Hyo Won Lee

Two-photon fluorescent trackers for monitoring of lipid droplets (LDs) would be highly effective for illustrating the critical roles of LDs in live cells or tissues. Although a number of one-photon fluorescent trackers for labeling LDs have been developed, their usability remains constrained in live sample imaging due to photo damage, shallow imaging depth, and auto-fluorescence. Recently, some two-photon fluorescent trackers for LDs have been developed to overcome these limitations. In this mini-review article, the advances in two-photon fluorescent trackers for monitoring of LDs are summarized. We summarize the chemical structures, two-photon properties, live sample imaging, and biomedical applications of the most recent representative two-photon fluorescent trackers for LDs. Additionally, the current challenges and future research trends for the two-photon fluorescent trackers of LDs are discussed.

The accumulation of hepatic lipid droplets (LDs) is a hallmark of non-alcoholic fatty liver disease (NAFLD). Appropriate degradation of hepatic LDs and oxidation of complete free fatty acids (FFAs) are important for preventing the development of NAFLD. Histone deacetylase (HDAC) is involved in the impaired lipid metabolism seen in high-fat diet (HFD)-induced obese mice. Here, we evaluated the effect of MS-275, an inhibitor of HDAC1/3, on the degradation of hepatic LDs and FFA oxidation in HFD-induced NAFLD mice. To assess the dynamic degradation of hepatic LDs and FFA oxidation in fatty livers of MS-275-treated HFD C57BL/6J mice, an intravital two-photon imaging system was used and biochemical analysis was performed. The MS-275 improved hepatic metabolic alterations in HFD-induced fatty liver by increasing the dynamic degradation of hepatic LDs and the interaction between LDs and lysozyme in the fatty liver. Numerous peri-droplet mitochondria, lipolysis, and lipophagy were observed in the MS-275-treated mouse fatty liver. Biochemical analysis revealed that the lipolysis and autophagy pathways were activated in MS-275 treated mouse liver. In addition, MS-275 reduced the de novo lipogenesis, but increased the mitochondrial oxidation and the expression levels of oxidation-related genes, such as PPARa, MCAD, CPT1b, and FGF21. Taken together, these results suggest that MS-275 stimulates the degradation of hepatic LDs and mitochondrial free fatty acid oxidation, thus protecting against HFD-induced NAFLD.

In an attempt to synthesize "Type 2" nicotinic acid crown ethers with a 3-carbinol appendage group, unexpected facile etherification was encountered during the cleavage of the tetrahydropyranyl (THP) group in acidic alcoholic media. The rationale for this reaction was provided by the examination of reactions with non-cyclic model compounds, and their THP derivatives in order to isolate any effects of the crown ether bridge. The 2,6-disubstituted dialkoxy groups were found to contribute to the stabilization of the carbonium ion derived from the protonation of the 3-pyridyl carbinols. Thus, nicotinic crown ether with a 3-carbinol group was prepared with the exclusion of alcoholic media. The preparation of "Type 2" nicotinic acid crown ethers with an aldehyde group was attempted. 3-Formylpyridino crown ether was obtained directly from the reaction of disodium pentaethylene glycolate and the diethoxy acetal derivative of 2,6-dichloro-3-formylpyridine. The syntheses of crown ethers, such as 18-crown-6 and 21-crown-7, with tripyridino moieties were accomplished. The Boekelheide rearrangement was employed to functionalize terminal pyridyl methyl groups to give the corresponding diol, which served as an important intermediate in subsequent cyclization procedures. The 1:1-ketalic macrocycles were transformed to 1,5-diketonic macrocycles, which then were converted to the corresponding tripyridino crown ethers. The X-ray crystal structure of a tripyridino macrocycle trihydrate indicated that a new type of mu-H-bonding for pyridine and water. The same method utilized for tripyridine macrocycles was applied to the synthesis of a pentapyridine macrocycle with a single ether bridge. The ether linkage was formed at the early stage of synthesis. The middle ring of the pentapyridine moiety was formed from an appropriate 1,5-diketone precursour at the last stage of synthesis. In order to prepare a cyclic hexapyridine the bisformyldipyridine was converted to a 1,3-dithiane derivative, which was cyclized with 1,3-dibromopropane. The macrocyclic bis(1,3-dithiane) was cleaved to a tetraketone. And finally cyclic hexapyridine was prepared from the reaction of the tetraketonic macrocycle and hydroxylamine under acidic condition.

Lipid droplets (LDs) are lipid-abundant organelles found in most cell lines and primarily consist of neutral lipids. They serve as a repository of various lipids and are associated with many cellular metabolic processes, including energy storage, membrane synthesis, and protein homeostasis. LDs are prominent in a variety of diseases related to lipid regulation, including obesity, fatty liver disease, diabetes, and atherosclerosis. To monitor LD dynamics in live samples, we developed a highly selective two-photon fluorescent tracker for LDs (LD1). It exhibited outstanding sensitivity with a remarkable two-photon-action cross section (Φδ_max > 600 GM), photostability, and low cytotoxicity. In human hepatocytes and in vivo mouse liver tissue imaging, LD1 showed very bright fluorescence with high LD selectivity and minimized background signal to evaluate the stages of nonalcoholic fatty liver disease. Interestingly, we demonstrated that the liver sinusoid morphology became narrower with increasing LD size and visualized the dynamics including fusion of the LDs in vivo. Moreover, real-time and dual-color TPM imaging with LD1 and a two-photon lysosome tracker could be a useful predictive screening tool in the drug development process to monitor impending drug-induced liver injury inducing drug candidates.

A two-photon fluorogenic probe with the untwisted intramolecular charge process which is comparable to commercial fluorophores has been successfully applied in observing hepatic steatosis.

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.

β-Galactosidase (β-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 β-gal enzyme activity in live cells and tissues. This ratiometric probe showed a fast emission color change (620–662 nm) in response to β-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 β-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.

Background: Endoscopy is the most important tool for gastric cancer diagnosis. However, it relies on naked-eye evaluation by endoscopists, and the histopathologic confirmation is time-consuming. We aimed to visualize and measure the activity of various enzymes through two-photon microscopy (TPM) using fluorescent probes and assess its diagnostic potential in gastric cancer.Methods: β-Galactosidase (β-gal), carboxylesterase (CES), and human NAD(P)H: quinone oxidoreductase (hNQO1) enzyme activities in the normal mucosa, ulcer, adenoma, and gastric cancer biopsy samples were measured using two-photon enzyme probes. The fluorescence emission ratio at long and short wavelengths (Ch2/Ch1) for each probe was comparatively analyzed. Approximately 8,000 – 9,000 sectional images in each group were obtained by measuring the Ch2/Ch1 ratio according to the tissue depth. Each probe was cross-validated by measuring enzymatic activity from a solution containing lysed tissue.Results: Total of 76 subjects were enrolled in this pilot study (normal 21, ulcer 18, adenoma 17, and cancer 20 patients, respectively). There were significant differences in the mean ratio values of β-gal (0.656 ± 0.142 vs. 1.127 ± 0.109, P < 0.001) and CES (0.876 ± 0.049 vs. 0.579 ± 0.089, P < 0.001) between the normal and cancer, respectively. The mean ratio value of cancer tissues was different compared to ulcer and adenoma (P < 0.001). The hNQO1 activity showed no significant difference between cancer and other conditions. Normal mucosa and cancer were visually and quantitatively distinguished through β-gal and CES analyses using TPM images, and enzymatic activity according to depth, was determined using sectional TPM ratiometric images. The results obtained from lysis buffer-treated tissue were consistent with TPM results.Conclusions: TPM imaging using ratiometric fluorescent probes enabled the discrimination of gastric cancer from normal, ulcer, and adenoma. This novel method can help in a visual differentiation and provide quantitative depth profiling in gastric cancer diagnosis.

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.

Imaging and photodynamic therapy through two‐photon excitation can be applied to medical application of deep inside the tissue by minimizing light absorption by the tissue. It is the best solution that can complement the shortcomings of conventional photodynamic therapy. In addition, auto‐fluorescence can be minimized, and damage to normal cells can be avoided through spatial and temporal control. To maximize the image realization and the effect of treatment, a single photosensitizer with high fluorescence quantum yields, two‐photon absorption value, and good¹O₂or reactive oxygen species production performance must be developed. Therefore, recent research related to this topic is summarized. In addition, two‐photon photosensitizers that approach selective imaging and treatment for target diseases and future challenges were discussed.