Vortex-induced silk fibroin gelation for encapsulation and delivery

Abstract

The present invention provided for a novel process of forming silk fibroin gels, and controlling the rate of β-sheet formation and resulting hydrogelation kinetics, by vortex treatment of silk fibroin solution. In addition, the vortex treatment of the present invention provides a silk fibroin gel that may be reversibly shear-thinned, enabling the use of these approach for precise control of silk self-assembly, both spatially and temporally. Active agents, including biological materials, viable cells or therapeutic agents, can be encapsulated in the hydrogels formed from the processes, and be used as delivery vehicles. Hence, the present invention provide for methods for silk fibroin gelation that are useful for biotechnological applications such as encapsulation and delivery of active agents, cells, and bioactive molecules.

1 . A method of forming silk fibroin gel, comprising vortexing a silk fibroin solution for a sufficient period of time to initiate intermolecular self-assembly of silk fibroin β-sheet structure, wherein substantial silk fibroin gelation occurs in less than about 16 hours after the vortexing. 
     
     
         2 . The method of  claim 1 , wherein said vortexing yields a solid phase and an aqueous phase, and wherein the method further comprises removing said solid phase and allowing gelation of the aqueous phase. 
     
     
         3 . The method of  claim 1  or  2 , wherein the silk fibroin in the solution has a concentration about 6 wt % or lower. 
     
     
         4 . The method of  claim 3 , wherein the silk fibroin in the solution has a concentration ranging from about 1.0 wt % to about 5.2 wt %. 
     
     
         5 . The method of  claim 1 , wherein the gelation time is controlled by adjusting one or more of (a) the time period of the vortex treatment; (b) the concentration of the silk fibroin in solution; or (c) the temperature of the silk fibroin solution after the vortex treatment. 
     
     
         6 . (canceled) 
     
     
         7 . The method of  claim 1 , further comprising
 introducing at least one active agent to the silk fibroin solution before substantial gelation occurs in the silk fibroin solution;   allowing the silk-fibroin to gel, forming a silk fibroin gel-encapsulated active agent.   
     
     
         8 . The method of  claim 7 , wherein the active agent is a therapeutic agent or a biological material, selected from the group consisting of cells, proteins, peptides, nucleic acids, nucleic acid analogs, nucleotides or oligonucleotides, peptide nucleic acids, aptamers, antibodies or fragments or portions thereof, antigens or epitopes, hormones, hormone antagonists, growth factors or recombinant growth factors and fragments and variants thereof, cell attachment mediators, cytokines, enzymes, antibiotics or antimicrobial compounds, viruses, toxins, prodrugs, chemotherapeutic agents, small molecules, drugs, and combinations thereof. 
     
     
         9 . The method of  claim 7 , wherein the active agent is a cell selected from the group consisting of progenitor cells or stem cells, smooth muscle cells, skeletal muscle cells, cardiac muscle cells, epithelial cells, endothelial cells, urothelial cells, fibroblasts, myoblasts, oscular cells, chondrocytes, chondroblasts, osteoblasts, osteoclasts, keratinocytes, kidney tubular cells, kidney basement membrane cells, integumentary cells, bone marrow cells, hepatocytes, bile duct cells, pancreatic islet cells, thyroid, parathyroid, adrenal, hypothalamic, pituitary, ovarian, testicular, salivary gland cells, adipocytes, precursor cells, and combinations thereof. 
     
     
         10 . The method of  claim 9 , the active agent further comprises a cell growth media. 
     
     
         11 . The method of  claim 7 , wherein the silk fibroin gel-encapsulated active agent is suitable for a biodelivery device. 
     
     
         12 . The method of  claim 7 , wherein the silk fibroin gel-encapsulated active agent is suitable for a medical implant or a tissue repair material. 
     
     
         13 . A method of delivering a reversibly shear-thinned silk fibroin gel to a target site, comprising:
 vortexing a silk fibroin solution for a sufficient period of time to initiate gelation, wherein the silk fibroin undergoes substantial gelation after the vortex treatment to form a silk fibroin gel;   introducing the silk fibroin gel through a shear-inducing delivery device to the target site while applying a shear force to shear-thin the silk fibroin gel; and   removing the shear force, whereupon the shear-thinned silk fibroin gel recovers from shear-thinning and re-gels.   
     
     
         14 . The method of  claim 13 , further comprising adding an active agent to said silk fibroin solution. 
     
     
         15 . The method of  claim 13  wherein said reversible shear-thinning silk fibroin gel is delivered locally to said target site with high spatial precision. 
     
     
         16 . The method of  claim 15 , wherein said reversible shear-thinning silk fibroin gel is delivered locally to said target site by injection through a needle. 
     
     
         17 . The method of  claim 15 , wherein the method is suitable for implanting a medical implant or a tissue repair material. 
     
     
         18 . A method for homogeneous delivery of at least one active agent to a target site, comprising:
 vortexing a silk fibroin solution for a sufficient period of time to initiate gelation;   introducing at least one active agent to the silk fibroin solution either before vortexing or before substantial gelation occurs in the silk fibroin solution, thereby forming a silk fibroin gel-encapsulated active agent that may be shear-thinned reversibly;   introducing to the target site the active agent-encapsulated silk fibroin gel through a shear-inducing delivery device to the target site while applying a shear force to shear-thin the agent-encapsulated silk fibroin gel; and   removing the shear force, whereupon the shear-thinned silk fibroin gel-encapsulated agent recovers gel form, thereby distributing the active agent in the gel form at the target site homogeneously.   
     
     
         19 . The method of  claim 18 , wherein said reversible shear-thinning silk fibroin gel is delivered locally to said target site with high spatial precision. 
     
     
         20 . The method of  claim 19 , wherein said reversible shear-thinning silk fibroin gel is delivered locally to said target site by injection. 
     
     
         21 . The method of  claim 18 , wherein the at least one silk fibroin gel-encapsulated active agent is delivered to the target site in vivo. 
     
     
         22 . (canceled) 
     
     
         23 . (canceled) 
     
     
         24 . (canceled) 
     
     
         25 . (canceled)