Flowview fv1000

To further confirm the PDT efficacy of TF-HA-CMC-PLGA NPs, the apoptosis assay was executed. Dual AO/EB staining was applied to detect apoptosis in A549 cell line. Briefly, A549 cells were seeded into the confocal dish at a cell density of 2 × 10⁵ cells/well. Twenty-four hours later, free HA, HA-CMC-PLGA NPs, or TF-HA-CMC-PLGA NPs (2 mL, equivalent to 0.05 μM HA) was added into each wells. The drug was removed and the cells were washed three times with PBS. Each well was added DMEM and exposed to a blue light emitting diode (470 nm, 90 mW/cm2) for 15 min. After 12 h incubation, 20 μL AO/EB (1:1) was added into each well according to the instruction by manufacturer. Laser confocal microscopy (Flowview FV1000, Olympus, Japan) was used to observe PDT-induced cell apoptosis in A549 cells. The fluorescence intensity was analyzed by Image J software. To observe the DNA breakage, chromosome aggregation, apoptotic body formation in A549 cells incubated with TF-HA-CMC-PLGA NPs (equivalent to 0.05 μM HA), the same procedure was performed as mentioned above, except for a longer incubation time (24 h).

CellRox^® oxidative stress reagents (Sigma-Aldrich) were used according to the manufacturer’s instructions to measure the intracellular ROS levels. BEAS-2B cells were seeded at a density of 2 × 10⁴ cells/well in an eight-well chamber slide and incubated for 24 h, followed by treatment with PM_2.5 or 8-OHdG for another 24 h. After washing twice with PBS, 5 μM CellROX^® green reagent was added to the cells, which were incubated for 30 min at 37 °C away from light. DAPI was used to stain the nuclei. Next, the cells were incubated in 3.7% paraformaldehyde for 15 min at 37 °C to perform cell fixation. Observations were made using an inverted microscope (Flowview FV1000, Olympus, Tokyo, Japan), and intracellular fluorescence values (excitation = 485 nm; emission = 520 nm) were measured using Image J software (version 1.53t, 24 August 2022 (upgrade)).