title: High-yield nontoxic gene transfer through conjugation of the CM₁₈-Tat₁₁ chimeric peptide with nanosecond electric pulses.
abstract: We report a novel nontoxic, high-yield, gene delivery system based on the synergistic use of nanosecond electric pulses (NPs) and nanomolar doses of the recently introduced CM18-Tat11 chimeric peptide (sequence of KWKLFKKIGAVLKVLTTGYGRKKRRQRRR, residues 1-7 of cecropin-A, 2-12 of melittin, and 47-57 of HIV-1 Tat protein). This combined use makes it possible to drastically reduce the required CM18-Tat11 concentration and confines stable nanopore formation to vesicle membranes followed by DNA release, while no detectable perturbation of the plasma membrane is observed. Two different experimental assays are exploited to quantitatively evaluate the details of NPs and CM18-Tat11 cooperation: (i) cytofluorimetric analysis of the integrity of synthetic 1,2-dioleoyl-sn-glycero-3-phosphocholine giant unilamellar vesicles exposed to CM18-Tat11 and NPs and (ii) the in vitro transfection efficiency of a green fluorescent protein-encoding plasmid conjugated to CM18-Tat11 in the presence of NPs. Data support a model in which NPs induce membrane perturbation in the form of transient pores on all cellular membranes, while the peptide stabilizes membrane defects selectively within endosomes. Interestingly, atomistic molecular dynamics simulations show that the latter activity can be specifically attributed to the CM18 module, while Tat11 remains essential for cargo binding and vector subcellular localization. We argue that this result represents a paradigmatic example that can open the way to other targeted delivery protocols.
authors.affiliation: NEST, Scuola Normale Superiore and Istituto Nanoscienze-CNR , Piazza San Silvestro 12, 56127 Pisa, Italy.
authors.forename: Fabrizio
authors.initials: F
authors.lastname: Salomone
authors.forename: Marie
authors.initials: M
authors.lastname: Breton
authors.forename: Isabelle
authors.initials: I
authors.lastname: Leray
authors.forename: Francesco
authors.initials: F
authors.lastname: Cardarelli
authors.forename: Claudia
authors.initials: C
authors.lastname: Boccardi
authors.forename: Daniel
authors.initials: D
authors.lastname: Bonhenry
authors.forename: Mounir
authors.initials: M
authors.lastname: Tarek
authors.forename: Lluis M
authors.initials: LM
authors.lastname: Mir
authors.forename: Fabio
authors.initials: F
authors.lastname: Beltram
mesh_terms: D002462:Cell Membrane / Q000378:metabolism; D002463:Cell Membrane Permeability / Q000502:physiology; D004247:DNA / Q000378:metabolism; D011992:Endosomes / Q000378:metabolism; D015696:Gene Products, tat / Q000378:metabolism*; D018014:Gene Transfer Techniques; D049452:Green Fluorescent Proteins / Q000378:metabolism; D056004:Molecular Dynamics Simulation; D010455:Peptides / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D011674:Pulse / Q000379:methods; D014162:Transfection / Q000379:methods; D053835:Unilamellar Liposomes / Q000378:metabolism
chemical_list: D015696:Gene Products, tat; D010455:Peptides; D053835:Unilamellar Liposomes; D049452:Green Fluorescent Proteins; D004247:DNA