单粒子微束亚细胞精确照射研究纳米金增敏质子生物效应的微观剂量学机制

Recent studies are indicating that gold nanoparticles(GNP)have significant radiosensitization effects,which may be utilized to further enhance the Relative Biological Effectiveness (RBE) of proton therapy that has a low RBE of ~1.1. However,current studies focus on photon irradiation and the macrodosimetric models of the radiosensitization effects have big underestimation, and the microdosimetric mechanism is still unclear. For proton therapy use of GNP radiosensitizer, the first essential step is to clarify the microdosimetric mechanism of the radiosensitization effect thus to establish an explicit dose-effectiveness relationship. In priliminary studies, 300nm gold film and 15nm GNPs were respectively used in alpha particle irradiation of peripheral blood lymph cells and Co60γirradiation of HepG2 cells,both produced distinct sensitization effects and the sensitivity enhancement ratios(SER) were higher than macroscopic dosimetric models, indicating a microscopic model is needed for stricter effect analysis; comparative experiments using free radical scvenger(DMSO) indicated that the sensitization effect of 15nm GNPs on γ-irradiated HepG2 cells was mainly from free radical contribution; meanwhile, microscopy observation indicated that 15nm GNPs mainly distributed in the cytoplasma outside nucleus, and MonteCarlo simulation result showed that the direct DNA damage by the secondary electrons from extranuclear GNPs can be mostly shielded by physical separation of nuclear memberane, with an average thickness of 50-100nm. Based on the priliminary results, a double-mechanism rediosensitization related with the subcellular(intra-and extranuclear) distribution of GNPs is proposed, as "direct local dose DNA damage by intranuclear GNPs" and "indirect free radical DNA damage by extranulcear GNPs". For a strict validation of the mechanism, the following studies will be carried out: (1) Monte-Carlo simulations of the collision ionization process of energetic protons and gold nanoparticles to establish a microscopic local dose enhancement model of the secondary electrons.(2)Local Effect Model(LEM)analysis of the direct DNA damage effect by the local dose of intranuclear gold nanoparticles, and validation of the direct radiosensitization mechanism by cell irradiation experiments using broad proton beam irradiation.(3)Subcellular single-proton microbeam irradiation for probing and validating of the indirect radiosensitization mechanism of free radical DNA damage by the local dose of extranuclear gold nanoparticles. With the proposed studies, the hypothesized double mechanism of radiosensitization effect on proton RBE by the local dose of endocytosed GNPs can be strictly validated and thus an explicit dose-response relationship may be established,as the necessary dosimetry model for the clinical use of GNPs radiosensitizer in proton radiotherapy.

纳米金放射增敏效应显著，有望克服质子治疗生物效应偏低的局限而显著提高肿瘤治疗比。但目前验证性研究多针对光子辐照且剂量学评价明显低估，而微观剂量学机制尚未阐明。 项目前期工作以纳米金增敏淋巴细胞α辐照和HepG2细胞γ辐照,均有明显增敏效应且增敏比大于宏观剂量增强比；自由基清除剂实验显示15nm纳米金增敏效应主要来自自由基效应；同时发现该尺度纳米金主要分布于核外细胞质而计算表明核外纳米金次级电子对DNA的直接损伤贡献很小。据此提出纳米金"核内局域剂量DNA直接损伤+核外自由基间接效应"双重增敏机制假说，拟通过蒙卡模拟建立纳米金的次级电子局域剂量模型；以局部效应模型原理计算核内纳米金局域剂量的DNA直接损伤增敏效应并以细胞实验验证；以亚细胞微束辐照方法验证核外纳米金间接增敏效应；由此阐明纳米金增敏质子生物效应的微观剂量学机制以建立准确量效关系，为探索其在质子治疗中的应用建立必需的剂量学基础。

【项目背景】针对质子治疗因生物效应偏低(RBE~1.1)而对乏氧/抗阻肿瘤疗效受限，以纳米金增敏提高质子治疗增益比极具潜力。目前主要问题：多数研究针对kVp/MV级光子射线，针对质子和重离子射线的增敏研究报道很少且进展有限；光子射线下纳米金增敏效应与物理剂量的计算结果差异很大，实际效应远高于计算结果，剂量学模型的缺陷阻碍了临床应用。因此针对质子治疗的纳米金增敏，首先需要对其在受照细胞中的放射增敏机制开展更为深入的微观剂量学机理研究。.【研究内容】提出纳米金位置关联的“核内局域剂量 DNA直接损伤+核外局域剂量自由基间接效应”双重增敏机制，采用局部效应模型结合亚细胞辐照/分析实验开展质子生物效应增敏的微观剂量学机理研究，主要包括：■蒙卡模拟纳米金质子碰撞电离的能量转移沉积机制，建立纳米金对质子径迹的微观剂量增强模型； ■局部效应模型(LEM)分析结合细胞辐照实验，研究核内纳米金局域剂量的DNA直接损伤增敏机制； ■亚细胞定位照射结合自由基清除实验对比，研究核外纳米金局域剂量的自由基间接损伤增敏机制。.【重要结果】■以PEG修饰的30nm纳米金增敏Hep-G2细胞，160kVpX射线和3MeV质子辐照下均获得显著增敏效应，但质子辐照下增敏效应较低，提示低能质子诱导的剂量增强效应低于光子辐照(~50%)；■实验观察30nm 纳米金实际上分布于核外细胞质，计算表明其表面剂量很难对细胞核内DNA产生直接损伤；■蒙卡计算放疗能量(2-200MeV)范围内质子辐照诱发的纳米金表面次级电子出射特性和微观剂量分布，结果显示质子仍可激发足够高的表面电子剂量(增强比~10)，但增强剂量的作用范围远小于光子激发的次级电子剂量(~100nm vs ~1um)，提示质子诱导的增敏效应极有可能是核外纳米金局域剂量作用于核外靶点导致细胞损伤(线粒体/溶酶体)，而不是次级电子剂量对DNA直接损伤的结果；■单粒子微束定位照射纳米金分布的细胞质部位，实验结果显示核外纳米金可导致细胞凋亡增加，但核内DNA双链断裂并没有明显增加。研究结果初步印证了以上推断，即质子辐照诱导的纳米金增敏效应与光子辐照下基于光电效应康普顿效应的二次电子及俄歇电子作用机制并 可导致DNA直接损伤不同，质子辐照下的增敏则是纳米金表面剂量的对核外靶点的损伤效应，该结论为后续发展GNP放射增敏的全细胞LEM模型建立了重要的理论实验依据。