Development of Drug Delivery Techniques for RNA Interference Therapy in Biomedical Applications


Student thesis: Doctoral Thesis

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Award date11 Oct 2019


RNA interference exists extensively in eukaryotic cells and different biomolecules play crucial roles in gene silencing, including small interfering RNA (siRNA), antisense RNA, and short hairpin RNA. Among these RNA molecules, siRNA is most commonly used to combat ‘undruggable’ diseases. However, the design and synthesis of efficient siRNA vehicles still require improvement. In the first part of thesis, we provided a brief review of the RNA interference (RNAi) technology and siRNA delivery techniques. The intracellular fate of siRNA delivery vehicles, including their interactions with the endocytic and autophagic pathways, was also discussed.

Although RNAi technology is widely applied for treatment of ‘undruggable’ diseases, one of the major obstacles impeding its progression of clinical trials is the shortage of efficient delivery vehicles. In the second part of thesis, we reported the development of a novel platform to manufacture lipid nanoparticles (LNPs) for both in vitro and in vivo use by solvent exchange method. After optimization of composition, the final LNP was around 50 nm in size and had a slightly negative charge, as measured by dynamic light scattering (DLS) approach. These LNPs were purified by Diethylaminoethyl (DEAE) beads and concentrated by ultracentrifugation and rotatory evaporation. The concentrated LNPs could be diluted in either Opti-MEM for in vitro transfection or saline for in vivo administration. HeLa cells with ectopic expression of EGFP protein were utilized to examine the gene silencing efficiency of LNPs. Cells transfected with LNP exhibited more than 90% viability and only ~30% of these cells were EGFP positive. Also, ectopic EGFP expression was significantly reduced, which was confirmed at both mRNA and protein levels. We also established an in vivo tumor model by subcutaneously injecting breast cancer 4T1 cells into nude mice. siRNA targeting PLK1 oncogene was packaged into LNP to test the anti-tumor effect in vivo. Two weeks post LNP administration, we observed a significant reduction of tumor size in siPLK1 treatment groups compared to the negative control group (siNC). The metastatic ability of 4T1 cells was assessed by counting nodes on lungs after whole lung staining with ink, and there were much fewer nodes in the siPLK1 treatment group than in siNC treatment group. After encapsulation of Cy5-labeled siRNA, the circulation and distribution of nanoparticles could be directly detected by animal live imaging. We found that LNP mainly accumulated in the liver 12 hours after intravenous injection, which was consistent with previous reports. This LNP synthesis platform could be improved for production of nanoparticles for both cellular transfection and animal studies.

Autophagy and endocytosis are significant pathways regulating macromolecule recycling and regeneration. Once nanoparticles are internalized, one of the major challenges is how to increase the intracellular release of siRNA while minimizing the interference of autophagy. In the third part of thesis, either LNP or Lipofectamine was used as a tool to explore the crosstalk between autophagy and endocytosis pathways and the effects of pathway interactions on siRNA-based gene knockdown. In our study, a series of small molecule inhibitorswere utilized to modulate these pathways, and their functions in perturbing gene silencing were tested. We especially focused on one drug, Vacuolin-1, which potently and reversibly inhibited autophagy by activating Rab5. In addition, its inhibitory effect on exocytosis was found in a variety of cell types. We discovered that Vacuolin-1 significantly reduced gene silencing caused by siRNA which was delivered by either Lipofectamine or lipid nanoparticles in HeLa cells. Vacuolin-1 exhibited the strongest inhibition effect among a few autophagy inhibitors, including Chloroquine, Wortmannin, and Bafilomycin A1. Quantification of siRNA indicated that siRNAs were trapped within cells instead of being secreted into the supernatant. Delivery of siRNA by electroporation confirmed that the general RNA interference machinery was not impaired after treatment with Vacuolin-1. siRNAs over-accumulated intracellularly and colocalized with endosome marker Rab5 or Rab7 in Vacuolin-1 treated cells, suggesting that Vacuolin-1 inhibited the cytoplasmic release of siRNA from late endosomes. Downregulation of key genes in autophagy (Atg5) or in endocytosis (Rab5) pathways rescued the decreased gene knockdown caused by Vacuolin-1 treatment. We proposed that Vacuolin-1 could potentially be used to regulate the intracellular release of siRNA delivered by lipid-based drug carriers for better therapeutic effects.

A series of studies related to lipid-based drug carriers have been described in the thesis, including LNP production, in vitro gene silencing by LNP, testing the antitumor effects of LNP in vivo, investigating the intracellular fate of lipid-based siRNA vehicles, and dissecting the signaling pathways involved. We hope that this thesis can serve as a reference for future development of siRNA delivery techniques.

    Research areas

  • lipid nanoparticle, siRNA, gene silencing, endocytosis, autophagy