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Molecules 2019, 24, 1388 10 of 14 were prepared by oil in water (o/w) emulsification followed by ionotropic gelation using a modified version of the described methods [20,22]. Briefly, 1 mL of ethanolic curcumin solution at various concentrations was added dropwise into 20 mL of Alg solution (0.6 mg/mL) containing various concentrations of Tween® 80 under vigorous stirring (1000 rpm) for 10 min. After sonication for 15 min, 4 mL of CaCl2 solution (0.67 mg/mL) was added dropwise into the resulting o/w emulsion and stirred continuously for 30 min, before addition of 4 mL of the CS solution. After being continuously stirred for 30 min, the resulting Cur-CS/Alg NP suspension was equilibrated overnight before characterization. The empty (unloaded) CS/Alg NPs were made in the same manner only without the addition of the curcumin. 3.3. Characterization of the Cur-CS/Alg NPs The particle size and zeta potential were determined using dynamic light scattering with a Nano-ZS Zeta-sizer (Malvern Instruments, Worcestershire, UK). The encapsulation efficiency (EE) and loading capacity (LC) of the CS/Alg NPs with curcumin were determined using an indirect method as previously described [22]. In brief, the Cur-CS/Alg NP suspension was separated from the aqueous media by ultracentrifugation at 105,000× g at 25 ◦C for 45 min. After freeze-drying at −85 ◦C for 12 h, the dry mass of the lyophilized NPs was recorded. Meanwhile, the amount of curcumin in the supernatant was determined using UV-vis spectroscopy (Agilent Cary 60, Agilent Technology Ltd., Santa Clara, CA, USA) to measure the absorbance at 425 nm. The EE (%) and LC (%) of the CS/Alg NPs were then calculated using Equations (5) and (6), respectively: EE (%) = 100 × (Cur in formulation − Cur in supernatant)/(Cur in formulation) (5) LC (%) = 100 × (Cur in formulation − Cur in supernatant)/(dry mass of NPs) (6) 3.4. Design and Optimization of the Cur-CS/Alg NP Formulation The Cur-CS/Alg NP formulation was designed and optimized using BBD and RSM, as provided by the Design-Expert® software (Trial-Version10.0.0, Stat Ease, MN, USA). The BBD was applied to determine the optimal level of factors (independent variables) that give responses (dependent variables). The independent variables were the curcumin concentration (X1), Tween® 80 concentration (X2), and CS/Alg mass ratio (X3). The dependent variables were the particle size (Y1), zeta potential (Y2), EE (Y3), and LC (Y4). The ranges of the independent variables and the constraints for the dependent variables are summarized in Table 5. Table 5. Variables and responses with their levels and constraints. Level Low Medium Independent variables (factors) High 1.5 2 0.2:1 Constraints 200–300 nm −30 mV to −20 mV Maximize Maximize X1 = Curcumin concentration (mg/mL) 0.5 X2 = Tween® 80 concentration (% w/v) 0.5 X3 = CS/Alg mass ratio 0.05:1 Dependent variables (responses) Y1 = Particle size (nm) Y2 = Zeta potential (mV) Y3 = EE (%) Y4 = LC (%) 3.5. Fabrication of the Blue LED Based Illumination Device 1 1 0.1:1 An LED based illumination device (Figure 5) specifically intended for the in vitro evaluation of the photodynamic effect of the Cur-CS/Alg NPs was developed based on a previous study [27]. The devicePDF Image | Synergistic Effects of Photo-Irradiation and Curcumin-Chitosan
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