Sulfated glycosaminoglycan biomaterials as proteoglycan mimics
Polymer conjugates are provided that are capable of mimicking functions of natural proteoglycans found in the extracellular matrix of connective tissues. The polymer conjugates of the invention have utility in treating a subject suffering soft tissue degenerative conditions. Also provided are simple and scalable chemical processes for the preparation of the polymer conjugates of the invention.
1. A method of preparing a water-soluble sulfated GAG conjugate, comprising the steps of:
i) contacting a sulfated GAG in aqueous solution at a concentration of 2 wt %-20 wt % with an effective amount of linking agent, wherein the molar ratio of GAG hydroxyl groups to linking agent is less than that required for gel formation, under conditions sufficient to react the linking agent with the sulfated GAG to form a solution comprising a soluble polymer characterized by pendant linking groups;
ii) adding additional sulfated GAG to the solution of step (i) in an amount sufficient to react with a portion of the pendant linking groups to form a soluble polymer conjugate with a branched architecture characterized by remaining pendant linking groups; and
iii) adding a modifier characterized by a reactive moiety under conditions sufficient to react the remaining pendant linking groups with the reactive modifier to form a water-soluble sulfated GAG branched conjugate bearing the modifier.
2. The method of claim 1 , wherein the product of step i) has a substantially linear structure.
3. The method of claim 1 , wherein the sulfated GAG conjugate product of step-iii has a molecular weight of 100,000 Da to 5,000,000 Da.
4. The method of claim 1 , wherein the sulfated GAG of step-i has a molecular weight of about 1,000 Da, 5,000 Da, 10,000 Da, 15,000 Da, 20,000 Da, 30,000 Da, 40,000 Da, 50,000 Da, 100,000 Da, or a range including any two of these numbers, preferably between 5,000 Da and 30,000 Da.
5. The method of claim 1 , wherein the ratio of GAG hydroxyl group equivalents, present in the solution of step i), to moles of linking agent is 0.8 to 4.0.
6. The method of claim 1 , wherein the sulfated GAG of step i) is identical to that added in step ii).
7. The method of claim 1 , wherein the pendant reactive group is vinyl.
8. The method of claim 1 , wherein the linking agent is divinyl sulfone.
9. The method of claim 1 , wherein pendant reactive groups are present in the soluble polymer formed in step ii) on between 2% and 30% of disaccharide repeat units, as determined by proton NMR.
10. The method of claim 1 , wherein the total amount of sulfated GAG added in steps i and ii is apportioned in a ratio between 15/85 and 80/20.
11. The method of claim 1 , wherein the reactive moiety of the modifier introduced in step-iii is an amine.
12. The method of claim 1 , wherein the modifier comprises a targeting moiety.
13. The method of claim 1 , wherein the modifier is lactosylamine.
14. The method of claim 1 , wherein the modifier comprises a hydrophobic moiety.
15. The method of claim 1 , wherein the water-soluble GAG conjugate comprises one or more residual unreacted linker groups capable of forming covalent bonds with mammalian tissue in situ.