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Molecules 2019, 24, 1388 12 of 14 3.9. Cell Viability Assay The MTT assay is based on the ability of mitochondrial dehydrogenase enzymes in living cells to convert the yellow water-soluble tetrazolium salt MTT into dark blue formazan crystals. The cells were washed and incubated in serum-free CM containing 0.5 mg/mL of MTT solution at 37 ◦C in a humidified atmosphere of 5% CO2/95% air for 4 h. The media was removed and 200 μL of DMSO was then added to each well to dissolve the formazan crystals. The absorbance of formazan was measured at 540 nm by a microplate reader (CLARIOstar, BMG Labtech, Ortenberg, Germany). Four replicates of each experiment were performed. 3.10. Physical Stability in Cell Culture Medium The physical stability of the Cur-CS/Alg NPs was determined in CM using the modified version of a previous protocol [22]. Briefly, 1 mL of freshly prepared Cur-CS/Alg NP suspension from the optimal formulation was dispersed in 9 mL of CM and incubated at 37 ◦C for 72 h. At specific time intervals, the particle size and zeta potential of the Cur-CS/Alg NPs were measured as previously described. 3.11. In Vitro Cellular Uptake HaCaT cells were seeded at a density of 5 × 103 cells per well and incubated at 37 ◦C in a humidified atmosphere of 5% CO2/95% air for 24 h. The cells were washed with serum-free CM and treated with curcumin or Cur-CS/Alg NPs at final Cur concentrations of 0.05 and 0.1 μg/mL. DMSO at 0.5% v/v and CS/Alg NPs at 1% v/v were used as controls. After incubation for 4 h, the cells were washed with PBS and subsequently imaged under a fluorescence microscope (Nikon, ECLIPSE TiU, Watertown, MA, USA) using a filter excitation and emission at 400 and 470 nm, respectively. Images were processed using Nikon software. 3.12. Statistical Analysis All numerical data in this study are presented as the mean ± standard deviation (SD). Statistical analysis was performed by one-way analysis of variance (ANOVA) followed by Tukey’s post hoc comparison, accepting significance at the p < 0.05 level. 4. Conclusions This study successfully optimized the components of the desired PDT system, a combination of Cur-CS/Alg NPs and blue LED light, for the treatment of TNF-α-induced psoriasis-like proliferation of keratinocytes. The optimal formulation of the Cur-CS/Alg NPs was 1.5 mg/mL curcumin, 0.5% (w/v) Tween® 80, and a CS/Alg mass ratio of 0.08:1, while the optimal conditions of the blue LED device were a blue light irradiation of 10 J/cm2 for 5 min at a distance of 18 cm from the LED array to the illumination area. In general, the administration of Cur-CS/Alg NPs with blue LED light could be potentially developed into an effective PDT system for the treatment of psoriasis. Therefore, further investigation of the anti-psoriasis properties of Cur-Cs/Alg NPs with blue LED light in animal models based on the in vitro results presented here is recommended. Author Contributions: Conceptualization, P.R. (Pornchai Rojsitthisak) and P.R. (Pranee Rojsitthisak); Methodology, C.G., C.M. and F.N.S.; Software, C.G. and F.N.S.; Validation, C.G., C.M. and F.N.S.; Formal Analysis, C.G. and F.N.S.; Investigation, C.G. and C.M.; Resources, J.W.; Data Curation, C.G., C.M. and F.N.S.; Writing-Original Draft Preparation, C.G., C.M. and F.N.S.; Writing-Review & Editing, P.R. (Pornchai Rojsitthisak) and P.R. (Pranee Rojsitthisak); Visualization, C.G. and C.M.; Supervision, P.R. (Pornchai Rojsitthisak) and P.R. (Pranee Rojsitthisak); Project Administration, P.R. (Pornchai Rojsitthisak) and P.R. (Pranee Rojsitthisak); Funding Acquisition, P.R. (Pornchai Rojsitthisak) and P.R. (Pranee Rojsitthisak). Funding: This research was funded by the Ratchadaphiseksomphot Endowment Fund of Chulalongkorn University for The Natural Products for Ageing and Chronic Diseases Research Unit. The Clinton Gomez was financially supported by the Scholarship for International Students in ASEAN Countries from the Graduate School of Chulalongkorn University. The APC was funded by Ratchadaphiseksomphot Endowment Fund of Chulalongkorn University.PDF Image | Synergistic Effects of Photo-Irradiation and Curcumin-Chitosan
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