Stepwise effects of formulation additives and process parameters on ethanol injection-prepared nanoliposomes for cancer delivery: a preformulation study

The ethanol injection method has emerged as a simple and rapid technique for the preparation of nanoliposomes. Since the size and polydispersity of nanoliposomes are crucial properties which directly affect their therapeutic effectiveness, the focus of this paper was to study the influence of different additives such as cholesterol, poloxamer 188, PEG 400, and cationic lipids, as well as process parameters on the particle size and polydispersity index (PDI) of nanoliposomes intended for cancer treatment. The optimum conditions were using phospholipid concentration 20 mg/mL in ethanol, injection of the ethanolic phase along the direction of aqueous phase, use of hand-press injection of the ethanolic solution, and the use of buffer as aqueous phase. The nanoliposomes had an average size of 91.12 ± 5.50 nm with a PDI of 0.15 ± 0.01. Their properties were influenced by cholesterol content and additives such as poloxamer 188, PEG 400, and cationic lipids. PEG 400 reduced particle size to 79.70 ± 1.80 nm, while cholesterol and cationic lipids increased it to 126.29 ± 8.39 nm and 130.34 ± 8.77 nm, respectively. The optimized formulation co-loaded with apigenin and gallic acid showed high entrapment efficiency (84.74 ± 2.33% and 60.06 ± 3.94%, respectively). It also demonstrated significantly higher cytotoxicity than free drugs, with a much lower IC50 (2.99 ± 0.2 µM vs. 25.97 ± 0.9 µM), highlighting the importance of formulation parameters. This suggests that preformulation studies are crucial to delineate the best process parameters and optimum additives and their concentrations.