Nitrobenzoxadiazole-Appended Cell Membrane Modifiers for Efficient Optoporation with Noncoherent Light.

NBD-BAM, bearing a nitrobenzoxadiazole (NBD) unit and an oleyl terminus conjugated via a poly(ethylene glycol) (PEG) spacer ( M = 2,000), was designed to fluorescently label cell membranes by docking its hydrophobic oleyl terminus. During laser scanning microscopy in a minimal essential medium (MEM), human hepatocellular carcinoma Hep3B cells labeled with NBD-BAM appeared to undergo optoporation at their plasma membrane. We confirmed this unprecedented possibility by a series of cellular uptake experiments using negatively charged and therefore membrane-impermeable quantum dots (QDs; D = 4.7 nm). Detailed studies indicated that the photoexcited NBD unit can generate singlet oxygen (O), which oxidizes the constituent phospholipids to transiently deteriorate the cell membrane. Reference membrane modifiers NBD-Oleyl and NBD-BAM having shorter or longer hydrophilic spacers between the NBD and oleyl units showed a little or substantially no optoporation. For understanding these results, one must consider the following contradictory factors: (1) The photosensitized O generation efficiently occurs only when the NBD unit is in aqueous media, and (2) the lifetime of O in aqueous media is very short (3.0-3.5 μs). As supported experimentally and computationally, the hydrophilic spacer length of NBD-BAM is optimal for compromising these factors. Further to note, the optoporation using NBD-BAM is not accompanied by cytotoxicity.