本文選題：磁共振成像 + 釓對比劑��； 參考：《山東大學(xué)》2014年碩士論文

【摘要】：癌癥是目前嚴重危害人類生命健康的重大疾病之一,導(dǎo)致了全球大約13%的死亡率,是僅次于心血管疾病的第二大殺手,近年來癌癥的發(fā)病率和死亡率仍然呈上升趨勢。早期診斷和及時治療,是提高腫瘤治愈率的關(guān)鍵。目前,在各種臨床上常用的影像學(xué)診斷技術(shù)中,磁共振成像(magnetic resonance imaging, MRI)因具有非侵入、無電離輻射、穿透深度不受限制、組織對比度強、具有較高的軟組織分辨率以及多參數(shù)、多序列、可任意斷面成像等優(yōu)勢,已成為臨床上腫瘤診斷的重要手段。MRI診斷的敏感性和特異性依賴于顯影對比劑的使用。對比劑通過縮短周圍質(zhì)子的弛豫時間,增強信號對比,從而提高成像質(zhì)量。臨床上目前廣泛使用的小分子對比劑存在弛豫率相對較低、體內(nèi)分布非特異性、腎清除迅速和顯影時間短等缺陷。 理想的MRI對比劑應(yīng)該滿足以下條件：(1)安全性好,低毒；(2)弛豫率高；(3)體內(nèi)滯留時間適當,利于臨床顯影診斷；(4)在靶組織或靶器官選擇性分布,有利于病變部位的特異性檢測。本課題選擇生物相容和生物可降解的天然多糖殼聚糖和透明質(zhì)酸為載體材料,分別采用前修飾和后修飾的方法,裝載小分子顯影對比劑釓,制備了載釓殼聚糖納米粒(Gd-CSNPs)和透明質(zhì)酸修飾的載釓殼聚糖納米粒(CS-DTPA-Gd/TPP/HA NPs),用作腫瘤靶向的MRI顯影對比劑,以期提高小分子對比劑的顯影效果、改善其體內(nèi)分布無特異性和腎清除迅速的缺陷。本課題對兩種載釓納米粒的理化性質(zhì)、體內(nèi)外顯影能力和安全性進行了系統(tǒng)評價,主要研究方法和結(jié)果如下： 1.載釓殼聚糖納米粒顯影對比劑 首先采用離子交聯(lián)法制備空白殼聚糖納米粒,并進行單因素考察,確定最優(yōu)處方；然后利用NHS-EDC反應(yīng)將二乙三胺五乙酸(diethylene triamine penlaacetic acid, DTPA)連接在空白殼聚糖納米粒表面,再與Gd螯合,從而制備了載釓殼聚糖納米粒(Gd-CSNPs)。采用透射電子顯微鏡觀察納米粒的外觀形態(tài),用激光粒徑和電位分析儀測定粒徑和zeta電位,用電感耦合等離子體發(fā)射光譜儀測定Gd-CSNPs的釓濃度,采用噻唑藍比色法(MTT法)測定納米粒的細胞毒性,使用磁共振儀考察納米粒的體外顯影能力和B16荷瘤小鼠模型的體內(nèi)顯影能力。 透射電鏡下觀察到Gd-CSNPs外觀呈球形或類球形,平均粒徑為153.0±7.5nm,電位為13.444±1.52mV；細胞毒性實驗結(jié)果顯示納米粒安全性良好；體外顯影評價結(jié)果顯示,Gd-CSNPs的縱向弛豫率是Magnevist的2.46倍,16μ.MMagnevist與4μM Gd-CSNPs的顯影信號強度相當,相同釓濃度的納米粒顯影強度明顯高于Magnevist;體內(nèi)顯影實驗表明,與Magnevist相比,B16荷瘤小鼠注射Gd-CSNPs后顯影強度明顯加強,腫瘤和肝臟部位強化明顯,且顯影時間長達4h。與小分子對比劑相比,將釓修飾在納米粒表面增加了對比劑的分子量,延長了釓的回旋時間,且釓分布在納米粒表面利于與水中質(zhì)子的交換,從而顯著提高了對比劑的弛豫率,增強了顯影效果；將小分子對比劑裝載于納米粒上,可利用納米粒的被動靶向作用,提高了對比劑在腫瘤部位的濃集,增強了腫瘤部位的顯影效果,提高了腫瘤診斷的靈敏度；此外,將小分子對比劑裝載于納米粒上,改善了其體內(nèi)腎清除迅速的特點,延長了對比劑在體內(nèi)的滯留時間,拓寬了顯影診斷的時間窗。 2.透明質(zhì)酸修飾的載釓殼聚糖納米粒顯影對比劑 本課題第二部分以殼聚糖和透明質(zhì)酸為載體材料,制備了透明質(zhì)酸修飾的載釓殼聚糖納米粒,用于腫瘤靶向顯影。為了進一步提高單個納米粒的載釓量,本部分采用了前修飾方法,即首先以殼聚糖為材料,通過化學(xué)連接的方法修飾一定比例的DTPA,并與Gd螯合,制備釓標記的殼聚糖,再通過靜電相互作用與HA、TPP交聯(lián)形成透明質(zhì)酸修飾的殼聚糖納米粒(CS-DTPA-Gd/TPP/HA NPs).采用紅外光譜和核磁共振氫譜對釓標記殼聚糖進行結(jié)構(gòu)驗證,用電感耦合等離子體發(fā)射光譜儀測定釓標記殼聚糖材料中的釓濃度,通過考察影響納米粒制備的主要影響因素確定最優(yōu)處方,并評價最優(yōu)處方的重現(xiàn)性,采用透射電子顯微鏡觀察納米粒的外觀形態(tài),用激光粒徑和電位分析儀測定粒徑、粒徑分布和zeta電位,采用MTT法測定納米粒對B16、HepG2和A549的細胞毒性,使用磁共振儀考察納米粒的體外顯影能力和B16荷瘤小鼠模型的體內(nèi)顯影能力,采用組織切片方法初步評價納米粒在小鼠體內(nèi)的安全性。 實驗結(jié)果表明,釓標記的殼聚糖材料成功合成,CS-DTPA-Gd/TPP/HA NPs外觀澄清,有淡藍色乳光,透射電鏡下呈球形或類球形,分散性良好,粒徑為213.8±2.6nm,多分散系數(shù)為0.219,粒徑分布較窄,電位為19.92±1.69mV；該納米粒對B16、HepG2和A549細胞的毒性很低；組織切片結(jié)果顯示,小鼠注射該納米粒后主要組織器官無明顯病理變化,體內(nèi)安全性良好；體外顯影結(jié)果表明,16μM Magnevist和2μM CS-DTPA-Gd/TPP/HA NPs的顯影強度相當,與相同釓濃度的Magnevist相比,納米粒溶液的顯影強度顯著提高；體內(nèi)顯影結(jié)果表明,B16荷瘤小鼠注射該納米粒后,與Magnevist相比,顯影信號強度大大提高,尤其是腫瘤和肝臟部位,對比劑的體內(nèi)滯留時間也顯著延長。 綜上所述,本課題制備的兩種載釓納米粒顯著提高了小分子釓對比劑的顯影能力,增強了體內(nèi)顯影效果,提高了其對腫瘤和肝臟的靶向效率,延長了體內(nèi)滯留時間,具有進一步的開發(fā)潛能和臨床應(yīng)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.

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R943;R445.2

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8 梁恒倫;腫瘤靶向性透明質(zhì)酸偶聯(lián)殼聚糖納米粒的制備及體外特性研究[D];南方醫(yī)科大學(xué);2011年

9 馮煒瑋;去甲斑蝥素—羥丙基殼聚糖納米粒的制備及其體外抗腫瘤性能研究[D];山東理工大學(xué);2012年

10 康衛(wèi)衛(wèi);5-FU-殼聚糖納米粒的制備、檢測及其對卵巢癌細胞的抑制作用[D];第四軍醫(yī)大學(xué);2013年

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