Thymoquinone-loaded mesenchymal stem cell-derived exosome as an efficient nano-system against breast cancer cells

Abstract

Objectives: Exosomes became the subject of extensive research in drug delivery approach due to their potential applicability as therapeutic tools for cancer therapy. Thymoquinone (Tq) is an anti-cancer agent due to its great anti-proliferative effect. However, poor solubility and weak bioavailability restrict its therapeutic applications. In this study, exosomes secreted from human adipocyte-derived mesenchymal stem cells (AdMSCs) were isolated and the efficacy of a novel encapsulation method for loading of Tq was investigated. Finally, the cytotoxic effect of Tq incorporated exosomes against cancer cells was evaluated.

Materials and methods: Exosomes secreted from AdMSCs were isolated via ultracentrifugation and characterized by electron microscopy and western blotting. Then, through a novel encapsulation approach, Tq was loaded into exosomes by the combination of three methods including incubation, freeze-thawing, and surfactant treatment. Then, the encapsulation efficiency, in vitro cellular uptake, and cytotoxicity of Tq incorporated exosomes (Tq@EXOs) in MCF7 and L929 cells were estimated.

Results: Tq loading into exosomes through our novel method caused a significant improvement in encapsulation efficiency of about 60%. The fluorescent microscopy and flow cytometry outcomes indicated the efficient uptake of Tq@EXOs-FITC by cells throughout 4 hr. Furthermore, MTT results displayed the ability of Tq@EXOs in effectively decreasing the cell viability of MCF7 without causing any obvious cytotoxicity on L929 as normal cells.

Conclusion: The results suggest that our approach provides effective loading of Tq into exosomes which offer a valuable and safe platform for drug delivery to cancer cells thus having a great potential for clinical studies.

Keywords: Adipose-derived; Breast cancer; Drug delivery system; Exosome; Thymoquinone; mesenchymal; stem cells.

Figure 1

Morphologic characterization of Adipocyte‐derived mesenchymal stem cells (AdMSCs). (A) The spindle shape of AdMSCs appeared at third day. (B-D) Homogenous population of cells developed with suitable confluency after three passage

Figure 2

Expression of cell surface antigens were confirmed in isolated AdMSCs using flowcytometry. Cells were positive for CD73 and CD90 which are popular for human adipocyte-derived mesenchymal stem cell markers and negative for CD45, CD34 and CD11b which are hematopoietic stem cell markers

Figure 3

Multilineage differentiation ability of AdMSCs. (A) Oil Red O staining of AdMSCs differentiated into adipocytes containing oil droplets. (B) Untreated cells as the control

Scheme 1

Schematic illustration of Tq loaded into EXOs using a novel technique based on the combination of three methods: incubation, freeze-thawing and surfactant treatment

Figure 4.

Field emission scanning electron microscopy (FE-SEM) image of exosomes

Figure 5

Western blot analysis of CD9, CD63 and CD81 as positive marker and Bcl2 as negative marker of isolated exosomes

Figure 6

The cytotoxic activity of free Tq against MCF7 and L929 cell line after 24 hr (A and C) and 72 hr (B and D). IC₅₀ of free Tq in MCF7 and L929 cells after 72 hr was obtained 76 µM and 52 µM, respectively. The experiment was done in triplicate for different time point

Figure 7

Cellular toxicity of Tq@EXOs in comparison with free Tq after 72 hr, in concentrations range of IC₅₀, IC₂₅ and IC₁₀ in L929 and MCF7 cells. Tq@EXOs could effectively decrease the MCF7 viability in all IC concentrations compare to L929. Mean± SD of three independent experiments using GraphPad Prism 6

Figure 8

Florescent microscopy and flow cytometry analysis of MCF7 cells upon 4 hr of exposure to Tq@EXOs-FITC and FITC dye. The qualitative analysis of MCF7 cells treated with Tq@EXOs-FITC and FITC dye after 4 hr (A). The quantitative analysis of flow cytometry experiments was also accomplished based on geometric mean florescence intensity of MCF7 cells incubated with either Tq@EXOs-FITC and FITC dye. Data are presented as mean± SD (n = 3) (B and C)