转铁蛋白导向脂质体核磁造影剂纳米粒子(TfNTR-LipNBD-Magnevist)——一种肿瘤靶向磁共振造影剂

造影剂辅助的核磁共振成像是目前肿瘤诊断的最好方法之一.但是由于核磁共振成像内在的低灵敏性以及造影剂的非特异性,导致肿瘤早期诊断较为困难.文章将一种新的肿瘤靶向核磁造影剂纳米粒子应用于早期肿瘤的影像诊断.这种新的肿瘤靶向核磁造影剂纳米粒子由配体转铁蛋白(Tf)、纳米水平的正电脂质体(Lip)载体和临床常用的造影剂Magnevist(TfNIR-LipNBD-Magnevist)三部分构成.另外转铁蛋白和脂质体粒子上,亦标记了荧光物质用于确定转铁蛋白-脂质体-造影剂纳米粒子的靶向性,以及肿瘤的光学影像诊断.在体外实验中,利用激光共聚焦显微镜和光学影像证明了靶向纳米粒子介导的细胞内吞和特异性结合.在裸鼠肿瘤模型中,造影剂纳米粒子TfNIR-LipNBD-Magnevist经尾静脉注入后,显著增强了肿瘤内信号与周围组织的对比度.由造影剂纳米粒子介导的肿瘤内信号显著强于单独Magnevist辅助的肿瘤内信号.同时,利用光学影像方法,在肿瘤内检测到特异的荧光信号.其结果进一步支持了转铁蛋白-脂质体-造影剂(TfNIR-LipNBD-Magnevist)纳米粒子的靶向性和肿瘤影像诊断的有效性.     

量子点光学成像技术在肿瘤诊断和治疗的应用

纳米技术在生物医学的进展使其在肿瘤的诊治中应用日益广泛。荧光纳米粒子中的量子点(Quantum Dots),具备光学成像特性在肿瘤中应用中显示出独特的优势。其作为一种荧光半导体纳米粒子,具有荧光强度高、稳定性强、激发波谱宽、发射波谱窄等光学特性。同时,它可以结合其他功能基团,包括靶向模式、治疗因素和成像探针,为临床肿瘤诊断和治疗提供了新的潜力。本文就量子点的类型和特点及量子点的肿瘤体外和体内成像进行综述。     

磁共振造影剂的研究进展

随着磁共振影像技术的快速发展,MRI在医学领域得到广泛应用,已成为目前临床常规影像诊断方法和手段之一.但MRI对信号探测的敏感性较低,因此需要某些介质在靶组织内大量聚集以达到信号扩增的目的,于是磁共振成像对比剂应用而生.磁共振造影剂(对比剂)可以提高成像分辨率,增加正常与病变组织的成像对比度,从而提高磁共振诊断疾病的敏感性和特异性,目前逐日成为众多学者研究关注的焦点之一.超顺磁性氧化铁纳米粒是一种新型的磁共振对比剂,它的有效成份为纳米级的Fe3O4或Fe2O3晶体核心,主要通过缩短组织中成像水质子的弛豫时间从而加快组织弛豫速率,得以提高正常组织和病灶组织的成像信号对比度,对肝、脾、淋巴结病变的成像效果好,安全性高,能够显著提高小病灶的检出,从而达到早期诊断发现疾病的目的.本文主要就磁共振造影剂的原理、分类及研究进展,尤其是超顺磁性氧化铁在肝脏疾病诊断中的应用进行了综述,并且对磁共振造影剂的未来发展趋势进行了展望.     

荧光磁性纳米粒子标记间充质干细胞靶向胃癌研究

目的:探索一种高效的早期胃癌诊断体系.方法:荧光磁性纳米粒子与间充质干细胞共培养,不同时间点检测干细胞存活率.利用荧光显微镜、普鲁士蓝染色、透射电子显微镜等方法观测干细胞被标记情况;建立裸鼠的胃癌模型,并将标记后的干细胞尾静脉注射入裸鼠体内,14d后用动物成像仪和核磁共振成像仪检测.结果:荧光磁性纳米粒子在低于 100μg/mL浓度下对间充质干细胞没有毒性作用,荧光磁性纳米粒子与干细胞共培养6h后,荧光显微镜、普鲁士蓝染色、透射电子显微镜检测结果显示粒子被内吞后定位于细胞质中,动物实验显示通过检测肿瘤部位的荧光信号和核磁信号,被标记的干细胞能够准确定位到胃癌的发生部位.结论:这种双模式检测体系为胃癌的早期诊断与治疗提供一种新的方法.     

白蛋白紫杉醇靶向制剂的制备与表征

二硫键断裂法,简便制备高载药量白蛋白紫杉醇纳米粒子。通过荧光光谱扫描法,监测纳米粒子的形成;透射电镜观察白蛋白紫杉醇纳米粒子的形貌;通过近红外成像的方法,观察纳米粒子在肿瘤部位的蓄积,评价粒子的肿瘤靶向性。结果:用3-巯基-2-丁酮断裂白蛋白的方法可以制备白蛋白紫杉醇纳米粒子,粒子大小约150nm,具有良好的肿瘤靶向性。     

近红外标记的维甲酸类造影剂用于骨肉瘤成像的研究

目的：开发一种具有＂找寻、治疗、可视＂功能的生物造影剂。方法：采用化学合成的方法得到近红外标记的维甲酸类造影剂,并进行骨肉瘤细胞的体外结合试验;皮下接种裸鼠,构建骨肉瘤的异种移植模型,持续10d对裸鼠进行体内光学成像,观察药物在体内的重新分布,并最终用免疫组织化学法对成像结果进行验证。结果：体外细胞结合试验证明,合成的维甲酸造影剂可以很好的与人的骨肉瘤细胞相结合,进而内化。近红外光学成像表明,该造影剂可用于骨肉瘤的早期和晚期诊断。全身成像显示了在肿瘤和肝脏的高信号强度。正电子发射断层显像（PET）显示肿瘤部位具有较高水平的18F-FDG代谢。剂量增加反应和毒性试验表明,高剂量的维甲酸造影剂必然与其全身毒性息息相关。免疫组化染色显示,发光组织中肿瘤细胞呈阳性。结论：合成的近红外标记的维甲酸造影剂可以用于检测人类骨肿瘤的异种移植,实现个体化分子诊疗的同时减少全身毒性。     

转铁蛋白修饰脂质体的制备及其肝癌靶向性研究

目的：肿瘤的靶向治疗是当前研究的热点,肝肿瘤细胞表面有大量的转铁蛋白受体表达,而正常组织较少,因此本研究制备转铁蛋白（TF）修饰的脂质体（TFLPs）,并对其肝肿瘤靶向性进行研究。方法：采用薄膜分散法制备普通脂质体,考察其形态,粒径,电位。通过体外血清稳定性模拟脂质体进入体内后的稳定性。通过HepG2肿瘤细胞对TFLPs的摄取实验考查脂质体与肝癌细胞的亲和力。构建荷瘤裸鼠模型,考查TFLPs在荷瘤裸鼠体内的分布。结果：所制备的TFLPs平均粒径为108．8±9．5nm,Zeta电位为．1．80±0．73mV。学期稳定性试验结果显示,TFLPs在24h内具有良好的血清稳定性。体外细胞摄取实验表明,HepG2细胞对TFLPs的摄取效率是普通长循环脂质体（LPs）的3．4倍。荷瘤裸鼠肝组织和肿瘤组织切片结果显示,TFLPs比LPs具有更好的肿瘤靶向性。结论：该脂质体制备方法简单,与LPs相比,经转铁蛋白修饰可显著提高肿瘤细胞对脂质体的摄取,TFLPs是一种潜在高效的肝癌靶向给药系统。     

Fe3O4磁性纳米粒子在生物医学领域的应用

Fe_3O_4磁性纳米粒子由于其良好的磁学性能,被广泛应用到了化学、生物、物理、环境保护等各个领域。尤其是在生物医学领域中的应用越来越受到研究者的关注。由于其所具有的优秀的超顺磁性性质,Fe_3O_4磁性纳米粒子可以作为造影剂,增强核磁共振成像的对比度和成像效果;也可以结合到纳米载药系统内用于药物的靶向输送;也可以包埋到蛋白内部用于蛋白的磁性分离;也可以用于基因治疗,提高靶细胞的转染效率;由于其在近红外光的作用下具有很好的光热转换效果,使温度升高,展现出的良好热疗效果,Fe_3O_4磁性纳米粒子又可以用于癌细胞的热疗。本文针对其在该领域中作为药物的靶向传递,蛋白的磁分离,核磁共振成像,热疗,以及基因治疗的载体等方面的研究应用进行了系统性的总结,阐述了Fe_3O_4磁性纳米粒子在生物医学领域中各种应用进展和优势。     

转铁蛋白受体——肿瘤的标志物和靶向治疗

转铁蛋白受体不仅介导细胞内铁的摄取和参与细胞生长调节,而且能够在肿瘤细胞表面高度表达,被认为是一种有效的肿瘤标志物,广泛应用于各类恶性肿瘤的靶向治疗。利用转铁蛋白或转铁蛋白受体的单克隆抗体与转铁蛋白受体的特异性结合作用,通过受体介导的内吞机制实现化疗药物、蛋白毒素或治疗性基因等的细胞摄入。随着对转铁蛋白受体的深入研究,靶向转铁蛋白受体的肿瘤药物将在基因治疗和跨血脑屏障运输等多方面得到进一步应用。     

新型造影剂在小鼠肝脏肿瘤Micro-CT活体成像中的应用

目的利用新型纳米颗粒造影剂结合Micro-CT成像技术,建立小鼠肝脏成像方法,并用于肝脏肿瘤的活体成像。方法 6只6-8周龄雄性C57BL/6J小鼠随机分成A组和B组,分别尾静脉注射纳米颗粒造影剂Exi Tron nano 12000 50μL和100μL;在注射前、注射后3 min、24 h、7 d、14 d、28 d和56 d对所有小鼠肝脏进行Micro-CT活体扫描;分别在小鼠肝左叶和肝右叶内选取感兴趣区（ROI）进行灰度值分析,比较不同时间点肝组织对比度的变化。确定合适的造影剂剂量,尾静脉注射至3只雄性16月龄HBV转基因肝癌模型小鼠（C组）,同上进行Micro-CT活体扫描,并于第56天全部安乐死后取肝脏观察病理学改变。结果 A组和B组小鼠在注射不同浓度造影剂后,冠状位重建图像及肝脏感兴趣区的平均灰度值结果显示：肝脏实质造影后均比注射前明显增强,24 h达到峰值,注射后56 d内,小鼠肝脏感兴趣区的平均灰度值与注射前相比仍维持在较高的水平,B组显著高于A组（P〈0.01）,确定后续实验采用B组造影剂剂量（100μL）。C组注射100μL造影剂后,各时间点均能比较清楚地看到肝脏癌性结节存在,病理学观察发现肝脏出现非典型增生,肿瘤细胞核大,染色质加深和肝细胞坏死。结论利用纳米颗粒造影剂结合Micro-CT成像技术,成功建立了小鼠肝脏活体成像方法,并可应用于肝脏肿瘤的活体成像研究。     

Dual probe with fluorescent and magnetic properties for imaging solid tumor xenografts

A dual probe with fluorescent and magnetic reporter groups was constructed by linkage of the near-infrared (NIR) fluorescent transferrin conjugate (Tf(NIR)) on the surface of contrast agent-encapsulated cationic liposome (Lip-CA). This probe was used for magnetic resonance imaging (MRI) and optical imaging of MDA-MB-231-luc breast cancer cells grown as a monolayer in vitro and as solid tumor xenografts in nude mice. Confocal microscopy, optical imaging, and MRI showed a dramatic increase of in vitro cellular uptake of the fluorescent and magnetic reporter groups from the probe compared with the uptake of contrast agent or Lip-CA alone. Pretreatment with transferrin (Tf) blocked uptake of the probe reporters, indicating the importance and specificity of the Tf moiety for targeting. Intravenous administration of the dual probe to nude mice significantly enhanced the tumor contrast in MRI, and preferential accumulation of the fluorescent signal was clearly seen in NIR-based optical images. More interestingly, the contrast enhancement in MRI showed a heterogeneous pattern within tumors, which reflected the tumor's morphologic heterogeneity. These results indicate that the newly developed dual probe enhances the tumor image contrast and is superior to contrast agent alone for identifying the tumor pathologic features on the basis of MRI but also is suitable for NIR-based optical imaging.     

MR Molecular Imaging of Prostate Cancer with a Small Molecular CLT1 Peptide Targeted Contrast Agent

Tumor extracellular matrix has abundance of cancer related proteins that can be used as biomarkers for cancer molecular imaging. In this work, we demonstrated effective MR cancer molecular imaging with a small molecular peptide targeted Gd-DOTA monoamide complex as a targeted MRI contrast agent specific to clotted plasma proteins in tumor stroma. We performed the experiment of evaluating the effectiveness of the agent for non-invasive detection of prostate tumor with MRI in a mouse orthotopic PC-3 prostate cancer model. The targeted contrast agent was effective to produce significant tumor contrast enhancement at a low dose of 0.03 mmol Gd/kg. The peptide targeted MRI contrast agent is promising for MR molecular imaging of prostate tumor.     

A tumor-targeted nanodelivery system to improve early MRI detection of cancer

The development of improvements in magnetic resonance imaging (MRI) that would enhance sensitivity, leading to earlier detection of cancer and visualization of metastatic disease, is an area of intense exploration. We have devised a tumor-targeting, liposomal nanodelivery platform for use in gene medicine. This systemically administered nanocomplex has been shown to specifically and efficiently deliver both genes and oligonucleotides to primary and metastatic tumor cells, resulting in significant tumor growth inhibition and even tumor regression. Here we examine the effect on MRI of incorporating conventional MRI contrast agent Magnevist into our anti-transferrin receptor single-chain antibody (TfRscFv) liposomal complex. Both in vitro and in an in vivo orthotopic mouse model of pancreatic cancer, we show increased resolution and image intensity with the complexed Magnevist. Using advanced microscopy techniques (scanning electron microscopy and scanning probe microscopy), we also established that the Magnevist is in fact encapsulated by the liposome in the complex and that the complex still retains its nanodimensional size. These results demonstrate that this TfRscFv-liposome-Magnevist nanocomplex has the potential to become a useful tool in early cancer detection.     

Improvement of MRI probes to allow efficient detection of gene expression

Recently, it has been demonstrated that magnetic resonance imaging (MRI) utilizing monocrystalline iron oxide nanoparticles (MIONs) targeted to an engineered transferrin receptor enables imaging of gene expression. However, the relatively high doses of iron oxides used indicated the need for improved MR imaging probes to monitor changes in gene expression in vivo. Using alternative conjugation chemistries to link targeting ligands and iron oxide nanoparticles, we present the development and characterization as well as improved receptor binding and MRI detection of a novel imaging probe. Iron oxide nanoparticles with a cross-linked dextran coat were conjugated to transferrin (Tf) through the linker molecule N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) to yield Tf-S-S-CLIO. The characteristics of this conjugate were evaluated in comparison to Tf-MION and Tf-CLIO generated by oxidative activation of the dextran-coat with subsequent reduction of Schiff's base. SPDP conjugation allowed approximately a 4-fold increase in the number of Tf molecules attached per iron oxide nanoparticle and resulted in a more than 10-fold improvement of binding and uptake by cells. This translated into an imaging probe that was 16 times better for imaging gene expression in a cellular MRI assay. This novel probe for MRI may substantially increase the sensitivity for the detection of endogenous or genetically induced transferrin receptor expression in small numbers of cells and may significantly reduce the imaging dose from over 100 mg/kg to doses of iron oxides that are currently used in clinical imaging.     

Targeted gadolinium-loaded dendrimer nanoparticles for tumor-specific magnetic resonance contrast enhancement

A target-specific MRI contrast agent for tumor cells expressing high affinity folate receptor was synthesized using generation five (G5) ofpolyamidoamine (PAMAM) dendrimer. Surface modified dendrimer was functionalized for targeting with folic acid (FA) and the remaining terminal primary amines of the dendrimer were conjugated with the bifunctional NCS-DOTA chelator that forms stable complexes with gadolinium (Gd III). Dendrimer-DOTA conjugates were then complexed with GdCl3 followed by ICP-OES as well as MRI measurement of their longitudinal relaxivity (T1 s(-1) mM(-1)) of water. In xenograft tumors established in immunodeficient (SCID) mice with KB human epithelial cancer cells expressing folate receptor (FAR), the 3D MRI results showed specific and statistically significant signal enhancement in tumors generated with targeted Gd(III)-DOTA-G5-FA compared with signal generated by non-targeted Gd(III)-DOTA-G5 contrast nanoparticle. The targeted dendrimer contrast nanoparticles infiltrated tumor and were retained in tumor cells up to 48 hours post-injection of targeted contrast nanoparticle. The presence of folic acid on the dendrimer resulted in specific delivery of the nanoparticle to tissues and xenograft tumor cells expressing folate receptor in vivo. We present the specificity of the dendrimer nanoparticles for targeted cancer imaging with the prolonged clearance time compared with the current clinically approved gadodiamide (Omniscan) contrast agent. Potential application of this approach may include determination of the folate receptor status of tumors and monitoring of drug therapy.     

Promising techniques for breast cancer detection,diagnosis, and staging using non-ionizing radiation imaging techniques

Traditional imaging for the diagnosis and staging of breast cancer has relied on the tissue morphology of cancers in the background of normal patterns of fibroglandular breast tissue. X-ray mammography and ultrasound have been the primary modalities for the diagnosis and the work-up of breast cancer. New modalities have been validated including magnetic resonance imaging (MRI) and positron emission tomography (PET). New pulse sequences in MRI combined with contrast enhancement kinetic perfusion curves have greatly enhanced detection of mammographically occult cancers. New modalities on the horizon include optical imaging, exploiting again the differential perfusion properties of cancers in a background of normal glandular tissue. Even more specificity can be ach eved with the addition of ductal or intravenous introduction of optical probes specific to tumor associated antigens such as the HER-2/neu receptor in aggressive breast cancers. Quantum dots and other fluorescent dyes coupled to peptides or other probes will greatly enhance our ability to detect cancers earlier and without ionizing radiation.     

HER2 targeted molecular MR imaging using a de novo designed protein contrast agent

The application of magnetic resonance imaging (MRI) to non-invasively assess disease biomarkers has been hampered by the lack of desired contrast agents with high relaxivity, targeting capability, and optimized pharmacokinetics. We have developed a novel MR imaging probe targeting to HER2, a biomarker for various cancer types and a drug target for anti-cancer therapies. This multimodal HER20targeted MR imaging probe integrates a de novo designed protein contrast agent with a high affinity HER2 affibody and a near IR fluorescent dye. Our probe can differentially monitor tumors with different expression levels of HER2 in both human cell lines and xenograft mice models. In addition to its 100-fold higher dose efficiency compared to clinically approved non-targeting contrast agent DTPA, our developed agent also exhibits advantages in crossing the endothelial boundary, tissue distribution, and tumor tissue retention over reported contrast agents as demonstrated by even distribution of the imaging probe across the entire tumor mass. This contrast agent will provide a powerful tool for quantitative assessment of molecular markers, and improved resolution for diagnosis, prognosis and drug discovery.     

A Liposomal Drug Platform Overrides Peptide Ligand Targeting to a Cancer Biomarker,Irrespective of Ligand Affinity or Density

One method for improving cancer treatment is the use of nanoparticle drugs functionalized with targeting ligands that recognize receptors expressed selectively by tumor cells. In theory such targeting ligands should specifically deliver the nanoparticle drug to the tumor, increasing drug concentration in the tumor and delivering the drug to its site of action within the tumor tissue. However, the leaky vasculature of tumors combined with a poor lymphatic system allows the passive accumulation, and subsequent retention, of nanosized materials in tumors. Furthermore, a large nanoparticle size may impede tumor penetration. As such, the role of active targeting in nanoparticle delivery is controversial, and it is difficult to predict how a targeted nanoparticle drug will behave in vivo. Here we report in vivo studies for α_vβ₆-specific H2009.1 peptide targeted liposomal doxorubicin, which increased liposomal delivery and toxicity to lung cancer cells in vitro. We systematically varied ligand affinity, ligand density, ligand stability, liposome dosage, and tumor models to assess the role of active targeting of liposomes to α_vβ₆. In direct contrast to the in vitro results, we demonstrate no difference in in vivo targeting or efficacy for H2009.1 tetrameric peptide liposomal doxorubicin, compared to control peptide and no peptide liposomes. Examining liposome accumulation and distribution within the tumor demonstrates that the liposome, and not the H2009.1 peptide, drives tumor accumulation, and that both targeted H2009.1 and untargeted liposomes remain in perivascular regions, with little tumor penetration. Thus H2009.1 targeted liposomes fail to improve drug efficacy because the liposome drug platform prevents the H2009.1 peptide from both actively targeting the tumor and binding to tumor cells throughout the tumor tissue. Therefore, using a high affinity and high specificity ligand targeting an over-expressed tumor biomarker does not guarantee enhanced efficacy of a liposomal drug. These results highlight the complexity of in vivo targeting.     

Primed Infusion with Delayed Equilibrium of Gd.DTPA for Enhanced Imaging of Small Pulmonary Metastases

Objectives

To use primed infusions of the magnetic resonance imaging (MRI) contrast agent Gd.DTPA (Magnevist), to achieve an equilibrium between blood and tissue (eqMRI). This may increase tumor Gd concentrations as a novel cancer imaging methodology for the enhancement of small tumor nodules within the low signal-to-noise background of the lung.

Methods

A primed infusion with a delay before equilibrium (eqMRI) of the Gd(III) chelator Gd.DTPA, via the intraperitoneal route, was used to evaluate gadolinium tumor enhancement as a function of a bolus injection, which is applied routinely in the clinic, compared to gadolinium maintained at equilibrium. A double gated (respiration and cardiac) spin-echo sequence at 9.4T was used to evaluate whole lungs pre contrast and then at 15 (representative of bolus enhancement), 25 and 35 minutes (representative of eqMRI). This was carried out in two lung metastasis models representative of high and low tumor cell seeding. Lungs containing discrete tumor nodes where inflation fixed and taken for haematoxylin and eosin staining as well as CD34 staining for correlation to MRI.

Results

We demonstrate that sustained Gd enhancement, afforded by Gd equilibrium, increases the detection of pulmonary metastases compared to bolus enhancement and those tumors which enhance at equilibrium are sub-millimetre in size (<0.7 mm²) with a similar morphology to early bronchoalveolar cell carcinomas.

Conclusion

As Gd-chelates are routinely used in the clinic for detecting tumors by MRI, this methodology is readily transferable to the clinic and advances MRI as a methodology for the detection of small pulmonary tumors.     

Conjugation of functionalized SPIONs with transferrin for targeting and imaging brain glial tumors in rat model

Currently, effective and specific diagnostic imaging of brain glioma is a major challenge. Nanomedicine plays an essential role by delivering the contrast agent in a targeted manner to specific tumor cells, leading to improvement in accurate diagnosis by good visualization and specific demonstration of tumor cells. This study investigated the preparation and characterization of a targeted MR contrast agent, transferrin-conjugated superparamagnetic iron oxide nanoparticles (Tf-SPIONs), for brain glioma detection. MR imaging showed the obvious contrast change of brain glioma before and after administration of Tf-SPIONs in C6 glioma rat model in vivo on T2 weighted imaging. Significant contrast enhancement of brain glioma could still be clearly seen even 48 h post injection, due to the retention of Tf-SPIONs in cytoplasm of tumor cells which was proved by Prussian blue staining. Thus, these results suggest that Tf-SPIONs could be a potential targeting MR contrast agent for the brain glioma.