CD44v6-O-MWNTS-Loaded Gemcitabine and CXCR4 siRNA Improves the Anti-tumor Effectiveness of Ovarian Cancer


Abstract

Ovarian cancer is one of the most common malignancies of the female reproductive system and the deadliest gynecologic cancer. CXCR4 is expressed in a variety of malignant tumors such as breast, prostate, and ovarian cancers. It is also closely related to the migration, invasion, and metastasis of tumor cells. Carbon nanotubes have great potential for targeted therapy of tumors. CD44v6 is not expressed in normal ovarian tissues but is highly expressed in ovarian epithelial carcinoma. In the present study, we applied small interfering RNA targeting the CXCR4 gene and the clinical treatment gemcitabine and oxaliplatin of ovarian cancer as the therapeutic drug, and organically integrated chemotherapy and gene therapy through carbon nanotubes, and used CD44v6 single chain antibody as the targeting moiety to explore its application in ovarian cancer treatment. Significantly, we successfully synthesized CD44v6-O-MWNTS/Gemcitabine/1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)/siRNA system and the results were observed by transmission electron microscope (TEM) and scanning electron microscope (SEM). CD44v6-O-MWNTS/Gemcitabine/DOTAP was able to fully load siRNA at the ratio of 1:2.5. The carbon nanotubes could protect the siRNA. The drug release analysis showed that O-MWNTS/drug/DOTAP/siRNA was able to effectively release the siRNA, and gemcitabine or oxaliplatin in a time-dependent manner. O-MWNTS/drug/DOTAP/siRNA was able to be effectively uptake by ovarian cancer cells. The cellular uptake of CD44v6-O-MWNTS/drug/DOTAP/siRNA mainly depends on lipid raft-mediated endocytosis. CD44v6-O-MWNTS/drug/DOTAP/siRNA improved the effect of siRNA on the inhibition of ovarian cancer cell viability and the induction of cell apoptosis. The expression of CXCR4 was decreased by CD44v6-O-MWNTS/drug/DOTAP/siRNA in ovarian cancer cells. Tumorigenicity analysis in nude mice showed that CD44v6-O-MWNTS/drug/DOTAP/siRNA significantly repressed the tumor growth of ovarian cancer cells in vivo. The levels of Ki-67 and CXCR4 were repressed by CD44v6-O-MWNTS/drug/DOTAP/siRNA in the system. Thus, we concluded that the obtained CD44v6-O-MWNTS could effectively load gemcitabine or oxaliplatin, and CXCR4 siRNA, internalized by cancer cells and realized notable in vitro and in vivo inhibitory function against ovarian cancer growth. Our study provides a promising nanomaterial for the co-delivery of siRNA and anti-tumor drugs for the therapy of ovarian cancer.

Keywords: CD44v6; CXCR4; MWNTs; gemcitabine; ovarian cancer.

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
CD44v6-O-MWNTS/Gemcitabine/DOTAP preparation and characterization. The scanning electron microscope (SEM) (left) image and transmission electron microscope (TEM) (right) image of CD44v6-O-MWNTS/Gemcitabine/DOTAP.
FIGURE 2
FIGURE 2
Assessment of siRNA-loading capacity and release. (A) The siRNA loading ability under diverse ratio of siRNA and CD44v6-O-MWNTS/Gemcitabine/DOTAP was detected by agarose gel electrophoresis. (B,C) The stability of CD44v6-O-MWNTS/Gemcitabine/DOTAP/siRNA in RNase A (B) and serum (C) was analyzed by agarose gel electrophoresis. (D) The drug release analysis was performed at pH 6.5 and pH 7.4. Release of gemcitabine and siRNA was detected by ultraviolet spectrophotometer. O/Gem/DO: O-MWNTS/Gemcitabine/DOTAP; O/Gem/DO/si: O-MWNTS/Gemcitabine/DOTAP/siRNA.
FIGURE 3
FIGURE 3
The cellular uptake of CD44v6-O-MWNTS/Gemcitabine/DOTAP/siRNA by SW626. (A,B) The cellular uptake of CD44v6-O-MWNTS/Gemcitabine/DOTAP/siRNA was analyzed by confocal fluorescence microscope (A) and flow cytometry (B) in SW626 cells. Fluorescence probe C6 was used to replace gemcitabine in CD44v6-O-MWNTS/C6/DOTAP/siRNA system for localization, and siRNA was labeled by Cy6 fluorescence probe. Five random areas were captured. (C) Cells were treated with low temperature, chlorpromazine hydrochloride [inhibitor of lattice (clathrin)-mediated endocytosis] or MβCD and dynasore [inhibitor of lipid-raft (lipid-raft)-mediated endocytosis]. The cellular uptake mechanism of CD44v6-O-MWNTS/Gemcitabine/DOTAP/siRNA was examined by intracellular drug concentration. **P < 0.01.
FIGURE 4
FIGURE 4
The effect of CD44v6-O-MWNTS/Gemcitabine/DOTAP/siRNA on SW626 cancer cell proliferation and apoptosis in vitro. (A–E) The SW626 cells were treated as the indicated labeling. (A) The cell viability was analyzed by MTT assays. (B,C) The cell proliferation was measured by colony formation assays. (D) The expression of Bax, Bid, Bim, Bcl-2, cleaved caspase-9 (c-caspase-9) was measured by Western blot analysis. (F) The expression of CXCR4 was detected by Western blot analysis. O/Gem/DO: O-MWNTS/Gemcitabine/DOTAP; O/Gem/DO/si: O-MWNTS/Gemcitabine/DOTAP/siRNA; 44-O/Gem/DO/si: CD44v6-O-MWNTS/Gemcitabine/DOTAP/siRNA. Data are presented as mean ± SD. Statistic significant differences were indicated: *P < 0.05, **P < 0.01.
FIGURE 5
FIGURE 5
The effect of CD44v6-O-MWNTS/Gemcitabine/DOTAP/siRNA on ovarian cancer cell growth in vivo. (A–E) The nude mice were injected with SW626 cells and were treated as the indicated labeling. (A) The body weight was remarketed. (B) HE staining of kidney, liver, and spleen. (C) The tumor growth curve was shown. (D) The tumor weight was shown. (E) The tumor size was shown. (F) The levels of Ki-67 and CXCR4 were measured by IHC. Gem: gemcitabine; O/Gem/DO/si: O-MWNTS/Gemcitabine/DOTAP/siRNA; 44-O/Gem/DO/si: CD44v6-O-MWNTS/Gemcitabine/DOTAP/siRNA. Data are presented as mean ± SD. Statistic significant differences were indicated: *P < 0.05, **P < 0.01.
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