Analysis of the capture and influence of nanoparticles on the cytotoxic effects of 7-ketocholesterol on cardiac cells: investigation by flow cytometry and by dynamic and spectral imaging microscopy associated with factor analysis of medical image sequences.
OBJECTIVE
To evaluate the capture of nanoparticles (quantum dots [QDs], fluorospheres) by nonbeating mouse cardiac cells (HL1-NB) cultured without or with 7-ketocholesterol (7KC) found at an increased level in the plasma of atherosclerotic patients and to simultaneously analyze their cytotoxic, proinflammatory and oxidative properties.


METHODS
Flow cytometry (FCM), confocal laser scanning microscopy and subsequent factor analysis image processing were used to characterize the uptake of nanoparticles and to define their cytotoxicity, evaluated by enhanced permeability to SYTOX Green, release of lactate dehydrogenase (LDH) and morphologic nuclear changes determined with Hoechst 33342. Proinflammatory effects were estimated by enzyme linked immunoassay to quantify IL-8 and MCP-1 secretion. The overproduction of reactive oxygen species (ROS) was determined by FCM with hydroethidine.


RESULTS
Whereas the nanoparticles had no cytotoxic or inflammatory effects, they could stimulate ROS production. QDs were not incorporated. When 7KC was used, LDH release was enhanced and QDs potentialized IL-8 secretion. The incorporation and exit dynamics of nanoparticles were visualized to differentiate the emission spectra of SYTOX Green and nanoparticles and to precisely determine the cellular localization of nanoparticles.


CONCLUSIONS
The selected nanoparticles, which accumulate at the inner or outer cytoplasmic membrane level, can induce biologic activities and are able to interfere with those of chemically defined molecules such as 7KC.
Kahn
Edmond
E
Institut National de la Santé et de la Recherche Médicale U678/Unité Mixte de Recherche-Santé, Universit6 Pierre et Marie Curie Paris 6, France. kahn@imed.jussieu.fr
Pelloux
Sophie
S
Baarine
Mauhamad
M
Khalfaoui
Taoufik
T
Ragot
Kévin
K
Frouin
Frédérique
F
Riedinger
Jean-Marc
JM
Tourneur
Yves
Y
Lizard
Gérard
G
D000818:Animals; D001562:Benzimidazoles; D045744:Cell Line, Tumor; D002463:Cell Membrane Permeability / Q000187:drug effects; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D018932:Chemokine CCL2 / Q000378:metabolism; D002863:Chromogenic Compounds; D004359:Drug Therapy, Combination; D004791:Enzyme Inhibitors / Q000633:toxicity*; D005163:Factor Analysis, Statistical; D005434:Flow Cytometry; D016209:Interleukin-8 / Q000378:metabolism; D007653:Ketocholesterols / Q000633:toxicity*; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D051379:Mice; D018613:Microscopy, Confocal; D036641:Microscopy, Fluorescence, Multiphoton; D032383:Myocytes, Cardiac / Q000187:drug effects* / Q000378:metabolism; D045663:Quantum Dots; D017382:Reactive Oxygen Species / Q000378:metabolism
D001562:Benzimidazoles; C491768:Ccl2 protein, mouse; D018932:Chemokine CCL2; D002863:Chromogenic Compounds; D004791:Enzyme Inhibitors; D016209:Interleukin-8; D007653:Ketocholesterols; D017382:Reactive Oxygen Species; D007770:L-Lactate Dehydrogenase; C003001:7-ketocholesterol; C017807:bisbenzimide ethoxide trihydrochloride