2.7. Cellular uptake and intracellular trafficking

The cellular uptake of different liposomes was further investigated by flow cytometry (BD, Franklin Lakes, NJ). Briefly, MCF-7 and MCF-7/MDR cells were seeded in 24-well plates at a density of 1 × 10⁵ cell/well and cultured for 24 h. Subsequently, cells were incubated with CL-RH123, PD-CL-RH123, TPD-CL-RH123 and HA-TPD-CL-RH123 for 1, 2, 4, 8, 12 and 24 h, and the fluorescence intensity was monitored by flow cytometry.

The real-time recording of the cellular internalization process of HA-TPD-CL-RH123 was assessed by confocal laser scanning microscopy (Carl Zeiss LSM 700, Germany). In brief, MCF-7/MDR cells were seeded in CLSM dish at a density of 5 × 10⁵ cells/well and cultured for 24 h. Afterward, cells were treated with HA-TPD-CL-RH123 for 1, 2, 4 and 8 h, and washed with PBS for three times. Then cells were fixed with 4% paraformaldehyde for 15 min and the cell nuclei were stained with 50 nM DAPI for 15 min. Finally, the cells were washed by PBS thrice and recorded by CLSM.

To quantitative study intracellular uptake, MCF-7/MDR cells were seeded in 6-well plates at a density of 1 × 10⁶ cells per well and cultured until a confluent monolayer of cell formed. Subsequently, different drug-loaded liposomes (PTX, 2 μg/mL) were added into each well and incubated with cells for 1, 2, 4 and 8 h, respectively. The original medium was then discarded and washed with PBS for three times. Thereafter, 150 μL of cell lysis buffer was added to fully lyse cells. The BCA Protein Assay Kit (Beyotime, China) was performed for determining the amount of protein, and the concentration of intracellular drug was detected by HPLC-MS-MS. The cellular uptake (Q_drug/Q_protein) was evaluated, where Q_drug and Q_protein represented the amount of drug and protein in MCF-7/MDR cells.

To further study the active targeting capability of HA-coated liposome, the CD44-overexpressing MCF-7/MDR cells were seeded in 6-well plates at a density of 1 × 10⁶ cells per well. After culturing for 24 h, the free HA (15 mg/mL) was added and incubated with cells for 2 h, followed by treatment with HA-TPD-CL-PTX/SOR for 6 h. Furthermore, the HA-coated liposome was pretreated with HAase (1 mg/mL) for 2 h, and then cells were incubated with the HAase-treated liposome for 6 h. Subsequently, the quantitative study of intracellular uptake was assessed by BCA Protein Assay Kit and analyzed by the same procedure as described above.

Confocal laser scanning microscopy (CLSM) was applied to further track the cellular transport process of different liposomes. In brief, MCF-7/MDR cells were seeded in a confocal microscope dish with 1 × 10⁵ cells/well density and cultured for 24 h. Then cells were treated with various RH123-loaded liposomes for 1 h and washed with cold PBS to remove the residual formulations. Subsequently, cells were further incubated with 1640 medium for another 0, 2 or 4 h and stained with Lyso-Tracker Red (Beyotime, China) for 90 min, and immediately imaged by CLSM.