Safe and Effective RNA-based Nanomedicines for Cancer Management

• Author / Creator

  Peng, Yi-Yang

• Small interfering ribonucleic acids (siRNA) effectively downregulate the specific gene expression from cleaving the associated messenger RNA (mRNA) in the cytoplasm, which is a promising strategy in cancer management as one of the origins of cancer is the unregulated expression of the oncogene. Over decades, nanotechnological research has focused on enhancing siRNA delivery to the cytoplasm of the target cell since the large molecular weight and anionic nature of siRNA impede the siRNA from permeating the cellular membrane, and the blood circulation time of free siRNA is really short. However, even after a tremendous amount of research on cationic polymers, a safe and effective non-viral vector is not yet available. Obstacles, such as opsonization, non-specific delivery, low cellular uptake, endosomal trap, intracellular release of siRNA and cytotoxicity, further make the development of gene therapy in cancer management more challenging. In this thesis, we report the development of three low toxic, nano-sized polymeric siRNA carriers with cytoplasmic triggered siRNA release capability. We also investigated their efficiency in mediating the knockdown of epidermal growth factor receptor (EGFR) in HeLa cells to determine their potential advantages in siRNA delivery.
  First, several acid degradable, galactose-based, cationic, and hyperbranched polymers with varying molecular weights (10 to 20 kDa) (MW) and compositions with 2-lactobioamidoethyl methacrylamide [LAEMA] and 2-aminoethyl methacrylamide hydrochloride [AEMA] at different ratios (2.0, 1.0, and 0.5) were prepared via reversible addition-fragmentation chain-transfer (RAFT) polymerization. Subsequently, the polymers were used to form polyplexes with siRNA, and the EGFR knockdown efficiency in cervical carcinoma was determined. By quantifying the EGFR expression for each treatment group by Western blot assays, 10 kDa polymer, which has a LAEMA:AEMA (L/A) ratio of 2.0, demonstrated a superior EGFR knockdown efficiency (~60%) than the others, and low toxicity levels. In addition, the polyplexes demonstrated to have excellent stability under physiological conditions for up to 2 days.
  Next, novel thermo-responsive and cationic hyperbranched polymers were prepared from (AEMA) and di(ethylene glycol) methyl ether methacrylate (DEGMA) via the RAFT polymerization for siRNA delivery. Thermo-responsiveness of polymers allows the formation of stable polyplexes in the human body environment. Non-degradable and acid-degradable hyperbranched polymers were synthesized using N,N′-methylene bis(acrylamide) (MBAm) and 2,2-dimethacroyloxy-1-ethoxypropane (DEP) cross-linkers, respectively. Both types of polymers were capable of forming stable nanosized polyplexes with siRNA. Epidermal growth factor receptor (EGFR) silencing of 95% was achieved with a cationic hyperbranched polymer that incorporated an acid-trigger release strategy, and no significant cytotoxicity was observed. Our results confirmed the high potency of using such hyperbranched polymers for the efficient protection and delivery of siRNA.
  Finally, redox-responsive galactose-based hyperbranched polymers (HRRP) were synthesized via RAFT polymerization from redox-responsive cross-linker N,N′-bis(methacryloyl)cystamine, LAEMA(L), and AEMA (A) with molecular weights of 10 and 20 kDa and L/A ratios of 1.5 and 1.0 were prepared. Disulfide-based cross-linker was chosen to selectively release the siRNA into the cytoplasm of the cancer cells by the trigger of elevating the level of glutathione (GSH) concentration in the tumor environment. 85% EGFR knockdown efficiency was achieved using 10 kDa HRRP with L/A ratio of 1.5 without triggering significant cellular death (around 95% cell viability).
  All proposed polymers showed enhanced EGFR knockdown efficiency (60% with the least efficient one) while maintaining a low toxicity level with cancer (HeLa) and normal (MRC-5) cell lines in vitro studies. All the polymers were capable of forming stable nano-sized polyplexes in the growth medium, even in the presence of serum. By employing passive targeting strategy, enhanced permeation and retention (EPR) effect, and extending the blood circulation time from having stealth property to escape reticuloendothelial system and renal clearance, the potential for success in vivo studies is noteworthy high. Before studying these materials in clinical trials, more in vivo studies will be required. Furthermore, we intend to use these polymers to deliver other kinds of therapeutic nucleic acid to treat additional cancer types, such as hepatocytes, which would potentially unfold the potential of these polymers as a new nanotechnological platform for nucleic acid delivery.

• Subjects / Keywords

  • Gene Delivery
  • Nanomedicine
  • Cancer Management
  • Cationic glycopolymers
  • Serum Stability
  • siRNA

• Graduation date

  Spring 2022

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/r3-pnr1-tt61

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.