The Potential of Optimized Liposomes in Enhancement of Cytotoxicity and Apoptosis of Encapsulated Egyptian Propolis on Hep-2 Cell Line

Abstract

Purpose: Development of pharmaceutical dosage forms of natural products has gained great interest recently. Propolis is a natural product with various active compounds and multiple pharmacological activities. Its resinous nature and low bioavailability were obstacles in the optimum use of this magnificent natural product.

Aim: This study evaluates the effect of using liposomes as a drug delivery system on the enhancement of the cytotoxic effect of propolis on squamous cell carcinoma cell lines (Hep-2) of head and neck.

Methods: An optimized liposomal formulation of propolis was prepared using the conventional thin film hydration method 1, 2. The prepared (Hep-2) cell line was treated with different concentrations of propolis and optimized propolis liposomes for 24 h. The effect of both propolis and propolis liposomes on cell line was investigated using MTT assay, cytological examination, and nuclear morphometric analysis. The effect of the drugs on the cell apoptosis was evaluated using Annexin V.

Results: The findings revealed that both propolis and propolis liposomes have a cytotoxic effect on Hep-2 cell line through induction of apoptosis. The effect was dose dependent. However, a statistically significant enhancement in propolis-mediated apoptosis on Hep-2 cells was elucidated due to encapsulation within the prepared liposomes.

Conclusion: Liposome is a powerful tool for enhancing the cytotoxicity of propolis against Hep-2 cell line.

Keywords: Hep-2; apoptosis; cytotoxicity; liposomes; propolis.

Figure 1

Transmission electron micrograph of the prepared liposomes P-lip (F4) when stained with uranyl acetate (2.5% w/v) with scale 0.5 µm.

Figure 2

In vitro release of flavonoids from the prepared propolis-encapsulated liposomes (F1–F5) in phosphate-buffered solution containing 0.5% tween 80 to maintain sink condition (PBS, pH 7.4, 37 °C). The results are represented as the means ± SD (n = 3).

Figure 3

The mean viability percentage of HEP-2 cells treated with different concentrations of propolis extract.

Figure 4

The mean viability percentage of HEP-2 cells treated with different concentrations of liposomal propolis and corresponding empty liposomes for 24 h.

Figure 5

Micrograph of untreated Hep-2 cells (control cells) at the power of 1000X oil.

Figure 6

Micrograph showing criteria of apoptosis in Hep-2 cells treated with increasing concentrations of propolis (half IC₅₀ of propolis (a), IC₅₀ of propolis (b), double IC₅₀ of propolis (c)) or propolis encapsulated-liposome (half IC₅₀ of propolis-encapsulated liposome (d), IC₅₀ of propolis-encapsulated liposome (e), and double IC₅₀ of propolis encapsulated-liposome (f)) for 24 h at the power of 1000X oil.

Figure 7

Annexin V-FITC and propidium iodide. A contour plot for cells treated with different concentrations of Propolis (half IC₅₀ of propolis (a), IC₅₀ of propolis (b), double IC₅₀ of propolis (c)) or propolis-encapsulated liposome (half IC₅₀ of propolis-encapsulated liposome (d), IC₅₀ of propolis-encapsulated liposome (e), and double IC₅₀ of propolis-encapsulated liposome (f)), and control cells (g).