Designing Nanomedicines for Breast Cancer Therapy: New Synthetic lipopolymers and Optimization of siRNA Formulations against Therapeutic Targets
Creators: Abbasi Dezfouli, Saba
Publisher: name: University of Alberta Library
Year: 2024

<p>In 2020, breast cancer became the most commonly diagnosed cancer worldwide. Conventional therapies like radiotherapy, chemotherapy and surgery have significant limitations and side effects that are mainly due to their inability to specifically target cancerous cells. Alternatively, gene therapy which can make use of both viral and non-viral carriers piqued the interest about 40 years ago. Viral carriers although available, are worrisome due to their potential insertional mutagenesis and high toxicity. Nonviral carriers, on the other hand, are preferred since they display lower immunogenicity and production costs. Short interfering RNAs (siRNA) carrying nanoparticles have a high potential to overcome the non-specificity of conventional therapies and reduce the expression of disease-associated proteins by mimicking the naturally occurring RNA interference mechanism. <br>
Effective siRNA delivery relies heavily on the selected delivery method due to several challenges associated with siRNA, such as its immunogenicity, primarily through antiviral innate immunity mechanisms involving pattern recognition receptors. Additionally, off-target effects occur when the siRNA&#39;s antisense strand partially matches unintended mRNA transcripts. Another non-sequence-specific limitation is the saturation of the RNA interference machinery. These topics are discussed in detail in the first section of the introduction. The second section delves into a range of delivery platforms explored in research, including lipids (like liposomes, micelles, emulsions, and lipid nanoparticles), polymers, peptides (such as cell-penetrating and membrane-perturbing peptides), and combination therapies (like peptide-polymer, liposome-peptide, and lipid-polymers). These platforms are favoured for their unique benefits, such as the presence of lipids in cell membrane composition, versatile chemical properties of polymers, and precise targeting capabilities, alongside peptides&#39; pH-responsive membrane disruption. A thorough explanation of the induced toxicities of delivery systems, such as immune cell recognition by B-cells or activation of complement cascade, modulation of cellular gene expression or membrane impairment through protein kinase C inhibition, and generation of reactive oxygen species is provided in this section. <br>
To improve cancer therapies, we are engineering novel non-viral lipid substituted PEI carriers specifically for the treatment of breast cancer. Our goal is to obtain high gene silencing through siRNA activity with the least amount of nonspecific toxicity. To address these, we hypothesize that lowering the lipopolymer ratio and adding negatively charged additives to our nanocomplexes will, respectively, reduce cytotoxicity and improve gene silencing ability by facilitating siRNA polyplexes uptake by the cells. To scrutinize our hypothesis, we investigated our nanocomplexes in Chapter 2 by experimenting with potential selected additives in various weight/weight ratios of polymer:additive:siRNA. The effect of different buffers for complex preparation was also explored to provide a better siRNA delivery environment. We first optimized our polyplexes in green fluorescent protein (GFP)+ MDA-MB-231 cells to effectively silence the GFP gene using GFP siRNA. After a series of screening experiments inclusion of phosphate pH 8.0 as complexation media and ratio 1 of lipopolymer proved to be the most effective formulation. Regarding the additives’ functionality, addition of either N- Lauroylsarcosine Sodium Salt (LS) or 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) in ratio 1 to siRNA was able to improve silencing by ~30% and achieve &gt;60% silencing with the least amount of undesired cytotoxicity. Furthermore, these effects were shown to be persistent for at least 6 days in the time course study. Anti-apoptotic Survivin gene, which was shown to play an important role in breast cancer, was then selected to test our complexes for endogenous gene silencing in MDA-MB-231 cells since there is no strong drug (i.e., small organic molecule) for inhibition of its oncogenic activity. qRT-PCR analysis and MTT assay revealed &gt;90% silencing and ~70% cell death by the same formulations. These formulations were then examined by flow cytometry assay during which they demonstrated ~97% FAM-siRNA uptake after 24 hours which did not decrease more than 1%, on day 2 post-treatment. During these experiments, we were able to identify two effective additives for siRNA-polyplex formulation. The results show improved formulations for the development of modified PEI-lipopolymers to target selected endogenous genes in wild-type MDA-MB-231 cells. </p>