Microfuge 16

Selamectin was tested on human erythrocytes of blood group 0 obtained from a healthy patient. The blood was centrifuged at 5000 rpm for five minutes (Microfuge 16, Beckman coulter, Brea, CA, USA) and the erythrocytes were washed 5 times with a solution (TBS) containing 50 Mm Tris-HCl (pH 7.6) and 0.15 M NaCl. After the washes, the erythrocytes were diluted 10-fold using the TBS solution. A volume of 50 µL of drug at the concentrations mentioned above (50 to 0.4 μg/mL) was added to 50 µL of cell suspension and incubated at 37 °C for 1 h. Two controls were included in the assay, which were dissolved drug solvent and 0.1% Triton X-100, used as CTR- and CTR+, respectively. After incubation, the plate was centrifuged at 500× g for 5 min and 50 μL of supernatant from each well was transferred to a new 96-well plate (Microfuge 16, Beckman coulter, Brea, CA, USA). The supernatants were used to measure the absorbance of the released hemoglobin at 540 nm. The hemolysis percentage of each sample was calculated using the following formula: $$\% \mathrm{Hemolysis}=\left[\frac{\left(\mathrm{Abs} 540 \mathrm{nm} \mathrm{of} \mathrm{the} \mathrm{test} \mathrm{sample}- \mathrm{Abs} 540 \mathrm{nm} \mathrm{of} \mathrm{CTR}-\right)}{(\mathrm{Abs} 540 \mathrm{nm} \mathrm{of} \mathrm{CTR}+-\mathrm{Abs} 540 \mathrm{nm} \mathrm{of} \mathrm{CTR}-}\right]\times 100$$

Before SEM examination, the microalgal samples were centrifuged at 5000 rpm (equivalent to relative centrifugal force of 1845× g) for 5 min (Microfuge 16, Beckman Coulter, Brea; CA, USA). The supernatant was decanted, and the pellet was fixed with 2.5% (v/v) glutaraldehyde for 4 to 6 h at 4 °C. The samples were washed three times, with 0.2% (v/v) sodium cacodylate buffer for 10 min each. The samples were then post-fixed with 1% (v/v) osmium tetroxide for 2 h at 4 °C and washed three times with sodium cacodylate buffer for 10 min each. After post-fixation, the samples were dehydrated gradually using a series of increasing acetone concentrations: 35%, 50%, 75%, 95% once for 10 min each, and 100% three times for 15 min each. After the drying process, the samples were mounted on aluminum stubs and coated with gold using a sputter coater. The samples were then observed using SEM (Model JEOL JSM-6400, Tokyo, Japan) with an accelerating voltage of 15.0  kV.

Peptide stability tests were performed according to a TCA stability assay based on the method used by Nguyen et al. [38 (link)]. Peptides were dissolved in water, mixed with human plasma* to a final concentration of 1.0 mg/mL, and incubated at 37 °C with agitation (300 rpm, Thermomixer, Eppendorf AG, Hamburg, Germany). After 0, 1, 2, 3, 6 and 24 h, 80 μL of the solution was taken and mixed with 15% TCA to obtain a final concentration of 3% (v/v). The samples were incubated in ice for 10 min and centrifuged for 10 min (12,000 rpm, Microfuge 16, Beckman Coulter, Brea, CA, USA). Supernatants were analyzed by RP-HPLC on column using a linear gradient of acetonitrile (5–100% over 15 min) with detection at 223 nm. Peptides were quantified by their peak areas relative to the initial peak areas (0 min). All stability tests were performed at least in triplicates. As control samples peptides dissolved only in water under the same conditions were used. To additionally assess the changes in the incubated samples (peptide degradation) mass spectrometry analysis was used. LC-MS experiments were performed using ESI-IT-TOF (Shimadzu, Kyoto, Japan).
* Blood was collected from healthy anonymous donors and EDTA was then used as an anticoagulant. The procedure was approved by the Independent Bioethics Commission for Research of the Medical University of Gdańsk (NKBBN/387/2014).