Mesenchymal Stem Cell Derived Exosomes as Nanodrug Carrier of Doxorubicin for Targeted Osteosarcoma Therapy via SDF1-CXCR4 Axis

Abstract

Purpose: The objective of this study was to investigate the antitumor activity, targeting capability, and mechanism of the developed nanodrug consisting of doxorubicin and exosome (Exo-Dox) derived from mesenchymal stem cells in vitro and in vivo.

Methods: The exosomes were isolated with Exosome Isolation Kit, and the Exo-Dox was prepared by mixing exosome with Dox-HCl, desalinizing with triethylamine and then dialyzing against PBS overnight. The exosome and Exo-Dox were examined by nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). The antitumor activity, targeting capability, and mechanism of the developed Exo-Dox were evaluated by cell viability assay, histological and immunofluorescence analysis and in vivo imaging system.

Results: NTA results showed the size of the exosomes had increased from 141.6 nm to 178.1 nm after loading with doxorubicin. Compared with free Dox, the Exo-Dox exhibited higher cytotoxicity against osteosarcoma MG63 cells, HOS cells, and 143B cells than free Dox, the half-maximal inhibitory concentrations (IC50) of Dox, Exo-Dox were calculated to be 0.178 and 0.078 μg mL^-1 in MG63 cells, 0.294 and 0.109μg mL^-1 in HOS cells, 0.315 and 0.123 μg mL^-1 in 143B cells, respectively. The in vivo imaging showed that MSC derived Exo could serve as a highly efficient delivery vehicle for targeted drug delivery. The immunohistochemistry and histology analysis indicated that compared with the free Dox group, the Ki67-positive cells and cardiotoxicity in Exo-Dox group were significantly decreased.

Conclusion: Our results suggested that MSC-derived Exo could be excellent nanocarriers used to deliver chemotherapeutic drug Dox specifically and efficiently in osteosarcoma, resulting in enhanced toxicity against osteosarcoma and less toxicity in heart tissue. We further demonstrated the targeting capability of Exo was due to the chemotaxis of MSC-derived exosomes to osteosarcoma cells via SDF1-CXCR4 axis.

Keywords: doxorubicin; exosome; nanocarrier; osteosarcoma; targeted therapy.

Scheme 1

Isolated exosomes were released from BM-MSC cells and loaded with doxorubicin to form a complex (Exo-Dox). We then evaluated the targeted antitumor effect of Exo-Dox both in vitro (A) and in vivo (B).

Figure 1

Characterization of exosomes: the size distributions of blank exosome (A) and exosome-doxorubicin (B) measured by NTA. The mean particle diameters were 141.6 nm for free exosome and 178.1 nm for exosome-doxorubicin. The morphology of blank exosome (C) and exosome-doxorubicin (D) as observed by TEM. (E) Western blotting analysis of the exosomal proteins CD81 and TSG101.

Figure 2

Cell viability of MG63, HOS, 143B and H9C2 cells treated with blank exosome (A), free doxorubicin and exosome-doxorubicin (B) detected by cell counting kit-8 (CCK-8). The inset shows the IC50 of free doxorubicin and exosome-doxorubicin, where the value was calculated according to (B). Mean values and standard deviations were obtained from three independent experiments.

Figure 3

(A) Transwell migration assay in MG63 cells treated with free Exo, free Dox (0.5 μg/mL-1), or Exo-Dox (0.5 μg/mL-1) for 24 h. (B) The quantitiation results of (A). Scale bar: 20 μm. Data are represented as the mean ± SD. ***P < 0.001. ****P < 0.0001.

Figure 4

In vivo efficacy evaluation of saline, free exosome, free doxorubicin and exosome-doxorubicin through intravenous injection in MG63 xenograft tumor-bearing nude mice. Images of tumor-bearing mice (A) and the xenograft tumor (B). Tumor volume changes (C) as well as the weight of the excised tumor tissues (D) from all groups. Mean ± SD, n = 5. ns, not significant. **p < 0.01, ***p < 0.001, ****p < 0.0001 when compared with the indicated groups.

Figure 5

The immunohistochemical staining of Ki-67 of the osteosarcoma tissues (A) and HE staining of heart tissue sections (B) from osteosarcoma-bearing mice treated with saline (control), free Exo, free Dox or Exo-Dox. Ki-67 positive cells were counted using Image Pro Plus 6.0 software.

Figure 6

The expression of CXCR4, SDF1 and tubulin proteins in 143B, MG63, HOS, H9C2, BM-MSCs cells and purified exosome measured by Western-blot analysis.

Figure 7

The biodistribution of DiR-labeled exosomes was examined by in vivo imaging of DiR fluorescence. (A) the expression of CXCR4 protein in MG63 cells after the knockout or overexpression of CXCR4 gene. (B) Nude mice bearing subcutaneous MG63, MG63 CXCR4+ and MG63 CXCR4- xenografts©received intravenous injection of saline, free DiR or Exo-DiR (50 μg/kg DiR) and were scanned at different times (1, 3, 6, and 12 h) after injection using an imaging system. (C) Fluorescence imaging of excised organs from tumor-bearing mice 12 h after IV injection with saline, free DiR or Exo-DiR. (D) Graphical representation of the fluorescence intensity of the tumor site at the selected time point in the five groups.  (E) Organs were removed and DiR dye signals in each organ were quantified after injection. The scale bar is in arbitrary units and is a colorimetric representation of the minimum and maximum signals; all the depicted images are reconstructed with the same scale. Mean ± SD, n = 5. ns, not significant. *p < 0.05, ****p < 0.0001 when compared with the indicated groups.