4.8. Fluorescence Observation of Nanoparticle Uptake into Cells

To investigate the cellular uptake of nanoparticles, HAsPBPE conjugates (20 mg) that were dissolved in 3 mL DMSO/water mixtures (2.5/0.5, v/v) and Ce6 (2 mg) dissolved in 1.0 mL of DMSO were added. This solution was dropped into 5 mL of deionized water and then magnetically stirred for 30 min. Then, this solution was introduced into the dialysis membrane (MWCO: 2000 Da) and then dialyzed against deionized water for 24 h with an exchange of water at 2~3 h intervals. Following this, the resulting nanoparticle solutions were lyophilized or used for analysis. The Ce6 contents in the nanoparticles were measured with the Infinite M200 pro microplate reader (Tecan Trading AG, Mannedorf, Switzerland) at 407 nm of excitation wavelength and 664 emission wavelength. The contents of Ce6 in the nanoparticles were calculated as follows: Drug contents = (Weight of Ce6/weight of nanoparticles) × 100. The Ce6 contents in the nanoparticles were 8.6% (w/w).

The Ce6-incorporated nanoparticles (0.1 mg as HAsPBPE conjugates) in the serum-free RPMI1640 media were treated to the cells. After 4 h, the cells were washed with PBS and then observed with a fluorescence microscope (Eclipse 80i, Nikon, Tokyo, Japan).