Quantitative Particle Uptake by Cells as Analyzed by Different Methods

Abstract

There is a large number of two-dimensional static in vitro studies about the uptake of colloidal nano- and microparticles, which has been published in the last decade. In this Minireview, different methods used for such studies are summarized and critically discussed. Supplementary experimental data allow for a direct comparison of the different techniques. Emphasis is given on how quantitative parameters can be extracted from studies in which different experimental techniques have been used, with the goal of allowing better comparison.

Keywords: bioanalytics; cells; nanoparticles; particle uptake.

Figure 1

Time‐dependent particle intensity I(t) versus the time to which cells have been exposed to particles given as function I(t)=I ₀ (1−exp(−t/T _1/2), with the two parameters I ₀ and T _1/2 (red curve: I ₀=1, T _1/2=7.5 h; blue curve: I ₀=1, T _1/2=15 h; green curve: I ₀=2, T _1/2=7.5 h). The kinetics of particle uptake (in terms of 1/T _1/2) are twice as fast for the red and green curves compared to the blue curve. Whereas, the total amount of internalized particles under saturation conditions (in terms of I ₀) is double for the green as compared to the red and blue curves. To determine the curves, at least two time points are required.

Figure 2

A) Sketch of the trajectory (x(t), y(t)) of a fluorescent particle, for which the fluorescence changes depending on the local pH, as measured by a ratiometric approach based on the fluorescence I _y(t) and I _r(t) recorded at two different emission wavelengths. The particle dose must be low enough to allow for the tracking of individual particles. B) Plot of the time‐dependent ratiometric read‐out I _r/I _y(t) originating from the traced particle over time, and the parameters which can be extracted from such traces. C) Example of 4 images from a time‐lapse series, in which HeLa cells had been incubated with capsules with integrated pH‐sensitive fluorophores as model particles, showing an overlay of phase contrast and the two (I _y, I _r) fluorescence channels in false colors (yellow, green). These data were obtained for positively charged capsules in the presence of serum for N _caps/cell(added)=5 capsules added per cell. For details see Table 1 and the Supporting Information. D) I _r/I _y(t) data obtained from the time‐lapse series shown in (C) for the indicated capsule. The following data were extracted for this particular trajectory: t _c=20 min, Δt _A=39 min, Δt _P50 %=139 min, Δt _P10 %=119 min, and |d(I _r/I _y)/dt)|_max=−0.107 min⁻¹.

Figure 3

A) The number of internalized particles per cell N _caps/cell is obtained individually for a number of different cells for each time point of incubation, for example, from overlays of bright field and fluorescence microscopy images. Labelling with pH‐sensitive dyes allows to distinguish internalized particles (shown in yellow) from extracellular particles (shown in red). B) From the counted particles per cell data, a histogram can be made for each time point, whereby f(N _caps/cell,t) corresponds for the frequency of cells with N _caps/cell internalized particles. The histograms can be converted into cumulative distribution function p(N _caps/cell,t). The mean number of internalized particles per cell can be either derived from the histograms (<N _caps/cell(t)>_(h)) or from the cumulative distribution function, where p=0.5 (<N _caps/cell(t)>_(p)).19a Plotting the mean number of internalized particles per cell versus time allows the maximum number of internalized particles per cell to be determined (i.e. under saturation conditions; <N _caps/cell(t)>_(sat,h) and <N _caps/cell(t)>_(sat,p)), as this is the time it takes cells to reach half saturation with the internalized particles (t _up(sat,h) and t _up(sat,p)). C) HeLa cells incubated with capsules with integrated pH‐sensitive fluorophores as model particles, showing an overlay of phase contrast and the two (I _y, I _r) fluorescence channels in false colors (yellow, green). These data were obtained for positively charged capsules in the presence of serum and for N _caps/cell(added)=10 capsules added per cell after 2 h of incubation. For details see Table 2 and the Supporting Information. D) Histograms and cumulative distribution functions for the positively charged capsules for which an image has been shown in (C), after 2 and 24 h of incubation. The following data were obtained from these graphs: <N _caps/cell(t=2 h)>_(h)=2.9, <N _caps/cell(t=24 h)>_(h)=6.0, <N _caps/cell(t=2 h)>_(p)=1.9, <N _caps/cell(t=24 h)>_(p)=4.8. The time‐dependent data are normalized to the maximum number of internalized capsules per cell. From these graphs the maximum number of internalized capsules per cell and the time it takes a cell to uptake the number of capsules under saturation conditions are derived. The values derived for the shown graphs are <N _caps/cell>_(sat,p)=5.6, t _up(sat,p)=3 h, t _up(1)=<1 h. The same scaling for the f(N _caps/cell,t) and p(N _caps/cell,t) is used to visualize that p(N _caps/cell,t) is formed by consecutively summing up the f(N _caps/cell,t) values.

Figure 4

A) The “signal” of particles in a cell, which can be fluorescence (I _y) or mass of element X (m _X) is detected per cell. Individual particles do not need to be laterally resolved for this purpose. B) The particle signal associated with cells (I _y in case of fluorescence, m _X in case of the elemental analysis of element X) is recorded for many cells at different time points t, leading to the average time‐dependent signal intensity per cell <I _y(t)>. From this, the maximum signal intensity <I _y>_(sat) under saturation conditions can be obtained, as well as the time t _up(sat) to reach half saturation. If the signal intensity per particle is known, then the particle signal per cell <I _y> can be converted into the number of particles per cell <N _caps/cell>. C) As an example, the internalization of capsules with integrated Au nanoparticles in their shells by HeLa cells is shown. These data were obtained for positively charged capsules in the presence of serum for N _caps/cell(added)=10 capsules added per cell. For details see Table 3 and the Supporting Information. The mean amount of elemental Au per cell <m _Au> was determined with ICP‐MS. As the amount of elemental Au per capsule is known, this could be converted into the mean number of internalized capsules per cell <N _caps/cell>. These data are plotted for different incubation times. The following data were obtained from this graph: <N _caps/cell>_(sat,.i)=3.6, t _up(sat,i)=<2 h. A summary of data obtained with this method is presented in Table 3.

Figure 5

A) Cells in a flow channel pass a fluorescence detector one at a time, and for each event the fluorescence intensity is recorded. For ratiometric pH‐sensitive fluorophores, fluorescence is detected in the yellow (I _y) and in the red (I _r) channel, corresponding to acidic and neutral local pH. B) All events are plotted, and different populations can be identified. Cells without associated particles have low fluorescence in both channels (depicted as “green” population). Cells with adherent and internalized particles have predominant red and yellow fluorescence due to neutral and acidic local pH, respectively, around the particles. The number of cells measured for each of the populations can be extracted as N _{cells w/o caps}, N _{cells w caps(adh)}, and N _{cells w caps(in)}. In practice, the populations may overlap and need to be distinguished by defining proper gatings. From the populations the mean fluorescence intensity of internalized particles per cell <I _y> can also be derived. B₁) By plotting the percentage of labelled cells versus time (N _{cells w caps(in)}(t)/N _cells) with N _cells=N _{cells w/o caps} + N _{cells w caps(adh)} + N _{cells w caps(in)}, the maximum percentage of labelled cells under saturation conditions (N _{cells w caps(in)}/N _cells)_(sat), and the time it needs to reach half saturation t _up(sat,f) can be calculated. B₂) The mean fluorescence per cell can also be plotted over time, leading to the fluorescence under saturation conditions <I _y>_(sat,c) and the time t _up(sat,c) it needs to reach half saturation. C) Population plot obtained for HeLa cells incubated with positively charged capsules in the presence of serum for N _caps/cell(added)=10 capsules added per cell. D) Plots of the percentage of labelled cells and the mean fluorescence intensity per cell versus time. From these graphs the following parameters were extracted: (N _{cells w caps(in)}/N _cells)_(sat,f)=65 %, t _up(sat,f)=4 h, <I _y>_(sat,c)=2040, and t _up(sat,c)=<2 h. The parameters of the total data set are presented in Table 3 and Table 4.