Quantifying the cellular uptake of semiconductor quantum dot nanoparticles by analytical electron microscopy

Abstract

SummarySemiconductor quantum dot nanoparticles are in demand as optical biomarkers yet the cellular uptake process is not fully understood; quantification of numbers and the fate of internalised particles are still to be achieved. We have focussed on the characterisation of cellular uptake of quantum dots using a combination of analytical electron microscopies because of the spatial resolution available to examine uptake at the nanoparticle level, using both imaging to locate particles and spectroscopy to confirm identity. In this study, commercially available quantum dots, CdSe/ZnS core/shell particles coated in peptides to target cellular uptake by endocytosis, have been investigated in terms of the agglomeration state in typical cell culture media, the traverse of particle agglomerates across U-2 OS cell membranes during endocytosis, the merging of endosomal vesicles during incubation of cells and in the correlation of imaging flow cytometry and TEM to measure the final nanoparticle dose internalised by the U-2 OS cells. We show that a combination of analytical TEM and serial block face SEM can provide a comprehensive description of the internalisation of an initial exposure dose of nanoparticles by an endocytically active cell population and how the internalised, membrane bound nanoparticle load is processed by the cells. We present a stochastic model of an endosome merging process and show that this provides a data driven modelling framework for the prediction of cellular uptake of engineered nanoparticles in general. 2 Lay summaryEngineered nanoparticles offer potential for improved medical diagnosis and treatment. The particles are small enough to enter cells and we can monitor this process using electron microscopy. The microscopy provides the resolution to count numbers of nanoparticles internalised by cells so that we can know the exact dose received by a cell or cell population and the final fate of the nanoparticles.

Nanoscopy for endosomal escape quantification

“…With a near atomic resolution (57), EM is an irreplaceable tool in studying the physio-chemical properties of NP and quantifying their voyage through the endo-lysosomal pathway (30,37,(58)(59)(60)(61)(62)(63)(64)(65)(66)(67)(68)(69)(70)(71)(72)(73)(74). EM can even detect a low number (few hundreds) of single nanoparticles escaping endosomal structures, and since it is a label-free method, it will localise and quantify NP generally untraceable by standard light microscopy methods.…”

Methodologies to investigate intracellular barriers for nucleic acid delivery in non-viral gene therapy

“…Due to the small de Broglie wavelength of accelerated electrons, electron microscopy (EM) provides superior spatial resolution (<0.2 nm for TEM; ± 1 nm for SEM) and is the technique of choice when it comes to resolving structures that are below the optical resolution limit [22,63,64]. Thus, EM is frequently used as a third technique to evaluate [65][66][67][68][69] or quantify [70][71][72] cellular uptake. Rothen-Rutishauser et al compared uptake of AuNPs in A549 cells via CLSM and TEM.…”

Nanomaterials

“…link to measured biological endpoints as discussed above and; 2. model the DDD relationship using a series of mathematical transformations (Figure 8(c)). 147,148 Step 1 will enable us to understand the key steps in the exposure pathway that may enhance a hazard presented by an NM type, for example, excessive particle uptake or gorging. 18 Step 2 will enable us to predict NM dose internalized by cells or organisms exposed to NM of a known primary particle size distribution in a given media.…”