Methods and compositions for preparing a silk microsphere

Abstract

Provided herein relates to methods and compositions for preparing a silk microsphere and the resulting silk microsphere. In some embodiments, the methods and compositions described herein are all aqueous, which can be used for encapsulating an active agent in a silk microsphere, while maintaining activity of the active agent during processing. In some embodiments, the resulting silk microsphere can be used for sustained delivery of an active agent encapsulated therein.

What is claimed is: 
     
         1 . A method of preparing a silk microsphere, the method comprising:
 inducing formation of beta-sheet structure of fibroin in a silk solution; and   inducing formation of a microsphere from the silk solution.   
     
     
         2 . The method of  claim 1 , wherein said formation of the beta-sheet structure of fibroin and the microsphere are induced simultaneously. 
     
     
         3 . The method of  claim 1  or  2 , wherein said formation of the beta-sheet structure of fibroin in the silk solution is induced by sonication. 
     
     
         4 . The method of any of  claims 1 - 3 , wherein said formation of the microsphere from the silk solution is induced by atomization of the silk solution. 
     
     
         5 . The method of  claim 2 , wherein said formation of the beta-sheet structure of fibroin and the microsphere are induced simultaneously by flowing the silk solution through a flow-through chamber that is ultrasonically activated or an ultrasonic atomizer. 
     
     
         6 . The method of  claim 5 , wherein the silk solution is flowed through the flow-through chamber or the ultrasonic atomizer at a flow rate of about 0.001 mL/min to about 5 mL/min. 
     
     
         7 . The method of  claim 6 , wherein the silk solution is flowed through the flow-through chamber or the ultrasonic atomizer at the flow rate of about 0.05 mL/min to about 0.3 mL/min. 
     
     
         8 . The method of any of  claims 3 - 7 , wherein the sonication is performed at a frequency of at least about 10 kHz, or about 20 kHz to about 40 kHz. 
     
     
         9 . The method of any of  claims 3 - 8 , wherein the sonication power output ranges from about 1 watt to about 50 watts, or from about 2 watts to about 20 watts. 
     
     
         10 . The method of any of  claims 1 - 9 , further comprising freezing the silk microsphere. 
     
     
         11 . The method of  claim 10 , wherein the silk microsphere can be frozen by exposing the silk microsphere to a sub-zero temperature. 
     
     
         12 . The method of  claim 10  or  11 , wherein the silk microsphere is exposed to the sub-zero temperature by collecting the silk microsphere in a container cooled by a cooling agent. 
     
     
         13 . The method of any of  claims 1 - 12 , further comprising subjecting the silk microsphere to lyophilization. 
     
     
         14 . The method of any of  claims 1 - 13 , wherein the silk microsphere has a porosity of at least about 30%. 
     
     
         15 . The method of any of  claims 1 - 14 , wherein the silk microsphere has a pore size of about 1 nm to about 500 μm, or 10 nm to about 50 μm. 
     
     
         16 . The method of any of  claims 1 - 15 , wherein the silk solution comprises silk fibroin at a concentration of about 1% (w/v) to about 30% (w/v). 
     
     
         17 . The method of  claim 16 , wherein the silk solution comprises silk fibroin at a concentration of about 5% (w/v). 
     
     
         18 . The method of any of  claims 1 - 17 , wherein the silk microsphere comprises an active agent. 
     
     
         19 . The method of  claim 18 , wherein the active agent includes a temperature-sensitive active agent. 
     
     
         20 . The method of  claim 18  or  19 , wherein the active agent is a therapeutic agent. 
     
     
         21 . The method of  claim 20 , wherein the therapeutic agent is selected from the group consisting of small organic or inorganic molecules; saccharides; oligosaccharides;
 polysaccharides; biological macromolecules, e.g., peptides, proteins, and peptide analogs and derivatives; peptidomimetics; nucleic acids; nucleic acid analogs and derivatives; antibodies and antigen binding fragments thereof; an extract made from biological materials such as bacteria, plants, fungi, or animal cells; animal tissues;   naturally occurring or synthetic compositions; and any combinations thereof.   
     
     
         22 . The method of  claim 20  or  21 , wherein the therapeutic agent includes bevacizumab, memantine, or a combination thereof. 
     
     
         23 . The method of any of  claims 18 - 22 , wherein the active agent is present in the silk microsphere in an amount of about 0.1% (w/w) to about 50%(w/w). 
     
     
         24 . The method of  claim 23 , wherein the active agent is present in the silk microsphere in an amount of about 1%(w/w) to about 30%(w/w). 
     
     
         25 . The method of any of  claims 18 - 24 , wherein the active agent is present in the silk solution. 
     
     
         26 . The method of any of  claims 1 - 25 , wherein the silk microsphere comprises silk in an amount of about 30%(w/w) to about 100%(w/w), of the total weight of the microsphere. 
     
     
         27 . The method of any of  claims 1 - 26 , wherein the silk solution further comprises an additive. 
     
     
         28 . The method of  claim 27 , wherein a weight ratio of the additive to silk in the silk solution is about 1:100 to about 100:1. 
     
     
         29 . The method of  claim 27  or  28 , wherein the weight ratio of the additive to silk in the silk solution is about 1:10 to about 10:1. 
     
     
         30 . The method of any of  claims 27 - 29 , wherein the additive is selected from the group consisting of a biopolymer, a porogen, a magnetic particle, a plasticizer, a detection label, and any combinations thereof. 
     
     
         31 . The method of any of  claims 27 - 30 , wherein the additive is a plasticizer. 
     
     
         32 . The method of  claim 30  or  31 , wherein the plasticizer induces formation of beta-sheet crystalline structure of fibroin in the silk. 
     
     
         33 . The method of any of  claims 30 - 32 , wherein the plasticizer is selected from the group consisting of glycerol, polyvinyl alcohol, collagen, gelatin, alginate, chitosan, hyaluronic acid, polyethylene glycol, polyethylene oxide, and any combinations thereof. 
     
     
         34 . The method of any of  claims 1 - 33 , further comprising subjecting the silk microsphere to a post-treatment. 
     
     
         35 . The method of  claim 34 , wherein the post-treatment further induces formation of beta-sheet crystalline structure of fibroin in the silk microsphere. 
     
     
         36 . The method of any of  claims 34 - 35 , wherein the post-treatment is selected from the group consisting of alcohol immersion, water vapor annealing, heat annealing, and any combinations thereof. 
     
     
         37 . The method of any of  claims 34 - 36 , wherein the silk microsphere prior to the post-treatment has a water solubility of less than 50%. 
     
     
         38 . The method of any of  claims 34 - 37 , wherein the silk microsphere prior to the post-treatment has a water solubility of less than 30%. 
     
     
         39 . The method of any of  claims 1 - 38 , wherein the silk microsphere has a size of about 10 μm to about 1000 μm. 
     
     
         40 . The method of any of  claims 1 - 39 , wherein the silk microsphere has a size of about 50 μm to about 100 μm. 
     
     
         41 . The method of any of  claims 4 - 40 , wherein the atomization comprises using a spray nozzle system of a droplet generator. 
     
     
         42 . The method of any of  claims 4 - 41 , wherein the atomization comprises syringe extrusion, coaxial air flow method, mechanical disturbance method, electrostatic force method, or electrostatic bead generator method. 
     
     
         43 . The method of any of  claims 4 - 42 , wherein the atomization comprises spraying the silk solution through a nozzle of an air driven droplet generating encapsulation unit. 
     
     
         44 . The method of any of  claims 1 - 43 , wherein a shape or a size of the silk microsphere is varied by varying one or more parameters selected from the group consisting of nozzle diameter; flow rate of the spray; pressure of the spray; distance of the container collecting the silk microsphere from the nozzle; concentration of the silk solution; power of sonication waves; sonication treatment time; and any combinations thereof. 
     
     
         45 . A silk microsphere prepared using the method of any of  claims 1 - 44 . 
     
     
         46 . The silk microsphere of  claim 45 , wherein the silk microsphere releases at least about 5% of the active agent loaded therein over a period of at least about 10 days. 
     
     
         47 . A pharmaceutical composition comprising the silk microsphere of any of  claims 45 - 46  and a pharmaceutically acceptable excipient. 
     
     
         48 . The composition of  claim 47 , wherein the composition is formulated to be injectable. 
     
     
         49 . A method of sustained delivery in vivo of a therapeutic agent comprising administering the pharmaceutical composition of any of  claims 47 - 48  to a subject in need thereof. 
     
     
         50 . A composition comprising a silk microsphere having a size of about 10 μm to about 2000 μm. 
     
     
         51 . The composition of  claim 50 , wherein the size of the silk microsphere is about 30 μm to about 1000 μm. 
     
     
         52 . The composition of  claim 50  or  51 , wherein the silk microsphere is water-insoluble. 
     
     
         53 . The composition of any of  claims 50 - 52 , wherein the water-insoluble silk microsphere has a beta sheet crystalline content of at least about 50% or higher. 
     
     
         54 . The composition of any of  claims 50 - 53 , wherein the silk microsphere further comprises an active agent. 
     
     
         55 . The composition of  claim 54 , wherein the active agent is solvent-sensitive and/or temperature-sensitive active agent. 
     
     
         56 . The composition of any of  claims 50 - 55 , wherein the active agent is selected from the group consisting of small organic or inorganic molecules; saccharides; oligosaccharides; polysaccharides; biological macromolecules, e.g., peptides, proteins, and peptide analogs and derivatives; peptidomimetics; nucleic acids; nucleic acid analogs and derivatives; antibodies and antigen binding fragments thereof; therapeutic agents; an extract made from biological materials such as bacteria, plants, fungi, or animal cells; animal tissues; naturally occurring or synthetic compositions; and any combinations thereof. 
     
     
         57 . The composition of  claim 56 , wherein the therapeutic agent comprises bevacizumab, memantine, or a combination thereof. 
     
     
         58 . The composition of any of  claims 54 - 57 , wherein the silk microsphere comprising the active agent has a release profile of about 1% release to about 50% release of the total loading of the active agent over a period of 5 days. 
     
     
         59 . The composition of  claim 58 , wherein the release profile comprises a sustained release. 
     
     
         60 . The composition of  claim 59 , wherein the release profile further comprises an immediate release. 
     
     
         61 . The composition of any of  claims 50 - 60 , wherein the active agent is present in the silk microsphere in an amount of about 0.1% (w/w) to about 50%(w/w). 
     
     
         62 . The composition of any of  claims 50 - 61 , wherein the silk microsphere comprises silk fibroin in an amount of about 10%(w/w) to about 100%(w/w), of the total weight of the microsphere. 
     
     
         63 . The composition of any of  claims 50 - 62 , wherein the silk microsphere further comprises an additive. 
     
     
         64 . The composition of  claim 63 , wherein a weight ratio of the additive to silk fibroin in the silk microsphere is about 1:100 to about 100:1. 
     
     
         65 . The composition of  claim 63  or  64 , wherein the additive is selected from the group consisting of a biopolymer, a porogen, a magnetic particle, a plasticizer, a detection label, and any combinations thereof. 
     
     
         66 . The composition of  claim 65 , wherein the additive comprises a plasticizer. 
     
     
         67 . The composition of  claim 66 , wherein the plasticizer induces formation of beta-sheet crystalline structure of fibroin in the silk. 
     
     
         68 . The composition of  claim 66  or  67 , wherein the plasticizer is selected from the group consisting of glycerol, polyvinyl alcohol, collagen, gelatin, alginate, chitosan, hyaluronic acid, polyethylene glycol, polyethylene oxide, and any combinations thereof. 
     
     
         69 . The composition of  claim 68 , wherein the additive comprises glycerol. 
     
     
         70 . The composition of  claim 69 , wherein the ratio of glycerol to silk fibroin the silk microsphere ranges from about 1:10 to about 10:1. 
     
     
         71 . The composition of any of  claims 50 - 70 , wherein the composition is injectable. 
     
     
         72 . The composition of any of  claims 50 - 71 , wherein the composition is a pharmaceutical composition. 
     
     
         73 . The composition of  claim 72 , further comprises a pharmaceutically acceptable excipient. 
     
     
         74 . The composition of  claim 72  or  73 , wherein the pharmaceutical composition is in a form of a tablet, a capsule, a lozenge, powder, paste, granules, a liquid, a solution, gel, or any combinations thereof. 
     
     
         75 . The composition of any of  claims 50 - 74 , wherein the silk microsphere is porous.