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. 2020 Aug 7:15:5701-5718.
doi: 10.2147/IJN.S257527. eCollection 2020.

CXCL12/CXCR4 Axis-Targeted Dual-Functional Nano-Drug Delivery System Against Ovarian Cancer

Jiyang Xue #  1 , Ruixiang Li #  2 , Dingding Gao  2 , Fenghua Chen  3 , Hongjuan Xie  1
Affiliations

CXCL12/CXCR4 Axis-Targeted Dual-Functional Nano-Drug Delivery System Against Ovarian Cancer

Jiyang Xue et al. Int J Nanomedicine. .

Abstract

Introduction: Traditional chemotherapy for ovarian cancer is limited due to drug resistance and systemic side effects. Although various targeted drug delivery strategies have been designed to enhance drug accumulation at the tumor site, simply improvement of targeting capability has not consistently led to satisfactory outcomes. Herein, AMD3100 was selected as the targeting ligand because of its high affinity to chemokine receptor 4 (CXCR4), which was highly expressed on ovarian cancer cells. Moreover, the AMD3100 has been proved having blockage capability of stromal cell-derived factor 1 (SDF-1 or CXCL12)/CXCR4 axis and to be a sensitizer of chemotherapeutic therapy. We designed a dual-functional targeting delivery system by modifying paclitaxel (PTX)-loaded PEGylation bovine serum albumin (BSA) nanoparticles (NPs) with AMD3100 (AMD-NP-PTX), which can not only achieve specific tumor-targeting efficiency but also enhance the therapeutic outcomes.

Methods: AMD3100 was chemically modified to Mal-PEG-NHS followed by reacting with BSA, then AMD-NP-PTX was synthesized and characterized. The targeting efficiency of AMD-NP was evaluated both in vitro and in vivo. The anticancer effect of AMD-NP-PTX was determined on Caov3 cells and ovarian cancer-bearing nude mice. Finally, the potential therapeutic mechanism was studied.

Results: AMD-NP-PTX was synthesized successfully and well characterized. Cellular uptake assay and in vivo imaging experiments demonstrated that NPs could be internalized by Caov3 cells more efficiently after modification of AMD3100. Furthermore, the AMD-NP-PTX exhibited significantly enhanced inhibition effect on tumor growth and metastasis compared with PTX, NP-PTX and free AMD3100 plus NP-PTX both in vitro and in vivo, and demonstrated improved safety profile. We also confirmed that AMD-NP-PTX worked through targeting CXCL12/CXCR4 axis, thereby disturbing its downstream signaling pathways including epithelial-mesenchymal transition (EMT) processes and nuclear factor κB (NF-κB) pathway.

Conclusion: The AMD-NP-PTX we designed would open a new avenue for dual-functional NPs in ovarian cancer therapy.

Keywords: AMD3100; CXCL12/CXCR4 axis; nanoparticle; ovarian cancer; paclitaxel.

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Conflict of interest statement

The authors report no conflicts of interest in this work.

Figures

Figure 1
Figure 1
Schematic diagram of AMD-NP-PTX preparation process.
Figure 2
Figure 2
NMR H spectra of Mal-PEG-NHS, AMD-PEG-NHS and NANO (AMD-NP-PTX). (a) – aromatic phenylene protons of AMD3100. (b) – methylene protons of PEG. (c) – Maleimide group of Mal-PEG-NHS.
Figure 3
Figure 3
Characterization of AMD-NP-PTX (A) Size distributions and Zeta potential of AMD-NP-PTX. (B) TEM characterization of AMD-NP-PTX. Scale bar=100 nm. (C) In vitro drug release curve of PTX solution and AMD-NP-PTX (pH 7.4). Data are presented as the mean±SD (n=3). (D) In vitro drug release profile of AMD-NP-PTX under different pH conditions (pH 6.8 and 7.4). Data are presented as the mean±SD (n=3). *p<0.05, **p<0.01 (significant difference between the two groups).
Figure 4
Figure 4
(A) Laser confocal assay was used to evaluate the uptake of Bodipy-labeled NP and AMD-NP by Caov3 cells and CHO cells in vitro. Blue represents the nucleus (DAPI) and red represents the fluorescent (Bodipy) labeled NPs. Scale bar=25 μm. (B) Competitive cell uptake study was performed by pre-addition of free AMD3100 with different concentrations using flow cytometry. (C) Quantitative analysis of flow cytometry. Data are presented as the mean±SD (n=3). **p<0.01.
Figure 5
Figure 5
The tumor accumulation study in vivo. (A) The whole-body imaging of Caov3 tumor-bearing mice at 1 h, 3 h, 6 h and 24 h after intravenous injection of Bodipy-labeled NP, AMD-NP. (B) Tissue distribution of Bodipy-labeled NP, AMD-NP imaged 24 h post-injection. (C) Quantitative analysis of Bodipy signal from NP and AMD-NP in different organs. Data are presented as the mean±SD (n=3). **p<0.01 compared with NP-PTX group.
Figure 6
Figure 6
Effects of AMD-NP-PTX on proliferation, apoptosis, and migration of Caov3 ovarian cancer cells (A) Cell viability. Data are presented as the mean±SD (n=3). (B) The histogram comparing the cell apoptosis in 5 groups. Data are presented as the mean±SD (n=3). (C) Cell apoptosis mapping measured by flow cytometry. (D) Images of Transwell migration results. Scale bar=200 μm. (E) Number of migrated cells among 6 groups. Data are presented as the mean±SD (n=3). #p<0.05, ##p<0.01 (significant difference compared with the control group); *p<0.05, **p<0.01 (significant difference between different treatment groups).
Figure 7
Figure 7
In vivo anticancer effect of AMD-NP-PTX on ovarian cancer-bearing nude mice. (A) The treatment regimen. (B) The curve of tumor volume change in each group. (C) The tumor nodules were dissected and photographed in each group after treatment. (D) The average tumor weight in each group. (E) TUNEL staining was used to detect the apoptosis of tumor tissue in nude mice. Scale bar=50 μm. (F) Tumor apoptosis cells assessed by counting the rate of TUNEL-positive cells. (G) Number of metastatic foci in different groups. Data are presented as the mean±SD (n=6). #p<0.05, ##p<0.01 (significant difference compared with the control group); *p<0.05, **p<0.01 (significant difference between different treatment groups).
Figure 8
Figure 8
In vivo toxicity evaluation. (A) Changes in body weight of nude mice during the treatment. (B) H&E stained images of major organs from mice treated with different formulations. Data are presented as the mean±SD (n=6). Scale bar=200 μm. #p<0.05, ##p<0.01 (significant difference compared to the initial body weight of tumor-bearing nude mice).
Figure 9
Figure 9
Study on the mechanism of AMD-NP-PTX against the growth and metastasis of ovarian cancer through Western blotting. (A) The relative expression of E-cadherin, N-cadherin, vimentin and NF-κB in tumor tissues of different groups measured by Western blotting. The quantification analysis of the expression of (B) E-cadherin, (C) N-cadherin, (D) vimentin and (E) NF-κB in tumor tissues measured by Western blotting. Data are presented as the mean±SD (n=3). #p<0.05, ##p<0.01 (significant difference compared with the control group); *p<0.05, **p<0.01 (significant difference between different treatment groups).
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Grants and funding

This study was financially supported by a grant from the National Natural Science Foundation of China (No. 81703420).