The introduction of nanoparticles, such as TiO 2, ZnO, SiO 2, and Ag, to cells significantly increases side scatter, leading to side scattering often being used to quantify nanoparticle uptake in mammalian cells 35,45,46 and bacteria. Forward and side scattering vary upon the size and complexity of the particles, respectively. Unlike other fluorescence-based methods such as microplate readers, it is non-destructive and measures individual cells rather than averaged signals from a population of cells. used flow cytometry and imaging 44 to identify molecular targets of drugs.Ī flow cytometer is a device that measures the scattering and fluorescence of single particles ( e.g. combined confocal microscopy with flow cytometry to precisely quantify the uptake of polystyrene particles, 5 and Valero et al. At present, there is no primary method to measure nanoparticle uptake, and therefore orthogonal methods often need to be complemented to achieve reliable results. While the ICP-based techniques have also been chosen for their more quantitative results, they are destructive, not easily accessible, and above all limited to metal particles. They have good spatial resolution but poor throughput, making them difficult to apply widely. 42,43 These can be broadly classified into three approaches: EM techniques such as transmission EM (TEM) and scanning EM (SEM), ICP-based techniques, and fluorescence-based techniques such as microplate reader, flow cytometry, and confocal imaging.Īmong them, TEM and confocal microscopy are the most widely used for nanoparticle uptake analysis. The analytical techniques employed to measure nanoparticle uptake are quite diverse 7,27 including transmission electron microscopy (EM), 28–30 inductively-coupled plasma mass spectrometry (ICP-MS), 31 ICP-atomic emission spectrometry, 26 microplate reader, 32,33 flow cytometry, 5,29,34–38 confocal microscopy, 5 transmission X-ray microscopy, 39 whole cell tomography, 40 confocal Raman microscopy, 41 and hyperspectral imaging. The regulation of cellular uptake is especially important in the delivery of nanomedicine, where researchers try to reduce non-specific uptake by precise surface modification. 23 The molecular mechanisms for uptake vary by the physicochemical properties of the nanoparticles, 24,25 among which surface charge is an important factor that regulates cellular interactions with the nanoparticles. 21,22 Temperature also plays an important role in nanoparticle uptake because the process is active and energy-dependent. 5,6–9 The cellular uptake of nanoparticles depends on physical properties such as size 10–13 and shape, 14,15 chemical properties such as surface charge 16 and modification, 17,18 and dispersion media characteristics such as the presence or absence of serum proteins, 19 heat inactivated serum, 20 and corona composition. Whether it is biomedical studies to verify the efficacy of nanomedicines or environmental studies to investigate the safety of engineered nanomaterials, quantifying the cellular uptake of nanoparticles is prerequisite to understanding the underlying mechanisms. Introduction Nanoparticles have drawn tremendous interest in diverse fields including medicine, 1 foods, 2 environmental science, 3 and cosmetics 4 because of their potential in related technologies. We further tested the assays with nine different types of mammalian cells and investigated the correlation between cell type/size and nanoparticle uptake.
We successfully applied the developed assays to more readily measure fluorescence-labelled silica nanoparticle uptake in A549 lung carcinoma cells in a quantitative rather than semi-quantitative manner.
The second approach was to measure the calibrated fluorescence intensity of the nanoparticle-treated cells in molecules of equivalent soluble fluorophore (MESF) values using calibration beads, which allows for comparisons between different sets of experiments. The first approach was to measure the percentage of nanoparticle-containing cells based on a cutoff fluorescence intensity as set from a histogram of control cells, which is a quick and easy way to relatively compare nanoparticle uptake in the same set of experiments. Here, we developed assays to quantify fluorescently labelled nanoparticle uptake in mammalian cells using a flow cytometer.
Most conventional quantification methods, such as electron microscopy or confocal imaging, are laborious and semi-quantitative and therefore not readily applicable to routine analyses. Reliable quantification of nanoparticle uptake in mammalian cells is essential to study the effects of nanoparticles in the fields of medicine and environmental science.