双光子蛋白质荧光探针及其在药物研究中的应用

蛋白质在生命现象和生命过程中起重要作用,在众多的蛋白质分析法中,荧光探针日益广泛地应用于药物筛选等领域.由于双光子荧光技术具有高分辨率、良好的组织穿透能力、极小的组织伤害性和极低的光漂白等特点,可以实现活体蛋白质功能的可视化研究,可为新药筛选提供服务.本文就对蛋白质荧光探针的研究现状进行综述,并展望双光子蛋白质荧光探针在药物研究中的应用前景.     

检测乙酰胆碱酯酶的双光子荧光探针的设计、合成及生物学评价

目的 设计并合成用于检测乙酰胆碱酯酶（acetylcholinesterase，AChE）的双光子荧光探针，并考察探针对AChE的成像检测性能。方法 经酯化、取代等多步有机反应合成探针，并通过¹H-NMR、¹³C-NMR和ESI-MS确认结构。考察探针与AChE发生响应后的荧光信噪比、灵敏度、特异性、酶动力学和双关子吸收截面积数值等指标；研究探针可否用于AChE抑制剂的体外筛选；利用单-双光子成像试验考察探针在细胞和组织水平上对AChE活力变化的检测能力。结果 通过¹H-NMR、¹³C-NMR和ESI-MS确认了产物的结构。体外试验表明探针与AChE响应后的荧光信噪比为15倍，检测限达到0.23 U·mL^–1，并具有很强的特异性和优良的酶亲合能力，在波长820 nm有最优的双光子吸收；探针可用于AChE抑制剂的体外筛选；探针通过单-双光子成像技术可对细胞和组织水平上的AChE活力变化进行成像检测，并且组织成像检测深度可达110 μm。结论 本研究成功开发了可用于AChE检测的双光子荧光探针，有潜力成为可用于活体中检测AChE活力的双光子成像试剂。     

活体肿瘤成像技术的研究进展

成像技术在肿瘤生物学研究和临床诊断治疗中是不可或缺的工具之一,活体动物成像实验平台的主要优点是无创伤、低成本、实时检测,而分子探针与活体荧光成像技术相结合对肿瘤的早期诊断具有重要意义。肿瘤的近红外荧光(NIRF)成像技术依赖于稳定、高特异性和敏感性的分子探针。综述活体肿瘤成像技术的研究进展以及该项技术与新的分子探针、显像剂、报告分子的协同作用,为进一步应用于临床肿瘤诊断和治疗打下基础。     

CyH-8近红外探针在活体大鼠棕色脂肪成像中的应用初探

目的 CyHF-8近红外探针能特异性聚集在棕色脂肪中，现用近红外荧光成像技术，探讨CyHF-8在活体大鼠模型中识别棕色脂肪的应用潜力。方法 筛选探针用于大鼠活体荧光成像合适的给药剂量(尾静脉注射0.5、1.0、2.0 mg·kg^-1);建立糖尿病模型大鼠，经尾静脉注射CyHF-8后，置小动物活体成像仪中进行活体荧光成像。结果 尾静脉注射0.5 mg·kg^-1 CyHF-8适用于SD大鼠活体棕色脂肪的荧光成像；探针聚集于大鼠棕色脂肪中，且荧光信号呈标志性三角形，皮肤和白色脂肪无信号，不干扰成像；与正常组比较，模型组活体棕色脂肪的荧光信号显著下降，与离体脂肪成像结果一致。结论 CyHF-8能有效聚集在活体大鼠体的内棕色脂肪中，并释放靶向荧光信号；近红外荧光成像技术可用于无创分析活体模型大鼠内的棕色脂肪。     

荧光检测技术在蛋白酪氨酸激酶抑制剂高通量筛选中的应用

介绍荧光共振能量转移技术、荧光偏振免疫分析技术、荧光素酶-激酶检测技术和毛细管电泳-激光诱导荧光技术在酪氨酸激酶抑制剂高通量筛选模型中的应用情况。蛋白酪氨酸激酶与多种疾病的发生和发展密切相关,通过高通量筛选寻找其抑制剂已成为新药研发的热点之一。荧光检测技术因具有灵敏、易于检测、适于微量化和安全性高等优势,在药物的高通量筛选中被广泛使用。     

显微成像技术在药物发现中的应用

显微成像技术通过帮助描述疾病过程及在此过程中可能进行干预的潜在治疗方法,可进一步加快药物发现过程。最近的一些新技术和对已有技术的改进集中解决了过去显微成像技术应用的技术难点,即增加了显微成像技术的空间分辨率,提高了组织穿透率、跨越机体组织障碍的能力,增加了实验通量。本文集中介绍最近显微成像技术一些值得注意的发展趋势,包括超显微技术的发展,多光子技术引入,纤维光学显微技术和自动成像技术以及应用于高内涵筛选的图像分析技术。总之,这些技术进步不仅仅加速了早期药物发现中已有的实验测定和疾病模型,还催生了许多新的测定方法和疾病模型。     

PKH26标记和分子荧光活体成像技术在软骨组织工程研究中的应用

目的 探讨PKH26荧光标记和分子荧光活体成像技术在软骨组织工程中的应用。方法 用PKH26荧光标记犬软骨细胞,种植到多孔支架上,体外培养1周后异位移植到裸鼠背部,4周后用分子荧光活体成像系统示踪,并与X线检查结果对比。然后处死裸鼠取材,与免疫组织化学染色和免疫荧光观察结果对比。结果 4周分子荧光活体成像系统观察裸鼠背部标本处呈圆形强荧光,表明组织工程软骨在裸鼠体内生长良好。组织学切片结果显示番红O染色、Ⅱ型胶原免疫组化染色和甲苯胺蓝染色阳性,荧光显微镜下观察结果显示组织工程化软骨中细胞均呈红色荧光,Ⅱ型胶原免疫荧光染色呈绿色荧光,叠加后呈黄色荧光。结论 PKH26荧光标记和分子荧光活体成像2种方法结合应用于软骨组织工程中,能够较理想地且大体无创伤性评估组织工程化软骨组织在体内的生长情况。     

近红外量子点在体内分析中的应用

半导体量子点作为新型高灵敏的荧光探针被广泛用于生物成像中。与传统可见光量子点相比,近红外量子点对深层组织和器官的检测具有更高的灵敏度和对比度,因而促进了活体动物实时荧光成像技术的发展并越来越受到人们的重视。总结介绍近红外量子点的光学特性及制备方法,及近红外量子点在活细胞标记、活体和组织成像包括淋巴结成像、血管造影与肿瘤定位等领域的研究进展及发展前景。     

光谱成像结合偏最小二乘判别法快速鉴别西洋参

目的：应用荧光光谱成像技术对不同市售来源的西洋参饮片及其伪品进行检测,结合偏最小二乘判别法区分其真伪,以期实现西洋参饮片的快速无损鉴别。方法：采用凝视式荧光光谱成像装置,对西洋参、人参、桔梗各30份样品进行了荧光光谱成像,提取了其特征光谱曲线。分别采用全光谱偏最小二乘判别（PLS-DA）与联合区间偏最小二乘判别（siPLS-DA）,对3种药材的光谱数据进行了建模分析。结果：相比于PLS-DA方法,siPLS-DA方法提高了模型质量和精度,其在训练集和验证集的识别率分别达到98.33%和96.67%。结论：所建立的模型精度高、预测性能优异,可实现西洋参饮片的快速无损鉴别。     

转基因玉米中外源基因表达红色荧光蛋白致敏性研究

红色荧光蛋白(red fluorescent protein, RFP)作为一种标记蛋白,可以被光激发产生荧光,将荧光蛋白与目的蛋白融合,在组织或细胞中表达,借助成像技术来进行观察,就可以确定带着荧光蛋白标签的蛋白质定位在细胞内或组织内某一区域,从而更加清晰的追踪这一蛋白质的亚细胞活动规律。1999年红色荧光蛋白首次被报道,因其激发和发射波长更长,在细胞内成像时背景低,已被广泛应用于生命科学、植物育种、石油勘探等各个领域[1-4]。     

Widespread functional and molecular imaging in drug development

The numbers of both large- and small-molecule drug candidates have increased substantially over the past decade, while overall and late-stage failure rates have hovered around 80 and 50% respectively. The corresponding rise in research and development expenditures relative to numbers of approved drugs has made it increasingly apparent that new methods are needed to assess potential efficacy in the earliest stages of drug development. It is generally not possible to power early-phase trials sufficiently to demonstrate efficacy using clinical end points. However, functional imaging techniques can often provide both the sensitivity to treatment effects and high reproducibility necessary to obtain statistically supportable evidence of treatment effect, even in relatively small Phase I trials. This article examines both the benefits and potential pitfalls associated with the inclusion of functional and molecular imaging in the drug development process.     

X-ray fluorescence elemental mapping and microscopy to follow hepatic disposition of a Gd-based magnetic resonance imaging contrast agent

1. Spatially resolved X-ray fluorescence (XRF) spectroscopy with synchrotron radiation is a technique that allows imaging and quantification of chemical elements in biological specimens with high sensitivity. In the present study, we applied XRF techniques at a macro and micro level to carry out drug distribution studies on ex vivo models to confirm the hepatobiliary disposition of the Gd-based magnetic resonance imaging contrast agent B22956/1. 2. Gd presence was selectively quantified allowing the determination of the time dependent disappearance of the drug from blood and its hepatic accumulation in mice after administration. Elemental mapping highlighted the drug distribution differences between healthy and diseased livers. XRF microanalyses showed that in CCl(4) -induced hepatitis, B22956/1 has greatly reduced hepatic accumulation, shown as a 20-fold reduction of Gd presence. Furthermore, a significant increase of Fe presence was found in steatotic compared with healthy livers, in line with the disease features. 3. The present results show that XRF might be useful in preclinical pharmacological studies with drugs containing exogenous elements. Furthermore, quantitative and high-sensitivity elemental mapping allows simultaneous detection of chemical variation, showing pathological conditions. This approach was useful in suggesting reduced B22956/1 accumulation in steatotic livers, thus opening possible new diagnostic perspectives for this drug.     

Positron emission tomography molecular imaging for drug development

Human in vivo molecular imaging with positron emission tomography (PET) enables a new kind of 'precision pharmacology', able to address questions central to drug development. Biodistribution studies with drug molecules carrying positron-emitting radioisotopes can test whether a new chemical entity reaches a target tissue compartment (such as the brain) in sufficient amounts to be pharmacologically active. Competition studies, using a radioligand that binds to the target of therapeutic interest with adequate specificity, enable direct assessment of the relationship between drug plasma concentration and target occupancy. Tailored radiotracers can be used to measure relative rates of biological processes, while radioligands specific for tissue markers expected to change with treatment can provide specific pharmacodynamic information. Integrated application of PET and magnetic resonance imaging (MRI) methods allows molecular interactions to be related directly to anatomical or physiological changes in a tissue. Applications of imaging in early drug development can suggest approaches to patient stratification for a personalized medicine able to deliver higher value from a drug after approval. Although imaging experimental medicine adds complexity to early drug development and costs per patient are high, appropriate use can increase returns on R and D investment by improving early decision making to reduce new drug attrition in later stages. We urge that the potential value of a translational molecular imaging strategy be considered routinely and at the earliest stages of new drug development.     

Multimodal molecular imaging in drug discovery and development

In addition to individual imaging techniques, the combination and integration of several imaging techniques, so-called multimodal imaging, can provide large amounts of anatomical, functional, and molecular information accelerating drug discovery and development processes. Imaging technologies aid in understanding the disease mechanism, finding new pharmacological targets, and assessment of new potential drug candidates and treatment response. Here, we describe how different imaging techniques can be used in different phases of drug discovery and development and highlight their strengths, related innovations, and future potential with a focus on the implementation of artificial intelligence (AI) and radiomics for imaging technologies.     

Enabling drug discovery and development through single-cell imaging

Introduction: Single-cell imaging-based assays are an area of active and growing investment in drug discovery and development. This approach offers researchers the capability to interrogate rare subpopulations of cells with minimal sample consumption and multiplexed readouts. Recent technological advances in the optical interrogation and manipulation of single cells have substantially increased the throughput and sensitivity of these assays.

Areas covered: In this review, the authors focus on three classes of single-cell imaging-based analyses: single-cell microscopy combined with microfluidics, mass spectrometric imaging for subcellular compound localization, and imaging mass cytometry (IMC). They provide an overview of each technology and recent examples of their utility in advancing drug discovery, based on the potential for scalability, multiplexing, and capability to generate definitive data on cellular heterogeneity and target engagement.

Expert opinion: Understanding target engagement and heterogeneity at the single-cell level will enable the development of safer and more effective therapies, particularly for new modalities like CAR-T cell therapies and gene editing approaches (AAV, CRISPR). Successful adoption of new single-cell imaging-based approaches in drug discovery will require tandem investment in advanced computational analysis and bioinformatic approaches, due to the complexity and multivariate nature of single-cell imaging data.     

高内涵筛选技术及其在药学研究中的应用

随着显微成像技术、数据分析软件及染色技术等的飞速发展,高内涵筛选已成为能够以细胞为研究对象,单次检测获取多靶点数据的检测系统,目前正逐渐被应用到细胞生物学多个领域,并将在未来的药物研发中发挥重要作用。介绍高内涵筛选的3个技术基础,概述目前该技术在药学研究中的应用情况,并对其发展前景及应用中存在的问题进行了讨论。     

红外光谱成像在药品质量控制中的应用

目的综述红外光谱成像在药品质量控制研究中的应用。方法参考文献,对红外光谱成像的原理、仪器组成以及在有效成分分布研究、高通量分析、制剂特征研究、过程分析、粉末混合均匀度、假药识别中的应用进行归纳和整理。结果与结论红外光谱成像是药品质量控制研究的一项新技术,是对现有药物分析技术手段的补充。     

Advances and challenges in high-throughput microscopy for live-cell subcellular imaging

Introduction: Fluorescence microscopy has seen a spectacular expansion in the biomedical sciences due to the advent of new probes, new contrast techniques and the development of super-resolution that allows imaging biological structure and function beyond the diffraction limit. By far, most current research microscopes are ‘low-throughput’ instruments and – in this respect – do not differ much from their counterparts a 100 years ago. In a context of costly clinical trials and depleted pipelines, compound design and candidate selection could benefit from microscopy-based ‘screening by imaging’ approaches.

Areas covered: The article identifies some of the obvious and sometimes not-so-evident bottlenecks for going ‘high-throughput’ with imaging-based protocols. The article focuses on recent (2007 – 2010) studies, with an emphasis on live-cell screening with subcellular resolution and takes both instrumentation and experimental design into consideration. Given that most screens from pharmaceutical high-throughput microscopy are never disclosed, this review is necessarily biased toward published (academic) work.

Expert opinion: Despite high expectations on imaging-based drug-discovery strategies, both robotic subcellular imaging itself and data mining still present major challenges that will need to be overcome to make high-throughput microscopy as versatile and as powerful as high-end research microscopes. Going high throughput while keeping the high information content of fluorescence microscopic imaging will need concerted developments of hardware, image analysis and segmentation software, but probably most important is the design of live-cell assays that generate interpretable data. Also the physiological and disease relevance of the biological models amenable to high-throughput microscopies must be critically evaluated.     

Promise and Progress for Functional and Molecular Imaging of Response to Targeted Therapies

Biomarkers to predict or monitor therapy response are becoming essential components of drug developer's armamentaria. Molecular and functional imaging has particular promise as a biomarker for anticancer therapies because it is non-invasive, can be used longitudinally and provides information on the whole patient or tumor. Despite this promise, molecular or functional imaging endpoints are not routinely incorporated into clinical trial design. As the costs of clinical trials and drug development become prohibitively more expensive, the need for improved biomarkers has become imperative and thus, the relatively high cost of imaging is justified. Imaging endpoints, such as Diffusion-Weighted MRI, DCE-MRI and FDG-PET have the potential to make drug development more efficient at all phases, from discovery screening with in vivo pharmacodynamics in animal models through the phase III enrichment of the patient population for potential responders. This review focuses on the progress of imaging responses to new classes of anti-cancer therapies targeted against PI3 kinase/AKT, HIF-1alpha and VEGF. The ultimate promise of molecular and functional imaging is to theragnostically predict response prior to commencement of targeted therapy.