Functionalized derivatives of hyaluronic acid, formation of hydrogels in situ using same, and methods for making and using same

Abstract

Methods for chemical modification of hyaluronic acid, formation of amine or aldehyde functionalized hyaluronic acid, and the cross-linking thereof to form hydrogels are provided. Functionalized hyaluronic acid hydrogels of this invention can be polymerized in situ, are biodegradable, and can serve as a tissue adhesive, a tissue separator, a drug delivery system, a matrix for cell cultures, and a temporary scaffold for tissue regeneration.

1 . A composition comprising derivatives of hyaluronic acid (ha) comprising disaccharide: subunits, wherein at least one of said disaccharide subunits is a substituted disaccharide subunit having a substitution at a carboxyl group, such that the substituted disaccharide subunit is of the formula:  
       
         
           
           
               
               
           
         
         wherein each of R′ and R″ is a side chain comprising one or more functional groups selected from the group consisting of hydrogen; bioactive peptide; alkyl; aryl; alkylaryl; arylalkyl; substituted alkylaryl containing an atom or atoms of oxygen, nitrogen, sulfur, or phosphorous; substituted arylalkyl containing an atom or atoms of oxygen, nitrogen, sulfur, phosphorous, halogen, or metal ion; and substituted heterocycle containing an atom or atoms of oxygen, nitrogen, sulfur, phosphorous, halogen or metal ion;  
         wherein said functional groups within or among each of said R′ or R″ side chains are either bound directly to each other or are separated by a member selected from the group consisting of ether, keto, amino, oxycarbonyl, sulfate, sulfoxide, carboxamide, alkyne and alkene; and  
         wherein each of said R′ and R″ side chains terminates with a terminal functional group selected from hydrogen, peptide, aldehyde, amine, hydrazide, maleimide, sulfhydryl, active ester, ester, carboxylate, imidoester, halogen and hydroxyl,  
         wherein said terminal functional groups of each of said R′ and R″ side chains may be bound directly to each other, with the proviso that when one of R′ or R″ is hydrogen, halogen or univalent metal ion, then R′ and R″ may not be bound directly to each other, and  
         wherein said derivatives of hyaluronic acid are covalently crosslinked via one of said terminal functional groups.  
       
     
     
         2 . The composition of  claim 1 , wherein at least one of said terminal functional groups is selected from peptide, aldehyde, amine, hydrazide, maleimide, sulfhydryl and active ester, whereby said composition is amenable to crosslinking.  
     
     
         3 . The composition of  claim 1 , wherein the molecular weight of said composition is at least 100,000 daltons.  
     
     
         4 . The composition of  claim 1 , wherein the molecular weight of said composition is at most 100,000 daltons.  
     
     
         5 . The composition of  claim 1 , wherein the molecular weight of said composition is at least 1,000,000 daltons.  
     
     
         6 . The composition of  claim 1 , wherein said composition is water soluble.  
     
     
         7 . A hydrogel of crosslinked HA derivatives, wherein said HA derivatives are compositions according to  claim 1 .  
     
     
         8 . The hydrogel of crosslinked HA derivatives of  claim 7 , wherein said hydrogel is biodegradable.  
     
     
         9 . A tissue adhesive comprising a hydrogel of  claim 7 , wherein the side chain is selected from activated ester, aldehyde and maleimide.  
     
     
         10 . A tissue adhesive comprising a hydrogel of  claim 7 , wherein the crosslinked HA derivatives are formed using a cross-linker selected from polyvalent active ester, aldehyde and maleimide.  
     
     
         11 . A tissue adhesive comprising a hydrogel of  claim 7 , wherein the crosslinked hydrogel is formed in the presence of at least one member selected from growth factors, cytokines, drugs, and bioactive peptides.  
     
     
         12 . The tissue adhesive of  claim 11 , wherein the crosslinked hydrogel is formed in the presence of a growth factor and wherein the growth factor is TGF-beta or BMP-2.  
     
     
         13 . A matrix for cell cultures comprising the hydrogel of  claim 7 , wherein the crosslinked HA-derivatives are formed using a cross-linker selected from polyvalent active ester, aldehyde, amine, arylazide, maleimide, and sulfhydryl.  
     
     
         14 . A matrix for cell cultures comprising the hydrogel of  claim 7 , wherein the crosslinked hydrogel is formed in the presence of at least one member selected from growth factors, cytokines, drugs, and bioactive peptides.  
     
     
         15 . The matrix of  claim 14 , wherein the crosslinked hydrogel is formed in the presence of a growth factor and wherein the growth factor is TGF-beta or BMP-2.  
     
     
         16 . A matrix for a scaffold comprising the hydrogel of  claim 7 , wherein the crosslinked HA-derivatives are formed using a cross-linker selected from polyvalent active ester, aldehyde, amine, maleimide and sulfhydryl.  
     
     
         17 . A matrix for a scaffold comprising the hydrogel of  claim 7 , wherein the crosslinked hydrogel is formed in the presence of at least one member selected from growth factors, cytokines, drugs, and bioactive peptides.  
     
     
         18 . The matrix of  claim 17 , wherein the crosslinked hydrogel is formed in the presence of a growth factor and wherein the growth factor is TGF-beta or BMP-2.  
     
     
         19 . The matrix of  claim 17 , wherein the matrix further comprises cells.  
     
     
         20 . A method of forming a crosslinked biodegradable material under physiological conditions from a composition comprising derivatives of hyaluronic acid comprising disaccharide subunits, wherein at least one of said disaccharide subunits is a substituted disaccharide subunit having a substitution at a carboxyl group, such that the substituted disaccharide subunit is of the formula:  
       
         
           
           
               
               
           
         
         wherein each of R′ and R″ is a side chain comprising one or more functional groups selected from the group consisting of hydrogen; bioactive peptide; alkyl; aryl; alkylaryl; arylalkyl; substituted alkylaryl containing an atom or atoms of oxygen, nitrogen, sulfur, or phosphorous; substituted arylalkyl containing an atom or atoms of oxygen, nitrogen, sulfur, phosphorous, halogen, or metal ion; and substituted heterocycle containing an atom or atoms of oxygen, nitrogen, sulfur, phosphorous, halogen or metal ion;  
         wherein said functional groups within or among each of said R′ or R″ side chains are either bound directly to each other or are separated by a member selected from the group consisting of ether, keto, amino, oxycarbonyl, sulfate, sulfoxide, carboxamide, alkyne and alkene; and  
         wherein each of said R′ and R″ side chains terminates with a terminal functional group selected from the group consisting of hydrogen, peptide, aldehyde, amine, arylazide, hydrazide, maleimide, sulfhydryl, active ester, ester, carboxylate, imidoester, halogen and hydroxyl; and  
         wherein said derivatives of hyaluronic acid are modified to an extent of more than 10%,  
       
     
     
         21 . The method of  claim 20 , wherein the material is formed in situ.  
     
     
         22 . A method of regenerating tissue or causing tissue adhesion, comprising contacting a tissue with a composition comprising derivatives of hyaluronic acid comprising disaccharide subunits, wherein at least one of said disaccharide subunits is a substituted disaccharide subunit having a substitution at a carboxyl group, such that the substituted disaccharide subunit is of the formula:  
       
         
           
           
               
               
           
         
         wherein each of R′ and R″ is a side chain comprising one or more functional groups selected from the group consisting of hydrogen; bioactive peptide; alkyl; aryl; alkylaryl; arylalkyl; substituted alkylaryl containing an atom or atoms of oxygen, nitrogen, sulfur, or phosphorous; substituted arylalkyl containing an atom or atoms of oxygen, nitrogen, sulfur, phosphorous, halogen, or metal ion; and substituted heterocycle containing an atom or atoms of oxygen, nitrogen, sulfur, phosphorous, halogen or metal ion;  
         wherein said functional groups within or among each of said R′ or R″ side chains are either bound directly to each other or are separated by a member selected from the group consisting of ether, keto, amino, oxycarbonyl, sulfate, sulfoxide, carboxamide, alkyne and alkene; and  
         wherein each of said R′ and R″ side chains terminates with a terminal functional group selected from the group consisting of hydrogen, peptide, aldehyde, amine, arylazide, hydrazide, maleimide, sulfhydryl, active ester, ester, carboxylate, imidoester, halogen and hydroxyl; and  
         wherein said derivatives of hyaluronic acid are modified to an extent of more than 10%.  
       
     
     
         23 . The method of  claim 22 , wherein the method comprising one or more of repairing cartilage, stemming hemorraging, reconstructing nerves and/or vessels, and anchoring skin, vascular or cartilage transplants or grafts.  
     
     
         24 . A method of delivering cells or bioactive materials, comprising delivering the cells or bioactive materials in a composition comprising derivatives of hyaluronic acid comprising disaccharide subunits, wherein at least one of said disaccharide subunits is a substituted disaccharide subunit having a substitution at a carboxyl group, such that the substituted disaccharide subunit is of the formula:  
       
         
           
           
               
               
           
         
         wherein each of R′ and R″ is a side chain comprising one or more functional groups selected from the group consisting of hydrogen; bioactive peptide; alkyl; aryl; alkylaryl; arylalkyl; substituted alkylaryl containing an atom or atoms of oxygen, nitrogen, sulfur, or phosphorous; substituted arylalkyl containing an atom or atoms of oxygen, nitrogen, sulfur, phosphorous, halogen, or metal ion; and substituted heterocycle containing an atom or atoms of oxygen, nitrogen, sulfur, phosphorous, halogen or metal ion;  
         wherein said functional groups within or among each of said R′ or R″ side chains are either bound directly to each other or are separated by a member selected from the group consisting of ether, keto, amino, oxycarbonyl, sulfate, sulfoxide, carboxamide, alkyne and alkene; and  
         wherein each of said R′ and R″ side chains terminates with a terminal functional group selected from the group consisting of hydrogen, peptide, aldehyde, amine, arylazide, hydrazide, maleimide, sulfhydryl, active ester, ester, carboxylate, imidoester, halogen and hydroxyl; and  
         wherein said derivatives of hyaluronic acid are modified to an extent of more than 10%.  
       
     
     
         25 . The method of  claim 24 , wherein the bioactive material is a growth factor.  
     
     
         26 . The method of  claim 24 , wherein the cells or bioactive material are delivered to treat a pathological wound condition.  
     
     
         27 . A method of generating tissue separation or preventing tissue adhesions, comprising contacting tissue with a composition comprising derivatives of hyaluronic acid comprising disaccharide subunits, wherein at least one of said disaccharide subunits is a substituted disaccharide subunit having a substitution at a carboxyl group, such that the substituted disaccharide subunit is of the formula:  
       
         
           
           
               
               
           
         
         wherein each of R′ and R″ is a side chain comprising one or more functional groups selected from the group consisting of hydrogen; bioactive peptide; alkyl; aryl; alkylaryl; arylalkyl; substituted alkylaryl containing an atom or atoms of oxygen, nitrogen, sulfur, or phosphorous; substituted arylalkyl containing an atom or atoms of oxygen, nitrogen, sulfur, phosphorous, halogen, or metal ion; and substituted heterocycle containing an atom or atoms of oxygen, nitrogen, sulfur, phosphorous, halogen or metal ion;  
         wherein said functional groups within or among each of said R′ or R″ side chains are either bound directly to each other or are separated by a member selected from the group consisting of ether, keto, amino, oxycarbonyl, sulfate, sulfoxide, carboxamide, alkyne and alkene; and  
         wherein each of said R′ and R″ side chains terminates with a terminal functional group selected from the group consisting of hydrogen, peptide, aldehyde, amine, arylazide, hydrazide, maleimide, sulfhydryl, active ester, ester, carboxylate, imidoester, halogen and hydroxyl; and  
         wherein said derivatives of hyaluronic acid are modified to an extent of more than 10%,.  
       
     
     
         28 . The method of  claim 27 , wherein the tissue separation or prevention of tissue adhesions is effective for joint lubrication, prevention of eye irritation or serving as a barrier to cells.  
     
     
         29 . A method of augmenting tissue, comprising contacting tissue with a composition comprising derivatives of hyaluronic acid comprising disaccharide subunits, wherein at least one of said disaccharide subunits is a substituted disaccharide subunit having a substitution at a carboxyl group, such that the substituted disaccharide subunit is of the formula:  
       
         
           
           
               
               
           
         
         wherein each of R′ and R″ is a side chain comprising one or more functional groups selected from the group consisting of hydrogen; bioactive peptide; alkyl; aryl; alkylaryl; arylalkyl; substituted alkylaryl containing an atom or atoms of oxygen, nitrogen, sulfur, or phosphorous; substituted arylalkyl containing an atom or atoms of oxygen, nitrogen, sulfur, phosphorous, halogen, or metal ion; and substituted heterocycle containing an atom or atoms of oxygen, nitrogen, sulfur, phosphorous, halogen or metal ion;  
         wherein said functional groups within or among each of said R′ or R″ side chains are either bound directly to each other or are separated by a member selected from the group consisting of ether, keto, amino, oxycarbonyl, sulfate, sulfoxide, carboxamide, alkyne and alkene; and  
         wherein each of said R′ and R″ side chains terminates with a terminal functional group selected from the group consisting of hydrogen, peptide, aldehyde, amine, arylazide, hydrazide, maleimide, sulfhydryl, active ester, ester, carboxylate, imidoester, halogen and hydroxyl; and  
         wherein said derivatives of hyaluronic acid are modified to an extent of more than 10%.  
       
     
     
         30 . The method of  claim 29 , wherein the tissue augmentation is effective for filling dermal creases or lip reconstruction.  
     
     
         31 . A method of sustaining drug release, comprising conjugating one or more pharmacological compounds to a composition comprising derivatives of hyaluronic acid comprising disaccharide subunits, wherein at least one of said disaccharide subunits is a substituted disaccharide subunit having a substitution at a carboxyl group, such that the substituted disaccharide subunit is of the formula:  
       
         
           
           
               
               
           
         
         wherein each of R′ and R″ is a side chain comprising one or more functional groups selected from the group consisting of hydrogen; bioactive peptide; alkyl; aryl; alkylaryl; arylalkyl; substituted alkylaryl containing an atom or atoms of oxygen, nitrogen, sulfur, or phosphorous; substituted arylalkyl containing an atom or atoms of oxygen, nitrogen, sulfur, phosphorous, halogen, or metal ion; and substituted heterocycle containing an atom or atoms of oxygen, nitrogen, sulfur, phosphorous, halogen or metal ion;  
         wherein said functional groups within or among each of said R′ or R″ side chains are either bound directly to each other or are separated by a member selected from the group consisting of ether, keto, amino, oxycarbonyl, sulfate, sulfoxide, carboxamide, alkyne and alkene; and  
         wherein each of said R′ and R″ side chains terminates with a terminal functional group selected from the group consisting of hydrogen, peptide, aldehyde, amine, arylazide, hydrazide, maleimide, sulfhydryl, active ester, ester, carboxylate, imidoester, halogen and hydroxyl; and  
         wherein said derivatives of hyaluronic acid are modified to an extent of more than 10%,  
         wherein the one or more pharmacological compounds are conjugated to the derivatives of hyaluronic acid.  
       
     
     
         32 . The method of  claim 31 , wherein the composition is in a free form.  
     
     
         33 . The method of  claim 31 , wherein the pharmacological compound is selected from anti-inflammatories, analgesics, steroids, cardiovascular agents, anti-tumor agents, immunosuppressants, sedatives, anti-bacterials, anti-fungals and anti-virals.