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Molecules 2019, 24, 1388 8 of 14 with a previous study that reported that LED irradiation with 412 to 426 nm blue light was toxic to keratinocytes only at higher light doses (33–100 J/cm2) [9]. In order to simulate a condition of hyperproliferative keratinocytes similar to that observed in psoriasis, HaCaT cells were treated with TNF-α for 24 h. The induction with TNF-α successfully increased the relative number of viable cells to 135.5% of that of the control group (Figure 2). Upon treatment with free curcumin at 0.05 μg/mL and 0.1 μg/mL, the relative number of viable TNF-α-induced cells decreased by 8.6% and 24.4%, respectively, (Figure 2A). With subsequent photo-irradiation of the free curcumin-treated cells, the viable cell number was further decreased by 30.8% and 42.7%, respectively (Figure 2A). On the other hand, treatment of the TNF-α-induced cells with Cur-CS/Alg NPs decreased the number of viable cells by about 26.0% and 41.0% at equivalent curcumin concentrations of 0.05 μg/mL and 0.1 μg/mL, respectively (Figure 2B). The loss in viable cell numbers of the Cur-CS/Alg NP treated cells was increased to 42.6% and 57.7%, respectively, after exposure to the blue LED light (Figure 2B). The desired effect of PDT is the reduction of the keratinocyte (here HaCaT cells) proliferation caused by TNF-α without decreasing the cell viability below that of untreated cells. The treatments that produced this desired effect were 0.1 μg/mL of free curcumin and 0.05 μg/mL of Cur-CS/Alg NPs followed by subsequent exposure to 10 J/cm2 of blue LED light. Treatment with 0.1 μg/mL of free curcumin or 0.05 μg/mL of Cur-CS/Alg NPs followed by photo-irradiation resulted in a decreased number of viable cells to 93.0% and 90.6%, respectively, which are significantly lower than that of the TNF-α-induced HaCaT cells, but not significantly different from the cell viability of the untreated normal HaCaT cells. These results suggest that Cur-CS/Alg NPs are more effective in reducing the cell proliferation induced by TNF-α than free curcumin, which might be due to the enhanced cellular uptake of the Cur-CS/Alg NPs compared to free curcumin. For example, it was previously demonstrated that encapsulation of hydrophobic molecules, like curcumin and its derivatives, may increase the drug effectiveness by improvement of its delivery into the cell [18–20,22]. The results also demonstrated that a further reduction in the number of viable cells was obtained when the treatment was combined with photo-irradiation. Specifically, the reduced HaCaT proliferation was more noticeable for the combination of blue LED light irradiation in the presence of Cur-CS/Alg NPs than in the presence of free curcumin. A possible explanation for the increased efficacy of Cur-CS/Alg NPs is the photoprotection provided to the encapsulated curcumin, and the low-level sustained release of curcumin from the Cur-CS/Alg NPs, as supported by a previous study [29]. This means that curcumin is protected from premature photodegradation during delivery and only the released curcumin inside the cells interacts with the blue light for an increased effect. Therefore, in summary, these effects are synergistic and the combination of Cur-CS/Alg NPs and photo-irradiation results in a higher loss of TNF-α-induced proliferation, at least in HaCaT cells. This observation suggests a potential approach in PDT for the PDT of psoriasis. 2.4. Physical Stability The physical stability of the optimized Cur-CS/Alg NPs in culture medium (CM) was evaluated at 37 ◦C for 72 h. The size and the zeta potential of Cur-CS/Alg NPs in CM were unchanged during incubation (Figure 3). In other words, there was no dissociation of Cur-CS/Alg NPs into small fragments or agglomeration of Cur-CS/Alg NPs, suggesting that no digestion of NPs into smaller particles occurred during the sample treatment of TNF-α-induced psoriasis-like proliferation of HaCaT cells.PDF Image | Synergistic Effects of Photo-Irradiation and Curcumin-Chitosan
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