RESUMEN
Neurotensin (NTS)-polyplex is a multicomponent nonviral vector that enables gene delivery via internalization of the neurotensin type 1 receptor (NTSR1) to dopaminergic neurons and cancer cells. An approach to improving its therapeutic safety is replacing the viral karyophilic component (peptide KPSV40; MAPTKRKGSCPGAAPNKPK), which performs the nuclear import activity, by a shorter synthetic peptide (KPRa; KMAPKKRK). We explored this issue and the mechanism of plasmid DNA translocation through the expression of the green fluorescent protein or red fluorescent protein fused with KPRa and internalization assays and whole-cell patch-clamp configuration experiments in a single cell together with importin α/ß pathway blockers. We showed that KPRa electrostatically bound to plasmid DNA increased the transgene expression compared with KPSV40 and enabled nuclear translocation of KPRa-fused red fluorescent proteins and plasmid DNA. Such translocation was blocked with ivermectin or mifepristone, suggesting importin α/ß pathway mediation. KPRa also enabled NTS-polyplex-mediated expression of reporter or physiological genes such as human mesencephalic-derived neurotrophic factor (hMANF) in dopaminergic neurons in vivo. KPRa is a synthetic monopartite peptide that showed nuclear import activity in NTS-polyplex vector-mediated gene delivery. KPRa could also improve the transfection of other nonviral vectors used in gene therapy.
Asunto(s)
Portadores de Fármacos/síntesis química , Técnicas de Transferencia de Gen , Vectores Genéticos/administración & dosificación , Neurotensina/administración & dosificación , Fragmentos de Péptidos/síntesis química , Transporte Activo de Núcleo Celular , Animales , Línea Celular , Núcleo Celular/metabolismo , Neuronas Dopaminérgicas/citología , Neuronas Dopaminérgicas/metabolismo , Terapia Genética/métodos , Vectores Genéticos/genética , Masculino , Ratones , Modelos Animales , Nanopartículas/química , Neurotensina/genética , Neurotensina/farmacocinética , Técnicas de Placa-Clamp , Plásmidos/genética , Ratas , Receptores de Neurotensina/metabolismo , Análisis de la Célula Individual , Técnicas EstereotáxicasRESUMEN
INTRODUCTION: Several strategies on the development of radiopharmaceuticals have been employed. Bifunctional chelators seem to be a promising approach since high radiochemical yields as well as good in vitro and in vivo stability have been achieved. To date, neurotensin analogs have been radiolabeled using the (99m)Tc-carbonyl approach and none was described employing the bifunctional chelating agent technique. AIM: The purpose of this study was to evaluate the radiochemical and biological behaviour of NT(8-13) analogue radiolabeled with (99m)Tc, using HYNIC and NHS-S-acetyl-MAG(3) as chelator agents. METHODS: Radiolabeling, in vitro stability toward cysteine and glutathione, partition coefficient and plasma protein binding were assessed for both radioconjugates. Biodistribution in healthy Swiss mice were carried out in order to evaluate the biological behaviour of the radiocomplexes. RESULTS: Radiochemical yields were higher than 97% and no apparent instability toward transchelant agents was observed for both radioconjugates. A higher lipophilic character was observed for the radioconjugate labeled via MAG(3). The chelators seem to have no effect on the percentage of the radioconjugate bound to plasma proteins. A similar biological pattern was observed for both radioconjugates. Total blood, bone and muscle values revealed a slightly slower clearance for the radiocomplex labeled via MAG(3). Moreover, a remarkable liver and intestinal uptake was observed for the radiocomplex labeled via MAG(3) even at the later time points studied. CONCLUSION: The high radiochemical yields achieved and the similar in vivo pattern found for both radioconjugates make them potential candidates for imaging tumors using nuclear medicine techniques.