Dual-reporter fluorescent probe for precise identification of liver cancer by sequentially responding to carboxylesterase and polarity.

Early treatment significantly improves the survival rate of liver cancer patients, so the development of early diagnostic methods for liver cancer is urgent. Liver cancer can develop from viral hepatitis, alcoholic liver, and fatty liver, thus making the above diseases share common features such as elevated viscosity, reactive oxygen species, and reactive nitrogen species. Therefore, accurate differentiation between other liver diseases and liver cancer is both a paramount practical need and challenging. Numerous fluorescent probes have been reported for the diagnosis of liver cancer by detecting a single biomarker, but these probes lack specificity for liver cancer in complex biological systems. Obviously, using multiple liver cancer biomarkers as the basis for judgment can dramatically improve diagnostic accuracy. Herein, we report the first fluorescent probe, LD-TCE, that sequentially detects carboxylesterase (CE) and lipid droplet polarity in liver cancer cells with high sensitivity and selectivity, with linear detection of CE in the range of 0-6 U/mL and a 65-fold fluorescence enhancement in response to polarity. The probe first reacts with CE and releases weak fluorescence, which is then dramatically enhanced due to the decrease in lipid droplet polarity in liver cancer cells. This approach allows the probe to enable specific imaging of liver cancer with higher contrast and accuracy. The probe successfully achieved the screening of liver cancer cells and the precise identification of liver cancer in mice. More importantly, it is not disturbed by liver fibrosis, which is a common pathological feature of many liver diseases. We believe that the LD-TCE is expected to be a powerful tool for early diagnosis of liver cancer.

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.

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.

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.

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.

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.

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.

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.

Two-photon imaging of the endoplasmic reticulum thiol flux in the brains of mice with depression phenotypes.

Depression is a common mental illness with high morbidity and mortality. Mounting evidence suggests that an imbalance of the oxidant-antioxidant defence system is strongly correlated with depression and the dysfunction of the endoplasmic reticulum (ER) is strongly related to the oxidative stress. Therefore, as vital and abundant antioxidants in the ER, biothiols may contribute to the etiology of depression. However, ideal two-photon (TP) fluorescent probes for in vivo imaging of ER-associated thiols in the brains of mice with depression phenotypes are still lacking. Hence, we describe a fluorescent probe (ER-SH) to visualize thiols in living systems. ER-SH displays high sensitivity, excellent ER-targeting ability, outstanding TP properties and low cytotoxicity. Using this ER-SH probe, we succeeded in revealing an increase in the endogenous thiol levels under ER stress induced by DTT. Significantly, TP in vivo imaging showed for the first time that the thiol levels are reduced in brains of mice with depression phenotypes. Collectively, this work can assist in further understanding the molecular mechanism of depression and offers a crucial dimension for diagnosis and anti-depression treatments.

Simultaneous Fluorescence Visualization of Endoplasmic Reticulum Superoxide Anion and Polarity in Myocardial Cells and Tissue.

Diabetic cardiomyopathy (DCM) is a critical complication of diabetes, the accurate pathogenesis of which remains elusive. It is widely accepted that endoplasmic reticulum (ER) stress and abnormal fluctuations of reactive oxygen species (ROS) are considered to be closely associated with progress of DCM. In addition, DCM-induced changes of myocardial tissue and ROS-derived oxidation of proteins will cause changes of the hydrophilic and hydrophobic domains and may further seriously alter the myocardial cell polarity. Thus, real-time detection of ROS and polarity in ER of live cells and in tissue will contribute to revealing the exact molecular mechanisms of DCM. In this article, we first present an ER-targetable fluorogenic probe termed ER-NAPC for sensitive and selective detection of superoxide anion (O2•-). ER-NAPC can precisely target ER and visualize the increase of O2•- level in a live H9c2 cardiomyocyte cell during ER stress. Meanwhile, by combining ER-NAPC with a polarity-sensitive probe, ER-P, we accomplish the simultaneous fluorescence visualization of O2•- and polarity in ER of live cells and diabetic myocardial tissue. The dual-color fluorescence imaging results indicate that the O2•- level and polarity will synergistically rise during ER stress in live cells and diabetic myocardial tissue. The proposed dual-color imaging strategy may offer a proven methodology for studying coordinated variation of different parameters during ER stress oriented disease.

Transferrin-navigation Nano Artificial Antibody Fluorescence Recognition of Circulating Tumor Cells.

Specific recognition of circulating tumor cells (CTCs) is of great significance for cancer diagnosis and personalized therapy. The antibodies and aptamer are commonly used for recognition of CTCs, but they often suffer from low stability and high cost. Therefore, chemically stable and low-cost artificial recognition elements are still highly demanded. Herein, we prepared nano artificial antibody based on molecular imprinting and applied for fluorescence recognition of CTCs. Surface imprinting was employed to construct a transferrin (TRA)-imprinted layer on the surface of rhodamine doped silica nanoparticles. Take advantage of the specific interaction between TRA and TRA receptor (overexpressed on cancer cells), the as-prepared TRA-imprinted artificial antibody was allowed for specific targeting cancer cells mediated by TRA. And the average recognition efficiency of the artificial antibody for the cancer cells was 88% through flow cytometry. Finally, the nano artificial antibody was successfully applied to specific identify mimetic CTCs, under the same conditions, the recognition ability of artificial antibody for CTCs was 8 times higher than the white blood cells.

The effect of selected food phytochemicals on breast cancer metastasis based on in vivo capture of circulating tumor cells.

The number of circulating tumor cells (CTCs) in a mouse tumor model fed with different dietary factors was first evaluated by in vivo CTC capture at different time points of cancer progression. The results showed that the number of CTCs reflected the degree of cancer progression. A new assessment method of dietary factor effects on cancer metastasis was established.

A new endoplasmic reticulum-targeted two-photon fluorescent probe for imaging of superoxide anion in diabetic mice.

Excessive or unfolded proteins accumulation in endoplasmic reticulum (ER) will cause ER stress, which has evolved to involve in various metabolic diseases. In particular, ER stress plays an important role in the pathogenesis of diabetes. Both ER stress and course of diabetes accompany oxidative stress and production of reactive oxygen species (ROS), among which superoxide anion (O2•-) is the first produced ROS and has been recognized as cell signaling mediator involved in the physiological and pathological process of diabetes. Hence, the development of effective monitoring methods of O2•- in live cells and in vivo is of great importance for ascertaining the onset and progress of related diseases. Herein, a new endoplasmic reticulum-targeted two-photon fluorescent probe termed ER-BZT is designed and synthesized for imaging of O2•-. The probe ER-BZT shows high sensitivity, selectivity, stability, and low cytotoxicity. Based on these superior properties, the rise of O2•- levels in endoplasmic reticulum induced with different stimuli is visualized by one- and two-photon fluorescence imaging. Most importantly, by utilizing ER-BZT, the two-photon fluorescence imaging results demonstrate that the endogenous O2•- concentration in abdominal or hepatic tissue of diabetic mice is higher than that in normal mice. Meanwhile, after treated with metformin, a broad-spectrum antidiabetic drug, the diabetic mice exhibit depressed O2•- level. The proposed two-photon probe, ER-BZT might serve as perfect tool to image the O2•- fluctuations and study the relevance between O2•- and various diseases in live cells and in vivo.

Molecularly imprinted polymer grafted paper-based method for the detection of 17ß-estradiol.

17ß-estradiol (17ß-E2) usually is used to promote the growth of animal. Abuse of 17ß-E2 has become a global food security problem, because the residues in foods can cause endocrine disorder through the food chain. A novel molecularly imprinted polymer (MIP) grafted paper-based method for the sensitive and specific detection of 17ß-E2 was reported in this work. Results showed that the MIP's optimum synthetic conditions were as follows: 12 mL of acetonitrile was chosen as the solvent; the molar ratio of template molecule, functional monomer and cross linker was 1:12:12. MIP synthesized had a good recognition ability, the limit of detection (LOD) of established detection method for milk and human urine samples could reach 0.25µgL-1.