Organic solvent free compositions comprising protein-polymer conjugates and uses thereof

Abstract

The present invention provides protein-polymer conjugates, and methods for generating biocompatible scaffolds formed of hydrogels comprising the conjugates and use of the scaffolds for tissue regeneration. The present invention provides improved processes for the preparation of the conjugates, wherein the conjugates of the invention are preferably produced in an environmentally friendly process avoiding polar organic solvents.

1 . A protein-polymer conjugate comprising an extracellular matrix protein covalently bound to a synthetic polymer, wherein said synthetic polymer contains at least one polymerizable group, and wherein said conjugate is produced by a process comprising performing a reaction between an ECM protein and said polymer thereby forming the conjugate; and concentrating the conjugate without precipitating the conjugate with a polar organic solvent such that the process is substantially free of polar organic solvents. 
     
     
         2 . The protein-polymer conjugate according to  claim 1 , wherein the process comprises the steps of:
 (a) providing a solution comprising at least one denatured extracellular matrix protein;   providing a solution. comprising a synthetic polymer having polymerizable groups;   (c) mixing the extracellular matrix protein solution of (a) with the solution comprising the synthetic polymer of (b) to create covalent conjugates between sulfhydryls of the protein and the polymerizable groups; and   (d) concentrating the crude reaction mixture of step (c) without the concentrating process utilizing polar organic solvents.   
     
     
         3 . The protein-polymer conjugate according to  claim 2 , wherein the extracellular matrix protein comprises fibrinogen of bovine or porcine source. 
     
     
         4 . The protein-polymer conjugate according to  claim 2 , wherein the extracellular matrix protein comprises purified or partially purified fibrinogen of human source. 
     
     
         5 . The protein-polymer conjugate according to  claim 1 , wherein the synthetic polymer is selected from the group consisting of polyethylene glycol (PEG), hydroxyapatite/polycaprolactone (HA/PLC), polyglycolic acid (PGA), poly-L-lactic acid (PLLA), polymethyl-methacrylate (PMMA), polyhydroxyalkanoate (PHA), poly-4-hydroxybutyrate (P4HB), polypropylene fumarate (PPF), polyethylene glycol-dimethacrylate (PEG-DMA), polyethylene glycol-diacrylate (PEG-DA), beta-tricalcium phosphate (beta-TCP) and nonbiodegradable polytetrafluoroethylene (PTFE). 
     
     
         6 . The protein-polymer conjugate according to  claim 5 , wherein the synthetic polymer is polyethylene glycol-diacrylate (PEG-DA). 
     
     
         7 . A composition comprising the protein-polymer conjugate according to  claim 1  further comprising the same unconjugated synthetic polymer, wherein the molar ratio of synthetic polymer to protein is between 40:1 to 400:1. 
     
     
         8 . The composition according to  claim 7 , wherein the molar ratio of synthetic polymer to protein is between 100:1 to 250:1. 
     
     
         9 . (canceled) 
     
     
         10 . A process of producing a protein-polymer conjugate composition comprising the steps of:
 (a) dissolving at least one denatured extracellular matrix protein under basic pH conditions providing strong protein denaturation and reducing conditions;   (b) providing a solution comprising a synthetic polymer having a polymerizable group;   (c) mixing the extracellular matrix protein solution of (a) with the solution comprising synthetic polymer of (b) to create covalent conjugates between sulfhydryls of the protein and the polymerizable groups; and   (d) concentrating the crude reaction mixture of step (c) without the concentrating process utilizing polar organic solvents.   thereby producing a conjugate composition devoid of polar organic solvents.   
     
     
         11 . A hydrogel composition comprising a cross-linked composition according to  claim 7 , and wherein said hydrogel composition is substantially free of polar organic solvents. 
     
     
         12 . A hydrogel comprising cross-linked conjugates according to  claim 1 , said cross-linked form comprising a plurality of said conjugate molecules, wherein the conjugate molecules are covalently cross-linked to one another upon polymerization of said polymerizable group, and wherein said composition is substantially free of polar organic solvents. 
     
     
         13 . The hydrogel composition of  claim 11 , wherein said hydrogel is substantially free of acetone and ethanol. 
     
     
         14 . (canceled) 
     
     
         15 . The hydrogel composition according to  claim 12  having a shear storage modulus (G′) in the range of 0.05 kPa to 35 kPa. 
     
     
         16 . A stable ready for use liquid formulation in a non-cross linked form, comprising a protein-polymer conjugate composition according to  claim 7  and at least one polymerization initiator agent. 
     
     
         17 . The ready for use liquid formulation according to  claim 16 , wherein the at least one polymerization initiator agent is selected from the group consisting of bis(2,4,6-trimethylbenzoyl) phenylphosphine oxide (BAPO), 2,2-dimethoxy-2-phenylacetophenone (DMPA), camphorquinone (CQ), 1 -phenyl-1,2-propanedione (PPD), Cp′Pt(CH 3 ) 3  (Cp=eta5-C 5 H 4 CH 3 ), 2-hydroxy-1-[4-(hydroxyethoxy)phenyl]-2-methyl-1-propanone (e.g., IRGACURE™ 2959), dimethylaminoethyl methacrylate (DMAEMA), 2,2-dimethoxy-2-phenylacetophenone, benzophenone (BP), flavin containing compounds, and a combination of triethanolamine, N -vinylpyrrolidone and eosin Y. 
     
     
         18 . The ready for use liquid formulation according to  claim 16 , wherein the liquid formulation is held in a container selected from a single vial aliquot or a prefilled syringe. 
     
     
         19 . The ready for use liquid formulation according to  claim 16 , wherein the liquid formulation is stored under UV-vis light protected conditions in a pre-gelated form prior to initiation of polymerization. 
     
     
         20 . (canceled) 
     
     
         21 . The ready for use liquid formulation according to  claim 16 , wherein the liquid formulation forms a hydrogel upon exposure to light. 
     
     
         22 . (canceled) 
     
     
         23 . (canceled) 
     
     
         24 . The ready for use liquid formulation according to  claim 1 , wherein said composition comprises less than 10 ppm of acetone. 
     
     
         25 . (canceled) 
     
     
         26 . The ready for use liquid formulation according to  claim 16 , wherein said composition comprises less than 100 ppm of ethanol. 
     
     
         27 . (canceled)