RESUMEN
The potential of RNAi therapies has been largely impeded by the inherent challenges in the functional delivery of siRNA to cells. Herein, we describe protocols for the synthesis and characterization of novel peptide-siRNA nanoparticles prepared from disulfide-constrained amphipathic peptides complexed with siRNA as promising siRNA delivery vectors. We also describe protocols for the application of these nanoparticles to the in vitro and in vivo delivery of siRNA to lung cells for the functional knockdown of lung proteins.
Asunto(s)
Disulfuros/química , Sistemas de Liberación de Medicamentos/métodos , Pulmón/efectos de los fármacos , Nanopartículas/química , Oligonucleótidos/química , Péptidos Cíclicos/administración & dosificación , Péptidos Cíclicos/química , ARN Interferente Pequeño/química , Células A549 , Línea Celular Tumoral , Técnicas de Transferencia de Gen , Humanos , Interferencia de ARN/fisiologíaRESUMEN
The promise of oligonucleotide therapeutic agents to perturb expression of disease-related genes remains unrealized, in part due to challenges with functional cellular delivery of these agents. Herein, we describe disulfide-constrained cyclic amphipathic peptides that complex with short-interfering RNA (siRNA) and affect functional cytosolic delivery and knockdown of target gene products in cell culture and in vivo to mouse lung. Reduction of the constraining disulfide bond and subsequent proteolytic clearance of the peptide are key design features that allow unmasking of the siRNA cargo and presentation to the RNA interference machinery.
RESUMEN
Hydrogel networks composed of rippled ß-sheet fibrils of coassembled D- and L-Ac-(FKFE)2-NH2 amphipathic peptides exhibit proteolytic stability and increased rheological strength compared to networks of self-assembled L-Ac-(FKFE)2-NH2 pleated ß-sheet fibrils. Modifying the ratios of l and d peptides in the coassembled rippled ß-sheet fibrils alters the degradation profiles of these hydrogel networks.