Tumor-targeted delivery and cell internalization of theranostic gadolinium nanoparticles for image-guided nanoparticle-enhanced radiation therapy

该项目的长期目标是克服阻碍临床应用的一些主要障碍 金属纳米颗粒（NP）放射增敏在放射治疗（RT）中的转化。研究表明 被动的、增强的渗透性和保留性（EPR）效应本身不足以递送所述量的 用可负担量的注射NP进行体内放射增敏所需的瘤内和细胞内NP 并且常规NP在体内被快速清除（~分钟）。成像体内NP生物分布， 定量RT治疗计划也是一个未解决的关键问题。积极定位和内化为 通过与pH低插入肽（pHLIPs）缀合的钆（Gd）NPs对癌细胞的抑制具有潜力， 用于增强肿瘤细胞中NP的摄取和通过MRI进行选择性定量成像的双重目的。 pHLIP-GdNPs可以主动靶向实体瘤不可避免的酸性微环境，这在肿瘤中不存在。 健康的组织因此，它上级于其他生物标记物靶向，例如抗体靶向，其可以 变得非特异性，并在处理期间通过选择非表达亚克隆而被避免。pHLIPs可以 还通过强的非内吞途径将结合的细胞不可渗透的货物递送到癌细胞内， 对于NP诱导的短程俄歇级联和光电子到达重要的细胞靶点至关重要， by experiments实验and simulations模拟.补充快速增加的MRI用于RT计划和机载 pHLIP-GdNP还可以解决用于治疗计划优化的成像问题。我们 初步MRI数据显示在pHLIP-GdNP注射后NP的长肿瘤保留（>9小时，可能是数天）。 具体目标：为了为更深入的转化和临床研究提供临床前基础，我们的目标是 （i）表征pHLIP-GdNP并评估其体外RT性质;（ii）开发生物物理机制， GdNPs的放射增敏模型，以阐明相关的生物学机制并促进定量RT 治疗计划;和（iii）研究pHLIP-GdNP的体内放射增敏和成像特性。 研究设计：（i）表征pHLIP-GdNP和内化，用显微镜成像细胞摄取， 荧光标记，在细胞培养物中用250 kVp和6 MV X射线进行克隆形成存活实验， 生成pH依赖性细胞存活曲线，并检查DNA损伤。(ii)使用蒙特卡罗粒子轨迹 结构模拟计算纳米粒子引起的微观剂量增强。DNA损伤将被建模 预测增感剂增强比，并与实验结果进行比较。(iii)调查的可行性 MR成像用于定量测定NP在体内的分布和肿瘤中的滞留-通过时间， 在小鼠模型的器官中，在注射pHLIP-GdNPs的小鼠中增强的体内放射增敏作用与 使用肿瘤生长延迟测定法对注射非靶向GdNPs的小鼠的肿瘤生长延迟进行测定，以及pHLIP-GdNPs的体内毒性。 影响：该项目可以导致一种新型的治疗诊断剂，提供改善的治疗率和成像。 NP递送和成像的这种新范例可以在图像引导的NP增强RT中具有广泛的影响。

The long-term objective of this project is to overcome some of the major hurdles that prevent the clinical translation of metallic nanoparticle (NP) radiosensitization in radiation therapy (RT). Studies have shown that the passive, enhanced permeability and retention (EPR) effect itself is not sufficient to deliver the amount of intratumoral and intracellular NPs needed for in vivo radiosensitization with an affordable amount of injected NPs and the conventional NPs are cleared rapidly (~minutes) in vivo. Imaging the in vivo NP biodistribution for quantitative RT treatment planning is also an unsolved critical issue. Actively targeting and internalization into cancer cells by gadolinium (Gd) NPs conjugated to pH-Low Insertion Peptides (pHLIPs) have the potential to serve the dual purpose of enhancing uptake of NPs in tumor cells and selective, quantitative imaging by MRI. pHLIP-GdNPs can actively target solid tumors’ unavoidable acidic microenvironment, which is not present in healthy tissues. Therefore, it is superior to other biomarker targeting, such as antibody targeting, which can become nonspecific and be evaded by selection of non-expressing subclones during treatment. pHLIPs can also deliver the conjugated cell-impermeable cargoes inside the cancer cells via a strong non-endocytic pathway, critical for NP-induced short-range Auger cascade and photoelectrons to reach the vital cellular targets as proved by experiments and simulations. Complementing the rapid increasing use of MRI for RT planning and on-board treatment-guidance, pHLIP-GdNPs can also solve the imageability problem for treatment plan optimization. Our preliminary MRI data shows long tumor retention of NPs (>9 hours, possibly days) post pHLIP-GdNPs injection. Specific Aims: To provide the pre-clinical foundation for more in-depth translational and clinical studies, we aim to (i) characterize pHLIP-GdNP and evaluate its RT properties in vitro; (ii) develop a mechanistic biophysical model of radiosensitization by GdNPs to elucidate relevant biolgocial mechanisms and facilitate quantitative RT treatment planning; and (iii) investigate the in vivo radiosensitization and imaging properties of pHLIP-GdNP. Research Design: (i) Characterize pHLIP-GdNP and internalization, microscopically image cellular uptake with fluorescent tags, conduct clonogenic survival experiments in cell culture with both 250 kVp and 6 MV X-rays, generate pH-dependent cell survival curves, and examine DNA damage. (ii) Use a Monte Carlo particle track structure simulation to calculate microscopic dose enhancement induced by NPs. DNA damage will be modeled to predict sensitizer enhancement ratios and compare with experimental results. (iii) Investigate the feasibility of MR imaging to determine quantitatively in vivo NP distribution and the residence-transit time in tumor and critical organs in mouse models, the enhanced radiosensitization in vivo in mice injected with pHLIP-GdNPs compared to mice injected with untargeted GdNPs using tumor growth delay assay, and the in vivo toxicity of pHLIP-GdNPs. Impact: This project can lead to a novel theranostic agent that offers improved therapeutic ratio and imageability. This new paradigm of NP delivery and imaging can a have broad impact in image-guided NP-enhanced RT.