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A highly efficient and tumor vascular-targeting therapeutic technique with size-expansible gadofullerene nanocrystals

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  • ReceivedSep 19, 2015
  • AcceptedSep 20, 2015
  • PublishedSep 30, 2015

Abstract

It has long been a dream to achieve tumor targeting therapy that can efficiently reduce the toxicity and severe side effects of conventional antitumor chemotherapeutic agents. Taking advantage of the abnormalities of tumor vasculature, we demonstrate here a new powerful tumor vascular-targeting therapeutic technique for solid cancers that applies advanced nanotechnology to cut off the nutrient supply of tumor cells by physically destroying the abnormal tumor blood vessels. Water soluble magnetic Gd@C82 nanocrystals of the chosen sizes are deliberately designed with abilities to penetrate into the leaky tumor blood vessels. By triggering the radiofrequency induced phase transition of gadofullerene nanocrystals while extravasating the tumor blood vessel, the explosive structural change of nanoparticles generates a devastating impact on abnormal tumor blood vessels, resulting in a rapid and extensive ischemia necrosis and shrinkage of the tumors. This unprecedented target-specific physiotherapy is found to work perfectly for advanced and refractory solid tumors.


Acknowledgment

This work was supported by the National Natural Science Foundation of China (51472248, 11179006 and 51372251) and the Key Research Program of the Chinese Academy of Sciences (KGZD-EW-T02 and XDA09030302). We thank Prof. Yan Li of Zhongnan Hospital of Wuhan University for help with tissues histology and biochemical analysis. We also thank Yongtao Li, Zhentao Zuo, and Yuqing Wang for developing the RF set-ups.


Interest statement

The authors declare that they have no conflict of interest.


Contributions statement

Wang C and Zhen M supervised the project and designed the experiments. Li J synthesized the GFNCs. Zhen M and Zhang G performed the in vivo experiments. Zhen M, Deng R and Zou T performed the in vitro characterization. Shu C and Wang T analyzed the nature of GFNCs. Fang F and Lei H performed the animal MRI studies. Wang C, Bai C and Luo Y contributed to the interpretation of the data. Zhen M, Luo Y and Wang C wrote the manuscript.


Author information

Mingming Zhen was born in 1987. She received her PhD degree in physical chemistry from the Institute of Chemistry, Chinese Academy of Sciences (ICCAS) in 2014. Currently, she is an assistant professor at ICCAS. Her research interests include biomedical applications of fullerenes and gadofullerenes.


Chunru Wang was born in 1965. He received his PhD degree in physical chemistry from Dalian Institute of Chemistry Physics, Chinese Academy of Sciences in 1992. Currently, he is a professor at ICCAS. His research interests include fullerenes and endohedral fullerenes, mainly focusing on their industrialization and applications. He discovered the metal carbide fullerenes for the first time, researched on high efficiency MRI contrast agents and developed a novel tumor vascular-targeting therapy technique using gadofullerenes.


Supplement

Supplementary information

Supporting data are available in the online version of the paper.


References

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  • Figure 1

    Characterizations of the GFNCs and the size expansion after RF irradiation. (a) As-synthesized GFNCs aqueous solution and the schematic diagram of functionalized GFNCs by hydroxylation with H2O2 in alkaline. (b) Cryo-TEM micrograph of GFNCs. The inset is the SAED pattern, in which the Debye ring demonstrates the crystalline form of GFNCs. (c) Powder DSC measurement for bulk Gd@C82 with the phase transition temperature at ca. 344 K (71°C). (d) Solution DSC measurement for GFNCs with 1 mmol L−1 Gd3+ concentration with the phase transition temperature at ca. 354 K (81°C). (e) The magnetization vs. field (M-H) curve of GFNCs. No hysteresis loop exists in the curve, indicating the superparamagnetism of the particles. (f) Average hydrodynamic diameter of GFNCs increases from 136 (±7.1) to 204 (±10.6) nm under the radiation of 200 MHz RF for 30 min.

  • Figure 2

    The dynamical MRI of tumor by 90 min RF-assisted GFNCs treatments. (a) The MR images of liver tumor-bearing mice pre-treatment. (b) The interception MRI of tumor area. (c) The pseudo MR images were analyzed using data software (Mricron). (d) The quantitative ratio of hypointensity volume (H) in the whole tumor volume (W).

  • Figure 3

    The evidences of excluding the hyperthermia effects. (a) Temperature changes of 0.9% NaCl, 1 mmol L−1 Gd-DTPA and 1 mmol L−1 GFNCs solution exposed under 200 MHz RF. (b and c) The temperature changes of tumor tissues monitored by an optical fiber temperature sensor during the treatment. (d–f) The cell viabilities of three kinds of cells incubated with GFNCs pre- and post-RF irradiations: (d) mice hepatic cancer cell H22; (e) human hepatic cancer line HepG2; (f) mice embryonic liver cell BNL (normal cells). The cells were incubated with 10–50 µmol L−1 GFNCs. The results indicate that GFNCs exhibit nontoxicity in vitro after RF irradiation.

  • Figure 4

    The observation of tumor blood vessels. (a–c) ESEM microphotography of tumor blood vessels treated (a) without and (b, c) with RF-assisted GFNCs. The endothelial cells were chipped off from the basilemma at 24 h after the treatment. (d–g) The MRA images of tumor vascular (yellow arrows) for (d) pre- and (e) 1.5 h post of control groups, and (f) pre- and (g) 1.5 h post RF-assisted GFNCs treatments.

  • Figure 5

    A schematic draw of the antitumor mechanism of RF-assisted GFNCs treatments. The GFNCs are first accumulated in the lining of the tumor blood vessels due to EPR effect, and then the sudden volume expansion and the rotation of the RF-assisted GFNCs induce the exfoliation of endothelia cells.

  • Figure 6

    Antitumor effects in vivo for liver cancer by RF-assisted GFNCs treatments. (a) The outline of tumor treatment procedure. (b) T1-weighted MRI of tumors (marked by the dotted circle) at pre- and post- injection of GFNCs, where the commercial Gd-DTPA was used as control. (c) Photographs of nude mice xenografted with H22 liver cancer after GFNCs and RF treatments. (d) The tumors change color from the center to the edge by ischemia and shrank dramatically after 24 h treatment, while the control group shows no change at all. (e) The images of long-term observation of tumor at 15 d after treatments. The tumors were entirely necrosis only remaining a crustlike scab compared with the control groups with huge tumor mass. (f) Liver tumor H&E staining of control group and therapy group one week after the therapy. The tumors treated with RF-assisted GFNCs were hemorrhagic necrosis due to the disrupting of tumor vasculature (×100 magnification), whereas the control groups are with intact blood vessel (as arrows indicated) and proliferous tumor cells.

  • Figure 7

    Anticancer therapy in 4T1 cancer model observed by bioluminescence. (a) Integrated bioluminescence emission in 10 min post of the injection of D-luciferin of breast cancer-bearing nude mice with RF-assisted GFNCs treatments using saline as control (yellow arrows labeled tumors). (b) Breast tumor relative growth of therapy group and that of control group with saline, characterized quantitatively by the intensity of bioluminescent of tumor cells. (c) Breast tumor H&E staining of control group and therapy group one week after the therapy (×200 magnification).

  • Figure 8

    The biodistribution of GFNCs in vivo. GFNCs were IV injected into H22 liver tumor-bearing mice and measured by 131I radiolabeled method, %ID/g ± standard deviation, n = 5.

  • Figure 9

    Tissue histology and biochemical analysis. (a) Brain, heart, liver, spleen, lung and kidney are collected from liver tumor-bearing nude mice one week after the treatment, showing negligible toxicities to the normal tissues, ×200 magnification. (b) Serum ALT, AST, BUN, TBIL, Cr and UA levels in normal nude mice and liver tumor-bearing nude mice associated with RF-assisted GFNCs treatments.

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