2.4. Characterization of Nanoparticles

The shape and surface morphology of the NPs were analyzed by scanning electron microscopy (SEM, Jeol JSM 7600F, Jeol Ltd., Tokyo, Japan). Samples were coated with a thin layer of colloidal gold applied in a cathodic vacuum evaporator before observation in a microscope.

The mean diameter and size distribution of the NPs were determined by laser diffraction using a Zetatrac^® Ultra 3500 system (Microtrac MRB, Montgomeryville, PA, USA). The diameter was expressed as volume diameter. The polydispersity index (PDI) was also calculated.

Zeta potential was determined by Laser-Doppler anemometry using a Malvern Zetasizer Nano S^® (Malvern Panalytical, Malvern, U.K.). Measurements were carried out at 25 °C in aqueous solution. For this, 5 mg of each formulation was suspended in 50 mL of distilled water and stirred for 1 min. Then, the aqueous dispersion of NPs at a concentration of 100 µg/mL was placed in a capillary cell (Cell Enhances Capillary^®, Malvern Panalytical, Malvern, U.K.) for zeta potential measurements. All formulations were analyzed in triplicate.

Encapsulation efficiency (EE%) of MH within the NPs was determined as follows: 10 mg of each formulation was dissolved in 1 mL of DCM. Then, 15 mL of methanol was added to this solution and centrifuged for 5 min at 5000 rpm. The supernatant (5 mL) was mixed with 5 mL of HCl 0.1 N and analyzed by HPLC [31]. The equipment used consisted of a Jasco chromatograph (Jasco International Co., Ltd., Tokyo, Japan) equipped with a PU-2080 pump and a UV/Vis 2070 detector. The chromatographic column selected was Gemini 5 μm NX-C18 (110 Å, 250 × 4.6 mm) (Phenomenex, Madrid, Spain). The mobile phase consisted of acetonitrile:water (27:73, v/v). The flow rate was 1.4 mL/min, and the volume injected was 50 µL. The detection wavelength was 256 nm. All analyses were carried out at 40 ± 0.5 °C. Drug loading (DL) was also determined by HPLC and expressed as the amount of MH incorporated by 100 mg of NPs.

To determine the EE% and DL of Rh-B, the same procedure was used. Quantification was performed by spectrophotometry at 555 nm.

In vitro release studies were carried out in a Memmert WB22 water bath (Memmert, Schwabach, Germany) at 37 ± 1 °C and constant agitation (100 rpm). For this, 20 mg of NPs were suspended in PBS at pH 7.4 (5 mL). At regular time intervals, samples were centrifuged with the supernatant withdrawn and filtered through 0.45 µm filters. Quantification of MH was performed by HPLC [31]. Quantification of Rh-B was conducted by spectrophotometry at 555 nm. All in vitro release tests were performed in triplicate.