基于多价适配体的循环肿瘤细胞的高效捕获和无损释放研究

The isolation and detection of rare circulating tumor cells (CTCs) is critical to cancer diagnosis and prognosis, personalized therapy, and disease monitoring. However, CTC are extremely rare in peripheral blood - as few as one CTC per billion blood cells, making their isolation and detection technically challenging. Here we will develop a multivalent aptamer-mediated, three-dimensional (3D) nanostructured substrate-based microfluidic device that is not only able to efficiently isolate CTC from unprocessed whole blood, but also able to non-destructive, temperature-mediated release and retrievale of cells. The multivalent aptamers will be prepared by two ways, i) the aptamer functional polyamidoamine (PAMAM) dendrimer will be synthesized by conjugating several aptamers to one PAMAM dendrimer molecule, ii) a long aptamer strand comprising repeating aptamer sequences will be synthesized by rolling circle amplification (RCA) technique. The multivalent aptamers will be used to further enhance the affinity of the aptamers with cells. The 3D nanostructured substrate, a silicon-nanopillar (SiNP) array, which allows for increasing cell/substrate contact frequency and duration, will be prepared by a wet chemical etching method. The cell capture substrates will be prepared by N-hydroxysuccinimide (NHS)/maleimide chemistry and streptavidin/biotin chemistry to introduce multivalent aptamer onto the SiNP substrates prior to the cell-capture experiments, which will vastly enhance the probability of the interaction between aptamers and CTC. Release of the captured cells is realized by a moderate temperature change, effected via integrated heaters and a temperature sensor, to reversibly disrupt the cell-aptamer interaction.The device parameters and the flow rate will be systemly investigated and optimized by studying their effects on the capture and release of prostate cancer cell lines, which is used as a model. Further, we will exploit the device for capture CTC from the peripheral blood of prostate cancer patients, according to the number of CTC, determine the stage of cancer development, prognosis, and further guidance individualized treatments.

循环肿瘤细胞（CTC）的分离和检测是建立癌症分期和预后、设计个性化治疗和疾病监测的关键。由于外周血中CTC数量极少，因此对其的分离和检测十分困难。本项目将多价适配体、三维纳米结构的基底与带有温度控制芯片的微流控芯片技术有机结合起来，发展一种CTC高效捕获和温控无损释放的全新方法。采用两种方法制备多价适配体，一是将适配体修饰到PAMAM树形高分子上，得到一个PAMAM上交联上多个适配体的多价适配体，二是利用滚环扩增技术合成包含多个适配体重复序列的长链结构的多价适配体。多价的适配体亲和分子可以提高其与靶细胞的亲和力，三维基底能够增强细胞与基底的接触频率和持续时间，温度控制芯片可以无损地释放捕获的细胞。以前列腺癌细胞系为模型，系统考察了所构建系统的捕获靶细胞的效果、纯度及温控释放的效果。本项研究将揭示出多价适配体、三维纳米结构基底、微流控芯片用于高效捕获和无损释放CTC的一般规律和内在机理。

循环肿瘤细胞（Circular Tumor Cells, CTC）是肿瘤细胞从瘤体脱落，进入血液循环系统，然后迁移到身体的其他组织部位，造成肿瘤的转移。CTC的分离和检测是建立癌症分期和预后、设计个性化治疗和疾病监测的关键。由于外周血中CTC数量极少，对其的分离和检测十分困难，因此迫切需要发展高灵敏和高特异性的CTC捕获技术。本研究围绕“CTC的高效捕获和无损释放”这一关键科学问题，借助不同的工艺手段，将三维纳米结构、抗粘附分子和多价适配体修饰、互补链或温度控制释放策略与微流控芯片技术有机结合起来，实现CTC的特异性捕获、响应性无损释放。本研究采用多种方法制备多价适配体，一是将适配体修饰到PAMAM树形高分子上，得到一个PAMAM上交联上多个适配体的多价适配体；二是利用滚环扩增技术合成包含多个适配体重复序列的长链结构的多价适配体；三是将适配体修饰到CBMA聚合物分子，得到一个多价适配体修饰的亲水捕获界面。并借助不同的工艺手段制备了多种三维纳米结构的基底，包括硅纳米柱，多尺度TiO2纳米棒阵列，壳聚糖纳米粒子和纳米纤维等。多价的适配体亲和分子可以提高其与靶细胞的亲和力，三维基底能够增强细胞与基底的接触频率和持续时间，微流控芯片技术可以提高捕获纯度，温度控制和互补短链的引入可以无损地释放捕获的细胞。以前列腺癌细胞系和乳腺癌细胞系为模型，系统考察了所构建系统的捕获靶细胞的效果、纯度及控制释放的效果，优化各种参数后，得到了较高的捕获效率和纯度，理想的释放效果。本项研究为揭示多价适配体、三维纳米结构基底、微流控芯片用于高效捕获和无损释放CTC的一般规律和内在机理提供了实验基础，并为稀少CTC的捕获和增殖提供了可行方案，将对以后的临床应用产生巨大的参考价值。