Combinational siRNA delivery using hyaluronic acid modified amphiphilic polyplexes against cell cycle and phosphatase proteins to inhibit growth and migration of triple-negative breast cancer cells.

Triple-negative breast cancer is an aggressive form of breast cancer with few therapeutic options if it recurs after adjuvant chemotherapy. RNA interference could be an alternative therapy for metastatic breast cancer, where small interfering RNA (siRNA) can silence the expression of aberrant genes critical for growth and migration of malignant cells. Here, we formulated a siRNA delivery system using lipid-substituted polyethylenimine (PEI) and hyaluronic acid (HA), and characterized the size, ζ-potential and cellular uptake of the nanoparticulate delivery system. Higher cellular uptake of siRNA by the tailored PEI/HA formulation suggested better interaction of complexes with breast cancer cells due to improved physicochemical characteristics of carrier and HA-binding CD44 receptors. The siRNAs against specific phosphatases that inhibited migration of MDA-MB-231 cells were then identified using library screen against 267 protein-tyrosine phosphatases, and siRNAs to inhibit cell migration were further validated. We then assessed the combinational delivery of a siRNA against CDC20 to decrease cell growth and a siRNA against several phosphatases shown to decrease migration of breast cancer cells. Combinational siRNA therapy against CDC20 and identified phosphatases PPP1R7, PTPN1, PTPN22, LHPP, PPP1R12A and DUPD1 successfully inhibited cell growth and migration, respectively, without interfering the functional effect of the co-delivered siRNA. The identified phosphatases could serve as potential targets to inhibit migration of highly aggressive metastatic breast cancer cells. Combinational siRNA delivery against cell cycle and phosphatases could be a promising strategy to inhibit both growth and migration of metastatic breast cancer cells, and potentially other types of metastatic cancer. STATEMENT OF SIGNIFICANCE: The manuscript investigated the efficacy of a tailored polymeric siRNA delivery system formulation as well as combinational siRNA therapy in metastatic breast cancer cells to inhibit malignant cell growth and migration. The siRNA delivery was undertaken by non-viral means with PEI/HA. We identified six phosphatases that could be critical targets to inhibit migration of highly aggressive metastatic breast cancer cells. We further report on specifically targeting cell cycle and phosphatase proteins to decrease both malignant cell growth and migration simultaneously. Clinical gene therapy against metastatic breast cancer with effective and safe delivery systems is urgently needed to realize the potential of molecular medicine in this deadly disease and our studies in this manuscript is intended to facilitate this endeavor.

Multiple siRNA delivery against cell cycle and anti-apoptosis proteins using lipid-substituted polyethylenimine in triple-negative breast cancer and nonmalignant cells.

Conventional breast cancer therapies have significant limitations that warrant a search for alternative therapies. Short-interfering RNA (siRNA), delivered by polymeric biomaterials and capable of silencing specific genes critical for growth of cancer cells, holds great promise as an effective, and more specific therapy. Here, we employed amphiphilic polymers and silenced the expression of two cell cycle proteins, TTK and CDC20, and the anti-apoptosis protein survivin to determine the efficacy of polymer-mediated siRNA treatment in breast cancer cells as well as side effects in nonmalignant cells in vitro. We first identified effective siRNA carriers by screening a library of lipid-substituted polyethylenimines (PEI), and PEI substituted with linoleic acid (LA) emerged as the most effective carrier for selected siRNAs. Combinations of TTK/CDC20 and CDC20/Survivin siRNAs decreased the growth of MDA-MB-231 cells significantly, while only TTK/CDC20 combination inhibited MCF7 cell growth. The effects of combinational siRNA therapy was higher when complexes were formulated at lower siRNA:polymer ratio (1:2) compared to higher ratio (1:8) in nonmalignant cells. The lead polymer (1.2PEI-LA6) showed differential transfection efficiency based on the cell-type transfected. We conclude that the lipid-substituted polymers could serve as a viable platform for delivery of multiple siRNAs against critical targets in breast cancer therapy. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 3031-3044, 2016.