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 di erent 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 e ects 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 e ect
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.