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
. 2013:8:919-31.
doi: 10.2147/IJN.S38749. Epub 2013 Mar 3.

Visualization of internalization of functionalized cobalt ferrite nanoparticles and their intracellular fate

Affiliations

Visualization of internalization of functionalized cobalt ferrite nanoparticles and their intracellular fate

Vladimir B Bregar et al. Int J Nanomedicine. 2013.

Abstract

In recent years, nanoparticles (NPs) and related applications have become an intensive area of research, especially in the biotechnological and biomedical fields, with magnetic NPs being one of the promising tools for tumor treatment and as MRI-contrast enhancers. Several internalization and cytotoxicity studies have been performed, but there are still many unanswered questions concerning NP interactions with cells and NP stability. In this study, we prepared functionalized magnetic NPs coated with polyacrylic acid, which were stable in physiological conditions and which were also nontoxic short-term. Using fluorescence, scanning, and transmission electron microscopy, we were able to observe and determine the internalization pathways of polyacrylic acid-coated NPs in Chinese hamster ovary cells. With scanning electron microscopy we captured what might be the first step of NPs internalization - an endocytic vesicle in the process of formation enclosing NPs bound to the membrane. With fluorescence microscopy we observed that NP aggregates were rapidly internalized, in a time-dependent manner, via macropinocytosis and clathrin-mediated endocytosis. Inside the cytoplasm, aggregated NPs were found enclosed in acidified vesicles accumulated in the perinuclear region 1 hour after exposure, where they stayed for up to 24 hours. High intracellular loading of NPs in the Chinese hamster ovary cells was obtained after 24 hours, with no observable toxic effects. Thus polyacrylic acid-coated NPs have potential for use in biotechnological and biomedical applications.

Keywords: internalization; intracellular fate; magnetic nanoparticles; scanning electron microscopy; transmission electron microscopy.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Dynamic light scattering analysis of Co-ferrite NPs. Notes: Distribution of the hydrodynamic diameters is shown for: NP cores in distilled water, NPs functionalized with PAA in distilled water, and functionalized NPs in cell culture medium with 10% FBS, which was used for all internalization experiments. Abbreviations: Co-ferrite, cobalt ferrite; NP, nanoparticle; PAA, polyacrylic acid; HAM, Ham’s tissue culture medium for mammalian cells; FBS, fetal bovine serum.
Figure 2
Figure 2
TEM micrograph of Co-ferrite NP cores. Notes: The average diameter of NPs is 10 nm. The Scale bar corresponds to 20 nm. Abbreviations: TEM, transmission electron microscopy; Co-ferrite, cobalt ferrite; NP, nanoparticle.
Figure 3
Figure 3
Exposure to functionalized Co-ferrite NPs induces membrane ruffling in CHO cells. CHO cells were incubated for 1 hour with distilled water (control) (A), or treated with NPs (B). (C-F), CHO cells were for 10 min incubated with distilled water (C), or with NPs and fixed. The presence of NPs induced perturbations of the plasma membrane, mostly in form of spherical membrane extensions (blebs) (D), circular ruffling, which look like cup-shaped projections (E), and lamellipodial ruffles, which appear as flat sheet-Iike projections on the celi surface (F). Notes: Arrows in panel (B) denote nantubes and in panel (F) NP aggregates. The scale bars correspond to 10 μm in (A) and (B), and to 1 μm in (CF). Abbreviations: Co-ferrite, cobalt ferrite; NP, nanoparticle; CHO, Chinese hamster ovary.
Figure 4
Figure 4
Interaction of functionalized Co-ferrite NPs with the cell membrane surface, observed with SEM. Notes: CHO cells were incubated with NPs for 30 minutes and fixed. NP aggregates, bound to the plasma membrane are denoted by arrows, and some NPs are found in a cavity in the outer cell membrane. Individual nanoparticle crystallites can be observed. The scale bar corresponds to 100 nm. Abbreviations: Co-ferrite, cobalt ferrite; NP, nanoparticle; SEM, scanning electron microscopy; CHO, Chinese hamster ovary.
Figure 5
Figure 5
Energy-dispersive X-ray spectroscopy confirmed the presence of NP aggregates on the membrane of CHO cells. CHO cells were incubated with NPs for 30 minutes and fixed. ln panel (A) there are marked and numbered areas, which correspond to sample regions in panel (B), values are represented as percentage of iron in the total elemental composition of the analysed region. Note: The scale bar corresponds to 500 nm. Abbreviations: NP, nanoparticle; CHO, Chinese hamster ovary.
Figure 6
Figure 6
Internalization of Co-ferrite NPs in CHO cells observed with TEM. CHO cells were incubated with NPs for 50 minutes and fixed. Membrane ruffles observed with TEM resemble membrane perturbations typical of macropinocytosis (A and B). Notes: NP aggregates are observed under membrane ruffles (big arrow) and in an already internalized vesicle (small arrow), most probably formed with macropinocytosis (A); membrane ruffles enclosing a small NP aggregate (big arrow) and already internalized NP aggregates in early endosomes (small arrows) and in a multivesicular body (asterisk) (B). The scale bars correspond to 1 μm. Abbreviations: Co-ferrite, cobalt ferrite; NP, nanoparticle; CHO, Chinese hamster ovary; TEM, transmission electron microscopy.
Figure 7
Figure 7
Functionalized Co-ferrite NPs enter CHO cells via clathrin-mediated endocytosis. Cells were incubated with NPs for 15 minutes and fixed. Small NP aggregates are present in a clathrin-coated pits on the cell surface (A), and in a clathrin-coated vesicle immediately after internalization (B). Notes: Presented figures are the most representative images of several clathrin-coated pits and vesicles observed. The scale bars correspond to 200 nm. Abbreviations: Co-ferrite, cobalt ferrite; NP, nanoparticle; CHO, Chinese hamster ovary.
Figure 8
Figure 8
TEM observation of Co-ferrite NPs after internalization. Cells were incubated for 1 hour and fixed. NPs are found in larger aggregates in bigger membrane bound vesicles (A), and in lysosomes (B). Note: The scale bars correspond to 200 nm. Abbreviations: TEM, transmission electron microscopy; Co-ferrite, cobalt ferrite; NP, nanoparticle.
Figure 9
Figure 9
Time dependent internalization of Co-ferrite NPs: cells under phase contrast microscope (A), RITC labelled NPs (B), late endosomes and lysosomes labelled with LysoTracker® Blue (C) and colocalization of RITC labelled NPs and Iysosomes (D) after 15 minutes, 75 minutes and 24 hours of incubation with NPs. Notes: NPs can be observed inside the cells already after 15 minutes, but they are mostly not yet colocalized with Iysosomes. After 75 minutes of incubation, colocalization of NPs and Iysosomes is observed. After 24 hours of incubation, fewer vesicles containing NPs are colocalized with LysoTracker® Blue staining, suggesting that these vesicles are mostly no longer acidic. Presented figures are images of live cells. The scale bars correspond to 25 μm. Abbreviations: RITC, rhodamine B isothiocyanate; Co-ferrite, cobalt ferrite; NP, nanoparticle.
Figure 10
Figure 10
TEM micrograph of CHO cells incubated with Co-ferrite NPs for 24 hours. Big NP aggregates are mostly bound with membrane (small arrows) (A and B) and accumulated in the perinuclear region (A). Some membrane ruffling can be observed (big arrow), suggesting internalization (A). Note: The scale bar corresponds to 2 μm in (A) and to 0.5 μm in (B). Abbreviations: TEM, transmission electron microscopy; CHO, Chinese hamster ovary; Co-ferrite, cobalt ferrite; NP, nanoparticle.
Figure 11
Figure 11
Viability and proliferation of cells after a 24-hour exposure to NPs, determined with MTS and PI viability assay. Notes: Cells were incubated with increasing concentrations of functionalized NPs for 24 hours. The standard MTS viability assay was performed; for the PI viability assay, cells were incubated for 5 minutes with PI to detect dead cells. The results are presented as percentage of viable cells compared with the number of cells in the control sample (PI) or percentage viability determined with spectrofluorimetry (MTS). Mean and standard error are shown for three independent experiments. Abbreviations: NP, nanoparticle; MTS, MTS viability assay; PI, propidium iodide.

Similar articles

Cited by

References

    1. Stone V, Johnston H, Clift MJ. Air pollution, ultrafine and nanoparticle toxicology: cellular and molecular interactions. IEEE Trans Nanobioscience. 2007;6(4):331–340. - PubMed
    1. Oberdörster G, Oberdörster E, Oberdörster J. Nanotoxicology: An emerging discipline evolving from studies of ultrafine particles. Environ Health Perspect. 2005;113(7):823–839. - PMC - PubMed
    1. Salata O. Applications of nanoparticles in biology and medicine. J Nanobiotechnology. 2004;2(1):3. - PMC - PubMed
    1. Zhang L, Gu FX, Chan JM, Wang AZ, Langer RS, Farokhzad OC. Nanoparticles in medicine: therapeutic applications and developments. Clin Pharmacol Ther. 2008;83(5):761–769. - PubMed
    1. De M, Ghosh PS, Rotello VM. Applications of nanoparticles in biology. Adv Mater. 2008;20(22):4225–4241.

Publication types

MeSH terms

LinkOut - more resources