Cosilencing Intestinal Transglutaminase-2 and Interleukin-15 Using Gelatin-Based Nanoparticles in an in Vitro Model of Celiac Disease.

In this study, we have developed a type B gelatin nanoparticle based siRNA delivery system for silencing of intestinal transglutaminase-2 (TG2) and interleukin-15 (IL-15) genes in cultured human intestinal epithelial cells (Caco-2) and murine alveolar macrophage cells (J774A.1). Small interfering RNA (siRNA) targeting the TG2 or IL-15 gene was encapsulated within gelatin nanoparticles using ethanol-water solvent displacement method. Size, charge, and morphology of gelatin nanoparticles were evaluated using a Zetasizer instrument and transmission electron microscopy. siRNA encapsulation efficiency was determined using an siRNA specific stem-loop quantitative polymerase chain reaction (qPCR) assay. Cellular uptake of siRNA-containing gelatin nanoparticles was determined using fluorescent microscopy and stem-loop qPCR assay. siRNA loading in the RISC (RNA-induced silencing complex) was determined by immunoprecipitation of argonaute 2 (AGO2) protein followed by stem-loop qPCR for siRNA quantification. Gene expression analysis of TG2, IL-15, and the proinflammatory cytokines, tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ), was performed using qPCR assays. Efficacy of silencing TG2 and IL-15 knockdown was evaluated in an in vitro model of celiac disease by utilizing immunogenic α-gliadin peptide p31-43 in cultured J774A.1 cells. siRNA-containing gelatin nanoparticles were spherical in shape with mean particle size and charge of 217 ± 8.39 nm and -6.2 ± 0.95 mV, respectively. siRNA loading efficiency within gelatin nanoparticles was found to be 89.3 ± 3.05%. Evaluations of cellular uptake using fluorescent microscopy showed rapid internalization of gelatin nanoparticles within 2 h of dosing, with cytosolic localization of delivered siRNA in Caco-2 cells. Gelatin nanoparticles showed greater intracellular siRNA exposure with a longer half-life, when compared to Lipofectamine-mediated siRNA delivery. Approximately 0.1% of total intracellular siRNA was associated in the RISC complex. A maximum knockdown of 60% was observed at 72 h post siRNA treatment for both TG2 and IL-15 genes, which corresponded to ∼200 copies of RISC associated siRNA. Further, efficacy of gelatin nanoparticle mediated knockdown of TG2 and IL-15 mRNA was tested in an in vitro model of celiac disease. Significant suppression in the levels of proinflammatory cytokines (TNF-α and IFN-γ) was observed in p31-43 stimulated J774A.1 cells upon either IL-15 or IL-15 + TG2 siRNA treatment. The results from this study indicate that gelatin nanoparticle mediated TG2 and IL-15 siRNA gene silencing is a very promising approach for the treatment of celiac disease.