RESUMEN
Endosomal escape and nuclear localization are two barriers to gene delivery that need to be addressed in the design of new nonviral gene delivery vehicles. We have previously synthesized low-toxicity polyethylene glycol (PEG)-based vehicles with endosomal escape functionalities, but it was determined that the transfection efficiency of PEG-based vehicles that escaped the endosome was still limited by poor nuclear localization. Two different nuclear localization signal (NLS) peptides, SV40 and TAT, were coupled to PEG-based vehicles with DNA-binding peptides (DBPs) to determine the effect of NLS peptides on the transfection efficiency of PEG-based gene delivery vehicles. Coupling one SV40 peptide, a classical NLS, or two TAT peptides, a nonclassical NLS, to PEG-DBP vehicles increased the transfection efficiency of PEG-DBP/DNA particles 15-fold and resulted in similar efficiency to that of a common cationic polymer vehicle, polyethylenimine (PEI). The transfection efficiency of both types of PEG-DBP-NLS particles was further increased 7-fold in the presence of chloroquine, suggesting that the transfection efficiency of PEG-DBP-NLS particles is limited by their ability to escape the endosome. To develop particles that could escape the endosome and target the nucleus, a mixture of PEG-DBP-NLS vehicles and PEG-based vehicles with DBPs and endosomal escape peptides were complexed with plasmid DNA to form multifunctional particles that had a transfection efficiency 2-3 times higher than that of PEI. Additionally, the PEG-based vehicles were less toxic and more resistant to nonspecific protein adsorption than PEI, making them an attractive alternative for nonviral gene delivery.
Asunto(s)
Endosomas/metabolismo , Señales de Localización Nuclear/química , Péptidos/química , Polietilenglicoles/química , Animales , Tampones (Química) , Células CHO , Núcleo Celular/metabolismo , Supervivencia Celular , Cricetinae , Cricetulus , Técnicas de Transferencia de Gen , Terapia Genética/métodos , Vectores Genéticos/administración & dosificación , Vectores Genéticos/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Peso Molecular , Señales de Localización Nuclear/metabolismo , Péptidos/síntesis química , Péptidos/aislamiento & purificación , Péptidos/metabolismo , Péptidos/farmacología , Fosfatos/química , Polietileneimina/química , Polietileneimina/metabolismo , TransfecciónRESUMEN
BACKGROUND: With recent progress in gene therapy clinical trials, there is an even greater demand to advance the development of nonviral gene delivery vehicles. We have previously developed poly(ethylene glycol) (PEG)-based vehicles with transfection efficiency similar to polyethyleneimine and low cytotoxicity. It was hypothesized that conjugating endosomal escape peptides (EEPs) to PEG-based vehicles would further increase their transfection efficiency. The present study aimed to determine how two different EEPs, INF7 and H5WYG, which destabilize the endosomal membrane at different pHs, affect the efficiency of PEG-based vehicles. METHODS: INF7 and H5WYG were conjugated to PEG-tetraacrylate (PEG-TA) via a Michael-type addition at the desired molar ratios. The pH-dependent membrane lytic activity, transfection efficiency, particle size, zeta potential, and endosomal escape kinetic rate constants were determined. RESULTS: Fusogenic peptides, INF7 and H5WYG, showed pH-dependent membrane lytic activity when conjugated to PEG-TA. The highest membrane lytic activity of PEG-INF7 and PEG-H5WYG conjugates occurred at pH 5 and 5.5, respectively. Coupling one INF7 peptide to PEG-DNA binding peptide (DBP) vehicles increased the transfection efficiency ten-fold and showed greater transfection efficiency than PEG-DBP vehicles coupled with H5WYG peptide. Fitting a first-order kinetic model to the average intracellular pH of the vehicle/DNA particles over time determined that coupling EEPs to PEG-DBP vehicles increased the endosomal escape rate constant by two orders of magnitude. CONCLUSIONS: Endosomal escape is a key step in nonviral cellular trafficking and thus the transfection efficiency of nonviral vehicles can be increased by targeting release of DNA from the endosome with EEPs.