Synthesis of a Novel Pyrazine-Pyridone Biheteroaryl-Based Fluorescence Sensor and Detection of Endogenous Labile Zinc Ions in Lung Cancer Cells

A small extent of endogenous labile zinc is involved in many vital physiological roles in living systems. However, its detailed functions have not been fully elucidated. In this study, we developed a novel biheteroaryl-based low molecular weight fluorescent sensor, 3-(phenylsulfonyl)-pyrazine-pyridone (5b), and applied it for the detection of endogenous labile zinc ions from lung cancer cells during apoptosis. The electron-withdrawing property of the sulfonyl group between the phenyl ring as an electron donor and the pyridone ring as a fluorophore inhibited the intramolecular charge transfer state, and the background fluorescence of the sensor was decreased in aqueous media. From the structure-fluorescence relationship analysis of the substituent effects with/without Zn 2+ , compound 5b acting as a sensor possessed favorable properties, including a longer emission wavelength, a large Stokes shift (over 100 nm),a large fluorescence enhancement in response to Zn 2+ under physical conditions, and good cell membrane permeability in living cells. Fluorescence imaging studies of human lung adenocarcinoma cells (A549) undergoing apoptosis revealed that compound 5b could detect endogenous labile zinc ions. These experiments suggested that the low molecular weight compound 5b is a potential fluorescence sensor for Zn 2+ toward understanding its functions in living systems

One-Pot Synthesis of 6-Substituted Amino-2,4-diaminopyrimidine Derivatives Using Ketene Dithioacetals with Amines and Guanidine Carbonate

6-Substituted amino-2,4-diaminopyrimidine derivatives were prepared by one-pot synthesis using ketene dithioacetals, amine compounds, and guanidine carbonate in pyridine. These pyrimidine products displayed blue fluorescence in the solid state

Effective intraperitoneal gene transfection system using nanobubbles and ultrasound irradiation

In this study, we demonstrate the low toxicity and highly efficient and spatially improvedtransfection of plasmid DNA (pDNA) with liposomal nanobubbles (bubble liposomes [BLs])using ultrasound (US) irradiation in mice. Naked pDNA with BLs was intraperitoneally injected, followed by US irradiation. The injection volume, the duration of US irradiation, and the dose of BLs were optimized. Both BLs and US irradiation were essential to achieve high transgeneexpression from naked pDNA. We observed transgene expression in the entire peritonealtissues, including the peritoneal wall, liver, spleen, stomach and small and large intestines. The area of transfection could be controlled with focused US irradiation. There were few changes in the morphology of the peritoneum, the peritoneal function or serum alanine aminotransferase levels, suggesting the safety of BLs with US irradiation. Using a tissue-clearing method, the spatial distribution of transgene expression was evaluated. BLs with US irradiation delivered pDNA to the submesothelial layer in the peritoneal wall, whereas transgene expression was restricted to the surface layer in the liver and stomach. Therefore, BLs with USirradiation could be an effective and safe method of gene transfection to the peritoneum

Characterization of transgene expression and pDNA distribution of the suctioned kidney in mice

We have previously developed an efficient and safe transfection method for the kidney in mice: renal suction-mediated transfection. In this study, we verified the detailed characteristics of transgene expression and plasmid DNA (pDNA) in mice to develop therapeutic strategies and application to gene function analysis in the kidney. After naked pDNA was administered intravenously, the right kidney was immediately suctioned by a tissue suction device. We examined the spatial distribution of transgene expression and pDNA in the suctioned kidney using tissue clearing by CUBIC, ClearT2, and Scale SQ reagents. Spatial distribution analysis showed that pDNA was transfected into extravascular cells and sufficiently delivered to the deep renal cortex. In addition, we revealed that transgene expression occurred mainly in peritubular fibroblasts of the suctioned kidney by tissue clearing and immunohistochemistry. Next, we confirmed the periods of pDNA uptake and activation of transcription factors nuclear factor-κB and activator protein 1 by luciferase assays. Moreover, the use of a pCpG-free plasmid enabled sustained transgene expression in the suctioned kidney. In conclusion, analyses of the spatial distribution and immunostaining of the section suggest that pDNA and transgene expression occurs mainly in peritubular fibroblasts of the suctioned kidney. In addition, we clarified some factors for efficient and/or sustained transgene expression in the suctioned kidney

亜鉛ｲｵﾝ応答性ﾋﾟﾘｼﾞﾝ-ﾋﾟﾘﾄﾞﾝ型蛍光ﾌﾟﾛｰﾌﾞを用いた細胞内亜鉛分布解析への挑戦(その1)

ｶｲｺ(Bombyx mori)の幼虫体液中にはｶｲｺ由来培養細胞の増殖を抑制する活性があることを見出した｡また､ｶｲｺ体液によって増殖を阻害された細胞中には脂肪滴の形成が認められた｡本研究ではこれらの細胞増殖抑制および脂肪蓄積促進活性の実体となるｶｲｺ体液因子の解明を行った｡ｶｲｺ体液を出発材料として､細胞増殖抑制活性を指標にﾀﾝﾊﾟｸ質の精製を行った｡6段階の精製過程の後､SDS-ﾎﾟﾘｱｸﾘﾙｱﾐﾄﾞｹﾞﾙ電気泳動において単一ﾊﾞﾝﾄﾞを示す画分を得た｡ｱﾐﾉ酸配列解析の結果､このﾀﾝﾊﾟｸ質はｶｲｺ Niemann-Pick disease type C2(BmNPC2)ﾀﾝﾊﾟｸ質であると同定された｡ﾘｺﾝﾋﾞﾅﾝﾄ BmNPC2 ﾀﾝﾊﾟｸ質は細胞増殖抑制活性を示したことから BmNPC2 ﾀﾝﾊﾟｸ質がｶｲｺ体液中の細胞増殖抑制活性の実体であることが示唆された｡さらに､ﾘｺﾝﾋﾞﾅﾝﾄ BmNPC2 ﾀﾝﾊﾟｸ質をｶｲｺ培養細胞に添加することによって､細胞内ﾄﾘｸﾞﾘｾﾘﾄﾞ量の増大が認められた｡以上の結果は､BmNPC2 ﾀﾝﾊﾟｸ質がｶｲｺにおいて細胞増殖と脂質代謝を制御する体液性因子であることを示唆している｡To detect the subcellular localization of labile zinc ions, we applied Bpy-NH2, a novel zinc responsive fluorescent probe that contains a unique pyridine-pyridone structure. Because of a compact size and physicochemical characteristics of Bpy-NH2, it passes through a cellular membrane and reach to any intracellular compartments where labile zinc ions are concentrated. In HeLa cells, Bpy-NH2 localized to the late endosomes and lysosomes as Fluo-Zin3 AM, a known fluorescent zinc probe, did. In the presence of pyrithione, a zinc ionophore, the fluorescent signal of Fluo-Zin3 AM still retained in the late endosomes and lysosomes, suggesting that this probe only can detect zinc ions localized to the acidic compartments. Intriguingly, in the presence of pyrithione, Bpy-NH2 detected the distribution of labile zinc ions to both the cytoplasm and nucleus. We propose that Bpy-NH2 may have an unbiased subcellular localization property and thus, it can detect bona fide intracellular localization of zinc ions

High-Affinity Ratiometric Fluorescence Probe Based on 6-Amino-2,2′-Bipyridine Scaffold for Endogenous Zn2+ and Its Application to Living Cells

Zinc is an essential trace element involved in many biological activities; however, its functions are not fully understood. To elucidate the role of endogenous labile Zn2+, we developed a novel ratiometric fluorescence probe, 5-(4-methoxyphenyl)-4-(methylsulfanyl)-[2,2′-bipyridin]-6-amine (6 (rBpyZ)) based on the 6-amino-2,2′-bipyridine scaffold, which acts as both the chelating agent for Zn2+ and the fluorescent moiety. The methoxy group acted as an electron donor, enabling the intramolecular charge transfer state of 6 (rBpyZ), and a ratiometric fluorescence response consisting of a decrease at the emission wavelength of 438 nm and a corresponding increase at the emission wavelength of 465 nm was observed. The ratiometric probe 6 (rBpyZ) exhibited a nanomolar-level dissociation constant (Kd = 0.77 nM), a large Stokes shift (139 nm), and an excellent detection limit (0.10 nM) under physiological conditions. Moreover, fluorescence imaging using A549 human lung adenocarcinoma cells revealed that 6 (rBpyZ) had good cell membrane permeability and could clearly visualize endogenous labile Zn2+. These results suggest that the ratiometric fluorescence probe 6 (rBpyZ) has considerable potential as a valuable tool for understanding the role of Zn2+ in living systems

Synthesis of a Novel Pyrazine–Pyridone Biheteroaryl-Based Fluorescence Sensor and Detection of Endogenous Labile Zinc Ions in Lung Cancer Cells

A small extent of endogenous labile zinc is involved in many vital physiological roles in living systems. However, its detailed functions have not been fully elucidated. In this study, we developed a novel biheteroaryl-based low molecular weight fluorescent sensor, 3-(phenylsulfonyl)-pyrazine–pyridone (5b), and applied it for the detection of endogenous labile zinc ions from lung cancer cells during apoptosis. The electron-withdrawing property of the sulfonyl group between the phenyl ring as an electron donor and the pyridone ring as a fluorophore inhibited the intramolecular charge transfer state, and the background fluorescence of the sensor was decreased in aqueous media. From the structure–fluorescence relationship analysis of the substituent effects with/without Zn2+, compound 5b acting as a sensor possessed favorable properties, including a longer emission wavelength, a large Stokes shift (over 100 nm), a large fluorescence enhancement in response to Zn2+ under physical conditions, and good cell membrane permeability in living cells. Fluorescence imaging studies of human lung adenocarcinoma cells (A549) undergoing apoptosis revealed that compound 5b could detect endogenous labile zinc ions. These experiments suggested that the low molecular weight compound 5b is a potential fluorescence sensor for Zn2+ toward understanding its functions in living systems

Peptide-Based Cancer-Targeted DDS and Molecular Imaging

argeting cancer cell-surface receptors is an attractive approach for cancer treatment and diagnosis. Peptides having high binding affinities to receptors overexpressed in cancer cells are useful because of their simple structure, low immunogenicity, and easy, cost-effective chemical synthesis. A number of peptide ligands have been developed for cancer cell-surface receptors and applied to nanoparticles with anticancer drugs, genes, small interfering RNAs (siRNAs), and molecular imaging agents. In particular, recent findings have revealed that peptide-modified PEGylated liposome-encapsulated drugs are effective in cancer-targeted therapy and cancer cell-specific imaging. This review discusses peptide-modified nanoparticles for drug delivery systems (DDS) and molecular imaging, focusing on peptide ligands for somatostatin receptors, integrin, transferrin receptor, human epidermal growth factor 2 (HER2), etc. In addition, methods to improve binding affinity or endosomal escape with spacer peptides and stimuli (internal and external) are discussed

Synthesis of 6-(4-diethylamino)phenyl-2-oxo-2H-pyran-3-carbonitorile derivatives and their fluorescence in solid state and in solutions

[[abstract]]One-pot synthesis of a new 2-pyrone dye (3a) by the reaction of 4-diethylamino-acetophenone (1) with methyl 2-cyano-3,3-bis(methylsulfanyl)acrylate (2) in the presence of sodium hydroxide as the base was carried out in DMSO. Compound 4a was synthesized by the replacement of methylsulfanyl group of 3a with dimethylamine at 4-position of pyrone ring. Similarly, compound 5a was prepared via the reaction of 3a with diethyl malonate. Compounds 3a–5a exhibited the following fluorescence in the solid state: red (3a), green (4a), and orange (5a). In addition, it was revealed that 2-pyrone dyes exhibit fluorescence in various solvents and show positive solvatochromism. Compounds 3a and 5a exhibited intense fluorescence in chloroform and dichloromethane (fluorescence quantum yield Φ: 0.94–0.95). In contrast, compound 4a exhibited intense fluorescence in polar solvents (methanol: Φ = 0.92). These 2-pyrone dyes have the potential for applications in various fields.[[notice]]補正完畢[[journaltype]]國外[[incitationindex]]SCI[[booktype]]紙本[[countrycodes]]GB

Ligand peptide-grafted PEGylated liposomes using HER2 targeted peptide-lipid derivatives for targeted delivery in breast cancer cells: The effect of serine-glycine repeated peptides as a spacer

Ligand peptide-grafted PEGylated liposomes have been widely studied for targeted drug delivery systems. Because ligand peptides are commonly grafted using PEG as a spacer on the surface of PEGylated liposomes, the interaction between ligand peptides and their corresponding receptors can be interrupted by steric hindrance of the PEG layer. Therefore, we aimed to develop ligand peptide-lipid derivatives to enhance the targeting efficiency of ligand peptide-grafted PEGylated liposomes, and designed a new ligand peptide-lipid derivatives having serine-glycine repeats (SG)n as a spacer based on the peptide length calculated by PyMol (v0.99). We selected KCCYSL (KCC) as the ligand peptide for binding to human epidermal growth factor receptor-2 (HER2). We synthesized new KCC-(SG)n-lipid derivatives (n = 3, 5, 7) and evaluated their cellular association in breast cancer cells. KCC-(SG)n/PEGylated liposomes dramatically increased cellular association on HER2-positive breast cancer cells. The results suggest that KCC can be grafted on the surface of KCC-(SG)n/PEGylated liposomes prepared from KCC-(SG)n-lipid derivatives (n = 3, 5, 7). In summary, we succeeded in developing KCC-(SG)n-lipid derivatives for the preparation of ligand peptide-grafted PEGylated liposomes