Nanoparticles containing plymeric nucleic acid homologs, pharmaceutical composition and articles of manufacture containing same and methods of use thereof

Abstract

A nanoparticle capable of delivery of an encapsulated molecule into a living cell. The nanoparticle includes an encapsulation media and an isolated nucleic acid homolog sequence. The encapsulation media is primarily polymeric. The nanoparticles release the encapsulated molecule over an extended period of time. Further disclosed are pharmaceutical compositions and articles of manufacture including nanoparticles and methods of preparing and using the nanoparticles.

1 . Nanoparticles for delivery of an encapsulated nucleic acid molecule into a living cell, the nanoparticles comprising:
 (a) poly(DL-lactide-co-glycolide) (PLGA);   (b) an isolated, bioactive nucleic acid molecule sequence encapsulated within the PLGA, wherein said nanoparticles are capable of releasing said isolated, bioactive nucleic acid molecule over an extended period of time.   
     
     
         2 . The nanoparticles of  claim 1 , further comprising a salt of a divalent cation. 
     
     
         3 . The nanoparticles of  claim 2 , wherein said divalent cation is calcium. 
     
     
         4 . The nanoparticles of  claim 1 , wherein said nucleic acid molecule is selected from the group consisting of RNA and DNA. 
     
     
         5 . The nanoparticles of  claim 4 , wherein the nucleic acid molecule includes phosphorothioate derivates at one or ore positions available for phosphothioate linkages. 
     
     
         6 . The nanoparticles of  claim 1 , further comprising an amino acid sequence that imparts cellular specificity. 
     
     
         7 . The nanoparticles of  claim 4 , wherein the nucleic acid molecule comprises a coding sequence that is transcribed in the cell. 
     
     
         8 . The nanoparticles of  claim 4 , wherein the nucleic acid molecule comprises an anti-sense RNA. 
     
     
         9 . The nanoparticles of  claim 4 , wherein the nucleic acid molecule comprises an oligonucleotide. 
     
     
         10 . The nanoparticles of  claim 1 , having an average particle size between 200 to 700 nm in diameter. 
     
     
         11 . The nanoparticles of  claim 1 , further comprising mannitol. 
     
     
         12 . The nanoparticles of  claim 1 , further comprising a liposome formulation of neutral and cationic lipids. 
     
     
         13 . The nanoparticles of  claim 1 , further comprising a liposome selected from the group consisting of: DOSPA, DOTAP and DOPE. 
     
     
         14 . The nanoparticles of  claim 1 , wherein the PLGA has a lactide-glycolide ratio of 50:50. 
     
     
         15 . The nanoparticles of  claim 13 , wherein the ratio of DOSPA or DTAP:DOPE is 1:1. 
     
     
         16 . A pharmaceutical composition comprising:
 (a) nanoparticles according to  claim 1 ; and   (b) a physiologically acceptable excipient.   
     
     
         17 . A method of making nanoparticles according to  claim 1 , the method comprising
 (a) mixing the nucleic acid molecule with a solution comprising PLGA under conditions such that the PLGA forms nanoparticles encapsulating the nucleic acid molecule; and   (b) isolating the nanoparticles of the solution.   
     
     
         18 . The method of  claim 17 , wherein the mixing comprises homogenizing the solution containing PLGA and the nucleic molecule. 
     
     
         19 . The method of  claim 17 , further comprising the step of emulsifying the PLGA-nucleic acid solution of step (a) with a PVA solution. 
     
     
         20 . The method of  claim 19 , wherein the PVA solution further comprises a salt of a divalent cation. 
     
     
         21 . The method of  claim 20 , wherein the divalent cation is calcium. 
     
     
         22 . The method of  claim 17 , wherein the isolating comprises evaporating the solution containing the nanoparticles. 
     
     
         23 . A method of delivering nanoparticles to a living cell, the method comprising
 (a) preparing nanoparticles according to  claim 17 ; and   (b) introducing the nanoparticles into the cell.   
     
     
         24 . The method of  claim 21 , wherein the introducing is accomplished by a method selected from the group consisting of injection, introduction of a sweating balloon, implantation of a stent, direct implantation of the nanoparticles within the subject and oral delivery. 
     
     
         25 . The method of  claim 17 , wherein the solution further comprises mannitol. 
     
     
         26 . A method of blocking expression of a specific sequence, the method comprising
 administering nanoparticles according to  claim 1 , wherein the nucleic acid molecule specifically hybridizes to at least a portion of the specific sequence so that a multistranded complex is formed between the nucleic acid molecule and the specific sequence.   
     
     
         27 . The method of  claim 26 , wherein the specific sequence is a genomic DNA sequence and the multistranded complex is a triple helix. 
     
     
         28 . The method of  claim 27 , wherein the specific sequence is a PNA sequence or an mRNA molecule. 
     
     
         29 . The method of  claim 26 , wherein the multistranded complex is enzymatically degraded.