In vitro Characterization of BSA NPs

The mean particle size, polydispersity index (PdI) and zeta potential of BSA-NPs were measured by Zetasizer (PCS Zetasizer^® Nano ZS Series DTS 1060, Malvern Instruments S.A., Worcestershire, UK) at a fixed angle at 25°C using a 4 mW He–Ne laser at 633 nm. Dispersions were diluted in deionized water and measurements were performed in triplicate.

The morphology of BSA-NPs and HA-BSA-NPs was examined by transmission electron microscopy (TEM) using JEOL, JEM-100 CX Electron Microscope, Tokyo, Japan. Before analysis, the selected dispersions were sprayed onto copper grids without staining²⁷ and shots were taken at x 30–50K magnification.

Entrapment efficiency (EE%) was calculated by determining the concentration of free (un-entrapped) Apa in the supernatant after separation of BSA-NPs by centrifugation. A high-performance liquid chromatographic assay (HPLC-UV) was used for Apa determination as reported by Lee et al.²² Agilent Technologies-1260 infinity; Santa Clara, CA, USA was used. Separation was carried out on reversed phase C18 column (Kinetex™; 250×4.6 mm, particle size 5 μm, pore size 100 μm). An isocratic eluent consisting of acetonitrile and distilled water in the ratio 45:55 v/v was used. The injection volume was 20 μL and the flow rate was adjusted to 0.5 mL/min. Peaks were detected at 347 nm using a UV detector. All solutions were protected from light and used within 24 h. Reversed-phase elution was carried out at room temperature. Apa concentration was calculated using calibration standards. Linearity was checked in the concentration range 50–5000 µg/mL with a determination coefficient 0.999. The method was validated for limit of detection (LOD) and limit of quantitation (LOQ) which were 5.77 and 19.25 µg/mL, respectively. Measurements were done in triplicate. The %EE of Apa in BSA-NPs was calculated from the difference between the initial drug concentration added and the free drug concentration in the supernatant using the following equation:

where C_i is the initial drug content and C_f is the free drug in the supernatant (unentrapped).

The in vitro release profile of Apa from BSA-NPs and HA-BSA-NPs was studied using the dialysis method. The NPs were suspended in 1 mL of PBS and placed in the dialysis bag (VISKING^® dialysis tubing MWCO 12,000–14,000) which was then suspended fully immersed in the release medium (PBS pH 7.4) maintaining sink conditions. The release of Apa was then determined at 37°C at 100 rpm in a thermostatically controlled shaking water bath (Köttermann GmbH, Hänigsen, Germany). Samples were collected at predetermined time intervals (0.25, 0.5, 1, 3, 5, 7 and 24 h) then filtered through 0.45µm Millipore^® syringe filters, followed by compensation with an equal volume of fresh release medium. The concentration of the drug was determined by the HPLC assay described above. The % Apa released was calculated in triplicate.

The mucoadhesiveness of BSA-NP and HA-BSA-NPs was assessed in vitro by measuring the changes in viscosity and ZP upon formulation incubation with mucin as reported previously.²⁸ Mucin was used in a concentration of 10% (w/v) with pH adjusted to 7.4 to mimic the ocular environment. Mucin was first hydrated with distilled water, followed by gentle stirring at room temperature for complete dispersion. Both viscosity and ZP measurements were done in triplicate.

BSA-NPs and HA-BSA-NPs were incubated with hydrated mucin in a 1:1 ratio for 30 minutes. NPs control samples were prepared by replacing mucin with distilled water, whereas mucin control sample was prepared similarly but with the NPs being replaced with distilled water. Viscosity of 0.5 mL samples was measured using cone and plate viscometer (Brookfield, DV2T, USA) following equilibrium for 30 min at 100 rpm using spindle 40 for 1 min, at 25 °C.

NPs interaction with mucin was also determined by measuring the change in ZP of NPs/mucin mixtures in the ratio 1:1 compared to NPs and mucin controls. Following incubation for 2 h at room temperature, ZP was measured as previously described.