Particles and compositions comprising the same for transfection

Abstract

The present invention relates to the localized delivery of nucleic acids to cells using polyelectrolyte assemblies in the form of particles that are prepared by layer-by-layer deposition of nucleic acid and specific polycation. It also relates to compositions comprising said particles and methods for the treatment of disorders or diseases by administration of such particles.

1 . A nanoparticle comprising a nanoparticle core and a multilayered shell, wherein:
 said multilayer shell is composed of a least one alternate bilayer of one positively charged layer comprising at least one chitosan derivative and one negatively charged layer comprising or consisting of at least one non-viral expression DNA vector,   where the outer layer of said multilayer shell is a positively charged layer comprising said at least one chitosan derivative, and   said chitosan derivatives of the multilayer shell are thiolated chitosan derivatives or a mixture of thiolated chitosan derivatives and non thiolated chitosan derivatives.   
     
     
         2 . The nanoparticle of  claim 1 , where the nanoparticle core is hollow or comprises an organic or inorganic material. 
     
     
         3 . The nanoparticle of  claim 1 , where the nanoparticle core is hollow or comprises a material selected from the group consisting of metals; metal-coated materials; metal oxides; plastics; ceramics; silicon; glasses;
 mica; graphite; hydrogels; polymers and combinations thereof, preferably gold, silver, platinum, and aluminum and combinations thereof, more preferably gold.   
     
     
         4 . The nanoparticle of  claim 1 , wherein said non-viral DNA vectors express at least one protein of interest or at least one interfering RNA capable of silencing a target protein. 
     
     
         5 . The nanoparticle of  claim 1 , where the DNA expression vectors further comprise a promoter specific of a target cell, preferably podocyte. 
     
     
         6 . The nanoparticle of  claim 1 , where the DNA expression vectors express at least one interfering RNA capable of silencing a mutated protein and express a corresponding functional protein which is encoded by a sequence to which said interfering RNAs do not bind. 
     
     
         7 . The nanoparticle of  claim 1 , where the said at least one non-viral expression DNA vector encodes at least one interfering RNA capable of silencing CMIP. 
     
     
         8 . The nanoparticle of  claim 1 , where the DNA expression vectors express at least one interfering RNA capable of silencing a mutated protein and express a corresponding functional protein which is encoded by a sequence to which said interfering RNAs do not bind, and wherein the protein is a protein of kidney slit diaphragm, preferably nephrin, podocine, or CD2AP. 
     
     
         9 . The nanoparticle of  claim 1 , wherein the nanoparticle size is in the range of 10 nm-1 μm. 
     
     
         10 . The nanoparticle of  claim 1 , wherein the number of negatively charged layers ranges from 1-15. 
     
     
         11 . A pharmaceutical composition comprising at least one nanoparticle and a pharmaceutically acceptable carrier, wherein the nanoparticle comprises a nanoparticle core and a multilayered shell, wherein:
 said multilayer shell is composed of a least one alternate bilayer of one positively charged layer comprising at least one chitosan derivative and one negatively charged layer comprising or consisting of at least one non-viral expression DNA vector,   where the outer layer of said multilayer shell is a positively charged layer comprising said at least one chitosan derivative, and   said chitosan derivatives of the multilayer shell are thiolated chitosan derivatives or a mixture of thiolated chitosan derivatives and non thiolated chitosan derivatives.   
     
     
         12 . (canceled) 
     
     
         13 . A method for the preparation of a nanoparticle comprising the steps consisting of:
 a. providing a core nanoparticle, as a template, that comprises or consists of a material selected from the group consisting of metals, metal-coated materials, metal oxides, plastics, ceramics, silicon, glasses, mica, graphite, hydrogels, polymers and combinations thereof, preferably the material is gold,   b. at least one step of layer-by-layer depositing alternatively a positively charged layer comprising at least one chitosan derivative and a negatively charged layer consisting of at least one non-viral expression DNA vector, on the surface of the core of step (a) to provide a polyelectrolyte assembly coating on the surface of the core, where the last deposited layer is a positively charged layer comprising said at least one chitosan derivative,    where said chitosan derivatives of the polyelectrolyte assembly are thiolated chitosan derivatives or a mixture of thiolated chitosan derivatives and non thiolated chitosan derivatives,   c. optionally, removal of the material of step (a).   
     
     
         14 . The method for the preparation of a nanoparticle of  claim 13 , where the first step of (b) is a step of depositing a positively charged layer comprising at least one chitosan derivative on the surface of the core of step (a), to provide a first layer coating on the surface of the core, followed by a step of depositing a negatively charged layer comprising at least one non-viral expression DNA vector, optionally said two steps can be reiterated alternatively, and where the last deposited layer is a positively charged layer comprising said at least one chitosan derivative. 
     
     
         15 . The method for the preparation of a nanoparticle of  claim 13 , where the thiolated chitosan derivatives are previously prepared by reacting chitosan derivatives with iminothiolane or by oxidation reaction of chitosan followed by reductive amination. 
     
     
         16 . A method for the treatment of a podocyte disease, wherein nanoparticles are administered to a subject in need of such treatment, wherein the nanoparticle comprises a nanoparticle core and a multilayered shell, wherein:
 said multilayer shell is composed of a least one alternate bilayer of one positively charged layer comprising at least one chitosan derivative and one negatively charged layer comprising or consisting of at least one non-viral expression DNA vector,   where the outer layer of said multilayer shell is a positively charged layer comprising said at least one chitosan derivative, and   said at least one chitosan derivative of the multilayer shell is a thiolated chitosan derivative or a mixture of thiolated chitosan derivatives and non thiolated chitosan derivatives; and   wherein the said at least one non-viral expression DNA vector encodes at least one interfering RNA capable of silencing CMIP.