Fluorescent Receptor Research Articles

A dual response UV-vis and fluorescence receptor based on acetylenic-indole conjoined silatrane for selective recognition of Co2+ and Cu2+ ions and in silico antidiabetic activity

This work involves the UV-vis and fluorescence study of a newly synthesized acetylenic-indole cojoined silatrane receptor for the selective recognition of Co2+ and Cu2+ ions and its molecular docking interaction with the α-glucosidase enzyme.

Recognition of d-Glucose in Water with Excellent Sensitivity, Selectivity, and Chiral Selectivity Using γ-Cyclodextrin and Fluorescent Boronic Acid Inclusion Complexes Having a Pseudo-diboronic Acid Moiety.

Fluorescence recognition of d-glucose in water with excellent sensitivity, selectivity, and chiral selectivity is desired because d-glucose is an essential component in biological and pathological processes. We report an innovative approach that exploits the 1:2 stoichiometric inclusion complexes of γ-cyclodextrin (γ-CyD) with two molecules of fluorescent monoboronic acid-based receptors, which form a pseudo-diboronic acid moiety as the recognition site for d-glucose in water. Two monoboronic acids (1F and 2N) were easily synthesized without heating or column purification. The 1:2 stoichiometric inclusion complexes (1F/γ-CyD and 2N/γ-CyD) were prepared in a mixture of dimethyl sulfoxide/water (2/98 in v/v) by mixing γ-CyD and the corresponding monoboronic acids. Both 1F/γ-CyD and 2N/γ-CyD exhibited strong turn-on response to d-glucose with excellent selectivity over nine other saccharides in the water-rich solvent at pH 7.4 owing to the ditopic recognition of d-glucose by the pseudo-diboronic acid moieties. The limits of detection of 1F/γ-CyD and 2N/γ-CyD for d-glucose were 1.1 and 1.8 μM, respectively, indicating the remarkable sensitivity for the detection of d-glucose at μM levels. 1F/γ-CyD and 2N/γ-CyD also demonstrated chiral-selective recognition of d-glucose, which is apparent from the 2.0- and 6.3-fold enhancement of fluorescence by the addition of d-glucose relative to l-glucose addition, owing to the chiral pseudo-diboronic acid moieties produced by the chiral γ-CyD cavity. To the best of our knowledge, 2N/γ-CyD has the highest d/l selectivity among hitherto reported fluorescent diboronic acid-based receptors.

Spatial quantification of single cell mRNA and ligand binding of the kappa opioid receptor in the rat hypothalamus

Detailed quantification of brain tissue provides a deeper understanding of changes in expression and function. We have created a pipeline to study the detailed expression patterns of the kappa opioid receptor in the rat hypothalamus using high resolution fluorescence microscopy and receptor autoradiography. The workflow involved structured serial sampling of rat hypothalamic nuclei, in situ detection of mRNA and receptor expression, and advanced image analysis. Our results demonstrate how maintaining spatial information can lead to increased understanding of RNA and protein expression. In addition, we show the detailed expression patterns of the kappa opioid receptor in the rat hypothalamus.

Citrate Functionalized Zirconium-Based Metal Organic Framework for the Fluorescent Detection of Ciprofloxacin in Aqueous Media.

Ciprofloxacin (CIP) is a commonly used antibiotic for the treatment of infectious diseases in humans and as a prophylactic agent in the livestock industry, leading to the environmental discharge of significant amounts of CIP. CIP is stable in aquatic systems leading to its pseudo-persistence. Constant exposure to these antibiotics results in the generation of antibiotic-resistant pathogens and potential toxicity/hypersensitivity in humans. Therefore, it is necessary to develop a convenient, rapid, and cost-effective method for the monitoring of ciprofloxacin in environmental samples. Rhodamine-based fluorescent receptors have the limitation of aqueous solubility. Therefore, in order to overcome this drawback, we designed a novel fluorescent receptor based on a zirconium-based metal organic framework (MOF-808). The precursor, MOF-808, was synthesized and functionalized by using sodium citrate to obtain a receptor called C-MOF-808. The C-MOF-808 was structurally characterized by XRD and spectroscopic analyses. Thus, this synthesized receptor can be used for the fluorescent detection of CIP in aqueous media with a detection limit of 9.4 µM. The detection phenomena of the receptor were studied by absorption as well as fluorescent spectra. The binding behavior of CIP with the receptor was studied by FT-IR and 1H-NMR analyses, and a binding mechanism is proposed.

Detection of endocrine and metabolism disrupting xenobiotics in milk-derived fat samples by fluorescent protein-tagged nuclear receptors and live cell imaging

Nuclear receptors (NRs) are ligand-modulated transcription factors that regulate multiple physiological functions in our body. Many NRs in their unliganded state are localized in the cytoplasm. The ligand-inducible nuclear translocation of NRs provides a valuable tool for studying the NR-ligand interactions and their downstream effects. The translocation response of NRs can be studied irrespective of the nature of the interacting ligand (agonist, antagonist, or a small molecule modulator). These nuclear translocation studies offer an advantage over promoter-reporter-based transcription assays where transcription response is observed only with the activating hormones or agonistic ligands. Globally, milk serves as a major dietary source. However, suspected presence of endocrine/metabolism-disrupting chemicals like bisphenols, parabens, organochlorine pesticides, carbamates, non-steroidal anti-inflammatory drugs, chloramphenicol, brominated flame retardants, etc. has been reported. Considering that these chemicals may impart serious developmental and metabolism-related health concerns, it is essential to develop assays suitable for the detection of xenobiotics present at differing levels in milk. Since milk samples cannot be used directly on cultured cells or for microscopy, a combination of screening strategies has been developed herein based on the revelation that i) lipophilic NR ligands can be successfully retrieved in milk-fat; ii) milk-fat treatment of cells is compatible with live-cell imaging studies; and finally, iii) treatment of cells with xenobiotics-spiked and normal milk derived fat provides a visual and quantifiable response of NR translocation in living cells. Utilizing a milk-fat extraction method and Green Fluorescent Protein (GFP) tagged NRs expressed in cultured mammalian cells, followed by an assessment of NR response proved to be an effective approach for screening xenobiotics present in milk samples. Highlights Diverse endocrine and metabolism-disrupting chemicals are suspected to contaminate milk. Nuclear receptors serve as ‘xenosensors’ for assessing the presence of xenobiotics in milk. Nuclear import of steroid receptors with (ant)agonist can be examined in live cells. Lipophilic xenobiotics are extracted and observed enriched in milk-fat fraction. A comprehensive cell-based protocol aids in the detection of xenobiotics in milk.

In vitro sepsis induces Nociceptin/Orphanin FQ receptor (NOP) expression in primary human vascular endothelial but not smooth muscle cells.

Sepsis is a dysregulated host response to infection that can cause widespread effects on other organs including cardiovascular depression, hypotension and organ failure. The receptor for Nociceptin/Orphanin FQ (N/OFQ), NOP is expressed on immune cells and these cells can release the peptide. Exogenous N/OFQ can dilate blood vessels and this peptide is increased in animal and human sepsis. We hypothesise that NOP receptors are present on vascular endothelial cells and therefore provide the target for released N/OFQ to cause vasodilation and hence hypotension. Using human umbilical vein endothelial cells (HUVEC) and human vascular smooth muscle cells (HVSMC) freshly prepared from umbilical cords and up to passage 4, we assessed NOP mRNA expression by Polymerase Chain Reaction (PCR), NOP surface receptor expression using a fluorescent NOP selective probe (N/OFQATTO594) and NOP receptor function with N/OFQ stimulated ERK1/2 phosphorylation. As an in vitro sepsis mimic we variably incubated cells with 100ng/ml Lipopolysaccharide and Peptidoglycan G (LPS/PepG). HUVECs express NOP mRNA and this was reduced by ~80% (n = 49) after 24–48 hours treatment with LPS/PepG. Untreated cells do not express surface NOP receptors but when treated with LPS/PepG the reduced mRNA was translated into protein visualised by N/OFQATTO594 binding (n = 49). These NOP receptors in treated cells produced an N/OFQ (1μM) driven increase in ERK1/2 phosphorylation (n = 20). One (of 50) HUVEC lines expressed NOP mRNA and receptor protein in the absence of LPS/PepG treatment. In contrast, HVSMC expressed NOP mRNA and surface receptor protein (n = 10) independently of LPS/PepG treatment. These receptors were also coupled to ERK1/2 where N/OFQ (1μM) increased phosphorylation. Collectively these data show that an in vitro sepsis mimic (LPS/PepG) upregulates functional NOP expression in the vascular endothelium. Activation of these endothelial receptors as suggested from in vivo whole animal work may contribute to the hypotensive response seen in sepsis. Moreover, blockade of these receptors might be a useful adjunct in the treatment of sepsis.

A Receptor Based on Diphenylaniline Donor Connected with Difuran and Pyridine as Acceptors: Synthesis, Crystal Structure and Selective Detection of Iron Ion

AbstractIn this work, the fluorescent receptor 4‐(2,6‐di(furan‐2‐yl)pyridin‐4‐yl)‐N,N‐diphenylaniline (DFPDA) was successfully synthesized overall in three steps, which bears three acceptor moieties one pyridine and two furans connected to a donor triphenylamine The DFPDA was fully characterized through 1H‐NMR, 13C‐NMR, ESI mass and elemental analysis. Further, single X‐ray structure confirm the intermediate and final DFPDA molecules. When we employed DFPDA molecule for sensing of metal ion, it shows selective detection of Fe3+ over other metal ions (Cu2+, K+, Cd2+, Co2+, Mn2+, Ni2+, Ba2+, Hg2+, Al3+, Pb2+, Zn2+, Ca2+, Fe2+ as their respective salts) dissolving in 1 : 1 water/DMSO solution with the binding constant 3.75×10−5 M. The limit of detection of DFPDA was found to be 52 nM which is much lower than the environmental protection agency guidelines (5.37 μM). Therefore, we believe that the DFPDA 1 fluorescent receptor may become outstanding chromophore for practical applications. Competing financial interests: The authors declare no competing interests.

TD-DFT investigation on anion recognition mechanism of anthraldehyde-based fluorescent thiosemicarbazone derivatives.

The mechanism of host-guest interaction of receptors towards fluoride ion has been investigated using computational methods. To distinguish the effect of aromaticity in host-guest interaction, we investigated unsubstituted (ATSC) and phenyl-substituted (APTSC) anthracene thiosemicarbazones towards different ions. In the ground state of receptor-fluoride complex, the added fluoride ion made hydrogen bond through N - H…F…H - N, whereas the intramolecular hydrogen bonding was through F - H…N in the excited state of receptor-fluoride complex. Experimental absorption and emission spectra were well reproduced by the calculated vertical excitation energies. The transition state (TS) calculations were performed to understand the thermodynamic features and mechanism of host-guest interaction. The natural bond orbital analyses show that the second perturbation energy for donor-acceptor interaction of F- with hydrogen is more than 300kcal/mol-1 at the excited state of receptor-fluoride complex, which indicates the strong single bond between fluoride and hydrogen atom. The PES scan confirms that deprotonation took place at the excited state of receptor-fluoride complex. The results indicate the excited-state proton transfer (ESPT) process from N-H group nearby the anthracene moiety. The APTSC is a better chemosensor than ATSC. This infers that the aromaticity will increase the efficiency of fluorescence receptor towards fluoride ion. A schematic representation of sensing mode of anthracene-based thiosemicarbazones toward fluoride ion. The fluoride ion first makes a hydrogen bond with NH proton nearby anthracene moiety. The excited state proton transfer mechanism was confirmed by PES and NBO studies.

A design of red emission CDs-based aptasensor for sensitive detection of insulin via fluorescence resonance energy transfer

We successfully designed an aptasensor based on the red emission carbon dots (R-CDs) and effectively detected insulin (INS) via fluorescence resonance energy transfer (FRET). In the process, the aptamer (apt) labeled with R-CDs (R-CDs@apt) was used as fluorescence donor and graphene oxide (GO) was used as fluorescence receptor. The successful detection due to the aptamer sequence has a certain affinity for Go and INS, while the affinity for INS is stronger than that of GO. When INS is not added to the detection system, the aptamer is adsorbed onto the surface of GO, shortening the distance between R-CDs@apt and GO, resulting in FRET and the quenching of fluorescence of R-CDs@apt. When INS was added to the detection system, the aptamer selectively bound INS and separated from the adsorption of GO, FRET gradually disappeared and the fluorescence of R-CDs@apt/GO/INS system was restored. By comparing the changes of fluorescence intensity before and after adding INS, the detection of INS was implemented. The aptasensor has a good linear curve with the detection limit of as low as 1.1 nM when the concentration of INS reached 1.3–150 nM. This method has excellent selectivity and anti-interference. Therefore, it is a potential method for detecting substances in biological fluids.

Fluorescent A2A and A3 adenosine receptor antagonists as flow cytometry probes.

Adenosine receptor (AR) ligands are beingdeveloped for metabolic, cardiovascular, neurological, and inflammatory diseases and cancer. The ease of drug discovery is contingent on the availability of pharmacological tools. Fluorescent antagonist ligands for the human A2A and A3ARs were synthesized using two validated pharmacophores, 1,3-dipropyl-8-phenylxanthine and triazolo[1,5-c]quinazolin-5-yl)amine, which were coupled toeight reporter fluorophores: AlexaFluor, JaneliaFluor (JF), cyanine, and near infrared (NIR) dyes. The conjugates were first screened using radioligand binding in HEK293 cells expressing one of the three AR subtypes. The highest affinities at A2AAR were Ki 144-316nM for 10, 12, and 19, and at A3AR affinity of Ki 21.6nM for 19. Specific binding of JF646 conjugate MRS7774 12 to the HEK293 cell surface A2AAR was imaged using confocal microscopy. Compound 19MRS7535, a triazolo[1,5-c]quinazolin-5-yl)amine containing a Sulfo-Cy7 NIR dye, was suitable for A3AR characterization in whole cells by flow cytometry (Kd 11.8nM), and its bitopic interaction mode with an A3AR homology model was predicted. Given its affinity and selectivity (11-fold vs. A2AAR, ~ 50-fold vs. A1AR and A2BAR) and a good specific-to-nonspecific binding ratio, 19 could be useful for live cell or potentially a diagnostic in vivo NIR imaging tool and/or therapy targeting the A3AR.

Comparative Study on the Performance of Three Detection Methods for the Quantification of Pacific Ciguatoxins in French Polynesian Strains of Gambierdiscus polynesiensis.

Gambierdiscus and Fukuyoa dinoflagellates produce a suite of secondary metabolites, including ciguatoxins (CTXs), which bioaccumulate and are further biotransformed in fish and marine invertebrates, causing ciguatera poisoning when consumed by humans. This study is the first to compare the performance of the fluorescent receptor binding assay (fRBA), neuroblastoma cell-based assay (CBA-N2a), and liquid chromatography tandem mass spectrometry (LC-MS/MS) for the quantitative estimation of CTX contents in 30 samples, obtained from four French Polynesian strains of Gambierdiscus polynesiensis. fRBA was applied to Gambierdiscus matrix for the first time, and several parameters of the fRBA protocol were refined. Following liquid/liquid partitioning to separate CTXs from other algal compounds, the variability of CTX contents was estimated using these three methods in three independent experiments. All three assays were significantly correlated with each other, with the highest correlation coefficient (r2 = 0.841) found between fRBA and LC-MS/MS. The CBA-N2a was more sensitive than LC-MS/MS and fRBA, with all assays showing good repeatability. The combined use of fRBA and/or CBA-N2a for screening purposes and LC-MS/MS for confirmation purposes allows for efficient CTX evaluation in Gambierdiscus. These findings, which support future collaborative studies for the inter-laboratory validation of CTX detection methods, will help improve ciguatera risk assessment and management.

Anion Binding by Fluorescent Ureido-Hexahomotrioxacalix[3]arene Receptors: An NMR, Absorption and Emission Spectroscopic Study.

Fluorescent receptors (4a–4c) based on (thio)ureido-functionalized hexahomotrioxacalix[3]arenes were synthesised and obtained in the partial cone conformation in solution. Naphthyl or pyrenyl fluorogenic units were introduced at the lower rim of the calixarene skeleton via a butyl spacer. The binding of biologically and environmentally relevant anions was studied with NMR, UV–vis absorption, and fluorescence titrations. Fluorescence of the pyrenyl receptor 4c displays both monomer and excimer fluorescence. The thermodynamics of complexation was determined in acetonitrile and was entropy-driven. Computational studies were also performed to bring further insight into the binding process. The data showed that association constants increase with the anion basicity, and AcO−, BzO− and F− were the best bound anions for all receptors. Pyrenylurea 4c is a slightly better receptor than naphthylurea 4a, and both are more efficient than naphthyl thiourea 4b. In addition, ureas 4a and 4c were also tested as ditopic receptors in the recognition of alkylammonium salts.

Folic acid-mediated fibrosis is driven by C5a receptor 1-mediated activation of kidney myeloid cells.

We have previously reported that increased expression and activation of kidney cell complement components play an important role in the pathogenesis of renal scarring. Here, we used floxed green fluorescent protein (GFP)-C5a receptor 1 (C5aR1) knockin mice (GFP-C5ar1fl/fl) and the model of folic acid (FA)-induced kidney injury to define the cell types and potential mechanisms by which increased C5aR1 activation leads to fibrosis. Using flow cytometry and confocal microscopy, we identified macrophages as the major interstitial cell type showing increased expression of C5aR1 in FA-treated mice. C5ar1fl/fl.Lyz2Cre+/- mice, in which C5aR1 has been specifically deleted in lysozyme M-expressing myeloid cells, experienced reduced fibrosis compared with control C5ar1fl/fl mice. Examination of C5aR1-expressing macrophage transcriptomes by gene set enrichment analysis demonstrated that these cells were enriched in pathways corresponding to the complement cascade, collagen formation, and the NABA matrisome, strongly pointing to their critical roles in tissue repair/scarring. Since C5aR1 was also detected in a small population of platelet-derived growth factor receptor-β+ GFP+ cells, we developed C5ar1fl/fl.Foxd1Cre+/- mice, in which C5aR1 is deleted specifically in pericytes, and found reduced FA-induced fibrosis. Primary cell cultures of platelet-derived growth factor receptor-β+ pericytes isolated from FA-treated C5ar1fl/fl.Foxd1Cre+/- mice showed reduced secretion of several cytokines, including IL-6 and macrophage inflammatory protein-2, compared with pericytes isolated from FA-treated control GFP-C5ar1fl/fl mice. Collectively, these data imply that C5a/C5aR1 axis activation primarily in interstitial cells contributes to the development of renal fibrosis.NEW & NOTEWORTHY This study used novel green fluorescent protein C5a receptor 1 floxed mice and the model of folic acid-mediated kidney fibrosis to demonstrate the pathogenic role of increased expression of this complement receptor on macrophages.

Hydrogen Peroxide and Hypochlorite Responsive Fluorescent Nanoprobes for Sensitive Cancer Cell Imaging.

Accurate diagnosis of cancer cells directly affects the clinical treatment of cancer and can significantly improve the therapeutic effect of cancer patients. Cancer cells have a unique microenvironment with a large amount of peroxide inside, effectively differentiated from relevant microenvironment normal cells. Therefore, designing the high-sensitive probes to recognize and distinguish the special physiological microenvironment of cancer cells can shed light on the early diagnosis of cancers. In this article, we design and construct a fluorescence (FL) contrast agent for cancer cell recognition and imaging analysis. Firstly, luminol-gold NPs (Lum-AuNPs) have been initially built, and then successfully loaded with the fluorescent receptor Chlorin e6 (Ce6) to prepare the luminescent nanoprobes (Ce6@Lum-AuNPs) with green synthesis, i.e., with biocompatible agents and mild temperature. The as-prepared fluorescent Ce6@Lum-AuNPs can efficiently and sensitively realize FL bioimaging of cancer cells. The relevant bio-sensing mechanism pertains to the presence of hypochlorite (ClO−); hydrogen peroxide (H2O2) in cancer cells could readily interact with luminol to produce chemiluminescence, which can activate the Ce6 component to emit near-infrared (NIR) FL. Therefore, this raises the possibility of utilizing the Ce6@Lum-AuNPs as efficient fluorescent nanoprobes for promising cancer early diagnosis and other relevant disease bioanalysis.

BOPPY-based novel fluorescent dopamine D2 and D3 receptor ligands

Dopamine is one of the crucial neurotransmitters in the human brain. Its out-of-range concentration can lead to various neurological diseases with special interest for dopamine D2 and D3 receptor subtypes. Although BODIPY is a highly versatile structural moiety for fluorescence labeling, we have looked out for structurally related pyridine-based moieties. We used BOPPY labelling of well-described D2R/D3R pharmacophores to obtain ligands with moderate to low nanomolar binding affinities as well as low to excellent quantum yields for bright fluorescence ligands. To best of our knowledge, this is the first report on the application of BOPPY fluorophores to GPCR ligands. This approach offers a general applicable way for fluorescence labelling via primary aliphatic amine elements.

Antipyrine-based Schiff base as fluorogenic chemosensor for recognition of Zn2+, Cu2+ and H2PO4− in aqueous media by comparator, half subtractor and integrated logic circuits

A new dual colorimetric and fluorescent receptor L, antipyrine-based chemosensor, was synthesized and characterized by UV–Vis absorption and fluorescence spectroscopic studies. The binding behavior of the chemosensor L toward Cu2+ and Zn2+ ions in an aqueous medium was investigated by UV–Vis absorption, 1HNMR and fluorescence spectroscopic studies. Benesi-Hildebrand plot is used to calculate the binding affinity. Based on the UV–Vis titration of receptor L, the 1:1 stoichiometry between L and M2+ (M2+= Cu2+ or Zn2+) is obtained. The detection limits (obtained by the 3σ method) of compound L are 4.23 × 10−6 mol L−1 for Cu2+ and 2.41 × 10−6 mol L−1 for Zn2+. The receptor L-Zn2+ can act as a highly selective sensor for H2PO4− ions over other anionic species (F−, Cl−, Br−, I−, NO3−, CN−, HSO4−, ClO4− and OAC−) in an aqueous medium. According to the changes in absorption and emission wavelengths and visual color changes with two sequential ionic inputs (Cu2+ and Zn2+), it is clear that the receptor behavior can mimic the arithmetic functions of XOR, INH1, INH2, XNOR, OR, NOR and IMP logic gates and complex logic circuits such as comparator and half subtractor with a single molecule. The ‘novel’ dinuclear Zn(II) complex could be used as a colorimetric fluorescent H2PO4− (Pi) chemosensor. By utilizing Cu2+, Zn2+ and H2PO4− ions as the inputs, a new combinational logic circuit with three different logic gates (OR, NOT, and AND) has been constructed.

Sensitive detection of patulin based on DNase Ⅰ-assisted fluorescent aptasensor by using AuNCs-modified truncated aptamer

Patulin (PAT) is a mycotoxin that exists in a wide range and is harmful to human health, which has aroused great attention to food safety. In this study, we established a novel assay to detect food samples contaminated by PAT. First of all, we proposed a truncation protocol based on the secondary structure of aptamer. By removing the stem-loop structure one by one, an optimized truncated PAT aptamer (P-30) was obtained. To the best of our knowledge, the mechanism between PAT and P-30 was elaborated in the assisting of molecular docking and circular dichroism for the first time. Furthermore, we developed a sensitive aptasensor platform to detect PAT in food samples. The aptamer modified with gold nanoclusters (AuNCs) was used as fluorescent probe, and BSA-directed synthesized MnO2 nanoflakes (MnO2 NFs) as fluorescence receptor. Additionally, fluorescence signal amplification strategy assisted by DNase Ⅰ successfully improved the sensitivity of aptasensor. The developed assay had a low detection limit of 8.5 ng/L, and can be effectively used in apple and grape juice samples. Thus, the established aptasensor shows a broad application prospect in PAT detection.

Excited-state dynamics of dipyrrolyldiketone difluoroboron complexes.

Anion-responsive photofunctional materials have been extensively studied because anions are important for biotic activity and constitute the building blocks of elegant supramolecular architectures. A number of fluorescent anion receptors that can probe anions in their environments have been reported, but the excited states of many of these molecules remain elusive. Studies on excited-state dynamics provide fruitful information for optimizing the emission properties, minimizing the photodegradation and photorelease of anions, and exploring novel photofunctions. In this study, we investigated the excited-state dynamics of an aryl-substituted dipyrrolyldiketone difluoroboron complex, a π-conjugated anion receptor, by time-resolved visible and infrared absorption spectroscopy and emission decay measurements combined with quantum chemical calculations. Anion binding was found to alter the radiative and nonradiative rate constants and the excited-state absorption of the anion receptor. In contrast, the molecular structures and binding abilities were similar in the S0 and S1 states.

Experimental Study of the In Vitro and In Vivo Functional Activity of NKG2D Chimeric Antigen Receptor

Aim. To study antitumor cytotoxic effect of CAR-T NKG2D and CAR-T anti-CD19 in vitro and in vivo in order to compare antitumor activity of chimeric antigen receptors (CAR) with different structural and functional properties. Materials & Methods. CAR constructions were produced by molecular cloning. CAR-T cell populations were obtained by transduction of healthy donor T-lymphocytes with recombinant lentiviral particles coding CAR NKG2D or CD19 target antigen CAR sequences. CAR-T cell proportion was assessed by FusionRed fluorescence and EGFR membrane receptor imaging. Specific in vitro cytotoxic activity of CAR-T effector cells was analyzed by Real-Time Cytotoxicity Assay (RTCA) during co-cultivation with HeLa_CD19 target cell line using xCELLigence. Interferon-Y (IFN-y) synthesis in vitro and in vivo along with the degree of cytotoxic effect were analyzed by immunoassay of culture medium of co-cultivated effector cells and target cells as well as isolated auto-plasma from the peripheral blood of mice. To assess the in vivo functional activity, CAR-T cell populations were infused into immunodeficient NSG-SGM3 mice (10 000 000 cells/mouse) 12 days after HeLa_CD19 cell injection and confirmation of engraftment and tumor growth. Upon euthanasia, tumors were removed and fixed in paraffin to prepare histological sections. CAR-T cell tumor infiltration was assessed by CD3 antigen immunohistochemical staining. Results. The highest ligand (molecules MICA, ULBP1/2/3/4/5/6) expression levels were detected in HeLa cell line. The obtained NKG2D CAR-T cells showed a considerable cytotoxic activity against HeLa_CD19 target line (cell index [CI] = 1.27), which was, however, twice as low as that of CAR-T anti-CD19 (CI = 0.60) (p = 0.0038). IFN-y level during co-cultivation of CAR-T anti-CD19 with HeLa_CD19 at the ratio of Е/Т = 1:1 was 64,852 pcg/mL, which was 3.5 times higher than IFN-y level during co-cultivation of CAR-T NKG2D with HeLa_CD19 (18,635 pcg/mL) (p = 0.0360). The degree of tumor infiltration by CAR-T anti-CD19 cells was higher than that by CAR-T NKG2D. The absence of NKG2D proliferating CAR-T cells in mice peripheral blood confirms their low persistence. IFN-y concentration in mice auto-plasma was 11.89 pcg/mL after CAR-T anti-CD19 infusion and 0.57 pcg/mL after CAR-T NKG2D infusion (p = 0.0079). The mean weight of tumor xenografts in experimental groups 10 days after CAR-T anti-CD19 injection was 0.72 g (p = 0.0142), after Т-lymphocyte and NKG2D CAR-T cell infusions it was 2.12 g and 1.2 g, respectively. Conclusion. CAR-T anti-CD19 cells are characterized by more pronounced cytotoxic effect under both in vitro and in vivo experimental conditions compared with CAR-T NKG2D cells. The degree of CAR-T anti-CD19 proliferation and their infiltration in mice xenograft models is considerably higher than the levels reached with NKG2D CAR-T cell injections. A single CAR-T NKG2D injection results only in short-term tumor reduction.

Synthesis of BINOL-xylose-conjugates as "Turn-off" fluorescent receptors for Fe3+ and secondary recognition of cysteine by their complexes.

A novel chiral fluorescence “turn-off” sensor was synthesised using the click reaction. The sensor was a BINOL–xylose derivative, modified at the 2-position and linked by 1,2,3-triazole. It was structurally characterized by 1HNMR, 13CNMR, ESI-MS and IR analysis. The selectivity of R-β-d-2 in methanol solution has been studied. Among the 19 transition metal ions, alkaline metal ions and alkaline earth metal ions studied, R-β-d-2 had a selective fluorescence quenching reaction for Fe3+. The detection limit of R-β-d-2 for Fe3+ was 0.91 μmol L−1. Complexation between R-β-d-2 and Fe3+ was investigated by ESI-MS and 1HNMR. The stoichiometric ratio of R-β-d-2 was 1 : 1. In addition, the R-β-d-2–Fe3+ complex was titrated with 20 naturally occurring amino acids and Hcy with GSH. It was found that the complex R-β-d-2–Fe3+ had a secondary recognition effect on Cys by switching to fluorescence.