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.
Bacterial cellulose nanocrystals (CNC) production
The bacterial cellulose membranes (BNC) were produced by Komagataeibacter xylinus (ATCC 53582), in Hestrin&Schramm broth, according to Jozala et al.33. The BNC were processed in a mechanical treatment with ultra-turrax and high-pressure homogenizer; and after, in a enzymatic treatment with cellulase for 72 h, to obtain CNC. The CNC were separated by centrifugation and filtration following the methodology developed by our research group (Soeiro et al.)35.
Preparation of alginate-amphotericin B (Alg-AmB) nanocomplexes
The Alg-AmB nanocomplexes were prepared according to Silva-Carvalho et al.5 with modifications. In our formulation, borate buffer (pH 11.0), sodium alginate and amphotericin B were utilized. The final concentration of AmB was 2.5 mg/mL in the solution. The nanocomplexes formulation were described in Table 1.
The nanocomplexes samples were kept under agitation at 4 °C for 48 h, inside temperature laboratory chamber, without interference from light. After that, the sample was run in dialysis membrane from 12,000 to 14,000 KDa. The membrane with sample was immersed in 5 L of distilled water under agitation at 4 °C, without light interference, for 30 h. During the dialysis process, the water was changed three times, until reaching pH value in the range of 5.5–5.7. Then, the samples were collected, stored at −80 °C for 24 h, and lyophilized for 72 h. The nanocomplexes yield was 73.72%.
Nanocomplexes covered by CNC
Alg and Alg-AmB nanocomplexes were covered with CNC according to Soeiro et al.35. Then, an amount of 10 mg of lyophilized nanocomplexes were dispersed in 10 mL water. After that, 1 mL of the nanocomplexes solution was added into 1 mL of 0.01% CNC suspension. The sample was kept on rotatory shaker (20 rpm) at 25 °C for 24 h.
Nanocomplexes physico-chemical characterization
The particles were characterized by the techniques of index of differential scanning calorimetry (DSC), size, polydispersity, zeta potential, infrared spectrometry by Fourier transform (FTIR) and UV–Vis spectrometry.
For the size, index of polydispersity and zeta potential were measured by the Zetasizer equipment (ZEN3600). The analysis was performed for six times, under an angle of 173º, at 25 °C.
The nanocomplexes stability was analysed by Differential Scanning Calorimetry (DSC). 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, Alg nanocomplexes, CNC, alginate + AmB (physical mixture without nanocomplexes process), Alg-AmB nanocomplexes and Alg-AmB nanocomplexes + CNC.
The chemical groups characterization was carried out by spectrometry in FTIR. The samples pure alginate, Alg nanocomplexes and Alg-AmB nanocomplexes were analysed by KBr tablets technique. The technique was performed utilizing 2 mg of the sample and 200 mg of the KBr. The FTIR 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.
In addition, the Alg nanocomplexes and Alg-AmB nanocomplexes were dispersed in distilled water (1 mg of sample/mL) and analysed by UV–Vis spectroscopy. The analyses were performed in the 300–450 nm range using a UV–Visible spectrophotometer (JASCO V-560) with 5 nm resolution and scanning speed of 400 nm/min.
In vitro toxicity
The in vitro toxicity analysis were run to evaluate the safety of the nanocomplexes compared with a commercial product, AMBISOME. AMBISOME is an injectable drug containing with a liposomal formulation. Its prescription is mentioned in several articles due to low toxicity6,7,34.
Hemolysis test
The hemolysis protocol and the animal’s blood were performed according Silva-Carvalho et al.5. The blood sample was collected in EDTA tube from a healthy dog with the owners’ consent. The blood sample was centrifuged for 10 min, at 4 °C and 1200 G. Then, the supernatant was discarded and the red blood cells were resuspended in phosphate buffer solution (pH 7.4).
The 450 µL of red blood cells at a concentration 1 × 108 cells / mL were placed in a 48-well plate in contact with 50 µL of the samples of AmB, Alg, Alg -AmB nanocomplexes, Alg-AmB nanocomplexes + CNC and AMBISOME. The samples were at concentrations of 1, 2, 4, 8, 16 and 32 µM. The 48-well plate was incubated under agitation for 30 min, at 37 °C and 120 rpm.
After the time, 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. The complete hemolysis was considered when hemoglobin was released with 1% triton X-100 (positive control).
Cytotoxicity
Human Embryonic Kidney (HEK) cell line was selected to perfume citotoxity due to the toxicity of AmB in renal cells. The HEK 293 T monolayer (1 × 104 cell/well) was incubated for 24 h (37 °C in a 5% CO2 atmosphere) with AmB, Alg, Alg-AmB, Alg-AmB + CNC and AMBISOME 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 h.
Fluorescence was measured (λ 560/λ 590) in a SpectraMAX GeminiXS microplate reader (Molecular Devices LLC, California, USA). The results were expressed as 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).
