Quantitative evaluation of the NIR-mediated photo-thermal ablation of tumor cells

描述（由申请人提供）：单壁碳纳米管（SWNTs）作为近红外（NIR）介导的肿瘤细胞热消融剂被广泛研究，因为它们有效地将吸收的近红外光转化为热量。尽管单壁碳纳米管具有治疗潜力，但目前还没有发表的研究表明，一个细胞需要多少单壁碳纳米管才能达到可测量的杀伤效果，或者什么是单壁碳纳米管杀死细胞最有效的亚细胞分布。这对于合理设计和测试策略以最终在体内向肿瘤细胞提供有效载荷具有重要意义。这种知识差距的主要原因是缺乏定量评估细胞中单壁碳纳米管数量和亚细胞位置的方法。我们最近开发了提取和测量少量细胞相关swnt的方法，并通过三维共聚焦拉曼显微镜直接成像其在细胞中的分布。该建议的总体目标是将nir介导的细胞死亡与体外细胞相关swnt的数量和位置联系起来，然后将这一信息用于小鼠肿瘤模型的初步研究。具体目的1，确定nir介导的靶细胞消融的功效与细胞相关swnt数量的关系。特异性目的1的目的是将细胞相关swnt的实际剂量与细胞死亡程度联系起来，这与通常的LD50曲线不同，LD50曲线将杀伤与介质中或注射到动物血液中的药物量联系起来。具体目的2，确定nir介导的靶细胞消融的功效与swnt亚细胞位置的关系。特定目的2的目的是生成nir介导消融的有效性与SWNT亚细胞位置（在质膜上或溶酶体囊泡内）相关的数据。特异性目的3，利用抗her -2- mab - swnt构建物消融NOD/SCID小鼠上生长的人BT-474肿瘤的初步研究。本研究的目的是了解在小鼠身上生长的人肿瘤体内模型中，靶向使质膜或溶酶体萎缩的swnt的数量是否会影响肿瘤细胞消融的程度。总之，本提案中的工作将深入了解剂量和亚细胞位置对swnt在nir介导的细胞消融中体外和体内肿瘤模型有效性的影响。

DESCRIPTION (provided by applicant): Single-walled carbon nanotubes (SWNTs) are being widely investigated as agents for the near infrared (NIR) - mediated thermal ablation of tumor cells because they efficiently convert absorbed NIR light into heat. Despite the therapeutic potential of SWNTs, there have been no published studies that correlate how many SWNTs need be associated with a cell to achieve a measured outcome of killing, or what is the most efficient subcellular distribution of SWNTs for killing cells. This is important information for the rational design and testing of strategies to eventually deliver an effective payload to tumor cells in vivo. The main reason for this knowledge gap has been the lack of methods for quantitatively assessing the amounts and subcellular location of SWNTs in cells. We have recently developed methods to extract and measure small amounts of cell-associated SWNTs and to directly image their distribution in cells by 3-dimensional confocal Raman microscopy. The overall objective of this proposal is to correlate NIR-mediated cell death with the quantity and location of cell-associated SWNTs in vitro and then carry this information to initial studies of a tumor model in mice. Specific aim 1, to determine the efficacy of NIR-mediated ablation of target cells as a function of the amount of cell-associated SWNTs. The objective of specific aim 1 is to correlate the actual dose of cell-associated SWNTs with the extent of cell death, distinct from the usual LD50 curves that relate killing to the amount of drug in the medium or injected into the blood of an animal. Specific aim 2, to determine the efficacy of NIR-mediated ablation of target cells as a function of the subcellular location of SWNTs. The objective of specific aim 2 is to generate data that correlates the effectiveness of NIR-mediated ablation with SWNT subcellular location, on the plasma membrane or within lysosomal vesicles. Specific aim 3, pilot studies on ablation of human BT-474 tumors growing on NOD/SCID mice using anti-Her-2-MAb-SWNT constructs. The objective of this study is to learn whether the amount of SWNTs targeted to wither the plasma membrane or lysosomes impacts the extent of tumor cell ablation in an in vivo model of human tumors growing on mice. Together, the work in this proposal will provide insight into the influence of dose and subcellular location on the effectiveness of SWNTs in NIR-mediated cell ablation with in vitro and in vivo tumor models. PUBLIC HEALTH RELEVANCE: Single-walled carbon nanotubes are a promising new material for cancer therapy because they have the ability to heat up and kill tumor cells when exposed to certain types of light. This proposal details quantitative studies to assess the effectiveness of single-walled carbon nanotubes as agents for the thermal destruction of tumor cells using in vitro and in vivo tumor models.