Inkubator 1000

The in vitro release of QCT and TAA from nanoliposomes was assessed by the dialysis method, using 5 mL of phosphate buffer with a pH of 7.0 and 150 mM aqueous medium under sink conditions. Therefore, volumes of 500 µL of the nanoliposomes were placed into a dialysis tube (VWR, modified polyethersulfone-PES cut-off 10 kDa, 500 µL, diameter: 0.96 cm) in triplicate and dialyzed at 37 °C for 16 h with constant stirring in an incubated orbital shaker (Inkubator 1000 with Unimax 1010, Heidolph Instruments, Schwalbach, Germany). Subsequently, the samples were taken from the external medium at intervals of 0, 5, 10, 20, 30, 60, 120, 180, 240, 360, and 996 min. The quantification of QCT and TAA was performed as described in the encapsulation efficiency section.

Turbidimetric solubility [23 (link),24 (link)] for PAM-18Na and PAM-18K with different ionization degrees were determined in triplicate, using a UV–Vis spectrophotometer coupled to a thermostat (Shimadzu 1800, Shimadzu Corporation, Kyoto, Japan). For this, the polymeric salts were independently dispersed in ultra-pure water at different concentrations depending on the ionization degrees. Low ID polyelectrolytes were prepared between 0.01 and 20.00 mg/mL, while high ID polyelectrolytes were prepared between 3.00 and 130.00 mg/mL. All systems were initially assisted with a homogenizer (Gehaka^® D-160, São Paulo, Brazil) at 4000 rpm for 10 min. Subsequently, each system was left on an orbital shaker (Unimax 1010, Inkubator 1000, Heidolph Instruments, Schwalbach, Germany) at 25 °C and 600 rpm for 24 h. Once the maximum agitation time was reached, the turbidimetric solubility value was immediately determined from measures of transmittance percentage (%T) at 600 nm, establishing like reference point, the value of concentration where the %T changed abruptly from 100% to a lower value. This value was determined through the evaluation of the first derivative of the %T vs polymer concentration graph. Thus, the turbidimetric solubility was taken as reference concentration for the preparation of the rest polymeric dispersions in the subsequent studies.

Growth assays were carried out on liquid medium. Yeast cell suspension was incubated for 12 h in a shaking incubator at 28 °C (Heidolph Inkubator 1000 at 1200 rpm). The growth was monitored turbidimetrically in the Anthos 2010 type 17550 microplate reader at 600 nm by performing measurements at 1 h intervals for 12 h.

The partition study of CBZ between SIO and ultrapure water was conducted in an incubated orbital shaker (Inkubator 1000 with Unimax 1010, Heidolph Instruments, Schwalbach, Germany). For this, 2.4 g CBZ was added to 50 mL of the oil phase (Mixture 1) with a stirring speed of 400 rpm for 45 min at 37 °C. Subsequently, 50 mL of ultrapure water was added to Mixture 1, forming a ternary mixture (CBZ, SIO and ultrapure water) which was stirred at 400 rpm for 45 min at 37 °C. Once the CBZ was distributed in the respective phases, it was left to rest for 3 min, during which a complete separation of SIO and ultrapure water occurred. Immediately after separation, 1 mL aliquots were taken from the oil and water phases (initial time), and the concentrations were determined using a UV/Vis spectrophotometer (UV-1800, Shimadzu, Milton Keynes, UK). Calibration curves of CBZ in SIO and ultrapure water were determined using serial concentrations of 1.0, 3.0, 5.0, 10.0, 15.0, 20.0, and 23.75 mg/mL. Finally, the ternary mixture was left to rest for 8 h (final time), after which 1 mL aliquots were taken from each respective phase and the concentration was measured by UV/Vis spectrophotometry.

The growth assay was performed on a liquid YPD medium. Yeast cell suspensions were incubated at 28 °C for 12 h with shaking (Heidolph Inkubator 1000 at 1200 rpm). The growth was monitored turbidimetrically in the Anthos 2010 type 17,550 microplate reader at λ = 600 nm by performing measurements at 2 h intervals for 12 h. The data represent the mean values from three independent experiments.