2.8. Cellular Uptake Studies

To assay cellular uptake of nanoparticles quantitatively, HT-29 cells at a density of 1 × 10⁴ cells per well were seeded in a 96-well microplate in a 5% CO₂, 95% air humidified incubator at 37 °C. After incubation for 24 h, the medium was withdrawn. The cells were washed three times with pre-warmed PBS, and then 100 µL of the DTX-loaded nanoparticles in PBS (concentration of 3.9–125.0 µg/mL) was pipetted into the wells. The cells were incubated at 37 °C for 0.5–4 h. At predetermined times, the supernatants of the wells were removed as samples (I_sample). The wells that retained the supernatant served as positive controls (I_positive) [50]. After withdrawing the supernatants, PBS was used to wash the cells three times. The cells then were solubilised with 100 µL of Triton X-100 (0.5% Triton X-100 in 0.2 N NaOH). Fluorescence intensities were measured using a microplate fluorescence reader (FLUOstar Omega, BMG LABTECH, Ortenberg, Germany). The excitation and emission wavelengths used were 485 and 520 nm, respectively. The uptake efficiency formula was calculated as follows:

where I_negative represents the fluorescence intensity retrieved from the wells containing the cells in PBS.

Fluorescent imaging of the cellular uptake of the nanoparticles was carried out according to a method described previously with some alterations [51]. Briefly, HT-29 cells were seeded onto a 6-well plate at 1 × 10⁵ cells per well and incubated in a 5% CO₂, 95% air humidified incubator at 37 °C. After 24 h of incubation, the medium was withdrawn, and PBS was used to wash the wells. Next, 2 mL of 50 µg/mL Alexa Fluor fWGA-labelled nanoparticles in PBS was pipetted into the wells and incubated for 2 h. The solution was then withdrawn, and PBS was used to wash the wells. The wells containing the cells were viewed under a fluorescence microscope (Olympus CKX41, Olympus, Tokyo, Japan) to observe the cells using bright field and a blue fluorescence filter under 40 × magnification with a numerical aperture of 0.6.

Coverslips were placed in the wells of a 6-well plate. HT-29 cells were seeded onto the coverslips at a density of 1 × 10⁵ cells per wells and incubated at 37 °C in a 5% CO₂, 95% air humidified incubator. After 24 h of incubation, the medium was withdrawn, and pre-warmed PBS was used to wash the wells. Next, 2 mL of 50 µg/mL nanoparticles in PBS was added in the wells and incubated for 2 h. The solution was withdrawn, and ice-cold PBS was used to wash the wells twice. Next, 2 mL of 4% PFA was used to fix the cells for 15 min, and ice-cold PBS was used to wash the cells twice [52]. Then, 2 mL of Triton X-100 (0.1% in PBS) was added to the cells and incubated for 15 min on ice. PBS was used to wash the cells three times. Next, 2 mL of blocking buffer (PBS containing 1% BSA and 0.2% Tween-20) was added to the cells, which then were incubated for 1 h. Primary and secondary antibodies were diluted at 1:200 with blocking buffer. The diluted primary antibody (anticlathrin antibody) was pipetted into the wells and incubated at 4 °C in a dark humidified chamber for 24 h. PBS was used to wash the cells five times. Subsequently, the diluted secondary antibody [goat antirabbit IgG H&L (Alexa Fluor^® 647)] was pipetted into the wells followed by 1 h of incubation in the dark at 4 °C. PBS was used to wash the cells five times. ProLong diamond antifade mountant with DAPI was used to mount the coverslips onto glass slides, and the slides were viewed under a confocal microscope (Zeiss CLSM 710, Zeiss, Jena, Germany) using an oil-immersion objective with 63 × magnification and a numerical aperture of 1.4 to observe the fluorescence emitted by the cells using DAPI, FITC, and A647 filters.