An indole-based fluorescent chemosensor targeting the autophagosome.

Autophagy is a process for the degradation and recycling of intracellular components and dysfunctional organelles. We developed an indole-embedded fluorescent naphthalimide for the selective imaging of autophagosomes in live cells. It was shown as intense puncta in the fluorescence confocal images and co-localizes with an autophagosome marker, LC3-RFP. In addition, it was applied to cellular autophagic models based on ER stress and starvation to verify its capability.

Chemistry of the carboxylic acid of dihydrofluorescein in oxidation and its application to fluorogenic ROS sensing.

The conjugation site of dihydrofluorescein (H2F) is important for the rational design of H2F-based reactive oxygen species (ROS) sensors. Despite the prevalence of H2F analogs detecting cellular ROS, the role of the carboxylic acid of H2F in oxidation is still unclear. To get insight into the conjugation site of H2F, we synthesized H2F diacetate (2) and its amide derivative (3). The absorption and emission spectra of deacetylated 2 and 3 in the presence of H2O2/hematin showed that the carboxylic acid of H2F plays a crucial role in the oxidation of H2F. NMR and HPLC analysis of the oxidation product of deacetylated 3 showed a quantitative and fast generation of non-fluorescent spirolactam (F-Lactam). As regards these observations, we untouched the carboxylic acid at the 3rd position and designed an H2F-based ROS sensor (7) that conjugated the lipophilic chain at the 5th position instead. A series of confocal microscopic experiments of 7 demonstrated that 7 prefers the ER location and that ROS are elevated in the cells by ER stress inducers.

Naphthalimide-4-(4-nitrophenyl)thiosemicarbazide: A Fluorescent Probe for Simultaneous Monitoring of Viscosity and Nitric Oxide in Living Cells.

Intracellular viscosity is a physicochemical factor that determines the outcomes of various biological processes, while nitric oxide (NO) is an essential signaling molecule that controls many cellular processes, including oxidative stress. Anticipating that both may be interrelated with a variety of pathologies, their simultaneous measurement would be highly valuable for the investigation of the pathological condition of cells. However, the development of a sensor for such simultaneous detection has not been attempted yet. Herein, we present the synthesis of naphthalimide-4-(4-nitrophenyl)thiosemicarbazide, probe 1, and its application to living cells under conditions of lipopolysaccharide or nystatin treatment, adopted as oxidative stress and altered intracellular viscosity models, respectively. The probe showed increased fluorescence in response to elevation of viscosity and NO levels at 470 and 550 nm, respectively, in the solution studies. When the probe was used for a confocal microscopic study of HeLa cells under stressed conditions, simultaneous monitoring of viscosity and NO level elevations was possible through fluorescence imaging using band-pass filters of 420-475 and 505-600 nm, respectively, upon excitation at a wavelength of 405 nm. Interestingly, both the cellular viscosity and NO levels increased together under lipopolysaccharide or nystatin treatment. Therefore, we suggest that probe 1 is a fluorescent chemical probe that enables the monitoring of alterations in intracellular viscosity and NO levels in living cells, which would be valuable in studies of various cellular damage models.

Binary Prodrug of Dichloroacetic Acid and Doxorubicin with Enhanced Anticancer Activity.

The inevitable challenge in conventional chemotherapy is to deliver the anticancer drugs to the dense population of tumors cells while minimizing the drug-associated side effects on the normal cells. Cancer cells' preference for glycolysis for energy production is well recognized. Intuitively, taking advantage of such cancer-associated metabolism would be a promising strategy for anticancer drug delivery with minimal side effects. In this investigation, we have designed a binary prodrug PDOX as a sequential drug delivery regimens to realize the combination therapy for cancer. As cancer cells exhibit abrupt metabolism with elevated pyruvate dehydrogenase kinase (PDK) activity, dichloroacetic acid (DCA, a well-known PDK inhibitor) was used in combination with anticancer drug doxorubicin (DOX). The designed molecular prodrug was activated selectively by cancer-associated esterase to deliver DCA and DOX, respectively, and induced synergetic effects. Hence, sequential targeted delivery of molecular prodrug PDOX offers a promising approach to overcome the offside drug toxicity, pharmacokinetics, and biodistribution of individuals and provide an alternative option for cancer treatment.

Self-Calibrating Bipartite Fluorescent Sensor for Nitroreductase Activity and Its Application to Cancer and Hypoxic Cells.

Aromatic nitro compounds are reduced to their corresponding amino derivatives by nitroreductases (NTR), while identification and characterization of the corresponding enzymes in mammalian systems are yet unrevealed. However, mammalian NTR activity has been considered as a favorable target in development of theranostic agents for cancer and hypoxia of solid tumors. Currently, small molecule-based fluorescent probes have emerged as a valuable assay tool for NTR activity. However, there has been a limit to comparing NTR activity between different cells, since most probes have relied on fluorescence changes that are affected by not only enzymatic activity but also nonenzymatic factors. Here, we developed a self-calibrating bipartite fluorescent probe, consisting of NTR-sensitive nitronaphthalimide and nonsensitive coumarin moieties. Thereby, it was possible to compare the relative NTR activity by monitoring fluorescence ratios in noncancerous and some cancerous cells and to demonstrate for certain that the elevated NTR activity is associated with cancer cells and hypoxia states.

Revealing Protein Aggregates under Thapsigargin-Induced ER Stress Using an ER-Targeted Thioflavin.

Endoplasmic reticulum-thioflavin T (ER-ThT), a thioflavin T-based fluorescent chemosensor, was developed to detect protein aggregates in the endoplasmic reticulum (ER) and was applied to live cells under various forms of ER stress. Upon dithiothreitol (DTT)-induced reductive denaturation of lysozyme and albumin, the intensity was increased in a protein concentration-dependent way, following a nonfluorescent lag phase. ER-ThT detects protein aggregates rather than unfolded proteins in solution, and the protein aggregation can be visualized in the presence of lipid membranes or native proteins. Within live HeLa cells, ER-ThT is localized in the ER and its fluorescence was dramatically increased upon ER stress induction by DTT, Thapsigargin, or Brefeldin A. Moreover, in the presence of ER stress modulators (tauroursodeoxycholic acid, trimethylamine N-oxide, or 4-phenylbutyric acid), also known as chemical chaperones, the fluorescence under Thapsigargin treatment was suppressed to the level of the control group. Thus, ER-ThT is capable of detecting the accumulation of protein aggregates under ER stress in living cells and acts as an in vitro screening tool for ER stress modulators, putative prodrugs against ER-related proteopathy. Overall, the results strongly suggest that protein aggregation is intricately involved in the activation of the unfolded protein response following ER stress.

A red-emitting styrylnaphthalimide-based fluorescent probe providing a ratiometric signal change for the precise and quantitative detection of H2O2.

A red-emitting and ratiometric fluorescence probe 1 for detecting H2O2, based on a styrylnaphthalimide-boronate ester was developed. Upon a H2O2-mediated hydrolysis of boronate ester, probe 1 was transformed to 2 with a ratiometric fluorescence change, decrease at 535 and increase at 640 nm. It was also found that the fluorescent reaction of 1 with H2O2 in solution could be completed within 10 min and the detection limit was estimated to be 0.30 µM. Moreover, this ratiometric change was highly selective for H2O2 over other redox species, metal ions, and anions. Also, this system was found to be capable of detecting H2O2 in the pH range of 6-9. Furthermore, probe 1 was preferentially accumulated into the endoplasmic reticulum (ER) in the live HeLa cells, and an increased H2O2 level in the presence of an ER stress inducer, thapsigargin was revealed.

Emerging two-dimensional monoelemental materials (Xenes) for biomedical applications.

The emergence of novel two-dimensional (2D) monoelemental materials (Xenes) has shown remarkable potential for their applications in different fields of technology, as well as addressing new discoveries in fundamental science. Xenes (e.g., borophene, silicene, germanene, stanene, phosphorene, arsenene, antimonene, bismuthene, and tellurene) are of particular interest because they are the most chemically tractable materials for synthetic exploration. Owing to their excellent physical, chemical, electronic and optical properties, Xenes have been regarded as promising agents for biosensors, bioimaging, therapeutic delivery, and theranostics, as well as in several other new bio-applications. In this tutorial review, we summarize their general properties including the classification of Xenes according to their bulk properties. The synthetic and modification methods of Xenes are also presented. Furthermore, the representative Xene nanoplatforms for various biomedical applications are highlighted. Finally, research progress, challenges, and perspectives for the future development of Xenes in biomedicines are discussed.

Dual-functional fluorescent molecular rotor for endoplasmic reticulum microviscosity imaging during reticulophagy.

The microviscosity change associated with reticulophagy is an important component for studying endoplasmic reticulum (ER) stress disorders. Here, a BODIPY-arsenicate conjugate 1-based fluorescent molecular rotor was designed to covalently bind vicinal dithiol-containing proteins in the ER, exhibiting a bifunction of reticulophagy initiation and microviscosity evaluation. Therefore, we could quantify the local viscosity changes during reticulophagy based on the fluorescence lifetime changes of probe 1.

Binary Drug Reinforced First Small-Molecule-Based Prodrug for Synergistic Anticancer Effects.

We developed a small-molecule-based binary drug delivery system (BDDS) with two anticancer drugs, SN-38 and 5'-DFUR. The drug release from the prodrug BDDS can be achieved upon its reaction with intracellular H2O2, overexpressed in cancer cells. The efficacy of BDDS was demonstrated by a comparative study along with that of a single drug conjugate (SDDS), bearing SN-38 alone.

High-depth fluorescence imaging using a two-photon FRET system for mitochondrial pH in live cells and tissues.

We developed a fluorescent pH probe (1) capable of two-photon excitation and far-visible-emission based on FRET, composed of naphthalimide-piperazine-rhodamine. It exhibited a pH-dependent reversible and fast ratiometric fluorescence change in the rhodamine emission. Probe 1 was applied to image the pH perturbations of mitochondria in living cells and tissues.

Glycyrrhetinic acid as a hepatocyte targeting unit for an anticancer drug delivery system with enhanced cell type selectivity.

Herein, we report the potential of glycyrrhetinic acid (GA) as an active targeting ligand for hepatocellular carcinoma (HCC) for the development of diagnosis/therapy using small-molecule based approaches. Our preliminary results demonstrated that GA-conjugation to diagnostic/therapeutic counterparts significantly enhanced their HCC targeting ability and excellent therapeutic efficacy.

Modulating the GSH/Trx selectivity of a fluorogenic disulfide-based thiol sensor to reveal diminished GSH levels under ER stress.

We synthesized a fluorogenic disulfide-based naphthalimide thiol probe (ER-Naph) with a hydrophilic endoplasmic reticulum (ER)-guiding glibenclamide unit. Its ER targeting ability and high selectivity to GSH over thioredoxin, a potent competitor, were clearly demonstrated, both in solution and in vitro. Finally, a confocal microscopic investigation revealed that GSH levels in the ER were dramatically decreased under thapsigargin, brefeldin A, and tunicamycin-induced ER stress models.

A Fluorescent Probe for Stimulated Emission Depletion Super-Resolution Imaging of Vicinal-Dithiol-Proteins on Mitochondrial Membrane.

Realizing the significant roles of vicinal-dithiol proteins (VDPs) in maintaining the cellular redox homeostasis and their implication in many diseases, we synthesized a smart arsenate based fluorescent probe 1 which can preferentially target the mitochondrial membrane-bound vicinal dithiol proteins (VDPs), especially voltage-dependent anion channel (VDAC2). The probe targetability was demonstrated by in vitro studies such as colocalization, stimulated emission depletion (STED) super-resolution imaging, proteomic MS/MS analysis, and Western blot analysis. The probe represents a rare example of fluorescence labeling of mitochondrial membrane-bound VDPs and can provide a new way to construct VDPs-specific fluorescent probes to gain deeper understanding of their roles in mitochondrial-related disorders.

Shedding light on tau protein aggregation: the progress in developing highly selective fluorophores.

Historically, in Alzheimer's disease research, a lot of attention has been paid to the development of highly selective fluorophores for beta amyloid plaques. With a shift in the understanding of the disease and the importance of a network of cross-talk interactions, the development of small-molecule fluorescent dyes with high selectivity for (hyperphosphorylated) tau protein aggregates in neurofibrillary tangles has been gaining increasing attention. Fluorescent dyes for the selective labelling of tau aggregates in histological AD brain sections have been described, spanning the entire visible range of the electromagnetic spectrum. Despite the relatively early stages of the development of the field, a large diversity in probe architectures has been reported. Importantly, a handful of near-infrared-emissive dyes have been described as well, and some of these have exhibited good pharmacological profiles, with a significant blood-brain-barrier permeability, and a demonstrated ability to label tau tangles in vivo in small-animal models of Alzheimer's disease and other tauopathies. The developments summarized in the current work are expected to aid the unravelling of the diverse set of players in the etiology of Alzheimer's disease. In this tutorial review, we seek to provide the reader with an overview of the most important recent developments and hope to provide some guidelines for the design of future probes.

Fluorogenic reaction-based prodrug conjugates as targeted cancer theranostics.

Theranostic systems are receiving ever-increasing attention due to their potential therapeutic utility, imaging enhancement capability, and promise for advancing the field of personalized medicine, particularly as it relates to the diagnosis, staging, and treatment of cancer. In this Tutorial Review, we provide an introduction to the concepts of theranostic drug delivery effected via use of conjugates that are able to target cancer cells selectively, provide cytotoxic chemotherapeutics, and produce readily monitored imaging signals in vitro and in vivo. The underlying design concepts, requiring the synthesis of conjugates composed of imaging reporters, masked chemotherapeutic drugs, cleavable linkers, and cancer targeting ligands, are discussed. Particular emphasis is placed on highlighting the potential benefits of fluorogenic reaction-based targeted systems that are activated for both imaging and therapy by cellular entities, e.g., thiols, reactive oxygen species and enzymes, which are present at relatively elevated levels in tumour environments, physiological characteristics of cancer, e.g., hypoxia and acidic pH. Also discussed are systems activated by an external stimulus, such as light. The work summarized in this Tutorial Review will help define the role fluorogenic reaction-based, cancer-targeting theranostics may have in advancing drug discovery efforts, as well as improving our understanding of cellular uptake and drug release mechanisms.

Rational Design of in Vivo Tau Tangle-Selective Near-Infrared Fluorophores: Expanding the BODIPY Universe.

The elucidation of the cause of Alzheimer's disease remains one of the greatest questions in neurodegenerative research. The lack of highly reliable low-cost sensors to study the structural changes in key proteins during the progression of the disease is a contributing factor to this lack of insight. In the current work, we describe the rational design and synthesis of two fluorescent BODIPY-based probes, named Tau 1 and Tau 2. The probes were evaluated on the molecular surface formed by a fibril of the PHF6 (306VQIVYK311) tau fragment using molecular docking studies to provide a potential molecular model to rationalize the selectivity of the new probes as compared to a homologous Aß-selective probe. The probes were synthesized in a few steps from commercially available starting products and could thus prove to be highly cost-effective. We demonstrated the excellent photophysical properties of the dyes, such as a large Stokes shift and emission in the near-infrared window of the electromagnetic spectrum. The probes demonstrated a high selectivity for self-assembled microtubule-associated protein tau (Tau protein), in both solution and cell-based experiments. Moreover, the administration to an acute murine model of tauopathy clearly revealed the staining of self-assembled hyperphosphorylated tau protein in pathologically relevant hippocampal brain regions. Tau 1 demonstrated efficient blood-brain barrier penetrability and demonstrated a clear selectivity for tau tangles over Aß plaques, as well as the capacity for in vivo imaging in a transgenic mouse model. The current work could open up avenues for the cost-effective monitoring of the tau protein aggregation state in animal models as well as tissue staining. Furthermore, these fluorophores could serve as the basis for the development of clinically relevant sensors, for example based on PET imaging.

A Mitochondria-Targeted Cryptocyanine-Based Photothermogenic Photosensitizer.

Cryptocyanine-based probes exhibit highly efficient photothermal conversion and represent a new class of photothermal agents for use in photothermal therapy (PTT). With the thermal susceptibility of mitochondria in mind, we have prepared a mitochondria-targeted, NIR-absorbing cryptocyanine probe (Mito-CCy) and evaluated its photophysical properties, photothermal conversion efficiency, biological compatibility, cytotoxicity, and mitochondrial localization in HeLa cells. Upon subjecting 0.5 mL of a PBS buffer solution (10 mM, pH 7.4, containing 50% DMSO) of Mito-CCy (0.5 mM) to 730 nm laser irradiation at 2.3 W/cm2, the temperature of the solution increased by 13.5 °C within 5 min. In contrast, the corresponding cryptocyanine (CCy) lacking the triarylphosphonium group gave rise to only an ∼3.4 °C increase in solution temperature under otherwise identical conditions. Mito-CCy also exhibited high cytotoxicity in HeLa cells when subject to photoirradiation. This light-induced cytotoxicity is attributed to the endogenous production of reactive oxygen species (ROS) induced under conditions of local heating. ROS are known to interfere with the mitochondrial defense system and to trigger apoptosis. By targeting the mitochondria, the present sensitizer-based photothermogenic approach is rendered more effective. As such, the system reported here represents the vanguard of what might be a new generation of organelle-targeted photothermal therapeutics.

Visualization of vesicular transport from the endoplasmic reticulum to lysosome using an amidine derived two-photon probe.

We report an amidine-based small molecule two-photon fluorescent probe (ELP1) for monitoring vesicle transport from the ER to lysosome in live cells. Two-photon microscopy imaging studies indicated that this probe initially localized in the ER and subsequently transferred to lysosomal compartments through vesicular transport. These results may provide an effective tool for studying trafficking-related biology and pathology in real-time.

An endoplasmic reticulum-selective ratiometric fluorescent probe for imaging a copper pool.

Hydrazide-linked naphthalimides undergo copper ion-selective hydrolysis with concomitant ratiometric fluorescence change. In addition, probe 1 is biocompatible and mainly localized to the endoplasmic reticulum (ER) in live HeLa cells. It thus can be used for imaging copper accumulation in the ER of live cells, as implied in copper-overloaded situations.