【作者】 张丽；

【导师】 张娜；

【作者基本信息】 山东大学 ， 药剂学， 2014， 硕士

【摘要】 癌症是目前严重危害人类生命健康的重大疾病之一,导致了全球大约13%的死亡率,是仅次于心血管疾病的第二大杀手,近年来癌症的发病率和死亡率仍然呈上升趋势。早期诊断和及时治疗,是提高肿瘤治愈率的关键。目前,在各种临床上常用的影像学诊断技术中,磁共振成像(magnetic resonance imaging, MRI)因具有非侵入、无电离辐射、穿透深度不受限制、组织对比度强、具有较高的软组织分辨率以及多参数、多序列、可任意断面成像等优势,已成为临床上肿瘤诊断的重要手段。MRI诊断的敏感性和特异性依赖于显影对比剂的使用。对比剂通过缩短周围质子的弛豫时间,增强信号对比,从而提高成像质量。临床上目前广泛使用的小分子对比剂存在弛豫率相对较低、体内分布非特异性、肾清除迅速和显影时间短等缺陷。理想的MRI对比剂应该满足以下条件：(1)安全性好,低毒；(2)弛豫率高；(3)体内滞留时间适当,利于临床显影诊断；(4)在靶组织或靶器官选择性分布,有利于病变部位的特异性检测。本课题选择生物相容和生物可降解的天然多糖壳聚糖和透明质酸为载体材料,分别采用前修饰和后修饰的方法,装载小分子显影对比剂钆,制备了载钆壳聚糖纳米粒(Gd-CSNPs)和透明质酸修饰的载钆壳聚糖纳米粒(CS-DTPA-Gd/TPP/HA NPs),用作肿瘤靶向的MRI显影对比剂,以期提高小分子对比剂的显影效果、改善其体内分布无特异性和肾清除迅速的缺陷。本课题对两种载钆纳米粒的理化性质、体内外显影能力和安全性进行了系统评价,主要研究方法和结果如下：1.载钆壳聚糖纳米粒显影对比剂首先采用离子交联法制备空白壳聚糖纳米粒,并进行单因素考察,确定最优处方；然后利用NHS-EDC反应将二乙三胺五乙酸(diethylene triamine penlaacetic acid, DTPA)连接在空白壳聚糖纳米粒表面,再与Gd螯合,从而制备了载钆壳聚糖纳米粒(Gd-CSNPs)。采用透射电子显微镜观察纳米粒的外观形态,用激光粒径和电位分析仪测定粒径和zeta电位,用电感耦合等离子体发射光谱仪测定Gd-CSNPs的钆浓度,采用噻唑蓝比色法(MTT法)测定纳米粒的细胞毒性,使用磁共振仪考察纳米粒的体外显影能力和B16荷瘤小鼠模型的体内显影能力。透射电镜下观察到Gd-CSNPs外观呈球形或类球形,平均粒径为153.0±7.5nm,电位为13.444±1.52mV；细胞毒性实验结果显示纳米粒安全性良好；体外显影评价结果显示,Gd-CSNPs的纵向弛豫率是Magnevist的2.46倍,16μ.MMagnevist与4μM Gd-CSNPs的显影信号强度相当,相同钆浓度的纳米粒显影强度明显高于Magnevist;体内显影实验表明,与Magnevist相比,B16荷瘤小鼠注射Gd-CSNPs后显影强度明显加强,肿瘤和肝脏部位强化明显,且显影时间长达4h。与小分子对比剂相比,将钆修饰在纳米粒表面增加了对比剂的分子量,延长了钆的回旋时间,且钆分布在纳米粒表面利于与水中质子的交换,从而显著提高了对比剂的弛豫率,增强了显影效果；将小分子对比剂装载于纳米粒上,可利用纳米粒的被动靶向作用,提高了对比剂在肿瘤部位的浓集,增强了肿瘤部位的显影效果,提高了肿瘤诊断的灵敏度；此外,将小分子对比剂装载于纳米粒上,改善了其体内肾清除迅速的特点,延长了对比剂在体内的滞留时间,拓宽了显影诊断的时间窗。2.透明质酸修饰的载钆壳聚糖纳米粒显影对比剂本课题第二部分以壳聚糖和透明质酸为载体材料,制备了透明质酸修饰的载钆壳聚糖纳米粒,用于肿瘤靶向显影。为了进一步提高单个纳米粒的载钆量,本部分采用了前修饰方法,即首先以壳聚糖为材料,通过化学连接的方法修饰一定比例的DTPA,并与Gd螯合,制备钆标记的壳聚糖,再通过静电相互作用与HA、TPP交联形成透明质酸修饰的壳聚糖纳米粒(CS-DTPA-Gd/TPP/HA NPs).采用红外光谱和核磁共振氢谱对钆标记壳聚糖进行结构验证,用电感耦合等离子体发射光谱仪测定钆标记壳聚糖材料中的钆浓度,通过考察影响纳米粒制备的主要影响因素确定最优处方,并评价最优处方的重现性,采用透射电子显微镜观察纳米粒的外观形态,用激光粒径和电位分析仪测定粒径、粒径分布和zeta电位,采用MTT法测定纳米粒对B16、HepG2和A549的细胞毒性,使用磁共振仪考察纳米粒的体外显影能力和B16荷瘤小鼠模型的体内显影能力,采用组织切片方法初步评价纳米粒在小鼠体内的安全性。实验结果表明,钆标记的壳聚糖材料成功合成,CS-DTPA-Gd/TPP/HA NPs外观澄清,有淡蓝色乳光,透射电镜下呈球形或类球形,分散性良好,粒径为213.8±2.6nm,多分散系数为0.219,粒径分布较窄,电位为19.92±1.69mV；该纳米粒对B16、HepG2和A549细胞的毒性很低；组织切片结果显示,小鼠注射该纳米粒后主要组织器官无明显病理变化,体内安全性良好；体外显影结果表明,16μM Magnevist和2μM CS-DTPA-Gd/TPP/HA NPs的显影强度相当,与相同钆浓度的Magnevist相比,纳米粒溶液的显影强度显著提高；体内显影结果表明,B16荷瘤小鼠注射该纳米粒后,与Magnevist相比,显影信号强度大大提高,尤其是肿瘤和肝脏部位,对比剂的体内滞留时间也显著延长。综上所述,本课题制备的两种载钆纳米粒显著提高了小分子钆对比剂的显影能力,增强了体内显影效果,提高了其对肿瘤和肝脏的靶向效率,延长了体内滞留时间,具有进一步的开发潜能和临床应用前景。更多还原

【Abstract】 Cancer, one of the major diseases presenting serious harm to human life and health, is the second killer after cardiovascular disease, which resulted in a mortality rate of about13percent worldwide. The incidence and mortality rate of cancer are still on the rise in recent years. Early diagnosis and timely treatment is essential for raising the cure rate and improving the survival rate of tumor patients. Among various imaging diagnostic techniques used in clinical currently, magnetic resonance imaging (MRI) has become a powerful tool for tumor diagnosis, due to the advantages of non-invasiveness, no ionizing radiation, infinite penetration depth, higher spatial resolution, and precise three-dimensional positioning ability. The sensitivity and specificity of MRI diagnosis depends on the using of imaging contrast agents. Contrast agents can enhance the signal intensity and improve the image contrast in local tissues that are magnetically similar but histologically distinct by shortening the relaxation time of water protons around. The small molecular contrast agents used in clinical presently have some defects such as relatively low relaxation rate, non-specific distribution in vivo, rapid renal clearance and short imaging time.An ideal MRI contrast agent shall meet the following conditions:(1) good safety and low toxicity;(2) high relaxation rate;(3) appropriate retention time in the body for imaging diagnosis;(4) selective distribution in target tissue and organs for specific detection of lesion site. In this paper, chitosan and hyaluronic acid, biocompatible and biodegradable natural polysaccharides, were chosen as the nanocarrier material for the preparation of gadolinium-loaded chitosan nanoparticles (Gd-CSNPs) and hyaluronic acid modified gadolinium-loaded chitosan nanoparticles (CS-DTPA-Gd/TPP/HA NPs) by post-modification and pre-modification methods, respectively. The Gd-loaded nanoparticles were expected to use as tumor targeted MRI contrast agents, in order to improve imaging contrast and its non-specific distribution in vivo, and overcome the drawback of rapid renal clearance of small molecular contrast agents. The physical and chemical properties, MRI imaging ability in vitro and in vivo, and the safety in cells and animals of the Gd-loaded nanoparticles were evaluated in this research systematically. The main research methods and results are as follows:1. Gd-loaded chitosan nanoparticles as molecular imaging contrast agentsChitosan nanoparticles (CSNPs) were prepared by ionic-cross linking method using sodium tripolyphosphate (TPP) as the linking agent. The formulation was optimized by single-factor experiments. Diethylene triamine penlaacetic acid (DTPA) was conjugated onto the surface of CSNPs using EDC-NHS reaction, then the DTPA linked CSNPs were incubated with GdCl3solution to obtain Gd-CSNPs. The morphology was observed using transmission electron microscope (TEM). The particle sizes and the zeta potentials were measured by laser particle size and potential analyzer, respectively. The Gd concentration of Gd-CSNPs solution was determined by inductively coupled plasma emission spectrometry. Cytotoxicity was tested by MTT method in B16cells. The MRI imaging abilities in vitro and in vivo were investigated by magnetic resonance instrument.The morphology of CSNPs and Gd-CSNPs were both approximately spherical; the average particle sizes and zeta potentials of Gd-CSNPs were153.0±7.5nm and13.44±1.52mV, respectively. The results of Cytotoxicity test showed that the safety of Gd-CSNPs was good. The results of the MRI imaging test in vitro showed that the longitudinal relaxation rate of Gd-CSNPs was2.46times than that of Magnevist. The imaging signal intensity of16μMMagnevist was same as that of4μM Gd-CSNPs. The results of MRI imaging in B16tumor bearing mice model suggested that the signal intensity were enhanced significantly by tail intravenous injection of Gd-CSNPs at40μM Gd/kg, especially in the tumor and liver. And the imaging time were prolonged to4h. Compared with small molecular contrast agents, Gd-CSNPs improved imaging contrast significantly by increasing the molecular weight of contrast agents, efficiently retarding the rotational motion of Gd complex, and increasing the exchange rate between Gd and water protons by modifying gadoliniumin on the surface of nanoparticles. And Gd-CSNPs also increased the distribution of contrast agents in tumor site by enhanced permeability and retention (EPR) effect, which was conductive to improve the sensitivity of the diagnosis of tumor. In addition, Gd-CSNPs improved the characteristics of rapid renal clearance in vivo of small molecular contrast agents and prolonged the residence time in the body, which widened the time window of imaging diagnosis.2. Gd-loaded chitosan nanoparticles modified with hyaluronic acid as molecular imaging contrast agentsIn the second part, Gd-loaded chitosan nanoparticles modified with hyaluronic acid (CS-DTPA-Gd/TPP/HA NPs) were prepared as molecular imaging contrast agents for tumor diagnosis. In order to further improve the gadolinium loads of a single nanoparticle, pre-modification method was adopted that DTPA was conjugated with chitosan first through chemical reaction and then incubated with free Gd ions to prepare gadolinium labeled chitosan. Then, CS-DTPA-Gd/TPP/HA NPs were prepared via electrostatic interaction using gadolinium labeled chitosan, HA and TPP. The successful synthesis of gadolinium labeled chitosan was verified by Fourier transform infrared spectrum and nuclear magnetic resonance hydrogen spectrum. The gadolinium concentration was determined by inductively coupled plasma emission spectrometer. The optimal prescription was determined by investigating the main influence factor of the preparation of nanoparticles, and its reproducibility was also studied. The morphology was observed by TEM. The particle sizes and the zeta potentials were measured by laser particle size and potential analyzer, respectively. Cytotoxicity was tested using MTT method in B16, HepG2and A549cells. The MRI imaging abilities in vitro and in vivo were investigated by magnetic resonance instrument. The safety of the nanoparticles in mice was evaluated by tissue section method preliminarily.The experimental results showed that gadolinium labeled chitosan was synthesized successfully; the appearance of CS-DTPA-Gd/TPP/HA NPs was clarify, with pale blue light; the morphology of the nanoparticles were approximately spherical, with good dispersion; the average particle sizes and zeta potentials were213.8Π2.6nm and19.92Π1.69mV, respectively; the polydispersity index was0.219, with a narrow particle size distribution. The results of cytotoxicity test showed that the safety of the nanoparticles was good. The results of the MRI imaging test in vitro showed that the imaging signal intensity of16μM Magnevist was same as that of2μM CS-DTPA-Gd/TPP/HA NPs. Compared with Magnevist of the same Gd concentration, the imaging signal intensity of the nanoparticles was improved significantly. The results of MRI imaging in B16tumor bearing mice model suggested that the signal intensity were enhanced significantly after tail intravenous injection of the nanoparticles, especially in the tumor and liver. And the imaging time were prolonged greatly.In conclusion, Gd-CSNPs and CS-DTPA-Gd/TPP/HA NPs increased the imaging ability of small molecule Gd-based contrast agents significantly, enhanced MRI imaging effect in vivo, improved the imaging in the tumor and liver, and prolonged the residence time in the body. They might be promising MRI contrast agents that has the potential of further development and clinical application prospects.更多还原