Combination Therapy Of Gene And Chemotheraneutic Drug By Lipid Nano-particles For Breast Cancer Stem Cell

One of the biggest obstacles towards treatment of breast cancer is breast cancer stem cells (BCSC), which have the properties of self-renewal and differentiation. Because of their resistance to anti-cancer therapeutics, it is hard to eliminate them. It has been proven that microRNA-200c (miR-200c) can restore chemosensitivity to microtubule-targeting drugs by reducing the expression of class III β-tubulin (TUBB3). In this study, combination therapy with miR-200c and Paclitaxel mediated by lipid nanoparticles has been investigated as an alternative strategy for achieving enhanced anticancer activity against BCSC.In this study a specific cationic lipid called DOTAP with positive charges is strategically selected to formulate solid lipid nanoparticles (SLN) for miRNA delivery. The size and zeta potential of SLN containing 20 wt% DOTAP were 28.4±4.2 nm and 18.4±1.3 mV. SLNs showed a decrease in size distribution and an increase in zeta potential after adding cationic DOTAP. The average diameter and zeta potential of SLN/miRNA nanoparticles (the weight ratio of SLN to miRNA is 90) was 36.1±3.0 nm nm and 14.7±0.9 mV, respectively. The gel retardation assay showed that the cationic SLN has the ability to condense anionic miRNAs via charge interactions to form SLN/miRNA complexes nanoparticles. The RNase protection assay showed that SLN can protect miRNAs from degradation by nucleases. The in vitro release of SLN/miRNAs was investigated as well. The release of miRNAs from SLN/miRNA showed an increase when the weight ratio of SLN is lower in the SLN/miRNA complexes.Based on our previous study, Nanostructured lipid nanoparticles (NLC) with 20 wt% oleic acid were prepared by aqueous solvent diffusion method. Mean particle sizes and zeta potential of NLC were 56.1+9.0 nm,-17.1±0.2 mV, respectively. And mean particle sizes and zeta potential of PTX loaded NLC were 64.5±3.3 nm nm and-16.4±0.3 mV. The drug encapsulation efficiency and drug loading of PTX loaded NLC were 73.33±2.03% and 3.51±0.07%. The in vitro drug release rate of PTX loaded NLC was investigated. The study showed that over 70% of drug was released in 48 h.We used multicellular spheroids, which had gained the characteristics as BCSCs, as a cell model. Real-Time PCR (PT-PCR) tests demonstrated that SLN/miRNAs presented relatively higher cellular uptake efficiency against BCSCs after incubation for 24 h, when compared with Lipofectamine 2000/miRNAs. The images obtained from CLSM and the results of colocalization of SLNs with miRNAs calculated by MetaMorph software were used to determine the intracellular miRNA release, which indicated that miRNA can be released in 12 h. Western Blot assay confirmed that SLN/miR-200c complexes could effectively down-regulate the TUBB3 expression. Images taken by confocal laser scanning microscope (CLSM) showed that ODA-FITC labeled NLC penetrated more and deeper into the multicellular spheroids when the incubation period was longer. In vitro celluar cytotoxicities tests confirmed that the blank SLN and NLC were low cytotoxic carriers. After BCSCs were transfected with SLN/miR-200c, the celluar cytotoxicities of Taxol and PTX loaded NLC against BCSCs were enhanced.