Enabling Combinatorial siRNA Delivery against Apoptosis-Related Proteins with Linoleic Acid and α-Linoleic Acid Substituted Low Molecular Weight Polyethylenimines.

PURPOSE: Short interfering RNA (siRNA) therapy promises a new era in treatment of breast cancers but effective delivery systems are needed for clinical use. Since silencing complementary targets may offer improved efficacy, this study was undertaken to identify non-viral carriers for combinatorial siRNA delivery for more effective therapy. METHODS: A library of lipid-substituted polymers from low molecular weight polyethyleneimine (PEI), linoleic acid (LA) and α-linoleic acid (αLA) with amide or thioester linkages was prepared and investigated for delivering Mcl-1, survivin and STAT5A siRNAs in breast cancer cells. RESULTS: The effective polymers formed 80-190 nm particles with similar zeta-potentials, but the serum stability was greater for complexes formed with amide-linked lipid conjugates. The LA and αLA substitutions, with the low molecular weight PEI (1.2 kDa and 2.0 kDa) were able to deliver siRNA effectively to cells and retarded the growth of breast cancer cells. The amide-linked lipid substituents showed higher cellular delivery of siRNA as compared to thioester linkages. Upon combinational delivery of siRNAs, growth of MCF-7 cells was inhibited to a greater extent with 2.0PEI-LA9 mediated delivery of Mcl-1 combined survivin siRNAs as compared to individual siRNAs. The qRT-PCR analysis confirmed the decrease in mRNA levels of target genes with specific siRNAs and 2.0PEI-LA9 was the most effective polymer for delivering siRNAs (either single or in combination). CONCLUSIONS: This study yielded effective siRNA carriers for combinational delivery of siRNAs. Careful choice of siRNA combinations will be critical since targeting individual genes might alter the expression of other critical mediators.

Breathing New Life into TRAIL for Breast Cancer Therapy: Co-Delivery of pTRAIL and Complementary siRNAs Using Lipopolymers.

Preclinical studies showed that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) therapy is safe and effective to combat cancers, but clinical outcomes have been less than optimal due to short half-life of TRAIL protein, insufficient induction of apoptosis, and TRAIL resistance displayed in many tumors. In this study, we explored co-delivery of a TRAIL expressing plasmid (pTRAIL) and complementary small interfering RNAs (siRNAs) (silencing Bcl2-like 12 [BCL2L12] and superoxide dismutase 1 [SOD1]) to improve the response of breast cancer cells against TRAIL therapy. It is desirable to co-deliver the pDNA along with siRNA using a single delivery agent, but this is challenging given different structures of long/flexible pDNA and short/rigid siRNA. Toward this goal, we identified an aliphatic lipid-grafted low-molecular weight polyethylenimine (PEI) that accommodated both pDNA and siRNA in a single complex. The co-delivery of pTRAIL with BCL2L12- or SOD1-specific siRNAs resulted more significant cell death in different breast cancer cells compared with separate delivery without affecting nonmalignant cells viability. Ternary complexes of lipopolymer with pTRAIL and BCL2L12 siRNA significantly retarded the growth of breast cancer xenografts in mice. The enhanced anticancer activity was attributed to increased in situ secretion of TRAIL and sensitization of breast cancer cells against TRAIL by the co-delivered siRNAs. The lipid-grafted PEIs capable of co-delivering multiple types of nucleic acids can serve as powerful carriers for more effective complementary therapeutics. Graphical Abstract [Figure: see text].

siRNA Library Screening to Identify Complementary Therapeutic Pairs in Triple-Negative Breast Cancer Cells.

The existence of tightly integrated cross talk through multiple signaling and effector pathways has been appreciated in malignant cells. The realization of the plasticity of such networks is stimulating the development of combinational therapy to overcome the limitations of one-dimensional therapies. Synergistic pairs of siRNAs or siRNA and drug combinations are the new frontiers in identifying effective therapeutic combinations. To elucidate effective combinations, we developed a versatile protocol to screen siRNA libraries in triple-negative breast cancer cell models. This protocol outlines the steps to identify synergistic combinations of siRNA-siRNA or siRNA-drug combinations using siRNA libraries via a robotic screen. By focusing on smaller functional siRNA libraries, we present methodologies to identify synergistic siRNA pairings against cancerous cell growth and molecular targets to augment the activity of pro-apoptotic TRAIL protein. Here, we summarize the critical steps to undertake such combinational target identification, emphasizing critical factors that affect the outcome of the screens. Our experience suggests that siRNA library screening is an efficient protocol to identify complementary therapeutic pairs of new or already-existing drugs. This protocol is simple, robust and can be completed within a 1-week working period.

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.

Effect of basic fibroblast growth factor in mouse embryonic stem cell culture and osteogenic differentiation.

Embryonic stem cells are actively explored as a cell source in tissue engineering and regenerative medicine involving bone repair. Basic fibroblast growth factor (bFGF) has been a valuable growth factor to support the culture of human stem cells as well as their osteogenic differentiation, but the influence of bFGF on mouse embryonic stem (mES) cells is not known. Towards this goal, D3 cells were treated with bFGF during maintenance conditions and during spontaneous and osteogenic differentiation. In feeder-free monolayers, up to 40 ng/ml of exogenous bFGF did not support self-renewal of mES without LIF during cell expansion. During spontaneous differentiation in high-density cultures, bFGF stimulated cell proliferation under certain conditions but did not influence differentiation, as judged by stage-specific embryonic antigen-1 expression. The addition of bFGF reduced the alkaline phosphatase (ALP) activity associated with osteoblast activity during differentiation induced by osteogenic supplements, although the extent of mineralization was unaffected by bFGF. The bFGF increased the mesenchymal stem cell marker Sca-1 in an mES cell population and led to an enhanced increase in osteocalcin and runx2 expression in combination with BMP-2. These results suggest that bFGF could be utilized to expand the cell population in high-density cultures in addition to enriching the BMP-2 responsiveness of mES cells.

Osteogenic differentiation of human mesenchymal stem cells cultured with dexamethasone, vitamin D3, basic fibroblast growth factor, and bone morphogenetic protein-2.

PURPOSE: Human mesenchymal stem cells (hMSCs) are pursued for cell-based therapies of bone defects. Successful use of hMSCs will require them to be osteogenically differentiated before transplantation. This study was intended to determine the optimal combination(s) of supplements needed for inducing osteogenesis in hMSCs. METHODS: The hMSCs were cultured with combinations of ß-glycerophosphate, dexamethasone (Dex), vitamin D3 (Vit-D3), basic fibroblast growth factor (bFGF), and bone morphogenetic protein-2 (BMP-2) to assess cell growth and osteogenesis. Osteogenic responses of the supplements were evaluated by alkaline phosphatase (ALP) activity, mineralization, and gene expression of ALP, Runx2, bone sialoprotein, and osteonectin. Adipogenesis was characterized based on Oil Red O staining, gene expression of peroxisome proliferator-activated receptor γ2, and adipocyte protein-2. RESULTS: Dex was found to be essential for mineralization of hMSCs. Cultures treated with Dex (100 nM), Vit-D3 (10/50 nM), and BMP-2 (500 ng/mL) demonstrated maximal calcification and up-regulation of ALP and bone sialoprotein expression. However, adipogenesis was up-regulated in parallel with osteogenesis in these cultures, as evident by the presence of lipid droplets and significant up-regulation of peroxisome proliferator-activated receptor γ2 and adipocyte protein-2 expression. An optimal condition was obtained at Dex (10 nM) and BMP-2 (500 ng/mL) for mineralization without increasing adipogenesis-related markers. The bFGF mitigated osteogenesis and enhanced adipogenesis. Vit-D3 appears essential for calcification only in the presence of bFGF. CONCLUSION: Treatment of hMSCs with appropriate supplements at optimal doses results in robust osteogenic differentiation with minimal adipogenesis. These findings could be used in the cultivation of hMSCs for cell-based strategies for bone regeneration.