EP1305355A1 - Biogelfilme sowie verfahren zu deren herstellung und nutzung - Google Patents

Biogelfilme sowie verfahren zu deren herstellung und nutzung

Info

Publication number
EP1305355A1
EP1305355A1 EP01957173A EP01957173A EP1305355A1 EP 1305355 A1 EP1305355 A1 EP 1305355A1 EP 01957173 A EP01957173 A EP 01957173A EP 01957173 A EP01957173 A EP 01957173A EP 1305355 A1 EP1305355 A1 EP 1305355A1
Authority
EP
European Patent Office
Prior art keywords
film
hydrogel
wound
films
polysaccharide
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP01957173A
Other languages
English (en)
French (fr)
Inventor
Yi Luo
Glenn D. Prestwich
Kelly R. Kirker
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Utah Research Foundation Inc
Original Assignee
University of Utah Research Foundation Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University of Utah Research Foundation Inc filed Critical University of Utah Research Foundation Inc
Publication of EP1305355A1 publication Critical patent/EP1305355A1/de
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
    • C08G65/32—Polymers modified by chemical after-treatment
    • C08G65/329—Polymers modified by chemical after-treatment with organic compounds
    • C08G65/336—Polymers modified by chemical after-treatment with organic compounds containing silicon
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0014—Skin, i.e. galenical aspects of topical compositions
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B11/00—Preparation of cellulose ethers
    • C08B11/20—Post-etherification treatments of chemical or physical type, e.g. mixed etherification in two steps, including purification
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B15/00—Preparation of other cellulose derivatives or modified cellulose, e.g. complexes
    • C08B15/005—Crosslinking of cellulose derivatives
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
    • C08B37/0045—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid alpha-D-Galacturonans, e.g. methyl ester of (alpha-1,4)-linked D-galacturonic acid units, i.e. pectin, or hydrolysis product of methyl ester of alpha-1,4-linked D-galacturonic acid units, i.e. pectinic acid; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
    • C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
    • C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
    • C08B37/0072—Hyaluronic acid, i.e. HA or hyaluronan; Derivatives thereof, e.g. crosslinked hyaluronic acid (hylan) or hyaluronates
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/002—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from unsaturated compounds
    • C08G65/005—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from unsaturated compounds containing halogens
    • C08G65/007—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from unsaturated compounds containing halogens containing fluorine

Definitions

  • This invention relates generally to hydrogel films and methods of making and using them.
  • Glycosaminoglycans are a class of biocompatible polymers.
  • hyaluronic acid which is a member of the GAG family, is a naturally-occurring biopolymer composed of repeating disaccharide units of N- acetyl-D-glucosamine (GlcNAc) D-glucuronic acid (GlcUA) found in the extracellular matrix of all higher animals.
  • Hyaluronic acid (HA) is a GAG.
  • HA and GAGs please see for example Laurent et al., 18 Acta Chem Scand 21 (1964), Yui et al, 22 /. Controlled Rel.
  • Hydrogel films have received attention as drug delivery vehicles due to their compatibility with most tissues and ability to manipulate their permeability in response to different solutes. All of the existing technologies present difficulties, however, because the alkaline conditions or high temperatures necessary to create hydrogel films with high mechanical strength are cumbersome and harsh. Additionally, many reactions employ an excess of small molecular crosslinking reagents which then require considerable purification in order to obtain material suitable for physiological use. The present invention addresses these problems.
  • HA forms highly viscous aqueous solutions, and it takes on an expanded random coil structure due to strong hydrogen bonding.
  • the coiled structure allows it to trap approximately 1000 times its weight in water.
  • HA has unique physiochemical properties as well as distinctive biological functions. These functions, relationships, and interactions are discussed for example in Laurent, T. C, Laurent, U. B. G., and Fraser, J. R. E. (1995) Functions of hyaluronan. Ann Rheum Dis 54, 429-432; Fraser, J. R. E., Laurent, T. C, and Laurent, U. B. G. (1997) Hyaluronan: Its nature, distribution, functions and turnover. J Intern Med 242, 27- 33; Dowthwaite, G.
  • CS chondroitin sulfate
  • GalNAc N-acetyl- galactosamine
  • CS is usually found bound to a core protein forming a proteoglycan, e.g. aggrecan or versican.
  • Aggregan is the primary proteoglycan in cartilage, and its primary function is to swell and hydrate the collagen fibril framework.
  • Versican is believed to play a role in intracellular signaling, cell recognition, and connecting ECM components to cell surface glycoproteins.
  • CS proteoglycans like neurocan and phosphacan play important roles in axon growth and pathfinding.
  • Wound healing is a complex and orderly sequence of events that involves a variety of cell-types and subcellular signals. Prompt wound repair is vital for patient recovery. It has been thought that GAG and HA may have a role in wound repair (Kearney, J. Wound healing. In Principles and Practice of Burns Management; J. A. D. Settle, Ed.; Churchill Livingstone: New York, 1996; pp 187-195 and Margolis, R. U., and Margolis, R. K. (1997) Chondroitin sulfate proteoglycans as mediators of axon growth and pathfinding. Cell Tissue Res 290, 343-348; Clark, R. A. F. Wound repair: Overview and general considerations. In The Molecular and Cellular
  • Severe bum injuries can cause extensive full-thickness skin loss and are accompanied by immunosuppression, which contributes to more than 10,000 fatalities and 100,000 hospitahzations each year.
  • the survival rates for patients with burn injuries have improved dramatically in tae past two decades.
  • the cited reasons for this improvement are better understanding of the resuscitation process, improved antibiotics (both systematic and topical), improved nutritional support for the hypermetabolic-catabolic effects of bum injuries, and perhaps most importantly, the recognition that early excision of devitalized tissue promotes closure of the burn wound and is the most effective method of preventing sepsis and multiple organ system failure.
  • the mortality rate for patients suffering from bums covering greater than 70% body surface area remains high.
  • hitergel FeHA, formerly Lubricoat
  • Hylagel which is an engineered hylan gel or membrane device
  • Seprafilm which is prepared by blending two anionic polymers, HA and carboxymefhylcellulose (CMC)
  • CMC carboxymefhylcellulose
  • GAG molecules have been chemically modified (Luo, Y., Kirker, K. R., and Prestwich, G. D. (2000) Cross-linked hyaluronic acid hydrogel films: new biomaterials for drag delivery. Journal of Controlled Release 69, 169-184; Pouyani, T., Harbison, G. S., and Prestwich, G. D. (1994) Novel hydrogels of hyaluronic acid: synthesis, surface morphology, and solid-state NMR. J Am Chem Soc 116, 7515-7522; and Pouyani, T., and Prestwich, G. D.
  • this invention in one aspect, relates to hydrogel films and methods of making and using these films.
  • Fig. 1 A shows structures of GAG and chondroitins.
  • Fig. IB shows preparation of HA hydrogel film by crosslinking hyaluronic acid converted to the adipic dihydrazide derivative and then crosslinked with poly(ethylene glycol)-propiondialdehyde.
  • Fig. 4 shows a diagram of an in vivo pig assay.
  • Fig. 5 shows a diagram of histology methods; (a) Front view of the division of one wound into four equal pieces, with the arrows indicating the edge sections for histological evaluation, (b) Side view of one piece of the wound. Dark represents the wound, while light represents the surrounding tissue.
  • Fig. 6 shows a diagram of the densitometry method.
  • Fig. 7 shows percent re-epithelialization of wounds treated with either an
  • Fig. 8 shows a bar graph of microvessel density.
  • Fig. 9 shows the total release of bFGF from an ethylene oxide sterilized HA film.
  • Fig. 10 shows the total release of bFGF from an unsterilized HA film.
  • Fig. 11 shows HA Oxidation of HA by Periodate (Note: these dialdehydes would be produced in multiple locations along the HA backbone, leading to a poly aldehyde species).
  • Fig. 12 shows a structural depiction of an HA-ADH chain (horizontal, top) cross-linked by an HA-polyaldehyde chain.
  • hydrogel film is intended to mean a macromolecular network which is capable of swelling in aqueous solution.
  • an isolated or biologically pure, protein or nucleic acid molecule is a compound that has been removed from its natural milieu.
  • isolated and biologically pure' do not necessarily reflect the extent to which the compound has been purified unless specific levels of purity are indicated.
  • An isolated compound of the present invention can be obtained from its natural source, can be produced using molecular biology techniques or can be produced by chemical synthesis.
  • a polysaccharide means one or more polysaccharides.
  • 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 embodiment 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 embodiment. 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.
  • the invention relates to a hydrogel film comprising a polymer, wherein the polymer has at least one unit having the formula I
  • X and Y are a polysaccharide residue
  • Z, Rl, R2, R3, R4, R5, R6, R7, and R8 are, independently, hydrogen, a polysaccharyl group, a substituted or unsubstituted hydrocarbyl group, a substituted or unsubstituted heterohydrocarbyl group, or a polyether group, wherein Z, R3, and R4 are not hydrogen.
  • the invention also relates to a hydrogel film comprising a compound having the formula JJ:
  • A is a glycosaminoglycan having at least one hydrazide group
  • B is a dialdehyde crosslinker
  • x is the number of glycosaminoglycan molecules, which is a whole number in a range of 1 to 100 molecules;
  • y is the number of dialdehyde crosslinker molecules, which is a whole number in the range of 1 to 10 molecules;
  • j is the number of crosslinked glycosaminoglycan-dialdehyde crosslinker- glycosaminoglycan units, which is a whole number in the range of 10 units to 100 million units.
  • the invention also relates to a hydrogel film produced by the process comprising reacting (1) a modified polysaccharide having at least one hydrazide group with (2) a polyaldehyde.
  • the invention yet further provides a method for producing a hydrogel film of the invention, comprising reacting (1) a modified polysaccharide having at least one hydrazide group with (2) a polyaldehyde.
  • the invention also provides a pharmaceutical composition comprising a pharmaceutically-acceptable compound and the hydrogel film of the invention.
  • the invention provides a method for producing a pharmaceutical composition, comprising admixing a pharmaceutically-acceptable compound with the hydrogel film of the invention.
  • the invention relates to a method for producing a pharmaceutical composition, comprising
  • step (a) reacting the modified polysaccharide in the admixture of step (a) with a polyaldehyde.
  • the invention also relates to a method for improving wound healing in a mammal in need of wound healing, comprising contacting the wound of a mammal with a hydrogel film of the invention.
  • the invention further provides a method for delivering at least one pharmaceutically-acceptable compound to a patient in need of such delivery, comprising contacting at least one tissue capable of receiving the pharmaceutically- acceptable compound with a pharmaceutical composition of the invention.
  • the invention also provides a method for purifying a modified polysaccharide having at least one hydrazide group, comprising performing a dialysis step.
  • A-D a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if it each is not individually recited each is individually and collectively contemplated meaning combinations, A- E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are considered disclosed. Likewise, any subset or combination of these is also disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E would be considered disclosed. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using.
  • Polysaccharides useful in the present invention should have at least one carboxylic acid group that can react with a dihydrazide to produce a modified polysaccharide.
  • the synthesis of modified polysaccharides is discussed below.
  • the polysaccharide is a GAG.
  • a 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. Any uronic acid containing natural or synthetic polymer is included within the scope of the present invention.
  • the number of disaccharide units may be any that are useful in the present invention.
  • 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 the present invention. 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 invention.
  • Hyaluronic acid is a non-sulfated GAG.
  • Hyaluronic acid 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 hyaluronic acid and salts thereof are well known.
  • Hyaluronic acid can be purchased from Clear Solutions Biotechnology, Inc. (Stonybrook, NY), Pharmacia Inc., Sigma Inc., and many other suppliers. For high molecular weight hyaluronic acid it is often in the range of 100-10,000 disaccharide units.
  • modified polysaccharide refers to a polysaccharide having at least one hydrazide group.
  • synthesis of the modified polysaccharides will be discussed below.
  • Dihydrazides that can be used to modify the polysaccharide are represented by formula UI:
  • E is hydrocarbyl such as alkyl, aryl, alkylaryl or arylalkyl or E is heterohydrocarbyl, which also includes oxygen, sulfur, and/or nitrogen atoms in addition to carbon atoms.
  • the alkyl group may be branched or unbranched and contain one to 20 carbons or other carbon-sized atoms, preferably 2 to 10, more preferably 4 to 8 carbons or carbon-sized heteroatoms, such as oxygen, sulfur or nitrogen.
  • the alkyl group may be fully saturated or may contain one or more multiple bonds.
  • the carbon atoms of the alkyl may be continuous or separated by one or more functional groups such as an oxygen atom, a keto group, an amino group, an oxycarbonyl group and the like.
  • the alkyl group may be substituted with one or more aryl groups.
  • the alkyl group may in whole or in part, be in form of rings such as cyclopentyl, cyclohexyl, and the like. Any of the alkyl groups described above may have double or triple bond(s).
  • any of the hydrocarbyl groups can be used as a heterocarbyl group, wherein the alkyl or aryl group contains a heteroatom such as oxygen, sulfur, or nitrogen incorporated within the chain or ring.
  • any of the carbon atoms of the alkyl group may be separated from each other or from the dihydrazide moiety with one or more groups such as carbonyl, oxycarbonyl, amino, and also oxygen and sulfur atoms singly or in a configuration such as -- S— S— , — O ⁇ CH 2 — CH 2 — O ⁇ , S— S-CH 2 ⁇ CH 2 - and NH(CH 2 ) n NH-, where n is from 1 to 20.
  • Aryl substituents are typically substituted or unsubstituted phenyl, but may also be any other aryl group such as pyrrolyl, furanyl, thiophenyl, pyridyl, thiazoyl, etc.
  • An inorganic, alkyl or other aryl group including halo, hydroxy, amino, thioether, oxyether, nitro, carbonyl, etc may further substitute the aryl group.
  • alkylaryl or arylalkyl groups may be a combination of alkyl and aryl groups as described above. These groups may be further substituted as described above.
  • H 2 N-NH-CO-NH-E-CO-NH-NH 2 can be hydrocarbyl, heterocarbyl, substituted hydrocarbyl substituted heterocarbyl and the like.
  • hydrocarbyl as used herein means the monovalent moiety obtained upon removal of a hydrogen atom from a parent hydrocarbon.
  • hydrocarbyl are alkyl of 1 to 20 carbon atoms, inclusive, such as methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, undecyl, decyl, dodecyl, octadecyl, nonodecyl, eicosyl, heneicosyl, docosyl, tricosyl, tetracosyl, pentacosyl and the isomeric forms thereof; aryl of 6 to 12 carbon atoms, inclusive, such as phenyl, tolyl, xylyl, naphthyl, biphenyl, tetraphenyl and the like; aralkyl of 7 to 12 carbon atoms, inclusive, such as benzyl, phenethyl, phenpropyl, phenbutyl, phenhexyl, napthoctyl and the like;
  • the hydrocarbyl group has 1 to 20 carbon atoms, inclusive.
  • substituted hydrocarbyl and heterocarbyl as used herein means the hydrocarbyl or heterocarbyl moiety as previously defined wherein one or more hydrogen atoms have been replaced with a chemical group, which does not adversely affect the desired preparation of the modified polysaccharide.
  • Representative of such groups are amino, phosphino, quaternary nitrogen (ammonium), quaternary phosphorous (phosphonium), hydroxyl, amide, alkoxy, mercapto, nitro, alkyl, halo, sulfone, sulfoxide, phosphate, phosphite, carboxylate, carbamate groups and the like.
  • E can be a polysaccharyl group or a polyether group.
  • dicarboxylic acids include, but are not hmited to, maleic acid, fumaric acid, and aromatic dicarboxylic acids, such as terephthalic acid and isophthalic acid.
  • aliphatic dihydrazides where E is an alkyl group, may have the formula IV:
  • n' can be any length but is preferably from 1 to 20.
  • adipic dihydrazide, suberic dihydrazide, and butandioic dihydrazide are used to prepare the modified polysaccharide.
  • Adipic dihydrazide can be purchased from Aldrich Chemical Co. (Milwaukee, WI).
  • the dihydrazides are at least partially soluble in water.
  • the dihydrazides are also weak bases or weak acids having a pK a for the protonated form, less than about 8, preferably in the range of 1 to 7 and most preferably 2 to 6.
  • pK a is used to express the extent of dissociation or the strength of weak acids, so that, for example, the pK a of the protonated amino group of amino acids is in the range of about 12-13 in contrast to the pK a of the protonated amino groups of the dihydrazides useful herein which is less than about 7.
  • the conversion of polysaccharide to the corresponding modified polysaccharides generally involves reacting the polysaccharide with a dihydrazide.
  • the dihydrazide reacts with a carboxylic acid group present on the polysaccharide.
  • the reaction is preferably carried out under mild conditions at a pH of about 2 to 8, preferably about 3 to 6.
  • the polysaccharide is dissolved in water, which may also contain water-miscible solvents including, but not limited to, dimethylformamide, dimethylsulfoxide, and hydrocarbyl alcohols, diols, or glycerols.
  • the number of dihydrazide groups present on the modified polysaccharide used will vary depending upon the amounts of dihydrazide and polysaccharide used, hi one embodiment, 1% to 99%, 10% to 90%, 20% to 80%, 30% to 70%, or 40%) to 50% of the carboxylic acid groups present on the polysaccharide are converted to the dihydrazide.
  • At least one molar equivalent of dihydrazide per molar equivalent of polysaccharide is added, hi other embodiments, for maximum percentage functionalization, a large molar excess of the dihydrazide (e.g., 10-100 fold) dissolved in water or aqueous-organic mixture is added and the pH of the reaction mixture is adjusted by the addition of dilute acid, e.g., HCI. A sufficient molar excess (e.g., 2 to 100 fold) of carbodiimide reagent dissolved in water, in any aqueous-organic mixture, or finely-divided in solid form is then added to the reaction mixture.
  • a large molar excess of the dihydrazide e.g., 10-100 fold
  • a sufficient molar excess e.g., 2 to 100 fold
  • An increase in pH may be observed after the addition of the carbodiimide and additional dilute HCI or other dilute acids may be added to adjust the pH.
  • the reaction is allowed to proceed at a temperature of about 0°C to about 100° C (e.g., just above freezing, 0° C, to just below boiling (100° C), preferably at or near ambient temperatures for purposes of convenience.
  • the time of the reaction is from about 0.5 to about 48 hours, preferably about one to about five hours with periodic testing and adjusting of the pH until no further change in pH is observed.
  • the modified polysaccharide comprises the reaction product between a GAG and a dihydrazide compound. In one preferred embodiment, the modified polysaccharide comprises the reaction product between adipic dihydrazide and hyaluronic acid (HA-ADH). In another preferred embodiment, the modified polysaccharide comprises the reaction product between adipic dihydrazide and chondroitin sulfate.
  • the modified polysaccharide Prior to reacting the modified polysaccharide with the polyaldehyde crosslinker, the modified polysaccharide can be further purified to enhance the crosslinking efficiency of the reaction with the polyaldehyde.
  • the invention provides a method for purifying the modified polysaccharide via dialysis. A series of dialysis steps can be performed in order to increase the overall purity of the modified polysaccharide.
  • the dialysis steps will vary depending upon the nature of the modified polysaccharide.
  • the solvent that is used to perform the dialysis step can increase the purity of the modified polysaccharide.
  • the solvent can be water, aqueous alcohol, or an aqueous salt. Any salt or alcohol known in the art is useful in this method, and will vary depending upon the modified polysaccharide.
  • a method for purifying HA-ADH comprises dialyzing HA-ADH against an aqueous salt, then successively against alternating solutions of aqueous alcohol and water.
  • the aqueous salt is NaCI and the alcohol is ethanol.
  • the invention also contemplates the optional steps of centrifuging the solution and lyophilizing the supernatant after the dialysis steps.
  • the method for preparing and purifying HA-ADH comprises the consecutive steps of: (1) dissolving hyaluronic acid in water; (2) adding adipic dihydrazide to hyaluronic acid while stirring; (3) adjusting the pH to a range of 4.5 to 5.0; (4) adding l-Ethyl-3-[3-(dimethylamino)- propyl]carbodiimide in solid form; (5) maintaining a pH range of 4.5 to 5.0; (6) stopping the reaction by raising the pH to 7.0; (7) transferring the reaction mixture to dialysis tubing with a molecular weight cut-off of 3,500 that has been soaked in water at room temperature for 3-4 hours and then rinsed; (8) dialyzing against 100 mM NaCI for 60 hours; (9) dialyzing against 1:3 EtOH-H 2 O (volume/volume); (10) dialyzing against pure water; and (11) dialyzing against 1:3 EtOH-H 2 O (volume/volume) .
  • a polyaldehyde crosslinker is reacted with the modified GAG to produce the hydrogel film of the invention.
  • a polyaldehyde is a compound that has two or more aldehyde groups [C(O)H]. In certain embodiments the aldehyde is a dialdehyde composition.
  • any compound possessing two or more aldehyde groups can be used in the present invention as the polyaldehyde crosslinker.
  • the polyaldehyde can be substituted or unsubstituted hydrocarbyl or substituted or unsubstituted heterohydrocarbyl.
  • the polyadlehyde can contain a polysaccharyl group or a polyether group.
  • the polyaldehyde can be a dendrimer or peptide.
  • a polyether dialdehyde such as poly(ethylene glycol) propiondialdehyde (PEG) is useful in the present invention.
  • PEG can be purchased from many commercial sources, such as Shearwater Polymers, Inc. (Huntsville, AL).
  • the polyaldehyde is glutaraldehyde.
  • polyaldehydes of the invention can be prepared by the oxidation of terminal polyols or polyepoxides possessing two or more hydroxy or epoxy groups, respectively, using techniques known in the art.
  • the method generally involves reacting the modified polysaccharide with the polyaldehyde crosslinker in the presence of a solvent.
  • the aldehyde group of the polyaldehyde reacts with the hydrazide group of a modified polysaccharide to produce a new carbon-nitrogen double bond, which is depicted in Scheme 1.
  • the second aldehyde group is then capable of reacting with the hydrazide group of a second modified polysaccharide to produce another carbon- nitrogen double bond to produce the unit depicted in Formula I.
  • the modified polysaccharides can be different or the same.
  • X and Y can be the same polysaccharide residue.
  • X and Y can be different polysaccharide residues.
  • the term "residue" is a section of pre-existing molecule that forms a portion of a new molecule.
  • the polysaccharide can be depicted as X-COOH, where X is the remainder of the polysaccharide molecule.
  • X-COOH reacts with a dihydrazide to produce the modified polysaccharide, X remains the same and is part of the modified polysaccharide.
  • X is the residue of the original GAG (X-COOH).
  • X and Y are, independently, a residue of chondroitin sulfate, dermatan, heparan, heparin, dermatan sulfate, heparan sulfate, alginic acid, pectin, or carboxymethylcellulose.
  • X and Y are a residue of hyaluronic acid.
  • the overall number of crosslinks and the number of different modified polysaccharides that are cross linked together are dependent on the number of reactive aldehyde groups in the polyaldehyde and dihydrazide groups present on the modified polysaccharide.
  • 1% to 100%), 10% to 90%), 30% to 80%, or 40% to 70% of the dihydrazide groups are crosslinked with the polyaldehyde.
  • hydrogel film has from 10 to 10,000,000 units having the formula I. In a preferred embodiment the hydrogel has from 10 to 10,000 units having the formula I.
  • adipic dihydrazide will crosslink when it modifies the uronic acid in l%-99% of the glycosaminoglycan or 1-50%.
  • the modification of the carboxylic acid containing polysaccharide such as GAG can contain 10-90% or 20-80%o or 30-70% or 40-60% or about 50% derivatization and the derivatized polysaccharide can contain greater than 10% or 20% or 30% or 40% or 50% or 60% or 70% or 80% or 90% or 99% crosshnking.
  • a hyaluronic acid (HA) with 5000 disaccharide units has 5000 carboxylic acid groups available.
  • a 1% modification means that there are 50 ADHs per HA molecule, 10% would be 500 ADH/HA, etc. Thus, even at low modification levels, there are some 50 sites per modified GAG molecule to form crosslinks.
  • Z is a polyether.
  • R 1 , R 2 , R 5 , R 6 , R 7 , and R 8 are hydrogen.
  • R 3 and R 4 are (CH ) n , wherein n is from 1 to 20, preferably 2 to 4.
  • X and Y are a residue of hyaluronic acid
  • Z is a polyethylene ether
  • R 1 , R 2 , R 5 , and R 6 are hydrogen
  • R 3 and R 4 is (CH 2 ) .
  • the hydrogel film is produced by reacting (1) a modified polysaccharide comprising the reaction product between adipic dihydrazide and hyaluronic acid and (2) a poly(ethylene glycol) propiondialdehyde.
  • the solvent present in the hydrogel film may be evaporated by any method known in the art such as air-drying, rotary evaporation at low pressure and/or lyophilization.
  • at least 80% of the solvent contained within the hydrogel film will evaporate. More preferred is a state where at least 90% of the solvent has evaporated from the hydrogel film.
  • the reaction solvent is water.
  • small amounts of water miscible organic solvents such as an alcohol or DMF or DMSO, may be used as well.
  • the cross-linking reaction be performed at room temperature, for example, 25 °C, but the cross-linking reaction can be performed within a range of temperatures from below 4 °C to above 90 °C but typically would be performed at between 4 °C and 60 °C, more typically between 4 °C and 50 °C, and more preferably at 4 °C or 30 or 37 degrees.
  • the reaction will also work at a variety of pHs between, for example, pH from 3 tolO, or pH from 4 to 9, or pH from 5 to 8, or preferably at neutral pH.
  • the hydrogel film may comprise 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 may act to control infection or inflammation, enhance cell growth and tissue regeneration, control tumor growth, and enhance bone growth, among other functions.
  • hydrogel films of the invention may contain combinations of two or more pharmaceutically-acceptable compounds. Hydrogel films which contain five or fewer pharmaceutically-acceptable compounds are preferred. More preferred are hydrogel films which contain two or fewer pharmaceutically- acceptable compounds. Hydrogel films which contain one pharmaceutically- acceptable compound are most preferred.
  • 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 metaprotemal 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, WI).
  • the pharmaceutically-acceptable compound is a steroid.
  • the pharmaceutical composition comprises a steroid and a hydrogel film comprised of the reaction product between hyaluronic acid/adipic dihydrazide (HA-ADH) and poly(ethylene glycol) propiondialdehyde.
  • compositions can be prepared using techniques known in the art.
  • the composition is prepared by admixing a hydrogel film of the invention 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 hydrogel film and pharmaceutically-acceptable compound. Covalent bonding to reactive therapeutic drugs, e.g., those having reactive carboxyl groups, can be undertaken on the hydrogel film.
  • 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 NH2 group of the dihydrazide-modified polysaccharide.
  • NHS N-hydroxysuccinimide
  • electrostatic or hydrophobic interactions can facilitate retention of a pharmaceutically-acceptable compound in the 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 pharmaceutically-acceptable compound is admixed with the modified polysaccharide, followed by reacting the modified polysaccharide with a polyaldehyde.
  • This embodiment also covers the possibility of the pharmaceutically-acceptable compound chemically reacting or physically interacting with the modified polysaccharide.
  • 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 mammal 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 of the present invention can be formulated in any excipient the biological system or entity can tolerate.
  • excipients include 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 may 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 may be formulated in a pharmaceutical composition.
  • Pharmaceutical compositions may include carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the molecule of choice.
  • Pharmaceutical compositions may also include one or more active ingredients such as antimicrobial agents, antiinflammatory agents, anesthetics, and the like.
  • the pharmaceutical composition may be administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated. Administration may 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 may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.
  • Formulations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders.
  • Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may 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.
  • the disclosed hydrogel films can be used for a variety of uses related to drug delivery, small molecule delivery, wound healing, bumhealing, 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 present hydrogel films and disclosed compositions can be placed directly in or on any biological system without purification as it is composed of only two biocompatible polymers.
  • sites the hydrogel film may be placed include soft tissue such as muscle or fat; hard tissue such as bone; 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; and other sites into or onto which the hydrogel film may be placed including a skin surface defect such as a cut, scrape or bum area.
  • the present hydrogel films are biodegradeable and naturally occurring enzymes will act to degrade them over time.
  • Components of the hydrogel film may be "bioabsorbable" in that the components of the hydrogel film will be broken down and absorbed within the biological system, for example, by a cell, tissue and the like. Additionally, the hydrogel films, especially hydrogel films that have not been rehydrated, may be applied to a biological system to absorb fluid from an area of interest.
  • the hydrogel films of this invention may 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 hydrogel film 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-con vulsants, 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 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), 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).
  • 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 preferred 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. These substances are all known in the art. Compounds are available from Sigma Chemical Company (St. Louis, MO).
  • therapeutic drugs 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 selected from the group consisting of 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 consisting 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 hydrogel film 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 ability of the hydrogel film to maintain a slow, sustained release of steroidal anti-inflammatories makes this invention extremely useful for wound healing after trauma or surgical intervention. Additionally, the hydrogel film can be used as a barrier system for enhancing cell growth and tissue
  • HA and the combination of HA with VEGF can enhance the process of neovasculogenesis in in vivo applications.
  • the present invention also provides methods to improve wound healing in a mammal in need of such improvement, comprising contacting a hydrogel film of the present invention with a wound of a mammal 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, comprising contacting at least one hydrogel film of the present invention 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 wound on the skin, a surgical incision, a bone defect, and the like.
  • 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, and the like.
  • the hydrogel film can also function as a barrier system for guided tissue regeneration by providing a surface over which cells can grow.
  • 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 may 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 hydrogel film may 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 hydrogel film may 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 hydrogel film may 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 hydrogel film When used to treat a defect on skin or other tissue, the hydrogel film 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 hydrogel film 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 tae like, to enhance the compatibility and/or performance or function of an implantable device with a body tissue in an implant site.
  • the hydrogel film can be used to coat the implantable device.
  • the hydrogel film 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 hydrogel film can also be used to enhance the performance or function of an implantable device.
  • the 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 tae void between the device and tissue and form a tight seal to reduce bacterial infection and loss of body fluid.
  • the disclosed compositions and methods can be applied to mammals in need of tissue regeneration.
  • cells may be incorporated into the hydrogel for implantation.
  • Preferred mammals to which the compositions and methods apply are mouse, porcine, bovine, ovine, and primates, including apes, chimpanzees, orangatangs, and humans.
  • the disclosed methods and compositions When being used in areas related to tissue regeneration such as wound or bum 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 the disclosed compositions and methods may 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 hydrogels 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.
  • Adipic Dihydrazide and l-Ethyl-3-[3-(dimethylamino)- propyl]carbodiimide(EDCI) were purchased from Aldrich Chemical Co. (Milwaukee, WI) and used as received.
  • Poly(ethylene glycol)-propiondialdehyde (PEG-diald) was obtained from Shearwater Polymers, Inc. (Huntsville, AL) and also used as received.
  • HA (6 g, 15 mmol) was dissolved in 1.2 L of water to give a 5 mg/ml solution.
  • Adipic dihydrazide 110 g, 0.63 mol was then added to the solution while stirring.
  • EDCI (lOg, 52 mmol) was added in solid form.
  • the pH of the reaction was maintained at 4.75 by the addition of 0.1N HCI.
  • the reaction was stopped by addition of 0.1 N NaOH, raising the pH of the reaction mixture to 7.0.
  • Dialysis tubing (MW cutoff 3,500) was soaked in water at room temperature for 3-4 hours and then rinsed.
  • the reaction mixture was transferred to the prewashed dialysis tubing and dialyzed exhaustively (60 hour against 100 NaCL) followed by dialysis against alternating solutions of 1:3 EtOH-H 2 O (v/v) and pure H 2 O. The solution was then centrifuged, and the supernatant lyophilized. The purity of the resulting HA-ADH was measured by GPC, and the degree of substitution by ADH was determined by H-NMR.
  • HA-ADH was dissolved in H O at a concentration of 5 mg/ml (Solution A).
  • PEG-dialdehyde was dissolved in H O at a concentration of 50 mg/ml (Solution B).
  • Solutions A and B were added to a small Petri dish in appropriate ratios to give equimolar equivalents of aldehyde and hydrazide functionalities and the solutions were mixed with gentle swirling.
  • a hydrogel film began to form within 60 seconds. Hydrogel films were successfully produced when the crosshnking agent (PEG- diald) was used in a molar ration of 0.25, 0.5, and 1 relative to ADH. The mixture was agitated on an orbital platform for an additional 24 hours to obtain a solid, uniform hydrogel film. Hydrogel films were stored in an open dish overnight at 37° C to allow solvent evaporation. Evaporation of solvent from the hydrogel film provided flexible, hydratable HA hydrogel film. 3.
  • DSC Differential Scanning Calorimeter
  • the thermal analysis profiles of the dried , hydrogel film samples (with or without loading) were obtained as the temperature was increased from room temperature to 55° C at a rate of 10° C/minute under nitrogen.
  • the DSC profiles show that after HA is converted into its ADH derivative, the endothermic and exothermic peak shifted, indicating an altered polymer microstmcture.
  • the crosshnking of the two polymers clearly produced a new material having a microstructure different from its components.
  • HA hydrogel films were gently rinsed with H 2 O and air-dried in an incubator at 37° C for 24 hours to give dried hydrogel samples. Swollen film samples were obtained by immersion of the HA hydrogel film disks in distilled H 2 O for 15 minutes, freezing quickly on dry ice, followed by lyophilization. HA hydrogel films subjected to enzymatic degradation were prepared by immersion of the films in pH 7.4 PBS buffer containing hyaluronidase (HAse) (100 U/mL) for 3 days. The films were removed from the HAse solution, rinsed gently with water and dried on PTFE surface at 37C.
  • HAse hyaluronidase
  • control HA hydrogel films were immersed in pH 7.4 PBS buffer without HAse at 37° C for 3 days and processed as for the enzyme- treated gels. Samples were gold-coated for conductance, and the surfaces of hydrogel films were examined with an SEM. Magnifications were at 6,000x and below.
  • Amaranth and acridine orange were purchased from Aldrich Chemical Co. (Milwaukee, WI) and used as received. b Preparation of HA-PEG hydrogel films with amaranth loading
  • Amaranth was loaded into the hydrogel films by an in situ polymerization method. Amaranth was dissolved in H O (10 mg/ml) and then mixed Solution A. The PEG-dialdehyde (50 mg/ml) Solution B was then added to create a hydrogel film containing the amaranth dye. The resulting hydrogel film was obtained by evaporation of solvent with amaranth-loading of 5.0% weight percentage relative to the dry macromolecular components.
  • a solution absorption method was used to load acridine orange onto the HA hydrogel film.
  • a HA hydrogel film was prepared and then immersed in a 1 mg/mL acridine orange/H 2 O solution for 60 seconds. The film was then washed with water, placed on a poly(tetrafluoroethylene (PTFE) surface, and dried at 37° C.
  • PTFE poly(tetrafluoroethylene
  • Swelling was quantified by using the cationic dye acridine orange to stain the hydrogel film in order to visualize the diameter size change during swelling under a microscope.
  • the average equilibrium SW of the hydrogel film in PBS at 37 C was 7.11+/- 0.56.
  • the equilibrium SW in 50 mM pH 7.0 phosphate buffer at 37 C was 7.83 +/- 1.75. No significant difference was observed among the equilibrium SW values in the different buffers, indicating the SW was largely unaffected by the changes in ionic strength.
  • the HA hydrogel films swelled rapidly and reached equilibrium within 100 seconds. The rate of swelling was independent of the buffer composition.
  • Hydrocortisone, dexamethasone, indomethacin, gentamycin, pilocarpine and diclofenac sodium were all obtained from Sigma Chemical Co. (St. Louis, MO) and used as received.
  • Hydrocortisone, dexamethasone and indomethacin were all dissolved in ethanol at a concentration of 10 mg/ml.
  • Pilocarpine and diclofenac sodium was dissolved in water at a concentration of 10 mg/ml.
  • An aliquot of each drug was mixed with Solution A, and the in situ polymerization method was followed to form a drag-containing hydrogel film.
  • Hydrogel films had drug loading of 5.0% weight percentage relative to their dry macromolecular components. All drag-loaded hydrogel films were obtained by agitating for 24 hours and dried slowly at 37° C.
  • a 10 mg/mL solution was again prepared which was added dropwise with agitation after Solution A was crosslinked by Solution B for 5 minutes. The film was then continuously agitated for 24 hours while the hydrogel film solidified and a film was obtained by drying as above.
  • the release rate of the drags from the HA hydrogel films was in direct correlation to the hydrophobicity of the drugs.
  • the rapidly released drags demonstrated almost complete release from the hydrogel film in 10 minutes following first-order kinetics, consistent with diffusion from the gel during hydration and concomitant swelling of the hydrogel film.
  • slow first-order release kinetics was observed for dexamethasone and prednisone.
  • Dexamethasone showed sustained release for 1 hour and prednisone showed sustained release for almost 24 hours.
  • the wound covered with a chondroitin-sulfate hydrogel film had a newly-grown epidermis layer covering the wound site and the non-regulated dermal tissue was filling in underneath the new epidermis.
  • the wound covered with Biobrane® demonstrated no epidermal coverage on the surface of the wound site.
  • Glycosaminoglycan (GAG) based hydrogel films were developed and evaluated for use as wound dressings.
  • Hyaluronic acid (HA) and chondroitin sulfate (CS) were first converted to the adipic dihydrazide derivative (ADH) and then crosslinked with the macromolecular homobifunctional reagent poly(ethylene glycol) propiondialdehyde (PEG-diald) to give a polymer network.
  • ADH adipic dihydrazide derivative
  • PEG-diald macromolecular homobifunctional reagent poly(ethylene glycol) propiondialdehyde
  • mice were purchased from Charles River Laboratories Wilmington, MA. Isoflurane was acquired from Abbot Laboratories North Chicago, IL. TegadermTM was obtained from 3M Health Care (St. Paul, MN). Curity® Non-Adhering Dressings and Curity® Sheer Bandages were purchased from Kendall Company (Mansfield, MA), and 9 mm Autoclips® were purchased from Becton Dickinson (Sparks, MD).
  • CS-ADH was obtained using tae same procedures, except CS was dissolved in H 2 O at 25 mg/mL and 5 and 2 molar equivalents of ADH and EDCI were used respectively.
  • GAG-PEG films were made as described (Luo, Y., Kirker, K. R., and Prestwich, G. D. (2000) Cross-linked hyaluronic acid hydrogel films: new biomaterials for drug delivery. Journal of Controlled Release 69, 169-184).
  • GAG- ADH was dissolved in H 2 O (5 mg/mL for HA-ADH and 25 mg/mL for CS-ADH) to form Solution A.
  • PEG-diald was dissolved in H2O at a concentration of 50 mg/mL
  • a wound approximately 1.0-cm in diameter, was created on the dorsal side of the mouse using curve blade surgical scissors. Both the epidermal and dermal layers were removed, creating a full thickness wound with minimal bleeding.
  • individual diameters of the wound site were marked and measured using digital calipers and averaged to determine the original wound diameter and area.
  • the wound was then dressed with one of three dressings: (1) TegadermTM (control); (2) ethylene oxide sterilized HA film and TegadermTM; or (3) ethylene oxide sterilized CS film and TegadermTM.
  • GAG films were cut into 13 -mm in diameter circular sheets that were rehydrated with sterile normal saline before use.
  • the TegadermTM was cut into 2-cm square pieces, pieces large enough to cover the entire wound and the surrounding area.
  • the dressed wound was then covered with a Curity® Non- Adhering Dressing, bandaged with a Curity® Sheer Bandage, sealed with two 9 mm Autoclips®, and allowed to heal.
  • the surgery was repeated multiple times to give a sample size of six mice per treatment per time point (described below).
  • the mouse On day 3, 5, 7, or 10 post-surgery, the mouse was anesthetized with isoflurane and sacrificed by cervical dislocation. The four wound diameters were measured again to determine the new wound area. The wound site was excised, and the tissue was processed for histological evaluation. Any wound that had been exposed to air, because the mouse had chewed off its bandage, was not used for analysis. The wound size measurements taken at the time of surgery and at the time of biopsy were used to calculate the percent wound contraction, using equation 1.
  • Histological endpoints included a quantitative measurement of the percent re-epithelialization, a qualitative measurement of the inflammatory response, and the presence of new vasculature.
  • the un-epithelialized wound diameter was measured using an eyepiece micrometer. This measurement, together with the original wound diameter was used in equation 2 to determine the percent re-epithelialization.
  • the average of all six sections from each wound site was calculated and determined to be the average of the percent re-epithelialization for that wound.
  • the inflammatory response was graded on a qualitative scale from 0-3 (0- no sign of inflammation, 1-minimal inflammation, 2-moderate inflammation, and 3- strong presence of inflammatory cells).
  • Wound contraction is a healing process whereby the edges of the wound pull inwards to reduce the overall wound area.
  • wound fibroblasts begin to assume a myofibroblast phenotype characterized by large bundles of actin containing microfilaments and the establishment of cell-cell and cell-matrix linkages (Clark, R. A. F. Wound repair: Overview and general considerations. In The Molecular and Cellular Biology of Wound Repair; R. A. F. Clark, Ed.; Plenum Press: New York, 1996; pp 3-50).
  • the fibroblasts link to extracellular fibronectin and collagen and to each other through adherens junctions.
  • These cell-cell, cell-matrix, and matrix-matrix links provide a network through which the traction of the fibroblasts can be transmitted across tae wound, thereby pulling the wound edges inward.
  • Re-epithelialization is the process through which new cutaneous tissue covers the wound defect. This process requires the uninjured keratinocytes along the wound edges to migrate laterally to cover the wound bed.
  • Both wound contraction (Fang, C.-H., Robb, E. C, Yu, G.-S., Alexander, J. W., and Warden, G. D. (1990) Observations on stability and contraction of composite skin grafts: Xenodermis or allodermis with an isograft overlay. Journal of Bum Care and Rehabilitation 11, 538-542; Joseph, H. L., Roisen, F. J., Anderson, G. L., Barker, J. H., Weiner, L. J., and Tobin, G. R.
  • Figures 2 and 3 show the results for both wound contraction and re- epithelialization, respectively. No significant difference in wound contraction was observed between either of the two assay groups (HA film or CS film treated wounds) and the control group (TegadermTM only) for any time point. On the other hand by day 5, wounds treated with CS film had significantly (p ⁇ 0.001) more epithelial tissue taan the controls. Additionally by day 7 post-surgery, wounds treated with either GAG film hand significantly more (p ⁇ 0.05) epithelial coverage.
  • mice of new Response completely blood re- vessels Dermis Muscle epithelialized (+/-)
  • Table 1 Histological evaluation of wounds for all three assay groups at all time points. Inflammatory response: (+++) strong, (++) moderate, (+) minimal, (-) none Comments: (a) little dermal regeneration, (b) film integrating into wound, (c) moderate dermal rengeration, (d) visual signs of dermal collagen organization
  • TegadermTM only treated wounds had no wound exudate, little dermal regeneration, and inflammatory response.
  • the TegadermTM dressing adhered to the wound bed, and removal resulted in the loss of tissue at the wound site. Therefore, all TegadermTM only dressing wounds were histologically processed with tae TegadermTM still in place.
  • An ideal wound dressing has many attributes. It should protect the wound from bacterial infection, control evaporative water loss and prevent dehydration, control permeability of oxygen and carbon dioxide, absorb wound exudate, and enhance the healing. Additionally, it should be composed of materials that are nontoxic, non-immunogenic, flexible, durable, and comfortable when worn.
  • the films described herein have significant advantages to the previously studied materials. They are hydrophilic and absorb wound exudate. They can be used immediately and do not require weeks of preparation.
  • the HA and CS films are biodegradable and non-immunogenic, and they can be used with an occlusive dressing to prevent infection. Additionally, the HA hydrogel films describe here have been shown to possess sustained release capabilities (Luo, Y., Kirker, K. R., and Prestwich, G. D. (2000) Cross-linked hyaluronic acid hydrogel films: new biomaterials for drug delivery. Journal of Controlled Release 69, 169-184) that may prove useful for delivering therapeutic agents at wound sites.
  • TegadermTM consists of a thin polyurethane membrane coated with a layer of an acrylic adhesive.
  • the dressing which is permeable to both water vapor and oxygen, is impermeable to microorganisms.
  • TegadermTM is used in the treatment of minor bums, pressure areas, donor sites, post-operative wounds, and a variety of minor injuries including abrasions, and lacerations.
  • mice Female pigs were purchased from Arnold's Hog Supply (Lehi, Ut). TegadermTM and VetWrap were obtained from 3M Animal Care Products (St. Paul, MN). Skin markers were purchased from DeRoyal (Powell, TN). Betasept® was acquired from Purdue Frederick (Norwalk, CT). Lap Sponges were obtained from Medical Action Industries (Ashville, NC). AutoSutureTM skin staples were purchased form United States Surgical Corporation (Norwalk, CT).
  • HA-ADH was made using slightly modified methods from those previously reported (Luo, Y., Kirker, K. R., and Prestwich, G. D. (2000) Cross-linked hyaluronic acid hydrogel films: new biomaterials for drug delivery. Journal of Controlled Release 69, 169-184).
  • 20 g of HA was dissolved in 2.5 L H 2 O by vortexing at 150 rpm at 37°C. After 3 hrs, the solution was moved to a mechanical stirrer and the pH of the solution was adjusted to 0.6-0.7 through the addition of concentrated HCI. The mixture was allowed to stir for an additional 24 hrs.
  • HA-ADH LMWHA solution was then used to prepare HA-ADH. 10 molar equivalents of ADH, and 2 molar equivalents of EDCI were added in solid form, while maintaining the pH at 4.75, with the addition of 1.0 N HCI. Raising the pH of reaction mixture to 7.0 stopped the reaction, and the reaction mixture was dialyzed thoroughly. The solution was then centrifuged, and the supernatant was lyophilized. The purity of HA-ADH was measured by GPC, and the degree of substitution by ADH was determined by 1 H-NMR. CS-ADH was obtained using the same procedures, except no adjustments were made to its molecular weight, it was dissolved in H O at 25 mg/mL, and 5 and 2 molar equivalents of ADH and EDCI were used respectively.
  • the mixture was agitated on an orbital platform for an additional 24 hr to obtain a solid, uniform hydrogel.
  • Hydrogel was then moved to a 37°C incubator and stored overnight to allow solvent evaporation.
  • the resulting film was then cut into 5 x 5 cm 2 pieces. Each piece was sterilized with ethylene oxide gas and stored separately at room temperature until use.
  • the wound healing characteristics of the GAG films were evaluated using a pig model ( Figure 4). All assays were completed with the approval of the University of Utah's Institutional Animal Care and Use Committee. To begin, a female pig, weighing approximately 40 kg was anesthetized with TKX (1-2 cc/50 kg) and Atropine (0.12-0.15 mg/kg) and intubated. The surgical area was shaved with an electric razor and prepped with iodine solution. Additional anesthesia (1-2% isoflurane), heart rate, and respiration were monitored with a surgical anesthesia machine.
  • a skin marker was used to outline the location of the 8 5 x 5 cm 2 desired surgical sites on the dorsal side of the pig.
  • 4 sites were located on each side of the pig with two wounds located medially and two laterally.
  • each intended site was lubricated with BetaseptTM, and a partial thickness wound was inflicted with a Padgett Electro-Dermatome from Padgett Instruments Kansas City, MO, creating wounds that were approximately 5 x 5 x 0.03 cm 3 . After haemostasis was reached, the exact wound dimensions were measured and the wounds were dressed.
  • the four wounds on one half of the pig were dressed with a GAG film plus TegadermTM, while the other half were dressed with TegadermTM alone. All dressed wounds were then covered with 4 Lap Sponges, wrapped with VetrapTM and a surgical stocking, and secured with skin staples. The surgery was repeated multiple times to give a sample size of three pigs per treatment per time point (described below).
  • the pig On day 3, 5, or 7 post-surgery, the pig was anesthetized with TKX (1-2 cc/50 kg) and Atropine (0.12-0.15 mg/kg) and sacrificed with Beuthanasia (20 cc). Each wound was excised in its entirety and analyzed histologically.
  • the inflammatory response was graded on a qualitative scale from 0-3 (0- no sign of inflammation, 1-minimal inflammation, 2-moderate inflammation, and 3- strong presence of inflammatory cells).
  • Dermal regeneration was determined using computer densitometry. For each tissue section, the density of dermal collagen (stained blue from the Masson's Trichrome stain) peripheral to the wound bed determined. Next, the density of new collagen in the wound bed was determined, and the ratio of densities was calculated and correlated to dermal regeneration (Figure 6).
  • the GAG- ADH was crosslinked with PEG-diald, which produced a bis- hydrazone functionality as the covalent crosslink.
  • the hydrogel began to form within 5 minutes after mixing of the GAG- ADH and PEG-diald solutions, but was agitated for 24 hrs. to assure the formation of a solid, uniform hydrogel. Evaporation of the solvent provided flexible, hydratable GAG hydrogel film.
  • the GAG- ADH films used in these investigations were 50% crosslinked (aldehyde: ADH ratio of 0.5:1).
  • GAG-PEG hydrogel films were made as described (Luo, Y., Kirker, K. R., and
  • the mixture was agitated on an orbital platform for an additional 24 hr to obtain a solid, uniform hydrogel.
  • Hydrogels were stored in an open dish overnight at 37°C to allow solvent evaporation and thus provide a flexible, hydratable HA hydrogel film.
  • human skin was also tested.
  • the tissue was thawed and cut into 50 mm x 7 mm sized samples.
  • Human epidermis samples were tested using the 2-lb. load and a crosshead speed of 0.8 in/min.
  • Example 9 Microvasculature growth stimulated by HA hydrogels
  • HA-ADH hydrogels were formed by cross-linking with PEG-dialdehyde (As discussed in Example 2), then dried to a film form by sitting at 37 °C for 24 hours. All steps were carried out under clean conditions. Three cases of films were formed:
  • 5mm x 5mm square samples were cut from the films and surgically implanted in a small pocket that was formed in a mouse ear immediately beneath its skin. These implants contained 1.0 mg of cross-linked HA, and the VEGF cases also contained 50ng of VEGF. Along with the animals receiving implants, a sham surgery was also performed in which a pocket was formed, but no implant was placed in it. Three animals were treated for each case.
  • the animals were sacrificed and both the implanted and contralateral ears were retrieved.
  • the ears were fixed in formalin, sectioned for microscopy and stained with hematoxylin and eosin.
  • bFGF basic Fibroblast Growth Factor
  • Solution A The bFGF solution was then mixed into Solution A.
  • 40.07 mg of PEG- diald was dissolved in H2O at a concentration of 25 mg/mL to form Solution B. Both solutions were cooled to 4°C, added to a 3.5 cm in diameter petri dish, and mixed with gentle vortexing.
  • a hydrogel formed within 5 minutes. The mixture was agitated on an orbital platform for an additional 24 hr to obtain a solid, uniform hydrogel. The hydrogel was then moved to a 37°C incubator and stored overnight to allow solvent evaporation and thus provide a flexible, hydratable HA hydrogel film. The resulting film was cut into 1-cm in diameter disks and stored separately. One half of the disks also received ethylene oxide gas sterilization.
  • a HA/bFGF disk was placed in a small glass vial. 1 ml of 100 U/ml of HAse in PBS was then added to the vial. The container was then placed on an orbital platform at 37°C and incubated. Periodically, the bathing solution was recovered and replaced with new solution. The recovered solution was stored at - 80°C until analyzed. The amount of bFGF in the collected solutions was quantified using the Quantikine® immunoassay by R&D Systems (Minneapolis, MN) and the methods described there in. (3) Preparation of GAG film with TGF- ⁇
  • TGF- ⁇ Human recombinant transforming growth factor beta 3
  • TGF- ⁇ Human recombinant transforming growth factor beta 3
  • 1.5 ng of TGF- ⁇ was resuspended as directed in sterile 4 mM HCI with 0.1 % BSA.
  • 109.5 mg of GAG-ADH was dissolved in H2O at 25 mg/mL to form Solution A.
  • the TGF- ⁇ solution was then mixed into Solution A.
  • a desired amount of PEG-diald (approximately 124 mg for either a HA or CS film) was dissolved in H2O at a concentration of PEG-diald
  • Solution B 25 mg/mL to form Solution B. Both solutions were cooled to 4°C, added to a 6 cm in diameter petri dish, and mixed with gentle vortexing. A hydrogel formed within 5 minutes. The mixture was agitated on an orbital platform for an additional 24 hr to obtain a solid, uniform hydrogel. The hydrogel was then moved to a 37°C incubator and stored overnight to allow solvent evaporation and thus provide a flexible, hydratable HA hydrogel film. The resulting film was cut into 1 x 2 cm 2 pieces, sterilized with ethylene oxide gas and stored separately.
  • TGF- ⁇ The release of TGF- ⁇ was observed using an in vivo mouse model.
  • a 2-cm midline laparotomy was created on a balb/c mouse. The incision was then closed with three sutures spaced 1-cm apart.
  • a GAG-film with TGF- ⁇ or a GAG-film without TGF- ⁇ was placed over the wound. The skin incision was closed with running sutures. All films were swollen with normal saline before use.
  • An additional control group consisted of laparotomies covered with no film at all. There were a total of 6 mice per group. After a week, the wounds were exposed and evaluated. Defect in lengths in between sutures were measured and histological samples were taken for evaluation. b) Results
  • LMWHA- ADH was crosslinked with PEG-diald, thereby physically trapping the bFGF in the hydrogel.
  • the hydrogel began to form within 5 minutes after mixing of the HA-ADH and PEG-diald solutions, but was agitated for 24 hrs to assure the formation of a solid, uniform hydrogel. Evaporation of the solvent provided flexible, hydratable GAG hydrogel film.
  • the HA-ADH films used in these investigations were 50% crosslinked (aldehyde: ADH ratio of 1:1 and 0.5:1).
  • TGF- ⁇ The release of TGF- ⁇ was observed using an in vivo mouse model. Gross evaluation of the wounds indicated that those films that released TGF- ⁇ , had considerably less defects than those not releasing the growth factor. Additionally, those receiving no film at all had the largest defects; some exhibiting hernias. All the films used in this Example were sterilized with ethylene oxide gas, yet the TGF- ⁇ was still active. This confirms the earlier results suggesting that gas sterilization can be used to sterilize GAG-ADH films with growth factors.
  • HA- ADH was dissolved in H 2 O at a concentration of 5 mg/mL (Solution A).
  • Oxidized HA was dissolved in H 2 O at a concentration of 10 mg/mL (Solution B).
  • Volumes of Solutions A and B were added to a small Petri dish to give various molar equivalents of aldehyde and hydrazide functionalities, and the solutions were mixed with gentle swirling.
  • a hydrogel began to form within 60 sec. The mixture was agitated on an orbital platform for an additional 24 hr to obtain a solid, uniform hydrogel.
  • Hydrogels were stored in an open dish overnight at 37 °C to allow solvent evaporation and thus provide a flexible, hydratable HA hydrogel film. Hydrogel films were successfully obtained with the molar ratio of aldehyde/hydrazide equal to 0.05, 0.1, 0.2, 0.5, or 1.0. Similar oxidation will occur with other polysaccharides, e.g. dextran, pectin, cellulose etc. The oxidized polysaccharides could be used as crosslinkers to prepare HA hydrogels. See Figures 11 and 12.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Polymers & Plastics (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Materials Engineering (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Inorganic Chemistry (AREA)
  • General Chemical & Material Sciences (AREA)
  • Dermatology (AREA)
  • Polysaccharides And Polysaccharide Derivatives (AREA)
  • Materials For Medical Uses (AREA)
  • Compositions Of Macromolecular Compounds (AREA)
  • Peptides Or Proteins (AREA)
EP01957173A 2000-07-17 2001-07-17 Biogelfilme sowie verfahren zu deren herstellung und nutzung Withdrawn EP1305355A1 (de)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US21872500P 2000-07-17 2000-07-17
US218725P 2000-07-17
PCT/US2001/022556 WO2002006373A1 (en) 2000-07-17 2001-07-17 Hydrogel films and methods of making and using therefor

Publications (1)

Publication Number Publication Date
EP1305355A1 true EP1305355A1 (de) 2003-05-02

Family

ID=22816250

Family Applications (1)

Application Number Title Priority Date Filing Date
EP01957173A Withdrawn EP1305355A1 (de) 2000-07-17 2001-07-17 Biogelfilme sowie verfahren zu deren herstellung und nutzung

Country Status (4)

Country Link
EP (1) EP1305355A1 (de)
AU (1) AU2001278943A1 (de)
CA (1) CA2416698A1 (de)
WO (1) WO2002006373A1 (de)

Families Citing this family (31)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003061626A1 (en) * 2002-01-18 2003-07-31 Control Delivery Systems, Inc. Polymeric gel system for the controlled delivery of codrugs
EP1539799B1 (de) 2002-06-21 2013-12-11 The University of Utah Research Foundation Quervernetzte verbindungen und verfahren zu deren herstellung und anwendung
AU2003284484A1 (en) * 2002-11-29 2004-06-23 Chugai Seiyaku Kabushiki Kaisha Drug-sustained release carrier
CN101137388A (zh) 2003-05-15 2008-03-05 犹他卅大学研究基金会 抗粘连复合材料及其应用方法
EP1694712A1 (de) 2003-12-04 2006-08-30 University of Utah Research Foundation Modifizierte makromoleküle und verfahren zu ihrer herstellung und verwendung
US8293890B2 (en) * 2004-04-30 2012-10-23 Advanced Cardiovascular Systems, Inc. Hyaluronic acid based copolymers
WO2006020231A2 (en) 2004-07-21 2006-02-23 Medtronic, Inc. Medical devices and methods for reducing localized fibrosis
ATE466602T1 (de) * 2005-02-14 2010-05-15 Yissum Res Dev Co Hydrogel-klebstoffe auf basis von depolymerisierten polysacchariden und verfahren zu dessen verwendung
DE602006019474D1 (de) * 2005-11-22 2011-02-17 Ard Sa Hren dafür und verwendungen davon
JP2009533455A (ja) * 2006-04-12 2009-09-17 マサチューセッツ インスティテュート オブ テクノロジー 癒着を阻害するための組成物および方法
EP1897543A1 (de) 2006-08-30 2008-03-12 Euro-Celtique S.A. Buprenorphine-Waffel zur Substitutionstherapie
DE102008005172A1 (de) * 2008-01-19 2009-07-23 Mike Ehrlich Pharmazeutische Zusammensetzung zur Verhinderung von post-operativen Adhäsionen
US8354274B2 (en) 2008-01-30 2013-01-15 Geron Corporation Synthetic surfaces for culturing cells in chemically defined media
US8329469B2 (en) 2008-01-30 2012-12-11 Geron Corporation Swellable (meth)acrylate surfaces for culturing cells in chemically defined media
EP2247718B1 (de) 2008-01-30 2019-01-16 Asterias Biotherapeutics, Inc. Synthetische oberflächen zur kultivierung von herzmuskelzellen aus stammzellen
SG188096A1 (en) 2008-01-30 2013-03-28 Geron Corp Synthetic surfaces for culturing stem cell derived oligodendrocyte progenitor cells
US8790701B2 (en) 2008-04-28 2014-07-29 Surmodics, Inc. Poly-α(1→4)glucopyranose-based matrices with hydrazide crosslinking
US8932622B2 (en) 2008-06-03 2015-01-13 Actamax Surgical Materials, Llc Tissue coating for preventing undesired tissue-to-tissue adhesions
CA2761909A1 (en) * 2009-05-14 2010-11-18 Central Michigan University Composition and method of preparation of polysaccharide gel-based artificial, biodegradable skin scaffolds
WO2010138074A1 (en) * 2009-05-29 2010-12-02 Hilborn Joens Hyaluronic acid based delivery systems
FR2972113A1 (fr) * 2011-03-02 2012-09-07 Philippe Zanchetta Utilisation d'un melange de polysaccharides specifiques comprenant de l'acide hyaluronique, de la chondroitine 6 sulfate, du dermatane sulfate et de l'heparine en cicatrisation cutanee
US10865383B2 (en) 2011-07-12 2020-12-15 Lineage Cell Therapeutics, Inc. Methods and formulations for orthopedic cell therapy
CN116059376A (zh) * 2014-05-30 2023-05-05 纽约州立大学研究基金会 促进骨形成的组合物和方法
KR102793214B1 (ko) 2019-01-30 2025-04-08 보오슈 앤드 롬 인코포레이팃드 가교화 중합체 망상구조물 및 이의 용도
CN110894302A (zh) * 2019-11-19 2020-03-20 福建医科大学孟超肝胆医院(福州市传染病医院) 一种基于亚胺键和酰腙键的抗菌水凝胶及其制备方法
CN113024845A (zh) * 2021-03-17 2021-06-25 广西医科大学 一种醛肼交联型抗菌水凝胶敷料的制备方法
CN114044548B (zh) * 2021-09-24 2023-08-11 杭州食疗晶元生物科技有限公司 一种快速降解水凝胶回收水资源的方法
CN114404648A (zh) * 2022-01-18 2022-04-29 张培华 促进糖尿病创面修复可降解抗菌止血水凝胶的制备方法
CN114486838B (zh) * 2022-02-18 2024-03-15 吉林大学 一种四环素类抗生素的检测方法
CN115252542A (zh) * 2022-07-29 2022-11-01 广州韵泽尚美生物科技有限公司 一种双疗程复合细胞生长因子水凝胶及其制备方法
CN119971127B (zh) * 2025-02-20 2025-10-28 金华市景迪医疗用品有限公司 一种创面修复用透明质酸钠凝胶复合物及其制备工艺

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
SE462454B (sv) * 1988-11-10 1990-06-25 Pharmacia Ab Maetyta foer anvaendning i biosensorer
SE8804074D0 (sv) * 1988-11-10 1988-11-10 Pharmacia Ab Sensorenhet och dess anvaendning i biosensorsystem
US6180288B1 (en) * 1997-03-21 2001-01-30 Kimberly-Clark Worldwide, Inc. Gel sensors and method of use thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO0206373A1 *

Also Published As

Publication number Publication date
CA2416698A1 (en) 2002-01-24
AU2001278943A1 (en) 2002-01-30
WO2002006373A1 (en) 2002-01-24

Similar Documents

Publication Publication Date Title
WO2002006373A1 (en) Hydrogel films and methods of making and using therefor
Kirker et al. Glycosaminoglycan hydrogel films as bio-interactive dressings for wound healing
Shi et al. Moldable hyaluronan hydrogel enabled by dynamic metal–bisphosphonate coordination chemistry for wound healing
Vercruysse et al. Hyaluronate derivatives in drug delivery
US8324184B2 (en) Anti-adhesion composites and methods of use thereof
US8691793B2 (en) Modified macromolecules and associated methods of synthesis and use
Kumar et al. Recent advances in the use of algal polysaccharides for skin wound healing
WO2008008859A2 (en) Macromolecules modified with electrophilic groups and methods of making and using thereof
Gao et al. One-pot synthesis of bioadhesive double-network hydrogel patch as disposable wound dressing
KR101379380B1 (ko) 생체적합성 히알루론산 가교물을 포함하는 약물 전달 조성물
EP3620186B1 (de) Biomaterialvorrichtungen zur geführten geweberegeneration
Mi et al. Fabrication of chondroitin sulfate‐chitosan composite artificial extracellular matrix for stabilization of fibroblast growth factor
CN111012803B (zh) 用于引导组织再生的生物材料装置和局部组合物
US9867909B2 (en) Multilayer implants for delivery of therapeutic agents
CN110624103B (zh) 用于治疗皮肤异常的生物材料装置和局部组合物
EP3620152A1 (de) Biomaterialvorrichtungen und topische zusammensetzungen zur behandlung von hautanomalien
US8563514B2 (en) Peptides and pharmaceutical compositions for treating connective tissue
Bebiano et al. Hydrogels as dynamic covalent networks for skin repair
Kirker Glycosaminoglycan hydrogels for wound healing
Ansari et al. Gellan gum–based wound dressings
Peng et al. Covalently crosslinked hyaluronan–polygalacturonic acid polymer as the drug carrier and its application in surgery
HK40020770A (en) Biomaterial devices and topical compositions for guided tissue regeneration
HK40019582A (en) Biomaterial devices and topical compositions for treatment of skin abnormalities

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20030217

AK Designated contracting states

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE TR

AX Request for extension of the european patent

Extension state: AL LT LV MK RO SI

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION HAS BEEN WITHDRAWN

18W Application withdrawn

Effective date: 20040519