In vitro cytotoxicity assay

First, the cytotoxicity of free drug combinations was studied by using the MTT assay. PC-3 cells were seeded in 96-well plates at a density of 5×10³ cells per well and incubated for 24 hours. The cells were treated with free DOX+TRI (the mass ratio of DOX to TRI was 1/0, 0.9/0.1, 0.8/0.2, 0.7/0.3, 0.6/0.4, 0.5/0.5, 0.4/0.6, 0.3/0.7, 0.2/0.8, 0.1/0.9, and 0/1) for 48 hours, and the final concentration of drug was uniform at 1 µg/mL. Subsequently, MTT stock solution (20 µL, 5 mg/mL in PBS) was added to each well, and cells were further incubated for 4 hours at 37°C. Finally, cells were dissolved in 200 µL of DMSO, and the absorbance was detected at 490 nm using a microplate reader (Thermo Multiskan MK3; Thermo Fisher Scientific, Waltham, MA, USA).

Then, the cytotoxicity of PEG-b-PLL was also evaluated by the same MTT method.

To investigate the pH-triggered charge reversal efference, DA-ss-DT, P-ss-DT, and SA-ss-DT were evaluated against PC-3 cells at pH 7.4 or 6.5 by the MTT assay. The cells were seeded in a 96-well plate at the density of 5×10³ cells per well. After incubation for 24 hours, the cells were treated with DA-ss-DT, P-ss-DT, or SA-ss-DT at pH 7.4 or 6.5. After 4 hours of incubation, the medium was removed, and the cells were washed twice with PBS and added to 200 µL fresh medium at pH 7.4 and incubated for 44 hours. Then, the cell viability was analyzed using the MTT method.

To investigate the sensitiveness of DA-ss-DT to reduction environment, we pretreated PC-3 cells with or without 10 mM GSH for 2 hours at 37°C. Then, the cells were washed with PBS to remove the GSH and incubated with DA-ss-DT or DA-cc-DT containing DOX and TRI at different concentrations (mass ratio of DOX to TRI was fixed at 4:1) for further 48 hours, followed by above-mentioned steps.

Six-week-old male BALB/c nude mice were subcutaneously injected with 4×10⁶ PC-3 cells at the right flank region.38 When the tumor size reached around 100 mm², the mice were randomly divided into seven groups (n=10) and intravenously injected with saline, free DOX, TRI, DOX+TRI, DA-ss-DT, DA-cc-DT, or SA-ss-DT every 3 days for three times. The tumor volume and mouse body weight were monitored every 3 days for up to 21 days after the first administration, and tumor size was calculated using the formula: Volume=0.5×(length)×(width).2

At the end of the experiment on antitumor efficacy, at day 21, PC-3 tumor-bearing nude mice were sacrificed, and tumor tissues were collected, fixed in 10% formalin, and embedded in paraffin blocks after dehydrating with gradient ethanol. Then, the tissues were cut into a slice and stained using H&E for histopathological evaluation.