硼10纳米胶束透过血脑屏障及脑胶质瘤细胞核靶向的实验研究

A successful boron neutron capture therapy (BNCT) requires selective boron tumor targeting to achieve boron concentrations (20μg /g tumor) and good tumor-to-normal tissue ratios, T/N, (>3). The tumor cell nucleus and DNA are especially attractive targets because the amount of boron required to produce a lethal effect may be substantially reduced to as less as ten percent if it is localized within or near the nucleus. High T/N ratios are easier to reach if the boron has good retention inside the tumor cells. In the present study, we try to construct 10B -loaded nanomicelles as 10B delivery vehicle for the purpose of cellular nucleus targeting and long cellular retention. The physicochemical properties of these micelles are to be investigated. Laser confocal microscopy and inductively coupled plasma-atomic emission spectrometer (ICP-AES) are to be engaged to determine the cellular 10B uptake, 10B cellular nucleus targeting and retention. The blood-brain barrier (BBB) penetration of 10B-nanomicelles was qualitatively and quantitatively studied by in vitro BBB model and animal BBB by Laser confocal microscopy and ICP-AES. Intracranial glioma models are to be established, 10B uptake and retention inside the glioma cells, in gross tumor, normal brain, skin and blood are to be studied by ICP-AES and Maestro EX in vivo imaging system. In vitro BNCT and intracranial glioma rat model BNCT will be conducted after treated with 10B-nanomicelles. We are investigating an approach of success BNCT by improving 10B cellular uptake, nucleus targeting and retention, as well as BBB penetration, with 10B-nanomicelles.

成功应用硼中子俘获治疗（BNCT）要求①每克肿瘤细胞摄取20μg硼（ 10B）；②肿瘤/正常组织（T/N）硼浓度比大于3。进入细胞核的硼，只要胞浆硼含量的10%就足于达到相同的致死性损伤；如硼在瘤内的滞留时间长，易获得高T/N比。这些科学问题尚待解决。为了解决硼的细胞核靶向与滞留，拟在我们前期硼运载和纳米研究结果基础上，制备硼-纳米胶束。激光共聚焦显微镜动态观察、感应耦合等离子体-原子发射光谱定量分析硼的细胞摄取、细胞核靶向与滞留；经典血脑屏障模型评价药物外排蛋白功能、表达和硼-纳米胶束透过血脑屏障能力；建立大鼠原位脑胶质瘤模型，活体荧光成像动态观察、光谱仪定量分析硼在鼠瘤细胞内、瘤体、病侧脑、对侧脑、血液等组织器官的分布与滞留；中子束对摄取硼的胶质瘤细胞和大鼠原位胶质瘤的BNCT效应。本研究从硼-纳米胶束的细胞摄取、细胞核靶向与滞留和透过血脑屏障能力的角度，探讨实现有效BNCT治疗新途径

恶性胶质瘤为中枢神经系统最常见肿瘤，绝大多数患者经过标准治疗后仍出现复发，探索新的治疗策略具有重要科学价值。本项目基于硼中子俘获疗法在肿瘤中的应用价值及尚缺理想硼俘获剂问题出发，旨在设计兼具亲肿瘤性和肿瘤细胞核高分布性的优良硼俘获剂，进一步提升硼中子俘获治疗效果。项目组经过数年反复探究及验证，成功制备了一系列功能性制剂（主要包括肿瘤靶向性纳米胶束，细胞核分布硼俘获剂和脑胶质瘤靶向性纳米脂质体）。纳米胶束外表面修饰iRGD穿透肽，形貌规整，粒径约25 nm，稳定性良好，体外A549细胞硼摄取量达209.83 ng 10B/106 cells，A549皮下瘤裸鼠体内硼分布参数T/B比值14.11，T/N比值19.49，另外在肿瘤微环境顽固性肿瘤B16F10荷瘤小鼠模型中，联用抗血管生成药物“恩度”可增加肿瘤组织硼分布量；细胞核分布硼俘获剂为阿霉素碳硼烷复合物，具有荧光激发特性，在共聚焦显微镜下可见胶质瘤细胞核内强荧光分布，ICP-MS分析结果亦显示过半硼量聚集胶质瘤细胞核内；纳米脂质体包载阿霉素碳硼烷复合物后与iRGD物理混合，形貌规整，大小约150 nm，呈电中性。实验过程中构建了GL261原位脑胶质瘤C57/BL6小鼠模型。活体荧光成像结果显示纳米脂质体在原位脑胶质瘤组织蓄积量很高，具备良好血脑屏障穿透性及脑肿瘤靶向性。本项目有效实现肿瘤病灶硼高富集、肿瘤细胞核硼高分布及脑胶质瘤硼高靶向计划目标，并应用国内小型加速器式中子管开展硼中子俘获治疗实验，基本完成原研究计划中关于脑胶质瘤及细胞核靶向性纳米载体应用于硼中子俘获治疗的研究工作。