The Comparison of In Vitro Photosensitizing Efficacy of Curcumin-Loaded Liposomes Following Photodynamic Therapy on Melanoma MUG-Mel2, Squamous Cell Carcinoma SCC-25, and Normal Keratinocyte HaCaT Cells

Abstract

The research focused on the investigation of curcumin encapsulated in hydrogenated soy phosphatidylcholine liposomes and its increased photoactive properties in photodynamic therapy (PDT). The goal of this study was two-fold: to emphasize the role of a natural photoactive plant-based derivative in the liposomal formulation as an easily bioavailable, alternative photosensitizer (PS) for the use in PDT of skin malignancies. Furthermore, the goal includes to prove the decreased cytotoxicity of phototoxic agents loaded in liposomes toward normal skin cells. Research was conducted on melanoma (MugMel2), squamous cell carcinoma (SCC-25), and normal human keratinocytes (HaCaT) cell lines. The assessment of viability with MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) evaluated cell death after exposure to blue light irradiation after 4 h of pre-incubation with free and encapsulated curcumin. Additionally, the wound healing assay, flow cytometry, and immunocytochemistry to detect apoptosis were performed. The malignant cells revealed increased phototoxicity after the therapy in comparison to normal cells. Moreover, liposome curcumin-based photodynamic therapy showed an increased ratio of apoptotic and necrotic cells. The study also demonstrated that nanocurcumin significantly decreased malignant cell motility following PDT treatment. Acquired results suggest that liposomal formulation of a poor soluble natural compound may improve photosensitizing properties of curcumin-mediated PDT treatment in skin cancers and reduce toxicity in normal keratinocytes.

Keywords: curcumin; liposomes; melanoma; natural photosensitizer; normal keratinocytes; photodynamic therapy; skin cancer treatment; squamous cell carcinoma.

Figure 1

Scheme of encapsulated curcumin in liposomes and mechanism of photodynamic therapy.

Figure 2

Results of cell viability after 4 h of incubation with 5, 10 µM curcumin and liposomal curcumin with and without the irradiation (2.5 J/cm²) evaluated by the MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) assay. (A) Viability-dependent bars for MUG-Mel2 cells after incubation with curcumin and liposomal curcumin without/with light. (B) Viability-dependent bars for SCC-25 cells after incubation with curcumin and liposomal curcumin without/with the light. (C) Viability-dependent bars for HaCaT cells after incubation with curcumin and liposomal curcumin without/with light. Encapsulated curcumin is significantly more cytotoxic than free curcumin in cancer cells. (D–F) Representative images of MUG-Mel-2, SCC-25, HaCaT cells morphology detected by phase-contrast microscopy. Results represent the mean from three different experiments. * p < 0.05.

Figure 3

Wound-healing assay in time point 0 h and 24 h of (A) HaCaT, (B) SCC-25, and (C) MUG-Mel2 cell line. Representative images show that, after 24 h, the scrap in control cells is minimal compared to cancer cells treated with liposomal curcumin in dose 10 µM with blue light (2.5 J/cm²). In treated cells, in the HaCaT control cell line, the scrap is smaller than in the other two cancer cells (D–F). (D) Quantification of cell migration for HaCaT, SCC-25, and MUG-Mel2 cells. Results are presented as the percentage of the wound surface. The initial wound area is expressed as 100% at 0 h. Results represent the mean from three different experiments. Scale bar = 50 µm. * p < 0.05.

Figure 4

Presenting immunocytochemical staining of the chosen apoptosis-related proteins Bax and Bcl-2 in HaCaT (A), MUG-Mel2 (B), and SCC-25 (C) cells lines in four conditions: control, control with blue light only, liposomal curcumin in dose 10 µM, and liposomal curcumin in dose 10 µM with a low dose of blue light (2.5 J/cm²). (D) Results of immunocytochemical analysis of HaCaT, MUG-Mel2, and the SCC-25 cell line and the chosen apoptosis-related protein expression calculated by the immunoreactivity score. Abbreviations: 0-no staining, 1-weak staining, 2-moderate staining, and 3-strong staining. Scale bar = 50 μm.

Figure 5

Effect of photodynamic therapy (PDT) with liposomal curcumin in dose 10 µM with blue light (2.5 J/cm2) on SCC-25 (A), MUG-Mel2 (B), and HaCaT (C) cells. Dot plots present alive-Q1, early apoptotic-Q2, late apoptotic-Q3, and dead-Q4 cells. After therapy, the cells were stained using Annexin-FICT /7-AAD Kit and were measured by flow cytometry. Bars represents the quantitative percentage of total apoptotic cells (early + late apoptosis) and necrotic cells in SCC-25, MUG-Mel2, and HaCaT cells. Results represent the mean from three different experiments. * p < 0.05.