Nanoparticle delivery vehicle

Abstract

A nanoparticle delivery vehicle, comprising a nanoparticle, an active agent and a nuclear localization signal and methods of modulating gene expression and protein expression employing the nanoparticle delivery vehicle. A representative method includes providing a nanoparticle delivery vehicle comprising a nanoparticle having a diameter of about 30 nm or less, an active agent and a nuclear localization signal; and contacting a target cell with the nanoparticle delivery vehicle, whereby an active agent is delivered to the nucleus of a target cell. Another representative method includes providing a nanoparticle delivery vehicle comprising a nanoparticle having a diameter greater than or equal to about 30 nm, an active agent and a nuclear localization signal; and contacting a target cell with the nanoparticle delivery vehicle, whereby an active agent is delivered to the cytoplasm of a cell.

1 - 49 . (canceled) 
     
     
         50 . A method of delivering an active agent to a cell, the method comprising:
 (a) providing a nanoparticle delivery vehicle comprising a nanoparticle scaffold, an active agent and a plurality of targeting agents, the plurality of targeting agents comprising a nuclear localization signal and one or more extracellular targeting agents; and   (b) contacting a target cell with the nanoparticle delivery vehicle, whereby an active agent is delivered to the target cell.   
     
     
         51 . (canceled) 
     
     
         52 . (canceled) 
     
     
         53 . The method of  claim 50 ,
 wherein the active agent is capable of interacting with a target nucleic acid sequence whose expression is to be modulated;   the target cell comprises the target nucleic acid sequence whose expression is to be modulated and   the method further comprises modulating the expression of the target nucleic acid sequence.   
     
     
         54 . The method of  claim 53 , further comprising determining a degree to which the target nucleic acid sequence is expressed. 
     
     
         55 . The method of  claim 54 , wherein the determining is by a technique selected from the group consisting of SDS-PAGE, enzyme activity assay, ELISA-based assay, spectroscopic assay, northern blot, Southern blot and radiological-based assay. 
     
     
         56 - 58 . (canceled) 
     
     
         59 . The method of  claim 50 ,
 wherein the active agent comprises a single stranded antisense nucleic acid sequence complementary to a nucleic acid sequence encoding a target protein;   the target cell comprises the nucleic acid sequence encoding the target protein and the method further comprises   modulating the expression of the target protein.   
     
     
         60 . The method of  claim 59 , further comprising determining a degree to which the target protein is expressed. 
     
     
         61 . The method of  claim 59 , wherein the determining is by a technique selected from the group consisting of SDS-PAGE, enzyme activity assay, ELISA-based assay, spectroscopic assay, northern blot, Southern blot and radiological-based assay. 
     
     
         62 - 64 . (canceled) 
     
     
         65 . The method of  claim 50 ,
 wherein the active agent comprises a ligand for which a wild-type transcription component has greater affinity than a natural ligand of the wild-type transcription component;   the cell comprises the wild-type transcription component and   the method further comprises modulating transcription in the cell.   
     
     
         66 . The method of  claim 65 , wherein the ligand for which a wild-type transcription component has greater affinity than a natural ligand of the wild-type transcription component comprises a morpholino oligonucleotide. 
     
     
         67 . The method of  claim 65 , wherein ligand for which a wild-type transcription component has greater affinity than a natural ligand of the wild-type transcription component comprises modified phosphodiester bonds. 
     
     
         68 . The method of  claim 65 , wherein the ligand for which a wild-type transcription component has greater affinity than a natural ligand of the wild-type transcription component has the ability to interact with a nucleic acid sequence encoding a regulatory protein to thereby form at least one of (a) an untranscribable three-dimensional structure and (b) untranslatable three-dimensional structure. 
     
     
         69 . The method of  claim 65 , wherein the nanoparticle delivery vehicle further comprises a tether sequence attached to, and disposed between, the ligand for which a wild-type transcription component has greater affinity than a natural ligand of the wild-type transcription component and the nanoparticle scaffold. 
     
     
         70 . The method of  claim 65 , further comprising determining a degree to which transcription is modulated. 
     
     
         71 . The method of  claim 70 , wherein the determining is by a technique selected from the group consisting of SDS-PAGE, enzyme activity assay, ELISA-based assay, spectroscopic assay, northern blot, Southern blot and radiological-based assay. 
     
     
         72 - 78 . (canceled) 
     
     
         79 . The method of  claim 50 ,
 wherein the active agent comprises a nucleic acid sequence known or suspected to alter the splicing pattern for a target gene;   the cell comprises the target gene and   the method further comprises modulating RNA splicing in the cell.   
     
     
         80 . The method of  claim 79 , wherein the nucleic acid sequence known or suspected to alter the splicing pattern for a target gene comprises a morpholino oligonucleotide. 
     
     
         81 . The method of  claim 79 , wherein the nucleic acid sequence known or suspected to alter the splicing pattern for a target gene comprises modified phosphodiester bonds. 
     
     
         82 . The method of  claim 79 , wherein the nanoparticle delivery vehicle further comprises a tether sequence attached to, and disposed between, the nucleic acid sequence known or suspected to alter the splicing pattern for a target gene and the nanoparticle scaffold. 
     
     
         83 . The method of  claim 79 , further comprising determining a degree to which the RNA splicing in a sample is modulated. 
     
     
         84 . The method of  claim 83 , wherein the determining is by a technique selected from the group consisting of SDS-PAGE, enzyme activity assay, ELISA-based assay, spectroscopic assay, northern blot, Southern blot and radiological-based assay. 
     
     
         85 - 90 . (canceled) 
     
     
         91 . The method of  claim 50 ,
 wherein the active agent is a single stranded nucleic acid sequence complementary to a nucleic acid sequence of an mRNA sequence encoding a protein of interest;   the cell comprises the mRNA sequence encoding a protein of interest and   the method further comprises modulating the translation of the mRNA sequence encoding a protein of interest in the cell.   
     
     
         92 . The method of  claim 91 , wherein the nanoparticle scaffold has a diameter of less than or equal to 1,000 nm. 
     
     
         93 . The method of  claim 91 , wherein the nanoparticle scaffold ranges in diameter from about 30 nm to about 1,000 nm. 
     
     
         94 . The method of  claim 91 , wherein the single stranded nucleic acid sequence complementary to the nucleic acid sequence of the mRNA sequence encoding a protein of interest comprises a morpholino oligonucleotide. 
     
     
         95 . The method of  claim 91 , wherein the single stranded nucleic acid sequence complementary to the nucleic acid sequence of the mRNA sequence encoding a protein of interest comprises modified phosphodiester bonds. 
     
     
         96 . The method of  claim 91 , wherein the single stranded nucleic acid sequence complementary to the nucleic acid sequence of the mRNA sequence encoding a protein of interest has the ability to interact with the nucleic acid sequence of the mRNA sequence encoding a protein of interest to thereby form at least one of (a) an untranscribable three-dimensional structure and (b) untranslatable three-dimensional structure. 
     
     
         97 . The method of  claim 91 , wherein the nanoparticle delivery vehicle further comprises a tether sequence attached to, and disposed between, the single stranded nucleic acid sequence complementary to the nucleic acid sequence of the mRNA sequence encoding a protein of interest and the nanoparticle scaffold. 
     
     
         98 . The method of  claim 91 , further comprising determining a degree to which the concentration of a regulatory protein in solution is modulated. 
     
     
         99 . The method of  claim 98 , wherein the determining is by a technique selected from the group consisting of SDS-PAGE, enzyme activity assay, ELISA-based assay, spectroscopic assay, northern blot, Southern blot and radiological-based assay. 
     
     
         100 - 108 . (canceled) 
     
     
         109 . The method of  claim 50 , wherein the nanoparticle delivery vehicle has a diameter that facilitates entry into the nucleus via a nuclear pore. 
     
     
         110 . The method of  claim 50 , wherein the nanoparticle scaffold has a diameter of about 30 nm or less. 
     
     
         111 . The method of  claim 50 , wherein the active agent is selected from the group consisting of a nucleic acid sequence, a nucleotide, a protein, a peptide sequence, and a small molecule. 
     
     
         112 . The method of  claim 111 , wherein the nucleic acid sequence is selected from the group consisting of a RNA, a DNA, a peptide nucleic acid sequence, and a chemically modified nucleic acid sequence. 
     
     
         113 . The method of  claim 111 , wherein the nucleic acid sequence is a full length gene or fragment thereof. 
     
     
         114 . The method of  claim 111 , wherein the nucleic acid sequence is an oligonucleotide. 
     
     
         115 . The method of  claim 111 , wherein the nucleic acid sequence has a length between about 20 and about 50 nucleotides. 
     
     
         116 . The method of  claim 111 , wherein the nucleic acid sequence is selected from a single stranded nucleic acid sequence and a double stranded nucleic acid sequence. 
     
     
         117 . The method of  claim 50 , wherein the active agent is selected from a chemotherapeutic; a toxin; a radiotherapeutic; a radiosensitizing agent; an imaging agent; a diagnostic agent; a gene therapy vector; an antisense nucleic acid construct; a transcription factor decoy; and an agent known to interact with one of an intracellular protein, a nucleic acid, a soluble ligand, and an insoluble ligand. 
     
     
         118 . The method of  claim 117 , wherein the antisense nucleic acid construct is an antisense oligodeoxynucleotide. 
     
     
         119 . The method of  claim 117 , wherein the active agent is an antisense nucleic acid construct and delivering an active agent to the cell prevents or delays one of infection, inflammation and tumor formation. 
     
     
         120 . The method of  claim 50 , wherein the nanoparticle delivery vehicle comprises two or more different active agents. 
     
     
         121 . The method of  claim 50 , wherein the nanoparticle delivery vehicle is disposed in a pharmaceutically acceptable diluent. 
     
     
         122 . The method of  claim 50 , wherein the nanoparticle delivery vehicle is in a pharmaceutical composition for administration to a subject or a sample. 
     
     
         123 . The method of  claim 122 , wherein the pharmaceutical composition is administered to the subject via parenteral administration, intravenous administration, or infusion directly into a desired target tissue. 
     
     
         124 . The method of  claim 123 , wherein the desired target tissue is a solid tumor or other neoplastic tissue. 
     
     
         125 . The method of  claim 50 , wherein the cell is suspended in a cell culture and the nanoparticle delivery vehicle is added to a cell culture medium. 
     
     
         126 . The method of  claim 50 , wherein the nanoparticle scaffold comprises a material selected from the group consisting of cadmium selenide, titanium, titanium dioxide, tin, tin oxide, silicon, silicon dioxide, iron, iron III  oxide, silver, nickel, gold, copper, aluminum, steel, cobalt-chrome alloy, titanium alloy, brushite, tricalcium phosphate, alumina, silica, zirconia, diamond, polystyrene, silicone rubber, a polypeptide, polycarbonate, polyurethanes, polypropylenes, polymethylmethacrylate, polyvinyl chloride, polyesters, polyethers, and polyethylene.