Biophysical characteristics of neurotensin polyplex for in vitro and in vivo gene transfection Martha L. Arango-Rodriguez a,c , Ivan Navarro-Quiroga d , Juan A. Gonzalez-Barrios a , Daniel B. Martinez-Arguelles a , Michael J. Bannon e , Juan Kouri b , Patricia Forgez f , William Rostene g , Refugio Garcia-Villegas a , Ismael Jimenez a , Daniel Martinez-Fong a, ⁎ a Departamento de Fisiología, Biofísica y Neurociencias, CINVESTAV, Apartado postal 14-740, México D.F. 07000, México b Departamento de Patología Experimental, CINVESTAV, Apartado postal 14-740, México D.F. 07000, México c Programa de Doctorado en Ciencias Biomédicas, Facultad de Medicina, UNAM, Apdo. Postal 70-250, México D.F. 04510, México d Biology Department, Wesleyan University, Middletown, CT 06459, USA e Department of Psychiatry and Behavioral Neurosciences and Pharmacology, Wayne State University School of Medicine, 2309 Scott Hall, 540 East Canfield Avenue, Detroit, MI 48201, USA f Fundamental and Clinical Oncology of Human Solid Tumors, INSERM U673, 75571 Paris Cedex 12, France g Chemokines and Their Receptors: Brain and Neuroendocrine Functions, INSERM U732, 184 rue du Faubourg Saint-Antoine, 75571 Paris Cedex 12, France Received 9 April 2005; received in revised form 9 February 2006; accepted 28 February 2006 Available online 3 April 2006 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 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 : [ 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 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. © 2006 Elsevier B.V. All rights reserved. Keywords: Gene delivery; Receptor-mediated endocytosis; DNA condensation; Fusogenic peptide; Nuclear localization signal; Karyophilic peptide 1. Introduction Nonviral gene-delivery systems are currently under devel- opment to discover a gene transfer-vector with the best efficiency, selectivity, and short- and long-term safety [1,2]. We have developed a neurotensin (NT)-vector consisting of the covalent binding of NT to poly-L-lysine (PLL), which binds plasmid DNA (pDNA) forming a complex known as NT- polyplex [3]. The NT was chosen for its ability to bind a membrane receptor and to be internalized in the perinuclear area Biochimica et Biophysica Acta 1760 (2006) 1009 – 1020 http://www.elsevier.com/locate/bba Abbreviations: BDNF, brain-derived neurotrophic factor; CFP, cyan- florescent protein; DMSO, dimethyl sulfoxide; DTT, dithiothreitol; FBS, fetal bovine serum; FP, hemagglutinin HA2 fusogenic peptide; GFP, green-florescent protein; hDAT, human-dopamine transporter; KP, Vp1 SV40 karyophilic peptide; LC-SPDP, N-succinimidyl 6-[3-(2-pyridyldithio) propionamido] hex- anoate; MM, molecular mass; MTT, (3-[4,5-dimethylthiazol-2-y]-2,5-diphenyl tetrazolium bromide); NT, neurotensin; NTS1, high-affinity NT receptor; PBS, phosphate-buffered saline solution; pDNA, plasmid DNA; PLL, poly-L-lysine; RT, room temperature; SNc, substantia nigra pars compacta; SDS, sodium dodecyl sulfate; TAE, TRIS . acetate-EDTA buffer; VTA, ventral tegmental area ⁎ Corresponding author. Tel.: +52 55 5061 3959; fax: +52 55 5061 3754. E-mail address: dmartine@fisio.cinvestav.mx (D. Martinez-Fong). 0304-4165/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagen.2006.02.021