Superior Drug Delivery Performance of Multifunctional Bilosomes: Innovative Strategy to Kill Skin Cancer Cells for Nanomedicine Application

Abstract

Purpose: Numerous failures in melanoma treatment as a highly aggressive form of skin cancer with an unfavorable prognosis and excessive resistance to conventional therapies are prompting an urgent search for more effective therapeutic tools. Consequently, to increase the treatment efficiency and to reduce the side effects of traditional administration ways, herein, it has become crucial to combine photodynamic therapy as a promising therapeutic approach with the selectivity and biocompatibility of a novel colloidal transdermal nanoplatform for effective delivery of hybrid cargo with synergistic effects on melanoma cells.

Methods: The self-assembled bilosomes, co-stabilized with L-α-phosphatidylcholine, sodium cholate, Pluronic^® P123, and cholesterol, were designated, and the stability of colloidal vesicles was studied using dynamic and electrophoretic light scattering, also provided in cell culture medium (Dulbecco's Modified Eagle's Medium). The hybrid compounds - a classical photosensitizer (Methylene Blue) along with a complementary natural polyphenolic agent (curcumin), were successfully co-loaded, as confirmed by UV-Vis, ATR-FTIR, and fluorescent spectroscopies. The biocompatibility and usefulness of the polymer functionalized bilosome with loaded double cargo were demonstrated in vitro cyto- and phototoxicity experiments using normal keratinocytes and melanoma cancer cells.

Results: The in vitro bioimaging and immunofluorescence study upon human skin epithelial (A375) and malignant (Me45) melanoma cell lines established the protective effect of the PEGylated bilosome surface. This effect was confirmed in cytotoxicity experiments, also determined on human cutaneous (HaCaT) keratinocytes. The flow cytometry experiments indicated the enhanced uptake of the encapsulated hybrid cargo compared to the non-loaded MB and CUR molecules, as well as a selectivity of the obtained nanocarriers upon tumor cell lines. The phyto-photodynamic action provided 24h-post irradiation revealed a more significant influence of the nanoplatform on Me45 cells in contrast to the A375 cell line, causing the cell viability rate below 20% of the control.

Conclusion: As a result, we established an innovative and effective strategy for potential metastatic melanoma treatment through the synergism of phyto-photodynamic therapy and novel bilosomal-origin nanophotosensitizers.

Keywords: A375 cells; Me45 cells; antitumor activity; human melanoma; nanovesicular carriers; phyto-photodynamic therapy.

Graphical abstract

Scheme 1

Comparison of the skin permeation mechanisms of the proposed functional bilosomes over conventional liposomes, with particular emphasis on their potential application in melanoma treatment.

Figure 1

Physicochemical properties of bilosomal formulations. Effect of storage time on particle size (Z-Ave) and polydispersity index (PdI) for empty and MB/CUR-bilosomes (A). TEM image of MB/CUR-bilosomes. The scale bar represents 50 nm (B). Time-dependent colloidal stability of the empty and double-loaded bilosomes- ζ-Potential changes over time (C). Fluorescence emission spectra before and after MB encapsulation in functional bilosomes (λ_exc=666 nm) (D).

Figure 2

Comparative ATR-FTIR spectra for empty bilosomes (grey line, A), pure MB (blue line, B), pure CUR (yellow line, C), and MB/CUR-bilosomes (green line, D) in the whole range region (4000–400 cm⁻¹ along with fingerprint regions of acyl chains (3000–2800 cm⁻¹) and polar head group (1300–900 cm⁻¹) of phospholipids for empty and double-loaded bilosomes.

Figure 3

Distribution of Z-Average (Z-Ave) and polydispersity index (PdI) as a function of incubation time in DMEM without (A) and supplemented with 10% FBS (B) for MB/CUR-loaded bilosomes.

Figure 4

Flow cytometry uptake comparison of the studied nanosystems (A) by human cancer A375, Me45, and normal HaCaT cell lines with the corresponding histograms (B) after 24 h incubation at 37 °C, ^##Statistical analysis: p ≤ 0.01.

Figure 5

Bioimaging and immunofluorescence study in Me45 and A375 cells (the final PS concentration was 2 µM). Holotomographic-HTM-visualization of the human melanoma morphology (A), immunofluorescent F-actin staining (B) of Me45 and A375 cells.

Figure 6

The dark cytotoxicity (without irradiation) and photocytotoxicity (phyto-PDT action) of the MB/CUR-loaded bilosomes compared to the free MB molecules (the final PS concentration was 2 µM), upon human melanoma A375 and Me45 cell lines, after incubation for 24 h post red light irradiation.