功能化纳米石墨烯在生物医学领域的应用
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摘要
在最近几年，功能化纳米石墨烯由于具有独特物理化学性质在包括生物医学在内的很多领域都引起了广泛的关注。在本博士论文中，我们系统地研究了功能化纳米石墨烯在生物医学尤其是肿瘤诊疗方面的潜在应用，并对这类材料的生物学效应和毒性学行为进行了探讨。我们通过聚乙二醇修饰得到具有优良的水溶性和生物相容性的纳米石墨烯用于肿瘤成像及光热治疗；我们进一步研究不同表面修饰和不同尺寸的纳米石墨烯在生物体内的行为，并利用优化的纳米石墨烯实现了动物体内的超低光功率肿瘤光热治疗；同时我们还以纳米石墨烯为基底，构建基于纳米石墨烯的功能复合物用于肿瘤多模式成像及成像指导下的肿瘤光热治疗；考虑到纳米材料的生物学效应和毒理学是重要的基础科学问题，我们还对纳米石墨烯在生物体内的分布、代谢、以及潜在毒性进行了系统的研究。主要的研究结果概括如下：
第一章:简要概述了功能化纳米石墨烯的制备、在生物医学方面的应用以及潜在毒性问题，并着重阐述了本论文的选题依据和研究重点。
第二章:我们使用聚乙二醇（PEG）修饰氧化石墨烯(GO)，从而制备具有良好生物相容性的nGO-PEG，然后使用荧光标记nGO-PEG进行活体肿瘤模型的荧光成像。小动物活体成像显示nGO-PEG在多种肿瘤模型中都有很高的富集，且具有较低的网状内皮系统吞噬。我们然后利用nGO-PEG在近红外的强吸收进行活体肿瘤的光热治疗，达到100%的肿瘤杀灭能力。
第三章:我们使用水合肼分别还原GO和nGO-PEG得到不同尺寸的还原石墨烯(RGO)和超小尺寸的还原石墨烯（nRGO），然后使用支链状PEG(C18PMH-PEG)非共价修饰RGO和nRGO，从而制备出具有生物相容性的RGO-PEG和nRGO-PEG。我们然后研究了不同尺寸和不同表面修饰的纳米石墨烯在生物体内的行为，并实现了超低功率的肿瘤光热治疗。
第四章:我们使用高温水热法制备还原石墨烯-四氧化三铁功能复合物（RGO-IONP），然后使用C18PMH-PEG非共价修饰得到RGO-IONP-PEG功能复合物。利用其固有的近红外强吸收、强磁性以及额外的荧光标记，我们实现了基于RGO-IONP-PEG功能复合物的肿瘤多模态成像（光声、磁共振和荧光成像），并首次实现了基于RGO-IONP-PEG功能复合物成像指导下的肿瘤光热治疗。
第五章:我们首次采用核素125I标记的方法来研究纳米石墨烯(nGO-PEG)在生物体内的长期分布以及潜在的长期毒性。通过尾静脉将125I标记的nGO-PEG注射入小鼠体内，我们发现nGO-PEG主要聚集在网状内皮系统包括肝和脾，但是可以通过尿液和粪便排出体外。我们还对实验小鼠的组织学和血液学进行分析，发现在我们使用的剂量（20mg/kg）下并没有对实验小鼠造成明显的毒性。
第六章:我们系统研究了GO及其衍生物分别经口服和腹腔给药后在体内的行为以及潜在的毒性。我们发现通过口服给药的nGO-PEG很少被器官，而经腹腔给药，除了GO，功能化的纳米石墨烯包括nGO-PEG，RGO-PEG和nRGO-PEG主要被肝脾吸收。虽然GO及其衍生物在小鼠体内停留很长时间，但是我们通过对组织学和血液学分析并没有实验小鼠造成明显的毒性。
总而言之，本论文对功能化纳米石墨烯以及功能复合物在生物医学上的应用，尤其在生物成像和肿瘤光热治疗等方面展开了较为系统的研究，并对纳米石墨烯的生物学效应和潜在毒性进行了较为深入的评价。我们的研究成果有力地推进基于纳米石墨烯这一新型二维纳米材料在生物医学上的应用，也为发展探索基于纳米材料的新型肿瘤治疗方法提出了新的思路。
In recent years, owing to their unique physical and chemical properties,nano-graphene has attracted tremendous interest in many different fields includingbiomedicine. In this dissertation, we have systematically studied the biomedicalapplications of functionalized nano-graphene, particularly for cancer diagnosis andtreatment, and explored the biological effect and potential toxicity of nano-graphene inanimals. PEGylated nano-graphene with excellent water-soluble and biocompatibility issynthesized and used for tumor imaging and photothermal therapy. How sizes andsurface chemistry affect the in vivo behaviors and photothermal therapeutic efficacy ofnano-graphene is then carefully investigated, realizing highly efficient in vivo tumorablation under an ultra-low laser power density using our optimized nano-grapheneformulation. Graphene-based magnetic nanocomposite is further developed and appliedfor tumor multimodal imaging and imaging guided cancer therapy. Moreover, the invivo biodistribution, excretion and the potential toxicity of nano-graphene with differentsizes and surface coatings are systematically investigated, suggesting thatnano-graphene with small sizes and well-designed surface coatings is not noticeablytoxic in vivo to animals. The main results of this dissertation are summarized asfollowing:
Chapter1: This chapter is an introductive overview that summarizes thefunctionalization, biomedical applications, and toxicology of nano-graphene.
Chapter2: We studied the in vivo behaviors of polyethylene glycol (PEG)functionalized nano-graphene oxide (nGO-PEG) by a near-infrared (NIR) fluorescentdye. In vivo fluorescence imaging revealed high tumor uptake of nGO-PEG in severalxenograft tumor mouse models and relatively low retention in reticuloendothelialsystems (RES). We then utilized the strong optical absorbance of nGO-PEG in the NIRregion for in vivo photothermal therapy, achieving100%tumor ablation in a mousetumor model.
Chapter3: We synthesized a number of graphene oxide (GO) derivatives with different sizes and surface coatings, and then studied how sizes and surface chemistrywould affect the in vivo behaviors of nano-graphene. It was found that ultra-small nanoreduced GO (nRGO) with covalent PEG coating showed greatly enhanced NIRabsorbance, prolonged blood circulation half-life, as well as increased tumor uptake.Using this optimized nGO-PEG, we realized highly efficient in vivo tumor ablation byusing an ultra-low laser power density in our mouse experiments.
Chapter4: We prepared reduced graphene oxide (RGO)-iron oxide (IONP)nanocomposite, which was noncovalently functionalized with PEG to render highstability in physiological solutions. Utilizing the intrinsic high NIR optical absorbanceand strong magnetic property of the obtained RGO–IONP–PEG, as well as externallabels, we realized in vivo multimodal photoacoustic tomography (PAT), magneticresonance (MR) and fluorescence imaging, based on which photothermal therapy wasdesigned and carried out. This work demonstrated the great promise of usinggraphene-based multifunctional nano-composites for cancer theranostic applications.
Chapter5: We for the first time studied the long-term biodistribution and potentialtoxicity of PEGylated nano-GO (nGO-PEG) labeled with125I. It was found thatnGO-PEG mainly accumulated in the reticuloendothelial system (RES) including liverand spleen after intravenous administration and could be gradually cleared out, likely byboth renal and fecal excretion. Moreover, nGO-PEG did not cause appreciable toxicityat our tested dose (20mg/kg) to the treated mice by hematology analysis andhistological examinations.
Chapter6: We systematically investigated the in vivo biodistribution and potentialtoxicity of nano-graphene and its derivatives via oral and intraperitoneal (i.p.)administration. We found that nGO-PEG labeled with125I showed no obvious tissueuptake via oral administration. In contrast, high accumulation of nGO-PEG，RGO-PEGand nRGO-PEG, but not as-made GO, in the reticuloendothelial (RES) system includingliver and spleen was observed after i.p. injection. Although GO and PEGylated GOderivatives would retain in the mouse body over a long period of time after i.p. injection,their toxicity to the treated animals was insignificant.
In summary, this dissertation, we have systematically studied the biomedicalapplications of nano-graphene, particularly for imaging and photothermal therapy ofcancer, and carefully investigated the toxicology of functionalized nano-graphene in vivo to animals. Our results greatly promote graphene-based biomedical research, andprovide helpful guidelines for the future explorations of other functional nanomaterialsin cancer theranostics.
第一章:简要概述了功能化纳米石墨烯的制备、在生物医学方面的应用以及潜在毒性问题，并着重阐述了本论文的选题依据和研究重点。
第二章:我们使用聚乙二醇（PEG）修饰氧化石墨烯(GO)，从而制备具有良好生物相容性的nGO-PEG，然后使用荧光标记nGO-PEG进行活体肿瘤模型的荧光成像。小动物活体成像显示nGO-PEG在多种肿瘤模型中都有很高的富集，且具有较低的网状内皮系统吞噬。我们然后利用nGO-PEG在近红外的强吸收进行活体肿瘤的光热治疗，达到100%的肿瘤杀灭能力。
第三章:我们使用水合肼分别还原GO和nGO-PEG得到不同尺寸的还原石墨烯(RGO)和超小尺寸的还原石墨烯（nRGO），然后使用支链状PEG(C18PMH-PEG)非共价修饰RGO和nRGO，从而制备出具有生物相容性的RGO-PEG和nRGO-PEG。我们然后研究了不同尺寸和不同表面修饰的纳米石墨烯在生物体内的行为，并实现了超低功率的肿瘤光热治疗。
第四章:我们使用高温水热法制备还原石墨烯-四氧化三铁功能复合物（RGO-IONP），然后使用C18PMH-PEG非共价修饰得到RGO-IONP-PEG功能复合物。利用其固有的近红外强吸收、强磁性以及额外的荧光标记，我们实现了基于RGO-IONP-PEG功能复合物的肿瘤多模态成像（光声、磁共振和荧光成像），并首次实现了基于RGO-IONP-PEG功能复合物成像指导下的肿瘤光热治疗。
第五章:我们首次采用核素125I标记的方法来研究纳米石墨烯(nGO-PEG)在生物体内的长期分布以及潜在的长期毒性。通过尾静脉将125I标记的nGO-PEG注射入小鼠体内，我们发现nGO-PEG主要聚集在网状内皮系统包括肝和脾，但是可以通过尿液和粪便排出体外。我们还对实验小鼠的组织学和血液学进行分析，发现在我们使用的剂量（20mg/kg）下并没有对实验小鼠造成明显的毒性。
第六章:我们系统研究了GO及其衍生物分别经口服和腹腔给药后在体内的行为以及潜在的毒性。我们发现通过口服给药的nGO-PEG很少被器官，而经腹腔给药，除了GO，功能化的纳米石墨烯包括nGO-PEG，RGO-PEG和nRGO-PEG主要被肝脾吸收。虽然GO及其衍生物在小鼠体内停留很长时间，但是我们通过对组织学和血液学分析并没有实验小鼠造成明显的毒性。
总而言之，本论文对功能化纳米石墨烯以及功能复合物在生物医学上的应用，尤其在生物成像和肿瘤光热治疗等方面展开了较为系统的研究，并对纳米石墨烯的生物学效应和潜在毒性进行了较为深入的评价。我们的研究成果有力地推进基于纳米石墨烯这一新型二维纳米材料在生物医学上的应用，也为发展探索基于纳米材料的新型肿瘤治疗方法提出了新的思路。
In recent years, owing to their unique physical and chemical properties,nano-graphene has attracted tremendous interest in many different fields includingbiomedicine. In this dissertation, we have systematically studied the biomedicalapplications of functionalized nano-graphene, particularly for cancer diagnosis andtreatment, and explored the biological effect and potential toxicity of nano-graphene inanimals. PEGylated nano-graphene with excellent water-soluble and biocompatibility issynthesized and used for tumor imaging and photothermal therapy. How sizes andsurface chemistry affect the in vivo behaviors and photothermal therapeutic efficacy ofnano-graphene is then carefully investigated, realizing highly efficient in vivo tumorablation under an ultra-low laser power density using our optimized nano-grapheneformulation. Graphene-based magnetic nanocomposite is further developed and appliedfor tumor multimodal imaging and imaging guided cancer therapy. Moreover, the invivo biodistribution, excretion and the potential toxicity of nano-graphene with differentsizes and surface coatings are systematically investigated, suggesting thatnano-graphene with small sizes and well-designed surface coatings is not noticeablytoxic in vivo to animals. The main results of this dissertation are summarized asfollowing:
Chapter1: This chapter is an introductive overview that summarizes thefunctionalization, biomedical applications, and toxicology of nano-graphene.
Chapter2: We studied the in vivo behaviors of polyethylene glycol (PEG)functionalized nano-graphene oxide (nGO-PEG) by a near-infrared (NIR) fluorescentdye. In vivo fluorescence imaging revealed high tumor uptake of nGO-PEG in severalxenograft tumor mouse models and relatively low retention in reticuloendothelialsystems (RES). We then utilized the strong optical absorbance of nGO-PEG in the NIRregion for in vivo photothermal therapy, achieving100%tumor ablation in a mousetumor model.
Chapter3: We synthesized a number of graphene oxide (GO) derivatives with different sizes and surface coatings, and then studied how sizes and surface chemistrywould affect the in vivo behaviors of nano-graphene. It was found that ultra-small nanoreduced GO (nRGO) with covalent PEG coating showed greatly enhanced NIRabsorbance, prolonged blood circulation half-life, as well as increased tumor uptake.Using this optimized nGO-PEG, we realized highly efficient in vivo tumor ablation byusing an ultra-low laser power density in our mouse experiments.
Chapter4: We prepared reduced graphene oxide (RGO)-iron oxide (IONP)nanocomposite, which was noncovalently functionalized with PEG to render highstability in physiological solutions. Utilizing the intrinsic high NIR optical absorbanceand strong magnetic property of the obtained RGO–IONP–PEG, as well as externallabels, we realized in vivo multimodal photoacoustic tomography (PAT), magneticresonance (MR) and fluorescence imaging, based on which photothermal therapy wasdesigned and carried out. This work demonstrated the great promise of usinggraphene-based multifunctional nano-composites for cancer theranostic applications.
Chapter5: We for the first time studied the long-term biodistribution and potentialtoxicity of PEGylated nano-GO (nGO-PEG) labeled with125I. It was found thatnGO-PEG mainly accumulated in the reticuloendothelial system (RES) including liverand spleen after intravenous administration and could be gradually cleared out, likely byboth renal and fecal excretion. Moreover, nGO-PEG did not cause appreciable toxicityat our tested dose (20mg/kg) to the treated mice by hematology analysis andhistological examinations.
Chapter6: We systematically investigated the in vivo biodistribution and potentialtoxicity of nano-graphene and its derivatives via oral and intraperitoneal (i.p.)administration. We found that nGO-PEG labeled with125I showed no obvious tissueuptake via oral administration. In contrast, high accumulation of nGO-PEG，RGO-PEGand nRGO-PEG, but not as-made GO, in the reticuloendothelial (RES) system includingliver and spleen was observed after i.p. injection. Although GO and PEGylated GOderivatives would retain in the mouse body over a long period of time after i.p. injection,their toxicity to the treated animals was insignificant.
In summary, this dissertation, we have systematically studied the biomedicalapplications of nano-graphene, particularly for imaging and photothermal therapy ofcancer, and carefully investigated the toxicology of functionalized nano-graphene in vivo to animals. Our results greatly promote graphene-based biomedical research, andprovide helpful guidelines for the future explorations of other functional nanomaterialsin cancer theranostics.
引文
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