Anticancer Prodrug Capable of Mitochondria-Targeting, Light-Triggered Release, and Fluorescence Monitoring.

Mitigating the adverse effects of anticancer agents requires innovative prodrug engineering. In this study, we showcase the potential of our o-quinone methide-based trigger-release-conjugation platform as a versatile tool for constructing advanced prodrug systems. Using this platform, we achieved the light-triggered release of an anticancer drug mechlorethamine, targeting mitochondrial DNA. The entire process was adeptly tracked through the emission of fluorescence signals, revealing notable effects across various cancer cell lines compared to a normal cell line. Exploring alternative cancer-associated triggers, including enzymes, and incorporating cancer/tumor-specific targeting elements could lead to effective prodrugs with reduced cytotoxicity.

Nitroreductase-Triggered Fluorophore Labeling of Cells and Tissues under Hypoxia.

Several reductases, including nitroreductase, are upregulated under hypoxic conditions characterized by an oxygen-deficient microenvironment. Given that hypoxia is a prominent feature of solid tumors, our investigation focused on developing a bioconjugative probe designed for staining tissue under hypoxic conditions, particularly activated by nitroreductase. This probe, developed using our trigger-release-bioconjugation system rooted in the ortho-quinone methide chemistry, exhibited selective activation by nitroreductase and fluorophore labeling within mitochondria and endoplasmic reticulum. As a result, it displayed sustained fluorescence that persisted even after washing steps in cells and tissues. We applied this innovative probe to stain mouse kidney tissue in an acute kidney injury model induced by inadequate oxygen supply. Among various organ tissues examined, only kidney tissue showed significantly higher fluorescence in the injury model compared with the control tissue, as revealed by two-photon microscopic imaging. This research presents a promising avenue for the development of practical staining agents for image-guided tumor surgery.

Polarity-Driven Two-Photon Fluorescent Probe for Monitoring the Perturbation in Lipid Droplet Levels during Mitochondrial Dysfunction and Acute Pancreatitis.

Lipid droplets (LDs) act as an energy reservoir in cancer cells; on the other hand, mitochondria are hyperactive to fulfill the energy demand to accelerate cell proliferation. We are interested in unfolding the relationship between the cellular energy reservoir and energy producer through fluorescence labeling. Thus, a dual organelle-targeted fluorescent probe MLD-1 has been rationally developed. It visualized the crosstalk between mitochondrial dysfunction and the fluctuation of LDs in live cells. Its two-photon ability allowed us to acquire deep tissue images. For the first time, we have shown that the probe has the ability to track the accumulation of LDs in different mouse organs during pancreatic inflammation. MLD-1, being a selectively polarity-driven, chemo- and photostable LD probe, may offer great possibilities for studying LD-associated biology in due course.

A Small-Molecule Fluorescence Probe for Nuclear ATP.

Fluorescence monitoring of ATP in different organelles is now feasible with a few biosensors developed, which, however, show low sensitivity, limited biocompatibility, and accessibility. Small-molecule ATP probes that alleviate those limitations thus have received much attention recently, leading to a few ATP probes that target several organelles except for the nucleus. We disclose the first small-molecule probe that selectively detects nuclear ATP through reversible binding, with 25-fold fluorescence enhancement at pH 7.4 and excellent selectivity against various biologically relevant species. Using the probe, we observed 2.1-3.3-fold and 3.9-7.8-fold higher nuclear ATP levels in cancerous cell lines and tumor tissues compared with normal cell lines and tissues, respectively, which are explained by the higher nuclear ATP level in the mitosis phase. The probe has great potential for studying nuclear ATP-associated biology.

Rapid Point-of-Care Quantification of Human Serum Albumin in Urine Based on Ratiometric Fluorescence Signaling Driven by Intramolecular H-Bonding.

Human serum albumin exerts multifunctions, such as maintaining the oncotic pressure of plasma, carrying hydrophobic molecules, and acting as the most important antioxidant in the blood. Lower serum albumin levels are linked to several cardiovascular diseases, and dysfunction of albumin reabsorption in the kidney is linked to liver disease, renal disorder, and diabetes. Albumin is thus a powerful diagnostic and prognostic marker; however, its quantification in urine by readily affordable tools is challenging owing to its very low concentration. To address this issue, we developed a ratiometric fluorescent probe with multiple advantages through a systematic structure variation of a benzocoumarin fluorophore and, further, a prototype of a smartphone-based point-of-care device. We determined albumin levels in urine and observed that a smoking person has notably higher urine albumin than a nonsmoking person. The cheap device provides a promising tool for albumin-associated disease diagnosis in communities with limited resources.

A π-Stacking Based Fluorescent Probe for Labeling of Flavin Analogues in Live Cells through Unusual FRET Process.

The flavin adenine dinucleotide (FAD) is an indispensable coenzyme in live cells. It acts as a catalyst in many redox responsive metabolic reactions, including oxidative phosphorylation in mitochondria. The real-time monitoring of flavin is important to understand the disorder in the metabolic process, redox system, etc. Thus, we have developed a fluorescent probe CPy-1 that noncovalently binds with flavin to exhibit the FRET process. 1H- NMR and docking study indicated that there is a strong hydrophobic interaction between flavins and CPy-1. Also, a π-π stacking between isoalloxazine ring in flavin and quinoline and coumarin moieties of CPy-1 favors self-assembly. The nontoxic probe CPy-1 could distinguish cancer cells from normal cells based on expressions of endogenous FAD.