RNA Interference in Vitro and in Vivo Using a Novel Chitosan/siRNA Nanoparticle System

Abstract

This work introduces a novel chitosan-based siRNA nanoparticle delivery system for RNA interference in vitro and in vivo. The formation of interpolyelectrolyte complexes between siRNA duplexes (21-mers) and chitosan polymer into nanoparticles, ranging from 40 to 600 nm, was shown using atomic force microscopy and photon correlation spectroscopy. Rapid uptake (1 h) of Cy5-labeled nanoparticles into NIH 3T3 cells, followed by accumulation over a 24 h period, was visualized using fluorescence microscopy. Nanoparticle-mediated knockdown of endogenous enhanced green fluorescent protein (EGFP) was demonstrated in both H1299 human lung carcinoma cells and murine peritoneal macrophages (77.9% and 89.3% reduction in EGFP fluorescence, respectively). In addition, Western analysis showed approximately 90% reduced expression of BCR/ABL-1 leukemia fusion protein while BCR expression was unaffected in K562 (Ph(+)) cells after transfection using nanoparticles containing siRNA specific to the BCR/ABL-1 junction sequence. Effective in vivo RNA interference was achieved in bronchiole epithelial cells of transgenic EGFP mice after nasal administration of chitosan/siRNA formulations (37% and 43% reduction compared to mismatch and untreated control, respectively). These findings highlight the potential application of this novel chitosan-based system in RNA-mediated therapy of systemic and mucosal disease.

A Novel Nanoparticle Formulation for Sustained Paclitaxel Delivery

“…5 and considered statistically significant when *p value is <0.05. The same mucoadhesive properties of chitosan have been shown effective in the delivery of various molecules in adenocarcinomas both in vitro and in vivo (13,18,42). Shikata and colleagues (42) demonstrated increased cellular internalization of drug loaded chitosan nanoparticles in squamous cell carcinomas (SCC-VII) and melanoma cells (B16F10) when compared to drug solutions alone.…”

“…Almost all human epithelial cell adenocarcinomas, nonepithelial cancer cell lines, hematological malignancies such as multiple myeloma, and some B-cell non-Hodgkin lymphomas exhibit an over expression of mucin-1 (16). Chitosan has been shown to have mucoadhesive properties both in vitro (12,15,17) and in vivo (13,(18)(19)(20)(21). Chitosan and glycerol monooleate (GMO) have been previously co-formulated together to sustain the delivery of PTX in an in situ gel with mucoadhesive properties (22).…”

Gene therapy progress and prospects: synthetic polymer-based systems

“…41 The mucoadhesive properties of chitosan were exploited for intranasal delivery of siRNA resulting in an effective knockdown of enhanced green fluorescent protein (EGFP) in bronchiolar epithelial cells of EGFP transgenic mice with no apparent toxicity. 42 Systemic delivery of siRNA chitosan polyplexes was also effective, as shown in a breast cancer xenograft model in mice. 43 Transferrin-receptor targeted, PEG-shielded siRNA polyplexes based on cyclodextrin-polycation conjugates were developed by Davis and colleagues 44 for intravenous delivery and therapy of subcutaneous tumors.…”

RNAi-based Cancer Therapeutics: Are we there yet?

“…Various promising nanoparticles (NPs) strategies that have been successful in depicting in vitro gene silencing and tumor reduction efficacy in animal models, safety in non-human primates and in few cases, have undergone clinical trial testing include, (A) Lipid based NPs: liposomes [16], lipoplexes [17,18], stable nucleic acid lipid nanoparticles (SNALP) [19,20] & lipidoid [21], polycation liposomes [22,23], (B) Polymeric NPs: both natural and synthetic, includes cyclodextrin [24], chitosan [25], PLGA [26], polymeric micelles [27], PEI [28] & dendrimers [29], (C) Inorganic NPs: calcium phosphate CaP [30], (D) Carbon-based materials, carbon nanotubes (SWNTs, MWNTs) [31,32], (E) Metal nanoparticles -Gold (Au) [33], Quantum dots (QDs) [34], silicon-based nanoparticles (MSNPs) [35] & super paramagnetic iron oxide nanoparticles (SPIONs) [36] etc. In addition to this, atelocollagen-based delivery strategy also proved to be successful in few cases [37,38].…”