Microwave Administration System Of SiRNA Based On Functionalized Chitosan Derivative Nanoparticles

Small interfering RNA (siRNA)-mediated knockdown of functional protein at the messenger RNA (mRNA) level offers an alternative therapeutic strategy to various diseases. However, the harsh environment in vivo leads to inefficient delivery of siRNA owing to the rapid degradation and poor penetration into the cells. Therefore, biodegradable carriers for administration were developed to encapsulate siRNA, protect siRNA from degradation and enhance the efficiency of gene silencing.Chitosan (CS), N-trimethyl chitosan (TMC), thiolated trimethyl chitosan (TC), or galactosylated trimethyl chitosan-cysteine (GTC) was used to encapsulate siRNA with sodium tripolyphosphate (TPP) or hyaluronic acid (HA) as crosslinkers. The resultant nanoparticles were characterized for particle size, Zeta potential, and siRNA gel retardation. Structural stabilities and siRNA protection in the intestinal fluids and homogenates were evaluated. In vitro assessment of cell binding, cellular uptake, cytotoxicity, and gene knockdown efficiency of GTC nanoparticles were carried out in lipopolysaccharide (LPS)-activated Raw264.7cells. In vivo RNAi efficiency of orally delivered GTC nanoparticles loaded with mitogen-activated protein kinase kinase kinase kinase4(Map4k4) siRNA (siMap4k4) was determined in mice suffering from dextran sulfate sodium (DSS)-induced ulcerative colitis. Among the various nanoparticles investigated, nanoparticles formed by crosslinking TC or GTC with TPP (TC/TPP NPs or GTC/TPP NPs) exhibited the preferable structural stability and siRNA protection in the intestinal tract. Cellular uptake levels of GTC/TPP NPs in activated macrophages were significantly enhanced owing to galactose receptor-mediated endocytosis. The in vitro and in vivo gene knockdown measurement showed that siMap4k4loaded GTC/TPP NPs could inhibit the production of a proinflammatory cytokine TNF-a. Orally administered siRNA loaded GTC/TPP NPs efficiently accumulated in the inflammatory colon site. Daily oral administration of GTC/TPP NPs containing siMap4k4significantly improved DSS-induced body weight loss, colon length shortening, and increase of myeloperoxidase activity.