Deciphering the mechanisms of cellular uptake of engineered nanoparticles by accurate evaluation of internalization using imaging flow cytometry

Abstract

BackgroundThe uptake of nanoparticles (NPs) by cells remains to be better characterized in order to understand the mechanisms of potential NP toxicity as well as for a reliable risk assessment. Real NP uptake is still difficult to evaluate because of the adsorption of NPs on the cellular surface.ResultsHere we used two approaches to distinguish adsorbed fluorescently labeled NPs from the internalized ones. The extracellular fluorescence was either quenched by Trypan Blue or the uptake was analyzed using imaging flow cytometry. We used this novel technique to define the inside of the cell to accurately study the uptake of fluorescently labeled (SiO2) and even non fluorescent but light diffracting NPs (TiO2). Time course, dose-dependence as well as the influence of surface charges on the uptake were shown in the pulmonary epithelial cell line NCI-H292. By setting up an integrative approach combining these flow cytometric analyses with confocal microscopy we deciphered the endocytic pathway involved in SiO2 NP uptake. Functional studies using energy depletion, pharmacological inhibitors, siRNA-clathrin heavy chain induced gene silencing and colocalization of NPs with proteins specific for different endocytic vesicles allowed us to determine macropinocytosis as the internalization pathway for SiO2 NPs in NCI-H292 cells.ConclusionThe integrative approach we propose here using the innovative imaging flow cytometry combined with confocal microscopy could be used to identify the physico-chemical characteristics of NPs involved in their uptake in view to redesign safe NPs.

Imaging flow cytometry assays for quantifying pigment grade titanium dioxide particle internalization and interactions with immune cells in whole blood

“…Nonetheless, flow imaging based assays, similar to conventional flow cytometric assays, are able to incorporate additional read outs such as staining for markers of immune activation and function thereby allowing direct associations to be made between TiO 2 interactions with single cells (adhesion or internalization) and the functional consequences of such interactions on the same cell. Like others, we found imaging cytometry a powerful technique for the analysis of particle localization, this was dependant on tailoring custom masks in order to obtain an accurate analysis 9, 10, 11, 12, 13, 14, 15. There is always potential for some particles analyzed to be attached externally to the front or back of the cells, the occurrence of false positive internalization events is random and at the same frequency within all data files as it is dependent only on the orientation of the cells as they are imaged.…”

Internalization and fate of silica nanoparticles in C2C12 skeletal muscle cells: evidence of a beneficial effect on myoblast fusion

“…These results indicated that the uptake of NPs in muscle cells was mainly an energy-dependent process, as observed in various cell types, including pancreatic, hepatic, and epithelial cells. 26,[29][30][31] In addition, even though ATP depletion considerably reduced NP uptake, it is not impossible that NPs enter cells via ATPindependent processes, as already proposed. 32,33 Accordingly, a physicochemical mechanism involving adhesion and membrane spreading around the NP surface has already been observed, with large unilamellar liposomes and red blood cells both lacking endocytic capabilities.…”

“…26 Shortly, the synthesis was based on the method described by Van Blaaderen. 27 In a first step, FITC (Thermo Fisher Scientific, Rockford, IL, USA) was covalently attached to a silane-coupling agent, (3-aminopropy1)triethoxysilane (APS), by the reaction of an amino group with an isothiocyanate group, leading to a thiourea link.…”

High throughput toxicity screening and intracellular detection of nanomaterials

“…Recently, SS signal analysis was successfully applied for detecting carboxylated nanodiamonds and tungsten carbide‐cobalt (WC‐Co) NMs in cells, results being validated by Raman and confocal microscopy associated with 3D reconstruction 82, 83. Finally, imaging flow cytometry (an integrative approach combining flow cytometry analyses with confocal microscopy) relates the physicochemical characteristics of NMs to their uptake, with a view to designing safe NMs 84. Flow cytometry appears to be a good alternative method to detect NM internalization compared to TEM which is very time‐consuming and requires heavy equipment.…”