用于肿瘤诊断的载钆壳聚糖纳米粒作为磁共振成像对比剂的研究

发布时间：2018-04-28 20:30

本文选题：磁共振成像 + 钆对比剂　；参考：《山东大学》2014年硕士论文

【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。
[Abstract]:Cancer is one of the major diseases which seriously endangers human life and health. It has caused the death rate of about 13% in the world. It is the second largest killer after cardiovascular disease. In recent years, the incidence and mortality of cancer are still on the rise. Early diagnosis and timely treatment are the key to raise the cure rate of high tumor. In common imaging diagnosis techniques, magnetic resonance imaging (MRI) has become an important means of clinical cancer diagnosis because of its non invasion, non ionizing radiation, unrestricted penetration depth, strong tissue contrast, high resolution of soft tissue, multi parameter, multi sequence and arbitrary section imaging. The sensitivity and specificity of the I diagnosis depend on the use of contrast media. Contrast agents enhance the signal contrast by shortening the relaxation time of the surrounding protons and enhancing the contrast of the signals, thus improving the imaging quality. The clinical current widely used small molecular contrast agents have relatively low relaxation rates, non specific distribution in the body, rapid renal clearance and short developing time. Depression.
Ideal MRI contrast agent should meet the following conditions: (1) good safety and low toxicity; (2) high relaxation rate; (3) proper retention time in the body, in favor of clinical development diagnosis; (4) selective distribution of target tissues or target organs, and the specific detection of lesion sites. This subject selects biocompatible and biodegradable natural polysaccharide chitosan Gadolinium loaded gadolinium loaded chitosan nanoparticles (Gd-CSNPs) and hyaluronic acid modified gadolinium chitosan nanoparticles (CS-DTPA-Gd/TPP/HA NPs) were prepared by pre modification and postmodification, and gadolinium loaded chitosan nanoparticles (CS-DTPA-Gd/TPP/HA NPs) modified by hyaluronic acid were used as the carrier materials, respectively. It was used as a contrast agent for tumor targeting MRI development. In this study, the physical and chemical properties of two kinds of gadolinium loaded nanoparticles, in vivo and in vitro development ability and safety were systematically evaluated. The main research methods and results are as follows:
1. carrier gadolinium chitosan nanoparticle contrast agent
First, the blank chitosan nanoparticles were prepared by the ionic crosslinking method, and the optimal formulation was determined by single factor investigation. Then the two ethyl three amine five acetic acid (diethylene triamine penlaacetic acid, DTPA) was connected to the surface of the blank chitosan nanoparticles and then chelated with Gd, thus preparing the gadolinium chitosan nanoparticles (Gd-CSNP). S). The appearance of nanoparticles was observed by transmission electron microscopy. The particle size and zeta potential were measured by the laser particle size and potential analyzer. The gadolinium concentration of Gd-CSNPs was measured by inductively coupled plasma emission spectrometer. The microcytotoxicity of nanoparticles was measured by the thiazolate Colorimetry (MTT method). The magnetic resonance apparatus was used to investigate the presence of nanoparticles in vitro. Shadow ability and B16 imaging ability in vivo.
Under transmission electron microscope, the appearance of Gd-CSNPs was spherical or spherical. The average particle size was 153 + 7.5nm and the potential was 13.444 + 1.52mV. The cytotoxicity test results showed that the nanoparticles were safe. The development of Gd-CSNPs in vitro showed that the longitudinal relaxation rate of Gd-CSNPs was 2.46 times of Magnevist, 16 mu.MMagnevist and 4 mu M Gd-CSNPs. The intensity of the nanoparticles with the same gadolinium concentration was significantly higher than that of Magnevist. In vivo development experiments showed that compared with Magnevist, the intensity of the development of B16 bearing mice was obviously enhanced, the enhancement of the tumor and liver was obvious, and the time of development of 4h. was increased to the surface of the nanoparticles as compared with the small fraction contrast agent. The molecular weight of the contrast agent prolongs the gyration time of gadolinium, and the distribution of gadolinium on the nanoparticles is beneficial to the exchange of protons in the water, thus significantly increasing the relaxation rate of the contrast agent and enhancing the development effect. The small molecule contrast agent is loaded on the nanoparticles, and the passive targeting of the nanoparticles can be used to improve the contrast agent in the tumor site. The enrichment of the tumor enhanced the development of the tumor site and increased the sensitivity of the tumor diagnosis. In addition, the small molecule contrast agent was loaded on the nanoparticles, which improved the characteristics of the rapid renal clearance in the body, extended the retention time of the contrast agent in the body and widened the time window for the development of the diagnosis.
2. hyaluronic acid modified gadolinium chitosan nanoparticles contrast agent
In the second part, the gadolinium chitosan nanoparticles modified by hyaluronic acid were prepared with chitosan and hyaluronic acid as the carrier materials. In order to further improve the gadolinium carrying capacity of a single nanoparticle, the pre modification method was adopted in this part, that is, the chitosan was first modified by chitosan as the material and modified by chemical connection. The ratio of DTPA and chelating with Gd to prepare gadolinium labeled chitosan and cross-linked by electrostatic interaction with HA and TPP to form hyaluronic acid modified chitosan nanoparticles (CS-DTPA-Gd/TPP/HA NPs). The structure of gadolinium labeled chitosan was tested by IR and NMR spectroscopy, and gadolinium was determined by ICP AES The concentration of gadolinium in chitosan was marked, and the optimal prescription was determined by investigating the main factors affecting the preparation of nanoparticles, and the reproducibility of the optimal prescription was evaluated. The appearance of nanoparticles was observed by transmission electron microscope. The particle size, particle size distribution and zeta potential were measured by the laser particle size and potential analyzer. The MTT method was used for the determination of nanoscale. The cytotoxicity of particles to B16, HepG2 and A549 was detected by magnetic resonance imaging (MRI), and the ability of the nanoparticles in vitro development and in vivo developing ability of the B16 tumor bearing mice model were investigated. The safety of nanoparticles in mice was evaluated by tissue section method.
The results showed that the gadolinium labeled chitosan material was successfully synthesized, CS-DTPA-Gd/TPP/HA NPs was clear in appearance, with light blue milk light, spherical or spherical under transmission electron microscope, with good dispersion, 213.8 2.6nm particle size, 0.219 dispersion coefficient, narrow particle size distribution and 19.92 + 1.69mV, and the nanoparticles were toxic to B16, HepG2 and A549 cells. The results showed that the main tissues and organs of the mice were no obvious pathological changes and the body was safe in vivo. The development in vitro showed that the developing intensity of 16 M Magnevist and 2 mu M CS-DTPA-Gd/TPP/HA NPs was equal, and the developing intensity of the nanoparticles was significantly higher than that of the same gadolinium concentration Magnevist. In vivo development results showed that after injection of the nanoparticles in B16 tumor bearing mice, the intensity of the developing signal was greatly increased compared with Magnevist, especially in the tumor and liver parts, and the retention time of the contrast agent in the body was also prolonged significantly.
To sum up, two kinds of gadolinium nanoparticles prepared by this project have significantly enhanced the developing ability of small gadolinium contrast agents, enhanced the effect of development in the body, improved its target efficiency for tumor and liver, extended the retention time in the body, and had further development potential and clinical application prospects.

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R943;R445.2

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