Design,synthesis and application in biological imaging of a novel red fluorescent dye based on a rhodanine derivative

A novel acceptor–donor–acceptor type molecule, namely 2-triphenylamine-1,3-dia[2-(3-ethyl-4-oxo-thiazolidin-2-ylidene)-malononitrile] (2RDNTPA), is designed and synthesized. 2RDNTPA exhibits a large Stokes shift of 244 nm and red fluorescence emission of 629 nm with a decent photoluminescence quantum yield of 13%. Furthermore, as a potential red fluorescent dye, 2RDNTPA can be applied in fluorescence imaging of living cancer cells (HepG2) with negligible cytotoxicity and a half maximal inhibitory concentration much more than 100 μM.

2RDNTPA can be applied in fluorescence imaging of living cancer cells (HepG2) with red emission of 620 nm and negligible cytotoxicity with a half maximal inhibitory concentration much more than 100 μM.

In recent years, imaging technology has achieved remarkable development, which can convert chemical and biological information into monitorable signals, making it possible to visually monitor different biological components and various physiological processes in living organisms.¹ Among various imaging technologies, fluorescence imaging has been considered as a promising technology for elucidating biological functions.^1–7 The usually utilized fluorescent dyes for fluorescence imaging are molecules with emission wavelength beyond 600 nm that allow for slight photo-damage on samples and low levels of auto fluorescence from biomolecules.^2,3 It is widely reported that heavy-metal complexes^8–10 and organic dyes are candidates for achieving red emission of fluorescence imaging. However, most heavy-metal complexes possess some drawbacks such as high cost, serious pollution and strong toxicity, which limit their applications.^8–10 Therefore, the development of fluorescent dyes with metal-free small organic dyes for efficient fluorescence imaging attracted more and more attention.In general, the core properties of an efficient dye for fluorescence imaging include the following: (1) a large Stokes shift between excitation and emission;³ (2) high photo-luminescence quantum yield (PLQY); (3) red or infrared emission to eliminate the backgrounds from biological environments; (4) good biocompatibility and low toxicity; (5) easy to synthesize. To date, some red or infrared light-emitting fluorophores such as Cy7-1,² AHGa,⁴ DCF-MPYM,⁵et al. showed great potentials in fluorescence imaging. However, compared with conventional fluorescence dyes, fluorophores for efficient fluorescence imaging are still limited in number. It remains a challenge to develop brand-new fluorescent dyes for fluorescence imaging.Rhodanine,^23–25 a five-membered S,N-heterocycle, can serve as a building block for organic dyes in biomedical fields such as biomolecule fluorescent labeling,¹¹ proteomics,¹² photo-dynamic therapy,^13–15 live cell imaging,^16–19 antitumor drugs,²⁰ DNA detection,⁸ pH probes,²¹ fluorescent sensors,²²etc. Owning to their strong electron-withdrawing properties, rhodanine derivatives have been broadly utilized as acceptors for the design of donor–acceptor (D–A) type photovoltaic materials in organic solar cells (OSCs).³⁹ However, up to now, there is no report on D–A type dyes based on rhodanine derivatives with efficient red emission for fluorescence imaging.Herein, we designed and synthesized a new acceptor–donor–acceptor (A–D–A) type molecule 2-triphenylamine-1,3-dia[2-(3-ethyl-4-oxo-thiazolidin-2-ylidene)-malononitrile] (2RDNTPA, Scheme 1), in which triphenylamine (TPA) and rhodanine derivative of 2-(3-ethyl-4-oxo-thiazolidin-2-ylidene)-malononitrile (RDN) act as the electron donor and acceptor group, respectively. TPA unit is widely used as electron donor in emitters for its suitable steric hindrance and strong electron-donating ability,^26,27 while RDN unit with two cyan groups possesses strong electron-accepting ability and high molecular rigidity. 2RDNTPA exhibits a large Stokes shift of 244 nm and red fluorescence emission of 629 nm with a decent PLQY of 13%. More importantly, it is demonstrated that 2RDNTPA can be applied in fluorescence imaging of the living cancer cells (HepG2) with negligible cytotoxicity.Open in a separate windowScheme 1Synthetic route of 2RDNTPA.Synthetic routes of 2RDNTPA were shown in Scheme 1 and the detailed procedure can be found in the ESI.^† First, the key intermediate 2-triphenylaminebenzene-1,3-dialdehyde (2AIDTPA) was prepared by Suzuki coupling reaction between 2-bromobenzene-1,3-dialdehyde and N,N-diphenyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline. Then 2AIDTPA was converted to 2RDNTPA via Knoevenagel condensation³⁰ with 2-(3-ethyl-4-oxo-thiazolidin-2-ylidene)-malononitrile. The intermediate and target compound were fully identified by ¹H NMR (Fig. S1 and S2, ESI^†), MS (Fig. S3, ESI^†) and elemental analysis. Due to the substituent effects on the double bonds between RDN groups and the central benzene cycle, 2RDNTPA may have three kinds of cis–trans isomers (e.g.: EE, ZZ and EZ). In general, E-isomer is more stable than the corresponding Z-isomer. In addition, the ¹H NMR of two RDN groups are the same (Fig. S2^†), indicating that 2RDNTPA possesses a symmetrical structure. As a result, we can conclude that the reported structure of 2RDNTPA belongs to an EE isomer (Scheme 1).Thermal gravimetric analysis showed the decomposition temperature was about 369 °C with 5% loss of weight (Fig. S4, ESI^†), while the glass transition temperature was not found during the second heating of differential scanning calorimetry measurement. The thermal analysis results clearly demonstrated the favorable thermal stability of 2RDNTPA for biological imaging.To predict the electronic distribution of 2RDNTPA, density functional theory (DFT) calculations were performed. As shown in Fig. 1a, 2RDNTPA displayed a highly twisted molecular structure with large torsion angles of 60° and 30° between RDN units and the central phenyl unit. As expected, the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) of 2RDNTPA were located on the donor and acceptor unit, respectively.³³ The HOMO and LUMO energy levels of 2RDNTPA were estimated to be −5.27 and −2.95 eV, respectively (Fig. 1b). Accordingly, the optical bandgap (E_g) of 2RDNTPA was calculated to be 2.32 eV.Open in a separate windowFig. 1(a) Molecular structure, (b) HOMO (left) and LUMO (right) distributions, and theoretically calculated HOMO, LUMO of 2RDNTPA.The photophysical properties of 2RDNTPA in powder were investigated. From the UV-vis absorption spectra (Fig. 2a), the absorption peak at 298 nm can be attributed to the intense absorption from TPA centered n–π* transition, while the broad absorption band in the range of 350–500 nm with peak at 385 nm originate from intramolecular charge transfer transition (ICT).^33–38 From the onset of the absorption spectra, E_g of 2RDNTPA was calculated to be 2.25 eV, which agrees well with the theoretical result. As shown in Fig. 2a, 2RDNTPA exhibited an obviously red emission peak at 629 nm with PLQY of 13% (Fig. S5^†). The Stokes shift of 2RDNTPA between excitation (385 nm) and emission (629 nm) was calculated to be 244 nm, which may facilitate the elimination of background interference effectively. From the fluorescence and phosphorescence spectra of 2RDNTPA at 77 K (Fig. 2b), the singlet energy level and triplet energy level of 2RDNTPA were estimated to be 2.25 and 1.99 eV, respectively. The transient PL decay curve of 2RDNTPA was also depicted in Fig. 2c, which exhibited two components decay with an average lifetime of 9.26 ns.Open in a separate windowFig. 2(a) Normalized UV-vis absorption and PL spectra (excitation of 385 nm) of compound 2RDNTPA in powder. (b) Normalized fluorescence (Fl) and phosphorescence (Phos) spectra with 0.1 s delay of compound 2RDNTPA in powder at 77 K. (c) Transient decay curves of compound 2RDNTPA in powder.We also measured UV-vis and fluorescence spectra of 2RDNTPA in different solvents. As shown in Fig. S6 and Table S1,^† polarity of the solvents has negligible influence on the UV-vis spectra of 2RDNTPA. In contrast, the emission spectra of 2RDNTPA is gradually red-shifted with increasing solvent polarity, indicating typical ICT feature (Fig. S7 and Table S1^†). To explore the aggregated luminescence properties, the PL intensity of 2RDNTPA in the mixture of chloroform and methanol with various methanol ratio fractions was measured. As shown in Fig. S8,^† the PL intensity showed an overall slow decline when methanol fractions (f_w) increased from 0% to 90%, showing obvious aggregation-caused quenching properties of 2RDNTPA in the chloroform/methanol system. To evaluate the light stability of 2RDNTPA, the absorption intensity in dimethyl sulfoxide solution with various irradiation times of 365 nm ultraviolet lamp was characterized. The absorption intensity showed slow decline when irradiation time increased from 0 to 24 h (Fig. S9^†). It was evident that 2RDNTPA proved to be highly photo-stable.The electrochemical properties of 2RDNTPA were investigated by cyclic voltammetry (CV) measurements. As depicted in Fig. S10,^† 2RDNTPA displayed quasi-reversible oxidation process assigned to the triphenylamine units.^36,37Based on the onset of oxidation from CV curve, the HOMO energy level was estimated to be −5.29 eV. From the equation E_LUMO = E_HOMO + E_g, here the E_g value was estimated from the edge of UV-vis absorption, the LUMO energy level was estimated to be −3.04 eV, which are in accordance with the calculated results.The unique photophysical properties of 2RDNTPA led us to investigate its application in biological imaging. We treated living HepG2 liver cancer cells with 2RDNTPA solution. The detailed procedure can be found in the ESI.^† Then confocal laser scanning microscopy (CLSM) imaging was carried out by utilizing the red emission of 2RDNTPA to investigate the cellular uptake and sub-organelle localization in cancer cells.^31,32 As shown in Fig. 3, CLSM imaging can clearly characterize the situation inside the cell with strong red fluorescence close to 620 nm, which agrees well with the excellent red light-emitting performance of 2RDNTPA. It is worth noting that the cells did not show obvious morphology change, indicating good biocompatibility of 2RDNTPA in living HepG2 cell. As we all know, the low cytotoxicity of organic dyes is a key criterion for live cell imaging.¹ So we conducted 2RDNTPA in vitro cytotoxicity experiments.^28,29 As shown in Fig. 4, when the concentration of 2RDNTPA reach to 100 μM, more than 95% cells still survive at no hν environment. According to U.S. National Cancer Institute recommends,⁴⁰ IC₅₀ (inhibitory concentrations) greater than 50 μM can be considered as invalid during cell toxicity assay. The results suggest that 2RDNTPA shows negligible toxicity in living HepG2 liver cancer cells whether under illuminated conditions or not. The good biocompatibility and low cytotoxicity of 2RDNTPA indicate that it can be utilized as an efficient dye for live cell imaging to replace traditional heavy-metal complexes with high cytotoxicity.⁴¹Open in a separate windowFig. 3(a) CLSM (excitation of 407 nm and emission of 620 nm) images of 2RDNTPA in living HepG2 cell (10 μM incubation overnight). (b) Bright field. (c) Merged image.Open in a separate windowFig. 4Cell toxicity assay of 2RDNTPA in living HepG2 cell.In summary, we have developed an A–D–A structured red fluorescent dye 2RDNTPA, in which TPA and rhodanine derivative RDN act as the electron donor and acceptor group, respectively. Based on theoretical calculations, photophysical and electrochemical tests, the photophysical properties of 2RDNTPA have been systematically investigated. 2RDNTPA exhibits a large Stokes shift of 244 nm and red fluorescence emission of 629 nm with a decent photoluminescence quantum yield of 13%. By using 2RDNTPA as a red dye in biological imaging of the living HepG2 cancer cells, we have obtained commendable imaging results with IC₅₀ of much more than 100 μM. The present work provides a new strategy for the design and application of organic fluorescent dyes.     

Genome Analysis of Cytochrome in Dinotefuran-Treated Apolygus lucorum (Meyer-Dür)

Bulletin of Environmental Contamination and Toxicology - In this study, two CYP genes, CYP395G1 and CYP4EY1, were analyzed in Apolygus lucorum (Hemiptera: Miridae). The expression pattern in...     

经腹肠道超声在克罗恩病术后复发及合并症中的诊断价值

目的评估经腹肠道超声在检测克罗恩病（CD）术后吻合口复发方面的准确性。 方法选取2015年5月至2018年1月北京协和医院就诊的CD术后患者32例，行肠道超声检查，以肠镜和（或）手术病理和（或）临床评分为"金标准"，将患者分为复发组与缓解组。采用χ²检验，评价超声征象在复发组与缓解组中的差异，采用四格表计算肠道超声诊断CD术后吻合口复发的敏感度、特异度、准确性阳性预测值和阴性预测值。 结果复发组患者30例，缓解组患者2例。以吻合口处肠壁厚度>0.3 cm及肠壁结构不清为诊断标准时，超声对CD复发的敏感度、特异度、阳性预测值和阴性预测值分别为97%、100%、100%、67%。确诊29例，超声诊断吻合口及其他部位肠瘘共15例（15/32）的准确性为100%，诊断吻合口狭窄共8例（8/32），准确性为100%。 结论肠道超声在CD术后吻合口临床复发有特征性表现，在CD术后复发的监测和随访上有应用价值。     

131I标记的抗甲状腺髓样癌人源单链抗体在荷瘤裸鼠的体内分布和放射免疫显像

目的 测定¹³¹I标记的抗甲状腺髓样癌(MTC)人源单链抗体(scFv)的放射化学纯度,并研究荷MTC细胞(TT cells)裸鼠¹³¹I-scFv体内分布和体外放射免疫(RII)显像特点。方法 氯胺T法对scFv进行¹³¹I标记,Sephadex G200纯化标记抗体,三氯醋酸沉淀法测定标记率,纸层析法测定标记抗体在1、6、12、24 h的放射化学纯度、室温稳定性和血清稳定性。构建荷TT细胞裸鼠模型,尾静脉注射¹³¹I-scFv,分析¹³¹I-scFv在裸鼠重要组织器官的分布情况,并于注射药物12、24、48、72 h行SPECT正位静态显像,观察肿瘤内放射性浓集情况,肿瘤组织显像清晰时行SPECT/CT融合图像。结果 标记抗体经纯化后,测得标记率为(78.60±0.08)%,放射化学纯度为(87.10±0.78)%,各个时间点的放射化学纯度均在90%以上。荷瘤裸鼠体内研究结果显示,¹³¹I-scFv的放射性瘤/血、瘤/肌肉比值随时间的增长逐渐增高,48 h达最高。RII显示,¹³¹I-scFv可选择性聚集于肿瘤组织,48 h时肿瘤显像与全身组织对比最清晰,此时融合图像可清晰显示肿瘤部位的放射性浓聚情况,体内分布和体外显像结果一致。结论 成功标记了抗MTC人源scFv,生物活性较高,荷瘤裸鼠肿瘤的RII较好。     

Estimating the relationship between dam water level and surface water area for the Danjiangkou Reservoir using Landsat remote sensing images

The Danjiangkou Reservoir (DJKR) is the freshwater source for the Middle Route of the South-to-North Water Diversion Project (MRSNWDP) in China. It is important to characterize the dam water level (DWL) and surface water area (SWA) of the DJKR for the MRSNWDP. In this study, 81 phases of time-series Landsat images are used to estimate the SWA of the DJKR from 1993 to 2015. SWA and in situ-observed DWL data are employed to investigate the relationship between DWL and SWA for the DJKR. The results show that polynomial functions can describe this relationship, and quantitative assessment and qualitative analysis indicate that a cubic polynomial function is the optimal relationship between the DWL and SWA of the DJKR. The coefficient of determination (R²) and root mean square error of the cubic polynomial function are 0.9879 and 0.6987, respectively. This relationship has implications for water resources management, flood control and environmental monitoring for the MRSNWDP.     

澳大利亚现行药师注册考试制度探讨

目的 为完善我国执业药师资格考试制度提供参考.方法 采用文献综述和综合分析法.结果与结论 澳大利亚药师注册考试准入资格严格,注册考试类别分层级,考试内容及题型注重药学理论与药学服务的融合,考试方式灵活多样,考试管理也推动了药师队伍的发展.     

阿托伐他汀对急性冠脉综合征患者hs-CRP和IL-6的影响

目的探讨阿托伐他汀对急性冠脉综合征（ACS）患者高敏C反应蛋白（hs-CRP）和白细胞介素-6（IL-6）的影响。方法 86例ACS患者随机分为2组,治疗组（46例）予常规药物治疗＋阿托伐他汀20 mg/d,对照组（40例）仅予以常规治疗,随访1个月。在入选时及观察结束后检测丙氨酸转氨酶（ALT）、天冬氨酸转氨酶（AST）、肌酸激酶（CK）、hs-CRP及IL-6。结果经1个月治疗后,两组患者治疗前后AST、ALT、CK差异无统计学意义（P〉0.05）;对照组hs-CRP及IL-6较治疗前有所降低（P〈0.05）;治疗组hs-CRP及IL-6较治疗前显著降低（P〈0.01）;与对照组比较,治疗后治疗组hs-CRP及IL-6均有所降低（P〈0.01）。结论阿托伐他汀可以降低ACS患者hs-CRP及IL-6水平,抑制冠状动脉粥样斑块的炎症反应。     

茵陈五苓糖浆质量标准研究

目的:完善茵陈五苓糖浆的质量标准.方法:采用TLC法鉴别方中桂枝、茵陈;采用HPLC法测定方中绿原酸含量.结果:薄层鉴别特征明显,专属性强.HPLC法测定绿原酸在0.033 ～0.602 μg范围内有良好的线性关系,r=0.999 8,平均回收率为99.8％,RSD=1.4％(n=6).结论:本方法简便、可靠、准确,可用于茵陈五苓糖浆的质量控制.     

保留棘突韧带复合结构椎管潜行减压治疗退变性腰椎管狭窄症

目的探讨保留棘突韧带复合结构椎管潜行减压治疗退变性腰椎管狭窄症的疗效。方法回顾采用保留棘突韧带复合结构椎管潜行减压方法治疗的87例患者术后1年及3年的疗效,采用JOA评分,分析术前与术后1年和3年的疗效。结果术后1年和术后3年后的平均改善率为分别(84.0±9.0)%和(85.4±8.1)%,疗效对比无明显差异。结论保留棘突韧带复合结构椎管潜行减压既可对腰椎管狭窄进行减压,又可减少对腰椎稳定性的影响。     

Cholera toxin induces malignant glioma cell differentiation via the PKA/CREB pathway

Malignant gliomas are one of the leading causes of cancer deaths worldwide, but chemoprevention strategies for them are few and poorly investigated. Here, we show that cholera toxin, the traditional biotoxin and well known inducer of accumulation of cellular cAMP, is capable of inducing differentiation on malignant gliomas in vitro with rat C6 and primary cultured human glioma cells. Cholera toxin-induced differentiation was characterized by typical morphological changes, increased expression of glial fibrillary acid protein, decreased expression of Ki-67, inhibition of cellular proliferation, and accumulation of cells in the G(1) phase of the cell cycle. Cholera toxin also triggered a significant reduction in the G(1) cell-cycle regulatory proteins cyclin D1 and Cdk2 along with an overexpression of cell-cycle inhibitory proteins p21(Cip1) and p27(Kip1). Abrogation of cAMP-dependent protein kinase A activity by protein kinase A inhibitor or silencing of cAMP-responsive element binding proteins by RNA interference resulted in suppressed differentiation. These findings imply the attractiveness of cholera toxin as a drug candidate for further development of differentiation therapy. Furthermore, activation of the protein kinase A/cAMP-responsive element binding protein pathway may be a key and requisite factor in glioma differentiation.