Covalent Chemistry on Nanosubstrates Enables Molecular Analysis of Purified Extracellular Vesicles in Hepatocellular Carcinoma

纳米基质上的共价化学使肝细胞癌中纯化的细胞外囊泡的分子分析成为可能

• 批准号: 10462534
• 负责人: Vatche Agopian
• 金额: $ 58万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-07 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10462534
• 关键词: Address; Adopted; Alcoholic Liver Diseases; Antibodies; Area; Bioinformatics; Biological Assay; Biological Markers; Biology; Biometry; Blood; Blood Circulation; Blood specimen; Cancer Biology; Cancer Etiology; Cells; Cessation of life; Chemistry; Cirrhosis; Clinical; Clinical Oncology; Couples; Coupling; Data; Development; Devices; Diagnosis; Diagnostic; Disease; Disease Progression; Disulfides; Early Diagnosis; Early Intervention; Etiology; Excision; Exhibits; Filtration; Goals; Hepatic; Hepatitis B; Hepatitis C; Heterogeneity; Incidence; Intervention; Joints; Liver; Liver Cirrhosis; Malignant Neoplasms; Mediating; Medical center; Messenger RNA; Methods; Microfabrication; Microfluidics; Minor; Modality; Molecular; Molecular Analysis; Molecular Profiling; Motivation; Nanostructures; Neoplasm Circulating Cells; Oncology; Operative Surgical Procedures; Patients; Performance; Phospholipids; Plasma; Postoperative Period; Precipitation; Primary carcinoma of the liver cells; Prognostic Marker; Quantitative Evaluations; Recovery; Recurrence; Research; Reverse Transcription; Risk; Sampling; Serum; Silicon; Surface; System; Technology; Time; Transcript; Tumor-Derived; Ultracentrifugation; Validation; Yang; alpha-Fetoproteins; anticancer research; assay development; base; cancer diagnosis; curative treatments; diagnostic algorithm; diagnostic biomarker; digital; early detection biomarkers; extracellular vesicles; in-vitro diagnostics; innovation; liquid biopsy; liver transplantation; microchip; molecular pathology; mortality; nano; nanomaterials; nanowire; neoplastic cell; non-alcoholic fatty liver disease; novel marker; particle; radiological imaging; tumor; tumor DNA; vesicular release

PROJECT SUMMARY Extracellular vesicles (EVs) are a heterogeneous group of phospholipid bilayer-enclosed particles with the biomolecular contents mirroring those of their parental cells. Since EVs are present in circulation at a relatively early stage of disease and persist across all disease stages, purification and characterization of tumor-derived EVs are expected to offer an opportunity for early cancer diagnosis. Hepatocellular carcinoma (HCC), the fourth most common cause of cancer-related deaths worldwide, is in dire need of diagnostic and prognostic biomarkers. Current clinical radiographic system and serum biomarkers (e.g., alpha-fetoprotein (AFP)) poorly discriminate early-stage HCC (where potentially curative therapies are available) from at-risk liver cirrhosis (where HCC surveillance is indicated). Moreover, sensitive biomarkers for HCC postoperative recurrence (where timely salvage treatment interventions can suppress disease progression) after curative-intent liver resection and liver transplantation remain a significant challenge for early-stage HCC. Therefore, exploiting the diagnostic potential of HCC EVs and EV cargo profiling for HCC early detection and postoperative recurrence holds great promise to significantly augment the ability of current diagnostic modalities. Conventional methods for isolating EVs, such as ultracentrifugation, filtration, and precipitation, are incapable of discriminating tumor-derived EVs from non-tumor-derived EVs. To address this unmet need, our team developed “EV Click Chips” for HCC EV purification. The innovation of our devices includes i) the covalent chemistry-mediated EV capture/release couples click chemistry-mediated EV capture and disulfide cleavage- driven EV release, ii) an optimized multi-marker cocktail targeting HCC-associated surface markers was adopted to overcome the heterogeneity of HCC EVs; iii) the incorporation of densely packed silicon nanowire substrates (SiNWS) dramatically increases the device surface areas for contacting/interacting with EVs; and iv) the microfluidic chaotic mixer facilitates repeated physical contact between SiNWS and the flow-through EVs, further enhancing the performance of EV purification. The purified HCC EVs can be characterized by quantifying a panel of 10 well-validated HCC-specific mRNA markers by incorporating Droplet Digital PCR (ddPCR) technology. The proposed research will conduct: i) an exploratory development and optimization of EV Click Chips for HCC EV purification, and ii) clinical validations of EV Click Chips for HCC early detection and postoperative recurrence using patient blood samples. Our long-term goal is to develop a new HCC EV purification system (i.e., EV Click Chips) by synergistically integrating four very powerful approaches, including covalent chemistry-mediated EV capture/release, multimarker antibody cocktails, nanostructured substrates, and microfluidic chaotic mixers. The purified HCC EVs will readily allow for quantitative cargo profiling to augment current HCC diagnostic algorithms.

项目摘要 细胞外蔬菜（EV）是一组异质的磷脂双层粘合颗粒 生物分子含量反映了其亲本细胞的含量。由于电动汽车存在于相对的循环中 疾病的早​​期阶段并在所有疾病阶段持续存在，肿瘤衍生的纯化和表征 预计EV将为早期癌症诊断提供机会。肝细胞癌（HCC）， 全球与癌症相关死亡的第四大原因是急需诊断和预后 生物标志物。当前的临床射线照相系统和血清生物标志物（例如，α-五蛋白蛋白（AFP））很差 处于危险的肝硬化，区分早期HCC（可以使用潜在的治疗疗法） （指示HCC监视的位置）。此外，HCC术后复发的敏感生物标志物 （及时救助治疗干预措施可以抑制疾病的进展） 对于早期HCC，切除和肝移植仍然是一个重大挑战。因此，利用 HCC EV和EV货物分析的诊断潜力，用于HCC早期检测和术后复发 保持着巨大的希望，可以显着增强当前诊断方式的能力。 隔离电动汽车的常规方法，例如超速离心，过滤和沉淀 将肿瘤衍生的电动汽车与非肿瘤衍生的电动汽车区分开。为了满足这种未满足的需求，我们的团队 开发了用于HCC EV净化的“ EV点击芯片”。我们设备的创新包括i）共价 化学介导的EV捕获/释放夫妇单击化学介导的EV捕获和二硫化裂解 - 驱动的EV释放，ii）优化的多标记鸡尾酒靶向HCC相关的表面标记是 通过克服HCC电动汽车的异质性； iii）掺入所挤压的硅纳米线 底物（SINW）大大增加了用于与电动汽车接触/相互作用的设备表面积；和 iv）微流体混沌搅拌机促进了SINW和流通的物理接触 电动汽车，进一步增强电动汽车净化的性能。纯化的HCC EV可以以 通过合并液滴数字PCR来量化10个验证的10次验证的HCC特异性mRNA标记 （DDPCR）技术。拟议的研究将进行：i）探索性开发和优化 EV点击芯片以进行HCC EV净化，ii）EV的临床验证hcc chips for HCC早期检测 并使用患者血液样本进行术后复发。我们的长期目标是开发新的HCC EV 通过协同整合四种非常强大的方法，包括 共价化学介导的EV捕获/释放，多标记抗体鸡尾酒，纳米结构底物， 和微流体混合混合物。纯化的HCC电动汽车将很容易允许定量货物分析 增强当前HCC诊断算法。