Intracellular trafficking of particles inside endosomal vesicles is regulated by particle size
Graphical abstract
Introduction
With the recent development of nanotechnology, nanoparticles have been used in a variety of fields, such as the food, cosmetics, and industries [1], [2], [3]. Recent studies in rodents have revealed that nanoparticles show greater tissue-penetration ability and internalization ability than conventional materials in various tissues (liver, spleen, and lung) [4], [5]. Therefore, nanoparticles such as mesoporous silica nanoparticles, gold nanoparticles, and fullerene are expected to be particularly useful as novel drug-delivery carriers and contrast agents in the medical field [6], [7], [8]. In addition, the cellular uptake and intracellular localization of nanoparticles change depending on the properties of these particles (size, charge, chemical composition, and surface modification) [9], [10], [11]. For example, some reports show that after nanoparticles enter the cell they are localized not only near the cell membrane but also at the perinuclear side of the cytoplasm and in organelles such as the nucleus; by carrying drugs to the perinuclear side of the cell they can thus improve therapeutic effects [12], [13]. These data suggest that nanoparticles have unique and therapeutically attractive behaviors in vivo and in vitro that is barely accomplished by conventional materials; their specific in vivo kinetics and intracellular dynamics give them potential as novel non-viral drug-delivery carriers. However, although assessment of the detailed intracellular dynamics of these particles—including distribution speed, spatiotemporal localization, intracellular processing, and excretion—is important if we are to understand the mechanisms of nanoparticle-specific intracellular dynamics, these mechanisms are still poorly understood.
Particle tracking by using real-time imaging is one of the most powerful methods of studying in detail the intracellular dynamics of particles such as protein aggregates, DNA aggregates, organelles, and viruses [14], [15]. Particle tracking is expected to reveal those intracellular movements that are difficult to unravel by using conventional methods such as localization analysis in fixed cells [16]. However, only limited numbers of studies have reported the trajectories of nanoparticles and the mean square displacement (MSD) of their intracellular movements [17], [18]. For this reason, the relationship between the properties and detailed intracellular dynamics of particles, including their motility (diffusion, velocity, and their mechanisms) has not yet been determined. Even the differences in intracellular motility between nanoparticles and sub-micron particles remain unclear.
Here, we investigated the intracellular trajectories and motilities of silica particles with diameters of 30, 50, 70, 100, 300, and 1000 nm inside the cell by using real-time imaging. We showed that silica nanoparticles (with diameters ≤ 100 nm) moved faster than sub-micron particles (with diameters > 100 nm). The silica particles were included within endosomal vesicles, which were then transported along the microtubules. Although silica nanoparticles and sub-micron particles were localized in early endosomes (EE), late endosomes (LE), and lysosomes (Ly) in different proportions, the differences in particle motility did not result from these differences in preferential localization. Surprisingly, regardless of the type of endosomal vesicle, those containing silica nanoparticles moved faster than those containing sub-micron particles. These results suggest that silica nanoparticles do not suppress the motility of endosomal vesicles, whereas sub-micron particles have suppressive effects on endosomal vesicle transport. The difference in endosomal motility between silica nanoparticles and sub-micron particles likely results from a difference in perturbation effects on endosomal vesicle transport. Our results reveal part of the mechanism of nanoparticle-specific intracellular dynamics and support a new hypothesis that differences in particle size influence the membrane trafficking of endosomal vesicles. These findings should help unravel the mechanisms of nanoparticle-specific dynamics, along with part of the cellular physiology of membrane trafficking of endosomal vesicles.
Section snippets
Nanoparticles and sub-micron particles
Fluorescence-labeled silica particles were purchased from Micromod Partikeltechnologie (Rostock/Warnemünde, Germany). Silica particles that had diameters of 70, 300, and 1000 nm (nSP70, SP300, and SP1000; catalog numbers 40-00-701, 40-00-302, and 40-00-103, respectively) and were labeled with orange fluorescence (Rhodamine B: excitation and emission wavelengths, 569 and 585 nm, respectively) were used. The silica particles were sonicated for 5 min and vortexed for 1 min before use.
Cell line, antibodies, plasmids, and reagents
A549 cells (human
Fluorescence images of silica particles in A549 cells
We used silica particles as model nanoparticles because they are among the most common nanoparticles, and mesoporous silica nanoparticles are expected to be useful as drug-delivery carriers [7]. Furthermore, silica nanoparticles show higher dispersibility and lower ionization tendency than metal nanoparticles such as zinc dioxide and iron oxide [21]. Therefore, there is less concern that the size of silica particles will be changed by aggregation and degradation during the tracking of
Discussion
Here, we assessed the intracellular movement of silica nanoparticles and sub-micron particles at the single-particle level in the living cell by using inclined illumination fluorescence microscopy. We first revealed that silica nanoparticles (nSP70) showed greater diffusion and faster active movement than silica sub-micron particles (SP300 and SP1000) (Fig. 2f, g, h). nSP70 and SP300 showed microtubule-dependent fast active movements similar to those of the endosomal vesicles, whereas SP1000
Conclusions
We showed here that endosomal vesicles containing 70-nm silica nanoparticle moved faster than those containing 300-nm sub-micron silica particle The differences in particle motility in the cells did not result from differences in preferential localization to endosomal vesicles; instead, they likely resulted from differences in perturbation effects on endosomal vesicle transport. Our results have revealed part of the mechanism of nanoparticle-specific intracellular dynamics and support a new
Competing financial interests
Y.Y. is employed by The Research Foundation for Microbial Diseases at Osaka University. All other authors declare no competing financial interests.
Acknowledgments
This study was supported by JSPS KAKENHI (No. JP25136712 to Y.Y., No. JP26-5340 to M.A., No. JP26242055 to Y.T. and No. JP15K12540 to Y.T.); by a Health Labour Sciences Research Grant from the Ministry of Health, Labour and Welfare, Japan (No. H25-kagaku-ippan-005 to Y.T.); and by The Uehara Memorial Foundation (to Y.Y.).
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