Fluorescence imaging sheds light on the immune evasion mechanisms of hepatic stellate cells mediated by superoxide anion.

Whether and how the reactive oxygen species generated by hepatic stellate cells (HSCs) promote immune evasion of hepatocellular carcinoma (HCC) remains mysterious. Therefore, investigating the function of superoxide anion (O2•-), the firstly generated reactive oxygen species, during the immune evasion become necessary. In this work, we establish a novel in situ imaging method for visualization of O2•- changes in HSCs based on a new two-photon fluorescence probe TPH. TPH comprises recognition group for O2•- and HSCs targeting peptides. We observe that O2•- in HSCs gradually rose, impairing the infiltration of CD8+ T cells in HCC mice. Further studies reveal that the cyclin-dependent kinase 4 is deactivated by O2•-, and then cause the up-regulation of PD-L1. Our work provides molecular insights into HSC-mediated immune evasion of HCC, which may represent potential targets for HCC immunotherapy.

Unveiling the Crucial Roles of O2•- and ATP in Hepatic Ischemia-Reperfusion Injury Using Dual-Color/Reversible Fluorescence Imaging.

Hepatic ischemia-reperfusion injury (HIRI) is mainly responsible for morbidity or death due to graft rejection after liver transplantation. During HIRI, superoxide anion (O2•-) and adenosine-5'-triphosphate (ATP) have been identified as pivotal biomarkers associated with oxidative stress and energy metabolism, respectively. However, how the temporal and spatial fluctuations of O2•- and ATP coordinate changes in HIRI and particularly how they synergistically regulate each other in the pathological mechanism of HIRI remains unclear. Herein, we rationally designed and successfully synthesized a dual-color and dual-reversible molecular fluorescent probe (UDP) for dynamic and simultaneous visualization of O2•- and ATP in real-time, and uncovered their interrelationship and synergy in HIRI. UDP featured excellent sensitivity, selectivity, and reversibility in response to O2•- and ATP, which rendered UDP suitable for detecting O2•- and ATP and generating independent responses in the blue and red fluorescence channels without spectral crosstalk. Notably, in situ imaging with UDP revealed for the first time synchronous O2•- bursts and ATP depletion in hepatocytes and mouse livers during the process of HIRI. Surprisingly, a slight increase in ATP was observed during reperfusion. More importantly, intracellular O2•-─succinate dehydrogenase (SDH)─mitochondrial (Mito) reduced nicotinamide adenine dinucleotide (NADH)─Mito ATP─intracellular ATP cascade signaling pathway in the HIRI process was unveiled which illustrated the correlation between O2•- and ATP for the first time. This research confirms the potential of UDP for the dynamic monitoring of HIRI and provides a clear illustration of HIRI pathogenesis.

Recent progress in small-molecule fluorescent probes for endoplasmic reticulum imaging in biological systems.

Endoplasmic reticulum (ER) is an indispensable organelle in eukaryotic cells involved in protein synthesis and processing, as well as calcium storage and release. Therefore, maintaining the quality of ER is of great importance for cellular homeostasis. Aberrant fluctuations of bioactive species in the ER will result in homeostasis disequilibrium and further cause ER stress, which has evolved to contribute to the pathogenesis of various diseases. Therefore, the real-time monitoring of various bioactive species in the ER is of high priority to ascertain the mysterious roles of ER, which will contribute to unveiling the corresponding mechanism of organism disturbances. Recently, fluorescence imaging has emerged as a robust technique for the direct visualization of molecular events due to its outstanding sensitivity, high temporal-spatial resolution and noninvasive nature. In this review, we comprehensively summarize the recent progress in design strategies, bioimaging applications, potential directions and challenges of ER-targetable small-molecular fluorescent probes.

Deep-Tissue Fluorescence Imaging Study of Reactive Oxygen Species in a Tumor Microenvironment.

Tumor microenvironment (TME) is the survival environment for tumor cells to proliferate and metastasize in deep tissue. TME contains tumor cells, immune cells, stromal cells and a variety of active molecules including reactive oxygen species (ROS). Inside the TME, ROS regulate the oxidation-reduction (redox) homeostasis and promote oxidative stress. Due to the rapid proliferation ability and specific metabolic patterns of the TME, ROS pervade virtually all complex physiological processes and play irreplaceable roles in protein modification, signal transduction, metabolism, and energy production in various tumors. Therefore, measurements of the dynamically, multicomponent simultaneous changes of ROS in the TME are of great significance to reveal the detailed proliferation and metastasis mechanisms of the tumor. Near-infrared (NIR) and two-photon (TP) fluorescence imaging techniques possess real-time, dynamic, highly sensitive, and highly signal-to-noise ratios with deep tissue penetration abilities. With the rationally designed probes, the NIR and TP fluorescence imaging techniques have been widely used to reveal the mechanisms of how ROS regulates and constructs complex signals and metabolic networks in TME. Therefore, we summarize the design principles and performances of NIR and TP fluorescence imaging of ROS in the TME in the last four years, as well as discuss the advantages and potentials of these works. This Review can provide guidance and prospects for future research work on TME and facilitate the development of antitumor drugs.

Demystifying Lysosomal α-l-Fucosidase in Liver Cancer-Bearing Mice by Specific Two-Photon Fluorescence Imaging.

Liver cancer is one of the most frequently diagnosed cancers and has high mortality. However, the early treatment and prognosis can greatly prolong the survival time of patients, which depends on its early detection. α-l-Fucosidase (AFU), as a vital lysosomal hydrolase, is considered to be an ideal biomarker for early stage liver cancer. So, in vivo monitoring of AFU is essential for the early and accurate diagnosis of liver cancer. Hence, we designed the first two-photon turn-on fluorescent reporter, termed HcyCl-F, which localized to lysosomes for fast imaging of AFU. The 2-chloro-4-phenyl-α-l-fucoside bond of HcyCl-F could be effectively hydrolyzed by AFU and released the hydroxyl on the benzene ring, eventually obtaining a strong conjugated compound (HcyCl-OH) with shiny fluorescence. We demonstrated that HcyCl-F was able to rapidly and accurately respond to AFU. Using a two-photon fluorescence microscope, we successfully visualized the fluctuation of AFU in lysosomes. More importantly, a fascinatingly strong fluorescence signal was observed in the tumor tissue of liver cancer-bearing mice. Of note, we confirmed that HcyCl-F could clearly detect liver tumors in stage I. Altogether, our work provides a simple and convenient method for deciphering the critical pathological function of AFU in depth and facilitates the nondestructive and effective diagnosis of liver cancer in the early stage.

Exploring the Changes of Peroxisomal Polarity in the Liver of Mice with Nonalcoholic Fatty Liver Disease.

Peroxisome proliferator-activated receptor alpha (PPAR-a) is a crucial nuclear transcription regulator of lipid metabolism, which is closely associated with the initiation and development of nonalcoholic fatty liver disease (NAFLD). Because PPAR-a can directly decide the level of peroxisomal metabolic enzymes, its changes might directly cause variations in peroxisomal polarity. Therefore, we developed a new two-photon fluorescence imaging probe, PX-P, in which the triphenylamine and cyanide moieties can real-time sense peroxisomal polarity changes. Using PX-P, we observed a prominent decrease in the peroxisomal polarity in the liver of mice with NAFLD for the first time. More importantly, we discovered that intracellular excessive peroxynitrite (ONOO-) and hydrogen peroxide (H2O2) underwent nitrification and oxidation, respectively, with various sites of PPAR-a. Interestingly, the key site of PPAR-a was nitrated by a low concentration of ONOO- rather than being oxidized by the high level of H2O2. These drastically reduced the activity of PPAR-a, accelerating the occurrence of NAFLD. Moreover, through activating PPARs with pioglitazone, peroxisomal polarity markedly increased compared with that of NAFLD. Altogether, our work presents a new approach for the early diagnosis of NAFLD and identifies potential therapeutic targets.

Rapid Two-Photon Fluorescence Imaging of Monoamine Oxidase B for Diagnosis of Early-Stage Liver Fibrosis in Mice.

Liver fibrosis could induce cirrhosis and liver cancer, causing serious damages to liver function and even death. Early diagnosis of fibrosis is extremely requisite for optimizing treatment schedule to improve cure rate. In early-stage fibrosis, overexpressed monoamine oxidase B (MAO-B) can serve as a biomarker, which greatly contributes to the diagnosis of early liver fibrosis. However, there is still a lack of desired strategy to precisely monitor MAO-B in situ. In this work, we established a two-photon fluorescence imaging method for in vivo detection of MAO-B activity counting on a simply prepared probe, BiPhAA. The BiPhAA could be activated by MAO-B within 10 min and fluoresced brightly. To our knowledge, this BiPhAA-based imaging platform for MAO-B is more rapid than other current detection methods. Furthermore, BiPhAA allowed the dynamic observation of endogenous MAO-B level changes in hepatic stellate cells (LX-2). Through two-photon fluorescence imaging, we observed six times higher fluorescence brightness in the liver tissue of fibrosis mice than that of normal mice, thus successfully distinguishing mice with liver fibrosis from normal mice. Our work offers a simple, fast, and highly sensitive approach for imaging MAO-B in situ and paves a way to the diagnosis of early liver fibrosis with accuracy.

ALP-Activated Chemiluminescence PDT Nano-Platform for Liver Cancer-Specific Theranostics.

Photodynamic therapy (PDT) is a promising therapeutic approach that has been extensively applied in curing cancers. However, the limited penetration depth of external light makes PDT only practical for some superficial tumor treatments. Moreover, an external light irradiation might cause damages to adjacent normal tissues. Additionally, the poor targeting ability of PDT can lead to side effects like skin phototoxicity. Therefore, a PDT strategy addressing these drawbacks is urgently exploited. Herein, we constructed a chemiluminescence theranostics platform named MSN@H6L@ß-CD@AMPPD NPs for liver cancer-specific, in situ diagnosis and therapy without an external light source. Through the interaction of host-guest, 3-[(2-spiroadamatane)-4-methoxy-4-(3-phosphoryloxy)-phenyl-1,2-dioxetane] dioxetane, a chemiluminescence substrate of the liver cancer biomarker alkaline phosphatase was integrated with ß-cyclodextrin. Then, the ß-cyclodextrin was covalently bound to the mesoporous silica loaded with (4-carboxyphenyl) porphyrin to finally obtain the MSN@H6L@ß-CD@AMPPD NPs. These NPs can be specifically hydrolyzed by the liver cancer alkaline phosphatase and lead to the liver cancer-targeting chemiluminescence. Subsequently, (4-carboxyphenyl) porphyrin was excited by the chemiluminescence through chemiluminescence resonance energy transfer and created both near-infrared fluorescence and 1O2. This strategy greatly promotes the penetration depth and targeting ability of the PDT. In brief, the platform accomplishes a PDT nano-theranostics for liver cancer and provides a method for the imaging, diagnosis, and therapy of tumors in deep tissue.

Oxidative Damage of Tryptophan Hydroxylase-2 Mediated by Peroxisomal Superoxide Anion Radical in Brains of Mouse with Depression.

Depression is intimately linked with oxidative stress in the brains. Peroxisome plays vital roles in the regulation of intracellular redox balance by keeping reactive oxygen species (ROS) homeostasis. Available evidence indicates a possible relationship between peroxisomal ROS and depression. Even so, the underlying modulation mechanisms of peroxisomal ROS in depression are still rudimentary due to the limitations of the existing detecting methods. Hence, we developed a two-photon fluorescent probe TCP for the real-time visualization of the first produced ROS superoxide anion radical (O2•-) in peroxisome. Using the two-photon fluorescence imaging, we found that peroxisomal O2•- rose during oxidative stress in the mouse brains, resulting in the inactivation of catalase (CAT). Subsequently, the intracellular H2O2 level elevated, which further oxidized tryptophan hydroxylase-2 (TPH2). Then the decrease contents of TPH2 caused the dysfunction of 5-hydroxytryptamine (5-HT) system in the mouse brains, eventually leading to depression-like behaviors. Our work provides evidence of a peroxisomal O2•- mediated signaling pathway in depression, which will conduce to pinpoint potential targets for the treatment of depression.

Observing Malondialdehyde-Mediated Signaling Pathway in Cerebral Ischemia Reperfusion Injury with a Specific Nanolight.

Cerebral ischemia reperfusion injury (CIRI) is closely related to lipid peroxidation. Malondialdehyde (MDA), as a biomarker of lipid peroxidation, is prone to addition with biomacromolecules, resulting in a secondary cerebral injury. However, desirable tools for in vivo-determining cerebral MDA are scarce. Thus, we devised innovative polymer carbon dots carbonized by benzoyl hydrazine and named them BH-PCDs. BH-PCDs covered with hydrazine groups directly form from one-pot synthesis. The functional nanoparticle specifically identifies MDA via a photoinduced electron transfer (PET) mechanism from other similar biological species, especially reactive carbonyl species. BH-PCDs afforded several valuable traits of a simple preparation, a large two-photon absorption cross section, and exceptional biocompatibility, as well as the ability of traversing the blood-brain barrier. Relying on BH-PCDs, we real-time portrayed the increased cerebral MDA under CIRI. Furthermore, combining with a commercial indicator of the superoxide anion (O2•-), an O2•--regulated MDA level under CIRI was visualized in vivo. Moreover, we demonstrated MDA inactivated glutamine synthetase under CIRI, mediating the glutamate level. Overall, we provide a perspective nanolight serviceable for treating CIRI, which could reveal the physiopathology mechanism of brain MDA.

Two-photon fluorescence visualization of lysosomal pH changes during mitophagy and cell apoptosis.

We herein develop a novel two-photon fluorescent probe termed L-pH for visualization of lysosomal pH within live cells. L-pH is composed of three moieties, including naphthalimide fluorophore as a fluorescence off-on response moiety, piperazine and morpholine groups as lysosomal targeting and pH responsive sites, as well as a reactive benzyl chloride segment for further lysosomal anchoring. The experimental results demonstrate that L-pH can instantaneously respond to various pH values with high sensitivity and selectivity, and has low cytotoxicity and excellent photostability. The one-photon and two-photon fluorescence imaging data indicate L-pH can preferably accumulate into lysosome and monitor the rise of lysosomal pH changes during myriad cell stress conditions, including heat shock, cell apoptosis and mitophagy. Moreover, L-pH was applied for imaging of pH difference in abdominal tissues of mice. L-pH will be a potential tool for monitoring lysosomal pH variation during lysosome-associated physiological and pathological states.

In situ visualization of peroxisomal viscosity in the liver of mice with non-alcoholic fatty liver disease by near-infrared fluorescence and photoacoustic imaging.

Non-alcoholic fatty liver disease (NAFLD) can gradually develop into hepatic failure, and early diagnosis is crucial to improve treatment efficiency. The occurrence of NAFLD is closely related to lipid metabolism. Peroxisomes act as the first and main site for lipid metabolism in the hepatocytes, so abnormal lipid metabolism might directly affect peroxisomal viscosity. Herein, we developed a new near-infrared fluorescence (NIRF) and photoacoustic (PA) imaging probe (PV-1) for the real-time visualization of peroxisomal viscosity in vivo. This PV-1 encompasses the malononitrile group as the rotor, which emits strong NIRF (at 705 nm) and PA (at 680 nm) signals when rotation is hindered as viscosity increases. Through dual-mode imaging, we discovered distinctly higher viscosity in the liver of NAFLD mice for the first time. We further found the remarkable amelioration of NAFLD upon treatment with N-acetylcysteine (NAC). Therefore, we anticipate that the PV-1 imaging method is promising for the early diagnosis and prognostic evaluation of NAFLD.

A Dual-Targeting Functionalized Graphene Film for Rapid and Highly Sensitive Fluorescence Imaging Detection of Hepatocellular Carcinoma Circulating Tumor Cells.

High recurrence and metastasis rates are the major causes of the high mortality of hepatocellular carcinoma (HCC). Circulating tumor cells (CTCs) disseminating into the bloodstream play an essential role in cancer metastasis. However, since HCC-CTCs are extremely rare, limitations of current detection methods impede accurate discerning of HCC-CTCs under complicated biological context. Here, a dual-targeting functionalized reduced graphene oxide film (DTFGF) for specifically identifying HCC-CTCs was created via coinstantaneous targeting epithelial cell adhesion molecule (EpCAM) and HCC cell-specific asialoglycoprotein receptor (ASGPR). Anti-EpCAM antibodies and galactose-rhodamine-polyacrylamide nanoparticles (Gal-Rh-PAA NPs) specifically recognizing ASGPR are modified on the surface of a graphene film that quenches the rhodamine fluorescence. HCC-CTCs can be captured by anti-EpCAM antibodies and endocytose Gal-Rh-PAA NPs, recovering the rhodamine fluorescence. Profiting from the accuracy of dual-targeting, less handling steps, and high resolution of fluorescence imaging, a simple, rapid, and low-cost HCC-CTC enumeration method is established with excellent sensitivity and selectivity than conventional methods. Using DTFGFs, as low as five HCC-CTCs were detected in a 1 mL blood sample. Further results revealed that larger HCC-CTC quantities indicate more advanced stages of HCC in patients. Overall, this work holds great promise for the early diagnosis, prognosis, and therapeutic evaluation of HCC.

A platform for primary tumor origin identification of circulating tumor cells via antibody cocktail-based in vivo capture and specific aptamer-based multicolor fluorescence imaging strategy.

Circulating tumor cells (CTCs) are expected to serve as a blood-based biomarker in the diagnosis of cancers at an early stage, providing an opportunity to increase the survival of cancer patients. Current techniques for CTC detection were designed for some particular types of cancer with confirmed primary tumor origin. In this work, a platform for the detection of two cancer types and the identification of the primary tumor origin of CTCs was established to meet the requirement of cancer diagnosis and clinical application. A combined strategy based on in vivo capture method using antibody cocktail and multicolor fluorescence imaging using aptamer was designed to achieve the high-efficiency capture of CTCs and the accurate location of the primary tumor. An antibody cocktail of epithelial cell adhesion molecule (EpCAM) and epidermal growth factor receptor (EGFR) was applied to capture breast cancer CTCs and hepatocellular CTCs in vivo. The capture efficiency of hepatocellular CTCs was significantly increased from 3.17% to 26.67% and the capture efficiency of breast cancer CTCs slightly increased from 27.00% to 29.84% compared with EpCAM-based capture of CTCs. Meanwhile, the primary tumor origins of breast cancer CTCs and hepatocellular CTCs were simultaneously distinguished by specific aptamer-based fluorescence probes without any signal crosstalk. The results of in vivo experiments using the dual tumor-bearing mouse model confirmed the feasibility of this method to capture CTCs and identify primary tumor origins. This simple and efficient approach has potential for future applications in cancer diagnosis and prognosis.

In situ photoacoustic imaging of cysteine to reveal the mechanism of limited GSH synthesis in pulmonary fibrosis.

We developed a photoacoustic and fluorescent dual-mode imaging probe, CCYS, for the detection of cysteine with high selectivity and sensitivity in a living system for the first time. By using CCYS, we found that the limited synthesis of glutathione in pulmonary fibrosis was not caused by cysteine deficiency.

Detection of Escherichia coli O157:H7 and Salmonella enterica serotype Typhimurium based on cell elongation induced by beta-lactam antibiotics.

Pathogenic bacteria such as Shiga toxigenic Escherichia coli and Salmonella can cause severe food-borne diseases. Rapid and sensitive detection of these foodborne pathogens is essential to ensure food safety. In this study, a novel method based on cell elongation induced by beta-lactam antibiotics for direct microscopic counting of Gram-negative bacteria was established. Combined with the sample preparation steps of membrane filtration and magnetic separation, the detection of E. coli O157:H7 and Salmonella enterica serotype Typhimurium was achieved by direct optical microscopic counting of the number of elongated bacteria. The limit of detection of E. coli O157:H7 and S. typhimurium could reach 20 CFU mL-1. The recovery tests for E. coli O157:H7 and S. typhimurium in water and milk samples showed acceptable recovery values between 93.6% and 106.2%. This method is sensitive, cost effective, and rapid (<2 h) and shows great potential for the detection of Gram-negative pathogens in various environmental and food samples.

Illuminating the Function of the Hydroxyl Radical in the Brains of Mice with Depression Phenotypes by Two-Photon Fluorescence Imaging.

Depression is intimately linked with oxidative stress. As one of the most reactive and oxidative reactive oxygen species that is overproduced during oxidative stress, the hydroxyl radical (. OH) can cause macromolecular damage and subsequent neurological diseases. However, due to the high reactivity and low concentration of . OH, precise exploration of . OH in brains remains a challenge. The two-photon fluorescence probe MD-B was developed for in situ . OH imaging in living systems. This probe achieves exceptional selectivity towards . OH through the one-electron oxidation of 3-methyl-pyrazolone as a new specific recognition site. MD-B can be used to map . OH in mouse brain, thereby revealing that increased . OH is positively correlated with the severity of depression phenotypes. Furthermore, . OH has been shown to inactivate deacetylase SIRT1, thereby leading to the occurrence and development of depression phenotypes. This work provides a new strategy for the future treatment of depression.

Observation of Acetylcholinesterase in Stress-Induced Depression Phenotypes by Two-Photon Fluorescence Imaging in the Mouse Brain.

Oxidative stress in depression is a prime cause of neurotransmitter metabolism dysfunction in the brain. Acetylcholinesterase (AChE), a key hydrolase in the cholinergic system, directly determines the degradation of neurotransmitters. However, due to the complexity of the brain and lack of appropriate in situ imaging tools, the mechanism underlying the changes in AChE activity in depression remains unclear. Hence, we generated a two-photon fluorescence probe (MCYN) for real-time visualization of AChE with excellent sensitivity and selectivity. AChE can specifically recognize and cleave the carbamic acid ester bond in MCYN, and MCYN emits bright fluorescence at 560 nm by two-photon excitation at 800 nm. By utilizing MCYN to monitor AChE, we discovered a significant increase in AChE activity in the brains of mice with depression phenotypes. Notably, with the assistance of a two-photon fluorescence imaging probe of the superoxide anion radical (O2•-), in vivo visualization for the first time revealed the positive correlation between AChE and O2•- levels associated with depressive behaviors. This finding suggests that oxidative stress may induce AChE overactivation, leading to depression-related behaviors. This work provides a new and rewarding perspective to elucidate the role of oxidative stress regulating AChE in the pathology of depression.