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
Silk fibroin (SF) films containing a peptide, neurotensin (NT), stimulated by iontophoresis were developed aiming to modulate the inflammatory process and prevent the growth of microorganisms typical of wounds. NT-loaded SF films composition shows predominance of ß-sheet structures that conferred adequate mechanical properties, transparency, moderate roughness and low swelling index to fibroin films. Infrared spectroscopy and thermal analysis suggested the presence of non-covalent interactions between NT and fibroin. Using the MALDI imaging technique, it was possible to visualize the homogeneous NT distribution throughout the film surface, in addition to its prolonged release for up to 72â¯h. In vitro studies in E. coli liposaccharide-stimulated macrophages showed a significant reduction of interleukins production after NT-loaded film application, whereas NT solution did not reduce them. Bi-laminated NT-loaded fibroin films containing silver electrodes provided a burst release of NT when anodic iontophoresis was applied, enabling a rapid onset of drug action. In addition, anodic iontophoresis presented a bacteriostatic effect against gram-positive microorganisms. Different iontophoresis densities, from 0.2 to 0.6â¯mA/cm2, did not significantly reduce fibroblast viability after 30â¯min of application. In conclusion, iontophoretic-stimulated peptide-loaded fibroin films could be a promising platform for the treatment of wounds.
Asunto(s)
Antibacterianos/administración & dosificación , Sistemas de Liberación de Medicamentos/métodos , Fibroínas/química , Neurotensina/administración & dosificación , Cicatrización de Heridas/efectos de los fármacos , Animales , Antibacterianos/química , Bacterias/efectos de los fármacos , Bacterias/metabolismo , Bombyx/química , Supervivencia Celular , Fibroblastos , Interleucinas/metabolismo , Iontoforesis/métodos , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Pruebas de Sensibilidad Microbiana , Neurotensina/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Synaptosomal membrane Na+, K+-ATPase is inhibited by neurotensin, an effect which involves its high affinity receptor (NTS1) [Lopez Ordieres MG, Rodriguez de Lores Arnaiz, G. Peptides 2000; 21:571-576.]. Herein, the effect of neurotensin on synaptosomal membrane Na+, K+-ATPase of rats 18 h after i.p. administration of antipsychotic haloperidol (2 mg/kg) or clozapine (10 mg/kg) was studied. Basal enzyme activity after these treatments did not differ from that in vehicle-treated rats. It was observed that 3.5 x 10(-6) M neurotensin reduced roughly 40% cerebral cortex Na+, K+-ATPase from vehicle-injected rats, produced no effect on the enzyme from rats injected with haloperidol but enhanced 26% that from rats injected with clozapine. The peptide decreased 40% striatal Na+, K+-ATPase from vehicle-injected rats or from rats injected with clozapine, whereas it failed to alter this enzyme activity from rats injected with haloperidol. Haloperidol and clozapine (1 x 10(-6) M) added in vitro failed to alter Na+, K+-ATPase activity in cerebral cortex synaptosomal membranes. Results obtained after antipsychotic administration may well offer an alternative explanation for the particular side effects recorded in therapeutics by typical (haloperidol) versus atypical (clozapine) antipsychotic drugs.
Asunto(s)
Antipsicóticos/administración & dosificación , Clozapina/administración & dosificación , Haloperidol/administración & dosificación , Neurotensina/administración & dosificación , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Sinaptosomas/enzimología , Animales , Química Encefálica/efectos de los fármacos , Corteza Cerebral/enzimología , Masculino , Neurotensina/metabolismo , Ratas , Ratas Wistar , Membranas Sinápticas/enzimologíaRESUMEN
Neurotensin (NT), an active neuropeptide, and bicuculline, a GABA-A receptor antagonist, were microinjected into the rat medial hypothalamus (MH) or the dorsal periaqueductal gray matter (DPAG). Bicuculline (80 pmol) produced behavioral activation which included jumping and NT (1-20 nmol) caused a dose-dependent behavioral activation accompanied by catalepsy rather than jumping. These results suggest that the behavioral activation produced by NT may be due to an interaction of the neuropeptide with specific receptors while its cataleptic effect may be attributed to the blockade of dopamine receptors.