Silk microspheres for encapsulation and controlled release

Abstract

A method was developed to prepare silk fibroin microspheres using lipid vesicles as templates to efficiently load therapeutic agents in active form for controlled release. The lipids are subsequently removed through the use of a dehydration agent, such as methanol or sodium chloride, resulting in β-sheet structure dominant silk microsphere structures having about 2 μm in diameter. The therapeutic agent can be entrapped in the silk microspheres and used in pharmaceutical formulations for controlled-release treatments.

We claim: 
     
         1 . A composition comprising a biomaterial encapsulated in crosslinked silk fibroin microspheres, wherein the microspheres contain lipid components. 
     
     
         2 . The composition of  claim 1 , wherein the microspheres contain less than about 5% lipids by weight. 
     
     
         3 . The composition of  claim 1 , wherein the microspheres have an average size of about 1.0 to about 3.0 μm in diameter. 
     
     
         4 . The composition of  claim 1 , wherein the microspheres have a beta sheet content of at least about 50%. 
     
     
         5 . The composition of  claim 1 , wherein at least 75% of the microspheres are spherically or substantially spherical. 
     
     
         6 . The composition of  claim 1 , wherein the composition comprise a scaffold. 
     
     
         7 . The composition of  claim 1 , wherein the biomaterial comprises a therapeutic agent. 
     
     
         8 . The composition of  claim 7 , wherein the activity of the therapeutic agent in the microsphere remains at least about 50% one month after administration to a patient. 
     
     
         9 . The composition of  claim 1 , wherein the lipid components predominantly form multilamellar structures in the microspheres. 
     
     
         10 . The composition of  claim 9 , wherein the therapeutic agent comprises a lipophilic drug, a hydrophilic drug or both. 
     
     
         11 . The composition of  claim 1 , wherein the lipid components predominantly form unilamellar structures in the microspheres. 
     
     
         12 . The composition of  claim 11 , wherein the therapeutic agent comprises a hydrophilic drug. 
     
     
         13 . The composition of  claim 1 , wherein the biomaterial and the silk fibroin are present in separate layers in the microspheres. 
     
     
         14 . The composition of  claim 1 , wherein the biomaterial and the silk fibroin are present in domains of the silk fibroin microspheres. 
     
     
         15 . The composition of  claim 1 , wherein the silk fibroin microspheres further comprise a biocompatible polymer. 
     
     
         16 . The composition of  claim 1 , wherein the microspheres are hydrated. 
     
     
         17 . A method of making silk fibroin microspheres, comprising:
 a. mixing a silk fibroin solution with lipid molecules in a pre-determined ratio, wherein the lipid molecules do not pre-form a liposome or a vesicle prior to the mixing; and   b. lyophilizing the mixture;   
       thereby forming silk fibroin microspheres. 
     
     
         18 . The method of  claim 17 , wherein the pre-determined ratio is selected to determine arrangement of the lipid molecules in the silk fibroin microspheres. 
     
     
         19 . The method of  claim 17 , wherein when the pre-determined ratio is above a threshold ratio, lipid molecules predominantly form a multilamellar structure in the silk fibroin microspheres. 
     
     
         20 . The method of  claim 17 , wherein when the pre-determined ratio is below a threshold ratio, lipid molecules predominantly form a unilamellar structure in the silk fibroin microspheres. 
     
     
         21 . The method of  claim 17 , wherein the silk fibroin solution further comprises a biomaterial. 
     
     
         22 . The method of  claim 21 , wherein the biomaterial comprises a therapeutic agent. 
     
     
         23 . The method of  claim 22 , wherein the therapeutic agent is present in an active form and remains in the active form upon encapsulation in the silk fibroin microspheres. 
     
     
         24 . The method of  claim 17 , further comprising at least a freeze-thaw step prior to said lyophilizing the mixture, wherein the freeze-thaw step promotes mixing among the lipid molecules and the silk fibroin. 
     
     
         25 . The method of  claim 24 , further comprising combining the lyophilized silk fibroin microsphere with a dehydration medium (e.g., methanol or salt) for a sufficient period of time to at least partially dehydrate the silk fibroin and induce beta-sheet structures in the silk fibroin. 
     
     
         26 . The method of  claim 25 , further comprising removing at least some of the lipids. 
     
     
         27 . The method of  claim 26 , wherein about 15% to about 20% of the total lipids remain the silk fibroin microspheres. 
     
     
         28 . The method of  claim 26 , wherein less than about 5% of the total lipids remain in the silk fibroin microspheres. 
     
     
         29 . The method of  claim 17 , wherein the silk fibroin microspheres are not produced by spray-dry methods.