Moreover, drug-loaded MSC-derived exosomes may also provide opportunities to target heterogeneity of the tumor including the presence of malignancy stem-like cells. Acknowledgments The authors are grateful to colleagues from Hannover Medical School including Thomas Roth?mel for support with the STR fragment analysis. in lung, liver, spleen, and kidney was reduced by 50% with MSC taxol exosomes, similar to the effects observed with taxol, even though concentration of taxol in exosomes was about 1000-fold reduced. Together, these findings in different malignancy cell populations and in vivo provide promising future perspectives for drug-loaded MSC-derived exosomes in efficiently targeting main tumors TRx0237 (LMTX) mesylate and metastases by reducing side effects. < 0.001) upon exposure to this chemotherapeutic compound, which is most likely related to enhanced cellular stress upon taxol TRx0237 (LMTX) mesylate treatment (Physique 2B). Indeed, cellular stress including warmth was associated with elevated production of doxorubicin-loaded exosomes . Further exosome analysis by the presence of tetraspanins in immunoblots revealed altered expression levels of the 26 kDa core protein and the 30C60 kDa glycosylated form of CD63 in all control and taxol-treated samples, with ImageJ quantification for relative intensities (Physique 2C). This is supported by Western blot analysis of previous work demonstrating the presence of exosome-associated CD63 tetraspanin molecules in MSC-derived exosome preparations . Together, these data substantiated isolation of EVs displaying stable exosomal properties. Determination and quantification of the amount of taxol in the MSC-derived exosomes delivered TRx0237 (LMTX) mesylate to the malignancy cells was assessed by LC-MS/MS. Representative histograms for the detection of taxol (paclitaxel) and its evaluation compared to the internal standard (docetaxel) are offered for 1.68 105 MSC290115GFP and exhibited 7.5 1.5 M taxol (= 3) in the cell culture medium Rabbit polyclonal to PCSK5 supernatant remaining after a 24 h treatment with 10 M taxol (Determine 3A,D). Moreover, cell-associated taxol of MSC290115GFP exhibited 1.17 0.01 M (= 3) after a 24 h stimulation with 10 M taxol (Figure 3B,D) and released exosomes isolated after further 24 h culture in serumfree medium of previously 24 h-treated MSC290115GFP with 10 M taxol revealed 74.9 3.9 nM (= 3) of this compound (Figure 3C,D). Open in a separate window Physique 3 Representative MSC290115GFP LC-MS/MS-chromatograms of paclitaxel (m/z: 854105, retention time: 6.3 min) and the internal standard docetaxel (m/z: 808226, retention time: 6.5 min) of (A) cell culture medium supernatant of 10 M taxol-treated MSC290115 after 24 h, (B) cell lysate of 10 M taxol-treated MSC290115 after 24 h, and (C) exosome lysate released after 24 h from previously 10 M taxol-treated MSC290115 for 24 h. The peak of the internal standard represents constant intensities in the range of 5 104 cps in all samples. Accordingly, paclitaxel intensities are varying in the different samples whereby in (A). 2.2 106 cps, in (B). 3.5 105 cps, and in (C). 2.5 104 cps were determined. (D) Quantification of taxol concentrations were performed TRx0237 (LMTX) mesylate by LC-MS/MS in the corresponding cell culture medium supernatants of 10 M taxol-treated four MSC populations (MSC290115GFP, MSC030816GFP, MSC060616GFP, and MSC280416GFP) (=taxol/medium), in the corresponding cell lysates of 10 M taxol-treated four MSC populations (=taxol/cells), and in the corresponding exosome lysates released after 24 h from previously 10 M taxol-treated four MSC populations for 24 h (=taxol/exosomes). Data represent the mean s.d. of three replicates. The minimalized heterogeneity of taxol incorporation into MSC by randomly choosing four different donors and passages is summarized in Figure 3D. Thus, 7.3 0.7 M taxol (= 4) remained in the medium supernatant of the four MSC cultures after a 24 h treatment with 10 M taxol. Accordingly, 1.4 0.4 M taxol (= 4) was found in the cell homogenates of the four taxol-treated MSC cultures demonstrating 14% incorporation of taxol. Furthermore, 123 0.7 nM taxol (= 4) was detectable in the different exosome preparations of the four taxol-exposed MSC populations equivalent to 1.23% incorporation of the initial taxol stimulation (Figure 3D). Equal aliquots from all four MSC-derived control or taxol exosomes were combined and incubated with different cancer cell populations, including A549 lung cancer cells, SK-OV-3 ovarian cancer cells, and MDA-hyb1 breast cancer cells. Cytotoxic effects were evaluated by fluorescence reduction in a fluoroscan assay whereby culture of the different cancer cell populations in control medium was set to 100%. Treatment with different amounts of taxol exhibited a concentration-dependent cytotoxicity of all cancer cells. Thus, exposure to 100 nM taxol for.