Reagents
Amphotericin B (AmB, molecular weight of 924.08 g/mol) powder from Streptomyces sp., resazurin Sodium salt, sodium alginate, triton X-100, sodium tetraborate decahydrate were purchased from Sigma-Aldrich (Missouri, USA). Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum (FBS) and penicillin-streptomycin were obtained from Merck Millipore (Massachusetts, USA). Roswell Park Memorial Institute (RPMI) 1640. Glutamax supplemented medium and L-glutamine (GlutaMAX™-I) was purchased from Gibco (Massachusetts, USA). Dimethylsulfoxide (DMSO ATCC® 4-X™) solution for cell culture was acquired from American Type Culture Collection (ATCC, Virginia, USA). Dialysis tubing with a molecular weight cut-off of 1000 Da was obtained from Orange Scientific (Braine-l'Alleud, Belgium). AmBisome® was kindly provided by Gilead Sciences.
Obtaining bacterial cellulose nanocrystals (NCC)
The production of the NCC was carried out following the methodology developed by our research group (In press). The five-step process uses the mechanical treatment of bacterial cellulose, obtained from the bacterium G. xylinus according to Jozala et al. (2014) (33), associated with enzymatic hydrolysis. The NCC had a size of 41.67 ± 5.55 nm and an index of polydispersity of 0.127 ± 0.005.
Preparation of nanocomplexes
The nanocomplexes were prepared according to the methodology of Silva-Carvalho et al. (2020) (5). In the formulation, borate buffer (pH 11), sodium alginate and amphotericin B were used, following the proportions described in table I.
The samples were kept under agitation at 4 °C for 48 hours without interference from light. In the next step, the dialysis process was carried out on a membrane from 12000 to 14000 KDa. The membrane with the sample was immersed in 5 L of distilled water under agitation at 4 °C, without light interference, for 30 hours. During the process, the water was changed three times to remove the salts, until reaching the pH value in the range of 5.5-5.7. Then, the samples were collected, frozen at -80 °C for 24 hours, and lyophilized. The final yield of the process was 73.72%.
Nanocomplex coating
The nanocomplexes Alg and Alg-AmB were covered with NCC. For that, the lyophilized nanocomplexes were dispersed in water in the proportion of 1 mg/mL. After dispersion, 1 ml of the nanocomplexes was added to 1 ml of 0.01% NCC suspension. This sample was kept on rotatory shaker (20 rpm) at 25 °C for 24 hours.
Physico-chemical characterization
For the physico-chemical characterization the following parameters were used: size, index of polydispersity, zeta potential, differential scanning calorimetry, infrared spectrometry by Fourier transform and UV-Vis spectrometry.
For the evaluation of the size, index of polydispersity and surface potential of the particles (zeta potential), the Zetasizer equipment (ZEN3600) was used, at an angle of 173º, at 25 °C for dynamic light scattering (DLS), Index of Polydispersity and zeta potential. The samples Alg-AmB and Alg-AmB + NCC were analysed in six times.
To characterize the stability of the nanocomplexes, Differential Scanning Calorimetry (DSC) was used. The characterization was performed by the DSC 6000 equipment (PERKIN ELMER | STEC INSTRUMENTS) in a nitrogen atmosphere with a flow of 20 mL/min, in the heating range of 25 – 250 °C with a heating rate of 10°C/min. The test was re-performed with the samples AmB, alginate (Alg), bacterial cellulose nanocrystals (NCC), alginate + AmB (physical mixture), Alg-AmB and Alg-AmB + NCC.
The characterization of the chemical groups was carried out by spectrometry in the infrared by Fourier transform (FTIR). The samples, nanocomplexes Alg and Alg-AmB, were analysed by the technique of KBr tablets with 2 mg of sample and 200 mg of KBr. The infrared spectra were obtained in the range of 4000–400 cm-1 in the Bruker Alpha II equipment, with a resolution of 4 cm-1 and 12 scans. The analyzed samples were: pure alginate, Alg and Alg-AmB.
In addition, the samples were analyzed by UV-vis spectroscopy in the 300-450 nm range using a UV-Visible visible spectrophotometer (JASCO V-560) with 5 nm resolution and scanning speed of 400 nm/min. The nanocomplexes Alg and Alg-AmB were dispersed in distilled water (1 mg of sample/mL). The material was diluted again to 10 µM in distilled water in order to avoid saturation of the test. AmB control was performed using 1082 µM diluted in borate buffer (0.1 M, pH 11). As with nanocomplexes, the solution was diluted to 10 µM with distilled water. To perform the measurement, 200 µL of the solutions were analyzed in a quartz cuvette. The absorbance ratio of peak I (315-350) to peak IV (408-410) was used to monitor the aggregation status of AmB and the results were evaluated for the quantification of AmB in the sample.
In vitro toxicity
The in vitro toxicity tests used a commercial sample of AmBisome® as a control. This drug is for injectable use and has a liposonal formulation containing AmB. The use of AmBisome® is mentioned in several articles in the literature due to its low toxicity (6,7,34).
Hemolysis test
The hemolysis test assesses the percentage of erythrocyte rupture. Both the protocol and the animal's blood were derived from the work of Silva-Carvalho et al. (2020) (5). Blood was collected from a healthy dog with the owners' consent on everything with EDTA. The whole blood was centrifuged for 10 min (4 °C, 1200 g) and the supernatant was discarded. Red blood cells were resuspended in phosphate buffer solution (pH 7.4).
The resuspended red blood cells were counted in a Neubauer chamber. In a 48-well plate, 450 µL of red blood cells at a concentration of 1 x 108 cells / mL were placed in contact with 50 µL of the samples, at concentrations of 1, 2, 4, 8, 16 and 32 µM AmB, Alg, Alg -AmB, Alg-AmB + NCC and AmBisome®. The plate was incubated with shaking for 30 minutes (37 °C, 120 rpm).
After the period, the solutions were collected and centrifuged for 10 min (4 ° C, 1200 G). The supernatants were collected and analyzed by UV-Vis spectrophotometry, with absorbance at 540 nm (referring to hemoglobin). Complete lysis (100%) was admitted by hemoglobin released with 1% triton X-100 (positive control).
Citotoxicity
This cell line was selected due to the toxicity of AmB in renal cells. A HEK293T monolayer (1 × 104 cell/well) was incubated for 24 h (37 °C in a 5% CO2 atmosphere) with AmB, AmBisome®, Alg and Alg-AmB, at concentrations of 0.78, 1, 17, 1.76, 2.63, 3.95, 5.93, 8.89, 13.33, 20, 30, 45 and 67.5 μM. After the incubation period, 10% (v / v) of a 2.5 mM resazurin solution was added to each well and the plates were incubated again under the same conditions as above for 4 hours.
Fluorescence was measured (λex 560 / λem 590) in a SpectraMAX GeminiXS microplate reader (Molecular Devices LLC, California, USA). The results were expressed as the mean percentage ± SD of viable cells in relation to the positive control condition considered to be 100% viable cells.
Statistical analysis
Data from toxicity tests were expressed as mean ± standard deviation. Analysis of Variance (ANOVA) followed by the Duncan test were used to verify differences among treatment protocols, and p values <0.05 were considered significant. Results were analyzed using Statistica® v. 8.0 (Dell, Round Rock, TX, USA) and GraphPad Prism® v. 6.0 (San Diego, CA, USA).