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

^FLNBD-BAM_PEG2k, bearing a nitrobenzoxadiazole (NBD) unit and an oleyl terminus conjugated via a poly(ethylene glycol) (PEG) spacer ( M_n = 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 ^FLNBD-BAM_PEG2k 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_h = 4.7 nm). Detailed studies indicated that the photoexcited NBD unit can generate singlet oxygen (¹O₂), which oxidizes the constituent phospholipids to transiently deteriorate the cell membrane. Reference membrane modifiers ^FLNBD-Oleyl and ^FLNBD-BAM_PEG8k 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 ¹O₂ generation efficiently occurs only when the NBD unit is in aqueous media, and (2) the lifetime of ¹O₂ in aqueous media is very short (3.0-3.5 μs). As supported experimentally and computationally, the hydrophilic spacer length of ^FLNBD-BAM_PEG2k is optimal for compromising these factors. Further to note, the optoporation using ^FLNBD-BAM_PEG2k is not accompanied by cytotoxicity.