2.4. In vivo nephrotoxicity studies

All experiments of this study were carried out in accordance with the principles of Laboratory Animal Care and approved by the Institutional Animal Care and Use Committee of the San Pablo CEU University (Madrid, Spain; Permit number: 64/13). In addition, studies were performed in compliance with national and European regulations.

All animal studies were performed in the animal center of San Pablo CEU University. BALB/c female mice aged 6–8 weeks (19–22 g) were acclimated at temperature 25 °C and humidity 60% with regular day/dark light cycles, starting from one week before the experiment to reduce the stress of adjusting to the new environment for the animals. The same conditions were used throughout the experiments.

Mice were divided into three groups of 5 to 8 animals each; the control group received a vehicle (5% glucose solution); the AmB-loaded NE group and the C-AmB group received a dose of 1 mg AmB per kg. All groups received intravenous (IV) injections over the course of 3 alternate days (three administrations). The weight of each animal was recorded before each dosing. After this procedure, the animals were euthanized 24 h after the last administration. Blood was obtained by cardiac puncture under deep CO₂ narcosis, and collected in tubes impregnated with anticoagulant (EDTA). After extraction, before they extinguished the effects of anaesthesia, humane euthanasia techniques (cervical dislocation) were applied. Plasma was separated from cellular components by centrifugation at approximately 2000g for 30 min at 4 °C. Immediately after, the plasma was divided into aliquots in 0.1 mL fractions (100 μL) and frozen at –80 °C, where it remained until the day of analysis.28

Blood urea nitrogen (BUN) and plasma creatinine levels were used as biochemical markers to evaluate the nephrotoxicity of the formulations. The analyses were performed using commercially available kits (Gold Analisa Diagnótica Ltda, Brazil).

The data were processed using Graph Prism 4 software. Analyses for the blood chemistry measurements and the urinary volume were evaluated using the one-way analysis of variance (ANOVA) test followed by Tukey's test, after log transformation. The difference was considered significant when the p value was less than 0.05.

Kidney tissue specimens were also collected and fixed in 10% neutral buffered formalin and subjected to tissue processing followed by embedding in paraffin. The sections were further sliced into layers with 5 μm thickness (Leica, Wetzlar, Germany) and stained with hematoxylin and eosin (H&E) for microscopic examination (Olympus, Tokyo, Japan).