Hydrogel Precursor Formulation and Production Process Thereof

Abstract

The present invention relates to a hydrogel precursor formulation, its process of production as well as a kit comprising said formulation and a method of production of a hydrogel using said formulation. The precursor formulation comprises at least one structural compound, preferably vinyl sulfone (acrylated branched) poly(ethylene glycol), and at least one linker compound, preferably a peptide with two cysteines, wherein said structural compound and said linker compound are polymerizable by a selective reaction between a nucleophile and a conjugated unsaturated bond or group. The precursor formulation is in the form of a powder.

1 - 22 . (canceled) 
     
     
         23 . A hydrogel precursor formulation comprising at least one structural compound and at least one linker compound, wherein said structural compound and said linker compound are polymerizable by a selective reaction between a nucleophile and a conjugated unsaturated bond or group, wherein the hydrogel precursor formulation is in the form of an unreacted powder. 
     
     
         24 . A hydrogel precursor formulation as claimed in  claim 23 , wherein the formulation additionally comprises at least one bioactive compound, preferably comprising an RGD peptide sequence, which is dimerizable with the structural compound through a selective reaction between a nucleophile and a conjugated unsaturated bond or group. 
     
     
         25 . A hydrogel precursor formulation as claimed in  claim 23 , wherein the structural compound is a multi-branched polyethylene glycol with vinyl sulfone end groups, preferably PEG-tri(vinyl sulfone) or PEG-tetra(vinyl sulfone). 
     
     
         26 . A hydrogel precursor formulation as claimed in  claim 23 , wherein the linker comprises at least two nucleophilic groups, preferably thiol groups. 
     
     
         27 . A hydrogel precursor formulation as claimed in  claim 23 , wherein the linker is a peptide comprising at least two cysteins, preferably located near the N- and C-terminus of the peptide. 
     
     
         28 . A precursor formulation as claimed in  claim 23 , wherein the structural compound and/or the linker compound are selected such that the selective reaction is hindered at or below pH 4.0. 
     
     
         29 . A precursor formulation as claimed in  claim 28 , wherein the reaction rate is at least twice as fast at pH 7.5 compared to pH 7.0. 
     
     
         30 . Process for the production of a hydrogel precursor formulation in form of a powder comprising the steps of:
 providing a first solution A of at least one structural compound;   providing a second solution B comprising at least one linker compound;   mixing of the solutions A and B; and   lyophilization of the resulting precursor solution   
       wherein the at least one structural compound and the at least one linker compound are polymerizable by a selective reaction between a nucleophile and a conjugated unsaturated bond or group, wherein the solutions A and B are mixed under conditions which hinder the selective reaction. 
     
     
         31 . Process as claimed in  claim 30 , wherein the solutions are mixed at or below pH 4.0, preferably at pH 3.5. 
     
     
         32 . Process as claimed in  claim 30 , wherein solution A comprises 5-10% w/v of the at least one structural compound, preferably 7.5% w/v. 
     
     
         33 . Process as claimed in  claim 30 , wherein solution B comprises 0.1-2% w/v of the at least one linker compound, preferably 1% w/v. 
     
     
         34 . Process as claimed in  claim 30 , wherein a solution C comprising at least one biologically active compound which is dimerizable with the structural compound by a selective reaction between a nucleophile and a conjugated unsaturated bond or group is added to solution A prior to the mixing of the solutions A and B. 
     
     
         35 . Process as claimed in  claim 34 , wherein solution C comprises 0.1-10% w/v of the at least one active compound, preferably 2%. 
     
     
         36 . Process as claimed in  claim 30 , wherein solution A, solution B and/or solution C are a solution of the at least one structural compound, the at least one linker compound or the at least one biologically active compound in distilled water. 
     
     
         37 . Process as claimed in  claim 30 , wherein the concentration of the compounds is selected such that the molar ratio of the nucleophile to the conjugated unsaturated bond or group is in the range of 0.8:1 to 1.3:1. 
     
     
         38 . Process as claimed in  claim 30 , wherein the precursor solution is subjected to filtration prior to the lyophilization step, preferably to sterile filtration. 
     
     
         39 . Process as claimed in  claim 30 , wherein the precursor solution is aliquoted and filled into containers, preferably under sterile conditions before the lyophilization step. 
     
     
         40 . Process as claimed in  claim 39 , wherein the containers are filled with sterile nitrogen gas and capped immediately after the lyophilization step. 
     
     
         41 . Kit of parts comprising at least one container filled with a hydrogel precursor formulation as claimed in  claim 23  and a container with a reaction buffer. 
     
     
         42 . Kit of parts as claimed in  claim 41 , wherein the reaction buffer has a pH of at least 7, preferably the reaction buffer has a pH between 7 and 8. 
     
     
         43 . Method of production of a hydrogel comprising the steps of
 Re-suspending a hydrogel precursor formulation as claimed in  claim 23  in a buffer having at least pH 7   Optionally adding a cell culture suspension to the precursor suspension   Casting of at least one gel with the precursor suspension   Polymerization of the at least one gel precursor for at least 30 minutes, preferably in an incubator at 37° C.