Neurotensin-SPDP-poly-L-lysine conjugate: a nonviral vector for targeted gene delivery to neural cells.
We report herein the synthesis of a novel DNA delivery system and in vitro evidence of its ability to transfect cell lines by binding to the high-affinity neurotensin receptor and subsequent internalization of ligand-receptor complexes. The targeting vehicle consisted of neurotensin crosslinked with poly-L-lysine via N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP). The SPDP-derivatives with either neurotensin or poly-L-lysine were purified by gel filtration. The conjugate resulting of the reaction of neurotensin-SPDP with HS-SPDP-poly-L-lysine was purified through Biogel A 1.5. The neurotensin-SPDP-poly-L-lysine conjugate was able to bind plasmidic DNAs (pSV2cat and pGreen Lantern-1) at optimal molar ratios of 1:5 and 1:6 (DNA: conjugate), respectively. The conjugate internalized those plasmids in the cell lines (N1E-115 and HT-29) bearing the high-affinity neurotensin receptor. Expression of the plasmid products, chloramphenicol acetyltransferase and green fluorescent protein, was observed in such cell lines. Both internalization and expression of the plasmids transferred by the neurotensin-SPDP-poly-L-lysine conjugate were prevented by neurotensin (1 microM) and SR-48692 (100 nM), a specific antagonist of the high-affinity neurotensin receptor. The neurotensin-SPDP-poly-L-lysine conjugate was unable to transfect cell lines lacking the neurotensin receptor (COS-7 and L-929). In rat brain, the high-affinity neurotensin receptor is expressed by specific neurons such as those of the nigrostriatal and mesolimbic dopaminergic systems. Therefore, the neurotensin-SPDP-poly-L-lysine conjugate could be a useful tool for gene delivery to those neuronal systems.
Martinez-Fong
D
D
Departamento de Fisiología, Biofísica y Neurociencias, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional de México, Apartado postal 14-740, 07000, Mexico D.F., Mexico. dmartine@fisio.cinvestav.mx
Navarro-Quiroga
I
I
Ochoa
I
I
Alvarez-Maya
I
I
Meraz
M A
MA
Luna
J
J
Arias-Montaño
J A
JA
D000818:Animals; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins / Q000737:chemistry; D004705:Endocytosis; D018390:Gene Targeting; D005815:Genetic Code; D005822:Genetic Vectors; D009447:Neuroblastoma / Q000737:chemistry; D009474:Neurons / Q000737:chemistry*; D009496:Neurotensin / Q000737:chemistry*; D011107:Polylysine / Q000737:chemistry*; D051381:Rats; D013388:Succinimides / Q000737:chemistry*; D014407:Tumor Cells, Cultured
D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D013388:Succinimides; D011107:Polylysine; C018151:N-succinimidyl 3-(2-pyridyldithio)propionate; D009496:Neurotensin; D015500:Chloramphenicol O-Acetyltransferase