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。     

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。     

核分子成像技术在基因治疗药物发现中的应用

医学成像的下一个发展方向是分子成像技术。分子成像技术具有很高的敏感性，非常适于研究靶标的分子结构。通过合适的报告基因．报告探针系统，核分子成像技术可以测量活体动物上的转基因表达。这种新方法学能够即时获得药物的体内分布和药理作用的定量数据，从而减少动物的消耗、缩短基因治疗药物的研发时间并能节约成本。     

采用活体成像技术观察肿瘤组织在裸鼠体内生长情况的研究

目的 采用活体成像技术监测稳定高表达荧光素酶报告基因的肿瘤细胞在裸鼠体内生长及转移情况,为肿瘤治疗药物的研发提供新的评价技术和方法.方法 采用Lipofectamine 2000介导的基因转染方法,将pGL4.17[luc2/neo]载体转染人肝癌细胞株HepG2,经G418抗性筛选及有限稀释法获得稳定高表达荧光素酶的单克隆细胞;采用活体成像的方法检测转染细胞在裸鼠体内的成瘤情况.结果 获得了可稳定高表达荧光素酶基因的单克隆细胞株,将单克隆细胞株植入裸鼠皮下.采用活体成像技术准确监测肿瘤细胞体内生长情况.结论 采用活体成像技术构建的肿瘤动物模型是拓展肿瘤体内生长、转移及治疗相关研究的理想模型.     

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。     

靶向HIV-1细胞受体基因CCR5的不对称siRNA的稳定性和体内分布研究

目的:探讨靶向HIV-1细胞受体基因CCR5的不对称siRNA (asiRNA)在血清和动物体内的稳定性和在动物体内分布情况.方法:用内皮抑素同脂质胶束联合递送asiRNA,琼脂糖凝胶电泳法检测asiRNA的血清稳定性,ELISA法和活体荧光成像法检测asiRNA的体内分布.结果:改变合适的实验条件和asiRNA的修饰,可提高asiRNA的血清稳定性.用ELISA法检测出静脉给药15 min后双链asiRNA在血液、肝、肺和肾内浓度较高,用活体荧光成像法检测出静脉给药24h后asiRNA向肾内汇集排泄,两种检测方法测得asiRNA在脾内浓度较低.结论:两种检测方法方便实用,灵敏度不同,均可用于阳离子脂质体递送asiRNA的体内分布检测.     

盐酸博安霉素注射用原位凝胶的体内分布研究

考察盐酸博安霉素(BAM)注射用原位凝胶在裸鼠体内的扩散情况以评价原位凝胶对盐酸博安霉素的阻滞作用。以荧光染料异硫氰酸荧光素(FITC)标记盐酸博安霉素,制备异硫氰酸荧光素-盐酸博安霉素偶联物(FITC-BAM),采用透析袋透析和Sephadex G25葡聚糖凝胶柱对偶联物进行分离纯化,利用基质辅助激光解析电离/飞行时间(MALDI-TOF)质谱检测偶联效果。建立裸鼠皮下肝癌移植瘤模型,应用动物体内活体光学成像系统定时检测盐酸博安霉素在裸鼠体内的特异性分布情况。MALDI-TOF质谱检测结果显示,FITC成功与BAM发生偶联,且二者偶联的分子比主要为1∶1或2∶1。活体动物成像系统观察显示,盐酸博安霉素注射用原位凝胶组FITC-BAM的扩散较普通注射液组明显延迟。研究表明,将盐酸博安霉素制备成注射用原位凝胶制剂,能够阻滞药物在体内的释放,延长作用时间。     

磁共振弥散加权成像对脑梗死的诊断价值

弥散加权成像(DWI)是一种新的磁共振(MR)功能成像技术,是目前唯一可以在活体内检测分子弥散运动的方法。目前,该技术已广泛应用于中枢神经系统,尤其对于缺血造成的脑内梗死性病灶诊断具有重要意义。本文通过观察78例脑梗死病灶在不同病程时期的变化特点,探讨DWI及表观弥散系数(ADC)值对脑梗死病灶的诊断价值。     

64层螺旋CT在亲属肾移植供体术前的应用

近年来,多层螺旋CT(MDCT)血管成像因其无创性及高度精确性越来越受到关注,已逐渐代替以往常规血管造影成为成像技术的首选。本研究对32例活体肾移植供体应用多层螺旋CT血管成像及后处理技术,探讨其在活体肾移植术前评估中的综合应用价值。1资料与方法1.1临床资料:对我中心自2007年1月至2009年7月完成的30例亲属活体肾移植手术中32例供体术前所进行的     

扩散加权成像的扩散计算模型及组织学基础研究进展

<正>磁共振扩散加权成像(diffusion weighted imaging,DWI)是MR功能成像技术,是迄今为止检测活体组织水扩散运动最有效的影像学方法,为建立活体影像学研究与组织学相关性提供了可能。扩散加权成像的组织学基础是水分子的扩散,由于分子运动的程度和方向是由微观结构和生物组织的影响,因此DWI可以间接描绘器官或组织的各种病理变化。目前这种成像特点受到广泛关注,近年来,随着DWI成像机制研究地不断深入,相继提出了多个新的扩散计算模型;另一方面,在组织学方面,目前认为水的跨膜转运有两种基本方式,即穿越膜脂质双分子层的简单扩散和水通道蛋白(aquaporin,AQP)介导的水分子转运[1]。AQP是膜蛋白,可促     

20(S)-原人参二醇对SMMC-7721细胞体内外作用的研究

目的观察不同剂量的20(S)-原人参二醇(Protopanaxadiol,PPD)在体内外对人肝癌细胞株SMMC-7721抗肿瘤作用。方法建立人肝癌裸鼠皮下移植瘤模型,观察20(S)-原人参二醇的肿瘤抑制作用。MTT比色法检测20(S)-原人参二醇对SMMC-7721细胞的增殖抑制作用,Ho-echst33342核染色观察细胞凋亡形态学改变,采用FITC-An-nexinⅤ/PI双染流式细胞术分析凋亡情况,同时检测Caspase-3活性。结果在体内,PPD可抑制SMMC-7721细胞裸鼠异种移植瘤生长;在体外,PPD对SMMC-7721细胞的增殖具有明显的抑制及诱导其凋亡作用,呈时间和剂量依赖性,Hoechst33342核染色可见凋亡小体,同时伴有Caspase-3活性的增加。结论20(S)-原人参二醇在体内外均可抑制SMMC-7721细胞增殖,并诱导其凋亡,其机制可能通过活化Caspase-3诱导细胞凋亡而发挥抗肿瘤作用。     

Doxorubicin Loaded pH-sensitive Micelle: Antitumoral Efficacy against Ovarian A2780/DOXR Tumor

PURPOSE: To evaluate pH-sensitive mixed micelles for multidrug resistant (MDR) ovarian tumor targeting and optical imaging of solid tumors. METHOD: Doxorubicin (DOX) encapsulated pH-sensitive mixed micelles composed of poly(L: -histidine)(MW 5K)-b-PEG(MW 2K) and poly(L: -lactic acid)(3K)-b-PEG (2K)-folate (PHSM-f) were prepared. Folate receptor-mediated endocytosis, drug uptake, endosomal disruption and cell viability were investigated at the cellular level. For in vivo tumor growth inhibition tests, multidrug resistant ovarian A2780/DOX(R) xenografted nude mice were used. Optical imaging was performed by using a Cy5.5 fluorescence dye-labeled mixed micelle system. Cy5.5 fluorescence intensity at the tumor site was measured in KB epidermoid xenografted nude mice. RESULTS: In vitro cell viability and drug distribution in the cytoplasm demonstrated the significantly superior efficacy of PHSM-f to free DOX and a control sample of DOX loaded pH-insensitive micelle composed of poly(L: -lactic acid)(3K)-b-PEG(2K)/poly(L: -lactic acid)(3K)-b-PEG(2K)-folate (80/20 wt/wt%) (PHIM-f). The mechanisms of these results were proved by folate receptor mediated endocytosis of micelle and endosomal disruption function by it. In addition, the optical imaging demonstrated the future application of the diagnositic area. PHSM-f inhibited the growth of multidrug resistant ovarian tumors efficiently in mice, with minimum weight loss. CONCLUSIONS: The pH-sensitive mixed micelle system demonstrates effective antitumor efficacy against the multidrug resistant ovarian tumor A2780/DOX(R).     

Octreotide-modified and pH-triggering polymeric micelles loaded with doxorubicin for tumor targeting delivery

A multifunctional mixed micelle was assembled for drug targeting delivery by combining two newly synthesized amphiphilic polymers, which were octreotide-polyethylene glycol-monostearate (OPMS) and N-octyl-N-succinyl-O-carboxymethyl chitosan (OSCC), respectively. The mixed micelle was designed to be characterized with long circulation, somatostatin receptors (SSTR)-mediated endocytosis and pH sensitivity. A series of assembling proportions of OPMS and OSCC was tested to reveal the effect of compositions on the functions. The particle size, zeta potential, drug loading and critical micelle concentration were examined. The dialysis test indicated a pH-triggering release behavior of the doxorubicin-loaded mixed micelle (DLMM), and faster release in acidic media (pH 4.0-6.0) in response to the protonation of carboxyl group. In addition, the PEG segments could efficiently protect the mixed micelle from plasma protein adsorption in vitro, and the DLMM composed of 20% OPMS and 80% OSCC provided the longest residence time after intravenous injection in rats in vivo. Due to SSTR mediated endocytosis, the significantly higher uptake of DLMM was observed in the tumor cells (SMMC-7721), compared with that in the normal cells (CHO) without SSTR expression. All the results suggested that the mixed micelle with multifunctional characteristics could be used as an effective approach for tumor treatment.     

Doxorubicin-Loaded Polymeric Micelles Based on Amphiphilic Polyphosphazenes with Poly( N -isopropylacrylamide-co- N , N -dimethylacrylamide) and Ethyl Glycinate as Side Groups: Synthesis, Preparation and In Vitro Evaluation

Purpose  To construct novel Doxorubicin-loaded polymeric micelles based on polyphosphazenes containing N-isopropylacrylamide copolymers and evaluate their various properties as well as in vitro anticancer effect. Methods  These amphiphilic graft polyphosphazenes PNDGP were synthesized via thermal ring-opening polymerization and subsequent two-step substitution reaction of hydrophilic and hydrophobic side groups. Micellization behavior in an aqueous phase was confirmed by fluorescence technique, DLS and TEM. Doxorubicin (DOX) was physically loaded into micelles by dialysis or O/W emulsion method. CLSM and MTT test were applied to observe intracellular drug distribution and determine cytotoxicity of drug-loaded micelles on Hela and HepG2 cells lines, respectively. Results  A series of PNDGPs with controlled substitution ratios were obtained. Poly(NIPAm-co-DMAA) can act as hydrophilic segments in micellular system since its LCST was over 37°C when PNIPAm was copolymerized with DMAA. The CMC value was decreased with the increase of Glyet content. In addition, more hydrophobic group content introduced into the polymer would facilitate DOX encapsulation into the micelle. DOX-loaded micelle could achieve comparative cytotoxicity as free drug via endocytosis and succedent drug release into cytoplasm of cancer cells. Conclusions  The results suggest that these polymers might be used as potential carriers of hydrophobic anti-tumor drug for cancer therapy.     

GLUT1 targeting and hypoxia-activating polymer-drug conjugate-based micelle for tumor chemo-thermal therapy

PurposeMitochondria are closely correlated with the proliferation and metastasis of tumor for providing suitable micro-environment and energy supply. Herein, we construct a glucose transporter 1 (GLUT1) targeting and hypoxia activating polyprodrug-based micelle (Glu-PEG-Azo-IR808-S-S-PTX) for mitochondria-specific drug delivery and tumor chemo-thermal therapy.ResultsThe micelle was characterized by hypoxia-sensitive PEG outer layer detachment, high photo-thermal conversion efficiency, and glutathione (GSH)-sensitive paclitaxel （PTX） release. It showed GLUT1 specifically cellular uptake and hypoxia-sensitive mitochondria targeting on A549 cell. In vivo fluorescence imaging confirmed the micelle also could selectively accumulate in tumor and its mitochondria on A549 tumor-bearing nude mice. Consequently, it not only exhibited higher cytotoxicity, apoptosis rate, and metastasis inhibition rate on A549 cells, but also better tumor growth and metastasis inhibition rate on tumor-bearing nude mice and lower whole-body toxicity. The mechanism might be caused by destroying mitochondria and down-regulating ATP production.ConclusionThis study provided a GLUT1 targeting, hypoxia, and reductive responsive nanomedicine that hold the potential to be exploited for tumor therapy.     

Polyplex Micelles from Triblock Copolymers Composed of Tandemly Aligned Segments with Biocompatible, Endosomal Escaping, and DNA-Condensing Functions for Systemic Gene Delivery to Pancreatic Tumor Tissue

Purpose  For systemic gene delivery to pancreatic tumor tissues, we prepared a three-layered polyplex micelle equipped with biocompatibility, efficient endosomal escape, and pDNA condensation functions from three components tandemly aligned; poly(ethylene glycol) (PEG), a poly(aspartamide) derivative with a 1,2-diaminoethane moiety (PAsp(DET)), and poly(l-lysine). Materials and Methods  The size and in vitro transfection efficacy of the polyplex micelles were determined by dynamic light scattering (DLS) and luciferase assay, respectively. The systemic gene delivery with the polyplex micelles was evaluated from enhanced green fluorescence protein (EGFP) expression in the tumor tissues. Results  The polyplex micelles were approximately 80 nm in size and had one order of magnitude higher in vitro transfection efficacy than that of a diblock copolymer as a control. With the aid of transforming growth factor (TGF)-β type I receptor (TβR-1) inhibitor, which enhances accumulation of macromolecular drugs in tumor tissues, the polyplex micelle from the triblock copolymer showed significant EGFP expression in the pancreatic tumor (BxPC3) tissues, mainly in the stromal regions including the vascular endothelial cells and fibroblasts. Conclusion  The three-layered polyplex micelles were confirmed to be an effective gene delivery system to subcutaneously implanted pancreatic tumor tissues through systemic administration.     

Pharmacokinetics and biodistribution of paclitaxel-loaded pluronic P105/L101 mixed polymeric micelles

A mixed polymeric micelle formulation of paclitaxel (PTX) has been developed with the purpose of improving the solubility and prolonging the time of blood circulation of PTX in comparison to current Taxol injection. The mixed micelles were prepared by thin-film method using a nonionic surfactant Pluronic P105, L101 and PTX. The mean size of PTX-loaded mixed micelles was 185 nm with narrow size distribution shown by a dynamic light scattering sizer and a transmission electron microscopy. The in vitro release profiles indicated that PTX release from the mixed micelles exhibited a sustained release behavior. A similar phenomenon was also observed in a pharmacokinetic assessment in rats, in which t(1/2beta) and AUC of the mixed micelle formulation were 5.5 and 4.9-fold higher than that of Taxol injection. The biodistribution study in mice showed that the PTX-loaded mixed micelles not only decreased drug uptake by liver, but also prolonged drug retention in blood, and increased distribution of the drug in lung, spleen and kidney. These results suggested that the mixed polymeric micelles may efficiently load, protect and retain PTX in both in vitro and in vivo environments, and could be a useful drug carrier for intravenous administration of PTX.     

99mTc‐tricarbonyl labeling of paclitaxel as an antimicrotubule agent and its evaluation in B16‐F10 melanoma tumor‐bearing mice

In the present study paclitaxel (taxol) was labeled with [^99mTc(CO)₃(H₂O)₃]⁺ core. Labeling was optimized, and radiochemical analysis was determined by thin layer chromatography and high performance liquid chromatography. Radiocomplex was evaluated and verified further as a tumor characterization agent in B16‐F10 melanoma tumor‐bearing mice. The [^99mTc(CO)₃(H₂O)₃]⁺‐paclitaxel complex with high specific activity (0.77 GBq/μmol) and labeling yield (96.8 ± 1.3) was obtained. No decrease in labeling was observed up to 6 hours, and the stability of the radiocomplex was found adequate. Our main achievement was high accumulation of radiolabeled paclitaxel in tumor (4.51 ± 0.65 percentage injected dose per gram [%ID/g] at 2‐h postinjection) followed by significant reduction (1.86 ± 0.27%ID/g) at 4‐hour postinjection. Because paclitaxel is a substrate for multidrug resistance, ^99mTc‐tricarbonyl‐paclitaxel imaging would be useful for tumor characterization rather than tumor detection.     

NGR配体修饰的紫杉醇PEG-PLGA胶束抗肿瘤作用的体外体内研究(英文)

本研究制备了NGR(asparagine-glycine-arginine)配体修饰的紫杉醇PEG-PLGA胶束(NGR-PM-PTX),并对其靶向肿瘤新生血管内皮细胞及肿瘤细胞所表达的氨肽酶N进行了研究。采用薄膜法制备NGR-PM-PTX胶束。通过针对人脐静脉内皮细胞(HUVEC),人纤维肉瘤细胞(HT1080)和人乳腺癌细胞(MCF-7)的流式细胞试验和激光共聚焦实验,在体外细胞水平上研究并证实了NGR配体修饰的聚合物胶束对于上述细胞的靶向效果。HT1080荷瘤裸鼠的体内药效学研究结果进一步证实NGR-PM-PTX的抗肿瘤药效。