Bioluminescence imaging (BLI) is a powerful imaging modality for monitoring biological phenomena both in vitro and in vivo. BLI is becoming a seamless imaging technology covering the range from cells...
Bioluminescence (BL) imaging, which utilizes light emitted through the enzymatic reaction of luciferase oxidizing its substrate luciferin, enables sensitive and noninvasive monitoring of life phenomena. Herein, we developed a series of caged furimazine (FMZ) derivatives by introducing a protective group at the C-3 position and a hydroxy group at the C-6 phenyl ring to realize long-term live-cell BL imaging based on the NanoLuc (NLuc)/ NanoKAZ (NKAZ)−FMZ system. The membrane permeability and cytotoxicity of the substrates were evaluated and related to their hydrophobicity. Among the series, the derivative with the bulkiest protective group (adamantanecarbonyl group) and a hydroxy substituent (named Ad-FMZ-OH) showed significantly prolonged and constant BL signal in cells expressing NLuc compared to the native FMZ substrate. This derivative enabled continuous BL imaging at the single-cell level for 24 h. Furthermore, we applied Ad-FMZ-OH to BL imaging of myocyte fusion and succeeded in the consecutive and sensitive monitoring at a single-cell level over a day. In summary, NLuc/NKAZ-caged FMZ derivatives have the potential to be applied to live-cell BL imaging of various life phenomena that require long-term observation.
A pH-responsive mixed-charge polymer consists of uniformly distributed cationic and anionic units in the polymer chain. Carboxylic acid monomers are generally used as anionic monomers. The pH responsive range is confined to a relatively narrow pH of ∼5, which is limiting the biological application of pH-responsive polymers. Therefore, we investigated the effect of a series of carboxylic acid and neutral monomers on the pKa value of pH-responsive mixed-charge polymers. We synthesized the polymers using [2-(methacryloyloxy)ethyl]trimethylammonium chloride, carboxylic acid monomers, and neutral monomers as the spacer moiety. As the monomer hydrophobicity and the composition ratio of neutral monomers increased, the pKa shifted to higher values. Finally, the polymer pKa values were tuned from 4.6 to 6.1. We confirmed that the pH-dependent interaction between anionic tissue constituent and pH-responsive mixed-charge polymers depends on their pKa.
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