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Molecules 2019, 24, 1388 6 of 14 diffusion into the layers of the skin [24]. From the results, it was found that the use of low to medium CS/Alg mass ratios produced NPs within the desired size range. The zeta potential is the electrostatic potential near the surface of the particles and is used as an indicator of the stability of NP suspensions [22]. From the results, it was observed that the use of medium to high CS/Alg mass ratios resulted in the production of Cur-CS/Alg NPs with a zeta potential from −20 mV to −30 mV, a range wherein improved stability is imparted to the NPs. The EE and LC of the CS/Alg NPs were relatively low because hydrophilic polymers (CS and Alg) were used to encapsulate a relatively hydrophobic molecule (curcumin). However, a sufficient EE and LC could be obtained even with the use of a low CS/Alg mass ratio, but increasing the CS/Alg mass ratio further decreased the EE and LC. Using RSM, the optimal formulation was derived as a CS/Alg mass ratio of 0.08:1, curcumin concentration of 1.5 mg/mL, and Tween® 80 concentration of 0.5% (w/v). To validate this optimal formulation, Cur-CS/Alg NPs were prepared using the optimized conditions and the observed values of the responses were compared with the predicted values. The results in Table 4 show that the observed and predicted values of the responses were acceptable with a low % error, which showed adequate precision for the prediction of the optimized condition. Table 4. Comparison of the observed and predicted responses of the optimized Cur-CS/Alg NP formulation. Optimized Formulation (X1, X2, X3) 15 mg/mL, 0.5% (w/v), 0.08:1 * Error was calculated as |observed value−predicted value| × 100 predicted value 2.2. Optimization of the Blue LED-Based Illumination Device Response Predicted Value Observed Value Error * 3.6 4.2 5.5 6.1 Y1 (nm) Y2 (mV) Y3 (%) Y4 (%) 254 −20.2 50.4 21.5 245 ± 11 −21.1 ± 1.2 47.6 ± 1.8 22.8 ± 0.5 The specific blue LED array device was fabricated as outlined in Section 3.5. The irradiance emitted by the LED array decreased with the increasing irradiation distance (light to plate distance) and can be continuously adjusted in the range from 28 to 181 mW/cm2. A better homogeneity of the irradiance was found for longer irradiation distances. Since the most commonly used irradiance for in vitro PDT studies is approximately 20 mW/cm2 [27], the optimal irradiation distance for the current system was set at 18 cm. In addition, the optimal operation for photodynamic therapy with this illumination device was as follows; 5 min prior to irradiation for machine equilibration, followed by 5 min of photo-irradiation. With this condition, the temperature for each experiment was controlled at 27 to 35 ◦C. Using the surrogate 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, we observed no change in the cell viability of keratinocyte after irradiation under this condition. Hence, this protocol provides an appropriate condition to investigate the effects of LED exposure in combination with other compounds (e.g., curcumin or Cur-CS/Alg NPs) in photodynamic therapy in vitro. 2.3. In Vitro Cytotoxicity/Anti-Proliferation Effect of the PDT on Normal and Tumor Necrosis Factor-Alpha (TNF-α)-Induced Cultured Human Kerlatinocyte (HaCaT) Cells The results of the MTT assay to evaluate the cytotoxic/anti-proliferative effects of photo-irradiation in combination with free curcumin dissolved in DMSO or loaded in CS/Alg NPs are shown in Figure 2. The stimulation of HaCaT cells with TNF-α resulted in an increased number of viable cells to ca. 135% relative to the unstimulated cells, confirming the responsiveness of HaCaT cells to TNF-α. The dimethyl sulfoxide (DMSO) and CS/Alg NPs controls (for free curcumin and Cur-CS/Alg NPs, respectively) showed no significant difference in the number of viable cells comparedPDF Image | Synergistic Effects of Photo-Irradiation and Curcumin-Chitosan
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