Nano Today
ReviewEndocytosis and intracellular transport of nanoparticles: Present knowledge and need for future studies
Graphical abstract
Research highlights
► Studies on the mechanism of interactions between cells and nanoparticles (NPs) must be improved. ► We discuss the possibilities and pitfalls in studies of endocytic pathways followed by NPs. ► We evaluate the use of inhibitors, mutated proteins, siRNAs and colocalization experiments in such studies. ► We discuss intracellular transport, recycling to cell exterior, disturbance of cell functions, and metabolism of NPs. ► Knowledge about cellular fate of NPs is essential to develop nanoparticles for clinical use.
Introduction
Nanoparticles have emerged as promising tools both for basic mechanistic studies of cells and animals, as well as for delivery of drugs or other substances in vitro and in vivo [1], [2], [3], [4], [5], [6]. The rate of uptake and intracellular localization of nanoparticles have been studied by many research groups, and several review articles summarizing the published data are available; see e.g. [7], [8], [9], [10], [11], [12], [13]. These reviews reveal that it is difficult to draw general conclusions about how to produce particles for optimal cellular uptake, as the rate and mechanism of uptake turns out to be cell-type dependent and vary between nanoparticles with different size, charge, and other surface properties. There are, however, several reports showing that nanoparticles of 20–50 nm are taken up more rapidly than smaller or larger particles [14], [15], [16]. Because particles with a positive charge will bind to the negatively charged cell surface, one would expect positively charged particles to be endocytosed more efficiently than negatively charged particles. In fact, a study in HeLa cells with positively and negatively charged nanoparticles of equal size (80 nm) showed a 2-fold higher uptake of the positively charged particles [17]. In contrast, a higher uptake of negatively charged nanoparticles has been reported in HEK cells [18]. As discussed below, many of the conclusions drawn about cellular uptake of nanoparticles need to be re-evaluated in light of the present knowledge of endocytic mechanisms.
Cell-type specific variation in handling of internalized particles can be expected, and significant differences in intracellular sorting, trafficking and localization of nonconjugated quantum dots (QDs) have been reported in three closely related human prostate cancer cells [19]. It is clear that for delivery of nanoparticles to heterogeneous tumours, differences in cellular uptake and sorting can have significant implications. Importantly, the polyvalent surface of nanoparticles may induce cross-linking of cellular receptors, start signalling processes, induce structural alterations at the cell surface, and interfere with normal cell function [15], [20]. Moreover, when studying cellular uptake of nanoparticles one should keep in mind that the rate of endocytosis may also depend on the cell density [21], [22].
So far most focus has been on uptake of nanoparticles into non-polarized cells. Importantly, polarized cells can have different endocytic mechanisms on the apical and basolateral pole [23]. Thus, a nonpolarized epithelial cell cannot be expected to correctly reflect the complexity found in epithelial cell layers where clathrin-independent endocytosis (CIE) is selectively regulated at the apical side and caveolae can be found exclusively at the basolateral side [23].
There is still a lot to learn about cellular uptake and intracellular transport of nanoparticles in order to interpret data from in vitro studies and to improve the in vivo use of the particles. Also, the recent report that caveosomes is an artifact in cells overexpressing caveolin [24] is important for re-interpretation of data already published regarding intracellular localization and degradation of nanoparticles.
In this article we present a summary of the present knowledge of different endocytic mechanisms and we describe how involvement of the various endocytic pathways in uptake of nanoparticles can be studied, including the pitfalls in performing such studies. We also shortly discuss some aspects of intracellular transport of particles, recycling to the cell exterior, metabolism and disturbances of cellular processes caused by nanoparticles.
Section snippets
Endocytic mechanisms
Cells use endocytosis for uptake of nutrients, down-regulation of growth factor receptors and as a master regulator of the signalling circuitry. There are several different types of endocytosis, all based on formation of intracellular vesicles following invagination of the plasma membrane or ruffling giving rise to larger vesicles [25], [26], [27], [28]. Phagocytosis (“cell eating”) is used for uptake of large particles such as bacteria, and is the first step in uptake and degradation of
Pharmacological inhibitors
Pharmacological inhibitors are often used to investigate which endocytic mechanism is responsible for cellular uptake of nanoparticles. This approach is far too often based on the assumption that these inhibitors have specific effects on a given endocytic mechanism, but as discussed below and summarized in Table 1, this is normally not the case.
Cholesterol depletion with methyl-β-cyclodextrin (mβCD) and perturbation of the cholesterol function by addition of the cholesterol-binding drugs
Delivery of nanoparticles to the cytosol
Delivery of nanoparticles into cells in vitro by using electroporation or microinjection is outside the main scope of the present discussion; for a review see [11]. The possibility to deliver substances directly into the cytosol by using positively charged “cell-penetrating peptides” has been an issue of much discussion for many years. Several authors have reported or discussed the option of coupling such peptides to nanoparticles [11], [59], [60]. Although different groups have come to
Disturbances of intracellular transport and other cellular processes induced by nanoparticles
Several review articles about the cellular toxicity of nanoparticles are available [69], [70], [71], [72], [73], [74], thus we will not discuss this issue in any detail. However, we would like to point out that several of the methods used to detect cellular toxicity require rather large effects on the cells, e.g. the trypan blue used to detect dead cells or the MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) used to assess mitochondrial activity by conversion of MTT
Cellular excretion and degradation of nanoparticles
There are a few articles describing exocytosis of nanoparticles (meaning recycling of the particles to the cell exterior) [14], [16], [77]. They all conclude that exocytosis of the particles is much slower than endocytosis. Whereas the rate of endocytosis seems to be fastest for particles of 20–50 nm, the rate of exocytosis decreases with increasing particle size [14], [16]. Chithrani and Chan [14] reported that the fraction of endocytosed nanoparticles varied for different cell lines and that
Summary
We have summarized different methods that can be used to investigate cellular uptake of nanoparticles and the pitfalls in such studies. The complexity, the combination of advanced chemistry and cell biology, makes it important that future research on nanoparticles is performed as a close collaboration between scientists with different backgrounds. This is important to prevent misleading/wrong interpretations and thus aid in bringing nanoparticles faster into clinical use.
Tore-Geir Iversen is a senior researcher and Project Leader at the Centre for Cancer Biomedicine, The Norwegian Radium Hospital in Oslo, Norway. He earned his Ph.D. at the Norwegian University of Science and Technology (NTNU), Trondheim in 1995, at that time studying microbial genetics. He joined the group of professor Sandvig in 1997, then studying endocytosis and intracellular transport of different protein toxins. In 2006 he turned his focus into studying how nanoparticles are endocytosed
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Tore-Geir Iversen is a senior researcher and Project Leader at the Centre for Cancer Biomedicine, The Norwegian Radium Hospital in Oslo, Norway. He earned his Ph.D. at the Norwegian University of Science and Technology (NTNU), Trondheim in 1995, at that time studying microbial genetics. He joined the group of professor Sandvig in 1997, then studying endocytosis and intracellular transport of different protein toxins. In 2006 he turned his focus into studying how nanoparticles are endocytosed and transported in cells. His group was the first to demonstrate that accumulation of nanoparticles within endosomes could induce changes in the normal intracellular transport of the cell. Current research interests also include more applied biological studies about nanoparticles and the criteria required for their clinical use in therapy and imaging.
Tore Skotland is a guest researcher at The Centre for Cancer Biomedicine, The Norwegian Radium Hospital in Oslo, Norway. He received his Ph.D. in biochemistry from the University of Bergen, Norway in 1980. After 11 years in basic research (protein chemistry and enzymology) he moved to pharmaceutical R&D in 1983 where he stayed for 26 years within the same company Nycomed/Amersham/GE Healthcare, i.e. one of the world leading companies in developing contrast agents for medical imaging. The last 20 years he was heading the work to characterize the metabolism and excretion of all types of contrast agents in the company (water soluble as well as particle based) for CT, MRI, SPECT, PET and optical imaging. He is co-author of approximately 75 publications.
Kirsten Sandvig is a group leader at The Centre for Cancer Biomedicine, The Norwegian Radium Hospital and professor at The University of Oslo in Norway. She received her Ph.D. in biochemistry at the University of Oslo in 1979. Professor Sandvig has for many years contributed significantly to our present knowledge about endocytosis and intracellular transport with approximately 250 publications in the field. She was the first to describe recycling of endocytosed material to the cell exterior, the first to describe retrograde transport from endosomes to ER, and also among the first to describe the existence of clathrin independent endocytosis. She has throughout her carrier used different types of protein toxins in her studies, which has resulted in numerous prizes for her scientific contributions.
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These authors contributed equally to this work.