Unique Synthetic Strategy for Probing in Situ Lysosomal NO for Screening Neuroinflammatory Phenotypes against SARS-CoV-2 RNA in Phagocytotic Microglia.

In the pathogenesis of microglia, brain immune cells promote nitrergic stress by overproducing nitric oxide (NO), leading to neuroinflammation. Furthermore, NO has been linked to COVID-19 progression, which has caused significant morbidity and mortality. SARS-CoV-2 infection activates inflammation by releasing excess NO and causing cell death in human microglial clone 3 (HMC3). In addition, NO regulates lysosomal functions and complex machinery to neutralize pathogens through phagocytosis. Therefore, developing lysosome-specific NO probes to monitor phagocytosis in microglia during the COVID-19 infection would be a significant study. Herein, a unique synthetic strategy was adopted to develop a NO selective fluorescent probe, PDM-NO, which can discriminate activated microglia from their resting state. The nonfluorescent PDM-NO exhibits a turn-on response toward NO only at lysosomal pH (4.5-5.5). Quantum chemical calculations (DFT/TD-DFT/PCM) and photophysical study revealed that the photoinduced electron transfer (PET) process is pivotal in tuning optical properties. PDM-NO demonstrated good biocompatibility and lysosomal specificity in activated HMC3 cells. Moreover, it can effectively map the dynamics of lysosomal NO against SARS-CoV-2 RNA-induced neuroinflammation in HMC3. Thus, PDM-NO is a potential fluorescent marker for detecting RNA virus infection and monitoring phagocytosis in HMC3.

A Photoinduced Electron Transfer-Based Hypochlorite-Specific Fluorescent Probe for Selective Imaging of Proinflammatory M1 in a Rheumatoid Arthritis Model.

The differentiation of the distinct phenotypes of macrophages is essential for monitoring the stage of inflammatory diseases for accurate diagnosis and treatment. Recent studies revealed that the level of hypochlorite (OCl-) varies from activated M1 macrophages (killing pathogens) to M2 (resolution of inflammation) during inflammation. Thus, we developed a simple and efficient fluorescent probe for discriminating M1 from M0 and M2. Herein, fluorescent-based imaging is applied as an alternative to immunohistochemistry, which is challenging due to the tedious process and high cost. We developed a hypochlorite-specific probe PMS-T to differentiate M1 and M2, employing a metabolism-oriented live-cell distinction. This probe enables the detection of inflammatory rheumatoid arthritis in an ex vivo mouse model. Thus, it can be a potential chemical tool for monitoring inflammatory diseases, including rheumatoid arthritis, that may overcome the existing barriers of immunohistochemistry.

Esterase Specific Fluorescent Probe: Mechanistic Understanding Using QM/MM Calculation and Cell States Discrimination.

Esterases enzymes regulate the body's homeostasis by catalyzing the hydrolysis of various esters. These are also involved in protein metabolism, detoxification, and signal transmission. Most importantly, esterase plays a significant role in cell viability and cytotoxicity assays. Hence, developing an efficient chemical probe is essential for monitoring the esterase activity. Several fluorescent probes for esterase have also been reported targeting cytosol and lysosomes. However, the ability to create efficient probes is constrained due to a lack of understanding of the esterase's active site for hydrolyzing the substrate. In addition, the fluorescent turn-on may limit efficient monitoring. Herein, we have developed a unique fluorescent probe, PM-OAc, to monitor mitochondrial esterase enzyme activity ratiometrically. This probe exhibited a bathochromic wavelength shift with esterase enzyme in alkaline pH (pH∼8.0) due to an intramolecular charge transfer (ICT) process. The phenomenon is well supported by TD-DFT calculation. Moreover, the substrate (PM-OAc) binding at the active site of esterase and its catalytic mechanism to hydrolyze the ester bond are elucidated by molecular dynamics (MD) simulation and QM/MM (Quantum mechanics/molecular mechanics) calculations, respectively. Fluorescent image-based analysis of the cellular environment reveals that our probe can distinguish between live and dead cells based on esterase enzyme activity.

Ratiometric fluorescent probes for pH mapping in cellular organelles.

The intracellular pH (pHi) in organelles, including mitochondria, endoplasmic reticulum, lysosomes, and nuclei, differs from the cytoplasmic pH, and thus maintaining the pH of these organelles is crucial for cellular homeostasis. Alterations in the intracellular pH (ΔpHi) in organelles lead to the disruption of cell proliferation, ion transportation, cellular homeostasis, and even cell death. Hence, accurately mapping the pH of organelles is crucial. Accordingly, the development of fluorescence imaging probes for targeting specific organelles and monitoring their dynamics at the molecular level has become the forefront of research in the last three decades. Among them, ratiometric fluorescent probes minimize the interference from the excitation wavelength of light, auto-fluorescence from probe concentration, environmental fluctuations, and instrument sensitivity through self-correction compared to monochromatic fluorescent probes, which are known as turn-on/off fluorescent probes. Small-molecular ratiometric fluorescent probes for detecting ΔpHi are challenging yet demanding. To date, sixty-two ratiometric pH probes have been reported for monitoring internal pH alterations in cellular organelles. However, a critical review on organelle-specific ratiometric probes for pH mapping is still lacking. Thus, in the present review, we report the most recent advances in ratiometric pH probes and the previous data on the role of mapping the ΔpHi of cellular organelles. The development strategy, including ratiometric fluorescence with one reference signal (RFRS) and ratiometric fluorescence with two reversible signals (RFRvS), is systematically illustrated. Finally, we emphasize the major challenges in developing ratiometric probes that merit further research in the future.

PET- and ICT-Based Ratiometric Probe: An Unusual Phenomenon of Morpholine-Conjugated Fluorophore for Mitochondrial pH Mapping during Mitophagy.

Mitochondrial functions are heavily influenced by acid-base homeostasis. Hence, elucidation of the mitochondrial pH is essential in living cells, and its alterations during pathologies is an interesting question to be addressed. Small molecular fluorescent probes are progressively applied to quantify the mitochondrial pH by fluorescence imaging. Herein, we designed a unique small molecular fluorescent probe, PM-Mor-OH, based on the lipophilic morpholine ligand-conjugated pyridinium derivative of "IndiFluors". The morpholine-conjugated fluorescent probe usually localized the lysosome. However, herein, we observed unusual phenomena of morpholine-tagged PM-Mor-OH that localized mitochondria explicitly. The morpholine ligand also plays a pivotal role in tuning optical properties via photoinduced electron transfer (PET) during internal pH alteration (ΔpHi). In the mitophagy process, lysosomes engulf damaged mitochondria, leading to ΔpHi, which can be monitored using our probe. It exhibited "ratiometric" emission at single wavelength excitation (ex. 488) and is suitable for monitoring and quantifying the ΔpHi using confocal microscope high-resolution image analysis during mitophagy. The bathochromic emission shifts due to intramolecular charge transfer (ICT) in basic pH were well explained by the time-dependent density functional theory (TD-DFT/PCM). Similarly, the change in the emission ratio (green/red) with pH variations was also validated by the PET process. In addition, PM-Mor-OH can quantify the pH change during oxidative stress induced by rapamycin, mutant A53T α-synuclein-mediated protein misfolding stress in mitochondria, and during starvation. Rapamycin-induced mitophagy was further elucidated by the translocation of mCherry Parkin to damaged mitochondria, which well correlates with our probe. Thus, PM-Mito-OH is a valuable probe for visualizing mitophagy and can act as a suitable tool for the diagnosis of mitochondrial diseases.

A Near-Infrared Probe Tracks and Treats Lung Tumor Initiating Cells by Targeting HMOX2.

Tumor initiating cells (TIC) are resistant to conventional anticancer therapy and associated with metastasis and relapse in cancer. Although various TIC markers and their antibodies have been proposed, it is limited to the use of antibodies for in vivo imaging or treatment of TIC. In this study, we discovered heme oxygenase 2 (HMOX2) as a novel biomarker for TIC and developed a selective small molecule probe TiNIR (tumor initiating cell probe with near infrared). TiNIR detects and enriches the functionally active TIC in human lung tumors, and through the photoacoustic property, TiNIR also visualizes lung TIC in the patient-derived xenograft (PDX) model. Furthermore, we demonstrate that TiNIR inhibits tumor growth by blocking the function of HMOX2, resulting in significantly increased survival rates of the cancer model mice. The novel therapeutic target HMOX2 and its fluorescent ligand TiNIR will open a new path for the molecular level of lung TIC diagnosis and treatment.

A Cyclometalated IrIII Complex as a Lysosome-Targeted Photodynamic Therapeutic Agent for Integrated Imaging and Therapy in Cancer Cells.

Organelle-targeted photosensitizers (PSs) having luminescence properties are potential theranostic agents for simultaneous luminescence imaging and photodynamic therapy. Herein, we report a water-soluble luminescent cyclometalated IrIII complex, Ir-Bp-Ly, as a lysosome-targeted theranostic probe. Ir-Bp-Ly exhibits exceptional photophysical properties, with good triplet-state quantum yield (0.90), singlet oxygen generation quantum yield (0.71 at pH 4), and long lifetime (1.47 µs). Interestingly, Ir-Bp-Ly localizes mostly in lysosomes due to the presence of morpholine units, suggesting its potential use as a lyso-tracker. Ir-Bp-Ly displays a notable PDT effect in C6 glioma cells, efficiently generating reactive oxygen species owing to close proximity between the energy levels of its triplet state and those of molecular oxygen (3 O2 ). The mechanism of cell death has been studied through caspase-3/7 and flow cytometry analyses, which clearly established the apoptotic pathway.

Real-Time In†Vivo Hepatotoxicity Monitoring through Chromophore-Conjugated Photon-Upconverting Nanoprobes.

Drug toxicity is a long-standing concern of modern medicine. A typical anti-pain/fever drug paracetamol often causes hepatotoxicity due to peroxynitrite ONOO- . Conventional blood tests fail to offer real-time unambiguous visualization of such hepatotoxicity in vivo. Here we report a luminescent approach to evaluate acute hepatotoxicity in vivo by chromophore-conjugated upconversion nanoparticles. Upon injection, these nanoprobes mainly accumulate in the liver and the luminescence of nanoparticles remains suppressed owing to energy transfer to the chromophore. ONOO- can readily bleach the chromophore and thus recover the luminescence, the presence of ONOO- in the liver leads to fast restoring of the near-infrared emission. Taking advantages of the high tissue-penetration capability of near-infrared excitation/emission, these nanoprobes achieve real-time monitoring of hepatotoxicity in living animals, thereby providing a convenient screening strategy for assessing hepatotoxicity of synthetic drugs.

A Diradical Approach towards BODIPY-Based Dyes with Intense Near-Infrared Absorption around λ=1100†nm.

A diradical approach to obtain stable organic dyes with intense absorption around λ=1100 nm is reported. The para- and meta-quinodimethane-bridged BODIPY dimers BD-1 and BD-2 were synthesized and were found to have a small amount of diradical character. These molecules exhibited very intense absorption at λ=1088 nm (ɛ=6.65×10(5)  M(-1) cm(-1) ) and 1136 nm (ɛ=6.44×10(5)  M(-1) cm(-1) ), respectively, together with large two-photon-absorption cross-sections. Structural isomerization induced little variation in their diradical character but distinctive differences in their physical properties. Moreover, the compounds showed a selective fluorescence turn-on response in the presence of the hydroxyl radical but not with other reactive oxygen species.

High-efficiency in vitro and in vivo detection of Zn2+ by dye-assembled upconversion nanoparticles.

Development of highly sensitive and selective sensing systems of divalent zinc ion (Zn(2+)) in organisms has been a growing interest in the past decades owing to its pivotal role in cellular metabolism, apoptosis, and neurotransmission. Herein, we report the rational design and synthesis of a Zn(2+) fluorescent-based probe by assembling lanthanide-doped upconversion nanoparticles (UCNPs) with chromophores. Specifically, upconversion luminescence (UCL) can be effectively quenched by the chromophores on the surface of nanoparticles via a fluorescence resonant energy transfer (FRET) process and subsequently recovered upon the addition of Zn(2+), thus allowing for quantitative monitoring of Zn(2+). Importantly, the sensing system enables detection of Zn(2+) in real biological samples. We demonstrate that this chromophore-UCNP nanosystem is capable of implementing an efficient in vitro and in vivo detection of Zn(2+) in mouse brain slice with Alzheimer's disease and zebrafish, respectively.

Chronological development of functional fluorophores for bio-imaging.

Functional fluorophores represent an emerging research field, distinguished by their diverse applications, especially in sensing and cellular imaging. After the discovery of quinine sulfate and subsequent elucidation of the fluorescence mechanism by Sir George Stokes, research in the field of fluorescence gained momentum. Over the past few decades, advancements in sophisticated instruments, including super-resolution microscopy, have further promoted cellular imaging using traditional fluorophores. These advancements include deciphering sensing mechanisms via photochemical reactions and scrutinizing the applications of fluorescent probes that specifically target organelles. This approach elucidates molecular interactions with biomolecules. Despite the abundance of literature illustrating different classes of probe development, a concise summary of newly developed fluorophores remains inadequate. In this review, we systematically summarize the chronological discovery of traditional fluorophores along with new fluorophores. We briefly discuss traditional fluorophores ranging from visible to near-infrared (NIR) in the context of cellular imaging and in vivo imaging. Furthermore, we explore ten new core fluorophores developed between 2007 and 2022, which exhibit advanced optical properties, providing new insights into bioimaging. We illustrate the utilization of new fluorophores in cellular imaging of biomolecules, such as reactive oxygen species (ROS), reactive nitrogen species (RNS), and proteins and microenvironments, especially pH and viscosity. Few of the fluorescent probes provided new insights into disease progression. Furthermore, we speculate on the potential prospects and significant challenges of existing fluorophores and their potential biomedical research applications. By addressing these aspects, we intend to illuminate the compelling advancements in fluorescent probe development and their potential influence across various fields.

Unraveling Ferroptosis Mechanisms: Tracking Cellular Viscosity with Small Molecular Fluorescent Probes.

Ferroptosis is a recently identified form of regulated cell death characterized by iron accumulation and lipid peroxidation. Numerous functions for ferroptosis have been identified in physiological as well as pathological processes, most notably in the treatment of cancer. The intricate balance of redox homeostasis is profoundly altered during ferroptosis, leading to alteration in cellular microenvironment. One such microenvironment is viscosity among others such as pH, polarity, and temperature. Therefore, understanding the dynamics of ferroptosis associated viscosity levels within organelles is crucial. To date, there are a very few reviews that detects ferroptosis assessing reactive species. In this review, we have summarized organelle's specific fluorescent probes that detects dynamics of microviscosity during ferroptosis. Also, we offer the readers an insight of their design strategy, photophysics and associated bioimaging concluding with the future perspective and challenges in the related field.

Light induced diversity-oriented synthesis (DOS) library of annulated indolizine fluorophores for imaging non-lysosomal lipid droplets (LDs).

We report the design, synthesis, and biological evaluation of a novel class of annulated indolizines as fluorescent probes. The compounds were generated through an eco-friendly, blue LED-induced domino reaction in ethyl acetate. A library of 24 coloured compounds exhibited tuneable emissions. One of the compounds (which we call DASS-fluor) proved to be an excellent polarity sensing probe. It is biocompatible, photostable, and detects specific types of lipid droplets (LDs in response to oleic acid, stress, and drug-induced autophagy in lungs and hepatic carcinoma cells). In comparison to Nile Red (a commercial probe), DASS-fluor can differentiate non-lysosomal LDs from lysosomal LDs and offers an advantage in precisely mapping drug-induced lipidosis caused by increased non-lysosomal LDs in cancerous cells. This unique probe could be a potential fluorescent marker for specific types of lipidosis induced by drugs.

A Ratiometric Fluorescent Probe for Hypochlorite and Lipid Droplets to Monitor Oxidative Stress.

Mitochondria are valuable subcellular organelles and play crucial roles in redox signaling in living cells. Substantial evidence proved that mitochondria are one of the critical sources of reactive oxygen species (ROS), and overproduction of ROS accompanies redox imbalance and cell immunity. Among ROS, hydrogen peroxide (H2O2) is the foremost redox regulator, which reacts with chloride ions in the presence of myeloperoxidase (MPO) to generate another biogenic redox molecule, hypochlorous acid (HOCl). These highly reactive ROS are the primary cause of damage to DNA (deoxyribonucleic acid), RNA (ribonucleic acid), and proteins, leading to various neuronal diseases and cell death. Cellular damage, related cell death, and oxidative stress are also associated with lysosomes which act as recycling units in the cytoplasm. Hence, simultaneous monitoring of multiple organelles using simple molecular probes is an exciting area of research that is yet to be explored. Significant evidence also suggests that oxidative stress induces the accumulation of lipid droplets in cells. Hence, monitoring redox biomolecules in mitochondria and lipid droplets in cells may give a new insight into cell damage, leading to cell death and related disease progressions. Herein, we developed simple hemicyanine-based small molecular probes with a boronic acid trigger. A fluorescent probe AB that could efficiently detect mitochondrial ROS, especially HOCl, and viscosity simultaneously. When the AB probe released phenylboronic acid after reacting with ROS, the product AB-OH exhibited ratiometric emissions depending on excitation. This AB-OH nicely translocates to lysosomes and efficiently monitors the lysosomal lipid droplets. Photoluminescence and confocal fluorescence imaging analysis suggest that AB and corresponding AB-OH molecules are potential chemical probes for studying oxidative stress.

A Systematic Design and Synthesis of PET-based Fluorescent Probes for Monitoring pH During Mitophagy.

Mitochondria are the powerhouse of the cell and function at pH ∼8.0. Dysfunctions of mitochondria, includes mitochondrial damage, leading to pH alteration. Hence, researchers aim to develop efficient pH probes for tracking mitochondrial pH dynamics. Herein, we developed a PET-based fluorescent probe for pH monitoring during mitochondrial dysfunctions. Three derivatives were synthesized with a variable spacer's length in pentacyclic pyridinium fluorophores (PM-C2, PM-C3, and PM-C6). An efficient electron transfers from the receptor (tertiary amine) was observed in the case of PM-C2 compared to the other two derivatives. This PET process was inhibited when tertiary amine was protonated in acidic pH. However, PM-C3 showed minimal fluorescence intensity at similar conditions and almost negligible change in case of PM-C6, suggesting poor PET process for both the derivatives. Furthermore, DFT/TD-DFT quantum chemical calculation well supported this optical phenomena and PET process. Biocompatible, photostable, and mitochondria-specific PM-C2 could monitor pH dynamics during mitochondrial damage which were engulfed by lysosome, also known as mitophagy. This mitophagy process were induced by rapamycin and starvation, which can be monitored by turn-on fluorescence enhancement. This process was further validated by tracking Parkin-protein translocation from cytoplasm to damaged mitochondria using our developed probe.

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.

Metal-free synthesis and study of glycine betaine derivatives in water for antimicrobial and anticancer applications.

A sustainable synthesis of interesting glycine betaine derivatives from cyclic 3°-amines viz. N-methyl morpholine (NMM), N-methyl piperidine (NMP), and 1,4-diazabicyclo[2.2.2]octane (DABCO) with numerous aryl diazoacetates 1 in water and under blue LED is reported. Generally, 3°-amines and metal carbenoids (from diazoacetates with transition metal catalysts) provide C-H insertion at the α-position of the amines. Computational comparison of the metal carbenoid with the singlet carbene (metal free and generated under blue LED) realized the difference in reactivity. Next, experimental results corroborated the preliminary findings. The products were isolated either by precipitation of the solid or gel-like final products from the aqueous reaction mixture without any chromatographic purification. The reaction mechanism was realized by control experiments. These compounds exhibit selective bactericidal properties against Gram-positive S. aureus, induce lipid droplets (LDs) formation in HePG2 cells and single crystal X-ray diffraction study of their halogenated analogs reveal interesting Hal … Hal contacts.

Activity-Based Fluorescent Probes for Sensing and Imaging of Reactive Carbonyl Species (RCSs).

This review explains various strategies for developing fluorescent probes to detect reactive carbonyl species (RCS). There are several mono and diacarbonyls among 30 varieties of reactive carbonyl species (RCSs) so far discovered, which play pivotal roles in pathological processes such as cancer, neurodegenerative diseases, cardiovascular disease, renal failure, and diabetes mellitus. These RCSs play essential roles in maintaining ion channel regulation, cellular signaling pathways, and metabolisms. Among RCSs, carbon monoxide (CO) is also utilized for its cardioprotective, anti-inflammatory, and anti-apoptotic effects. Fluorescence-based non-invasive optical tools have come out as one of the promising methods for analyzing the concentrations and co-localizations of these small metabolites. There has been a tremendous eruption in developing fluorescent probes for selective detection of specific RCSs within cellular and aqueous environments due to their high sensitivity, high spatial and temporal resolution of fluorescence imaging. Fluorescence-based sensing mechanisms such as intramolecular charge transfer (ICT), photoinduced electron transfer (PeT), excited-state intramolecular proton transfer (ESIPT), and fluorescence resonance energy transfer (FRET) are described. In particular, probes for dicarbonyls such as methylglyoxal (MGO), malondialdehyde (MDA), along with monocarbonyls that include formaldehyde (FA), carbon monoxide (CO) and phosgene are discussed. One of the most exciting advances in this review is the summary of fluorescent probes of dicarbonyl compounds.

An efficient PeT based fluorescent probe for mapping mitochondrial oxidative stress produced via the Nox2 pathway.

The human innate immune system eliminates invading pathogens through phagocytosis. The first step of this process is activating the nicotinamide adenine dinucleotide phosphate oxidase (Nox2) that utilizes NADPH to produce superoxide anion radicals and other reactive oxygen species (ROS). These ROS then alter the mitochondrial membrane potential and increase peroxide in the mitochondria. The peroxide reacts with myeloperoxidase (MPO) and chloride ions to produce pro-inflammatory oxidant hypochlorous acid (HOCl), which causes oxidative stress leading to cell death. The adverse effects of HOCl are highly associated with cardiovascular disease, neurodegenerative disorders, acute lung injuries, inflammatory diseases, and cancer. Therefore, mapping HOCl in the Nox2 pathway is crucial for an in-depth understanding of the innate immune system. Herein, we developed a unique pentacyclic pyridinium probe, PM-S, that exhibited efficient photoinduced electron transfer (PeT) with HOCl triggered methyl(phenyl)sulfane. PM-S showed several advantages, including better chemical stability, large Stokes shifts (>6258 cm-1), high sensitivity (∼50 nM) and specificity to mitochondria, compared to its parent pyrylium PY-S derivative. This probe is also efficient in studying the HOCl produced via the Nox2 pathway in HepG2 and HeLa cells. Analysis using a simple microplate reader and FACS analysis with various inhibitors and inducers supported the mechanistic understanding of Nox2, which can offer an advanced platform for monitoring the inflammatory process more efficiently.

Depletion Driven Assembly of Ultrasmall Metal Nanoclusters: From Kinetically Arrested Assemblies to Thermodynamically Stable, Spherical Superclusters.

Nanoscale assembly of ultrasmall metal nanoclusters (MNCs) by means of molecular forces has proven to be a powerful strategy to engineer their molecule-like properties in multiscale dimensions. By leveraging depletion attraction as the guiding force, herein, we demonstrate the formation of kinetically trapped NCs assemblies with enhanced photoluminescence (PL) and excited state lifetimes and extend the principle to cluster impregnated cationic nanogels, nonluminescent Au(I)-thiolate complexes, and weakly luminescent CuNCs. We further demonstrate a thermal energy driven kinetic barrier breaking process to isolate these assemblies. These isolated assemblies are thermodynamically stable, built from a strong network among several discrete, ultrasmall AuNCs and exhibit several unusual properties such as high stability in various pH, strong PL, microsecond lifetimes, large Stocks shifts, and higher accumulation in the lysosome of cancer cells. We anticipate our strategy may find wider use in creating a large variety of MNC-based assemblies with many unforeseen arrangements, properties, and applications.