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.

1 . A method of preparing silk fibroin microspheres, comprising:
 a. mixing a silk fibroin solution with a lipid composition;   b. lyophilizing the mixture;   c. combining the lyophilized material with a dehydration medium for a sufficient period of time to at least partially dehydrate the silk fibroin solution and induce β-sheet structures in the silk fibroin; and   d. removing at least a portion of the lipids to form silk fibroin microspheres.   
   
   
       2 . The method of  claim 1 , wherein the dehydration medium is selected from the group consisting of methanol, ethanol, propanol, acetone, chloroform, polyethylene glycol solutions, and salt solutions. 
   
   
       3 . The method of  claim 2 , wherein the dehydration medium is methanol or a solution of sodium chloride. 
   
   
       4 . The method of  claim 1 , further comprising the step of freeze-thawing the silk fibroin-lipid composition mixture before the lyophilization step. 
   
   
       5 . The method of  claim 1 , further comprising the step of suspending the microspheres in water or a buffer. 
   
   
       6 . The method of  claim 1 , wherein all or substantially all of the removable lipids have been removed. 
   
   
       7 . The method of  claim 1 , wherein about 15 to about 20% of the total lipids remain in the silk fibroin microspheres. 
   
   
       8 . The method of  claim 1 ; wherein less than about 5% of the total lipids remain in the silk fibroin microspheres. 
   
   
       9 . The method of  claim 1 , wherein the lipid composition is selected from the group consisting of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC); 1,2-dioleoyl-sn-glycero-3-phophoethanolamine (DOPE); 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC); and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). 
   
   
       10 . The method of  claim 1 , wherein the silk fibroin solution contains at least one therapeutic agent. 
   
   
       11 . The method of  claim 10 , wherein the therapeutic agent is encapsulated in the silk fibroin microspheres. 
   
   
       12 . The method of  claim 11 , wherein the therapeutic agent is in an active form and capable of administering a therapeutic effect upon release from the microsphere. 
   
   
       13 . The method of  claim 10 , wherein the therapeutic agent is selected from the group consisting of proteins, peptides, nucleic acids, peptide nucleic acids, aptamers, antibodies, growth factors, cytokines, enzymes, small molecules, and combinations thereof. 
   
   
       14 . The method of  claim 10 , wherein the therapeutic agent is selected from the group consisting of morphogenetic protein 2 (BMP-2), insulin-like growth factor I and II (IGF-I and II), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), fibroblast growth factors (FGFs), transforming growth factors-β (TGFs-β), transforming growth factors-α, erythropoietin (EPO), interferon α and γ, interleukins, tumor necrosis factor α and β, insulin, antibiotics, adenosine, and combinations thereof. 
   
   
       15 . A pharmaceutical formulation comprising the silk fibroin microspheres produced by the method of  claim 11 . 
   
   
       16 . The formulation of  claim 15 , wherein the average size of the microspheres is less than about 2.0 μm. 
   
   
       17 . A method of administering a therapeutic agent to a patient in need thereof, comprising the step of administering the pharmaceutical formulation of  claim 15  to the patient. 
   
   
       18 . The method of  claim 17 , wherein less than about 5% of the therapeutic agent is released into the patient from the microspheres over a period of one month. 
   
   
       19 . The method of  claim 17 , wherein at least about 5% of the therapeutic agent remains in the microspheres 10 days after administration. 
   
   
       20 . The method of  claim 17 , wherein the activity of the therapeutic agent remains at least at 50% one month after administration. 
   
   
       21 . A drug delivery composition comprising a therapeutic agent encapsulated in crosslinked silk fibroin microspheres, wherein the microspheres contain lipid components. 
   
   
       22 . The composition of  claim 21 , wherein the lipid components assist in controlling the release of the therapeutic agent from the microspheres. 
   
   
       23 . The composition of  claim 21 , wherein the microspheres contain less than about 5% lipids by weight. 
   
   
       24 . The composition of  claim 21 , wherein the weight percentage of microspheres in the total silk is at least 50%. 
   
   
       25 . The composition of  claim 21 , wherein the average size of the microspheres is less than about 2.0 μm. 
   
   
       26 . The composition of  claim 21 , wherein the crosslinking of the silk fibroin microspheres was induced by exposing the silk fibroin to methanol or sodium chloride. 
   
   
       27 . A method of encapsulating a biomaterial in silk fibroin microcapsules, comprising:
 a. mixing a solution comprising silk fibroin and a biomaterial with a lipid composition;   b. lyophilizing the mixture;   c. dehydrating the lyophilized material in a dehydration medium for a sufficient period of time to at least partially dehydrate the silk fibroin solution and induce β-sheet structures in the silk fibroin; and   d. removing at least a portion of the lipids to produce a biomaterial that has been encapsulated in silk fibroin microspheres.   
   
   
       28 . The method of  claim 27 , wherein the dehydration medium is selected from the group consisting of methanol, ethanol, propanol, acetone, chloroform, polyethylene glycol solutions, and salt solutions. 
   
   
       29 . The method of  claim 28 , wherein the dehydration medium is methanol or a solution of sodium chloride. 
   
   
       30 . The method of  claim 27 , further comprising the step of freeze-thawing the silk fibroin-lipid composition mixture before the lyophilization step. 
   
   
       31 . The method of  claim 27 , wherein the biomaterial is a therapeutic agent. 
   
   
       32 . The method of  claim 27 , wherein the biomaterial is an enzyme or enzyme-based electrode. 
   
   
       33 . The method of  claim 32 , wherein the enzyme or enzyme-based electrode is used in a field selected from the group consisting of tissue engineering, biosensors, the food industry, environmental control, and biomedical applications. 
   
   
       34 . A silk fibroin microsphere composition, comprising a therapeutic agent encapsulated in crosslinked silk fibroin microspheres, wherein at least 75% of the microspheres are spherical or substantially spherical, and wherein at least 75% of the microspheres have a diameter ranging from about 1.0 to about 3.0 microns. 
   
   
       35 . The composition of  claim 34 , wherein at least 90% of the microspheres are spherical or substantially spherical. 
   
   
       36 . The composition of  claim 34 , wherein at least 90% of the microspheres have a diameter ranging from about 1.0 to about 3.0 microns. 
   
   
       37 . The composition of  claim 36 , wherein at least 95% of the microspheres have a diameter ranging from about 1.0 to about 3.0 microns.