In vitro Cellular Uptake and Intracellular Drug Release

To investigate the ability for the NAG-targeted formulations to enhance cellular uptake and intracellular trafficking (distribution in cytoplasm), FITC-labeled PTXL-ss-PMAGP-GEM/NAG NLCs and FITC-labeled PTXL-ss-PMAGP-GEM NLCs were prepared. Briefly, 50 mg of each NLCs and FITC (5 mg) were dissolved in DMF and reacted at ambient temperature (about 20°C) for 24 h, followed by dialyzing and freeze-drying.

To quantify cellular uptake, A549 and LTEP-a-2 cells (which overexpress glucose receptors) were plated in six-well plates at a density of 2 × 10⁵ cells/well, and treated with FITC-labeled PTXL-ss-PMAGP-GEM/NAG NLCs or FITC-labeled PTXL-ss-PMAGP-GEM NLCs with total drug concentrations of 5, 10, and 20 μmol/L, respectively. Untreated cells served as a control. Following incubation of 0.5, 2 and 4 h, respectively, cells were detached and washed with PBS three times. The cells were spread into 0.5 mL PBS and tested by flow cytometry (Epics XL; Beckman Coulter, Brea, CA, USA). Similarly, control cells L929 fibroblasts (with little or no glucose receptors) representing normal cells were also treated with 10 μmol/L FITC-PTXL-ss-PMAGP-GEM/NAG NLCs.

To evaluate the cellular uptake visually, confocal laser scanning microscopy (CLSM) images of A549 cells were acquired. A549 cells were seeded in six-well plates and incubated with FITC-labeled PTXL-ss-PMAGP-GEM/NAG NLCs and FITC-labeled PTXL-ss-PMAGP-GEM NLCs (10 and 20 μmol/L), respectively. After exposure for 1, 2 and 4 h, cells were washed and fixed by paraformaldehyde. After washing again, Hoechst 33342 (10 mg/mL) was added to stain cell nuclei, and the images of cells were observed via confocal microscopy (TCS SPE; Leica Microsystems, Wetzlar, Germany).