Cationic shell-crosslinked knedel-like nanoparticles for highly efficient gene and oligonucleotide transfection of mammalian cells.

In this work, a robust synthetic nanostructure was designed for the effective packaging of DNA and it was shown to be an efficient agent for cell transfection. An amphiphilic block copolymer, poly(acrylamidoethylamine)(128)-b-polystyrene(40) (PAEA(128)-b-PS(40)), was synthesized, micellized in water and shell-crosslinked using a diacid-derivatized crosslinker, to give cationic shell-crosslinked nanoparticles (cSCKs) with a mean hydrodynamic diameter of 14 +/- 2 nm. A series of discrete complexes of the cSCKs with plasmid DNA (pDNA) was able to be formed over a broad range of polymer amine:pDNA phosphate ratios (N/P ratio), 2:1-20:1. The sizes of the complexes and their ability to fully bind the pDNA were dependent upon the N/P ratio, as characterized by dynamic light scattering (DLS), transmission electron microscopy (TEM) and gel retardation assay. A luciferase activity assay and EGFP expression were used to evaluate intracellular delivery of a splice-correcting phosphorothioate and genetic material, respectively, by the cSCKs, which indicated that an N/P ratio of 6:1 gave the highest transfection. It was shown by both luciferase activity assay (48 h) and EGFP transfection data that high transfection efficiencies were achieved for HeLa cells transfected by cSCK/CCUCUUACCUCAGUUACA and cSCK/pEGFP-N1 plasmid, respectively. The cSCK/pEGFP-N1 plasmid transfection efficiency of 27% far exceeded the performance of Polyfect (PAMAM dendrimers), which achieved only 12% transfection efficiency, under the same conditions. Cytotoxicities for the cSCKs were evaluated for HeLa and CHO cells.

[1]  S. Fukushima,et al.  Biocompatible micellar nanovectors achieve efficient gene transfer to vascular lesions without cytotoxicity and thrombus formation , 2007, Gene Therapy.

[2]  Kinam Park,et al.  Polycation gene delivery systems: escape from endosomes to cytosol , 2003, The Journal of pharmacy and pharmacology.

[3]  Yu Matsumoto,et al.  Polyplex micelles with cyclic RGD peptide ligands and disulfide cross-links directing to the enhanced transfection via controlled intracellular trafficking. , 2008, Molecular pharmaceutics.

[4]  Kai Qi,et al.  64Cu-labeled folate-conjugated shell cross-linked nanoparticles for tumor imaging and radiotherapy: synthesis, radiolabeling, and biologic evaluation. , 2005, Journal of nuclear medicine : official publication, Society of Nuclear Medicine.

[5]  Shinsuke Sando,et al.  A quantum dot conjugated sugar ball and its cellular uptake. On the size effects of endocytosis in the subviral region. , 2004, Journal of the American Chemical Society.

[6]  D. Scherman,et al.  A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[7]  Kazunori Kataoka,et al.  Charge-conversion ternary polyplex with endosome disruption moiety: a technique for efficient and safe gene delivery. , 2008, Angewandte Chemie.

[8]  Daniel G. Anderson,et al.  Structure/property studies of polymeric gene delivery using a library of poly(beta-amino esters). , 2005, Molecular therapy : the journal of the American Society of Gene Therapy.

[9]  Daniel G. Anderson,et al.  Structure/property studies of polymeric gene delivery using a library of poly(β-amino esters). , 2005, Molecular therapy : the journal of the American Society of Gene Therapy.

[10]  Daniel W. Pack,et al.  Design and development of polymers for gene delivery , 2005, Nature Reviews Drug Discovery.

[11]  E. Furth,et al.  A pilot study of in vivo liver-directed gene transfer with an adenoviral vector in partial ornithine transcarbamylase deficiency. , 2002, Human gene therapy.

[12]  J. L. Turner,et al.  Shell Cross-Linked Nanoparticles Designed To Target Angiogenic Blood Vessels via αvβ3 Receptor−Ligand Interactions , 2004 .

[13]  J. Feijen,et al.  Novel bioreducible poly(amido amine)s for highly efficient gene delivery. , 2007, Bioconjugate chemistry.

[14]  K. Luger,et al.  Crystal structure of a nucleosome core particle containing the variant histone H2A.Z , 2000, Nature Structural Biology.

[15]  Warren C W Chan,et al.  Elucidating the mechanism of cellular uptake and removal of protein-coated gold nanoparticles of different sizes and shapes. , 2007, Nano letters.

[16]  J. L. Turner,et al.  Synthesis of Gadolinium‐Labeled Shell‐Crosslinked Nanoparticles for Magnetic Resonance Imaging Applications , 2005 .

[17]  Robert Langer,et al.  A Combinatorial Polymer Library Approach Yields Insight into Nonviral Gene Delivery , 2008 .

[18]  J. L. Turner,et al.  Folic acid-conjugated nanostructured materials designed for cancer cell targeting. , 2003, Chemical communications.

[19]  S. Wise.  Nanocarriers as an emerging platform for cancer therapy , 2007 .

[20]  K. Wooley,et al.  Solution and Surface Charge Properties of Shell Cross-Linked Knedel Nanoparticles , 1999 .

[21]  T. Kowalewski,et al.  Packaging of DNA by shell crosslinked nanoparticles. , 1999, Nucleic acids research.

[22]  S. Fukushima,et al.  A PEG‐Based Biocompatible Block Catiomer with High Buffering Capacity for the Construction of Polyplex Micelles Showing Efficient Gene Transfer toward Primary Cells , 2006, ChemMedChem.

[23]  T. Richmond,et al.  Crystal structure of the nucleosome core particle at 2.8 Å resolution , 1997, Nature.

[24]  F. Ledley.  Non-viral gene therapy. , 1994, Current opinion in biotechnology.

[25]  S. W. Kim,et al.  Novel biodegradable poly(disulfide amine)s for gene delivery with high efficiency and low cytotoxicity. , 2008, Bioconjugate chemistry.

[26]  Kazunori Kataoka,et al.  A protein nanocarrier from charge-conversion polymer in response to endosomal pH. , 2007, Journal of the American Chemical Society.

[27]  Leaf Huang,et al.  Nonviral gene therapy: promises and challenges , 2000, Gene Therapy.

[28]  K. Kataoka,et al.  Non-viral gene therapy: Gene design and delivery , 2005 .

[29]  Chung-Yuan Mou,et al.  The effect of surface charge on the uptake and biological function of mesoporous silica nanoparticles in 3T3-L1 cells and human mesenchymal stem cells. , 2007, Biomaterials.

[30]  Andreas M. Nyström,et al.  Facile, efficient approach to accomplish tunable chemistries and variable biodistributions for shell cross-linked nanoparticles. , 2008, Biomacromolecules.

[31]  R. Kole,et al.  Up-regulation of luciferase gene expression with antisense oligonucleotides: implications and applications in functional assay development. , 1998, Biochemistry.

[32]  T. Reineke,et al.  General structure-activity relationship for poly(glycoamidoamine)s: the effect of amine density on cytotoxicity and DNA delivery efficiency. , 2008, Bioconjugate chemistry.

[33]  K. Wooley,et al.  The preparation of t‐butyl acrylate, methyl acrylate, and styrene block copolymers by atom transfer radical polymerization: Precursors to amphiphilic and hydrophilic block copolymers and conversion to complex nanostructured materials , 2000 .

[34]  Ke-Xin Zhang,et al.  Shape effects of nanoparticles conjugated with cell-penetrating peptides (HIV Tat PTD) on CHO cell uptake. , 2008, Bioconjugate chemistry.

[35]  Craig J. Hawker,et al.  Strategies for Optimized Radiolabeling of Nanoparticles for in vivo PET Imaging , 2007 .

[36]  I. Verma,et al.  Gene therapy - promises, problems and prospects , 1997, Nature.

[37]  Huajian Gao,et al.  Mechanics of receptor-mediated endocytosis. , 2005, Proceedings of the National Academy of Sciences of the United States of America.