10 research outputs found
A Delicate Balance When Substituting a Small Hydrophobe onto Low Molecular Weight Polyethylenimine to Improve Its Nucleic Acid Delivery Efficiency
A Delicate Balance When Substituting a Small Hydrophobe onto Low Molecular Weight Polyethylenimine to Improve Its Nucleic Acid Delivery Efficiency
High
molecular weight (HMW) polyethylenimine (PEI) is one of the most versatile
nonviral gene vectors that was extensively investigated over the past
two decades. The cytotoxic profile of HMW PEI, however, encouraged
a search for safer alternatives. Because of lack of cytotoxicity of
low molecular weight (LMW) PEI, enhancing its performance via hydrophobic
modifications has been pursued to this end. Since the performance
of modified PEIs depends on the nature and extent of substituents,
we systematically investigated the effect of hydrophobic modification
of LMW (1.2 kDa) PEI with a short propionic acid (PrA). Moderate enhancements
in PEI hydrophobicity resulted in enhanced cellular uptake of polyplexes
and siRNA-induced silencing efficacy, whereas further increase in
PrA substitution abolished the uptake as well as the silencing. We
performed all-atom molecular dynamics simulations to elucidate the
mechanistic details behind these observations. A new assembly mechanism
was observed by the presence of hydrophobic PrA moieties, where PrA
migrated to core of the polyplex. This phenomenon caused higher surface
hydrophobicity and surface charge density at low substitutions, and
it caused deleterious effects on surface hydrophobicity and cationic
charge at higher substitutions. It is evident that an optimal balance
of hydrophobicity/hydrophilicity is needed to achieve the desired
polyplex properties for an efficient siRNA delivery, and our mechanistic
findings should provide valuable insights for the design of improved
substituents on nonviral carriers
Fibronectin-modified surfaces for evaluating the influence of cell adhesion on sensitivity of leukemic cells to siRNA nanoparticles
A Systematic Comparison of Lipopolymers for siRNA Delivery to Multiple Breast Cancer Cell Lines: \u3cem\u3eIn vitro\u3c/em\u3e studies
Small interfering RNA (siRNA) therapy is a promising approach for treatment of a wide range of cancers, including breast cancers that display variable phenotypic features. To explore the general utility of siRNA therapy to control aberrant expression of genes in breast cancer, we conducted a detailed analysis of siRNA delivery and silencing response in vitro in 6 separate breast cancer cell models (MDA-MB-231, MDA-MB-231-KRas-CRM, MCF-7, AU565, MDA-MB-435 and MDA-MB-468 cells). Using lipopolymers for siRNA complexation and delivery, we found a large variation in siRNA delivery efficiency depending on the specific lipopolymer used for siRNA complexation and delivery. Some lipopolymers were effective in all cell types used in this study, indicating the possibility of universal carriers for siRNA therapy. The delivery efficiency for effective lipopolymers was not correlated with dextran uptake in the cells tested, which indicated a receptor-mediated internalization for siRNA complexes with lipopolymers, unlike fluid-phase transfer associated with dextran uptake. Consistent with this, specific inhibitors involved in clathrin- and caveolin-mediated endocytosis significantly (\u3e50%) reduced the internalization of siRNA complexes in all cell types. Using JAK2 and STAT3 silencing in MDA-MB-231 and MDA-MB-468 cells, a general correlation between the uptake and silencing efficiency at the mRNA level was evident, but it appeared that the choice of the target rather than the cell type was more critical for consistent silencing. We conclude that siRNA therapy with lipopolymers can be undertaken in multiple breast cancer cell phenotypes with similar efficiency, indicating the general applicability of non-viral RNAi in clinical management of molecularly heterogeneous breast cancers. Statement of significance The manuscript investigated the efficacy of siRNA carriers across multiple breast cancer cell lines. The lipopolymeric carriers were capable of delivering effective dose of siRNA to a range of breast cancer cells. Despite some differences in uptake efficiency among cell types, the mechanism of delivery was similar, with CME and CvME significantly involved in the internalization of polyplexes, while fluid-phase endocytosis was not significant. Specific target silencing was correlated to delivery efficiency, but we did notice the presence of lipopolymers that achieved high silencing with minimal siRNA delivery. Silencing specific targets in different cell types were more uniformly achieved as compared to targeting different targets in the same cells. Our studies enhance the feasibility of delivering siRNA to different types of breast cancer cells
Multiple siRNA Delivery Against Cell Cycle and Anti-Apoptosis Proteins Using Lipid-substituted Polyethylenimine in Triplenegative Breast Cancer and Non-Malignant 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 non-malignant 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 non-malignant 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
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 with 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