Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016 Nov 29;13(1):63.
doi: 10.1186/s12989-016-0173-1.

Endocytosed nanoparticles hold endosomes and stimulate binucleated cells formation

Lin Xia  1 , Weihong Gu  1 , Mingyi Zhang  1 , Ya-Nan Chang  1 , Kui Chen  1 , Xue Bai  1 , Lai Yu  1 , Juan Li  1 , Shan Li  2   3 , Gengmei Xing  4
Affiliations

Endocytosed nanoparticles hold endosomes and stimulate binucleated cells formation

Lin Xia et al. Part Fibre Toxicol. .

Abstract

Background: Nanotechnology developed rapidly in cellular diagnosis and treatment, the endocytic system was an important pathway for targeting cell. In the research of developing macrophages as drug carriers or important therapeutic targets, an interesting phenomenon, internalized nanoparticles induced to form binucleated macrophages, was found although the particles dose did not cause obvious cytotoxicity.

Results: Under 25 μg/ml, internalized 30 nm polystyrene beads(30 nm Ps nanoparticles) induced the formation of binucleated macrophages when they entered into endosomes via the endocytic pathway. These internalized 30 nm Ps nanoparticles (25 μg/ml) and 30 nm Au-NPs (1.575 ng/ml) also induced markedly rise of binucleated cell rates in A549, HePG-2 and HCT116. This endosome, aggregated anionic polystyrene particles were dispersed and bound on inner membrane, was induced to form a large vesicle-like structure (LVLS). This phenomenon blocked transport of the particles from the endosome to lysosome and therefore restricted endosomal membrane trafficking through the transport vesicles. Early endosome antigen-1 and Ras-related protein-11 expressions were upregulated; however, the localized distributions of these pivotal proteins were altered. We hypothesized that these LVLS were held by the internalized and dispersed particles decreasing the amount of cell membrane available to support the completion of cytokinesis. In addition, altered distributions of pivotal proteins prevented transfer vesicles from fusion and hampered the separation of daughter cells.

Conclusions: 30 nm Ps nanoparticles induced formation of LVLS, blocked the vesicle transport in endocytic system and the distributions of regular proteins required in cytokinesis which led to binucleated cells of macrophages. Markedly raised binucleated rate was also observed in human lung adenocarcinoma epithelial cell line(A549), human hepatoma cell line(HePG-2) and human colorectal cancer cell line(HCT116) treated by 30 nm Ps nanoparticles and Au-NPs.

Keywords: Binucleated cell; Endocytic; Endosome; Large vesicle-like structures; Nanoparticles.

PubMed Disclaimer

Figures

Fig. 1
Fig. 1
Nanoparticles were internalized by cells. a: SEM photographs were 30 nm Ps particles with ultrasonic or not. Utrasonic raised the monodispersion of 30 nm Ps particles obviously. b: Zeta potential of 30 nm Ps particels varied from -14 mV to -50 mV with ultrasonic. Potential detector checked the Ps 30 nm particles which were negative on surface. c: 30 nm Ps particles labelled with FITC entered the cell and were present in cytoplasm (red rectangle). d: 30 nm Ps particles gathered in large vesicle-like structures (LVLS) with time going along (white arrows)
Fig. 2
Fig. 2
Nanoparticles induced binucleated cells formation. a: After co-culture for 12 h, the binuclear cells were present (red rectangle, white arrows) and LVLS generated in cytoplasm. A magnified image of binucleated cell showed the 30 nm Ps particles bound on the inner surface of the LVLS. b: Video of the process of binuclear cell formation (white arrow). c: Flow cytometer assay indicated that 30 nm Ps particles increased the percent of binuclear cells (black line was treated cell and red line was control cell). d: The percent of binuclear cells reached 9.97 % in treated cells and was 0.83 % in control cells. The difference of the percent of binuclear cells in treated cell and control cell was significance (p < 0.05)
Fig. 3
Fig. 3
Influence of 30 nm Ps particles on human tumor cells. a, binuclear A549. b, binuclear HePG-2 c: binuclear HCT116. In these binucleated cells, vesicles with green fluorescence of Ps nanoparticles presented in cytoplasm. d: the percent of binuclear A549, HePG-2 and HCT116 cells were 5.37 %, 7.12 % and 5.18 % in treated cells to 0.51 %, 0.63 % and 0.49 % in control respectively. The difference of the percent of binuclear cells in treated cell and control cell was significance (p < 0.05)
Fig. 4
Fig. 4
Intracellular transport and distribution of the nanoparticles. EEA1: The co-locations (yellow) of EEA1 (red) and 30 nm Ps particles (green) were present at 10 min, the yellow spots were magnified at right and left superior corners. The co-locations decreased at 30 min, there was hardly co-location and the LVLS generated at 50 min. Rab7: Rab7 co-located hardly with 30 nm Ps particles at 10 min, 30 min and 50 min, the LVLS were also present in the cell at 50 min. LAMP-1: LAMP-1 didn’t co-locate with 30 nm Ps particles from 10 min to 50 min. The co-locations of EEA1 and 30 nm Ps particles at 10 min indicated that the particles entered the cell by endocytic transport. Following that, the particles didn’t co-locate with Rab7 and LAMP-1. That indicated that the particles were not transported through late endosome to lysosome. It meant that the 30 nm Ps particles induced the LVLS formation in early endosome
Fig. 5
Fig. 5
Interference with membrane vesicles distribution. a: 3D-reconstruction of control cell and treated cell, red spots of transferrin were present in control (a), red spots and green spots of Ps particles were present in treated cell (b). The red spots of transferrin accumulated at midbody in control, but were short in treated cell. b: Surface Plot of transferrin in control (a) and treated cell (b), the fluorescence of transferrin distributed and gathered at midbody and poles regions of control cell. But in treated cell, the transferrin dispersed in cytoplasm randomly. c. Transferrin located closely to the LVLS contained 30 nm Ps particles (green) in 3D space. A magnified image showed the close tethers of transferrin vesicles and LVLS. d: The returning time of transferrin from cytoplasm to cell surface in treated cell delayed obviously. The red fluorescent intensity reduction in cytoplasm of control cell was faster than that in treated cell. The t1/2 of control cells was 2.5 min and the t1/2 of treated cells was 4.9 min. The transferrin vesicles were tethered by LVLS contained 30 nm Ps particles
Fig. 6
Fig. 6
Expressions and distributions of Rab11 and EEA1. a: Western blotting revealed that the amount of proteins of Rab11 and EEA1 increased along the co-culturing time (from 10 min, 30 min to 50 min). b: (a) Red fluorescent spots of Rab11 accumulated at the midbody in control cell of mitosis telophase, (b) the red fluorescent spots accumulated to large clusters beside the midbody in treated cell of mitosis telophase. c: (a) Red fluorescent spots of EEA1 mainly focused at the midbody in control cell of mitosis telophase, (b) the red fluorescent spots were less at the midbody and accumulated to large clusters at the poles of treated cell of mitosis telophase. d: Fluorescence intensities of Rab11 and EEA1 at midbody were lower obviously after co-cultured with 30 nm Ps particles. Comparted with control, the difference of Rab11 was significance (p < 0.05) and EEA1 was without significance in treated cell. Distributions of these key regulators (EEA1 and Rab11) which regulate the organizing of contractile ring and cytokinesis were disturbed obviously
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Meng H, et al. Aspect Ratio Determines the Quantity of Mesoporous Silica Nanoparticle Uptake by a Small GTPase-Dependent Macropinocytosis Mechanism. ACS Nano. 2011;5(6):4434–4447. doi: 10.1021/nn103344k. - DOI - PMC - PubMed
    1. Yan Y, Such GK, Johnston AP, Best JP, Caruso F. Engineering particles for therapeutic delivery: prospects and challenges. ACS Nano. 2012;6(5):3663–3669. doi: 10.1021/nn3016162. - DOI - PubMed
    1. Yang G, Kang Z, Ye X, Wu T, Zhu Q. Molecular simulation of flavin adenine dinucleotide immobilized on charged single-walled carbon nanotubes for biosensor applications. Biomaterials. 2012;33(34):8757–8770. doi: 10.1016/j.biomaterials.2012.08.053. - DOI - PubMed
    1. Mckay HF, Burgess DR. Life is a Highway’: Membrane Trafficking During Cytokinesis. Traffic. 2011;12(3):247–251. doi: 10.1111/j.1600-0854.2010.01139.x. - DOI - PMC - PubMed
    1. Schiel JA, Prekeris R. Membrane dynamics during cytokinesis. Curr Opin Cell Biol. 2013;25(1):92–98. doi: 10.1016/j.ceb.2012.10.012. - DOI - PMC - PubMed

Publication types

LinkOut - more resources