Carrier-free biologically-active protein nanostructures

Abstract

The present disclosure provides compositions and methods for efficient and effective protein delivery in vitro and in vivo. In some aspects, proteins are reversibly crosslinked to each other and/or modified with functional groups and protected from protease degradation by a polymer-based or silica-based nanoshell.

What is claimed is: 
     
         1 . A method of producing a nanostructure the method comprising:
 (a) contacting a plurality of proteins with a degradable linker under conditions that permit reversible covalent crosslinking of the proteins to each other through the degradable linker, thereby producing a nanostructure.   
     
     
         2 . The method of  claim 1 , further comprising:
 (b) contacting the nanostructure with a polymer under conditions that permit conjugation of the polymer to proteins of the nanostructure.   
     
     
         3 . The method of  claim 1 , wherein the conditions of (a) include contacting the proteins with the degradable linker in an aqueous buffer:
 (i) at a temperature of 4° C. to 25° C.; and/or   (ii) for 30 minutes to one hour.   
     
     
         4 . The method of  claim 2 , wherein the conditions of (b) include contacting the nanostructure with the polymer in an aqueous buffer:
 (i) at a temperature of 4° C. to 25° C.; and/or   (ii) for 30 minutes to one hour.   
     
     
         5 . (canceled) 
     
     
         6 . The method of  claim 1 , wherein the conditions of (a) do not include one or more selected from the group consisting of:
 (i) contacting the proteins with the degradable linker at a temperature of greater than 30° C., and   (ii) contacting the proteins with the degradable linker in an organic solvent.   
     
     
         7 . The method of  claim 2 , wherein the conditions of (b) do not include one or more selected from the group consisting of:
 (i) contacting the nanostructure with the polymer at a temperature of greater than 30° C.; and   (ii) contacting the nanostructure with the polymer in an organic solvent.   
     
     
         8 . The method of  claim 1 , wherein the degradable linker degrades under physiological conditions to release the crosslinked proteins. 
     
     
         9 .- 13 . (canceled) 
     
     
         14 . The method of  claim 1 , wherein the degradable linker is a redox responsive linker that comprises a disulfide bond. 
     
     
         15 . The method of  claim 2 , wherein the polymer is a hydrophilic polymer. 
     
     
         16 . The method of  claim 15 , wherein the hydrophilic polymer comprises polyethylene glycol (PEG), poly(ethylene oxide), polylactic acid, poly(lactic-co-glycolic acid), polyglutamate, or polylysine. 
     
     
         17 . (canceled) 
     
     
         18 . The method of  claim 1 , wherein the dry size of the nanostructure is less than 100 nm in diameter, or the dry size of the nanostructure is 50-60 nm in diameter. 
     
     
         19 . (canceled) 
     
     
         20 . The method of  claim 1 , wherein the hydrodynamic size of the nanostructure is less than 100 nm in diameter, or the hydrodynamic size of the nanostructure is 80-90 nm in diameter. 
     
     
         21 . (canceled) 
     
     
         22 . The method of  claim 3 , wherein the concentration of the protein in the aqueous buffer is 10 mg/mL to 50 mg/mL. 
     
     
         23 . The method of  claim 1 , wherein the nanostructure is monodispersed. 
     
     
         24 . The method of  claim 1 , wherein the nanostructure does include albumin. 
     
     
         25 . The method of  claim 1 , wherein the weight percentage of protein in the nanostructure is at least 75%, at least 80%, at least 85%, or at least 90%. 
     
     
         26 .- 107 . (canceled) 
     
     
         108 . The method of  claim 1 , wherein the proteins are immunostimulatory proteins. 
     
     
         109 . The method of  claim 108 , wherein the immunostimulatory proteins are immunostimulatory cytokines, antigens, or immunostimulatory antibodies or antibody fragments. 
     
     
         110 . The method of  claim 109 , wherein immunostimulatory proteins are immunostimulatory cytokines, and the immunostimulatory cytokines are IL-2, IL-7, IL-15, IL-15 superagonist, IFN-gamma, IFN-alpha, GM-CSF, or FLT3-ligand. 
     
     
         111 . The method of  claim 108 , wherein the immunostimulatory proteins are fusion proteins. 
     
     
         112 . The method of  claim 2 , wherein the conditions permit covalent conjugation of the polymer to the proteins of the nanostructure. 
     
     
         113 . The method of  claim 2 , wherein the conditions permit non-covalent conjugation of the polymer to the proteins of the nanostructure. 
     
     
         114 . The method of  claim 2 , wherein the conditions permit surface conjugation of the polymer to the nanostructure.