SCIENCE CHINA Materials, Volume 58, Issue 6: 467-480(2015) https://doi.org/10.1007/s40843-015-0059-9

Altering the response of intracellular reactive oxygen to magnetic nanoparticles using ultrasound and microbubbles

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  • ReceivedMay 9, 2015
  • AcceptedJun 5, 2015
  • PublishedJun 19, 2015


Engineered iron oxide magnetic nanoparticles (MNPs) are one of the most promising tools in nanomedicine-based diagnostics and therapy. However, increasing evidence suggests that their specific delivery efficiency and potential long-term cytotoxicity remain a great concern. In this study, using 12 nm γ-Fe2O3 MNPs, we investigated three types of uptake pathways for MNPs into HepG2 cells: (1) a conventional incubation endocytic pathway; (2) MNPs co-administrated with microbubbles under ultrasound exposure; and (3) ultrasound delivery of MNPs covalently coated on the surface of microbubbles. The delivery efficiency and intracellular distribution of MNPs were evaluated, and the cytotoxicity induced by reactive oxygen species (ROS) was studied in detail. The results show that MNPs can be delivered into the lysosomes via classical incubation endocytic internalization; however, microbubbles and ultrasound allow the MNPs to pass through the cell membrane and enter the cytosol via a non-internalizing uptake route much more evenly and efficiently. Further, these different delivery routes result in different ROS levels and antioxidant capacities, as well as intracellular glutathione peroxidase activity for HepG2 cells. Our data indicate that the microbubble–ultrasound treatment method can serve as an efficient cytosolic delivery strategy to minimize long-term cytotoxicity of MNPs.


This investigation was financially funded by the project of National Key Basic Research Program of China (2011CB933503 and 2013CB733804), the National Natural Science Foundation of China (NSFC) (31370019 and 61127002), Jiangsu Provincial Special Program of Medical Science (BL2013029). Partial funding also came from the Author of National Excellent Doctoral Dissertation of China (201259), as well as from the Fundamental Research Funds for the Central Universities.

Interest statement

The authors declare that they have no conflict of interest.

Contributions statement

Yang F, Zhang Y and Gu N developed the initial concept. Yang F, Li M and Chen Z designed the experiments. Yang F, Li M and Cui H performed the experiments and data analysis. Chen Z and Wang T cultured the cells. Song L and Zhang Y prepared and characterized the magnetic nanoparticles. Yang F, Li M, Cui H, Gu Z and Gu N co-wrote the manuscript. Yang F and Gu N supervised the study. All authors discussed the results and commented on the manuscript.

Author information

Fang Yang was born in 1979. She received her PhD degree in biomedical engineering from the School of Biological Science and Medical Engineering, Southeast University, Nanjing, China, in 2009. Currently, she works at the School of Biological Science and Medical Engineering, Southeast University. Her research interests mainly focus on ultrasound multi-modal imaging; ultrasound molecular imaging, and imaging (ultrasound, magnetic resonance, optical, CT, etc.) guided drug delivery system, etc.

Ning Gu was born in 1964. He received his PhD degree in biomedical engineering from the Department of Biomedical Engineering, Southeast University, Nanjing, China, in 1996. Currently he is a Changjiang Scholar Professor and NSFC Outstanding Young Investigator Fund Winner at the School of Biological Science and Medical Engineering, Southeast University. He also serves as the president of Jiangsu Society of Biomedical Engineering, the director of the Research Center for Nanoscale Science and Technology of Southeast University. His research interests include biomaterials, nanobiology, medical imaging, advanced instrument development, etc.


Supplementary information

Supplementary data include the efficiency of magnetic nanoparticles entering into the cells after different cellular uptake pathways; AFM scans of the outer cell membrane after ultrasound exposure with microbubbles; intracellular ROS level, activity of GPx, and T-AOC of HepG2 cells treated with only microbubbles, only ultrasound, and phosphate buffered saline, respectively; cell viability after different cellular uptake pathways. These materials are available free of charge via the online version of this paper


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  • Figure 1

    (a) TEM image of γ-Fe2O3 nanoparticles and (d) the mean hydrodynamic size of γ-Fe2O3. The microscopy images and mean size distribution of microbubbles with (b, e) unbound and (c, f) bound γ-Fe2O3 nanoparticles. Insets in (b, c) are the TEM images of microbubbles with unbound and bound γ-Fe2O3 nanoparticles, and the scale bar is 500 nm.

  • Figure 2

    (a) The statistic results of the amplitudes for the fundamental, second, and third harmonics. The γ-Fe2O3-coated microbubbles possess stronger (about 5 or 8 dB) second and third harmonics than the microbubbles without nanoparticles. (b) Disrupted oscillating microbubble detection vs. ultrasound exposure time for microbubbles with unbound and bound nanoparticles. (c) Cell viability after ultrasound treatment with microbubbles with unbound and bound nanoparticles.

  • Figure 3

    Microscopy and TEM images of γ-Fe2O3 nanoparticle distribution in the cells using different intracellular pathways. (a, d) Cell incubation with nanoparticles for 12 h. (b, e) Cells treated with microbubbles and free MNPs for 40 s ultrasound exposure. (c, f) Cells treated with MNP-coated microbubbles for 40 s ultrasound exposure. The images labeled (a1), (b1), (c1), (d1), (e1), and (f1) are the corresponding enlarged images. The results show that different uptake routes result in different nanoparticle localization and distribution within the cells.

  • Figure 4

    The intracellular ROS level produced by MNP uptake per living cell in HepG2 cells treated with different uptake pathways after (a) 0.5, 2, 6, and 12 h and (b) 24 h. Hydrogen peroxide (500 µM, 10 min) was used as a positive control in these experiments. The columns represent the mean ± standard deviation (SD) from at least three independent experiments. Significant differences were found with respect to the control group at *p< 0.05 and **p< 0.01.

  • Figure 5

    (a) The activity of GPx in HepG2 cells and (b) T-AOC of HepG2 cells treated with different MNP uptake pathways after 24 h. The columns represent the mean ± SD from at least three independent experiments per sample. Significant differences with respect to the control group were found at *p< 0.05 and **p< 0.01. Results are expressed as mU GPx per mg proteins for GPx activity and as mM trolox-equivalent antioxidant capacity per mg protein.

  • Figure 6

    Microscopy images of HepG2 cells after 24 h treated with MNPs using different delivery routes: (a) cells without any treatment, (b) cell incubation with MNPs, (c) MNPs delivered into the cells with the aid of microbubbles and ultrasound, and (d) MNPs loaded on the surface of microbubbles then delivered into the cells by ultrasound exposure. The lysosomes were stained with Lyso-Tracker Red. (e) The median intracellular red fluorescence intensity as a function of the particular MNP uptake pathway per living cell in HepG2 cells treated after 24 h. The columns represent the mean ± SD from at least three independent experiments per sample. Significant differences with respect to the control group were found at *p< 0.05. The scale bar is 10 µm.

  • Figure 7

    Schematic of different intracellular uptake pathways of MNPs: (a) incubation of MNPs with cells, which displays internalization and aggregation in lysosomes. (b) and (c) Sonoporation induced by ultrasound and microbubbles with unbound and bound nanoparticles, respectively, results in the MNPs accessing the cytosol of cells. The uniform distribution and neutral cytosolic microenvironment would decompose H2O2 into oxygen through catalase-like activity, which would avoid the long-term cytotoxicity in the intracellular therapy and imaging applications.

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