Size-dependent cellular uptake and localization profiles of silver nanoparticles

Abstract

Purpose:

Silver nanoparticles (AgNPs) have been widely applied in various fields as excellent antibacterial reagents over the past decades. Although the particle size is considered as the most crucial factor influencing cellular uptake, transportation, and accumulation behaviors, there are still many controversies regarding the correlation between size and uptake of AgNPs. In this study, size-dependent cellular uptake of AgNPs with different diameters was investigated in B16 cells.

Methods:

The uptake of AgNPs was investigated by inductively coupled plasma-mass spectrometry (ICP-MS) and transmission electron microscopic (TEM) imaging in B16 cells.

Results:

Twenty nanometer and 100 nm AgNPs had the lowest and highest uptake efficiency at both 12 hours and 24 hours, respectively. Smaller AgNPs crossed the plasma membrane faster with uniform distribution: 5 nm AgNPs were detected in both cytoplasm and nucleus at 0.5 hours after incubation. Larger AgNPs were extremely difficult to migrate: 100 nm AgNPs were detected in the nucleus at 12 hours after incubation. Internalization of AgNPs was directly observed, mainly within membrane-bound structures, such as intracellular vesicles and late endosomes. The uptake of all four-sized AgNPs (5 nm, 20 nm, 50 nm, 100 nm) decreased significantly after the pre-treatment with chlorpromazine hydrochloride, which can specifically inhibit the clathrin-mediated endocytosis. The internalization efficiencies of AgNPs (5 nm, 20 nm, 50 nm) were markedly reduced by methyl-β-cyclodextrin, a specific caveolin-mediated endocytosis inhibitor, whereas 5-(N-ethyl-N-isopropyl) amiloride as an inhibitor of macropinocytosis inhibited the uptake of larger sizes of AgNPs (50 nm and 100 nm).

Conclusion:

The results suggest that the size of AgNPs can not only affect the efficiency of cellular uptake, but also the type of endocytosis. The clathrin-mediated endocytosis may be the most common endocytic pathway for AgNPs in B16 cells, and AgNPs at each size were likely to enter cells by a major internalization pathway.

Keywords:

• silver nanoparticles
• size-dependence
• cellular uptake
• B16 cells

Acknowledgments

This study is financially supported by the National Natural Science Foundation of China (grant number 31600814) and “Strategic Priority Research Program” of Chinese Academy of Sciences (grant number XDA09040300).

Disclosure

The authors report no conflicts of interest in this work.

Supplementary material

Figure S1 UV analysis of AgNPs at the concentration of 10 µg/mL.

Abbreviation: AgNPs, silver nanoparticles.

Figure S2 Cell viability of B16 cells with AgNPs treatment. After 24 hours incubation, cells were harvested and detatched by a MTS assay kit. This assay were performed three times, and representative results areshown here (Mean value±SD, n=4 for each group; *P<0.05, significantly different from the control.

Abbreviation: AgNPs, silver nanoparticles.