Model 3k 30

BSA NPs were prepared by a modified nano-emulsification technique [38 (link)]. Briefly, BSA (30, 50, and 60 mg) was dissolved in 2 mL deionized water, and the pH of the solution was adjusted between 5.6 and 5.9 using 10% w/v citric acid. To prepare FLUR-loaded BSA NPs, FLUR (15, 25, and 40 mg) was dispersed separately in 0.5 mL of chloroform then added dropwise to the prepared BSA solution under vigorous stirring (800 rpm) via a magnetic stirrer (IKA Labortechnik, Staufen, Germany) for at least an hour. The mixture was then homogenized for 5 min at 15,000 rpm in an ice bath using a high shear homogenizer (Ultra Turrax; IKA Labortechnik, Staufen, Germany). NPs were formed upon exposure to high shear stress. The NPs suspension was centrifuged at 13,000 rpm, 4° for 30 min (Model 3K-30; Sigma Laborzentrifugen GmbH, Osterode, Germany), the supernatant was then removed, and the pellet of NPs was re-dispersed in 0.9% w/v normal saline.

The amount of HA coated on BSA NPs was determined by a turbidimetric method which is based on the formation of turbidity between HA and CTAB reagent [39 (link)]. First, the calibration curve of HA was prepared where 2 mL of CTAB reagent (2.5 g in 100 mL of 0.2 mol/L NaCl solution) was added to 1 mL of different serial dilutions of standard HA (1 mg/mL) and shaken gently to ensure complete mixing. The mixture was left for 9 min and then the absorbance was read at the 10th minute at 400 nm wavelength using an ultraviolet and visible spectrophotometer (Cary 60 UV-Vis Spectrophotometer, Agilent, Santa Clara, CA, USA). For HA-BSA NPs, the NPs were centrifuged at 13,000 rpm, 4 °C for 20 min (Model 3K-30; Sigma Laborzentrifugen GmbH, Osterode, Germany), and the supernatant was treated as mentioned earlier in the calibration curve. The concentration of free HA was calculated based on the calibration curve of HA and the amount of HA-coated onto the surface of NPs. This was determined by difference using Equation (1): $$Amount of HA coat=Total amount of HA added-Free HA in the supernatant$$

Briefly, the NPs dispersion was centrifuged at 13,000 rpm, 4 °C for 30 min (Model 3K-30; Sigma Laborzentrifugen GmbH, Osterode, Germany). The concentration of unentrapped free FLUR in the supernatant was measured spectrophotometrically at 247 nm using an ultraviolet and visible spectrophotometer (Cary 60 UV-Vis Spectrophotometer, Agilent, Santa Clara, CA, USA). Samples were measured in triplicates and represented as mean value ± SD. %EE and %LC were calculated using Equations (2) and (3): $$\%EE=\frac{\left(Total FLUR concentration-Concentration of free unentrapped FLUR\right)}{Total FLUR concentration}\times 100$$
$$\% LC=\frac{Concentartion of entrapped FLUR}{Weight of NPs}\times 100$$