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. 2013:8:1551-62.
doi: 10.2147/IJN.S40011. Epub 2013 Apr 19.

Photodynamic therapy of a 2-methoxyestradiol tumor-targeting drug delivery system mediated by Asn-Gly-Arg in breast cancer

Jinjin Shi  1 , Zhenzhen Wang, Lei Wang, Honghong Wang, Lulu Li, Xiaoyuan Yu, Jing Zhang, Rou Ma, Zhenzhong Zhang
Affiliations

Photodynamic therapy of a 2-methoxyestradiol tumor-targeting drug delivery system mediated by Asn-Gly-Arg in breast cancer

Jinjin Shi et al. Int J Nanomedicine. 2013.

Abstract

Fullerene (C60) has shown great potential in drug delivery. In this study we exploited modified fullerene (diadduct malonic acid-fullerene-Asn-Gly-Arg peptide [DMA-C60-NGR]) as an antitumor drug carrier in order to build a new tumor-targeting drug delivery system. We also investigated the synergistic enhancement of cancer therapy using photodynamic therapy (PDT) induced by DMA-C60-NGR and 2-methoxyestradiol (2ME). Cytotoxicity tests indicated that DMA-C60-NGR had no obvious toxicity, while our drug delivery system (DMA-C60-2ME-NGR) had a high inhibition effect on MCF-7 cells compared to free 2ME. The tumor-targeting drug delivery system could efficiently cross cell membranes, and illumination induced the generation of intracellular reactive oxygen species and DNA damage. Furthermore, DMA-C60-2ME-NGR with irradiation had the highest inhibition effect on MCF-7 cells compared to the other groups. DMA-C60-NGR combined with 2ME showed a good synergistic photosensitization effect for inhibiting the growth of MCF-7 cells, demonstrating that DMA-C60-2ME-NGR may be promising for high treatment efficacy with minimal side effects in future therapy.

Keywords: drug delivery system; fullerene; photodynamic therapy; tumor targeting.

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Figures

Figure 1
Figure 1
Scheme of DMA-C60-2ME-NGR. Abbreviations: DSPE-PEG, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyet hylene glycol)-2000]; NGR, (Asn-Gly-Arg) peptide; 2ME, 2-methoxyestradiol.
Figure 2
Figure 2
Scheme of preparation of DMA-C60. Abbreviation: DPM, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[metho xy(polyethylene glycol)-2000]-maleimide; DMA-C60, diadduct malonate derivative of C60; NGR, (Asn-Gly-Arg) peptide.
Figure 3
Figure 3
FT-IR spectrum. Notes: Upper, pristine C60; lower, DMA-C60. Abbreviation: DMA-C60, diadduct malonate derivative of C60.
Figure 4
Figure 4
Characterization of DMA-C60-NGR. (A) Photos of DMA-C60-NGR in PBS buffer; (B) thin layer silica gel chromatography image of 1-(NGR), 2-(NGR mechanical mixing with the DMA-C60), and 3-(DMA-C60-NGR); (C) size and zeta potential of DMA-C60-NGR; (D) AFM images of DMA-C60-NGR. Abbreviations: DMA-C60, diadduct malonate derivative of C60; NGR, (Asn-Gly-Arg) peptide.
Figure 5
Figure 5
Characterization of DMA-C60-NGR-2ME. (A) Photos of DMA-C60-NGR-2ME in water; (B) UV spectrum of DMA-C60-NGR-2ME, DMA-C60-NGR and 2ME. Abbreviations: DMA-C60, diadduct malonate derivative of C60; NGR, Asn-Gly-Arg peptide; 2ME, 2-methoxyestradiol.
Figure 6
Figure 6
Characterization of DMA-C60-2ME-NGR. (A) Size of DMA-C60-2ME-NGR; (B) zeta potential of DMA-C60-2ME-NGR; and (C) AFM images of DMA-C60-2ME-NGR. Abbreviations: DMA-C60, diadduct malonate derivative of C60; NGR, Asn-Gly-Arg peptide; 2ME, 2-methoxyestradiol.
Figure 7
Figure 7
Viability of MCF-7 cells exposed to visible light for 1 hour after incubation for 24 hours. Note: Data are presented as mean ± standard deviation (n = 3). Abbreviations: DMA-C60, diadduct malonate derivative of C60; NGR, Asn-Gly-Arg peptide.
Figure 8
Figure 8
Fluorescence microscopic images of MCF-7 cells incubated with intracellular ROS probe H2DCFDA and illuminated with 532 nm laser for 5 minutes. (A) H2DCFDA without DMA-C60-NGR; (B) DMA-C60-NGR (5 μg/mL) + H2DCFDA. Abbreviations: DMA-C60v diadduct malonate derivative of C60; NGR Asn-Gly-Arg peptide H2DCFDA 5-(and-6)-chloromethyl-2′ -7′ -dichlorodihydrofluoresceindiacetate.
Figure 9
Figure 9
Images of single cell gel electrophoresis for DNA damage. (A) Untreated cells; (B) cells exposed to visible light alone for 1 hour; (C) cells treated with 5 μg/mL of DMA-C60-NGR alone for 24 hours; and (D) cells treated with 5 μg/mL of DMA-C60-NGR for 24 hours and then exposed to visible light for 1 hour. Abbreviations: DMA-C60, diadduct malonate derivative of C60; NGR, Asn-Gly-Arg peptide.
Figure 10
Figure 10
Fluorescence photomicrographs of MCF-7 cells stained with Hoechst-33258. (A) Untreated cells; (B) cells exposed to visible light alone for 1 hour; (C) cells treated with 5 μg/mL of DMA-C60-NGR alone for 24 hours; and (D) cells treated with 5 μg/mL of DMA-C60-NGR for 24 hours and then exposed to visible light for 1 hour. Abbreviations: DMA-C60, diadduct malonate derivative of C60; NGR, Asn-Gly-Arg peptide.
Figure 11
Figure 11
Fluorescence microscopic images of MCF-7 cells. (A) Control group; (B) FITC alone; (C) DMA-C60-FITC; and (D) DMA-C60-NGR-FITC at 1, 2, and 3 hours. Abbreviations: DMA-C60, diadduct malonate derivative of C60; NGR, Asn-Gly-Arg peptide; FITC, fluorescein isothiocyanate.
Figure 12
Figure 12
Cytotoxicity of DMA-C60-NGR and DMA-C60 on MCF-7 cells for 72 hours. Note: Data are presented as mean ± standard deviation (n = 3). Abbreviations: DMA-C60, diadduct malonate derivative of C60; NGR, Asn-Gly-Arg peptide.
Figure 13
Figure 13
Inhibition efficacy of 2ME, DMA-C60–2ME, and DMA-C60-2ME-NGR on MCF-7 cells. Notes: Upper panel, 24 hours; middle panel, 48 hours; lower panel, 72 hours. Data are presented as mean ± standard deviation (n = 3). Abbreviations: DMA-C60, diadduct malonate derivative of C60; NGR, Asn-Gly-Arg peptide; 2ME: 2-methoxyestradiol.
Figure 14
Figure 14
Inhibition efficacy of DMA-C60-2ME-NGR and DMA-C60-2ME-NGR-irradiated group on MCF-7 cells for 24 hours. Note: Data are presented as mean ± standard deviation (n = 3). Abbreviations: DMA-C60, diadduct malonate derivative of C60; NGR, Asn-Gly-Arg peptide; 2ME: 2-methoxyestradiol.
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