Uv 1900 spectrophotometer

In vitro dissolution studies were performed according to our previously published study [19 (link)]. In brief, a drug-loaded fiber sample (25–30 mg) was incubated in 30 ml of PBS with pH 5.7 + 1% SLS at 35°C under magnetic stirring for 72 h. Aliquots of samples (3 ml) were taken from the release medium at specific time intervals, filtered and evaluated for A/S mixture content spectrophotometrically at 516 nm (UV–1900 spectrophotometer, Hitachi, Japan), while the same volume of phosphate buffer was replaced to maintain sink conditions. Drug released from the fiber mats at different timepoints was estimated through a calibration curve of various concentrations (n = 9) of the A/S mixture in the release medium vs absorbance values. Three independent fiber mats were analyzed each time for their dissolution profile in triplicate (n = 9 for each sample) and the average values (±SD) were calculated.
Drug concentration (mg/mL) = 0.0392 × absorbance + 0.0001; (R2=1)(6)

For the estimation of entrapment efficiency (% EE), 2 ml of sample SGNCs suspension was ultracentrifuged (Heraeus Multifuge X3R, Thermo Scientific, Reinach, Switzerland) at 15,000 rpm at 4 °C for 30 min. The clear supernatant was immediately analyzed for free drug content. The supernatant was diluted and analyzed by UV spectrophotometer (UV–1900 spectrophotometer, Hitachi, Tokyo, Japan) to determine the content of free drugs in the clear supernatant solution. The exact concentration of free drugs was determined from the standard curve of gliclazide. Each experiment was performed in triplicate^{63 (link)}.
The % EE was calculated using the equation as below: $$\%\mathrm{EE}=\frac{\mathrm{Initial}\mathrm{total}\mathrm{drug}-\mathrm{Free}\mathrm{drug}}{\mathrm{Initial}\mathrm{total}\mathrm{drug}}$$

The actual amount of the A/S mixture (API) incorporated in the polymeric nanofibrous matrix (both inside and onto the surface of the fibers) compared to the initial amount of drug used –hereinafter referred to as entrapment efficiency – was determined by UV/Vis spectrophotometry (mod. UV-1900 spectrophotometer, Hitachi, Japan) at λ_max = 516 nm. In specific, drug-loaded CA and PCL nanofibers were weighed (~ 5 mg per sample) and dissolved (under vortexing) in 4 mL chloroform to dissolve the fiber structure, thus releasing the drug in the organic solvent. Subsequently, the amount of A/S mixture present in the organic phase was measured spectrophotometrically (control: chloroform; calibration curve presented in the supplementary file).
The entrapment efficiency and drug loading were calculated based on the Eqs. (3) and (4), respectively:
$$\mathit{\text{Entrapment efficiency}}\left(\%\right)=\left(\frac{\mathit{\text{weight of drug entrapped in the fibrous matrix}}}{\mathit{\text{weight of drug added in the polymer solution}}}\right)\times 100$$ $$\mathit{\text{Drug loading}}\left(\%\right)=\left(\frac{\mathit{\text{weight of drug entrapped in the fibrous matrix}}}{\mathit{\text{weight of fiber}}\mathit{mat}}\right)\times 100$$
In order to assess the distribution of the drug across each fiber mat, three independent meshes were selected from random parts of the electrospun membrane and analyzed as described above. The measurement was performed in three replicates each time (n = 9 for each sample) and the average values (± SD) have been estimated.