Cellular uptake assay

The cellular qualitative uptake assay was performed by a P-gp substrate with fluorescence, R123.37,38 Specifically, MCF-7 and MCF-7/PTX cells were seeded into a 12-well plate (1×10⁵ cells/well) in growth medium solution with a glass coverslip/well. After 24 h of incubation, the original medium was replaced with free R123, FFSSTP/R123, FFTP/R123, or FFP/R123 growth medium solution (R123 concentration was 100 μg/mL in each preparation) and incubated at 37°C for 4 h. Then, coverslips with cells were taken off, washed with ice-cold PBS, placed in empty wells, and treated with 4% paraformaldehyde (1 mL) for 15 min. Cell nucleus was counterstained with Hoechst 33342 for 5 min after washing with ice-cold PBS. The final samples (washed with ice-cold PBS to remove the residual Hoechst 33342) were analyzed under a confocal laser scanning microscope (Nikon A1; Nikon Corporation, Tokyo, Japan).

A liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was used to determine the cellular quantitative accumulation and retention. MCF-7 and MCF-7/PTX cells were seeded in 12-well plates at a density of 1×10⁵ cells/well. In time-dependent uptake investigation, cells were incubated by FFSSTP/PTX, FFTP/PTX, FFP/PTX, or PTX growth medium solution at a PTX concentration of 5 μg/mL for 1, 2, 3, and 4 h at 37°C after removing the old medium. Cell uptake was terminated through repeated washing with ice-cold PBS, and the residual PTX was also removed from the cell surface. Then, the cells were lysed with RIPA lysis buffer (100 μL), and PTX was extracted with acetonitrile (200 μL/sample), sonicated in ice bath, and centrifuged (12,000× g, 5 min). The supernatant of extracts was collected and analyzed by LC–MS/MS. Moreover, the protein content in each sample was determined by a BCA protein assay kit. The chromatographic column was a Hypersil GOLD C₁₈ column, and the mobile phase was consisted of 0.1% formic acid (A) and acetonitrile containing 0.1% formic acid (B). The column temperature was 20°C, the flow rate was 0.4 mL/min, and the run time was 5.5 min. The sample injection volume was 10 μL. The gradient elution was delivered at the start of run 80/20% of A/B; from 1.5 to 5.5 min, the gradient starts at 20% A and ramps to 80% A. An electronic spray ion-positive mode was selected to operate the mass spectrometer. The selected reaction monitoring transitions used for quantification were performed at m/z 854.25/509.12 (collision energy: 15 eV) for PTX and m/z 830/527.14 (collision energy: 25 eV) for the internal standard, docetaxel. The weighted (1/X²) least-squares linear regression of calibration curves based on peak area ratios of PTX to docetaxel versus actual concentrations was used to determine the linearity (5–5,000 ng/mL). The cellular accumulation of PTX was normalized with total protein content. The following equation was used to calculate the uptake index (UI):

where C and P were the PTX and protein concentration in the cell lysis solution, respectively.

In intracellular retention study, MCF-7 and MCF-7/PTX cells were cultured with FFSSTP/PTX, FFTP/PTX, FFP/PTX, or PTX growth medium solution for 4 h and washed with ice-cold PBS, followed by incubation with culture medium at 37°C for additional 1, 2, 3, and 4 h. Cells were lysed, the concentrations of PTX in cell lysate were measured, and the intracellular retention ratio was calculated by the following equation:

where UI_(t) and UI₍₀₎ were the values of UI at different additional incubation times or the values of UI before additional incubation, respectively.