Quantitative analysis is an important but still largely unexplored issue in the study of nanomedicine procedures, in particular at the cellular and subcellular levels. Many phenomena were discovered by which nanoparticles enhance the cancer cell mortality or facilitate the action of other cell-killing factors [1–4]. However, the potential modulation of these phenomena for procedures such as radiotherapy [5–9] or drug delivery [7, 10–13] requires clarifying a number of issues, many of them quantitative.
Such issues are not simple since each cell line interacts differently with nanoparticles [14–16]. Furthermore, the specific chemistry and morphology of each type of nanoparticles influence the interaction mechanisms leading to nanoparticle uptake [17–23]. Quantitative features are specifically important since they can affect internalization processes (endocytosis, pinocytosis, free membrane trafficking, etc.) [24–27], the optimization of nanomedicine procedures (in particular the maximum nanoparticle uptake by each cell line [28–30]) and the conditions to avoid toxicity.
An effective quantitative analysis should include not only average properties but also the statistical distributions for the level of uptake and for the size of the clusters formed by aggregated nanoparticles. Furthermore, it would be preferable to identify the location of the internalized nanoparticles and clusters with respect to the different organelles in cells for their different functions.
The procedure presented here meets these requirements and stems from an extensive previous work to develop suitable instruments and methods. In recent years, we introduced a series of imaging approaches for biosystems based on the high brightness and coherence of x-ray synchrotron sources [31–37]. Such methods reached sufficient spatial resolution for subcellular analysis , thus enabling us to harvest valuable and reliable quantitative information.
The results presented below show that the extraction of detailed quantitative data on nanoparticle cellular uptake is entirely feasible. Although so far validated for the specific case of gold nanoparticles (AuNPs) on two cell lines, the method can have much broader applications - for example, to all nanoparticles containing high-Z elements. The approach is non-destructive and reaches high spatial resolution.
The procedure started with the acquisition of transmission hard-x-ray micrographs with an instrument that can reach a 30-nm spatial resolution [38, 39]. We collected either individual projection micrographs or sets of projection images at different angles for tomographic 3D reconstruction. The high penetration of our hard-x-rays (8 keV photon energy) made it possible to work with 3D samples, i.e., cell cultures in gel.
Large cell collections could be simultaneously imaged as required for quantitative analysis. Staining with heavy metals (uranium or osmium acetate) was used in specific cases to reveal specific intracellular (organelle) details. Zernike phase contrast was also exploited for visualizing nanoparticle clusters smaller than ~100 nm.
From the microimages, we extracted quantitative data on the number and size of uptaken nanoparticle clusters and information on the cluster positions in the cells. The procedure was first tested on bare (uncoated) AuNPs with average size ~15 nm prepared by a recently developed method [40–43]. This is based on x-ray irradiation of precursor solutions and produces nanoparticle colloids with high density and excellent stability. Although the sizes of these nanoparticles are smaller than the currently achieved resolution of X-ray microscopy, the aggregation of the nanoparticles after internalization by cells forms clusters of size large enough to be imaged and quantitatively analyzed.
The tests were then extended to AuNPs coated with polyetheleneglycol (PEG), prepared with a similar irradiation method . We tested both types of nanoparticles on two different cancer cell lines, EMT-6 and HeLa cell, detecting the significant quantitative differences discussed below.
One interesting issue analyzed in our tests was the quantitative relation between the nanoparticle uptake and the cell survival. The image analysis results were cross-checked with those of cell viability bioassays. The corresponding conclusions are interesting on their own considering the present open issues on the cellular effects of AuNPs.
Specifically, we found that both naked and PEG-coated AuNPs cause cell death at high concentrations. Quantitative uptake, quantitative cell death rate and colloid concentration appear all correlated.
Quite interestingly, no particle uptake was found at cell nuclei locations. This indicated that the nuclear membrane selectivity remained unchanged in the presence of nanoparticles.