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

Description

(NBD)-N-FL-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 (NBD)-N-FL-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 (O-1(2)), which oxidizes the constituent phospholipids to transiently deteriorate the cell membrane. Reference membrane modifiers (NBD)-N-FL-Oleyl and (NBD)-N-FL-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 O-1(2) generation efficiently occurs only when the NBD unit is in aqueous media, and (2) the lifetime of O-1(2) in aqueous media is very short (3.0-3.5 mu s). As supported experimentally and computationally, the hydrophilic spacer length of (NBD)-N-FL-BAM(PEG2k) is optimal for compromising these factors. Further to note, the optoporation using (NBD)-N-FL-BAM(PEG2k) is not accompanied by cytotoxicity.

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