磁性碳量子点探针的构建及其在肿瘤诊疗中的应用研究
发布时间：2019-06-17 20:19
【摘要】：癌症的早期诊断已引起人们的高度重视,分子影像学检测作为癌症早期诊断的重要工具,为肿瘤诊断提供更准确和完整的信息,以达到早期诊断、早期治疗的目的。基于多功能纳米粒子的分子成像技术已在肿瘤检测的灵敏度和特异性方面取得了一定进展,量子点、荧光染料分子和磁性纳米粒子等已经作为成像探针运用到了活体荧光成像和磁共振成像中。然而,这些探针有毒而且水溶液稳定性差,灵敏度欠佳,极大的限制了其在体内的应用。因此,构建一种体系简单、生物友好、成像灵敏的多功能靶向探针是分子成像技术亟待解决的问题。基于此,本课题通过微波辅助法将氯化钆(GdCl3)以钆离子Gd3+的形式掺杂到碳点CDs的里面,同时PEI对CDs表面进行氨基化修饰,我们得到了高量子产率的掺钆碳量子点Gd-CDs,即能够在可见光照射下发出明亮的绿光,它具有荧光强度高、光学稳定性和生物相容性好。以去铁蛋白纳米笼为载体,去铁蛋白笼AFn独特的笼状结构特性即在pH=2蛋白笼支解成多个亚甲基结构使得笼打开,在pH=7蛋白笼重新自组装从而实现封装药物阿霉素。Gd-CDs作为一个荧光兼磁性的探针偶联载药体系AFn包裹DOX。在EDC和NHS催化剂的协同下将靶头叶酸FA连接到AFn上赋予其肿瘤靶向能力。Gd-CDs/AFn(DOX)/FA在叶酸介导下特异性靶向肿瘤细胞以及肿瘤病变组织。实现体内和体外的双模式(荧光/核磁共振)成像及细胞追踪,用于肿瘤的早期诊断及治疗。采用X射线光电子能谱技术(XPS)对制备的Gd-CDs进行元素分析,Gd元素轨道的变化表明Gd离子被掺进去CDs中,通过透射电镜观察Gd-CDs的粒径约6 nm,多分散系数PDI为0.201,Zeta电位测其电位约9.05 mV。Gd-CDs的正电性表明其表面修饰上氨基。通过扫描Gd-CDs光谱图,制备的Gd-CDs具有明显的上、下转换发光性质。通过对制备的Gd-CDs/AFn(DOX)/FA进行物理化学性质等表征,其平均粒径为112±3.5 nm,电位约-23.7±2.1 mV。采用紫外可见分光光度计测定载药系统中DOX的包封率,结果表明其包封率达86.6%。采用透析法分别测定了制剂在pH 5.0和pH 7.4时DOX的体外释放,分析结果显示通过调节pH值能较好的控制药物释放。在偏酸性环境pH 5.0时累计释药率为79.2%。Gd-CDs/AFn(DOX)/FA因含有钆离子具有T1成像能力,MRI体外实验表明Gd-CDs/AFn(DOX)/FA在T1磁场下,具有明显的浓度依赖性,而且随着浓度的增大呈现白化效应。本研究选择人源的乳腺癌细胞(MCF-7)作为其体外抗肿瘤活性评价的模型,考察了Gd-CDs/AFn(DOX)/FA细胞毒作用机制,包括细胞内摄取实验、抑制率等。采用MTT法分别考察了Gd-CDs、Gd-CDs/AFn的细胞毒性,结果显示,设定浓度为0~200μg/mL,Gd-CDs和Gd-CDs/AFn两者均表现出低毒性,细胞存活率均为86%以上。且Gd-CDs键合到AFn表面后,相比Gd-CDs组Gd-CDs/AFn组的细胞存活率明显增强。MCF-7细胞摄取实验中通过观察Gd-CDs自身的绿色荧光,Gd-CDs能够较好的标记载药系统的细胞摄取情况,与Gd-CDs/AFn(DOX)相比,Gd-CDs/AFn(DOX)/FA有较高的细胞摄取量,表明连有叶酸靶头的去铁蛋白给药系统更有利于包裹药物快速进入肿瘤细胞。体外抗肿瘤活性实验表明:Gd-CDs/AFn(DOX)/FA可以有效的靶向MCF-7细胞,并高效的穿透细胞膜,携带更多的DOX进入细胞内部。去铁蛋白笼在弱酸环境下笼渐渐离解打开,从而缓慢释放出药物DOX,从而诱导MCF-7细胞凋亡,最终抑制MCF-7细胞的生长和增殖。采用鼠源腹水瘤S180细胞株,建立KM雌性小白鼠荷瘤作为其体内抗肿瘤活性评价的模型。体内的抗肿瘤活性实验表明:Gd-CDs/AFn(DOX)/FA具有较低的系统毒性。在叶酸介导下,能将更多的Gd-CDs/AFn(DOX)/FA纳米粒子滞留在肿瘤部位,在肿瘤组织偏酸性环境下,去铁蛋白笼逐渐打开使药物DOX缓慢从Gd-CDs/AFn(DOX)/FA释放出来,从而大量的药物更易于滞留在肿瘤部位。通过DOX嵌入DNA抑制核酸的合成从而诱导肿瘤细胞的凋亡,最终抑制肿瘤的生长。DOX在小鼠血浆的药代动力学实验结果表明,纳米复合物Gd-CDs/AFn(DOX)/FA能够延长DOX在体内的循环停留时间。具有叶酸靶向的Gd-CDs/AFn(DOX)/FA使药物更多的聚集在肿瘤部位,进一步证明构建的纳米给药系统在体内的靶向能力。此外,MRI体内实验显示注射Gd-CDs/AFn(DOX)/FA后,观察到核磁成像中小鼠肿瘤部位T1场信号强度得到增强,说明Gd-CDs/AFn(DOX)/FA可作为肿瘤核磁成像诊断的对比剂。
[Abstract]:Early diagnosis of cancer has attracted the attention of people, the molecular imaging detection is an important tool for early diagnosis of cancer, and provides more accurate and complete information for tumor diagnosis, so as to achieve the purpose of early diagnosis and early treatment. Molecular imaging techniques based on multi-function nanoparticles have made some progress in the sensitivity and specificity of tumor detection, and quantum dots, fluorescent dye molecules and magnetic nanoparticles have been used as imaging probes in vivo fluorescence imaging and magnetic resonance imaging. However, these probes are toxic and the aqueous solution has poor stability and poor sensitivity, which greatly limits its application in vivo. Therefore, to construct a multi-function targeting probe with simple system, bio-friendly and sensitive imaging is a problem to be solved in molecular imaging. On the basis of this, by microwave-assisted method, the chlorinated carbon (GdCl3) is doped into the inner surface of the carbon point CDs in the form of a fluorescent ion Gd3 +, and the PEI is subjected to the amination modification on the surface of the CDs, so that the doped carbon quantum dot Gd-CDs with high quantum yield is obtained, That is, the bright green light can be emitted under the irradiation of visible light, and has the advantages of high fluorescence intensity, good optical stability and good biocompatibility. And taking the ferritin nano-cage as a carrier, the unique cage-like structure characteristic of the ferritin cage AFn is that the cage is opened when the pH = 2 protein cage is resolved into a plurality of methylene structures, and the packaging drug adriamycin is realized by the self-assembly of the pH = 7 protein cage. Gd-CDs is used as a fluorescent and magnetic probe to couple the drug-carrying system AFn to the DOX. The target-head folic acid FA is connected to the AFn for the tumor targeting ability under the coordination of the EDC and the NHS catalyst. Gd-CDs/ AFn (DOX)/ FA specifically targeted tumor cells and tumor lesion tissues with folate-mediated. The dual-mode (fluorescence/ nuclear magnetic resonance) imaging and cell tracking in vivo and in vitro are realized for early diagnosis and treatment of tumors. The elements of Gd-CDs were analyzed by X-ray photoelectron spectroscopy (XPS). The change of Gd-element orbit showed that the Gd-ions were doped in the CDs. The particle size of Gd-CDs was observed by transmission electron microscope. The particle size of Gd-CDs was about 6 nm, and the multi-dispersion coefficient PDI was 0.201. The potential of Zeta potential is about 9.05 mV. The positive electrical property of Gd-CDs indicates that its surface is modified with amino group. The Gd-CDs prepared by scanning the Gd-CDs have obvious superior and lower conversion luminescence properties. The average particle size of the prepared Gd-CDs/ AFn (DOX)/ FA was 112-3.5 nm and the potential was about-23.7-2.1mV. The encapsulation rate of DOX in the drug-loaded system was determined by an ultraviolet-visible spectrophotometer. The results showed that the encapsulation rate was 86.6%. The in vitro release of DOX was determined by dialysis method at pH 5.0 and pH 7.4, and the results showed that the release of the drug was controlled by adjusting the pH value. The results showed that Gd-CDs/ AFn (DOX)/ FA had a significant concentration dependence under the T1 magnetic field and the whitening effect with the increase of the concentration. In this study, human-derived breast cancer cells (MCF-7) were selected as the model of their in vitro anti-tumor activity evaluation, and the mechanism of Gd-CDs/ AFn (DOX)/ FA (DOX)/ FA was investigated. The cytotoxicity of Gd-CDs and Gd-CDs/ AFn was studied by MTT method. The results showed that the concentration of Gd-CDs and Gd-CDs and Gd-CDs/ AFn showed low toxicity and the cell survival rate was over 86%. And the cell survival rate of the Gd-CDs/ AFn group in the Gd-CDs group was obviously enhanced after the Gd-CDs were bonded to the surface of the AFn. Compared with Gd-CDs/ AFn (DOX), the cell uptake of Gd-CDs/ AFn (DOX)/ FA was higher than that of Gd-CDs/ AFn (DOX). It is shown that the drug delivery system with the folic acid target head is more beneficial to the rapid entry of the packaged drugs into the tumor cells. In vitro anti-tumor activity experiments show that Gd-CDs/ AFn (DOX)/ FA can effectively target MCF-7 cells and penetrate cell membranes efficiently and carry more DOX into the cells. The cage gradually dissociates and opens in a weak acid environment, so that the drug DOX is slowly released, thereby inducing the apoptosis of the MCF-7 cell, and finally inhibiting the growth and the proliferation of the MCF-7 cell. The model of tumor-bearing activity of KM female mice was established by using mouse-derived ascites S180 cell line. The anti-tumor activity in vivo showed that Gd-CDs/ AFn (DOX)/ FA had lower systemic toxicity. In the presence of folic acid, more of the Gd-CDs/ AFn (DOX)/ FA nanoparticles can be retained in the tumor site. In the acidic environment of the tumor tissue, the de-ferritin cage is gradually opened to release the drug DOX slowly from the Gd-CDs/ AFn (DOX)/ FA, so that a large amount of the drug can be more easily retained in the tumor site. The DNA is embedded into the DNA to inhibit the synthesis of the nucleic acid, thereby inducing the apoptosis of the tumor cells, and finally inhibiting the growth of the tumor. The results of the pharmacokinetics of DOX in mouse plasma show that the nano-complex Gd-CDs/ AFn (DOX)/ FA can prolong the cyclic residence time of DOX in the body. The addition of folic acid-targeted Gd-CDs/ AFn (DOX)/ FA to the tumor site further demonstrates the targeted ability of the constructed nano-drug delivery system in vivo. In addition, after the injection of Gd-CDs/ AFn (DOX)/ FA in the MRI, the signal intensity of the T1 field in the tumor site of the mouse was enhanced and the Gd-CDs/ AFn (DOX)/ FA could be used as the contrast agent for the diagnosis of tumor nuclear magnetic resonance imaging.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：O657.3;R730.4
[Abstract]:Early diagnosis of cancer has attracted the attention of people, the molecular imaging detection is an important tool for early diagnosis of cancer, and provides more accurate and complete information for tumor diagnosis, so as to achieve the purpose of early diagnosis and early treatment. Molecular imaging techniques based on multi-function nanoparticles have made some progress in the sensitivity and specificity of tumor detection, and quantum dots, fluorescent dye molecules and magnetic nanoparticles have been used as imaging probes in vivo fluorescence imaging and magnetic resonance imaging. However, these probes are toxic and the aqueous solution has poor stability and poor sensitivity, which greatly limits its application in vivo. Therefore, to construct a multi-function targeting probe with simple system, bio-friendly and sensitive imaging is a problem to be solved in molecular imaging. On the basis of this, by microwave-assisted method, the chlorinated carbon (GdCl3) is doped into the inner surface of the carbon point CDs in the form of a fluorescent ion Gd3 +, and the PEI is subjected to the amination modification on the surface of the CDs, so that the doped carbon quantum dot Gd-CDs with high quantum yield is obtained, That is, the bright green light can be emitted under the irradiation of visible light, and has the advantages of high fluorescence intensity, good optical stability and good biocompatibility. And taking the ferritin nano-cage as a carrier, the unique cage-like structure characteristic of the ferritin cage AFn is that the cage is opened when the pH = 2 protein cage is resolved into a plurality of methylene structures, and the packaging drug adriamycin is realized by the self-assembly of the pH = 7 protein cage. Gd-CDs is used as a fluorescent and magnetic probe to couple the drug-carrying system AFn to the DOX. The target-head folic acid FA is connected to the AFn for the tumor targeting ability under the coordination of the EDC and the NHS catalyst. Gd-CDs/ AFn (DOX)/ FA specifically targeted tumor cells and tumor lesion tissues with folate-mediated. The dual-mode (fluorescence/ nuclear magnetic resonance) imaging and cell tracking in vivo and in vitro are realized for early diagnosis and treatment of tumors. The elements of Gd-CDs were analyzed by X-ray photoelectron spectroscopy (XPS). The change of Gd-element orbit showed that the Gd-ions were doped in the CDs. The particle size of Gd-CDs was observed by transmission electron microscope. The particle size of Gd-CDs was about 6 nm, and the multi-dispersion coefficient PDI was 0.201. The potential of Zeta potential is about 9.05 mV. The positive electrical property of Gd-CDs indicates that its surface is modified with amino group. The Gd-CDs prepared by scanning the Gd-CDs have obvious superior and lower conversion luminescence properties. The average particle size of the prepared Gd-CDs/ AFn (DOX)/ FA was 112-3.5 nm and the potential was about-23.7-2.1mV. The encapsulation rate of DOX in the drug-loaded system was determined by an ultraviolet-visible spectrophotometer. The results showed that the encapsulation rate was 86.6%. The in vitro release of DOX was determined by dialysis method at pH 5.0 and pH 7.4, and the results showed that the release of the drug was controlled by adjusting the pH value. The results showed that Gd-CDs/ AFn (DOX)/ FA had a significant concentration dependence under the T1 magnetic field and the whitening effect with the increase of the concentration. In this study, human-derived breast cancer cells (MCF-7) were selected as the model of their in vitro anti-tumor activity evaluation, and the mechanism of Gd-CDs/ AFn (DOX)/ FA (DOX)/ FA was investigated. The cytotoxicity of Gd-CDs and Gd-CDs/ AFn was studied by MTT method. The results showed that the concentration of Gd-CDs and Gd-CDs and Gd-CDs/ AFn showed low toxicity and the cell survival rate was over 86%. And the cell survival rate of the Gd-CDs/ AFn group in the Gd-CDs group was obviously enhanced after the Gd-CDs were bonded to the surface of the AFn. Compared with Gd-CDs/ AFn (DOX), the cell uptake of Gd-CDs/ AFn (DOX)/ FA was higher than that of Gd-CDs/ AFn (DOX). It is shown that the drug delivery system with the folic acid target head is more beneficial to the rapid entry of the packaged drugs into the tumor cells. In vitro anti-tumor activity experiments show that Gd-CDs/ AFn (DOX)/ FA can effectively target MCF-7 cells and penetrate cell membranes efficiently and carry more DOX into the cells. The cage gradually dissociates and opens in a weak acid environment, so that the drug DOX is slowly released, thereby inducing the apoptosis of the MCF-7 cell, and finally inhibiting the growth and the proliferation of the MCF-7 cell. The model of tumor-bearing activity of KM female mice was established by using mouse-derived ascites S180 cell line. The anti-tumor activity in vivo showed that Gd-CDs/ AFn (DOX)/ FA had lower systemic toxicity. In the presence of folic acid, more of the Gd-CDs/ AFn (DOX)/ FA nanoparticles can be retained in the tumor site. In the acidic environment of the tumor tissue, the de-ferritin cage is gradually opened to release the drug DOX slowly from the Gd-CDs/ AFn (DOX)/ FA, so that a large amount of the drug can be more easily retained in the tumor site. The DNA is embedded into the DNA to inhibit the synthesis of the nucleic acid, thereby inducing the apoptosis of the tumor cells, and finally inhibiting the growth of the tumor. The results of the pharmacokinetics of DOX in mouse plasma show that the nano-complex Gd-CDs/ AFn (DOX)/ FA can prolong the cyclic residence time of DOX in the body. The addition of folic acid-targeted Gd-CDs/ AFn (DOX)/ FA to the tumor site further demonstrates the targeted ability of the constructed nano-drug delivery system in vivo. In addition, after the injection of Gd-CDs/ AFn (DOX)/ FA in the MRI, the signal intensity of the T1 field in the tumor site of the mouse was enhanced and the Gd-CDs/ AFn (DOX)/ FA could be used as the contrast agent for the diagnosis of tumor nuclear magnetic resonance imaging.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：O657.3;R730.4
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