Quantification of Nanoparticle Dose and Vesicular Inheritance in Proliferating Cells

Abstract

Assessing dose in nanoparticle–cell interactions is inherently difficult due to a complex multiplicity of possible mechanisms and metrics controlling particle uptake. The fundamental unit of nanoparticle dose is the number of particles internalized per cell; we show that this can be obtained for large cell populations that internalize fluorescent nanoparticles by endocytosis, through calibration of cytometry measurements to transmission electron microscopy data. Low-throughput, high-resolution electron imaging of quantum dots in U-2 OS cells is quantified and correlated with high-throughput, low-resolution optical imaging of the nanoparticle-loaded cells. From the correlated data, we obtain probability distribution functions of vesicles per cell and nanoparticles per vesicle. Sampling of these distributions and comparison to fluorescence intensity histograms from flow cytometry provide the calibration factor required to transform the cytometry metric to total particle dose per cell, the mean value of which is 2.4 million. Use of the probability distribution functions to analyze particle partitioning during cell division indicates that, while vesicle inheritance is near symmetric, highly variable vesicle loading leads to a highly asymmetric particle dose within the daughter cells.

Bimodal in vivo imaging provides early assessment of stem-cell-based photoreceptor engraftment

“…We chose QT for its reported long-term retention within target cells 12 and its long-term photo-stability and low toxicity; 13,14 the latter, a result of it being sequestered within intracellular vesicles. 15 The following surgical procedure was done under a dual-headed ophthalmic surgical microscope (Zeiss, Oberkochen, Germany), which also served as our ophthalmoscope. Recipients were anesthetized with isoflurane, host-eye anesthetized with proparicaine (Alcon, Fort Worth, TX, USA) preceding insertion of lid-retractors, and pupils dilated with 1% tropicamide (Bausch and Lomb, Rochester, NY, USA).…”

Quantifying Nanoparticle–Cell Interactions

“…This was achieved by assuming groups of quantum dots within vesicles act as discrete and stable fluorescent sources. We have prepared standard thin sections of these cells for correlation of TEM with the optical results [4]. TEM imaging can identify and quantify the number of quantum dot loaded vesicles per cell section [4] and the number of dots per vesicle (Figure 1).…”

“…We have prepared standard thin sections of these cells for correlation of TEM with the optical results [4]. TEM imaging can identify and quantify the number of quantum dot loaded vesicles per cell section [4] and the number of dots per vesicle (Figure 1). Nanoparticles used for in vitro studies such as these quantum dots are often coated with polymers or proteins to facilitate entry into cells.…”

“…Quantitative analysis of the serum-quantum dot solution used in [3] and [4] shows a heterogeneous dispersion of dots with a high frequency of monodisperse dots plus a significant (volume) fraction of agglomerates. Quantitative TEM imaging of cellular uptake shows a similar distribution of uptake in vesicles and we have correlated this distribution to the distribution identified by optical metrology [3].…”

“…location and number of vesicles per 2-D cell section plus the number of quantum dots per vesicle) and serial sectioning (i.e. location, size and number of vesicles in whole cells (3-D)) with that provided by optical imaging, we have quantified nanoparticle dose and dose dispersion following of cellular uptake by targeted quantum dots [4]. Figure 1.…”