually due to reduction of disulfide by DTF. As the gels dissolved, a model drug (blue dextran M w 200,000) that had been non-covalendy entrapped in the hydrogel films was released.
  • the enzyme HAse also accelerated the release of model drug (blue dextran) from films.
  • model drug blue dextran
  • the release percentage of blue dextran from air-oxidized HA-DTPH film in PBS at 37° C. at 300 rpm was less than 7%, while under the same conditions in PBS with 100 U/mL Hase, 30% of the blue dextran was released with concomitant partial degradation of the film.
  • approximately 36% of the film had been lost due to enzymatic digestion, as determined gravimetrically.
  • Murine fibroblasts (L-929, ATCC, Manassas, Va.) were cultured in a triple flask (Fisher, Springfield, N.J.) until 90% confluence, and then trypsinized and mixed with HA-DTPH solution to a final concentration of 2 ⁇ 10 6 /mL. Next, 0.5 mL of the HA-DTPH solution was added into each well of a 12-well plate. The cell-loaded plates were incubated (37° C., 5% CO 2 , 4 h) until a solid hydrogel formed, and then 2 mL of DMEM/F-12 medium with 10% of newborn calf serum (GIBCO, Rockville, Md.) was added into each well. The plates were transferred to an incubator (37° C., 5% CO 2 , three days) without a change of medium.
  • F-DA fluorescein diactate
  • PI propidium iodide
  • the number of viable cells in each hydrogel was determined using a biochemical assay (Cell-Titer 96 Proliferation Kit, Promega, Madison, Wis.) as previously described (Lutolf, M. P.; Tirelli, N.; Cerritelli, S.; Cavalli, L.; Hubbell, J. A. Bioconjugate Chem. 2001, 12, 1051-1056).
  • a tetrazolium salt is reduced by the mitochondria of living cells into a colored formazan product whose presence can be detected spectrophotometrically.
  • the hydrogels in 12-well plates were rinsed twice with PBS buffer, then 900 ⁇ l of DMEM/F-12 medium with 5% of newborn calf serum and 180 ⁇ L of Cell Titer 96 Proliferation Kit solution were added into each well. After 2 h of incubation with gentle shaking (37° C., 5% CO 2 ), a 125- ⁇ L aliquot of each of the solutions was transferred individually into a 96-well plate and read at 550 nm with a OPTI Max microplate reader (Molecular Devices). The absorbance reading was converted into a cell number based on standard curves generated from the assay of known numbers of cells. Data sets were compared using two-tailed, unpaired t-tests. P-values less than 0.05 were considered to be significant.
  • HA-DTPH solution under physiological conditions exhibits potential utility for many biomedical applications, e.g., wound healing, defect filling, prevention of post-surgical adhesions, and cell encapsulation for tissue repair.
  • Murine fibroblasts were entrapped within a crosslinking HA-DTPH hydrogel, and the encapsulated cells were examined after 24 h and 96 h of culture. Viable cells, indicated by green fluorescence upon F-DA staining, were evident after 96 h of culture. Fewer than 5% dead cells were observed as red fluorescence from PI staining (data not shown).
  • the fibroblasts in the hydrogel maintained a round shape. In addition, clumps of cells, as well as individual cells, were observed in hydrogel.
  • HA-DTPH thiolated HA
  • gelatin-DTPH thiolated gelatin
  • SD degree of substitution
  • free thiols on the side chain of HA-DTPH and gelatin-DTPH were determined by 1 H NMR and by a modified Ellman method (Butterworth P R W, Baum H, and Porter J W. A modification of the Ellman procedure for the estimation of protein sulfhydryl groups. Arch Biochem Biophys 1967;1 18:716-723).
  • pKa determination The pKa values for the thiols in HA-DTPH and gelatin-DTPH were determined spectrophotometrically based on the UV absorption of thiolates (Benesch R and Benesch RE. Thiolation of protein. Proc Nat Acad Sci USA 1958;44:848-853; Lutolf M P, Tirelli N, Cerritelli S, Cavalli L, and Hubbell J A. Systematic modulation of Michael-type reactivity of thiols through the use of charged amino acids. Bioconjugate Chem 2001; 12:1051-1056). Solutions of HA-DTPH and gelatin-DTPH (ca. 5 mg each) were dissolved in 100 ml of 0.001 N HCl containing 0.1 N NaCl (stable ionic strength). UV scans from 190-300 nm were recorded for freshly-prepared solutions,
  • Turbidimetric titration The electrostatic interactions of HA-DTPH and gelatin-DTPH were investigated by turbidimetric titration (Shu X Z, Zhu K J, and Song W. Novel pH-sensitive citrate crosslinked chitosan film for drug crontrolled release. Int J Pharm 2001;212: 19-28; Park J M, Muhoberac B B, Dubin P L, and Xia J. Effects of protein charge heterogeneity in protein-polyelectrolyte complexatiom Macromolecules 1992;25:290-295).
  • a solution of 1.0 mg/ml of either HA-DTPH or LMW HA and 1.0 mg/ml of either gelatin-DTPH or unmodified gelatin was prepared at pH 1.5, and aliquots of a stock NaCl solution were added to adjust the ionic strength.
  • Titrant 0.01-0.2 N NaOH
  • Titrant was delivered using a microburette into the solution with gentle stirring at 30 plus/minus 0.5° C., and the pH was monitored by a digital pH meter with a precision of plus/minus 0.01.
  • Changes in turbidity were monitored at 420 nm with an WV-vis spectrophotometer and reported as (100-T)%, which is linearly proportional to the true turbidity measurements when T>0.9.
  • the time interval between turbidity measurements was ca. 4 min.
  • HA-DTPH and gelatin-DTPH were dissolved in 0.02 M PBS (pH 6.5) to give 3.0% (w/v) solutions.
  • the pH of each solution was adjusted to 7.4 by the addition of 1.0 N NaOH, and then the solutions were mixed according to volume ratio of HA-DTPH:gelatin-DTPH of 100:0, 80:20, 60:40, 40:60, 20:80, and 0:100.
  • the transmittance of the solutions was monitored at 550 nm.
  • This phenomenon was evaluated for the thiolated derivatives of HA and gelatin, which still have numerous unmodified carboxylates (1.58 mmol/g for HA-DTPH, 0.65 mmol/g for gelatin-DTPH) and amine groups (0.35 mmol/g for gelatin-DTPH).
  • Turbidometric titration indicated that similar electrostatic interactions occurred in the mixed solutions of HA-DTPH and getatin-DTPH, but over a broader pH region due to the shift to higher pI for gelatin-DTPH resulting from conversion of >40% of the carboxylates to thiols.
  • HA-DTPH and gelatin-DTPH were dissolved in 0.02 M PBS, and the pH was adjusted to 7.4 to give clear solutions.
  • solutions containing various ratios of HA-DTPH and gelatin-DTPH became translucent, and phase separation occurred immediately due to their electrostatic interactions ( FIG. 8 ).
  • This effect precluded fabrication of homogeneous, transparent hydrogel films from blends of HA-DTPH and gelatin-DTPH.
  • the ionic strength of the solutions was increased to mask the electrostatic binding. Indeed, turbidimetric titration revealed that this binding was completely prevented by 3.0% (w/v) NaCl (data not shown).
  • HA-gelatin hydrogel films crosslinked by disulfide bond Preparation of HA-gelatin hydrogel films crosslinked by disulfide bond.
  • HA-DTPH and gelatin-DTPH (3.0 g each ) were separately dissolved in 100 ml of 20 mM PBS buffer (pH 6.5) containing 1.0% (w/v) NaCl, and then the pH of each solution was adjusted to 7.4 by the addition of 1.0 N NaOH. Then, HA-DTPH and gelatin-DTPH solutions were combined in volume ratios of 100:0, 80:20, 60:40, 40:60, 20:80, and 0:100, and thoroughly mixed by gentle vortexing. The mixed solutions (30 ml) were poured into 9-cm petri-dishes and allowed to crosslink in air and to dry at room temperature.
  • air-crosslinked films were obtained and cut into 6, 8, or 1.6-mm diameter discs.
  • the film discs were then further oxidized by immersion in 0.1% H 2 O 2 for 1 h.
  • the film discs were then rinsed with distilled water and dried at ambient pressure and temperature for one day, and then at 1 mm Hg for one week.
  • the blended hydrogel films were obtained by pouring 30 ml of mixed HA-DTPH-gelatin-DTPH solutions containing 1.0% NaCl (w/v) into 9-cm petri-dishes. Air oxidation and drying at room temperature produced disulfide-crossslinked films. Crosslinking density in these films was increased by further oxidation with 0.1% (w/v) H 2 O 2 ; films were then rinsed and dried in vacuo.
  • the disulfide content of the HA-gelatin hydrogel films was determined by NTSB after exhaustive acidic hydrolysis ( FIG. 9 ). In agreement with previous results (Nicolas F L and Gagnieu C H. Denatured thiolated collagen II. Crosslinking by oxidation. Biomaterials 1997;18:815-821), only 25-50% of the thiols were oxidized to disulfides. Since no free thiols were detectedby DTNB (Ellman G L. A calorimetric method for determining low concentrations of mercaptans.
  • Electrostatic attraction between HA-DTPH and gelatin-DTPH also facilitated disulfide formation; blended films had more disulfide bonds than the HA-DTPH film (p ⁇ 0.01, except for HA-DTPH:gelatin-DTPH of 80:20).
  • the equilibrium swelling ratio of the hydrogel films in PBS is shown in FIG. 10 .
  • the swelling ratio decreased from 3.27 to 2.33. This ratio is determined only by the crosslinking density, but is also related to the bulk properties of the films.
  • Disulfide content determination Film discs with diameter of 6 mm were degraded by acid hydrolysis (0.1 N HCl, 37° C., 150 rpm for 10 days).
  • the total sulfur content (S ⁇ S+SH) was measured using 2-nitro-5thiosulfobenzoate (NTSB) (Thannhauser T W, Konishi Y, and Scheraga H A. Analysis for disulfide bonds in peptides and proteins. Methods In Enzymology 1987;143:115-119), and the free thiol content was measured by the Ellman method (Ellman G L. A colorimetric method for determining low concentrations of mercaptans. Arch Biochem Biophys 1958;74:443-450). Disulfide content, equivalent to crosslinking density, was calculated as the difference between total sulfur content and free thiol content.
  • HA-gelatin hydrogel film In vitro degradation of HA-gelatin hydrogel film.
  • the degradation of disulfide-crosslinked HA-gelatin films was performed using collagenase and HAse. Film discs with diameter of 8 mm were incubated in a glass bottle containing 3 ml medium with 300 U/ml collagenase or HAse, and placed in an incubator at 37° C., 150 rpm. The medium was changed every two days. At predetermined intervals, the films were washed five times with distilled water and dried under vacuum.
  • the buffer used for collagenase was 100 mM Tris-HCl buffer (pH 7.4) containing 5 mM CaCl 2 and 0.05 mg/ml sodium azide (Choi Y S, Hong S R, Lee Y M, Song K W, Park M H, and Nam Y S. Studies on gelatin-containing artifical skin:II. preparation and characterization of crosslinked gelatin-hyaluronate sponge. J Biomed Mater Res (Appl Biomater) 1999;48:631-639). HAse digestions were performed in 30 mM citric acid, 150 mM Na 2 HPO 4 , 150 mM NaCl (pH 6.3) (Bulpitt P and Aeschlimann D.
  • weight loss after 7 days for an HA-gelatin (60% gelatin) film was 18% with 300 U/ml collagenase and 5% with HAse; with both enzymes combined, weight loss was as high as 50% (FIG 11 b ).
  • Balb/c 3T3 fibroblasts were seeded on the surface of the HA-gelatin films of different compositions and cultured in vitro for 24 h, and then the live cells were stained with F-DA to give green fluorescence.
  • a morphological study revealed that only a very small number of cells with spherical shape were attached to the surface of HA-DTPH hydrogel film that lacked a protein component ( FIG. 12 a ).
  • Addition of gelatin-DTPH significantly improved the cell attachment ( FIG. 12 b - 12 d ), even at 20% (w/v).
  • the majority of cells adopted a spindle-shaped morphology and spread uniformly on the hydrogel surface ( FIG. 12 b - 12 d ).
  • FIG. 13 shows that while cells on the HA-DTPH hydrogel surface failed to proliferate, increasing percentages of gelatin in the films result in accelerated cell proliferation.
  • PEG-diacrylate (PEGDA), PEG-dimethacrylate (PEGDM), PEG-diacrylamide (PEGDAA) and PEG-imethacrylainide (PEGDMA) were synthesized from PEG (Mw 3400 KDa, Aldrich) or PEG-diamine (M w 3400, Shearwater Polymers) as described with minor modifications. Briefly: PEG (or PEG-diamine) molecular weight 3400 (10 g, 5.88 mmol of functional group) was azeotropically distilled with 400 ml of toluene under argon, removing ca. 100 ml of toluene.
  • Conjugate addition The relative reactivity of conjugate addition of ⁇ -unsaturated esters and amides of poly(ethylene glycol) to thiols was first evaluated using cysteine as a model. The conjugate addition of cysteine to each of the four electrophilic species is shown in FIG. 15 . Cysteine (2.5 mg) and PEG-diacrylate (PEGDA), PEG-dimethacrylate (PEDMA), PEG-diacrylamide (PEGDAA) or PEG-dimethacrylamide (PEGDMA) were dissolved in 5 ml of 0.1 N PBS, pH 7.4 (ratio of double bond/SH 2/1).
  • Crosslinking was evaluated in detail for both thiolated HA deriviatives with PEGDA as the homobifunctional crosslinker. After 1 h, a mixture of each thiolated HA (HA-DTPH and HA-DTBH) and PEGDA had completely gelled. The resulting hydrogels were then incubated in medium (pH 4.5 or 1.0) to quench the crosslinking addition, and the crosslinking efficiency was determined by measuring the remaining free PEG electophile and the remaining free thiols and performing the calculations indicated below.
  • the quantity of free PEGDA in the hydrogel was determined by GPC with monitoring of the eluent at 233 nm. Briefly, the hydrogel (0.1 ml) was ground into small particles and suspended in 2 ml of 0.1 M acetate buffer (pH 4.5). After stirring for 4 h at room temperature, the amount of residual PEG derivatives was determined using a standard calibration curve. No free thiolated HA was detected by GPC at 210 nm.
  • the free thiols in the hydrogel were determined using either the DTNB or NTSB assay. Briefly, a 0.05-ml fragment of hydrogel was suspended in 0.5 ml of 0.1 N HCl solution. After 48 h at room temperature with agitation at 150 rpm, the hydrogel had dissociated. Next, 2.0 ml of either NTSB or DTNB reagent was added to each gel, and the number of free thiols in the hydrogel was determined spectrophotometrically at 412 nin. Thiolated HA solutions alone were used as reference materials, and the disulfide formation during hydrogel preparation (1 h) under nitrogen protection was negligible.
  • the extent of effective crosslinking (i.e., double-end anchorage), unreacted pendent double bond groups during the coupling reaction (i.e., single-end anchorage) was calculated from the total PEGDA used (A), the unreacted PEGDA (B), the total thiols (C) and the free thiols in hydrogel (D).
  • Single-end anchorage equals to the theoretical consumed thiols (2(A-B)) minus the actually consumed thiols (C-D). Subtraction of single-end anchorage from the experimentally measured consumed thiols (C-D) reveals the extent of double-end anchorage.
  • Table 3 shows the crosslinking efficiencies
  • Table 4 shows the crosslinking densities, equilibrium swelling ratios, and gelation times for the gels obtained by the reaction of HA-DTPH and HA-DTBH.
  • TABLE 3 Crosslinking efficiency of PEGDA to HA-DTPH and HA-DTBH Crosslinking efficiency (%) Molar ratio of PEGDA of thiols to Double-end Single-end double bonds anchorage anchorage Unreacted HA-DTPH:PEGDA 1:1 76.2 9.7 14.1 2:1 93.7 6.3 0 3:1 100.0 0 0 HA-DTBH:PEGDA 1:1 48.3 19.3 32.4 2:1 60.0 12.7 27.3 3:1 73.8 8.3 17.9
  • Hydrogels were placed in PBS buffer at 37° C. for 48 h and the medium was changed frequently.
  • the swelling ratio (Q) was defined as a ratio of the weight of swollen gel to the weight of dry gel.
  • the weight of the dry gels was determined by washing the hydrogel with distilled water 5 times and then drying the gel under vacuum (1 mm Hg) at room temperature for 3 days.
  • Hydrogel discs (0.5 ml) were prepared from HA-DTPH and PEGDA as described above by crosslinking in the bottom of a 6-mm diameter vial.
  • Hyaluronidase (HAse) solutions (0, 50, 150 and 250 U/ml) were prepared in 30 mM citric acid, 150 mM Na 2 HPO 4 , 150 mM NaCl (pH 6;3); 5 ml of enzyme solution was added to each vial containing the hydrogel, and vials were incubated at 37° C. with orbital agitation at 150 rpm.
  • FIG. 17 shows the digestion of a HA-DTPH/-PEGDA hydrogel by HAse, showing that at lower concentrations of enzyme the gel remains largely intact for several days in vitro.
  • a 4.5% PEGDA solution was prepared by dissolving PEGDA in PBS buffer and sterilized by filtration with 0.45 ⁇ m syringe filter.
  • T31 human pharyngeal fibroblasts that had been cultured in triple flasks (175 cm 2 ) and trypsinized with 0.25% sterile trypsin in 0.05% EDTA, were suspended in freshly prepared HA-DTPH solution at concentration of 10 6 cells/ml.
  • To four volumes of the cell suspension was added one volume of the PEGDA stock solution, and the mixture was vortexed gently.
  • 300 ⁇ l of the mixture of the fibroblast-seeded HA-DTPH-PEGDA mixture was poured into each well of 12-well plate and gelation was allowed to occur (1 h).
  • complete DMEM/F-12 medium was added into each well and the plate was incubated for at 37° C. in a 5% CO 2 incubator. The medium was changed every three days without damaging the gel.
  • the seeded hydrogels were used to determine in vitro cell viability and proliferation and for transplantation in vivo into nude mice for fibrous tissue generation.
  • Cell proliferation was determined at day 0, 3, 6, 14, and 28.
  • four fibroblast-seeded HA-DTPH-PEGDA hydrogels were transferred into each well of a 12-well plate, and rinsed twice with PBS buffer.
  • 900 ⁇ L of DMEM/F-12 medium with 5% newborn calf serum and 180 ⁇ l of CellTiter 96 Proliferation Kit (Promega, Madison, Wis.) were added into each well of a 12-well plate.
  • 125 ⁇ l of the solution was transferred into each of six wells of a 96-well plate.
  • HA-DTPH-PEGDA hydrogels were minced with 22 gauze needles, digested in 5% cyanogen bromide (CNBr, Sigma) in 70% formic acid (Sigma) for 8 h at 35° C., diluted with same volume of distilled water, and lyophilized overnight.
  • the lyophilized samples were dissolved in PBS buffer and read with Cary 3E spectrophotometer (Varian, Inc., Walnut Creek, Calif.) at 280 nm to determine the protein concentration.
  • Sample buffer containing ⁇ -mercaptoethanol was added (50 ⁇ g of sample per 20 ⁇ l of sample buffer) and aliquots were separated on a 10% PAGE/SDS at 80 v for 8 h plus 300 v. for 3 h.
  • the gel was silver stained and collagen peptide fragments were analyzed by comparison with standard collagen type I fragments. The collagen typing of these cultured fibroblasts showed that even after 28 days of in vitro culture, the cells retained the same phenotype as characterized by collagen type I production.
  • the cell number decreased following two weeks culture in vitro.
  • T31 fibroblast increased tenfold after 28 days itt vitro culture, which indicated the injectable hydrogel described here was excellent candidate for tissue regeneration.
  • Thiol-modified HA was prepared using the hydrazide technology described above. Briefly, low molecular weight HA (200 k Da) was reacted with 3,3′-dithiobis propanoic hydrazide (DTPH) at pH 4.75 by the carbodiimide-catalysed reaction. The gel like product was reduced by solid form DTT, after dialysis, the thiolated HA derivatives were prepared with different loadings. HA-DTPH was dissolved in PBS buffer to the concentration of 1.25% (w/v). Modified MMC was dissolved in minimal ethanol and added into the HA-DTPH solution. The theoretical MMC loading to the disaccharides was 0.5%, 1% and 2% respectively. The procedure was conducted under N 2 protection and the final pH of the mixture was adjusted to 8.0. The reaction was processed for three hours with stirring. FIG. 22 depicts the reaction sequence.
  • HA-MMC solution was adjusted to pH 7.4 after the coupling reaction.
  • PEG diacrylate was dissolved in PBS buffer to the concentration of 4.5% (w/v). The two solutions were mixed together and vortexed for one minute.
  • the reaction mixture was removed by Eppendorf® Combitips and added to 2 cm ⁇ 2 cm dishes, 2 mL/dishes. The hydrogels were formed in about half hour and were evaporated in air to dryness for several days to form the films.
  • FIG. 23 depicts the reaction sequence.
  • MMC release experiment Dried hydrogel films were cut into 2 cm squares. The square gel film and the cut off margin were weighed separately, and the MMC contained in each square film was calculated. Each film was dipped into 5 mL 100 mM PBS buffer and shaken gently at 37° C. At each time point, 0.5 mL solution was removed and 0.5 mL fresh PBS buffer was added. The solution containing released MMC was detected at a wavelength of 358 nm. The accumulated concentration of released MMC was plotted as a function of the time.
  • FIGS. 25 a and b show the results of in vitro MMC release results.
  • FIG. 25 a shows the absolute released concentration.
  • the released MMC is proportional to the MMC contained in the hydrogel.
  • the relative release pattern is shown in FIG. 25 b after repoltting the data.
  • HA films with 1% and 2% MMC loadings have similar release profiles. At the first half hour, about 13% MMC was released from the hydrogel, which may come from two sources: one was the uncoupled MMC, the other was hydrolyzed MMC. Then a slow release pattern was observed with a half-life around 48 hours. The release of MMC continued for 5 days until reaching a platform. There were still a considerable amount of MMC embeded in the film after 8 days. These results indicate that the newly synthesized HA-MMC-PEG hydrogel has similar hydrolysis kinetics as the described MMC-TA conjugate.

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US7928069B2 (en) 2011-04-19
US20090105193A1 (en) 2009-04-23
EP1539799A2 (fr) 2005-06-15
AU2003299509A8 (en) 2004-05-13
CA2489712C (fr) 2016-07-12
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AU2003299509A1 (en) 2004-05-13
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US8859523B2 (en) 2014-10-14

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