Abstract
Objective:
To evaluate the cytotoxicity of iron nanoparticles on cardiac cells and to determine whether they can modulate the biological activity of 7-ketocholesterol (7KC) involved in the development of cardiovascular diseases. Nanoparticles of iron labeled with Texas Red are introduced in cultures of nonbeating mouse cardiac cells (HL1-NB) with or without 7-ketocholesterol 7KC, and their ability to induce cell death, pro-inflammatory and oxidative effects are analyzed simultaneously.
Study design:
Flow cytometry (FCM), confocal laser scanning microscopy (CLSM), and subsequent factor analysis image processing (FAMIS) are used to characterize the action of iron nanoparticles and to define their cytotoxicity which is evaluated by enhanced permeability to SYTOX Green, and release of lactate deshydrogenase (LDH). Pro-inflammatory effects are estimated by ELISA in order to quantify IL-8 and MCP-1 secretions. Pro-oxidative effects are measured with hydroethydine (HE).
Results:
Iron Texas Red nanoparticles accumulate at the cytoplasmic membrane level. They induce a slight LDH release, and have no inflammatory or oxidative effects. However, they enhance the cytotoxic, pro-inflammatory and oxidative effects of 7KC. The accumulation dynamics of SYTOX Green in cells is measured by CLSM to characterize the toxicity of nanoparticles. The emission spectra of SYTOX Green and nanoparticles are differentiated, and corresponding factor images specify the possible capture and cellular localization of nanoparticles in cells.
Conclusion:
The designed protocol makes it possible to show how Iron Texas Red nanoparticles are captured by cardiomyocytes. Interestingly, whereas these fluorescent iron nanoparticles have no cytotoxic, pro-inflammatory or oxidative activities, they enhance the side effects of 7KC.
Keywords:
7-ketocholesterol; FAMIS; SYTOX Green; cardiomyocytes; confocal microscopy; iron nanoparticles.