Biophysical characteristics of neurotensin polyplex for in vitro and in vivo gene transfection.
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.
Arango-Rodriguez
Martha L
ML
Departamento de Fisiología, Biofísica y Neurociencias, CINVESTAV, Apartado postal 14-740, México D.F. 07000, México.
Navarro-Quiroga
Ivan
I
Gonzalez-Barrios
Juan A
JA
Martinez-Arguelles
Daniel B
DB
Bannon
Michael J
MJ
Kouri
Juan
J
Forgez
Patricia
P
Rostene
William
W
Garcia-Villegas
Refugio
R
Jimenez
Ismael
I
Martinez-Fong
Daniel
D
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
D019913:Nuclear Localization Signals; D011720:Pyrazoles; D011804:Quinolines; C079087:SR 48692; D009496:Neurotensin; D004298:Dopamine