Multifunctional Theranostic Probe Based on the Pim-1 Kinase Inhibitor with the Function of Tracking pH Fluctuations during Treatment.

Currently, kinase inhibitors have been applied in the diagnosis or treatment of cancer with their unique advantages. It is of great significance to develop some comprehensive theranostic reagents based on kinase inhibitors to improve the performance of reagents for biomedical applications. Besides, tracking changes in the intracellular environment (e.g., pH) during cancer development and drug delivery is also critical for cancer research and treatment. Therefore, it is an urgent desire to design some novel multifunctional reagents based on kinase inhibitor strategies that can trace changes in the microenvironment of cancer cells. In this paper, a multifunctional theranostic reagent based on Pim-1 kinase inhibitor 5-bromobenzofuran-2-carboxylic acid is proposed. The theranostic probe binds to tumor-specific Pim-1 kinase, releases strong fluorescence, and produces cytotoxicity, thus achieving cell screening and killing effects. Furthermore, the probe can specifically target lysosomes and sensitively respond to pH. It can be used to track the pH changes in the intracellular environment under conditions of autophagy and external stimulation, as a visual tool to monitor pH fluctuations during cancer treatment. In conclusion, this simple but multifunctional theranostic reagent proposed in this work is expected to provide a promising method for cancer diagnosis and therapy.

H2S-activated fluorescent probe enables dual-channel fluorescence tracking of drug release in tumor cells.

Nowadays, the selective release of therapeutic drugs into tumor cells has become an important way of tumor treatment due to the high side effects of chemotherapy drugs. As one of the gas mediators, hydrogen sulfide (H2S) is closely related to cancer. Due to the high content of H2S in tumor cells, it can be used as a signaling molecule that triggers the release of drugs to achieve the selective release of therapeutic drugs. In addition, dual-channel fluorescence imaging technology can be better applied to monitor the drug delivery process and distinguish the state before and after drug release, so as to better track the effect of drug therapy. Based on this, we used NBD amines (NBD-NHR) as the recognition group of H2S and connected the tyrosine kinase inhibitor crizotinib to construct an activated dual-channel fluorescent probe CZ-NBD. After the probe enters the tumor cells, it consumes H2S and releases crizotinib, which is highly toxic to the tumor cells. Importantly, the probe displays significant fluorescence changes in different cells, enabling not only the screening of tumor cells, but also tracking and monitoring drug release and tumor cell activity. Therefore, the construction of probe CZ-NBD provides a new strategy for drug release monitoring in tumor cells.

A novel GSH-activable theranostic probe containing kinase inhibitor for synergistic treatment and selective imaging of tumor cells.

Theranostic probe is becoming a powerful tool for diagnosis and treatment of cancer. Although some theranostic probes have been successfully developed, there is still a great room for improvement in sensitive diagnosis and efficient treatment. Herein, we developed a novel GSH-activable theranostic probe NC-G, which uses 1,8-naphthalimide-4-sulfonamide as a fluorescence imaging group and crizotinib as a highly toxic kinase inhibitor to tumor cells. The probe not only has high sensitivity (DL = 74 nM) and specificity, but also can detect GSH sensitively in cells and zebrafish. In addition, probe NC-G can not only show more obvious fluorescence in tumor cells to achieve sensitive diagnosis of tumor cells, but also release the inhibitor crizotinib to achieve high toxicity to tumor cells. It is worth noting that the consumption of GSH can cause oxidative stress response of cells and the release of SO2 can induce cell apoptosis during the recognition process of the probe and GSH. Thus, the synergistic effect of crizotinib, GSH depletion, and SO2 release provides a highly effective therapeutic feature for tumor cells. Therefore, probe NC-G can serve as an excellent theranostic probe for sensitive imaging and highly effective treatment of tumor cells.

Novel amphiphilic hydroxyethyl starch-based nanoparticles loading camptothecin exhibit high anticancer activity in HepG2 cells and zebrafish.

Camptothecin is a naturally occurred anticancer drug but exhibits limitations including poor aqueous solubility, low bioavailability, and high level of adverse drug reactions on normal organs. To overcome these problems, this paper developed a novel amphiphilic Lau-Leu-HES carrier using hydroxyethyl starch, lauric acid, and L-leucine as starting materials. The carrier was successfully applied to prepare Lau-Leu-HES nanoparticles loading camptothecin. The drug loading efficiency and encapsulation efficiency of the nanoparticles were calculated to be 29.04% and 81.85%, respectively. The nanoparticles exhibited high zeta potential (-15.51 mV) and small hydrodynamic diameter (105.4 nm). Camptothecin in nanoparticles could be rapidly released under acidic condition (pH = 4.5), thereby indicating the high sensitivity under cancer microenvironments. Anticancer investigation revealed that the nanoparticles could inhibit the proliferation of HepG2 cells in vitro. Compared with commercial available drug doxorubicin, the nanoparticles could significantly inhibit the expression of krasv12 oncogene in transgenic Tg (EGFP-krasV12) zebrafish. These results indicate that the camptothecin-loaded Lau-Leu-HES nanoparticles are expected to be a potential candidate for cancer therapy.

A novel ratiometric fluorescent probe for the detection of nickel ions in the environment and living organisms.

Nickel resources are abundant in the world, and the application of nickel in production and life is more and more extensive. However, excessive nickel entering the environment will not only cause environmental pollution but also seriously endanger plants, animals and human health. Nickel compounds are carcinogenic and have been classified as a class 1 carcinogen. Nickel mainly exists in the form of divalent ions in the environment. However, there are few simple and effective methods for the detection of nickel ions, and these methods still have certain limitations. At present, the mechanisms of nickel influence in organisms are also unclear. Therefore, we constructed a ratiometric fluorescent probe Ra-Ni, which can achieve its own self-calibration and avoid the interference of other factors, thereby realizing the specific identification of nickel ions. The probe can detect nickel ions sensitively with a detection limit as low as 26.2 nM and can respond in a short time (< 2 min), which proves the great potential of the probe in the detection of nickel ions. At the same time, Ra-Ni has also been successfully used for imaging nickel ions in living cells and zebrafish, providing an effective tool for the study of physiological and pathological processes. The detection effect of nickel ions in actual water sample is also satisfactory, which further demonstrates the practicability of Ra-Ni in environmental applications.

A new-type HOCl-activatable fluorescent probe and its applications in water environment and biosystems.

The outbreak and spread of Corona Virus Disease 2019 (COVID-19) has led to a significant increase in the consumption of sodium hypochlorite (NaOCl) disinfectants. NaOCl hydrolyzes to produce hypochlorous acid (HOCl) to kill viruses, which is a relatively efficient chlorine-based disinfectant commonly used in public disinfection. While people enjoy the convenience of NaOCl disinfection, excessive and indiscriminate use of it will affect the water environment and threaten human health. Importantly, HOCl is an indispensable reactive oxygen species (ROS) in human body. Whether its concentration is normal or not is closely related to human health. Excessive production of HOCl in the body contributes to some inflammatory diseases and even cancer. Also, we noticed that the concentration of ROS in cancer cells is about 10 times higher than that in normal cells. Herein, we developed a HOCl-activatable biotinylated dual-function fluorescent probe BTH. For this probe, we introduced biotin on the naphthalimide fluorophore, which increased the water solubility and enabled the probe to aggregate in cancer cells by targeting specific receptor overexpressed on the surface of cancer cell membrane. After reacting to HOCl, the p-aminophenylether moiety of this probe was oxidatively removed and the fluorescence of the probe was recovered. As expected, in the PBS solution with pH of 7.4, BTH could give full play to the performance of detecting HOCl, and it has made achievements in detecting the concentration of HOCl in actual water samples. Besides that, BTH had effectively distinguished between cancer cells and normal cells through a dual-function discrimination strategy, which used biotin to enrich the probe in cancer cells and reacted with overexpressed HOCl in cancer cells. Importantly, this dual-function discrimination strategy could obtain the precision detection of cancer cells, thereby offering assistance for improving the accuracy of early cancer diagnosis.

Concise Biothiol-Activatable HPQ-NBD Conjugate as a Targeted Theranostic Probe for Tumor Cells.

Cancer, as a malignant tumor, seriously endangers human health. The study of cancer diagnosis and therapy has great practical significance. The development of theranostic agents has become a very important research topic. Nevertheless, some existing agents still have imperfections, such as complex structures and difficult syntheses. Therefore, it is urgent for researchers to develop simple novel theranostic agents. In this study, the precipitated fluorophore HAPQ was used as a simple drug molecule for the first time and combined with NBD-Cl to construct a simple and efficient theranostic probe (HAPQ-NBD). The theranostic probe can distinguish between tumor cells and normal cells based on the higher levels of biothiol in tumor cells. In addition, the probe can use biothiol as a control switch to release higher levels of precipitated fluorophore HAPQ in tumor cells, leading to selective high toxicity to tumor cells, thus achieving the goal of selectively killing tumor cells. The construction of probe HAPQ-NBD provides a practical tool for the diagnosis and therapy of cancer. It is expected that the development and utilization of precipitated fluorophore will provide a new method and strategy for cancer diagnosis and therapy.

A new isothiocyanate-based Golgi-targeting fluorescent probe for Cys and its bioimaging applications during the Golgi stress response.

When the cell environment changes or is stimulated, the Golgi apparatus will respond to the corresponding stress, through the opening of related pathways, the expression of corresponding substances can be promoted or inhibited to achieve the purpose of controlling cell redox homeostasis and reducing cytotoxicity. Intuitive analysis of the changes in the content of various substances in the process of stress has important guiding value for the further study of stress response, drug evaluation and clinical diagnosis. Therefore, for the Cys overexpressed during the oxidative stress of the Golgi apparatus, we developed a specific and sensitive fluorescent probe (Gol-NCS) to visually monitor the biologically important Cys in real time. The probe has low cytotoxicity and shows great potential in cell and zebrafish imaging, it can detect the changes of endogenous and exogenous cysteine. It is important to explore the synthetic pathway of Cys during Golgi stress by using the Golgi targeting performance of the probe Gol-NCS. It is confirmed by fluorescence imaging for the first time that the activity of CSE enzyme plays a decisive role in the formation of Cys. Therefore, probe Gol-NCS with excellent photochemical properties is expected to provide help for the research on the involvement of Cys in Golgi stress.

An Integrated Fluorescent Probe for Ratiometric Detection of Glutathione in the Golgi Apparatus and Activated Organelle-Targeted Therapy.

Cancer is a serious threat to human health, and there is an urgent need to develop new treatment methods to overcome it. Organelle targeting therapy, as a highly effective and less toxic side effect treatment strategy, has great research significance and development prospects. Being an essential organelle, the Golgi apparatus plays a particularly major role in the growth of cancer cells. Acting as an indispensable and highly expressed antioxidant in cancer cells, glutathione (GSH) also contributes greatly during the Golgi oxidative stress. Therefore, it counts for much to track the changes of GSH concentration in Golgi for monitoring the occurrence and development of tumor cells, and exploring Golgi-targeted therapy is also extremely important for effective treatment of cancer. In this work, we designed and synthesized a simple Golgi-targeting fluorescent probe GT-GSH for accurately detecting GSH. The probe GT-GSH reacting with GSH decomposes toxic substances to Golgi, thereby killing cancer cells. At the same time, the ratiometric fluorescent probe can detect the concentration changes of GSH in Golgi stress with high sensitivity and selectivity in living cells. Therefore, such a GSH-responsive fluorescent probe with a Golgi-targeted therapy effect gives a new method for accurate treatment of cancer.

A novel cell membrane-targeting fluorescent probe for imaging endogenous/exogenous formaldehyde in live cells and zebrafish.

Formaldehyde (FA), an economically important chemical, has become a global pollutant and poses a threat to human health. As a kind of reactive carbonyl species, the abnormal production and degradation of FA in cells are related to many diseases. Therefore, it is of great significance to detect FA on the cell membrane and identify the internal and external sources of FA to analyse the causes of FA-induced physiological and pathological changes. In this work, a novel fluorescent probe Mem-FA was constructed by combining a dodecyl chain to target the cell membrane. Based on photoinduced electron transfer (PET), the probe relies on hydrazine as the receptor for FA recognition. Through this mechanism, the probe can detect FA sensitively, selectively and quantitatively. In addition, the probe Mem-FA can detect FA in vivo, especially the endogenous FA produced by tetrahydrofolate in a one-carbon cycle. More importantly, the probe Mem-FA can sensitively detect and distinguish the internal and external sources of FA on the cell membrane. Therefore, Mem-FA is capable of specifically tracing the fluctuations of FA-induced diseases.

A novel p-dimethylaminophenylether-based fluorescent probe for the detection of native ONOO- in cells and zebrafish.

Peroxynitrite (ONOO-) is a highly reactive substance, and plays an essential part in maintaining cellular homeostasis. It is crucial to monitor the ONOO- level in cells in normal and abnormal states. We introduced a p-dimethylaminophenylether-based fluorescent probe PDPE-PN, which could be synthesized readily. The new probe had prominent sensitivity and specificity, and a fast response towards ONOO-. The spectral performance of probe PDPE-PN was outstanding and the limit of detection was 69 nM. Probe PDPE-PN with low toxicity was applied to detect endogenous/exogenous ONOO- in RAW 264.7 macrophages and zebrafish. Importantly, successful application of the new receptor opens up new ideas for the design of ONOO- probes.

Treatment of Parkinson's disease in Zebrafish model with a berberine derivative capable of crossing blood brain barrier, targeting mitochondria, and convenient for bioimaging experiments.

Berberine is a famous alkaloid extracted from Berberis plants and has been widely used as medications and functional food additives. Recent studies reveal that berberine exhibits neuroprotective activity in animal models of Parkinson's disease (PD), the second most prevalent neurodegenerative disorders all over the world. However, the actual site of anti-PD action of berberine remains largely unknown. To this end, we employed a fluorescently labeled berberine derivative BBRP to investigate the subcellular localization and blood brain barrier (BBB) permeability in a cellular model of PD and zebrafish PD model. Biological investigations revealed that BBRP retained the neuroprotective activity of berberine against PD-like symptoms in PC12 cells and zebrafish, such as protecting 6-OHDA induced cell death, relieving MPTP induced PD-like behavior and increasing dopaminergic neuron loss in zebrafish. We also found that BBRP could readily penetrate BBB and function in the brain of zebrafish suffering from PD. Subcellular localization study indicated that BBRP could rapidly and specifically accumulate in mitochondria of PC12 cells when it exerted anti-PD effect. In addition, BBRP could suppress accumulation of Pink1 protein and inhibit the overexpression of LC3 protein in 6-OHDA damaged cells. All these results suggested that the potential site of action of berberine is mitochondria in the brain under the PD condition. Therefore, the findings described herein would be useful for further development of berberine as an anti-PD drug.

Toxicity of different zinc oxide nanomaterials and dose-dependent onset and development of Parkinson's disease-like symptoms induced by zinc oxide nanorods.

With the increasing applications in various fields, the release and accumulation of zinc oxide (ZnO) nanomaterials ultimately lead to unexpected consequences to environment and human health. Therefore, toxicity comparison among ZnO nanomaterials with different shape/size and their adverse effects need better characterization. Here, we utilized zebrafish larvae and human neuroblastoma cells SH-SY5Y to compare the toxic effects of ZnO nanoparticles (ZnO NPs), short ZnO nanorods (s-ZnO NRs), and long ZnO NRs (l-ZnO NRs). We found their developmental- and neuro-toxicity levels were similar, where the smaller sizes showed slightly higher toxicity than the larger sizes. The developmental neurotoxicity of l-ZnO NRs (0.1, 1, 10, 50, and 100 µg/mL) was further investigated since they had the lowest toxicity. Our results indicated that l-ZnO NRs induced developmental neurotoxicity with hallmarks linked to Parkinson's disease (PD)-like symptoms at relatively high doses, including the disruption of locomotor activity as well as neurodevelopmental and PD responsive genes expression, and the induction of dopaminergic neuronal loss and apoptosis in zebrafish brain. l-ZnO NRs activated reactive oxygen species production, whose excessive accumulation triggered mitochondrial damage and mitochondrial apoptosis, eventually leading to PD-like symptoms. Collectively, the developmental- and neuro-toxicity of ZnO nanomaterials was identified, in which l-ZnO NRs harbors a remarkably potential risk for the onset and development of PD at relatively high doses, stressing the discretion of safe range in view of nano-ZnO exposure to ecosystem and human beings.

Visualization of the cysteine level during Golgi stress using a novel Golgi-targeting highly specific fluorescent probe.

A novel Golgi-targeting Cys-specific fluorescent probe (Gol-Cys) was synthesized. Probe Gol-Cys is not only sufficiently sensitive to native Cys in living cells and zebrafish, but also can be used for monitoring the Cys level during Golgi stress.

A Simple Long-wavelength Fluorescent Probe for Simultaneous Discrimination of Cysteine/Homocysteine and Glutathione/Hydrogen Sulfide with Two Separated Fluorescence Emission Channels by Single Wavelength Excitation.

Small molecular biothiols, such as cysteine (Cys), homocysteine (Hcy), reduced glutathione (GSH), and hydrogen sulfide (H2S), play crucial parts in regulating the redox balance of life activities, regulating normal physiological activities and preventing various diseases. Quantitative analysis of these important small molecular substances is very important for revealing their diverse physiological and pathological effects. Although many fluorescent probes have been reported to detect biothiols in cells, it is still not sufficiently advanced to detect biothiols with separated fluorescence emission peak by same wavelength excitation. In our work, we designed a simple conjugate of Nile red and NBD (7-nitro-1,2,3-benzoxadiazole) as long-wavelength fluorescent probe NR-NBD for the simultaneous discrimination of these biothiols at single wavelength excitation. Probe NR-NBD could efficiently discriminate Cys/Hcy, GSH and H2S by two separated fluorescence emission channels and absorption spectra. Importantly, probe NR-NBD has excellent specificity and sensitivity towards the monitoring of endogenous/exogenous Cys/Hcy and GSH/H2S in living cells and zebrafish.

Two novel non-holostane type glycosides from the viscera of sea cucumber Apostichopus japonicus.

Two novel glycosides, apostichoposide A1 (1) and B1 (2), were isolated from the viscera of Chinese sea cucumbers (Apostichopus japonicus, Selenka) collected in the Bohai sea. Both the isolated glycosides were characterized by non-holostane type aglycones having 18(16)-lactone and 7(8)-double bond. Cytotoxic activities of the two compounds were evaluated against three human cancer cell lines. Compound 1 had adequate cytotoxic activity against MGC-803 and PC-3M cell lines. Our results indicated that glycosides present in A. japonicus viscera are an important high value resource for biotechnological applications.

A novel highly sensitive fluorescent probe for bioimaging biothiols and its applications in distinguishing cancer cells from normal cells.

In recent years, targeting drugs made by physical loading or chemical bonding of drugs on small molecular carriers have shown a very wide application prospect in the field of tumor and cancer treatment. How to achieve the release of drugs in cancer cells has become the core of this research. One of the most important bases for drug localization is to use the difference of small molecular biothiol concentration between cancer cells and normal cells. Details of the changes of biothiol levels in the growth and reproduction of cancer cells are still poorly understood, and the main reason is the lack of sensitive real-time imaging tools for biothiols in cancer cells. In this work, we reasonably designed and synthesized the combination of 4-hydroxy-1,8-naphthalimide and NBD-Cl as a concise fluorescent probe HN-NBD for imaging biothiols in live cells and zebrafish. In addition, due to the advantages of HN-NBD design, it is sufficiently sensitive to biothiols, and further imaging can distinguish cancer cells from normal cells. Probe HN-NBD would be of great significance to biomedical researchers for the study of biothiol-related diseases, the screening of new anticancer drugs, and the early diagnosis and treatment of cancers.

A novel water-soluble fluorescent probe with ultra-sensitivity over a wider pH range and its application for differentiating cancer cells from normal cells.

A novel pH-sensitive fluorescent probe has been designed and synthesized for sensing intracellular pH. This probe showed excellent water solubility, it was sensitive toward the pH range from 4 to 12, and it was especially sensitive in alkaline environments. During the pH changes from acidic to alkaline environments, the color of the solution turned from yellow to purple, thus the difference in color can be used to distinguish between acidic and alkaline systems. The other major features of probe pH-DCN including high selectivity, low toxicity, good reversibility and stability allowed pH-DCN to visualize fluctuations of the pH in live cells. Moreover, probe pH-DCN has successfully discriminated cancer cells from normal cells by monitoring their different intracellular pH levels.

Chemical constituents from Alismatis Rhizoma and their anti-inflammatory activities in vitro and in vivo.

Six new compounds, including a new compound with an unusual 2, 4, 6-cycloheptatrien ketone skeleton (1), two new diphenylpropanoid ethers (2, 3), a new protostane-type triterpenoid (4), two new norsesquiterpene (5a, 5b), and two new natural products (6, 7), together with eleven known compounds (8-18) were isolated from the aqueous extract of Alismatis Rhizoma (AR). Their structures were elucidated by a combination of 1D and 2D NMR (1H and 13C NMR, COSY, HSQC, HMBC, and NOESY), HRESIMS spectroscopic data, experimental and calculated electronic circular dichroism (ECD) spectra. Some of the compounds were evaluated for their inhibitory effects on nitric oxide (NO) production in LPS-induced RAW 264.7 cells. Two protostane-type triterpenoids, compounds 4 and 17, exhibited potent inhibitory activities with the IC50 values of 39.3 and 63.9 µM compared with indomethacin. In the meanwhile, their anti-inflammatory effects were also confirmed by acute inflammation model induced by CuSO4 in zebrafish.

Development of a Concise Rhodamine-Formylhydrazine Type Fluorescent Probe for Highly Specific and Ultrasensitive Tracing of Basal HOCl in Live Cells and Zebrafish.

Hypochlorous acid (HOCl) has received special attention by virtue of its pivotal antimicrobial nature, and the appropriate amount of HOCl is beneficial to innate immunity of host to cope with microbial invasion. However, the uncontrollable accumulation of HOCl is implicated in many human diseases and even cancers. Thus, to determine its deeper biological functions, it is significantly important to specifically monitor intracellular HOCl in biosystems. Herein, we rationally designed a simple fluorescent probe FH-HA on the basis of the formylhydrazine recognition receptor and rhodamine B fluorophore. It is worth noting that the formylhydrazine moiety for the first time is adopted as the recognition receptor for specifically recognizing HOCl. Additionally, probe FH-HA also exhibited excellent performance in many areas including satisfactory water-solubility, high specificity, and excellent sensitivity. Notably, probe FH-HA could quickly respond to HOCl (within 3 s), which facilitates the tracing of transient HOCl. More importantly, probe FH-HA was capable of specifically tracing the fluctuations of endogenous HOCl in living cells and zebrafish, and it could monitor basal HOCl in cancer cells to distinguish cancer cells from normal ones.