High-sensitivity in vivo imaging for tumors using a spectral up-conversion nanoparticle NaYF4: Yb3+, Er3+ in cooperation with a microtubulin inhibitor

Fluorescein has been used for in vivo imaging to identify tumors. However, this technique presents several limitations, mainly due to its limited targeting efficiency, tissue autofluorescence and poor light penetration in tissue. In the present study, an alternative fluorescence imaging technique to localize tumors has been developed by using up-conversion nanoparticles (UCNs) and enhanced targeting approaches. A folic acid molecule is conjoined with UCNs (NaYF(4): Yb(3+), Er(3+)) to improve the tumor-specificity; the UCN is also loaded with the microtubule inhibitor CA4P, to further improve the local delivery of particles in the tumor. The proposed imaging technique combines several well-established individual concepts into one novel integrated procedure and significantly improves its tumor-imaging capability: the near-infrared excitation for UCNs minimizes tissue autofluorescence and allows imaging into deeper tissue; the improvement in the signal to noise ratio (SNR) is at least a magnitude better than that of a conventional fluorescence imaging technique, and the modification of UCNs with folic acid significantly improves the tumor targeting efficiency by utilizing its affinity for the folic acid receptor that is often over expressed in tumors. The loading of CA4P further helps UCNs to cross blood vessel walls to reach tumor cells by depolymerizing the microtubules of endothelial cells. The integrated nanoparticle possesses the near-infrared-identical optical properties of UCNs alone, thus achieving a highly effective fluorescence imaging probe. The results demonstrated that the proposed method provides an excellent alternative for tumor localization and a potential traceable vehicle for highly efficient drug delivery.     

Ratiometric Detection of Glutathione Based on Disulfide Linkage Rupture between a FRET Coumarin Donor and a Rhodamine Acceptor

Abnormal levels of glutathione, a cellular antioxidant, can lead to a variety of diseases. We have constructed a near-infrared ratiometric fluorescent probe to detect glutathione concentrations in biological samples. The probe consists of a coumarin donor, which is connected through a disulfide-tethered linker to a rhodamine acceptor. Under the excitation of the coumarin donor at 405 nm, the probe shows weak visible fluorescence of the coumarin donor at 470 nm and strong near-infrared fluorescence of the rhodamine acceptor at 652 nm due to efficient Forster resonance energy transfer (FRET) from the donor to the acceptor. Glutathione breaks the disulfide bond through reduction, which results in a dramatic increase in coumarin fluorescence and a corresponding decrease in rhodamine fluorescence. The probe possesses excellent cell permeability, biocompatibility, and good ratiometric fluorescence responses to glutathione and cysteine with a self-calibration capability. The probe was utilized to ratiometrically visualize glutathione concentration alterations in HeLa cells and Drosophila melanogaster larvae.     

Systemic Delivery of Protein Nanocages Bearing CTT Peptides for Enhanced Imaging of MMP-2 Expression in Metastatic Tumor Models

Matrix metalloproteinase 2 (MMP-2) in metastatic cancer tissue, which is associated with a poor prognosis, is a potential target for tumor imaging in vivo. Here, we describe a metastatic cancer cell-targeted protein nanocage. An MMP-2-binding peptide, termed CTT peptide (CTTHWGFTLC), was conjugated to the surface of a naturally occurring heat shock protein nanocage by genetic modification. The engineered protein nanocages showed a binding affinity for MMP-2 and selective uptake in cancer cells that highly expressed MMP-2 in vitro. In near-infrared fluorescence imaging, the nanocages showed specific and significant accumulation in tumor tissue after intravenous injection in vivo. These protein nanocages conjugated with CTT peptide could be potentially applied to a noninvasive near-infrared fluorescence detection method for imaging gelatinase activity in metastatic tumors in vivo.     

One-Pot Synthesis of Coumarin–Indomethacin Hybrids as COX-2 Targeting Probes for Cancer Imaging

Facile synthesis of 6- or 7-substituted coumarin-indomathacin hybrids (Coum-IDM) has been developed for specific cyclooxygenase-2 (COX-2) binding along with their intrinsic fluorescent properties. A mild and rapid condensation/dehydrative cyclization of 2-hydroxy benzaldehyde with activated indomethacin was carried out in one step under ultrasound irradiation. Coum-IDM₄ was found to be the best of this series as it presented significant binding to COX-2 and exhibited higher fluorescent intensity in cancer cells than in normal cells. Therefore, in the light of drug development tools, this new hybrid system could be a potential targeted probe for COX-2-overexpressed inflammation and cancer-cell tracking.     

甘氨酸-氟硼荧近红外荧光分子探针的合成及性能测试

将一种具有近红外荧光性能的氟硼荧染料（BDDIPY）作为显影基团,制成新型的检测肿瘤细胞的近红外荧光分子探针。首先以氟硼二吡咯烷（BOD）为起始原料,通过缩合反应,将具有强供电子作用的N-苯基亚氨基二苄引入到氟硼荧中,得到了荧光光谱处于近红外区的氟硼荧化合物,接着用N-羟基琥珀酰亚胺为活化剂,将具有肿瘤亲和力的甘氨酸引入到荧光基团-氟硼荧（BDDIPY）,最终得到甘氨酸-氟硼荧近红外荧光探针,并对其结构进行了核磁和质谱表征,并进行了光学和生物学性能测试。实验结果显示：甘氨酸-氟硼荧近红外荧光分子探针的荧光发射波长大于700 nm,与肺癌细胞（GLC-82）具有较好的亲和力,达到23.65,是一个具有良好应用前景的检测肿瘤细胞的近红外荧光分子探针。     

A sensitive and selective near-infrared fluorescent probe for mercuric ions and its biological imaging applications

A new mercury(II) near-infrared region fluorescent probe 3,9-dithia-6-monoazaundecane-tricarbocyanine has been designed and synthesized. It consists of two functional moieties: the tricarbocyanine performs as the near-infrared region fluorophore, and the 3,9-dithia-6-monoazaundecane acts as the selected binding site for metal ions. The near-IR excitation and emission profiles of the probe can minimize cell and tissue damage and avoid native fluorescence from natural cellular species. It exhibits fluorescence increase upon the binding of the Hg(2+) based on the inhibition of the photoinduced electron transfer quenching mechanism. Excellent sensitivity and selectivity for mercuric ions are observed with this probe. The value of the system is demonstrated by its use in monitoring the real-time uptake of Hg(2+) within HepG2 cells and five day old zebrafish. The synthesis and remarkable properties of it help to extend the development of metal ions fluorescent probes for biological applications.     

Combined effects of cyclooxygenase-1 and cyclooxygenase-2 selective inhibitors on ovarian carcinoma in vivo

The present study was designed to investigate the combined effects of cyclooxygenase (COX)-1 and COX-2 selective inhibitors on human ovarian SKOV-3 carcinoma cells xenograft-bearing mice. The animals were treated with 3 mg/kg SC-560 (a COX-1 selective inhibitor) alone, 25 mg/kg celecoxib (a COX-2 selective inhibitor) alone, or SC-560/celecoxib by gavage, twice a day for three weeks. To test the mechanism of inhibition of tumor growth by COX selective inhibitors, the index of proliferating cells in tumor tissues was determined by immunostaining and the index of apoptotic cells by the terminal-deoxynucleotidyl-transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) method. The inhibitory rate on tumor growth in the combination group was 35.54% which is significant statistically compared with that of the control group (P < 0.05). In the combination group, the index of cell proliferation and apoptosis were 12.40% and 51.03% respectively, which are significant statistically compared with those of the control group (22.56%, 19.07%, all P < 0.05). These studies indicate that synergism between two COX inhibitors and inhibitor combination treatment has particular potential for chemoprevention of ovarian cancer growth.     

Preparation of waterborne polyurethane with outstanding fluorescence properties and programmable emission intensity

The hard segment content and hydrophilic group content of a fluorescent waterborne polyurethane (FWPU) were used to optimize the polymer structure which can influence the fluorescence properties. The synthesis of FWPU was based on 2,2′‐((4‐([3,3′:6′,2″‐terpyridine]‐4′‐yl)phenyl)azanediyl)diethanol (TPPDA) as fluorophore. The structure of FWPU was characterized using Fourier transform infrared and ¹H NMR spectroscopies, and the polymer molecular weight and polydispersity were measured using gel permeation chromatography. The relationship between fluorescence properties and FWPU structure was studied using luminescence spectrometry. Compared to TPPDA, the maximum emission wavelength of FWPU showed a hypsochromic shift, and the fluorescence emission intensity and quantum yield of FWPU were increased obviously. Moreover, increasing the hard segment content led to an enhancement of the fluorescence lifetime of FWPU, and to the fluorescence intensity increasing at first and then decreasing. The fluorescence emission intensity and quantum yield of FWPU were increased nearly 11 and 7 times, respectively, compared to TPPDA when the hard segment content was 65%. With an increase of hydrophilic group content, the fluorescence emission intensity of FWPU increased. The fluorescence emission intensity of FWPU exhibited a response to pH, which facilitated its employment as a fluorescence probe. FWPU with excellent solid fluorescence is very suitable for processing into optical devices, with increasing hard segment content and hydrophilic group content both enhancing the solid fluorescence emission intensity of FWPU. © 2016 Society of Chemical Industry     

A Phosphinate-Containing Fluorophore Capable of Selectively Inducing Apoptosis in Cancer Cells

Chemotherapeutic agents generally suffer from off-target cytotoxicity in noncancerous cell types, leading to undesired side effects. As a result, significant effort has been put into identifying compounds that are selective for cancerous over noncancerous cell types. Our laboratory has recently developed a series of near-infrared (NIR) fluorophores containing a phosphinate functionality at the bridging position of a xanthene scaffold, termed Nebraska Red (NR) fluorophores. Herein, we report the selective cytotoxicity of one NR derivative, NR₇₄₄, against HeLa (cervical cancer) cells versus NIH-3T3 (noncancerous fibroblast) cells. Mechanistic studies based on the NIR fluorescence signal of NR₇₄₄ showed distinct subcellular localization in HeLa (mitochondrial) versus NIH-3T3 (lysosomal) that resulted from the elevated mitochondrial potential in HeLa cells. This study provides a new, NIR scaffold for the further development of reagents for targeted cancer therapy.     

Striking Antitumor Activity of a Methinium System with Incorporated Quinoxaline Unit Obtained by Spontaneous Cyclization

A novel pentamethinium salt was synthesized with an unforeseen expanded conjugated quinoxaline unit directly incorporated into a pentamethinium chain. The compound exhibited high fluorescence intensity, selective mitochondrial localization, high cytotoxicity, and selectivity toward malignant cell lines, and resulted in remarkable in vivo suppression of tumor growth in mice.     

L-Type Ca2+ Channel Inhibition Rescues the LPS-Induced Neuroinflammatory Response and Impairments in Spatial Memory and Dendritic Spine Formation

Ca²⁺ signaling is implicated in the transition between microglial surveillance and activation. Several L-type Ca²⁺ channel blockers (CCBs) have been shown to ameliorate neuroinflammation by modulating microglial activity. In this study, we examined the effects of the L-type CCB felodipine on LPS-mediated proinflammatory responses. We found that felodipine treatment significantly diminished LPS-evoked proinflammatory cytokine levels in BV2 microglial cells in an L-type Ca²⁺ channel-dependent manner. In addition, felodipine leads to the inhibition of TLR4/AKT/STAT3 signaling in BV2 microglial cells. We further examined the effects of felodipine on LPS-stimulated neuroinflammation in vivo and found that daily administration (3 or 7 days, i.p.) significantly reduced LPS-mediated gliosis and COX-2 and IL-1β levels in C57BL/6 (wild-type) mice. Moreover, felodipine administration significantly reduced chronic neuroinflammation-induced spatial memory impairment, dendritic spine number, and microgliosis in C57BL/6 mice. Taken together, our results suggest that the L-type CCB felodipine could be repurposed for the treatment of neuroinflammation/cognitive function-associated diseases.     

A FRET-Based Near-Infrared Fluorescent Probe for Ratiometric Detection of Cysteine in Mitochondria

We report a near-infrared fluorescent probe A for the ratiometric detection of cysteine based on FRET from a coumarin donor to a near-infrared rhodamine acceptor. Upon addition of cysteine, the coumarin fluorescence increased dramatically up to 18-fold and the fluorescence of the rhodamine acceptor decreased moderately by 45 % under excitation of the coumarin unit. Probe A has been used to detect cysteine concentration changes in live cells ratiometrically and to visualize fluctuations in cysteine concentrations induced by oxidation stress through treatment with hydrogen peroxide or lipopolysaccharide (LPS). Finally, probe A was successfully applied for the in vivo imaging of Drosophila melanogaster larvae to measure cysteine concentration changes.     

Chloro‐Functionalized Photo‐crosslinking BODIPY for Glutathione Sensing and Subcellular Trafficking

Glutathione (GSH) is one of major antioxidants inside cells that regulates oxidoreduction homeostasis. Recently, there have been extensive efforts to visualize GSH in live cells, but most of the probes available today are simple detection sensors and do not provide details of cellular localization. A new fluorescent probe (pcBD2‐Cl), which is cell permeable and selectively reacts with GSH in situ, has been developed. The in situ GSH‐labeled probe (pcBD2–GSH) exhibited quenches fluorescence, but subsequent binding to cellular abundant glutathione S‐transferase (GST) recovers the fluorescence intensity, which makes it possible to image the GSH–GST complex in live cells. Interactions between probe and GST were confirmed by means of photo‐crosslinking under intact live‐cell conditions. Interestingly, isomers of chloro‐functionalized 4,4‐difluoro‐4‐bora‐3a,4a‐diaza‐s‐indacene (BODIPY) compounds behaved very distinctively inside the cells. Following co‐staining imaging with MitoTracker and mitochondria fractionation upon lipopolysaccharide‐mediated reactive oxygen species induction experiments showed that pcBD2–GSH accumulated in mitochondria. This is the first example of a live‐cell imaging probe to visualize translocation of GSH from the cytosol to mitochondria.     

Synthesis of CdTe quantum dot-conjugated CC49 and their application for in vitro imaging of gastric adenocarcinoma cells

The purpose of this experiment was to investigate the visible imaging of gastric adenocarcinoma cells in vitro by targeting tumor-associated glycoprotein 72 (TAG-72) with near-infrared quantum dots (QDs). QDs with an emission wavelength of about 550 to 780 nm were conjugated to CC49 monoclonal antibodies against TAG-72, resulting in a probe named as CC49-QDs. A gastric adenocarcinoma cell line (MGC80-3) expressing high levels of TAG-72 was cultured for fluorescence imaging, and a gastric epithelial cell line (GES-1) was used for the negative control group. Transmission electron microscopy indicated that the average diameter of CC49-QDs was 0.2 nm higher compared with that of the primary QDs. Also, fluorescence spectrum analysis indicated that the CC49-QDs did not have different optical properties compared to the primary QDs. Immunohistochemical examination and in vitro fluorescence imaging of the tumors showed that the CC49-QDs probe could bind TAG-72 expressed on MGC80-3 cells.     

The GTPase Arf1 Is a Determinant of Yeast Vps13 Localization to the Golgi Apparatus

VPS13 proteins are evolutionarily conserved. Mutations in the four human genes (VPS13A-D) encoding VPS13A-D proteins are linked to developmental or neurodegenerative diseases. The relationship between the specific localization of individual VPS13 proteins, their molecular functions, and the pathology of these diseases is unknown. Here we used a yeast model to establish the determinants of Vps13′s interaction with the membranes of Golgi apparatus. We analyzed the different phenotypes of the arf1-3 arf2Δ vps13∆ strain, with reduced activity of the Arf1 GTPase, the master regulator of Golgi function and entirely devoid of Vps13. Our analysis led us to propose that Vps13 and Arf1 proteins cooperate at the Golgi apparatus. We showed that Vps13 binds to the Arf1 GTPase through its C-terminal Pleckstrin homology (PH)-like domain. This domain also interacts with phosphoinositol 4,5-bisphosphate as it was bound to liposomes enriched with this lipid. The homologous domain of VPS13A exhibited the same behavior. Furthermore, a fusion of the PH-like domain of Vps13 to green fluorescent protein was localized to Golgi structures in an Arf1-dependent manner. These results suggest that the PH-like domains and Arf1 are determinants of the localization of VPS13 proteins to the Golgi apparatus in yeast and humans.     

Multifunctional Cyanine-Based Theranostic Probe for Cancer Imaging and Therapy

Cancer is one of the leading causes of death in the world. A cancer-targeted multifunctional probe labeled with the radionuclide has been developed to provide multi-modalities for NIR fluorescence and nuclear imaging (PET, SPECT), for photothermal therapy (PTT), and targeted radionuclide therapy of cancer. In this study, synthesis, characterization, in vitro, and in vivo biological evaluation of the cyanine-based probe (DOTA-NIR790) were demonstrated. The use of cyanine dyes for the selective accumulation of cancer cells were used to achieve the characteristics of tumor markers. Therefore, all kinds of organ tumors can be targeted for diagnosis and treatment. The DOTA-NIR790 labeled with lutetium-111 could detect original or metastatic tumors by using SPECT imaging and quantify tumor accumulation. The β-emission of ¹⁷⁷Lu-DOTA-NIR790 can be used for targeted radionuclide therapy of tumors. The DOTA-NIR790 enabled imaging by NIR fluorescence and by nuclear imaging (SPECT) to monitor in real-time the tumor accumulation and the situation of cancer therapy, and to guide the surgery or the photothermal therapy of the tumor. The radionuclide-labeled heptamethine cyanine based probe (DOTA-NIR790) offers multifunctional modalities for imaging and therapies of cancer.     

Conjugated polyelectrolyte-cisplatin complex nanoparticles for simultaneous in vivo imaging and drug tracking

A molecular brush based on conjugated polyelectrolyte (CPE) grafted with dense poly(ethylene glycol) (PEG) chains was successfully complexed with an anticancer agent, cisplatin, to form cisplatin-loaded nanoparticles (CPE-PEG-Pt). The obtained nanoparticles have high far-red/near-infrared fluorescence and are able to release the drug in a continuous and slow manner. These nanoparticles have not only been used to visualize HepG2 cancer cells, but also served as an in vivo fluorescent imaging probe that simultaneously tracks the in vivo drug distribution in nude mice upon intravenous administration.     

A NIR Emitting Cyanine with Large Stokes’ Shift for Mitochondria and Identification of their Membrane Potential Disruption

An NIR emitting (λ_em≈730 nm) cyanine probe ExCy was synthesized in good yields by extending the π-conjugation length (i. e., with furan moiety) to the donor-accepter system. ExCy exhibited a large Stokes’ shift (Δλ≈100 nm) due to strong intramolecular charge transfer (ICT), and high fluorescence quantum yield (Φ_fl≈0.47 in DCM). Due to its low fluorescence in an aqueous environment (Φ_fl≈0.007 in H₂O), the probe exhibited the potential of achieving a large fluorescence turn-on upon entering a hydrophobic cellular environment. Fluorescence confocal microscopy studies revealed that ExCy was readily excitable with a far-red laser line (i. e., 640 nm) while the corresponding emission was collected in the NIR region. ExCy exhibited excellent selectivity towards live cell mitochondria according to the co-localization studies. The probe also exhibited high photostability, long-term imaging ability and wash-free staining ability, when being applied to live cells. Our studies indicated that the mitochondrial localization of ExCy was dependent on the membrane potential of the mitochondria. ExCy was successfully utilized as a mitochondrial membrane potential dysfunction indicator to visually identify cells with mitochondrial dysfunction via fluorescence confocal microscopy. ExCy was further examined for potential in vivo imaging of zebrafish.     

鼠肝高尔基体双向凝胶电泳的建立

目的分离纯化鼠肝高尔基体,初步建立一套相对完善、分辨率和重复性均较高的高尔基体双向凝胶电泳技术。方法应用蔗糖密度梯度超离心法分离纯化高尔基体,通过电镜观察和标志酶分析鉴定其形态及纯度,双向凝胶电泳分离高尔基体蛋白质,用PDQuest8.0软件分析电泳图谱。结果电镜观察显示大部分为高尔基体,纯化高尔基体标志酶TPP酶比活力提高了120倍,得到了分辨率和重复性均较好的双向凝胶电泳图谱。结论已成功建立了鼠肝高尔基体双向凝胶电泳技术。     

Fluorescence response and detection of Cu2 + with 2-(2-hydroxyphenyl)benzimidazole in aqueous medium

We present a simple but highly selective and sensitive fluorescence probe for Cu^2 + in aqueous medium: 2-(2-hydroxyphenyl)benzimidazole (L1). The probe can be applied to the monitoring of Cu^2 + with wide pH range from 6 to 11 and used in biological imaging for the detection of Cu^2 + in living cells.