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. 2017 Dec 22:13:143-159.
doi: 10.2147/IJN.S148960. eCollection 2018.

Novel functionalized nanoparticles for tumor-targeting co-delivery of doxorubicin and siRNA to enhance cancer therapy

Yu Xia  1 , Tiantian Xu  1 , Changbing Wang  1 , Yinghua Li  1 , Zhengfang Lin  1 , Mingqi Zhao  1 , Bing Zhu  1
Affiliations

Novel functionalized nanoparticles for tumor-targeting co-delivery of doxorubicin and siRNA to enhance cancer therapy

Yu Xia et al. Int J Nanomedicine. .

Abstract

Human homeobox protein (Nanog) is highly expressed in most cancer cells and has gradually emerged as an excellent target in cancer therapy, owing to its regulation of cancer cell proliferation, metastasis and apoptosis. In this study, we prepared tumor-targeting functionalized selenium nanoparticles (RGDfC-SeNPs) to load chemotherapeutic doxorubicin (DOX) and Nanog siRNA. Herein, RGDfC peptide was used as a tumor-targeting moiety which could specifically bind to αvβ3 integrins overexpressed on various cancer cells. The sizes of RGDfC-SeNPs@DOX nanoparticles (~12 nm) were confirmed by both dynamic light scattering and transmission electron microscopy. The chemical structure of RGDfC-SeNPs@DOX was characterized via Fourier-transform infrared spectroscopy. The RGDfC-SeNPs@DOX was compacted with siRNA (anti-Nanog) by electrostatic interaction to fabricate the RGDfC-SeNPs@DOX/siRNA complex. The RGDfC-SeNPs@DOX/siRNA complex nanoparticles could efficiently enter into HepG2 cells via clathrin-associated endocytosis, and showed high gene transfection efficiency that resulted in enhanced gene silencing. The in vivo biodistribution experiment indicated that RGDfC-SeNPs@DOX/siRNA nanoparticles were capable of specifically accumulating in the tumor site. Furthermore, treatment with RGDfC-SeNPs@DOX/siRNA resulted in a more significant anticancer activity than the free DOX, RGDfC-SeNPs@DOX or RGDfC-SeNPs/siRNA in vitro and in vivo. In summary, this study shows a novel type of DOX and siRNA co-delivery system, thereby providing an alternative route for cancer treatment.

Keywords: Nanog siRNA; doxorubicin; drug delivery; nanoparticles; tumor targeting.

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Conflict of interest statement

Disclosure The authors report no conflicts of interest in this work.

Figures

Figure 1
Figure 1
(A) TEM image of RGDfC-SeNPs@DOX. (B) The magnified TEM image of RGDfC-SeNPs@DOX. (C) Size distributions of RGDfC-SeNPs@DOX. (D) EDX analysis of RGDfC-SeNPs@DOX. (E) FTIR spectra of SeNPs, RGDfC, DOX and RGDfC-SeNPs@DOX. Abbreviations: DOX, doxorubicin; EDX, energy-dispersive X-ray spectroscopy; FTIR, Fourier-transform infrared spectroscopy; RGDfC, Arg-Gly-Asp-D-Phe-Cys peptide; SeNPs, selenium nanoparticles; TEM, transmission electron microscopy.
Figure 2
Figure 2
(A) The binding between RGDfC-SeNPs@DOX and siRNA was examined by agarose gel electrophoresis at various N/P ratios. (B) In vitro pH-triggered release of DOX from the RGDfC-SeNPs@DOX/siRNA nanoparticles. Abbreviations: DOX, doxorubicin; N, nitrogen; NPs@DOX/siRNA, RGDfC-SeNPs@DOX/siRNA; P, phosphorus; RGDfC, Arg-Gly-Asp-D-Phe-Cys peptide; SeNPs, selenium nanoparticles.
Figure 3
Figure 3
(A) Cellular uptake of RGDfC-SeNPs@DOX/siRNA nanoparticles was visualized by red fluorescence from DOX in HepG2 cells. (B) TEM image showed the internalization of RGDfC-SeNPs and RGDfC-SeNPs@DOX/siRNA nanoparticles after 8 h of incubation in HepG2 cells. (C) Effects of endocytosis inhibitors and temperature on the internalization of RGDfC-SeNPs@DOX/siRNA. (D) The analyses of cells after treatment with naked FAM-siRNA, Lipofectamine 2000/FAM-siRNA and RGDfC-SeNPs@DOX/FAM-siRNA for 24 h were performed by flow cytometry. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; DOG, 2-deoxy-D-glucose; DOX, doxorubicin; FAM-siRNA, Lipofectamine 2000/FAM-siRNA; Lipo, Lipofectamine 2000; NPs@DOX/FAM-siRNA, RGDfC-SeNPs@DOX/FAM-siRNA; NPs@DOX/siRNA, RGDfC-SeNPs@DOX/siRNA; NPs, nanoparticles; PBS, phosphate buffer saline; RGDfC, Arg-Gly-Asp-D-Phe-Cys peptide; SeNPs, selenium nanoparticles; TEM, transmission electron microscopy.
Figure 4
Figure 4
(A) Inhibition of Nanog mRNA expression levels was quantified by qPCR. **p<0.01, vs the control group. (B) The Nanog expression levels of HepG2 cells treated with RGDfC-SeNPs@DOX/siNC, naked siRNA, RGDfC-SeNPs@DOX/siRNA and Lipofectamine 2000/siRNA were evaluated by Western blotting. Abbreviations: DOX, doxorubicin; Lipo, Lipofectamine 2000; NPs@DOX/siNC, RGDfC-SeNPs@DOX/siNC; NPs@DOX/siRNA, RGDfC-SeNPs@DOX/siRNA; NPs, nanoparticles; qPCR, quantitative real-time PCR; RGDfC, Arg-Gly-Asp-D-Phe-Cys peptide; SeNPs, selenium nanoparticles; siNC, negative control siRNA.
Figure 5
Figure 5
(A) In vitro cytotoxicity of RGDfC-SeNPs/siNC, naked siRNA, RGDfC-SeNPs/siRNA and Lipofectamine 2000/siRNA on HepG2 cells. **p<0.01 vs the untreated group. (B) In vitro cytotoxicity of free DOX, RGDfC-SeNPs@DOX and RGDfC-SeNPs@DOX/siRNA on HepG2 cells. **p<0.01 vs the untreated group. Abbreviations: DOX, doxorubicin; Lipo, Lipofectamine 2000; NPs@DOX, RGDfC-SeNPs@DOX; NPs@DOX/siRNA, RGDfC-SeNPs@DOX/siRNA; NPs/siNC, RGDfC-SeNPs/siNC; NPs/siRNA, RGDfC-SeNPs/siRNA; NPs, nanoparticles; RGDfC, Arg-Gly-Asp-D-Phe-Cys peptide; SeNPs, selenium nanoparticles; siNC, negative control siRNA.
Figure 6
Figure 6
(A) The scratching width was observed at 24 h after the treatment with free DOX, RGDfC-SeNPs@DOX, RGDfC-SeNPs/siRNA and RGDfC-SeNPs@DOX/siRNA. The scale bars are 200 μm. (B) The cell motility of (a) control, (b) free DOX, (c) RGDfC-SeNPs@DOX, (d) RGDfC-SeNPs/siRNA and (e) RGDfC-SeNPs@DOX/siRNA groups was quantitatively analyzed. (C) The effect of free DOX, RGDfC-SeNPs@DOX, RGDfC-SeNPs/siRNA and RGDfC-SeNPs@DOX/siRNA on the migration of HepG2 cells. The scale bar is 200 μm. (D) The inhibition rate of cell migration of (a) control, (b) free DOX, (c) RGDfC-SeNPs@DOX, (d) RGDfC-SeNPs/siRNA and (e) RGDfC-SeNPs@DOX/siRNA was quantitatively analyzed. Abbreviations: DOX, doxorubicin; NPs, nanoparticles; NPs@DOX, RGDfC-SeNPs@DOX; NPs@DOX/siRNA, RGDfC-SeNPs@DOX/siRNA; NPs/siRNA, RGDfC-SeNPs/siRNA; RGDfC, Arg-Gly-Asp-D-Phe-Cys peptide; SeNPs, selenium nanoparticles.
Figure 7
Figure 7
(A) Effects of free DOX, RGDfC-SeNPs@DOX, RGDfC-SeNPs/siRNA and RGDfC-SeNPs@DOX/siRNA on cell apoptosis and cell cycle distribution in HepG2 cells. (B) Effects of free DOX. Abbreviations: DOX, doxorubicin; NPs, nanoparticles; RGDfC-SeNPs@DOX, RGDfC-SeNPs/siRNA and RGDfC-SeNPs@DOX/siRNA on cell apoptosis in HepG2 cells. NPs@DOX, RGDfC-SeNPs@DOX; NPs@DOX/siRNA, RGDfC-SeNPs@DOX/siRNA; NPs/siRNA, RGDfC-SeNPs/siRNA; RGDfC, Arg-Gly-Asp-D-Phe-Cys peptide; SeNPs, selenium nanoparticles.
Figure 8
Figure 8
Effects of RGDfC-SeNPs@DOX/siRNA on protein expression levels of MMP2, MMP9, caspase-3 and PARP in HepG2 cells. Abbreviations: DOX, doxorubicin; MMP2, matrix metalloproteinase-2; MMP9, matrix metalloproteinase-9; RGDfC, Arg-Gly-Asp-D-Phe-Cys peptide; SeNPs, selenium nanoparticles.
Figure 9
Figure 9
In vivo fluorescence imaging of tumor-bearing mice, excised tumor and organs after administration of RGDfC-SeNPs@DOX/cy5.5-siRNA at 4 h. Abbreviations: DOX, doxorubicin; NPs, nanoparticles; NPs@DOX/siRNA, RGDfC-SeNPs@DOX/cy5.5-siRNA; RGDfC, Arg-Gly-Asp-D-Phe-Cys peptide; SeNPs, selenium nanoparticles; T, tumor; H, heart; L, liver; S, spleen; Lu, lung; K, kidney.
Figure 10
Figure 10
(A) The body weight change of tumor-bearing mice receiving different therapy. (B) Tumor growth inhibition of tumor-bearing mice after treatment with saline, free DOX, RGDfC-SeNPs@DOX, RGDfC-SeNPs/siRNA and RGDfC-SeNPs@DOX/siRNA. (C) Photograph of tumors stripped from mice administered intravenously with saline, free DOX, RGDfC-SeNPs@DOX, RGDfC-SeNPs/siRNA and RGDfC-SeNPs@DOX/siRNA, respectively. (D) H&E and immunohistochemical analysis of the tumor tissues from tumor-bearing mice after treatment with saline or RGDfC-SeNPs@DOX/siRNA. *p<0.05 and **p<0.01, vs the saline group. Magnification ×400. Abbreviations: DOX, doxorubicin; NPs, nanoparticles; NPs@DOX, RGDfC-SeNPs@DOX; NPs@DOX/siRNA, RGDfC-SeNPs@DOX/siRNA; NPs/siRNA, RGDfC-SeNPs/siRNA; RGDfC, Arg-Gly-Asp-D-Phe-Cys peptide; SeNPs, selenium nanoparticles; H&E, hematoxylin and eosin.
Scheme 1
Scheme 1
Schematic illustration of the formation of RGDfC-SeNPs@DOX/siRNA. Abbreviations: DOX, doxorubicin; RGDfC, Arg-Gly-Asp-D-Phe-Cys peptide; SeNPs, selenium nanoparticles; Vc, ascorbic acid.
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