Invert fluorescence microscope

To evaluate the internalization of exosomes in vitro, the PL-exo and PL-exo-ALN were labeled with the DiD firstly as mentioned above. Then, BMSCs were exposed to the labeled exosomes for 12 h. After that, the cells were fixed with 4% paraformaldehyde (PFA) for 30 min at room temperature, followed by permeabilized in 0.1% Triton-X 100 in PBS for 5 min, and stained cytoskeleton and nucleus with FITC labeled phalloidin (Yeasan, Shanghai, China) and 4′,6-diamidino-2- phenylindole (DAPI, Beyotime Biotechnology, China) respectively. The images were obtained using an invert fluorescence microscope (Olympus, Japan).
The bone targeting effect of PL-exo-ALN was evaluated in vivo by assessing the biodistribution of the intravenous delivery of DiD labeled exosomes. Six female Sprague–Dawley rats (200–250 g, 8-week-old) were obtained from the Animal Center of the Chinese Academy of Science (Shanghai, China). The rats were divided into two groups: PL-exo and PL-exo-ALN (n = 3 in each group). After anesthesia with sodium pentobarbital, the rats were received 100 μg exosomes (dissolved in 200 μL of PBS) via tail vein injection. Six hours later, the rats were sacrificed and the femur along with major organs (femur and heart, liver, spleen, lung, kidney) were collected for IVIS imaging (Lumina Series III, PerkinElmer, USA).

The comet assay was performed as described previously [48 (link)] to evaluate DNA strand breakdown. Cells were treated as described above and then suspended in PBS (1 × 10⁵ cells/mL). Briefly, 1% low melting point agarose (LMA, 80 µL) was added to the cells (200 cells/µL, 20 µL), which were then put on a slide pre-coated with 0.8% normal agarose (NMA, 100 µL) dissolved in PBS, and covered immediately with a coverslip (4 °C, 15 min). After solidification, the coverslip was cautiously removed. The cells were lysed in a pre-chilled lysis solution containing 2.5 M NaCl, 100 mM Na₂EDTA, 10 mM Tris, 200 mM NaOH, 1% sodium lauroyl sarcosinate, and 1% Triton X-100 at pH 10 (4 °C; 90 min). The slides were placed in an electrophoresis chamber with an alkaline electrophoresis solution (200 mM NaOH and 1 mM Na₂EDTA, pH > 13) for unwinding and expression of alkali-labile sites (30 min) and were subsequently electrophoresed (25 V and 300 mA, 20 min) with aim to draw the negatively charged DNA toward the anode. After neutralization, the cells were stained with DAPI (50 µg/mL, 20 µL) in the dark for 5 min. They were then observed under an invert fluorescence microscope (Olympus, Tokyo, Japan) the DNA damage (average tail length) was quantified using the CASP software to analyze the comet images.