Cancer-Targeted Azo Dye for Two-Photon Photodynamic Therapy in Human Colon Tissue.

Inappropriate cancer management can be prevented by simultaneous cancer diagnosis, treatment, and real-time assessment of therapeutic processes. Here, we describe the design of a two-photon (TP) photosensitizer (PS), ACC-B, for high temporal and spatioselective near-infrared cancer therapy. ACC-B consisting of a biotin unit significantly enhanced the cancer sensitivity of the PS. Upon TP irradiation, ACC-B generated reactive oxygen species (ROS) through the type I photodynamic therapy (PDT) process and triggered highly selective cancer ablation. In addition, fluorescence microscopy images revealed that ACC-B-loaded live human colon tissues showed a marked difference in ACC-B uptake between normal and cancer tissues, and this property was used for real-time imaging. Upon 770 nm TP treatment, ACC-B generated ROS efficiently in live colon cancer tissues with high spatial selectivity. During PDT, ACC-B can provide in situ spatioselective visualization of cellular behavior and molecular information for therapeutic assessment in specific regions.

Highly Stable Red-Emissive Ratiometric Probe for Monitoring ß-Galactosidase Activity Using Fluorescence Microscopy and Flow Cytometry.

ß-Galactosidase (ß-gal), well known as a useful reporter enzyme, is a potent biomarker for various diseases such as colorectal and ovarian cancers. We have developed a highly stable red-emissive ratiometric fluorescent probe (CCGal1) for quantitatively monitoring the ß-gal enzyme activity in live cells and tissues. This ratiometric probe showed a fast emission color change (620-662 nm) in response to ß-gal selectively, which was accompanied by high enzyme reaction efficacy, cell-staining ability, and outstanding stability with minimized cytotoxicity. Confocal fluorescence microscopy ratiometric images, combined with fluorescence-activated cell sorting flow cytometry, demonstrated that CCGal1 could provide useful information for the diagnosis, prognosis, and treatment of ß-gal enzyme activity-related diseases such as colorectal and ovarian cancers. Further, it may yield meaningful strategies for designing and modifying multifunctional bioprobes with different biomedical applications.

A red-emissive two-photon fluorescent probe for mitochondrial sodium ions in live tissue.

A cyclocyanine (CC)-based organic small molecule two-photon (TP) fluorescent probe (CCNa1) was developed for mitochondrial sodium ion sensing. CCNa1 exhibits a low solvatochromic shift and strong TP fluorescence enhancement at 575 nm upon binding to Na+ and is insensitive to other metal ions and to pH. CCNa1 demonstrated fast cell loading ability, biocompatibility, and sensitive response to mitochondrial Na+ influx in live cells and mouse brain tissue.

Near-Infrared Ratiometric Two-Photon Probe for pH Measurement in Human Stomach Cancer Tissue.

Stomach cancer is a global health issue because of its incidence and mortality rates worldwide. We developed a near-infrared (NIR) emissive ratiometric two-photon (TP) probe (HCC1) for the quantitative analysis of pH in live cells and human stomach tissues. The probe design is based on a restrained hemicyanine core that controls the intramolecular charge transfer from 2-naphthol, with a suitable pKa value (7.50) under physiological conditions. The probe exhibited improved quantum yield, stability, and TP activity under physiological conditions. In addition, intracellular pH titration (pH 4.0 to 10.0) of HCC1 revealed an ideal intracellular pKa of approximately 7.2, negligible cytotoxicity, and TP excited fluorescence in situ, thereby allowing direct imaging of the cellular pH in live cells and tissues. Ratiometric two-photon microscope imaging with HCC1 of human stomach tissue revealed a clear intratissue pH variation among normal, adenoma, and cancer tissues. Our results demonstrate that HCC1 is useful as an NIR imaging probe for in situ pH-related studies and in cancer research.

Two-Photon Fluorescent Probes for Detecting Enzyme Activities in Live Tissues.

Enzyme regulation is crucial in living organisms to catalyze various biosyntheses to maintain several physiological functions. On the contrary, abnormal enzyme activities can affect bioactivities leading to various serious disorders including cancer, Alzheimer's disease, Parkinson's disease, heart disease, and so on. This biological significance led to the development of various techniques to map specific enzyme activities in living systems to understand their role and distribution. Two-photon microscopy (TPM) in particular has emerged as a promising system for in situ real-time bioimaging owing to its robustness, high sensitivity, and noninvasiveness. It was achieved through the use of a two-photon (TP) light source of an optical window (700-1450 nm) beneficial in deeper light penetration and extraordinary spatial selectivity. Therefore, developing enzyme sensors utilized in TPM has significance in obtaining in vivo enzyme activities with minimal perturbation. The development of an efficient detection tool for enzymes has been continuously reported in the previous literature; here, we meticulously review the TP design strategies that have been attempted by researchers to develop enzyme TP fluorescent sensors that are proving very useful in providing insights for enzyme investigation in the biological system. In this review, the representative TP enzymatic probes that have been made in the past 5 years and their applications in tissue imaging are discussed in brief. In addition, the prospects and challenges of TP enzymatic probe development are also discussed.

Screening of Drug-Induced Steatosis and Phospholipidosis Using Lipid Droplet-Selective Two-Photon Probes.

Lipid droplets (LDs) are organelles that play a major role in regulating the storage of neutral lipids. Dysregulation of LDs is associated with metabolic disorders, such as fatty liver diseases, obesity, diabetes, and atherosclerosis. We have developed LD-selective small-molecule fluorescence probes (probes 3 and 4) that are available for both one- and two-photon microscopy, employing live or fixed cells. We found that probes 3 and 4 sensitively detect the increased LDs in response to oleic acid or endoplasmic reticulum stress, both in cells and tissues of the liver. The narrow absorption and emission bands of probes 3 and 4 allow multicolor imaging for the study of the role of LDs in pathophysiology and LD-associated signaling by the coapplication of the probes for different organelles or antibodies against specific proteins. In addition, we show here, for the first time, that two-photon microscopy imaging using our LD-selective probes with LysoTracker provides a novel method for screening drugs to potentially induce steatosis and/or phospholipidosis.

Combining hydrophilic and hydrophobic environment sensitive dyes to detect a wide range of cellular polarity.

Intracellular polarity is an important parameter of pathological and biological phenomena of cells; abnormal polarities are associated with diabetes, neurological diseases, and cancer. However, previously reported polarity probes have issues with quantitatively detecting intracellular polarities, can measure only a limited range of polarities, and can only detect specific intracellular regions. Here, we developed a novel two-dye system, RPS-1, that contains a new "turn-on" polarity probe (Dye1) based on a spiropyran intramolecular ring closing-opening system activated in polar protic solvents, and a benzothiadiazole containing dye (Dye3), which emits only in non-polar solvents with a large stoke shift. Individually, Dye1 and Dye3 selectively localized to lysosome and lipid droplets, respectively; however, combining these dyes, which have completely different characteristics, via a piperazine linker resulted in the staining of various intracellular organelles. Therefore, as Dye1 and Dye3 have the same absorption but different emissions, combining them resulted in a ratiometric polarity probe that could quantitatively measure a wider polarity range inside the cell using a single excitation source. In addition, ratiometric imaging using our RPS-1 probe to quantitatively detect the distribution of polarity in different cell lines indicated that lysosomes were the most polar organelles in the cell.

An azo dye for photodynamic therapy that is activated selectively by two-photon excitation.

Two-photon photodynamic therapy (TP-PDT) is a promising approach for the treatment of cancer because of its better penetration depth and superior spatial selectivity. Here, we describe an azo group containing cyclized-cyanine derivatives (ACC1 and ACC2) as a two-photon activated, type I based photosensitizer (PS). These small-molecule and heavy atom-free organic dyes showed marked reactive oxygen species (ROS)-generating ability under physiological conditions, as well as fast loading ability into the cells and negligible dark toxicity. Live cell analyses with one- and two-photon microscopy revealed that these dyes showed higher ROS generation ability upon two-photon excitation than upon one-photon excitation via the type I process. The PSs have superior PDT properties compared to conventional Visudyne and 5-ALA under mild conditions. These characteristics allowed for precise PDT at the target region in mimic tumor spheroids, demonstrating that the developed TP PS could be useful in efficient PDT applications and in designing various PSs.

Pyroacm Resin: An Acetamidomethyl Derived Resin for Solid Phase Synthesis of Peptides through Side Chain Anchoring of C-Terminal Cysteine Residues.

The design, synthesis and utilization of an efficient acetamidomethyl derived resin for the peptide synthesis is presented using established Fmoc and Boc protocols via side chain anchoring. Cleavage of the target peptide from the resin is performed using carboxymethylsulfenyl chloride under mild conditions which gave in situ thiol-sulfenyl protection of the cysteine residues. The utility of the resin is successfully demonstrated through applications to the syntheses of model peptides and natural products Riparin 1.1 and Riparin 1.2.

Novel 3-arylethynyl-substituted thieno[3,4-b]pyrazine derivatives as human transglutaminase 2 inhibitors.

In the process of optimization, we developed a novel core skeleton of thieno[3,4-b]pyrazine via GK-13. The derivatives synthesized were shown to inhibit TGase 2 activity in cancer cells. Some of the hit compounds such as the arylethynyl group-coupled thieno[3,4-b]pyrazine derivatives were shown to exhibit promising activity for use as potential therapeutic small-molecules in renal cancer by inhibiting TGase 2 activity.