论文总字数：21022字

摘 要

目的：众所周知，癌症正在成为全球公共健康的主要威胁之一，应对癌症的治疗方法日益成熟，但癌症早期症状的发现仍然是一个难题，阻碍着癌症的早期精准确诊，使得治疗难度增加。目前临床上常用电子计算机断层扫描(CT)、磁共振成像(MR)、单光子发射计算机断层成像(SPECT)和正电子发射断层成像(PET)对肿瘤进行即时诊断，为了得到更为全面的成像分析，构建多模态纳米成像造影剂已成为目前的研究热点。

方法：本文将超支化聚乙烯亚胺（PEI）高分子聚合物作为纳米平台构建SPECT/CT双模态造影剂，用于癌症的早期诊断。根据超支化聚乙烯亚胺的结构特点，表面可进行功能化修饰，内部可包裹金纳米颗粒，构建酸性微环境响应型锝-99m标记金纳米粒分子探针，对产物进行基本的定性及定量表征、粒径分析，并在体外进行MTT细胞毒性测试。

结果：本实验结果显示，制备的酸性微环境响应型锝-99m标记金纳米粒分子探针，每个PEI表面修饰有13.28个mPEG-COOH，14.2个MAL-PEG-SVA，6.2个DTPA，4.8个FI，5.3个APAS，并包裹纳米金颗粒在其内部，通过紫外可见吸收光谱证明形成了金纳米颗粒，且该纳米复合材料水溶性良好。透射电镜显示，金纳米颗粒粒径约为3.3nm，MTT实验表明该纳米分子探针在一定浓度范围内对细胞无毒害作用，具有良好的生物相容性。

结论：酸性微环境响应型锝-99m标记金纳米分子探针具备作为SPECT/CT双模态造影剂的优良条件，有望极大的提高其在肿瘤处的摄取量从而提高癌症早期诊断准确度，为癌症患者带来福音。

关键词： SPECT/CT成像 聚乙烯亚胺 酸性微环境响应 表征 生物相容性

Construction of Acidic Microenvironment Responsive Technetium-99m Labeled Gold Nanoparticles Molecular Probes and Their Imaging Applications in vitro

ABSTRACT

Objective: As is known to all, cancer is becoming one of the major threats to global public health, and treatment methods for cancer are increasingly mature. However, the detection of early symptoms of cancer is still a difficult problem, which hinders the early precise diagnosis of cancer and makes the treatment more difficult. At present, Computed Tomography (CT), Magnetic Resonance Imaging (MR), Single Photon Emission Computed Tomography (SPECT) and Positron Emission Tomography (PET) are commonly used in clinical practice for immediate diagnosis of tumor. In order to obtain more comprehensive imaging analysis, the construction of multimode nano-imaging contrast agent has become a research hotspot.

Methods: Here，using hyperbranched polyethyleneimide (PEI) polymer as nano-platform to construct SPECT/CT dual mode contrast agent for early diagnosis of cancer. Based on the structural characteristic of hyperbranched polyethyleneimine, which can be modified with functional group on the surface and entrapped gold nanoparticle inside. Through this way，technetium-99m(^99mTc) labeled gold nanoparticle molecular probe was constructed. Via basic qualitative and quantitative characterization, size analysis to evaluate the product obtained. Then MTT assay was utilized to evaluate the cytotoxicity in vitro.

Results: The results of this experiment showed that every polyethyleneimine modified with 13.28 mPEG-COOH, 14.2 MAL-PEG-SVA, 6.2 DTPA, 4.8 FI, 5.3 APAS on the surface of polyethyleneimine respectively, then entrapping gold nanoparticle inside of the prepared acidic microenvironment responsive technetium-99m labeled gold nanoparticles molecular probes. The gained gold nanoparticle can be confirmed by Ultra Violet-Visible absorption spectroscopy. And the nanocomposites owned good water solubility. Transmission electron microscope result indicates the size of the gold nanoparticle is approximately 3.3 nm. MTT experiment showed that the constructed molecular probe had no toxic effect on cancer cells within a certain concentration range, which demonstrate the good biocompatibility of the molecular probe.

Conclusion: Technetium-99m-labeled gold nanomolecular probe with acid microenvironment responsive function can be a good SPECT/CT dual mode contrast agent, which may greatly improve the cellular uptake at tumor site, then improve the accuracy of early diagnosis of cancer and bring good news to cancer patients.

Key words: SPECT/CT imaging; Polyethyleneimine; Acid microenvironment responsive; Characterization; Biocompatibility

目 录

摘要 I

ABSTRACT II

第一章 文献综述 1

1.1早期肿瘤监测手段 1

1.2聚乙烯亚胺 2

1.2.1 PEI的表面修饰 2

1.2.2 PEI作为金纳米颗粒载体 3

1.2.3 功能化PEI作为肿瘤探针 3

1.3 pH敏感两性离子 3

1.3.1 肿瘤组织特异性 3

1.3.2 两性离子的肿瘤靶向性 4

1.4酸性微环境响应型纳米分子探针的构建及应用 5

1.4.1 纳米探针的构建 5

1.4.2 纳米探针的应用 6

1.5酸性微环境响应型纳米探针研究意义 6

1.5.1本论文主要内容 6

1.5.2本论文的研究意义 7

第二章 实验方法 8

2.1 实验试剂 8

2.2 实验仪器 9

2.3 纳米探针的制备 9

2.3.1 合成PEI-mPEG 9

2.3.2 合成PEI-FI-mPEG-SVA-PEG-MAL 10

2.3.3 Au PENPs-FI的制备 10

2.4 纳米探针的表征 11

2.4.1 ¹HNMR分析 11

2.4.2 紫外测试 11

2.4.3 粒径测量 11

2.5 生物相容性实验 11

2.5.1 MTT法简介 11

2.5.2 MTT法具体操作 12

2.5.3 MTT法注意事项 12

第三章 结果与讨论 14

3.1 纳米探针的表征 14

3.1.1 APAS的表征 14

3.1.2 ¹HNMR谱图 15

3.1.3紫外可见吸收光谱图 17

3.1.4合成纳米探针的粒径分析 18

3.2 纳米探针生物相容性评价 20

第四章 结论与展望 21

4.1 结论 21

4.2 课题研究展望 21

参考文献 22

致谢 24

第一章 文献综述

近年来,癌症已成为全球公共健康的主要威胁之一，就在WHO近期公布的最新癌症数据显示，2018年全球有约960万人口死于癌症。针对癌症的治疗方式日益成熟，但癌症早期症状难以发现，等到确诊，一半以上的癌症患者已经发展到中晚期，给治疗带来极大困难。因此，癌症的早期发现和早期治疗就变得至关重要。

纳米技术的出现使得我们有更多的选择来构建肿瘤造影剂，开发多模态造影技术，提高早期肿瘤诊断的精确度、灵敏性。纳米粒子在体内可表现出循环时间长、靶向性、安全性高等优点，作为造影剂载体其拥有广阔的应用前景。通过对纳米粒进行修饰，改变其表面性质，从而装载金属离子、金属氧化物等造影元素，实现对肿瘤早期诊断。

请支付后下载全文，论文总字数：21022字