Exosome mimetics derived from bone marrow mesenchymal stem cells ablate neuroblastoma tumor in vitro and in vivo

Abstract

Purpose: To develop exosome-mimetics derived from bone marrow mesenchymal stem cells (EM) as a novel nanoscale drug delivery system(nanoDDS) with improved tumor targeting activity, therapeutic effect, and biosafety, and to evaluate the therapeutic effect of doxorubicin loaded EM (EM-Dox) on neuroblastoma (NB) in vitro and in vivo.

Methods: EM was prepared by serial extrusion of bone marrow mesenchymal stem cells (BMSCs), ammonium sulfate gradient method was used to promote the active loading of doxorubicin, and EM-Dox was obtained after removal of free doxorubicin by dialysis. The obtained EM and EM-Dox were characterized by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), Western Blot assay(WB), and the yield of exosomes and EM was further compared. Confocal fluorescent microscopy was used to verify the uptake of EM-Dox and free doxorubicin (Free-Dox) by NB cells. CCK-8 assay, cell cycle assay, and cell apoptosis assay were used to evaluate the antitumor effect of EM-Dox on NB cells in vitro. In addition, the targeted therapeutic effect and biosafety of EM-Dox against NB were evaluated in tumor-bearing nude mice.

Results: TEM, NTA, and WB verified that both EM and EM-Dox feature highly similar morphology, size and marker protein expression in comparison with naturally occurred exosomes, but the particle size of EM-Dox increased slightly after loading doxorubicin. The protein yield and particle yield of EM-Dox were 16.8 and 26.3-folds higher than those of exosomes, respectively. Confocal fluorescent microscopy showed that EM and doxorubicin had a definite co-localization. EM-Dox was readily internalized in two well-established human NB cell lines. The intracellular content of doxorubicin in cells treated with EM-Dox was significantly higher than that treated with Free-Dox. CCK-8 assay and flow cytometry confirmed that EM-Dox could inhibit NB cell proliferation, induce G2/M phase cell cycle arrest, and promote NB cell apoptosis in vitro. In vivo bioluminescence imaging results demonstrated that EM-Dox effectively targets NB tumors in vivo. Compared with Free-Dox, EM-Dox had a significantly increased inhibitory effect against NB tumor proliferation and progression in vivo, without inducing any myocardial injury.

Conclusions: EM-Dox showed significantly increased anti-tumor activity in comparison with free doxorubicin in vitro and in vivo, and scalable EMs may represent a new class of NanoDDS that can potentially replace naturally occurred exosomes in preclinical or clinical translations.

Keywords: Drug delivery; Exosome mimetic; Neuroblastoma; Targeted therapy.