The STING (stimulator of interferon genes) pathway is one of the pathways that regulate innate immunity, and the extracellular hydrolytic enzyme ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) has been identified as its dominant negative regulator. Since activation of the innate immune system is a promising strategy for the treatment of various infectious diseases and cancers, ENPP1 inhibitors have attracted great attention as candidate drugs. We have previously identified small-molecule ENPP1 inhibitors having a [1,2,4]triazolo[1,5-a]pyrimidine scaffold by means of chemical screening using a fluorescence probe, TG-mAMP. In this study, we evaluated the structure-activity relationships of the hit and lead compounds in detail, and succeeded in developing compounds that strongly and selectively inhibit ENPP1 not only in vitro, but also in cellular systems.

UNASSIGNED: Indocyanine Green (ICG) as an agent for photodynamic therapy (PDT) of melanoma cancer has low quantum yield, short circulation half-life, poor photo-stability, and tendency to aggregation.
UNASSIGNED: N-doped carbon quantum dot (CQD) nanoparticle was applied to encapsulate ICG and overcome ICG obstacle in PDT with simultaneous cell imaging property.
UNASSIGNED: CQD was prepared using hydrothermal method. Cell culture study and In vivo assessments on C57BL/6 mice containing melanoma cancer cells was performed.
UNASSIGNED: Results showed that CQD size slightly enhanced from 24.55 nm to 42.67 nm after ICG loading. Detection of reactive oxygen species (ROS) demonstrated that CQD improved ICG photo-stability and ROS generation capacity upon laser irradiation. Cell culture study illustrated that ICG@CQD could decrease survival rate of melanoma cancer cells of B16F10 cell line from 48% for pure ICG to 28% for ICG@CQD. Confocal microscopy images approved more cellular uptake and more qualified cell imaging ability of ICG@CQD. In vivo assessments displayed obvious inhibitory effect of tumor growth for ICG@CQD in comparison to free ICG on the C57BL/6 mice. In vivo fluorescence images confirmed that ICG@CQD accumulates remarkably more than free ICG in tumor region. Finally, ICG@CQD was proposed as an innovative nanocarrier for PDT and diagnosis.

Selective detection of a metal ion with high selectivity is of great importance to understand its existence and its role in many chemical and biological processes. We report here the synthesis, characterization and Al3+ sensing properties of two rhodamine-based isomers, (E)-2-((2-(allyloxy)benzylidene)amino)ethyl)-3\',6\'-bis(ethylamine)-2\',7\'-dimethylspiro[isoindoline-1,9\'-xanthen]-3-one (L-2-oxy) and (E)-2-((4-(allyloxy)benzylidene)amino)ethyl)-3\',6\'-bis(ethylamine)-2\',7\'-dimethylspiro[isoindoline-1,9\'-xanthen]-3-one (L-4-oxy). L-2-oxyand L-4-oxy show pink coloration, significant enhancement in absorbance at 530 nm and fluorescence intensity at 553 nm in the presence of Al3+ among several cations. Quantum yield and lifetime of the probes increase in the presence of Al3+. LOD values have been determined as low as ∼1.0 nM for both the isomers. DFT study suggests that the cation induces opening of spirolactam ring resulting in the changes of the rhodamine dyes. Additional reason could be Chelation Enhanced Fluorescence (CHEF) effect due to the subsequent chelation of the metal ion. Between two isomers, L-2-oxy displays better sensing ability towards Al3+ in terms of fluorescence enhancement, limit of detection, lifetime enhancement. Both the probes have been utilized in cell imaging studies using rat skeletal myoblast cell line (L6 cell line).

The development of compact and highly active plasmonic nanotags tuned on the first transparency window of biological tissues is under demand for cell imaging applications. The optical activity of bare plasmonic nanoparticles is determined by morphology but the more complex core-shell systems require experimental verification as a shell may change the expected trends. A comparative study of fluorescence core-shell nanotags with different morphology of gold core is presented in this work. Four types of gold nanoparticles (nanostars, nanobones, short and long nanorods), differing in the surface roughness were used for preparation of complex nanotags with a polymer shell containing cyanine 5.5 dye inside and surface functionalized with folic acid as a model delivery vector. The obtained core-shell nanotags were characterized with transmission electron microscopy, UV-Vis absorption spectroscopy and zeta potential measurements. Imaging performance of the obtained nanotags was studied with a fluorescence microscope on human pancreatic cancer cells, indicating a successful internalization of all nanotags by cancer cells and fluorescence intensity depending on the spectral overlap between the dye, plasmonic band of gold core and laser wavelength. The tags based on gold nanorods showed the brightest fluorescence among the studied systems. Scanning electron microscopy of the cells incubated with nanotags proved their internalization in membrane and cytoplasm. The cell viability assay showed reduced cytotoxicity and good biocompatibility up to the concentration enough for cell imaging. The obtained results suggested that compact core-shell nanotags can be used for targeting the folate receptor positive tumor cells.

Taking curcumin as the starting point, β-cyclodextrin was introduced on both sides, and lipid-soluble curcumin was coated by acrylic resin using oil-in-water strategy. Four different types of curcumin fluorescent complexes EPO-Curcumin (EPO-Cur), L100-55-Curcumin (L100-55-Cur), EPO -Curcumin-β-cyclodextrin (EPO-Cur-β-cd) and L100-55-Curcumin-β-cyclodextrin (L100-55-Cur-β-cd) were prepared to solve their own solubility and biocompatibility issues. The prepared curcumin fluorescent complexes were characterized and tested by spectroscopy. The characteristic peaks of 3446 cm-1 (hydroxyl group), 1735cm-1(carbonyl group) and 1455 cm-1 (aromatic group) were determined in the infrared spectrum. In the fluorescence emission spectrum, it was found that the emission intensity of different curcumin fluorescent complexes in polar solvents reached hundreds of times. Through the transmission electron microscopy shows that acrylic resin tightly coats curcumin into rods or clusters. In order to observe their compatibility with tumor cells more directly, live cell fluorescence imaging was carried out, and it was found that all four kinds of curcumin fluorescence complexes had good biocompatibility. In particular, the effect of EPO-Cur-β-cd and L100-55-Cur-β-cd is better than that of EPO-Cur and L100-55-Cur.

Natural Killer (NK) cells play a pivotal role in the elimination of tumor cells. The interactions that NK cells can establish with cancer cells in the tumor microenvironment (TME) are crucial for the outcome of the anti-tumor response, possibly resulting in mechanisms able to modulate NK cell effector functions on the one side, and to modify tumor cell phenotype and properties on the other side. This chapter will describe two different experimental approaches for the evaluation of NK-tumor cell interactions. First, a detailed protocol for the setting up of NK-tumor cell co-cultures will be illustrated, followed by information on cell imaging techniques, useful for assessing cell morphology and cytoskeletal changes. The second part will be focused on the description of a proteomic approach aimed at investigating the effect of this crosstalk from another point of view, i.e., characterizing the cellular and molecular pathways modulated in tumor cells following interaction with NK cells. The chapter centers on the interaction between NK and melanoma cells and refers to experimental approaches we set up to study the effects of this cross-talk on the process of the Epithelial-to-Mesenchymal Transition (EMT). Nevertheless, the described protocols can be quite easily adapted to study the interaction of NK cells with adherent tumor cell lines of different origin and histotype, as in our original study, we also analyzed possible NK-induced morphologic changes in the cervix adenocarcinoma HeLa cells and the colon cancer HT29 cells.

The selective detection of hydrogen sulfide in physiological and pathological processes has gained substantial attention in recent years. However, the real-time detection of hydrogen sulfide remains an elusive goal. In this work, a new type of hemicyanidin-based fluorescent \"turn-on\" probe NTR-HS (Ex = 680 nm, Em = 760 nm) was developed to detected H2S in a very short time (3 min). The fluorescence quantum yield is 0.15 and accompanied with a noticeable color change from violet to blue that can be used to detect H2S in the range 1.04 × 10-7-4 × 10-5 M with a limit of detection of 1.04 x 10-7 M. The NTR-HS probe was also used for imaging of endogenous hydrogen sulfide and mitochondrial localization in HCT116 and HeLa cells. The detection mechanism was studied through fluorescence, UV-Vis, NMR, and mass analysis. Notably, the probe was successfully used to imaging H2S in mice and locating hydrogen sulfide in the large intestine of mice.

Selective detection of H2 S in the cellular systems using fluorescent CPs/MOFs is of great scientific interest due to their outstanding aqueous stability, biocompatibility and real-time detection ability. Fabrication of such materials using complete biologically essential elements and applying them as an efficient biosensor is still quite challenging. In this context, two newly synthesized CPs containing biologically essential metal ion (Zn) and nitro/azido functional groups into the framework to sense extracellular and intracellular H2 S by reducing into respective amines are presented. The CP-1 containing the azide group acted as an efficient fluorescent turn-on probe with the lowest detection limit (7.2 μM) and shortest response time (30 s) among the Zn-based probes reported till date. Moreover, CP-1 exhibited green luminescence in live cells after imaging a very low concentration of H2 S, whereas the nitro analogue CP-2 could not detect the target analyte due to its framework disruption.

Monoaromatic molecules are a category of molecules containing a single aromatic ring which generally emit light in the ultraviolet (UV) region. Despite their facile preparation, the UV emission greatly limits their application as organic probes. In this study, we developed a general method to red shift the emission of monoaromatic molecules. Significant fluorescence red-shift (∼100 nm per intramolecular hydrogen bonding) can be achieved by introducing intramolecular hydrogen bonding units to benzene, a typical monoaromatic molecule. Upon increasing the number of hydrogen bonding units on the benzene ring, UV, blue, and green emissions are screened, which are switchable by simply breaking/restoration the intramolecular hydrogen bonding. As a demonstration, with the breaking of one intramolecular H-bonding, the green emission (λemmax = 533 nm) of 2,5-dihydroxyterephthalic acid (DHTA) changed to cyan (λemmax = 463 nm) upon the formation of its phosphorylated form (denoted as PDHTA), which, in the presence of alkaline phosphatase (ALP), hydrolyzed and recovered the green emission. By taking advantage of the switchable emission colors, ratiometric in vitro and endogenous ALP sensing was achieved. This general approach offers a great promise to develop organic probes with tunable emissions for fluorescence analysis and imaging by different intramolecular hydrogen bonding.

Zika virus (ZIKV) infection during pregnancy can result in congenital Zika syndrome which is characterized by microcephaly and other neurodevelopmental disorders. In this chapter, we describe methods to model ex vivo ZIKV infection in astrocytes and tissue explants from human fetal brain. These cell- and tissue-based platforms have been useful to elucidate mechanisms of ZIKV persistence and might lead to important clues about virus-induced neuropathogenesis. In addition, these ex vivo model systems allow researchers to conduct drug discovery and development experiments in more representative settings of the developing human brain.