Box-Behnken design assisted approach in optimizing lipid composition for cationic liposome formulation as gene carrier

Cationic liposomes represent a promising non-viral carrier platform for gene delivery. The successful intracellular delivery of genes to the target cell is highly influenced by lipid compositions in the liposomal formulation. In the present study, a Box-Behnken design was applied to investigate the optimal lipid composition for the liposome-based transfection agent. The concentrations of DOTAP, DSPE-PEG, and cholesterol were set as independent factors. A total of 15 lipid compositions were generated and tested for specific responses, including particle size, encapsulation efficiency, cell viability, and cell transfection. The data were then analyzed to predict the optimal composition using response surface methodology (RSM). The results for particle size, encapsulation efficiency, cell viability and fluorescence intensity ranged from 158.7 to 2064 nm, 48.19–95.72%, 81.50–122.67%, and 0.0–9.08, respectively. Compositions of liposome-based transfection agent without DOTAP, those without cholesterol, and those containing DSPE-PEG2000 with a molar ratio equal to or greater than that of cholesterol tended to exhibit low encapsulation efficiency. The ability of the liposome to complex DNA, as determined through electrophoresis gel retardation assay, showed that the composition without DOTAP produced DNA bands, indicating that the prepared liposomes had a less ability to complex DNA. The cytotoxicity test results indicated that all lipid compositions were considered non-toxic, as they exhibited >80% cell viability. The cell transfection assay demonstrated that the lipid composition containing a combination of DOTAP and cholesterol was able to transfect DNA into cells. According to response analysis, RSM predicted that the optimal lipid composition consisted of 2.75 μmol DOTAP and 0.91 μmol cholesterol, with a desirability value of 0.85. Although the equation model is still acceptable for predicting the optimal lipid composition, further study is needed to obtain a model with higher desirability, such as by using more lipid compositions, increased replications, and different variable responses. •Gene delivery to target cell by liposomes is highly influenced by lipid composition•We used Box-Behnken design to optimize lipid composition in liposome formulation.•The concentrations of DOTAP, DSPE-PEG, and cholesterol were set as independent factors.•Particle size, encapsulation efficiency, cell viability, and cell transfection were selected as specific responses.•RSM p