Sorvall stratos centrifuge

PLGA nanoparticles (NP) were prepared by a single/double emulsion solvent evaporation technique with some modifications [38 (link)]. Briefly, 200 mg of PLGA was dissolved in 2 mL of DCM and added drop wise to 5 mL of 0.3% PVA solution under constant vortex. The solution was emulsified in ice bath for 2 min by using Ultra-Sonicator (Thermo Fisher Scientific, Fair Lawn, NJ, USA) at 40% amplitude with 10 s on/off pulse mode. Then, the emulsified solution was transferred into 45 mL of 0.3% PVA solution and the mixture was stirred for 3 h to allow the solvent evaporation and particle hardening. The particles were collected by Sorvall Stratos Centrifuge (Thermo Fisher Scientific, Fair Lawn, NJ, USA) at 10016× g for 30 min at 4 °C and washed three times with DI water. The purified PLGA NPs were lyophilized for 2 days by using Freezone Freeze Dryer (Labconco, Kansas City, MO, USA) and stored at −20 °C.
For drug encapsulation, the diclofenac drug solution was mixed with polymer solution and the mixture was sonicated for 30 s before mixing with PVA emulsifier as above. The chitosan coated samples were prepared by mixing the chitosan solution (in 1% Acetic acid) with PVA solution before adding the emulsified polymer solution for stirring.

Gelatin was extracted from flounder bones according to the method of Ahmad et al.^{41 (link)} with some modifications. The following operations were performed at room temperature unless otherwise specified. The treated bones were cut into small segments (~1 cm). Bone segments were soaked in 0.1 M NaOH solution (1:30 w/v) to remove noncollagenous proteins and the solution was changed every 2 h three times. Then, the bones were defatted with 10% isopropanol solution (1:30 w/v) for 12 h with slight stirring. The treated bones were soaked in 0.5 M EDTA-2Na solution (1:30 w/v)^{42 (link)} to decalcify them and the solution was changed every 6 h five times at 4 °C. The decalcified bones were soaked in 0.1 M glacial acetic acid solution (1:10 w/v) for 4 h with slight stirring to swell the collagen in the flounder bone and returned to neutral pH (6.5~7.0) (pH meter, Hangzhou Special Paper Co., Ltd, Zhejiang, China). The swollen flounder bones were soaked in distilled water at 55 °C for 8 h with successive stirring to extract gelatin. The mixture was centrifuged (Sorvall Stratos Centrifuge, Thermo Fisher Scientific, Waltham, MA, USA) at 8000 g for 20 min and the sediment was discarded. The gelatin supernatant was freeze-dried using a vacuum freeze dryer (FreeZone 2.5 L, Labconco, Palo Alto, CA, USA).

The back bones were rinsed with distilled water, and the gelatin extraction was done as described by Mahmoodani et al. (2014 (link)) with slight modifications. Briefly, the bones were soaked in 0.1 mol/L NaOH at a ratio of 1:15 (w/v) to remove pigments and non-collagenous proteins, with magnetic stirring at room temperature (25°C) for 4 H. The above solution was changed every h. After that, the samples were washed with distilled water until a neutral pH (7.0) using a pH meter (PHS-3C, Shanghai Yidian Technology Instrument Co., Ltd., Shanghai, China) and the liquid was removed by cotton gauze. After the alkaline treatment, the bones were mixed with 0.2 mol/L EDTA for 12 h to decalcify. Then the bones were mixed with 0.1 mol/L HCl at a ratio of 1:15 (w/v) and stirred at room temperature for 1 h. The acid treatment can destroy ionic bonds between collagen molecules and increase the extraction rate (Hattrem et al., 2015 (link)). The bones were washed with distilled water to neutral pH. Subsequent gelatin extraction was done in distilled water at 55–70°C for 4 h, increasing 5°C every h. The extracted solution was centrifuged at 10,000 × g (Sorvall Stratos Centrifuge, Thermo Fisher Scientific, Waltham, MA, USA) for 30 min at 4°C and the supernatant was dried in a freeze-dryer (LG-1.0, Xinyang Quick-freezing Equipment Manufacturing Co., Ltd., Shenyang, China) for 72 h.