Biophysical characteristics of neurotensin polyplex for in vitro and in vivo gene transfection

Martha L. Arango-Rodriguez, Ivan Navarro-Quiroga, Juan A. Gonzalez-Barrios, Daniel B. Martinez-Arguelles, Michael J. Bannon, Juan Kouri, Patricia Forgez, William Rostene, Refugio Garcia-Villegas, Ismael Jimenez, Daniel Martinez-Fong

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24 Scopus citations

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 [125I]-NT, [3H]-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 μM 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 : [125I]-NT-[3H]-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 μM 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.

Original languageEnglish
Pages (from-to)1009-1020
Number of pages12
JournalBiochimica et Biophysica Acta - General Subjects
Volume1760
Issue number7
DOIs
StatePublished - Jul 2006
Externally publishedYes

Keywords

  • DNA condensation
  • Fusogenic peptide
  • Gene delivery
  • Karyophilic peptide
  • Nuclear localization signal
  • Receptor-mediated endocytosis

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