RESUMEN
The remarkable regenerative capacity of the zebrafish has made it an important model organism for studying heart regeneration. However, current loss-of-function studies are limited by a lack of conditional-knockout and effective gene-knockdown methods for the adult heart. Here, we report a novel siRNA knockdown method facilitated by poly(ethylene glycol)-b-poly(D,L-lactide) (PEG-PLA) nanoparticles. The siRNA-encapsulated nanoparticles successfully entered cells and resulted in remarkable gene-specific knockdown in the adult heart. This effect was demonstrated by down-regulation of the Aldh1a2 and Dusp6 proteins after intrapleural delivery of nanoparticle-encapsulated siRNAs. Furthermore, siRNA-mediated knockdown of Aldh1a2 was sufficient to inhibit myocardial proliferation and decrease the numbers of Gata4-positive cardiomyocytes after ventricular resection. Therefore, the results of this work demonstrate that nanoparticle-facilitated siRNA delivery provides an alternative tool for loss-of-function studies of genes in the adult heart in particular and other organs in general in the adult zebrafish.
Asunto(s)
Técnicas de Silenciamiento del Gen/métodos , Miocardio/metabolismo , Nanopartículas/metabolismo , Polietilenglicoles/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Pez Cebra/genética , Familia de Aldehído Deshidrogenasa 1 , Animales , Proliferación Celular/genética , Proliferación Celular/fisiología , Fosfatasa 6 de Especificidad Dual/genética , Isoenzimas/genética , Miocardio/citología , ARN Interferente Pequeño/genética , Retinal-Deshidrogenasa/genéticaRESUMEN
Cationic polymers constitute an important class of materials in development of delivery vehicles for nucleic acid-based therapeutics. Among them, polyethylenimine (PEI) has been a classical cationic carrier intensively studied for therapeutic delivery of DNA, RNA, and short RNA molecules to treat diseases. However, the development of PEI for in vivo applications has been hampered by the inherent problems associated with the material, particularly its cytotoxicity and the instability of the nucleic acid complexation systems formed via electrostatic interactions. Here, we demonstrate a strategy to modify PEI polymers via hydrazidation to create neutralized, stable, and multifunctional system for delivering siRNA molecules. Through substitution of the primary amino groups of PEI with neutral hydrazide groups, cross-linked nanoparticles with surface decorated with a model targeting ligands were generated. The neutral cross-linked siRNA nanoparticles not only showed favorable biocompatibility and cell internalization efficiency in vitro but also allowed for significant tissue uptake and gene silencing efficiency in zebrafish heart in vivo. Our study suggests transformation of conventional branched PEI into a neutral polymer that can lead to a new category of nonviral carriers, and the resulting functional delivery systems may be further explored for development of siRNA therapeutics for treating cardiovascular disease/injury.