Development of ;;Smart;; Particles for Silencing Anti-apoptotic Bel-2 Protein Expression in Epithelial Cancer Cells.

Gene Delivery;Biomedical Engineering;Engineering;Biomedical Engineering

Lin, Yen-LingMycek, Mary-Ann ;

University of Michigan

关键词: Gene Delivery;    Biomedical Engineering;    Engineering;    Biomedical Engineering;   

Others  :  https://deepblue.lib.umich.edu/bitstream/handle/2027.42/97922/ccling_1.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

B-cell lymphoma 2 (Bcl-2) is an anti-apoptotic protein that is over-expressed in head and neck cancer cells and results in increased radio- and chemo-resistance. Short interfering RNA (siRNA) inhibits Bcl-2 expression causing enhanced cancer cell death and reduction of tumor growth. Transforming anti-Bcl-2 siRNA into a viable therapy with a defined dosage regimen requires a biocompatible carrier that can shuttle a large dose of siRNA into the cytoplasm of cancer cells. This dissertation describes the development of degradable, pH-sensitive, membrane-destabilizing, comb- and star-shaped polymers to condense anti-Bcl-2 siRNA into ;;smart” nanoparticles, which bypassed the endosomal membrane and delivered the cargo into the cytoplasm of cancer cells resulting in efficient knockdown of Bcl-2 gene expression. Specifically, comb-like polymers were synthesized by grafting copolymers of cationic trimethyl aminoethyl methacrylate (TMAEMA) and hydrophobic hexyl methacrylate (HMA) monomers from a diblock linear backbone via acid-labile hydrazone linkages. Similarly, β-cyclodextrin (β-CD) was used as a core to synthesize star-shaped polymers where pH-sensitive dimethyl aminoethyl methacrylate (DMAEMA) and hydrophobic HMA monomers were grafted from the secondary face of β-CD via hydrazone linkages to form β-CD-P(HMA-co-DMAEMA) polymers. Both comb- and star-shaped polymers condensed siRNA molecules into ;;smart” particles that were stable at physiologic pH but rapidly degraded into membrane-active fragments in acidic endosomal pH. We systematically evaluated the effect of hydrophobic/hydrophilic balance (HMA/DMAEMA ratio), percentage of DMAEMA monomers quaternized into TMAEMA, and molecular weight of grafts on the ability of star polymers to achieve functional delivery of anti-Bcl-2 siRNA. Results show that star-shaped polymers incorporating P(HMA-co-DMAEMA-co-TMAEMA) grafts with the MW of 25 kDa, 50/50 HMA/DMAEMA monomers, and 50% of DMAEMA monomers transformed to TMAEMA exhibit the highest transfection efficiency. These star-shaped polymers delivered anti-Bcl-2 siRNA into UM-SCC-17B cells causing 50-75% reduction in Bcl-2 mRNA and protein levels. Further, combining ;;smart” particles loaded with anti-Bcl-2 siRNA with AT-101(a Bcl-2 small molecule inhibitor) synergistically inhibited the proliferation of cancer cells by 63% while increasing cancer cell apoptosis by 12-14%. These results confirm the successful development of a new family of degradable, pH-sensitive, membrane-destabilizing star-shaped polymers that enhance the cytoplasmic delivery of anti-Bcl-2 siRNA into head and neck cancer cells.