Fig. 3: Characterizations of M1EVs based formulations and evaluations of the penetration capacity and synergistic anti-tumor efficacy in vitro. | Signal Transduction and Targeted Therapy

Fig. 3: Characterizations of M1EVs based formulations and evaluations of the penetration capacity and synergistic anti-tumor efficacy in vitro.

From: Exploration and functionalization of M1-macrophage extracellular vesicles for effective accumulation in glioblastoma and strong synergistic therapeutic effects

Fig. 3

a. TEM image of M1EVs. Scale bar: 100 nm. b. ProteinSimple® capillary immunoassay (Wes) analysis of CD9, CD81, ALIX, TSG101, iNOS, F4/80, and GAPDH in M1 macrophages and M1EVs. c. Confocal laser scanning microscopy (CLSM) images of AQ4N-M1EVs (Top, green: M1EVs; red: AQ4N) and Ce6-M1EVs (bottom, green: M1EVs; red: Ce6). All images have the same scale of 1 μm. d. Representative flow cytometry analysis images of M1EVs (top) and TA-M1EVs (M1EVs containing AQ4N and TRMRA in place of Ce6 due to the overlayed spectrum with AQ4N) (bottom). e. Production of ROS with Ce6, CPPO/Ce6, CC-M1EVs, and CCA-M1EVs in buffers with different H2O2 concentrations, where A0 and A were the absorbance of ABDA at 399 nm before and after H2O2 addition (n = 3). f. Cumulative AQ4N release profiles of CCA-M1EVs before and after H2O2 treatment in PBS buffer (n = 3). g. Consumption of oxygen with different formulations after H2O2 treatment in PBS buffer (n = 3). h. Quantification of the AQ4/AQ4N ratio after different treatments based on high-performance liquid chromatography (HPLC) analysis. i. Illustration of in vitro BBB and TME model. The TranswellTM co-culture system containing bEnd.3 cells in the upper chamber and a combination of U87MG glioma cells and macrophages in the bottom chamber under hypoxic condition. j. CLSM images of bEnd.3 cells with different treatments. Scale bar: 5 μm. (green: ZO-1, red: EVs). k. Accumulative penetration efficiency of M1EVs, CC-M1EVs, A-M1EVs, and CCA-M1EVs labeled with DiD through a monolayer bEnd.3 layer at different time points (n = 3). l. Flow cytometry analysis of the M2/M1 ratio in the lower chamber after incubation with different EV designs (n = 3). m. Production of H2O2 with different treatments in the lower chamber (Amplex Red Hydrogen Peroxide Assay Kit) (n = 3). n. Assessment of intracellular ROS (labeled by DCFH-DA) of U87MG cells in the lower chamber (n = 3). o. Flow cytometry analysis of the cell-death-inducing effect of different formulations on U87MG cells in the lower chamber (Annexin V and PI in the dead cell apoptosis kit) (n = 3). Statistical significance was calculated via one-way ANOVA with a Kruskal-Wallis test (e, g, l, m, n, and o) or unpaired two-tailed Student’s t-test (f). ns, not significant

Back to article page