60s evolution

Samples were centrifuged at 16,000× g for 60 min at 4 °C using the centrifugal filter (Merck Millipore, Darmstadt, Germany, Microcon, −10 kDa with Ultracel-10 membrane). After centrifugation, the filtered solution was analyzed to determine drug concentration using the UV–VIS spectrophotometry (Thermo Fisher Scientific, 60S Evolution, Madison, WI, USA) method at 239 nm, utilizing the equation from the fitted standard curve plot constructed previously [24 (link)]. The encapsulation efficiency (EE) was calculated by using Equation (1), where the total drug amount added (Drug_total) and the non-entrapped drug (Drug_free) in the filtered solution were considered [36 (link)].
$\mathrm{EE} (\%)=\frac{\left[\mathrm{Drug}\right]\mathrm{total}-\left[\mathrm{Drug}\right]\mathrm{free}}{\left[\mathrm{Drug}\right]\mathrm{total}}\times 100$

The dissolution profile of samples was performed in the simulated gastrointestinal medium [29 ], using a dissolution equipment 299 model (Ethiktechnology, Sao Paulo, Brazil). The hard gelatin capsules containing the formulations were placed in basket apparatus, using 1000 mL of hydrochloric acid solution 1% (v/v) as dissolution medium at 37 ± 0.5 °C, under 100 rpm agitation. At specific intervals, aliquots of 10 mL of dissolution medium were removed, filtered through a 0.45 µm cellulose acetate, and analyzed by UV spectrophotometry at 290 nm, previously validated in a spectrophotometer 60S Evolution (Thermo Fisher Scientific Inc, Madison, WI, USA). All experiments were performed six times (n = 6) and the cumulative percentage of released propranolol was plotted versus time.
The effect of the experimental variations on the drug release profile of the different samples was evaluated by comparisons using the independent-model of similarity factor (f2), according to Equation (7).

where, n is the number of experimental intervals, t is the interval time, D1t is the drug ratio dissolved at a specific interval time for formulation (1), and D2t is the drug ratio dissolved at specific interval time for formulation (2). Formulations with drug dissolution profiles that are statistically similar present F2 values in the range of 50 to 100.

The stoichiometry of complexes was evaluated by the continuous variation method (Job’s method) [23 ] and further confirmed by ITC. Solutions of DX and SBE-β-CD were prepared at 0.06 mM in acetate buffer (pH 5.0) and mixed at different volume ratios. The samples were analyzed by UV/Vis spectroscopy (Thermo Fisher Scientific, 60S Evolution, USA) at 275 nm and the difference in absorbance between the solutions with and without SBE-β-CD was related to “R” (Δabs × R), calculated by Equation (1), with substances in mM: $$\mathrm{R}=\frac{\left[\mathrm{D}\mathrm{X}\right]}{\left[\mathrm{D}\mathrm{X}\right]+[\mathrm{S}\mathrm{B}\mathrm{E}-\mathsf{\beta }-\mathrm{C}\mathrm{D}]}$$