Cck 8 assay

Cell viability was assessed using the CCK-8 assay (Biosharp, China, BS350A). Briefly, cells were seeded in 96-well plates (1 × 10³ cells per well) and grown for 24 h in a incubator at 37℃ and 5% CO2 using RPMI 1640 medium with 10% fetal bovine serum. Then, the cells were incubated with the CCK-8 solution for 2 h (final concentration: 10%). The absorbance at 450 nm was measured using a Synergy HT Multi-Mode Microplate Reader (BioTek, USA) every 12 h for 2 days.

After transfection and H/R treatment, the CCK-8 assay (BioSharp, Hefei, China) was used to detect cell viability. The transfected and treated H9C2 cells were cultured in a 96-well plate supplemented with a 10 µl CCK‐8 solution. After 1 h of co-culture at 37 °C, the absorbance was assessed at 450 nm using a microplate reader (Bio-Rad, Hercules, CA, US).

Cell viability was assessed using the CCK-8 assay (Biosharp, Hefei, China) as per the instructions provided by the manufacturer to confirm the cytotoxic effects of chitosan, PAP, and chitosan@PAP. Briefly, the MC3T3-E1 cells were placed in a 96-well dish at a concentration of 3 × 10⁴ cells/mL and left to incubate for a period of 1 d. The MC3T3E1 cells were then exposed to chitosan, PAP, or chitosan@PAP in a culture medium for a duration of 3 d. Subsequently, 10 µL of CCK-8 was added to the culture and incubated at 37 °C in the absence of light for 3 h. A microplate reader was then used to measure the absorbance at 450 nm.

3 × 10³ cells were inoculated in each well of a 96-well plate and incubated for 2 h at 37 °C after 12 h with 10 μL of CCK-8 assay (Biosharp, China) and the optical density (OD) values at 450 nm and 630 nm were read by an enzyme-labeled instrument^{15 (link)}. The test was then carried out every 24 h for three consecutive times. The OD values at 450 nm minus 630 nm were calculated. 1000 cells were inoculated in each well of a six-well plate and changed every four days^{16 (link)}. 2 weeks later 4% paraformaldehyde was fixed, and photographed after crystalline violet staining.

Cell proliferation was evaluated by CCK‐8 assay (Biosharp) according to the manufacturer's protocol. Briefly, cells were seeded in 96‐well plates with 100 μl of medium and 10 μl of CCK‐8. After incubation for 2 h at 37°C, the absorbance of each well was measured with a microplate reader at 450 nm. Transwell assays with Matrigel were used to investigate the cell invasion ability. Approximately 20 000 cells were seeded into each well (24‐well plates), which had been pre‐coated with 100 μl of diluted Matrigel with medium (1:6). Medium with 10% FBS was added to the lower chambers while serum‐free medium was added to the upper chambers. After incubation in 37°C for 24 or 48 h, the cells were stained with 0.5% crystal violet and observed under a microscope. A wound healing assay was performed to evaluate the migration ability of NSCLC cells.

Cytotoxicity toward the Huh‐7 and Hepa1‐6 cells was determined quantitatively in vitro by the cell counting kit‐8 (CCK‐8) assay. Huh‐7 and Hepa1−6 cells were distributed in a 96‐well culture plate (5000 cells per well) and incubated overnight. Then, the original medium was discarded and different drug formulations were added in different concentrations for different incubation times. The blank culture medium was treated as control. After 4 h coincubation, the cells were irradiated with a 760 nm laser (1 W cm⁻², 4 min) while the other groups were placed in the dark. The cells were treated with ultrasound at 1 MHz, 2.0 W cm⁻² for 60 s. Then, the cells were further incubated for 12 h. Subsequently, cytotoxicity was evaluated by CCK‐8 assay (Biosharp, Beijing, China) according to the manufacturer's instructions. Cell viability was detected by the standard CCK‐8 protocol. Each assay was repeated in triplicate.

The effect of T140-KLA EVs on cell proliferation was assessed by CCK8 assay (Biosharp, China). Briefly, MOLM13 cells seeded in 24-well plates (50,000 cells/0.5 mL) received 50 μg of RBCEVs and were maintained under optimal growing conditions. After 24, 48, 72 and 96 hours of treatment with RBCEVs, 10% (vol/vol) of CCK8 reagent was added to the plate for 2 hours at 37 °C, protected from light. The absorbance of collected supernatants was read at 450 nm using a SynergyTM H1 microplate reader (BioTek, USA) or a Spark 10 M microplate reader (Tecan, Switzerland). To assess the viability of breast cancer cells, 10,000 CA1a or 4T1-tdTomato-hEGFR cells were seeded in 96-well plates 24 hours prior to addition of miR-125b-ASO-loaded RBCEVs. Following 3 days of incubation with EVs, cell viability was assessed as described above.
Annexin V-FITC and propidium iodide (PI) staining (Nanjing Vazyme Biotech, China) were used to determine apoptosis of treated cells. Briefly, after 96 hours of treatment with RBCEVs, MOLM13 cells were collected and washed twice with cold Annexin V binding buffer, then incubated with 100 μL of binding buffer containing 5 μL of annexin-V-FITC and 5 μL of PI for 10 minutes at room temperature, protected from light. The cells were subsequently washed twice with cold binding buffer and analyzed by flow cytometry.