Targeted vault nanoparticles engineered with an endosomolytic peptide deliver biomolecules to the cytoplasm.

Vault nanoparticles were engineered to enhance their escape from the endosomal compartment by fusing a membrane lytic peptide derived from adenovirus protein VI (pVI) to the N-terminus of the major vault protein to form pVI-vaults. We demonstrate that these pVI-vaults disrupt the endosomal membrane using three different experimental protocols including (1) enhancement of DNA transfection, (2) co-delivery of a cytosolic ribotoxin, and (3) direct visualization by fluorescence. Furthermore, direct targeting of vaults to specific cell surface epidermal growth factor receptors led to enhanced cellular uptake and efficient delivery of vaults to the cytoplasm. This process was monitored with fluorescent vaults, and morphological changes in the endosomal compartment were observed. By combining targeting and endosomal escape into a single recombinant vault, high levels of transfection efficiency were achieved using low numbers of vault particles. These results demonstrate that engineered vaults are effective, efficient, and nontoxic nanoparticles for targeted delivery of biomaterials to the cell cytoplasm.

[1]  C. Sword,et al.  Effects of Listeria monocytogenes Hemolysin on Phagocytic Cells and Lysosomes , 1970, Infection and immunity.

[2]  J. Northrop,et al.  Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[3]  M. Uhlén,et al.  A synthetic IgG-binding domain based on staphylococcal protein A. , 1987, Protein engineering.

[4]  M. Marquet,et al.  Genetic Engineering of Structural Protein Polymers , 1990, Biotechnology progress.

[5]  N. Kedersha,et al.  Vaults. III. Vault ribonucleoprotein particles open into flower-like structures with octagonal symmetry , 1991, The Journal of cell biology.

[6]  V. Kickhoefer,et al.  Vault ribonucleoprotein particles from rat and bullfrog contain a related small RNA that is transcribed by RNA polymerase III. , 1993, The Journal of biological chemistry.

[7]  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.

[8]  K. Mechtler,et al.  Activation of the complement system by synthetic DNA complexes: a potential barrier for intravenous gene delivery. , 1996, Human gene therapy.

[9]  P. Felgner.  Improvements in cationic liposomes for in vivo gene transfer. , 1996, Human gene therapy.

[10]  During,et al.  Adeno-associated virus as a gene delivery system. , 1997, Advanced drug delivery reviews.

[11]  V. Kickhoefer,et al.  Vaults Are Up-regulated in Multidrug-resistant Cancer Cell Lines* , 1998, The Journal of Biological Chemistry.

[12]  D. Lauffenburger,et al.  EGF receptor regulation of cell motility: EGF induces disassembly of focal adhesions independently of the motility-associated PLCgamma signaling pathway. , 1998, Journal of cell science.

[13]  V. Kickhoefer,et al.  The 193-Kd Vault Protein, Vparp, Is a Novel Poly(Adp-Ribose) Polymerase , 1999, The Journal of cell biology.

[14]  L. Smith,et al.  Peptide-based gene delivery. , 1999, Current opinion in molecular therapeutics.

[15]  V. Kickhoefer,et al.  Vaults and Telomerase Share a Common Subunit, TEP1* , 1999, The Journal of Biological Chemistry.

[16]  A. Wells.  EGF receptor. , 1999, The international journal of biochemistry & cell biology.

[17]  H. Yamasaki,et al.  Bystander effect in herpes simplex virus-thymidine kinase/ganciclovir cancer gene therapy: role of gap-junctional intercellular communication. , 2000, Cancer research.

[18]  S. Simões,et al.  Cationic lipid-DNA complexes in gene delivery: from biophysics to biological applications. , 2001, Advanced drug delivery reviews.

[19]  P. Sonneveld,et al.  Multiple Human Vault RNAs , 2001, The Journal of Biological Chemistry.

[20]  V. Kickhoefer,et al.  Assembly of Vault-like Particles in Insect Cells Expressing Only the Major Vault Protein* , 2001, The Journal of Biological Chemistry.

[21]  K. Kataoka,et al.  Polyion complex micelles as vectors in gene therapy – pharmacokinetics and in vivo gene transfer , 2002, Gene Therapy.

[22]  Ernst Wagner,et al.  Targeting tumors with non-viral gene delivery systems. , 2002, Drug discovery today.

[23]  P. Sonneveld,et al.  Structural domains of vault proteins: a role for the coiled coil domain in vault assembly. , 2002, Biochemical and biophysical research communications.

[24]  K. Suprenant.  Vault ribonucleoprotein particles: sarcophagi, gondolas, or safety deposit boxes? , 2002, Biochemistry.

[25]  K. Lundstrom.  Latest development in viral vectors for gene therapy. , 2003, Trends in biotechnology.

[26]  K. Kataoka,et al.  Size‐Controlled Formation of a Calcium Phosphate‐Based Organic–Inorganic Hybrid Vector for Gene Delivery Using Poly(ethylene glycol)‐block‐poly(aspartic acid) , 2004 .

[27]  D. Fischer,et al.  A Novel Non-Viral Vector for DNA Delivery Based on Low Molecular Weight, Branched Polyethylenimine: Effect of Molecular Weight on Transfection Efficiency and Cytotoxicity , 1999, Pharmaceutical Research.

[28]  Jindrich Kopecek,et al.  Intracellular Processing of Poly(Ethylene Imine)/Ribozyme Complexes Can Be Observed in Living Cells by Using Confocal Laser Scanning Microscopy and Inhibitor Experiments , 2002, Pharmaceutical Research.

[29]  V. Kickhoefer,et al.  Cryoelectron microscopy imaging of recombinant and tissue derived vaults: localization of the MVP N termini and VPARP. , 2004, Journal of molecular biology.

[30]  L G Griffith,et al.  Quantitative comparison of polyethylenimine formulations and adenoviral vectors in terms of intracellular gene delivery processes , 2005, Gene Therapy.

[31]  Toshihiro Akaike,et al.  Bio-functional inorganic materials: an attractive branch of gene-based nano-medicine delivery for 21st century. , 2005, Current gene therapy.

[32]  M. Eisenstein.  Fast, cheap and under control , 2005, Nature Methods.

[33]  G. Nemerow,et al.  Adenovirus Protein VI Mediates Membrane Disruption following Capsid Disassembly , 2005, Journal of Virology.

[34]  Jing C. Zhou,et al.  Engineering of vault nanocapsules with enzymatic and fluorescent properties. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[35]  J. Swanson,et al.  Membrane perforations inhibit lysosome fusion by altering pH and calcium in Listeria monocytogenes vacuoles , 2006, Cellular microbiology.

[36]  G. Kozlov,et al.  Solution structure of a two-repeat fragment of major vault protein. , 2005, Journal of molecular biology.

[37]  V. Kickhoefer,et al.  The vault exterior shell is a dynamic structure that allows incorporation of vault-associated proteins into its interior. , 2006, Biochemistry.

[38]  Vault nanocapsule dissociation into halves triggered at low pH. , 2007, Biochemistry.

[39]  V. Kickhoefer,et al.  Draft Crystal Structure of the Vault Shell at 9-Å Resolution , 2007, PLoS biology.

[40]  C. Rooney,et al.  Dendritic cell function after gene transfer with adenovirus-calcium phosphate co-precipitates. , 2007, Molecular therapy : the journal of the American Society of Gene Therapy.

[41]  L. Rome,et al.  Encapsulation of semiconducting polymers in vault protein cages. , 2008, Nano letters.

[42]  L. Rome,et al.  Reversible pH lability of cross-linked vault nanocapsules. , 2008, Nano letters.

[43]  V. Kickhoefer,et al.  Targeting vault nanoparticles to specific cell surface receptors. , 2009, ACS nano.

[44]  V. Kickhoefer,et al.  A Vault Nanoparticle Vaccine Induces Protective Mucosal Immunity , 2009, PloS one.

[45]  V. Kickhoefer,et al.  Vault nanoparticles containing an adenovirus-derived membrane lytic protein facilitate toxin and gene transfer. , 2009, ACS nano.

[46]  Hideaki Tanaka,et al.  The Structure of Rat Liver Vault at 3.5 Angstrom Resolution , 2009, Science.

[47]  V. Kickhoefer,et al.  Structural stability of vault particles. , 2009, Journal of pharmaceutical sciences.

[48]  V. Kickhoefer,et al.  Vaults are dynamically unconstrained cytoplasmic nanoparticles capable of half vault exchange. , 2010, ACS nano.

[49]  Min Huang,et al.  Novel CCL21-Vault Nanocapsule Intratumoral Delivery Inhibits Lung Cancer Growth , 2011, PloS one.

[50]  G. Nemerow,et al.  Functional Genetic and Biophysical Analyses of Membrane Disruption by Human Adenovirus , 2011, Journal of Virology.

[51]  Drug Delivery: Vaults Engineered for Hydrophobic Drug Delivery (Small 10/2011) , 2011 .