title: Biophysical characteristics of neurotensin polyplex for in vitro and in vivo gene transfection.
abstract: Previously we improved the neurotensin (NT)-polyplex by the coupling of HA2 fusogenic peptide (FP) and Vp1 SV40 karyophilic peptide (KP). We now report the proportion of [(125)I]-NT, [(3)H]-FP, and poly-l-lysine (PLL) in the NT-polyplex, and some of its biophysical properties. We concluded that the most efficient NT-polyplex comprised 1 NT, 4 FP, and 2 PLL molecules. Electrophoresis revealed that high acidity is detrimental for NT-polyplex stability. Electron microscopy and electrophoresis studies showed that 6 muM KP and 1% serum condensed the plasmid DNA (pDNA) before the appearance of toroid structures. Four plasmids were used to evaluate the transfection efficiency. In vitro, maximum expression was produced at molar ratios (pDNA : [(125)I]-NT-[(3)H]-FP-PLL conjugate) of 1:34 for pEGFP-N1 and 1:27 for pECFP-Nuc. Cotransfection of those plasmids was attained at their optimum molar ratios. In vivo, maximum expression of the pDAT-BDNF-flag in dopamine neurons was produced at a 1:45 molar ratio, whereas that of pDAT-EGFP was at 1:20. The NT-polyplex in the presence of 1 muM SR-48692, an NT-receptor specific antagonist, and untargeted polyplex did not cause transfection in vivo demonstrating the specificity of gene transfer via NT-receptor endocytosis. This information is essential for synthesizing an efficient NT-polyplex that can provide a useful tool for specific gene transfection.
authors.affiliation: Departamento de Fisiología, Biofísica y Neurociencias, CINVESTAV, Apartado postal 14-740, México D.F. 07000, México.
authors.forename: Martha L
authors.initials: ML
authors.lastname: Arango-Rodriguez
authors.forename: Ivan
authors.initials: I
authors.lastname: Navarro-Quiroga
authors.forename: Juan A
authors.initials: JA
authors.lastname: Gonzalez-Barrios
authors.forename: Daniel B
authors.initials: DB
authors.lastname: Martinez-Arguelles
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Bannon
authors.forename: Juan
authors.initials: J
authors.lastname: Kouri
authors.forename: Patricia
authors.initials: P
authors.lastname: Forgez
authors.forename: William
authors.initials: W
authors.lastname: Rostene
authors.forename: Refugio
authors.initials: R
authors.lastname: Garcia-Villegas
authors.forename: Ismael
authors.initials: I
authors.lastname: Jimenez
authors.forename: Daniel
authors.initials: D
authors.lastname: Martinez-Fong
mesh_terms: D000818:Animals; D001703:Biophysics / Q000379:methods; D045744:Cell Line, Tumor; D004298:Dopamine / Q000378:metabolism; D005821:Genetic Techniques; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D008297:Male; D009474:Neurons / Q000378:metabolism; D009496:Neurotensin / Q000737:chemistry*; D019913:Nuclear Localization Signals; D011720:Pyrazoles / Q000737:chemistry; D011804:Quinolines / Q000737:chemistry; D051381:Rats; D017208:Rats, Wistar; D014162:Transfection
chemical_list: D019913:Nuclear Localization Signals; D011720:Pyrazoles; D011804:Quinolines; C079087:SR 48692; D009496:Neurotensin; D004298:Dopamine