Figure 2 Response surface for the effects of independent variables on the size of EGCG nanoliposomes. The effects of phosphatidylcholine-to-cholesterol ratio and Tween 80 concentration were shown in (A) (EGCG concentration = 5 mg/mL and rotary evaporation temperature = 35°C); the effects of EGCG concentration and rotary evaporation temperature were shown in (B) (phosphatidylcholine-to-cholesterol ratio = 4

and Tween 80 concentration = 1 mg/mL). The effect of the EGCG concentration and rotary evaporation temperature on the nanoliposome size is given in Figure  2B. The rotary evaporation temperature had an effect on the size of the liposomes. Zhou et al. reported that during the preparation, the lipid solution Apoptosis inhibitor temperatures

are critical parameters for the character of the gemcitabine liposome injection [37]. Besides, it has also been cited that different EGCG concentrations have an effect on the particle size and dispersion of the liposome. Similar trend has been reported for paclitaxel magnetic nanoparticle liposome [38]. Optimization After the effects of PC/CH, EGCG concentration, Tween 80 concentration, and rotary evaporation temperature on the formulation of EGCG nanoliposomes were investigated, the optimum ranges for each independent variable were found to generate EGCG nanoliposomes with the highest EE and AG-014699 datasheet small size. The optimum formulation conditions were as follows (Table  3): phosphatidylcholine-to-cholesterol ratio of 4.00, EGCG concentration of 4.88 mg/mL, Tween 80 concentration of 1.08 mg/mL, and rotary evaporation temperature of 34.51°C. The conditions gave the highest encapsulation efficiency (85.79% ± 1.65%) with the low value of the particle size (180 nm ± 4 nm), and the experimental values were close to the predicted values (Table  4), which indicated that the optimized preparation conditions were very reliable.

17-DMAG (Alvespimycin) HCl EGCG nanoliposomes of optimized formulation were used for the determination of particle size distribution (Figure  3). The results indicated that the model used can identify operating conditions for preparing EGCG nanoliposomes. Table 3 Predicted optimum conditions for the preparation of EGCG nanoliposomes Factor Low High Optimum Phosphatidylcholine/cholesterol 3 5 4 EGCG concentration (mg/mL) 4 6 4.88 Tween 80 concentration (mg/mL) 0.5 1.5 1.08 Rotary evaporation temperature (°C) 30 40 34.51 Table 4 Predicted and experimental values of the responses obtained at optimum conditions Response Predicted value Experimental value EE (%) 85.14 85.79 ± 1.65 Size (nm) 181 180 ± 4 Results are shown as the mean ± SD (n = 3). Figure 3 The particle size of the optimized EGCG nanoliposomes. Malondialdehyde value Phospholipid was used as the major component of liposomal membrane, containing partially polyunsaturated fatty acid residues sensitive to oxidative free radicals [39]. The MDA, which is a final product of fatty acid peroxidation, was evaluated in the study.