Lipid nanoprobe integrated microdevice for extracellular vesicle isolation and duplex sequencing based mutation detection for non-invasive lung cancer diagnosis

用于细胞外囊泡分离和基于双重测序的突变检测的脂质纳米探针集成微器件用于非侵入性肺癌诊断

基本信息

批准号：
10686287
负责人：
Siyang Zheng
金额：
$ 34.35万
依托单位：
CARNEGIE-MELLON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-08-06 至 2024-07-31
项目状态：
已结题

项目摘要

PROJECT SUMMARY In this proposed research, we will develop a lipid nanoprobe (LNP) integrated microdevice, LiEVchip, for isolation of extracellular vesicles (EVs) from blood plasma of patients with non-small cell lung cancer (NSCLC). The proposed LiEVchip features high-throughput sample processing, one-step enrichment, and low cost. It is expected to isolate EVs from 1 ml of unprocessed plasma in 20 minutes with over 80% isolation efficiency. Furthermore, combination with duplex sequencing based ultrasensitive DNA mutation detection platform, the LiEVchip will significantly prompt EV-based cancer diagnostics in an invasive way. In our previous study, we developed a two-component LNP system for rapid EV isolation and comprehensively EV molecular analyses. Particularly, we successfully identified EV DNA mutations from four patients with advanced NSCLC. The LNP system overcomes low throughput, low purity and other common shortcomings in EV isolation, showing great potential for clinical use. Hence, we proposed to develop a highly integrated one- component LiEVchip for rapid and efficient EV isolation. Firstly, we will design and fabricate the proposed LiEVchip. The LiEVchip integrates the LNP to capture EVs based on their lipid membrane envelope. The EV isolation efficiency is boosted by the design of microchannel curvature to enhance flow-surface interaction by Dean flow, and the empowerment of electrokinetic enhanced EV isolation by the nanoelectrode array. Then, we will optimize its operational parameters to make a balance between EV isolation efficiency and purity. After comprehensive optimization, we will rigorously validate its performance with spike samples by analyzing EV content, including RNA, DNA, and proteins, and compare with those isolated by the prevalent EV isolation method, OptiPrep density gradient ultracentrifugation (odgUC). Subsequently, we will develop duplex sequencing based DNA mutation detection method with 3rd generation high-throughput sequencer (PacBio). The detection sensitivity of point/deletion mutations and complex structural variations (SVs) will be validated with spiked-in samples. Finally, in a cohort clinical study, we will first recruit 40 NSCLC patients at stage IV to validate the overall technology. EV from plasma will be isolated with LiEVchip, and a panel of 20 most common NSCLC mutated genes will be assayed simultaneously by duplex sequencing. We will further perform EV isolation and duplex sequencing in the additional 120 samples covering stage I-III NSCLC to investigate the potential of this technology in early NSCLC diagnosis. Besides, we will use the developed technology to monitor mutation status of 20 NSCLC patients undergoing targeted therapy to explore its clinical utility in treatment monitoring. The cohort clinical studies not only can testify whether LiEVchp combined with duplex sequencing can be routinely applied to detect diverse malignancies in NSCLC, but also can demonstrate the clinical diagnostic values of EV DNA. Altogether, the successful completion of this proposed project will pave the way for clinical translation of EV diagnostics.

项目概要在这项拟议的研究中，我们将开发一种脂质纳米探针（LNP）集成微器件LiEVchip，用于从非小细胞肺癌（NSCLC）患者的血浆中分离细胞外囊泡（EV）。所提出的 LiEVchip 具有高通量样品处理、一步富集和低成本的特点。这是预计可在 20 分钟内从 1 毫升未经处理的血浆中分离出 EV，分离效率超过 80%。此外，结合基于双链测序的超灵敏DNA突变检测平台， LiEVchip 将显着促进基于 EV 的侵入性癌症诊断。在我们之前的研究中，我们开发了一种双组件 LNP 系统，用于快速 EV 隔离和全面 EV分子分析。特别是，我们成功地从四名患有此病的患者中鉴定出了 EV DNA 突变。晚期非小细胞肺癌。 LNP系统克服了低通量、低纯度等常见缺点 EV分离，显示出巨大的临床应用潜力。因此，我们建议开发一种高度集成的单 LiEVchip 组件可实现快速高效的 EV 隔离。首先，我们将设计和制造建议的 LiEV芯片。 LiEVchip 集成了 LNP，可根据脂质膜包膜捕获 EV。电动汽车微通道曲率设计提高了隔离效率，通过以下方式增强流-表面相互作用 Dean 流，以及纳米电极阵列增强动电隔离的能力。然后，我们将优化其运行参数，以在电动汽车隔离效率和纯度之间取得平衡。后全面优化，我们将通过分析EV，用尖峰样本严格验证其性能内容，包括 RNA、DNA 和蛋白质，并与通过流行的 EV 分离分离的内容进行比较方法，OptiPrep 密度梯度超速离心 (odgUC)。后续我们将开发复式基于测序的 DNA 突变检测方法，采用第三代高通量测序仪 (PacBio)。将验证点/缺失突变和复杂结构变异（SV）的检测灵敏度与加标样品。最后，在一项队列临床研究中，我们将首先招募 40 名 IV 期 NSCLC 患者来研究验证整体技术。来自等离子体的 EV 将通过 LiEVchip 进行隔离，并且面板包含 20 个最常见的 NSCLC 突变基因将通过双链测序同时进行检测。我们将进一步开展EV 对涵盖 I-III 期 NSCLC 的另外 120 个样本进行分离和双重测序，以研究该技术在早期 NSCLC 诊断中的潜力。此外，我们将利用已开发的技术监测20例接受靶向治疗的NSCLC患者的突变状态，探讨其临床应用治疗监测。队列临床研究不仅可以证明LiEVchp联合双链体是否有效测序可常规用于检测 NSCLC 中的多种恶性肿瘤，而且还可以证明 EV DNA的临床诊断价值。总而言之，该拟议项目的成功完成将为 EV诊断的临床转化之路。

项目成果

期刊论文数量（6）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Enrichment of extracellular vesicles with lipid nanoprobe functionalized nanostructured silica.

DOI：
10.1039/c8lc01359d
发表时间：
2019-07
期刊：
Lab on a chip
影响因子：
6.1
作者：
Y. Wan;Mackenzie Maurer;Hongzhang He;Yiqiu Xia;S. Hao;Wen-Long Zhang;N. Yee;Si-Yang Zheng
通讯作者：
Y. Wan;Mackenzie Maurer;Hongzhang He;Yiqiu Xia;S. Hao;Wen-Long Zhang;N. Yee;Si-Yang Zheng

Small extracellular vesicles in cancer.