近红外荧光染料IR-783介导的肿瘤成像

目的评估近红外荧光染料IR-783在犬自发性肿瘤中的特异性成像。方法将IR-783染料(5μmol/kg)腹腔注射荷瘤裸鼠,通过活体成像仪检测IR-783的代谢周期,在此基础上,将IR-783染料(1.5μmol/kg)通过后肢静脉注射入自发肿瘤犬体内,5 d后手术切除肿瘤组织,分别进行荧光成像、组织切片HE染色、冰冻切片荧光观察。结果 IR-783染料注射荷瘤裸鼠后可以在肿瘤部位检测到特异性荧光,连续观察8 d,仍有较强的荧光。IR-783注射自发肿瘤犬5 d后,可在肿瘤组织中检测到特异性荧光。结论近红外荧光染料IR-783能够被肿瘤组织特异性吸收,可用于犬自发性肿瘤的特异性诊断,具有重要的临床应用前景。     

FTS与NIRF共轭化合物用于小鼠肿瘤模型的成像和治疗

目的研究法尼基硫代水杨酸(farnesylthiosalicylic Acid,FTS)与七甲川菁(heptamethine carbocyanine)近红外(near infrared,NIR)荧光染料共轭化合物的肿瘤靶向性及其在活体成像中的应用,明确该化合物对肿瘤生长的抑制作用。方法将人乳腺癌细胞MCF-7、胶质瘤细胞U251和前列腺癌细胞PC3培养至对数生长期后,分别加入不同浓度的FTS和FTS-IR783,观察两种化合物对肿瘤细胞的生长抑制作用;培养的三种肿瘤细胞中加入FTS-IR783(20μmol/L),荧光显微镜下观察荧光染料在肿瘤细胞中的聚集;将三种肿瘤细胞(每只1×10~6个)皮下移植裸鼠,两周后荷瘤鼠腹腔注射FTS-IR783(每只10 nmol/L),活体成像分别测定肿瘤部位近红外荧光信号和肿瘤体积的相关性。结果与FTS相比较,FTS-IR783可显著抑制MCF-7、U251和PC3的生长;三种肿瘤细胞可特异性识别FTS-IR783,呈现近红外荧光集聚;皮下荷瘤模型注射FTS-IR783后,活体成像显示肿瘤部位荧光强度与生物发光强度相关性分别达到0.987,0.998和0.971。结论 FTS与近红外荧光染料IR-783共轭结合后可特异性识别肿瘤细胞,用于肿瘤模型的活体成像,同时该化合物具有的肿瘤靶向性可显著抑制肿瘤细胞的生长,有望成为新型的靶向药物。     

近红外荧光染料在胃癌人源性肿瘤组织异种移植模型研究中的应用

目的建立胃癌人源性肿瘤组织异种移植(patient-derived tumor xenograft,PDX)裸鼠模型,探讨近红外荧光(near infrared fluorescence,NIRF)染料IR-783在胃癌PDX模型活体成像研究中的应用。方法取临床胃癌新鲜手术切除标本,裸鼠肾包膜移植建立PDX模型,HE染色比较移植肿瘤组织与患者肿瘤组织形态结构的一致性。将移植肿瘤组织裸鼠皮下接种,20 d后,荷瘤小鼠腹腔注射NIRF染料IR-783(每只10 nmol/L),活体成像连续测定肿瘤部位近红外荧光强度并分析肿瘤体积与荧光强度的相关性;免疫组织化学检测移植肿瘤组织中HIF1α与OATP1B3的表达强度。结果成功建立了3个人胃癌PDX模型,移植肿瘤组织较好的保持了原发肿瘤的特征,NIRF活体成像早期检测到肾包膜部位荧光信号。PDX模型中肿瘤体积与荧光强度的相关性均在98%以上。移植肿瘤组织中HIF1α与OATP1B3表达呈强阳性。结论 NIRF染料IR-783可在PDX模型肿瘤部位特异性聚集,用于PDX模型的早期检测,这种肿瘤靶向性可能与HIF1α和OATP1B3的表达相关。     

七甲川菁近红外荧光染料对胃癌原位移植模型的靶向识别

目的研究七甲川菁近红外荧光(NIRF)染料在胃癌原位移植模型活体成像中的应用效果。方法将标记荧光酶素的人胃癌细胞系Hep G2原位移植裸鼠建立肿瘤模型,同时诱发制备胃溃疡模型;对上述模型分别采用生物发光成像和NIRF成像,观察胃癌组织对近红外荧光染料的吸收;探索缺氧和阴离子转运肽(OATP)对胃癌组织吸收NIRF染料的影响,明确NIRF染料靶向识别肿瘤细胞的特异性。结果 NIRF信号与生物发光信号在胃癌原位移植模型活体成像中具有较好的相关性。胃癌组织部位可获得较强的NIRF荧光信号,而胃溃疡部位未检测到荧光信号。缺氧能够增强胃癌细胞对NIRF染料的吸收,而阴离子转运肽特异性抑制剂磺溴酞钠(BSP)能够显著降低肿瘤细胞对NIRF染料的吸收。结论七甲川菁近红外荧光染料能够靶向识别胃癌原位移植模型。     

新型七甲川菁染料用作肿瘤模型活体成像的研究进展

一种近红外荧光(NIRF)七甲川菁染料不需要化学修饰,可直接被肿瘤细胞吸收呈特异性聚集,从而可用于肿瘤活体成像。这种染料在肿瘤细胞与正常细胞之间的摄取差异可能是由于特异型有机阴离子转运肽的作用,并受到低氧条件控制。这些特性将会拓展NIRF类染料在肿瘤成像研究中的应用。     

七甲川花菁染料作为肿瘤靶向和光动力治疗载体的研究进展

七甲川花菁近红外荧光染料(NIRF)可直接被肿瘤细胞特异性吸收,具有肿瘤靶向性。与化疗药物偶联后,该类染料可通过血脑屏障将药物转运至肿瘤部位,不仅可以减少化疗药物使用剂量,降低药物的毒副作用,也可通过近红外荧光成像实现对肿瘤治疗的实时监控。七甲川花菁染料所展示的线粒体毒性和光敏特性,可直接杀死肿瘤细胞,抑制肿瘤新生血管的形成。通过纳米包裹,能够显著增强该类染料的肿瘤靶向能力,实现实时跟踪药物释放情况。七甲川花菁染料特异性识别肿瘤细胞的能力与有机阴离子转运肽的作用密切相关,缺氧和线粒体膜电位也参与了染料吸收的调控。这些发现有利于将近红外荧光染料应用于肿瘤的靶向治疗。     

叶酸受体靶向的金纳米棒用于细胞内光声成像

目的 分子成像技术具有“早期检测”的特点,由于分子水平上的畸变早于解剖水平上的变化。本研究采用细胞内光声分子成像（PMI）方法,对靶向到癌细胞上的叶酸-金纳米棒（FA-AuNRs）精确定位成像。方法 本文合成了FA-AuNRs,并对其性质包括形貌、吸收光谱和生物相容性进行了研究。修饰叶酸赋予FA-AuNRs特异性靶向到叶酸受体高表达癌细胞的能力。然后,通过PMI实验研究FA-AuNRs对癌细胞的靶向特异性。结果 FA-AuNRs呈棒状,在~800 nm处有一近红外吸收峰。在癌细胞的细胞质中观察到强光声信号,而在正常细胞中只有弱光声信号,表明FA-AuNRs通过叶酸受体介导的内吞作用被癌细胞选择性摄取。这项研究证明了PMI能够实现对靶向到癌细胞上的FA-AuNRs精确定位成像。结论 借助特异性靶向作用,可以通过PMI获得癌细胞表面分子信息。该方法有望实现在细胞和分子水平上对生物过程进行可视化、表征和量化。     

鸦胆子油乳对膀胱癌影响的实验分析

目的通过实验分析鸦胆子油乳对膀胱癌的影响。方法在人体外培养人膀胱癌细胞(BIU-87),并将不同浓度的鸦胆子油乳加入其中,之后观察人膀胱癌细胞的生长、组织结构和细胞周期。对ICR小鼠进行亚硝胺的膀胱癌诱导,再给ICR小鼠膀胱灌注鸦胆子油乳,在光镜和电镜下观察膀胱癌的进展或抑制情况。结果鸦胆子油乳对人膀胱癌细胞产生了抑制作用,并破坏了膀胱癌细胞的微结构和超微结构,改变了膀胱癌细胞的性质并致其坏死,同时它还能够阻止人膀胱癌细胞由G0期向S期进展以及抑制DNA的合成。ICR小鼠实验结果表明,亚硝胺诱导的膀胱癌在膀胱灌注鸦胆子油乳之后得到有效的抑制。结论鸦胆子油乳能够抑制膀胱癌细胞的生长与繁殖,临床中可加以应用。     

重组腺相关病毒小鼠骨骼肌中近红外荧光蛋白表达及活体成像

近红外荧光蛋白因激发光和发射光波长位于近红外区,在动物组织中光吸收和光散射最低,更适宜于动物活体组织的深层成像.构建了一种携带近红外荧光蛋白(near-infrared fluorescent protein,iRFP)713基因的重组表达质粒pAAV-iRFP713,将重组表达质粒与辅助质粒共转染AAV-293细胞,包装重组腺相关病毒(recombinant adeno-associated virus,rAAV)rAAV-iRFP713.重组腺相关病毒表达载体感染体外培养的癌细胞,48h后,荧光显微镜检测显示近红外荧光蛋白在癌细胞中高效表达,荧光明亮.重组腺相关病毒表达载体注射小鼠骨骼肌,48h后,用近红外荧光活体成像系统检测证明近红外荧光蛋白在小鼠骨骼肌中表达较强, 活体组织成像清晰.实验结果表明近红外荧光蛋白在体内体外均能很好地表达并荧光成像,为动物活体组织标记和成像的研究提供新方法.     

胃癌血管靶向肽GX1修饰的人血清白蛋白用于胃癌近红外活体成像

目的:以肿瘤血管靶向肽GX1修饰的人血清白蛋白(HSA)作为吲哚菁绿(ICG)的载体,合成近红外荧光探针GX1-HSA-ICG,研究其作为近红外荧光探针在荷人胃癌裸鼠活体中的靶向成像能力。方法:以HSA作为ICG的载体,通过化学修饰与GX1共价连接,合成GX1-HSA-ICG纳米颗粒探针;使用SDS-PAGE对探针合成进行鉴定;采用探针与脐静脉内皮细胞HUVEC以及与肿瘤细胞共培养的脐静脉内皮细胞Co-HUVEC进行结合和竞争抑制试验,验证探针和Co-HUVEC细胞结合的特异性;利用小动物活体成像系统对皮下荷胃癌小鼠进行近红外荧光活体成像,验证探针在体内的胃癌靶向性。结果:成功合成GX1-HSA-ICG。细胞结合与竞争抑制实验显示GX1-HSA-ICG可与Co-HUVEC细胞特异性结合;荷瘤小鼠活体成像也显示出GX1-HSA-ICG较ICG有更长体内的循环时间,并且胃癌组织局部较HSA-ICG有更强的聚集。结论:本研究成功合成了胃癌血管靶向肽GX1修饰的HSA为荧光染料载体的胃癌血管靶向探针,成功对荷胃癌裸鼠进行了活体成像。使用HSA为载体的探针较单纯使用ICG的肿瘤局部滞留能力显著提高,GX1增加了探针的胃癌靶向特异性。该探针在胃癌的早期诊断和抗肿瘤血管生成治疗评估中具有潜在的应用价值。     

双模态纳米诊疗一体药物在肝癌动物模型中的应用

摘要 目的：构建一种肿瘤诊断和治疗一体化药物，并利用肝癌动物模型开展诊疗效能评价。方法：利用多孔金属有机骨架材料ZIF-8，通过配位作用同时对化疗药物阿霉素（DOX）和近红外荧光染料IR-820进行负载。而后，利用超声的方法在ZIF-8-IR-820-DOX表面修饰了红细胞膜以提高载药体系的生物安全性和稳定性，得到具有生物伪装特性的pH响应型ZIF-8-IR-820-DOX@RM纳米颗粒。最后，通过对该药物体系的粒径、表面电位、形貌等理化性质进行表征，并利用肝癌动物模型验证该药物的诊断和治疗效能。结果：成功构建了一款红细胞伪装的金属框架纳米诊疗一体试剂，该试剂具有较好的pH相应性，在肿瘤pH 5.5 条件下，药物的释放率达到98.4 %，而在机体正常pH 7.4条件下，释放率仅为15.3 %。在小鼠肝癌动物模型的诊疗过程中，能通过近红外荧光较好的识别肿瘤的位置和大小，且对小鼠肿瘤具有较好的治疗效果。结论：本研究所构建的ZIF-8-IR-820-DOX @RM能通过肿瘤处增强的渗透性和保留（EPR）效应，精准的到达肿瘤部位，并利用pH响应性，在肿瘤酸性环境中精准释放携带的抗肿瘤药物和近红外荧光成像试剂，实现对肿瘤的诊断和治疗一体化设计。为肿瘤治疗的相关研究提供了一种思路和借鉴。     

Engineering of near IR fluorescent albumin nanoparticles for in vivo detection of colon cancer

ABSTRACT: BACKGROUND: The use of near-infrared (NIR) fluorescence imaging techniques has gained great interest for early detection of cancer because water and other intrinsic biomolecules display negligible absorption or autofluorescence in this region. Novel fluorescent nanoparticles with potential to improve neoplasm detection sensitivity may prove to be a valuable tool in early detection of colon tumors. METHODS: The present study describes the synthesis and use of NIR fluorescent albumin nanoparticles as a diagnostic tool for detection of colon cancer. These fluorescent nanoparticles were prepared by a precipitation process of human serum albumin (HSA) in aqueous solution in the presence of a carboxylic acid derivative of the NIR dye IR-783 (CANIR). Tumor-targeting ligands such as peanut agglutinin (PNA), anti-carcinoembryonic antigen antibodies (anti-CEA) and tumor associated glycoprotein-72 monoclonal antibodies (anti-TAG-72) were covalently conjugated to the albumin nanoparticles via the surface carboxylate groups by using the carbodiimide activation method. RESULTS AND DISCUSSION: Leakage of the encapsulated dye into PBS containing 4% HSA or human bowel juice was not detected. This study also demonstrates that the encapsulation of the NIR fluorescent dye within the HSA nanoparticles reduces the photobleaching of the dye significantly. Specific colon tumor detection in a mouse model was demonstrated for PNA, anti-CEA and anti-TAG-72 conjugated NIR fluorescent HSA nanoparticles. These bioactive NIR fluorescent albumin nanoparticles also detected invisible tumors that were revealed as pathological only subsequent to histological analysis. CONCLUSIONS: These results may suggest a significant advantage of NIR fluorescence imaging using NIR fluorescent nanoparticles over regular colonoscopy. In future work we plan to broaden this study by encapsulating cancer drugs, such as paclitaxel and doxorubicin, within these biodegradable NIR fluorescent HSA nanoparticles, in order to use them for both detection as well as therapy of colon cancer and others.     

A new optical and nuclear dual-labeled imaging agent targeting interleukin 11 receptor alpha-chain

Optical imaging has great potential for studying molecular recognitions both in vivo and in vitro, yet nuclear imaging is the most effective clinical molecular imaging modality. The combination of optical and nuclear imaging modalities may provide complementary information for improving diagnosis and management of diseases. In this study we developed an optical and nuclear dual-labeled imaging agent, 111In-DTPA-Bz-NH-SA-K(IR-783-S-Ph-CO)-c(CGRRAGGSC)NH2, called DLIA-IL11Ralpha. 111In-DTPA-Bz-NH-SA is the radiotracer moiety; a near-infrared dye IR-783-S-Ph-COOH serves as the fluorescent emitter; and the cyclic peptide c(CGRRAGGSC), which is known to target interleukin 11 receptor alpha-chain (IL-11Ralpha), delivers the desired imaging agent to its target. Experiments revealed that the cyclic peptide c(CGRRAGGSC) continued to possess the targeting capability to IL-11Ralpha after the conjugation of the optical and nuclear tracers. Furthermore, the presence of the metal isotope chelator did not cause quenching of fluorescence emission. The cross validation and direct comparison of optical and nuclear imaging of a tumor was achieved using a single injection, and the preliminary results show the conjugate has tumor targeting capabilities in vivo.     

Synthesis and characterization of a novel near-infrared fluorescent probe for applications in imaging A549 cells

Objective

To design and synthesize a novel near-infrared (NIR) fluorescent probe based on indocyanine Green (ICG), that can be applied in imaging living cells.

Results

A highly fluorescent novel NIR fluorescent probe (IR-793) was synthesized in two steps. IR-793 had better fluorescence and optical stability than ICG. In addition, no obvious cytotoxicity effect of IR-793 was observed and cell viability was above 75% at the maximum concentration (120 nM). IR-793 also exhibited good performance in imaging living A549 cells.

Conclusion

IR-793, a novel NIR fluorescent probe that is stable, low-cost, highly fluorescent and low cytotoxicity, has been designed and synthesized for imaging living cells.

     

肿瘤微环境中ROS介导IgG表达对膀胱癌细胞增殖和侵袭能力的影响

摘要 目的：探讨肿瘤微环境（TME）中活性氧（ROS）介导免疫球蛋白G（IgG）表达对膀胱癌EJ细胞增殖、迁移和侵袭能力的影响。方法：临床收集的18例膀胱癌患者样本,通过Western blot法检测膀胱癌和癌旁正常组织样本中IgG表达量。利用免疫荧光染色（IF）技术分别对膀胱癌组织和癌旁正常组织中ROS和IgG分子进行共定位和相对定量分析。将活性氧清除剂N-乙酰基-L-半胱氨酸（NAC）加入膀胱癌细胞EJ中,实验分为3组：空白组（EJ细胞）、阴性对照组（EJ+PBS）、实验组（EJ+PBS+NAC）,10 mM NAC药物处理48小时后,运用DHE-ROS荧光探针技术和Western blot实验检测药物NAC对ROS和IgG相对表达水平的影响;通过克隆集落形成实验、划痕实验、Transwell实验检测去除ROS后对膀胱癌细胞增殖、迁移和侵袭的影响。结果：人体膀胱癌组织中ROS和IgG分子表达水平显著高于癌旁正常组织（P<0.001）;荧光显微镜显示膀胱肿瘤组织中正常膀胱尿路上皮细胞组织被肿瘤细胞严重破坏,结构紊乱不规则,IgG和ROS表达水平均升高,而癌旁组织膀胱尿路上皮组织的结构均匀规则;NAC药物处理EJ细胞后,与空白组和阴性对照组相比ROS和IgG表达显著降低,同时实验组细胞的增殖、迁移和侵袭能力明显下降（P<0.01）。结论：ROS和IgG在临床膀胱癌组织和体外膀胱癌细胞株EJ中均显著高表达,在肿瘤微环境中ROS通过调控IgG表达,从而促进膀胱癌细胞的增殖、迁移、侵袭。ROS和IgG可能成为膀胱癌早期诊断和生物治疗的临床新靶点。     

The application of anti‐ESAT‐6 monoclonal antibody fluorescent probe in ex vivo near‐infrared fluorescence imaging in mice with pulmonary tuberculosis

Here, we aimed to assess the feasibility of anti‐ESAT‐6 monoclonal antibody (mAb) coupling with IR783 and rhodamine fluorescent probe in the detection of ESAT‐6 expression in tuberculosis tissue of mice using near‐infrared fluorescence imaging. IR783 and rhodamine were conjugated to the anti‐ESAT‐6 mAb or IgG. Mice in the experimental group were injected with fluorescence‐labeled mAb probe, and mice in the control group were injected with fluorescence‐labeled non‐specific IgG antibody. Twenty‐four hours later, the lung tissue of mice was examined using ex vivo near‐infrared fluorescence imaging. In addition, the contrast‐to‐noise ratio (CNR) was calculated by measuring the signal intensities of the pulmonary lesions, normal lung tissue and background noise. The frozen lung tissue section was examined under fluorescence microscopy and compared with hemoxylin and eosin (HE) staining. The ex vivo near‐infrared fluorescence imaging showed that the fluorescence signal in the lung tuberculosis lesions in the experimental group was significantly enhanced, whereas there was only a weak fluorescence signal or even no fluorescence signal in the control group. CNR values were 64.40 ± 7.02 (n = 6) and 8.75 ± 3.87 (n = 6), respectively (t = 17.01, p < 0.001). The fluorescence accumulation distribution detected under fluorescence microscopy was consistent with HE staining of the tuberculosis region. In conclusion, anti‐ESAT‐6 mAb fluorescent probe could target and be applied in specific ex vivo imaging of mice tuberculosis, and may be of further use in tuberculosis in living mice. Copyright © 2013 John Wiley & Sons, Ltd.     

Evaluation of the cytotoxicity of a two photon absorbing fluorescence compound on human HepG2 cells and its application to tracking human hepatic cancer cells in mice

Abstract

Small organic dyes have been applied widely in fluorescence imaging techniques for biomedical research. We investigated the cytotoxicity of a novel fluorescent dye, trans-4-(N-2-hydroxyethyl-N-ethyl amino)-4′-(dimethyl amino) stilbene (DMAHAS), on human hepatocellular carcinoma (HepG2) cells using methyl thiazolyl tetrazolium(MTT), a neutral red assay, a Coomassie brilliant blue assay, and flow cytometric analysis. Our results showed that DMAHAS had live cell permeability, stable cytosolic localization and no significant cytotoxicity to HepG2 cells. We explored its application further for tumor cell tracking in a human liver tumor xenograft mouse model. Tumor xenografts were examined by fluorescence imaging and conventional histological methods. In addition, a method based on DMAHAS release was developed for tumor-specific cytotoxicity analysis. Our study indicated that DMAHAS is a reliable probe for tumor tracking and fluorescence imaging.     

Targeting bladder tumor cells in vivo and in the urine with a peptide identified by phage display

Bladder cancer is one of the most common tumors of the genitourinary tract. Here, we use phage display to identify a peptide that targets bladder tumor cells. A phage library containing random peptides was screened for binding to cells from human bladder tumor xenografts. Phage clones were further selected for binding to a bladder tumor cell line in culture. Six clones displaying the consensus sequence CXNXDXR(X)/(R)C showed selective binding to cells from primary human bladder cancer tissue. Of these, the CSNRDARRC sequence was selected for further study as a synthetic peptide. Fluorescein-conjugated CSNRDARRC peptide selectively bound to frozen sections of human bladder tumor tissue, whereas only negligible binding to normal bladder tissue was observed. When the fluorescent peptide was introduced into the bladder lumen, in a carcinogen-induced rat tumor model, it selectively bound to tumor epithelium. Moreover, when the peptide was intravenously injected into the tail vein, it homed to the bladder tumor but was not detectable in normal bladder and control organs. Next, we examined whether the peptide can detect tumor cells in urine. The fluorescent peptide bound to cultured bladder tumor cells but not to other types of tumor cell lines. Moreover, it bound to urinary cells of patients with bladder cancer, while showing little binding to urinary cells of patients with inflammation or healthy individuals. The CSNRDARRC peptide may be useful as a targeting moiety for selective delivery of therapeutics and as a diagnostic probe for the detection of bladder cancer.     

Lactose substituted zinc phthalocyanine: A near infrared fluorescence imaging probe for liver cancer targeting

A near infrared fluorescence probe, lactose substituted zinc phthalocyanine, [2,9(10),16(17),23(24)-tetrakis((1-(β-d-lactose-2-yl)-1H-1,2,3-triazol-4-yl)methoxyl)phthalocyaninato] zinc(II), was synthesized via click reaction. Structural characterization and optical experiment demonstrated its excellent biocompatibility and fluorescence imaging ability. Near infrared fluorescence imaging in vivo for liver cancer, lung cancer and melanoma cancer with tumor bearing nude mice as models demonstrated that lactose substituted zinc phthalocyanine has specifically targeting ability to liver cancer while no targeting to lung cancer or melanoma, which implied its potential in liver cancer diagnosis as a near infrared optical probe.