Optimising RNA delivery into renal tubular epithelial cells

Abstract

BACKGROUND: RNA-based drugs are a promising therapeutic option for treating kidney disease. However, one main challenge is selectively targeting these oligonucleotides to one kidney cell type only, without off target effects. My PhD sought to optimise extracellular RNA delivery into renal proximal tubular epithelial cells (rpTECs) using nanoparticles composed of chitosan, which is a biodegradable and biocompatible polysaccharide-based polymer that is a derivative of chitin. Additionally, this body of work also aimed to ascertain if the uptake of chitosan nanoparticles by rpTECs was in-part dependent on a large endocytic protein called megalin.

METHOD: By combining RTqPCR and polyacrylamide gel electrophoresis, two immortalised proximal tubular cell lines were probed to ascertain if megalin was expressed at the transcriptional and protein level. After phenotyping different potential cell lines for megalin expression, an immortalised porcine proximal tubular cell line (LLC-PK1) was selected for downstream nanoparticle uptake experiments. Chitosan nanoparticles were produced using the ionic gelation method, in which sodium tripolyphosphate was added dropwise to chitosan solution, at different concentrations, under continuous magnetic stirring. Dynamic light scattering was then used to measure the particle size, polydispersity index (PDI), and zeta potential (surface charge) of chitosan nanoparticles and their morphology assessed using scanning electron microscopy. Chitosan nanoparticles were used to entrap Cy3-tagged small interfering RNA (siRNA), and the nanocomplex used to transfect porcine proximal tubular LLC-PK1 cells. By assessing the fluorescence intensity of Cy3-signal, a microplate reader was used to measure the transfection efficiency of chitosan nanoparticles loaded with Cy3-tagged siRNA; and to ascertain potential cytotoxicity induced by chitosan nanoparticles and their siRNA cargo. In order to detect whether target-specific siRNA delivered by chitosan nanoparticles was able to mediate gene silencing, II developed a reporter assay using fluorescent mCherry protein. To ascertain if the endocytosis of chitosan nanoparticles was in-part megalin dependent, mCherry stably expressing LLC-PK1 cells were transfected with siRNA-loaded chitosan nanoparticles in the presence or absence of cilastatin, a drug known to block megalin receptors.

RESULTS: Unlike human renal proximal tubular cells immortalised using human telomerase reverse transcriptase (rpTEC-TERT1), porcine proximal tubular LLC-PK1 cells demonstrated significant expression of the LRP2 transcript that encoded megalin and produced high molecular weight megalin bands on gel electrophoresis: indicative of megalin expression. Small chitosan nanoparticles (~100nm) with a PDI of 50%, and overall surface charge of ± 30 mV were produced at a chitosan to sodium tripolyphosphate ratio of 2-2.5 to 1. On scanning electron microscopy, chitosan nanoparticles were asymmetrical in shape. The loading of these chitosan nanoparticles with Cy3-tagged siRNA achieved an encapsulation efficiency of ~98%. Additionally, there was significant intracellular uptake of chitosan-siRNA loaded nanoparticles by LLC-PK1 cells as early as 4.5 hours post cell transfection. Furthermore, LLC-PK1 cells exposed to chitosan nanoparticles demonstrated increased viability and proliferation. Chitosan nanoparticles loaded with Cy3-tagged target-specific siRNA induced significant gene silencing in a stable polyclone of mCherry expressing LLC-PK1 cells. However, chitosan nanoparticles were less efficient at mediating gene silencing than the liposomal transfection reagent, lipofectamine™ RNAiMAX. In the presence of cilastatin, chitosan nanoparticles loaded with target-specific siRNA still facilitated gene silencing even with megalin receptor blockade, which suggested that the endocytosis of chitosan nanoparticles was not entirely megalin dependent.

CONCLUSION: Taken together, the work presented in this thesis contributes to our mechanistic understanding of how chitosan nanoparticles are taken up by renal proximal tubular cells. The major outcome of my PhD could help to inform further research into the development of kidney-targeting delivery vehicles for RNA therapeutics.