新型荧光碳点的研制及其在脑肿瘤光学影像中的应用

发布时间：2018-05-20 18:49

本文选题：荧光碳点 + 光学影像探针　；参考：《复旦大学》2014年博士论文

【摘要】：肿瘤是威胁人类生命和健康的重大疾病,尤其是脑肿瘤；导致脑肿瘤高死亡率的一个重要原因在于其诊断的滞后性。传统的医学影像虽然可以在解剖学基础上发现肿瘤,但肉眼可以辨别的肿瘤往往已经处于中、晚期,或者发生转移,失去了最佳的治疗时机。因此,肿瘤尤其是脑肿瘤的影像诊断具有极为重要的临床意义。目前,常规的医学影像在临床上发挥着重要作用,但仍存在诸多不足。其中磁共振影像(MRI)的空间分辨率高,但灵敏度偏低；单光子发射计算机断层成像(SPECT)和正电子发射计算机断层扫描(PET)的灵敏度高,但空间分辨率较低。光学影像是一种新兴的影像技术,其在灵敏度和分辨率方面显示出极大的优势和发展前景；尤其是基于纳米技术的影像探针的应用,使其在肿瘤光学影像,特别是早期肿瘤的影像诊断领域具有良好的发展前景和科学研究价值。近年,纳米技术广泛应用于生物医药领域。荧光碳点(CDs)是继量子点之后的又一种新型荧光纳米材料。其具备诸多优点：抗光漂白性强、无“光闪烁”现象,光稳定性高；生物相容性好,毒性低；表面易于功能化修饰等。目前,国际上CDs作为光学影像探针的研究尚处于初期阶段,主要在体外细胞的荧光影像方面有一些报道,而在体内肿瘤光学影像中的应用仍少有研究。关键问题主要是：①大多数CDs的发射光波长处于400nm-500 nm的较短波长可见光区,对机体组织的穿透性差,限制其体内深层组织的光学影像；同时还存在生物体自发荧光的干扰。因此,希望研制在红光区或近红外光区发光的CDs.②CDs在生物体内的分布没有选择性,无法浓集于病灶如肿瘤部位,致使肿瘤组织与正常组织边界的对比度弱,影像效果差。因此,希望设计具有肿瘤靶向性的CDs以提高其肿瘤信噪比。③CDs的粒径尺度和表面性质致使其体内生物半衰期较短,进一步削弱了影像效果。为了解决上述关键问题,本文拟针对脑肿瘤,首先通过选择不同的碳源材料,逐步优化CDs的光学性质,使之发射波长趋于红光区。其次在CDs表面进行功能化的PEG修饰,以延长生物半衰期。最后修饰两类靶向分子：①脑毛细血管内皮细胞和肿瘤细胞均高表达受体——低密度脂蛋白受体相关蛋白(LRP)的高亲和配体Angiopep-2(ANG),通过主动双级脑靶向作用首先透过血脑屏障,进而靶向进入脑肿瘤组织和肿瘤细胞；或者②肿瘤新生血管和肿瘤细胞均高表达受体——整合素αvβ3的高亲和配体c[RGDyK]环肽(RGD),通过主动双级肿瘤靶向作用首先透过肿瘤新生血管,进而靶向进入肿瘤细胞。从而增加CDs在肿瘤部位的富集和信噪比,提高脑肿瘤光学影像效果。本文的研究内容分为三章：第一章以蜘蛛丝为新型的有机碳源材料,采用水热法制备了粒径约5 nm的蜘蛛丝荧光碳点CDs-1,其最大发射峰波长为447 nm。考察了碳点浓度和孵育时间对其细胞摄取的影响,结果表明CDs-1能够被人源脑胶质瘤U87细胞摄取,且呈浓度依赖性；观察了在脑肿瘤细胞中的共定位情况,溶酶体和线粒体是其细胞内运行的可能途径。CDs-1具有良好的生物相容性和较低的细胞毒性,有利其作为体外肿瘤细胞光学影像探针。CDs-1的缺点是发射波长处于短波长可见光区,组织穿透性差、生物体本底荧光干扰大,尚不能用于体内肿瘤的光学影像,有待进一步提高和完善。第二章以甘氨酸为碳源材料,采用高温热解法制备了粒径约5 nm的甘氨酸荧光碳点CDs-2,其最大发射峰波长红移至500 nm。考察了碳点浓度和孵育时间对其细胞摄取的影响,结果表明鼠源脑胶质瘤C6细胞摄取CDs-2具有显著的时间和浓度依赖性；脑肿瘤细胞中的共定位结果表明,CDs-2细胞摄取后能够迅速进入溶酶体和线粒体,并且快速从细胞器中逸出。荷脑肿瘤裸鼠模型的体内肿瘤光学影像观察表明,CDs-2能够迅速浓集于脑部,并且主要聚集于脑肿瘤接种部位。此外还证实了其良好的生物相容性和细胞低毒性。综上所述,CDs-2能够用于肿瘤的体外细胞影像,也具有一定的体内肿瘤影像效果。但因其发射波长仍处于较短波长的可见光区,致使体内肿瘤影像信噪比不高,仍需进一步提高和完善。第三章通过氨基酸和糖种类的选择以及两者配比的优化,使荧光碳点的发射波长进一步红移。优化处方以谷氨酸和蔗糖按10：1复配为碳源材料,采用高温热解法制备了粒径约5 nm的谷氨酸-蔗糖复合荧光碳点CDs-3,其有效发射波长达600 nm。为了延长生物半衰期,制备了PEG-CDs-3;并进一步设计了两种双级脑肿瘤靶向的荧光碳点：ANG-PEG-CDs-3和RGD-PEG-CDs-3。考察碳点浓度和孵育时间对其细胞摄取的影响,结果显示修饰后的CDs-3被肿瘤细胞摄取具有显著的时间和浓度依赖性,且ANG或RGD修饰有利于其细胞摄取,提高了肿瘤细胞靶向性；其在肿瘤细胞中的共定位结果表明,修饰后的CDs-3通过溶酶体途径进入细胞。碳点的血浆蛋白稳定性实验结果表明PEG修饰可以明显降低血浆蛋白的吸附,有助于延长其体内长循环。血液相容性和细胞毒性评价结果表明,ANG-PEG-CDs-3和RGD-PEG-CDs-3具有良好的生物相容性和低细胞毒性,适合于体内影像应用。进一步评价了ANG-PEG-CDs-3、RGD-PEG-CDs-3和PEG-CDs-3在荷脑胶质瘤裸鼠模型和荷乳腺癌皮下瘤裸鼠模型中的组织分布及肿瘤主动靶向性,评估了各碳点的体内肿瘤光学影像效果。结果表明,ANG-PEG-CDs-3能够用于体内脑肿瘤的光学影像,效果优于RGD-PEG-CDs-3; RGD-PEG-CDs-3能够用于乳腺癌皮下瘤的光学影像,效果优于其脑肿瘤影像。组织器官和细胞水平评价结果进一步验证了碳点经ANG或RGD修饰有利于提高其肿瘤主动靶向性,增加碳点在肿瘤部位的浓集和信噪比,提高脑肿瘤和皮下瘤的光学影像效果。综上所述,本文创新性地通过复合碳源材料优选及多种策略的协同设计,研制了两种脑肿瘤靶向的荧光碳点ANG-PEG-CDs和RGD-PEG-CDs-3,增加其在肿瘤部位的富集和信噪比,提高脑肿瘤和皮下瘤的光学影像效果,有望使该领域的研究由体外拓展至体内；同时也为肿瘤的光学影像,尤其是早期影像诊断提供有价值的影像探针和研究资料。
[Abstract]:Cancer is a major disease that threatens human life and health, especially brain tumors. One of the important reasons for the high mortality of brain tumors is the hysteresis of the diagnosis. Although traditional medical images can find tumors on the basis of anatomy, the tumor that can be identified by the naked eye is already in the middle, late, or metastasize. Therefore, the imaging diagnosis of tumor, especially the brain tumor, has a very important clinical significance. At present, the conventional medical imaging plays an important role in clinical, but there are still many shortcomings. The spatial resolution of magnetic resonance imaging (MRI) is high, but the sensitivity is low; the single photon emission computer fault is formed. SPECT and positron emission computed tomography (PET) have high sensitivity, but low spatial resolution. Optical imaging is a new imaging technique, which shows great advantages and prospects in sensitivity and resolution, especially the application of nanotechnology based imaging probes to make it special in tumor optical images. In recent years, nanotechnology has been widely used in the field of biomedicine. CDs is another new kind of nanomaterial after quantum dots. It has many advantages: strong photobleaching, no "light scintillation", and high photostability. The biocompatibility is good, the toxicity is low, and the surface is easy to function modification. At present, the research of CDs as an optical image probe is still in the early stage. There are some reports on the fluorescence image of the cells in vitro, but the application of the optical imaging in the body is still less. The key issues are: (1) most of the CDs hair. The light wavelength in the shorter wavelength of the 400nm-500 nm is visible, the penetration of the body is poor and the optical images of the deep tissue in the body are limited. At the same time, there are also the interference of the spontaneous fluorescence of the organism. Therefore, the distribution of CDs. CDs in the red and near infrared light regions is not selective and cannot be concentrated. The contrast between the tumor tissue and the normal tissue boundary is weak and the image effect is poor. Therefore, it is hoped that the tumor targeting CDs can be designed to improve the tumor signal-to-noise ratio. (3) the size and surface properties of CDs make the biological half-life in the body shorter, and further weaken the image effect. The key problem is to optimize the optical properties of CDs by selecting different carbon source materials to make the emission wavelengths tend to be red area. Secondly, the functional PEG modification on the surface of CDs is used to prolong the biological half-life. Finally, two kinds of target molecules are modified: the high surface of the brain capillary endothelial cells and tumor cells The high affinity ligand Angiopep-2 (ANG) of the receptor, low density lipoprotein receptor related protein (LRP), first penetrates the blood brain barrier through the active two-stage brain targeting, and then targets the brain tumor tissue and tumor cells, or the high affinity ligand C of the neovascularization and tumor cells of the tumor, the high affinity ligand C of integrin alpha v beta 3 [RGDyK] cyclic peptide (RGD), through the targeted action of the active two-stage tumor, first penetrates the neovascularization of the tumor and then targets the tumor cells, thus increasing the enrichment and signal to noise ratio of CDs at the tumor site, and improving the optical image effect of the brain tumor. The research contents of this paper are divided into three chapters: the first chapter is a new type of organic carbon source material with spider silk, which is used as a new type of organic carbon source. The fluorescence carbon point CDs-1 of the spider silk with a particle size of about 5 nm was prepared by hydrothermal method. The maximum emission wavelength was 447 nm., and the effects of carbon point concentration and incubation time on the cell uptake were investigated. The results showed that CDs-1 was able to be absorbed by human glioma U87 cells and was concentration dependent. The co localization of the brain tumor cells and the lysase were observed. Body and mitochondria are the possible pathways of its intracellular operation..CDs-1 has good biocompatibility and low cytotoxicity. The disadvantage of.CDs-1 as an optical image probe for tumor cells in vitro is that the emission wavelength is in the short wavelength visible light area, the tissue penetration is poor, the biological background fluorescence interference is large, and it is not yet used in the body tumor. The second chapter uses glycine as carbon source material in the second chapter to prepare the glycine fluorescent carbon point CDs-2 with a particle size of about 5 nm by high temperature pyrolysis. The maximum emission peak wavelength is red to 500 nm., and the effect of carbon point concentration and incubation time on the cell uptake is investigated. The results show that rat brain glioma C6 cells The uptake of CDs-2 has a significant time and concentration dependence; the co localization results in brain tumor cells indicate that CDs-2 cells can quickly enter lysosomes and mitochondria and escape from the organelles quickly. The tumor optical image of the nude mice model of the brain tumor bearing tumor of the brain of the nude mice shows that CDs-2 can quickly concentrate in the brain. In addition to the good biocompatibility and low cytotoxicity, CDs-2 can be applied to the cell image of the tumor in vitro and the tumor imaging effect in vivo. However, the signal to noise ratio of the tumor image in the body is not high because its emission wavelength is still in the short wavelength visible light region. The third chapter, through the selection of amino acids and sugars and the optimization of their ratio, further redshift the emission wavelengths of the fluorescent carbon points. The optimized formulation was made of glutamic acid and sucrose as carbon source materials according to 10:1, and a high temperature pyrolysis method was used to prepare the glutamate sucrose compound fluorescent carbon point CDs-3 with a particle size of about 5 nm. The effective emission wavelength was 600 nm. in order to prolong the biological half-life, and PEG-CDs-3 was prepared, and the target fluorescence carbon points of two kinds of two-stage brain tumors were further designed. ANG-PEG-CDs-3 and RGD-PEG-CDs-3. were used to investigate the effects of carbon point concentration and incubation time on their cell uptake. The results showed that the modified CDs-3 was significantly absorbed by the tumor cells. The time and concentration dependence, and the ANG or RGD modification was beneficial to the cell uptake and enhanced the targeting of the tumor cells. The co localization results in the tumor cells showed that the modified CDs-3 entered the cells through the lysosome pathway. The experimental results of plasma protein stability in the carbon point showed that the PEG modification could significantly reduce the adsorption of plasma protein. The results of blood compatibility and cytotoxicity show that ANG-PEG-CDs-3 and RGD-PEG-CDs-3 have good biocompatibility and low cytotoxicity and are suitable for the application of imaging in vivo. Further evaluation of ANG-PEG-CDs-3, RGD-PEG-CDs-3 and PEG-CDs-3 in the nude mice model of glioma and the subcutaneous of breast cancer The tissue distribution and active targeting of tumor in nude mice model were used to evaluate the optical image effect of the tumor in vivo. The results showed that ANG-PEG-CDs-3 could be used in the optical image of brain tumor in vivo, and the effect was better than that of RGD-PEG-CDs-3; RGD-PEG-CDs-3 could be used in the optical image of the subcutaneous tumor of breast cancer, and the effect was better than that of the brain tumor image. The evaluation results of the weave and cell level further verify that the ANG or RGD modification can improve the active targeting of the tumor, increase the concentration and signal-to-noise ratio of the carbon point at the tumor site, and improve the optical image effect of the brain tumor and subcutaneous tumor. The fluorescent carbon points ANG-PEG-CDs and RGD-PEG-CDs-3 targeted by two kinds of brain tumors are designed and developed. The enrichment and signal to noise ratio of the tumor sites are increased and the optical image effects of brain tumors and subcutaneous tumors are improved. It is expected that the research in this field will be extended from in vitro to the body, and it is also provided for the optical image of the swelling tumor, especially the early imaging diagnosis. Valuable imaging probes and research materials.
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R943

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