Transmission electron microscopy

The proliferation of BMSCs in response to Fe₃O₄ nanoparticles and SMF was measured by the Cell Counting Kit-8 assay (CCK-8; Dojindo, Tokyo, Japan). Briefly, 5×10³ cells/well (four replicates per group) were seeded into 96-well plates and then treated with different concentrations of Fe₃O₄ nanoparticles (400, 200, 100, 50 and 25 µg/mL). A group without nanoparticles served as the control. After culturing for 1, 3, and 5 days, CCK-8 solution (10 μL) and 90 μL of culture medium were added to each well and incubated at 37 °C for 1 h. The absorbance was observed at 450 nm by using a microplate reader (Varioskan Flash, Thermo Scientific™, USA), and the optical density was recorded for cell viability. Then, we cultured the BMSCs at the optimal concentration of Fe₃O₄ nanoparticles combined with different SMF strengths (50, 100, 150 mT), to measure cell viability as above.
The nanoscale morphology of the Fe₃O₄ nanoparticles taken up by BMSCs was observed by transmission electron microscopy (TEM) (Hitachi, Tokyo, Japan). According to the results of cell viability, we selected the best synergistic magnetic environment (concentration of Fe₃O₄ nanoparticles and strength of SMF) to stimulate the secretion of mag-BMSC-Exos.

The BLZ-954/NLG-919 co-loaded hybrid micelles (HM) were prepared using chloroform containing mixture polymer (PEG_5k-hyd-PCL_3k, 25 mg; PEI_3k-PCL_3k, 12.5 mg) and BLZ-945 (0.2 mg, dissolved in DMSO) and NLG-919 (3 mg, dissolved in DMSO). After evaporating in a rotary evaporator at 60 °C, the dispersion was added to 70 °C deionized water and further stirred for 1 h at room temperature. Subsequently, the micelles solution was dialyzed against deionized water for 12 h to remove organic solvent with membrane dialysis tubing (molecular weight cutoff of 3.5 kDa, Spectrum Laboratories, Inc. USA) and an ultrafiltration tube (molecular weight of 2K) was used to remove free BLZ-945/NLG-919, resulting BN@HM. Afterwards, 12.5 mg OVA was bounded with PEI through electrostatic adherence by vibrating at 4 °C for 1 h, resulting nanovaccine BN@HM-OVA. The sizes and zeta potentials of the formed BN@HM-OVA nanovaccine were measured with Zetasizer Nano ZS90 (Malvern, UK). The morphology of the nanovaccine was observed with transmission electron microscopy (TEM) (Hitachi, Japan).

To characterize the morphology of nEVs and CNVs, 10 μl of samples were loaded on the 400 mesh formvar coated copper grid and allowed to incubate for 3 min at room temperature (RT). Samples were drained out with a filter paper and stained with 1% filtered uranyl acetate solution for 1 min. Prepared samples were imaged with Hitachi transmission electron microscopy (TEM) at an acceleration voltage of 100 kV.^{33 (link)} The size distribution and concentration of nEVs and CNVs were detected by Nanosight NS300. Three 30-s videos were taken. Data was analyzed by the build-in algorithm of a NS300 machine and represented as mean ±SD. Classical EV protein biomarkers, including CD81, TSG101, and GAPDH, in nEVs and CNVs were analyzed with western blot. Briefly, 200 μL of nEVs or CNVs lysates were prepared by adding 50 μL of RIPA lysis buffer on ice. Samples were further mixed with 5× loading buffer and placed at 95 °C for 20 min. Gels were running at 60 V for stacking and 100 V for separating. The proteins were transferred to PVDF membrane using Bio-rad mini blotting system at 25 V for 7 min. The membranes were blocked for 1 h in 5% skimmed milk dissolved in TBS. The proteins were detected by incubation with primary antibody conjugated with HRP (CD81: sc-166029, TSG101: sc-7964, GAPDH: sc-32233, Santa Cruz). The membranes were washed thrice prior to imaging.