Optimization of a cationic liposome-based gene delivery system for the application of miR-145 in anticancer therapeutics

Abstract

Abstract. In order to improve the delivery efficiency of microRNA (miRNA or miR)-145, the present study examined several factors which may affect cationic liposome (CL)-based transfection, including the hydration medium used for the preparation of liposomes, the quantity of the plasmid, the molar ratio of N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTAP)/cholesterol (chol), or DOTAP/chol, and the weight ratio of DOTAP/DNA. In order to enhance the transfection efficiency, protamine was selected as a DNA-condensing agent to form liposome-protamine-DNA (LPD) ternary complexes. An agarose gel retardation assay was used to examine the DNA binding affinity of the CLs. Following transfection, GFP fluorescence images were captured and flow cytometry was performed to determine the transfection efficiency. Furthermore, an MTT assay was performed to determine the cytotoxicity of the liposome complexes. The final optimal conditions were as follows: 5% glucose as the hydration medium, a molar ratio of DOTAP/chol at 3:1 for the preparation of CLs, a weight ratio of DOTAP/protamine/DNA of 3:0.5:1, with 8 µg plasmid added for the preparation of the LPD complexes. In vitro, the LPD complexes exhibited an enhanced transfection efficiency and low cytotoxicity, which indicated that the presented LPD vector enhanced the transfection efficiency of the CLs. The HepG2 cells were found to have the lowest expression levels of miR-145out of the cell lines tested (A549, BGC-823, HepG2, HeLa, LoVo and MCF-7). Following the transient transfection of the HepG2 cells with miR-145, the results revealed that the overexpression of miR-145 inhibited the proliferation of the HepG2 cells and downregulated the expression of cyclin-dependent kinase 6 (CDK6), cyclinD1, c-Myc, and Sp1 transcription factor (Sp1). In conclusion, in this study, we optimized a liposome-based delivery system for the efficient delivery of miR-145 into cancer cells. This may provide a foundation for further research into the use of miR-145 in anticancer therapeutics.

Improved delivery of miR-1296 loaded cationic nanoliposomes for effective suppression of triple negative breast cancer

“…The high loading of miR-1296 was confirmed by a gel retardation assay at an n/p ratio of 3 by way of no migrated free miR-1296 band was detected. Our findings are consistent with cationic NL made of DOTAP:chol, that an n/p ratio of 3 showed complete retardation of miR-145 ( Tao et al, 2016 ).…”

Polyprenol-Based Lipofecting Agents for In Vivo Delivery of Therapeutic DNA to Treat Hypertensive Rats

“…The in vitro tests of PTAI-based lipofecting reagents were designed to evaluate if PTAI lipoplexes could be adjusted to in vivo administration in 5% glucose solution for single injections and for prolonged administration in osmotic pumps. Obtained results showing that 5% glucose solution is a suitable medium for PTAI-based lipoplexes preparation are in accordance with data previously shown in the literature that this solution is favorable for in vivo and in vitro nucleic acids delivery systems (Phua et al 2013;Tao et al 2016). Moreover, some data were also showing that glucose solution causes formation of smaller and more effective (especially in the presence of serum) lipoplexes (Sakaguchi et al 2008).…”

Sphingomyelin-Based Nanosystems (SNs) for the Development of Anticancer miRNA Therapeutics

“…Results evidenced again the superior performance of the formulation that encapsulates Lpx (SNs-Lpx), probably due to a protective effect of the miRNA145 ( Figure 4B). Importantly, results obtained with SNs-Lpx were superior to those recently published with miRNA145-loaded PLGA/PEI/HA nanoparticles in HTC-116 cells (11-fold increase), miRNA-145-loaded magnetic nanoparticles in AsPC-1cells (19-fold increase) and HPAF-II cells (4-fold increase), miRNA145-loaded cationic liposomes in HepG2 cells (9-fold increase), miRNA145 associated to a lentiviral vector in SW620 cells (8.2-fold increase), and miRNA145-loaded protamine nanocapsules in SW480 cells (33-fold increase) [15][16][17]49,50]. In relation to nanometric porous metal-organic frameworks (nanoMOFs), they show a similar efficiency in SW480 cells, superior to lipofectamine, as recently published by our group [51].…”