Bioactive Hydrogel

Abstract

The invention relates to a bioactive hydrogel as a hybrid material of heparin and star-branched polyethylene glycol with functionalized end groups, wherein the heparin is bound directly by reaction of the carboxyl groups activated with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimides/N-hydroxysulfosuccinimide (EDC/s-NHS) with the terminal amino groups of the polyethylene glycol covalently by amide bonds.

What is claimed is: 
     
         1 . A bioactive hydrogel comprising a hybrid material of heparin and star-branched polyethylene glycol with functionalized end groups, wherein the heparin is bound covalently to the star-branched polyethylene glycol by amide bonds, by direct reaction of carboxyl groups of heparin activated with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimides/N-hydroxysulfosuccinimide (EDC/s-NHS) with terminal amino groups of the polyethylene glycol. 
     
     
         2 . The bioactive hydrogel according to  claim 1  which is in the form of a tubular structure. 
     
     
         3 . The bioactive hydrogel according to  claim 1  which is in the form of a film. 
     
     
         4 . The bioactive hydrogel according to  claim 3 , wherein the film is from about 80 to 2000 μm thick. 
     
     
         5 . The bioactive hydrogel according to  claim 1  which is in the form of a 2D cell culture support, wherein the hydrogel is covalently coupled to an inorganic carrier via a layer of reactive polymer. 
     
     
         6 . The bioactive hydrogel according to  claim 1 , wherein signal molecules are coupled reversibly, electrostatically to the heparin. 
     
     
         7 . The bioactive hydrogel according to  claim 6 , wherein the signal molecules are growth factors. 
     
     
         8 . The bioactive hydrogel according to  claim 7 , wherein the growth factors are bFGF or VEGF. 
     
     
         9 . The bioactive hydrogel according to  claim 1  comprising heparin with a chain length of 4000 to 14 000 MW. 
     
     
         10 . The bioactive hydrogel according to  claim 1  comprising star-PEG with a molecular weight of 10 000 to 19 000 MW. 
     
     
         11 . A tubular structure comprising the hydrogel according to  claim 1  inside a tubular carrier material. 
     
     
         12 . The bioactive hydrogel according to  claim 1  comprising heparin modified with an adhesion protein of sequence cycloRGDyK. 
     
     
         13 . A film comprising the hydrogel according to  claim 1  prepared by
 dropwise application of a defined amount of liquid gel materials on carrier surfaces which have been rendered hydrophobic, 
 gel formation after covering the carrier surfaces with cover surfaces which have been rendered hydrophobic, 
 transfer of the carrier surfaces to a washing solution and 
 removal of the films after swelling of the hydrogels of the carrier surfaces. 
 
     
     
         14 . A cell culture support comprising the hydrogel according to  claim 1  covalently coupled to an inorganic carrier via a layer of reactive polymer. 
     
     
         15 . A method of production of the bioactive hydrogel according to  claim 1 , wherein
 a) the components heparin,
 1-ethyl-3-(3-dimethylaminopropyl) carbodiimides EDC, 
 sulfo-N-hydroxysulfosuccinimide s-NHS and 
 star-shaped polyethylene glycol star-PEG are dissolved separately, after which 
   b) EDC and s-NHS are mixed together as activation reagents for the carboxyl groups of heparin, and   c) the heparin is activated by adding EDC and s-NHS and then   d) star-PEG is added and the mixture is homogenized and   e) gel formation then takes place, and   f) after that, the finished formed gel is washed.   
     
     
         16 . The method according to  claim 15 , wherein
 a) heparin,
 1-ethyl-3-(3-dimethylaminopropyl) carbodiimides EDC, 
 sulfo-N-hydroxysulfosuccinimide s-NHS and 
 star-shaped polyethylene glycol star-PEG are dissolved separately in deionized water at 4° C., after which 
   b) EDC and s-NHS are mixed together as activation reagents for the carboxyl groups of heparin in the ratio of two to one, and   c) activation of heparin takes place after adding EDC and s-NHS for 15 min at 4° C. and then   d) star-PEG is added and the mixture is homogenized at 8° C. for 15 min and   e) gel formation takes place for a period of 1 to 14 h at room temperature, and   f) then the finished formed gel is washed with phosphate-buffered sodium chloride solution or alternately in acid or basic salt solutions and in phosphate-buffered sodium chloride solution.   
     
     
         17 . The method according to  claim 15 , wherein a ratio of EDC and s-NHS relative to amino groups of star-PEG is 1.75 to 1, and a ratio of star-PEG to heparin is from 1 to 1 to 6 to 1. 
     
     
         18 . The method according to  claim 15  further comprising modifying the hydrogel after step f) with an adhesion protein cycloRGDyK, wherein the washed hydrogel is activated with a solution of EDC/s-NHS in 1/15 M phosphate buffer at pH=5 for 30 min at 4° C., after which the solution is replaced with a solution of 100 mM borate buffer of pH=8 containing 0.2 mg/ml cycloRGDyK and is immobilized for 2 h at room temperature, after which the modified hydrogel is rinsed with PBS. 
     
     
         19 . The method according to  claim 15  further comprising loading the hydrogel with growth factors b-FGF or VEGF, wherein the hydrogel is incubated with a solution of b-FGF or VEGF with a concentration of 1-5 μg/ml in PBS for 4 to 24 h at room temperature and is then washed in PBS. 
     
     
         20 . The method according to  claim 15 , wherein the heparin is modified with an adhesion protein or the finished formed gel of step (f) is modified with an adhesion protein.