Modified Spraying Technique and Response Surface Methodology for the Preparation and Optimization of Propolis Liposomes of Enhanced Anti-Proliferative Activity against Human Melanoma Cell Line A375

Abstract

Propolis is a honeybee product that contains a mixture of natural substances with a broad spectrum of biological activities. However, the clinical application of propolis is limited due to the presence of a myriad of constituents with different physicochemical properties, low bioavailability and lack of appropriate formulations. In this study, a modified injection technique (spraying technique) has been developed for the encapsulation of the Egyptian propolis within liposomal formulation. The effects of three variables (lipid molar concentration, drug loading and cholesterol percentage) on the particle size and poly dispersity index (PDI) were studied using response surface methodology and the Box-Behnken design. Response surface diagrams were used to develop an optimized liposomal formulation of the Egyptian propolis. A comparative study between the optimized liposomal formulation prepared either by the typical ethanol injection method (TEIM) or the spraying method in terms of particle size, PDI and the in-vitro anti-proliferative effect against human melanoma cell line A375 was carried out. The spraying method resulted in the formation of smaller propolis-loaded liposomes compared to TEIM (particle sizes of 90 ± 6.2 nm, and 170 ± 14.7 nm, respectively). Furthermore, the IC50 values against A375 cells were found to be 3.04 ± 0.14, 4.5 ± 0.09, and 18.06 ± 0.75 for spray-prepared propolis liposomes (PP-Lip), TEIM PP-Lip, and propolis extract (PE), respectively. The encapsulation of PE into liposomes is expected to improve its cellular uptake by endocytosis. Moreover, smaller and more uniform liposomes obtained by spraying can be expected to achieve higher cellular uptake, as the ratio of liposomes or liposomal aggregates that fall above the capacity of cell membrane to "wrap" them will be minimized.

Keywords: enhanced cellular uptake; liposomes; propolis; response surface methodology; spraying technique.

Figure 1

Schematic diagram showing the details of the spray based preparation method of PP-Lip. This figure was created using Biorender.com.

Figure 2

Macroscopic examination of the typical ethanol injection method (TEIM)-prepared and spray-prepared PP-Lip.

Figure 3

(a) 3D plot showing the effect of CH% and LMC on particle size. (b) Perturbation blot of factors affecting particle size.

Figure 4

(a) 3D plot showing the effect of CH% and LMC on PDI. (b) Perturbation plot of factors affecting PDI.

Figure 5

Top: particle size distribution of liposomes prepared using (a) TEIM and (b) spray method. Transmission electron microscope (EM) image of the (c) TEIM-prepared and (d) spray-prepared PP-Lip.

Figure 6

(a) Dose–response curves representing the cytotoxic activity of propolis extract (PE), TEIM-prepared PP-Lip and spray-prepared PP-Lip against A375 human melanoma cells, and (b) IC50 values (μg/mL) of different formulations. Values are average and n = 3. In the bottom figure, A represents a significant difference from PE (p < 0.01), and B means significantly different from TEIM PP-Lip (p < 0.05).