Ultrastructural changes of erythrocytes in whole blood after exposure to prospective in silico-designed anticancer agents: a qualitative case study

Abstract

BackgroundNovel, in silico-designed anticancer compounds were synthesized in our laboratory namely, 2-ethyl-3-O-sulphamoyl-estra-1,3,5(10),15-tetraen-17-ol (ESE-15-ol) and 2-ethyl-3-O-sulphamoyl-estra-1,3,5(10)16-tetraene (ESE-16). These compounds were designed to have improved bioavailability when compared to their source compound, 2-methoxyestradiol. This theoretically would be due to their increased binding affinity to carbonic anhydrase II, present in erythrocytes. Since the novel compounds under investigation are proposed to be transported within erythrocytes bound to carbonic anhydrase II, the morphological effect which they may exert on whole blood and erythrocytes is of great significance. A secondary outcome included revision of previously reported procedures for the handling of the whole blood sample.The purpose of this study was twofold. Firstly, the ultrastructural morphology of a healthy female’s erythrocytes was examined via scanning electron microscopy (SEM) after exposure to the newly in silico-designed compounds. Morphology of erythrocytes following exposure to ESE-15-ol and ESE-16 for 3 minutes and 24 hours at 22°C were described with the use of SEM. The haemolytic activity of the compounds after 24 hours exposure were also determined with the ex vivo haemolysis assay. Secondly, storage conditions of the whole blood sample were investigated by determining morphological changes after a 24 hour storage period at 22°C and 37°C.ResultsNo significant morphological changes were observed in the erythrocyte morphology after exposure to the novel anticancer compounds. Storage of the whole blood samples at 37°C for 24 hours resulted in visible morphological stress in the erythrocytes. Erythrocytes incubated at 22°C for 24 hours showed no structural deformity or distress.ConclusionsFrom this research the optimal temperature for ex vivo exposure of whole blood samples to ESE-15-ol and ESE-16 for 24 hours was determined to be 22°C. Data from this study revealed the potential of these compounds to be applied to ex vivo study techniques, since no damage occurred to erythrocytes ultrastructure under these conditions. As no structural changes were observed in erythrocytes exposed to ESE-15-ol and ESE-16, further ex vivo experiments will be conducted into the potential effects of these compounds on whole blood. Optimal incubation conditions up to 24 hours for whole blood were established as a secondary outcome.

In vitro assessment of a computer-designed potential anticancer agent in cervical cancer cells

“…Since these sulphamoylated compounds are also hypothesized to be transported in the blood bound to carbonic anhydrase II, their influence on morphology of erythrocytes is noteworthy. Ex vivo exposure of 180 nm ESE-15-ol to whole blood for 24 h revealed no significant erythrocyte morphological damage and support future potential for in vivo studies [15]. …”

Ex vivoDetermination of an Estradiol Analogue-Induced Changes on Platelet Morphology and Angiogenic Biomarkers

“…With the use of SEM, possible morphological changes were studied on the platelets of healthy female individuals after exposure to ESE-16. Results showed no morphological changes in the platelets after exposure when compared with the relevant controls (Repsold et al, 2014 a , 2014 b ).…”

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“…The principle of this test was when erythrocytes are hemolyzed, free hemoglobin released to the surrounding medium. Free hemoglobin can be assessed spectrophotometrically at 540 nm according to Repsold and colleagues, 2014 15 . Fifty microliters of whole blood were incubated with two milliliters of normal saline containing either metformin or rosuvastatin or their controls at 37 ºC.…”