Evolution 300 uv visible spectrophotometer

¹H- (400, 500, and 600 MHz), and ¹³C-NMR (100, 125, and 150 MHz) and 2D-NMR spectra were recorded on Bruker Avance-NMR spectrometers (France) in CD₃OD, CD₃COCD₃ or DMSO-d₆. Melting points were recorded on Buchi M-560 apparatus (Japan). EI- and HREI-MS were recorded on JEOL JMS-600H (Japan). Optical rotations of all isolated compounds were measured on JASCO P-2000 polarimeter (Japan) in chloroform or methanol. IR analyses were performed on Bruker Vector 22 FT-IR spectrometer (France). Evolution 300 UV-visible spectrophotometer was used to record the UV spectra (Thermo Scientific, England). Single-crystal X-ray diffraction data was collected on Bruker APEXII D8 Venture diffractometer, fitted with PHOTON 100 detector (CMOS technology), and fine-focus sealed tube having X-ray source [Cu Kα radiation α = 1.54178 Å]. Reflection intensities were integrated using SAINT software. Absorption correction was done on M-multi-scan. Structures were solved on SHELXTL [30 –31 ].
Crystallographic data for compounds 1, 2, 4, and 8 were deposited with the Cambridge Crystallographic Data Center and can be obtained free of charge from the Cambridge Crystallographic Data Center viawww.ccdc.cam.ac.uk/data_request/cif.

For the quantification of LEV on each NGI stage, the solutions obtained from the NGI were diluted, and the amount of levofloxacin was determined with an Evolution 300 UV-Visible-Spectrophotometer (ThermoFisher Scientific, Waltham, MA, USA) at a wavelength of 292 nm. The limit of detection and limit of quantification with this method were determined to be around 0.6 µg/mL and 1.8 µg/mL, respectively. Due to the variation in the literature data, the solubility of crystalline LEV was analysed at three relevant pH values of 6.6, 6.8 and 7.2 by measuring the supernatant of saturated solutions with the above method. The apparent solubility of the amorphous material was investigated by dissolving LEV-ARG masses corresponding to defined degrees of supersaturation in 2M HEPES buffer. The supersaturated solutions were observed until precipitation occurred.

The encapsulation efficiency (EE) and loading capacity (LC) of the vesicles were estimated by an indirect assay based on the quantification of the free drug in the external medium that surrounds the vesicles. Each sample was diluted 1:10 (v/v) with PBS buffer and then transferred to a VIVASPIN^® 500 centrifuge tube (10 KDa, Sartorius, Goettingen, Germany) with a subsequent centrifugation at 14,000× g for 40 min in a Hermle Z323 K centrifuge (Hermle LaborTechnik, Wehingen, Germany). Afterwards, the supernatant, corresponding to the non-loaded IBU fraction, was diluted in PBS buffer (1.5:8.5) and the absorbance was determined at 222 nm using an Evolution^® 300 UV/Visible spectrophotometer (Thermo Scientific, Hertfordshire, UK). After calculating the concentration of IBU in the non-loaded fraction ([IBU_NL]) based on a previously determined calibration curve, it was possible to calculate the EE and LC, knowing the total concentration of IBU added to prepare the vesicles ([IBU_T]), as well as the total concentration of soy phosphatidylcholine used to prepare the vesicles ([PC_T]), according to the equations below: $$\%\mathrm{EE}=\frac{\left[{\mathrm{IBU}}_{\mathrm{T}}\right]-\left[{\mathrm{IBU}}_{\mathrm{NL}}\right]}{\left[{\mathrm{Ibu}}_{\mathrm{T}}\right]}\times 100$$
$$\%\mathrm{LC}=\frac{\left[{\mathrm{IBU}}_{\mathrm{T}}\right]-\left[{\mathrm{IBU}}_{\mathrm{NL}}\right]}{\left[{\mathrm{PC}}_{\mathrm{T}}\right]}\times 100$$