表面功能化的磁性荧光纳米颗粒的制备、表征及其在胰腺癌靶向双模态成像中的应用研究

发布时间：2018-03-24 22:30

本文选题：磁性纳米颗粒　切入点：胰腺癌　出处：《苏州大学》2014年硕士论文

【摘要】：胰腺癌早期症状不显著，早期诊断缺乏特异性，因此死亡率高。近年来，分子影像学发展迅速，其以分子生物学为基础，在活体状态下从分子、基因水平对胰腺癌进行更早期的诊断。磁共振成像（magnetic resonance imaging，，MRI）具有空间分辨率高、组织对比度好等优点，因此在分子影像中应用广泛，但敏感性较低，因此限制了其在肿瘤早期诊断的作用。近红外荧光成像（Near-infrared fluorescence imaging, NIRF）具有灵敏度高、无射线辐射、对人体无害、可重复曝光等优点，但是空间分辨率较差，背景噪声高。同时利用MRI和荧光成像对同一物体进行成像以获得补充信息，即MRI/NIRF双模态成像，可以实现优势互补，其研究的重点是双模态成像探针的制备。故研制新型的磁性荧光纳米颗粒，并探讨其性能特征及其在胰腺癌双模态成像中的应用，是本文的主要工作。本论文主要内容分为以下两个部分：（1）制备双模态探针的前体-磁性纳米颗粒（2）制备双模态探针并探讨其生物安全性、体内生物分布及其在胰腺癌的靶向成像效果。第一部分主要是制备磁性纳米颗粒。首先是油性的超顺磁性氧化铁纳米颗粒（superparamagnetic iron oxide nanoparticles, SPIONs）的制备，实验以Fe(acac)3为前驱体，以油酸、油胺、1,2-十六烷以及二苄醚的混合物为表面活性剂，在有机溶液中，用高温热解法制备油溶性的SPIONs。并用TEM、XRD、VSM等方法对其进行表征，结果表明所制备的油溶性SPIONs具有良好的形貌、均一的尺寸，并具有超顺磁性，无剩磁。然后用两种不同的方法对所合成的油溶性SPIONs进行表面改性。第一种方法基于双亲性改性，先用反相旋蒸法使亲水性大分子PMAO取代油溶性SPIONs表面的疏水性配体，然后通过2,2'-(乙烯二氧)双(乙胺)和PMAO的开环聚合反应，得到足够的氨基、羧基用于后期的荧光染料及抗体的偶联。第二种方法是配体交换，先利用NOBF4将油溶性SPIONs转换为易溶于DMF等的亲水性颗粒，然后用BSA通过配体交换将亲水性纳米颗粒转换为水溶性的BSA·SPIONs。TEM、胶体稳定性及弛豫率测试表明这两种方法修饰合成的纳米颗粒均具有良好的水溶性、胶体稳定性及较高的弛豫率。第二部分通过MTT实验和组织HE染色考察BSA·SPIONs的生物安全性，结果表明该纳米粒子对人肝正常上皮L02细胞的毒性较低，具有良好的生物相容性。纳米粒子在体内的生物分布，是通过小鼠尾静脉注射BSA·SPIONs后，在不同时间点取出相应的组织器官，测定其内Fe的含量来进行的。研究结果显示该纳米颗粒进入体内后主要分布于肝、脾、大肠，于注射后6-12h达到高峰，其后逐渐下降至正常水平。BSA·SPIONs偶联近红外荧光染料Cy5及靶向胰腺癌的抗Plec-1抗体后，即靶向的双模态探针dyeBSA·SPION-mAb，用其进行细胞水平的MRI、近红外荧光成像及在体MRI，验证了该探针较低的非特异性吸附、良好的靶向性及用于MRI/NIRF双模态成像的可行性。综上，本文用两种方法制成了具有良好的生物相容性及稳定性的磁性纳米颗粒，选用BSA修饰的磁性颗粒偶联荧光染料后，初步探究了该探针的生物安全性及体内生物分布。将该探针偶联靶向抗体后，可用作肿瘤早期探测，并通过体外、体内靶向双模态成像的研究，证明了该新型磁性荧光双模态探针的生物医学应用价值，说明其广阔的应用前景。
[Abstract]:The early symptoms of pancreatic cancer is not significant, early diagnosis and lack of specificity, therefore the mortality rate is high. In recent years, the rapid development of molecular imaging, which is based on molecular biology, in vivo from molecular gene level for diagnosis of pancreatic cancer earlier. Magnetic resonance imaging (magnetic resonance, imaging, MRI) with spatial resolution the advantages of high tissue contrast and so on, so the application of molecular imaging in wide range, but the sensitivity is low, thus limiting its role in early diagnosis of cancer. Near infrared fluorescence imaging (Near-infrared fluorescence, imaging, NIRF) has the advantages of high sensitivity, no radiation, harmless to the human body, can be repeated exposure and other advantages, but the space low resolution, high background noise. At the same time using MRI and fluorescence imaging imaging of the same object in order to obtain additional information, namely MRI/NIRF dual mode imaging, can achieve complementary advantages, its research The emphasis is focused on the preparation of dual mode imaging probes. Therefore, the development of new magnetic fluorescent nanoparticles and their performance characteristics and their application in dual mode imaging of pancreatic cancer are the main works of this paper.
The main contents of this paper are divided into two parts: (1) preparation of dual modal probe precursor magnetic nanoparticles (2) to prepare bimodal probes and explore their biological safety, in vivo biodistribution and targeted imaging effect in pancreatic cancer.
The first part is the preparation of magnetic nano particles. The first is oily superparamagnetic iron oxide nanoparticles (superparamagnetic iron oxide nanoparticles, SPIONs) were prepared based on Fe (ACAC) 3 as precursor, oleic acid, oil amine, 1,2- sixteen alkyl benzyl ether mixture and two as surfactant in the organic solution, using high temperature pyrolysis preparation of oil soluble SPIONs. and TEM, XRD, VSM and other methods were characterized. Results show that the preparation of oil soluble SPIONs has good appearance, uniform size, and superparamagnetism, no residual surface modification of the synthetic oil soluble SPIONs and then using two different methods. The first method is based on amphiphilic modified the hydrophilic macromolecular PMAO to replace the oil soluble hydrophobic ligands of SPIONs surface by rotary evaporation method, and then 2,2'- (two ethylene oxide) - (ethylamine) open loop and PMAO The polymerization reaction, get enough amino, carboxyl for coupling fluorescent dye and later antibody. The second method is ligand exchange, using NOBF4 oil soluble SPIONs converted into hydrophilic particles easily dissolve in DMF, and ligand exchange hydrophilic nanoparticles convert soluble BSA - SPIONs.TEM by water with BSA, colloid stability and relaxation rate test showed that the two kinds of methods to modify the synthesis of nanoparticles have good water solubility, colloid stability and higher relaxation rate.
The second part was the biological safety of SPIONs BSA was investigated by MTT experiment and HE, results showed that the toxicity of nanoparticles on human normal liver epithelial L02 cells is low, has good biocompatibility. Nanoparticles in vivo biodistribution, through tail vein injection of BSA SPIONs, remove the corresponding organization organ at different time points, the determination of Fe in the paper. The results of research showed that the nano particles into the body mainly distributed in liver, spleen, intestine, after injection of 6-12h reached the peak, then decreased gradually to the normal level of.BSA and SPIONs coupling near infrared fluorescent dye Cy5 and anti Plec-1 targeting pancreatic cancer the antibody, targeting the dual mode probe dyeBSA - SPION-mAb cell level with its MRI, near-infrared fluorescence imaging and in vivo MRI, verified the nonspecific adsorption of the probe is low, targeted and good The feasibility of MRI/NIRF dual mode imaging.
In summary, this paper use two methods made of magnetic nanoparticles has good biological compatibility and stability of magnetic particles modified by BSA coupling fluorescent dye, preliminary study of the probe biological safety and biodistribution. The probe coupling targeting antibodies, can be used for early tumor detection, and through in vitro, to study the dual mode imaging in vivo, proved that the new biomedical applications of magnetic fluorescent double mode probe value shows its broad application prospects.

【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R735.9;R445.2

【参考文献】