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Re-engineering Tumor Cell-Derived Extracellular Vesicles with Exogenous Tags for Downstream Analysis

用外源标签重新设计肿瘤细胞衍生的细胞外囊泡用于下游分析

基本信息

批准号：
10451108
负责人：
Santosh Aryal
金额：
$ 40.54万
依托单位：
UNIVERSITY OF TEXAS TYLER
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-09-10 至 2024-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10451108
关键词：
Re engineering Tumor Cell Derived

项目摘要

Extracellular vesicles (EVs) are phospholipid and protein constructs continuously secreted by cells and carry parent cell-specific genetic materials, proteins, and lipids, which can be selectively taken up by neighboring or distant target cells far from their release. EVs derived from cells with abnormal conditions such as viral infections, neurodegenerative diseases, and cancer have specific cargos which can be exploited as a potential biomarker for the respective disease condition. Moreover, the reports suggest that tumor EVs play a role as metastatic site surveyors and are crucial to create a favorable microenvironment or niche for metastatic cells. Therefore, in order to elucidate the mode of action of EVs and their constituents, tracking EVs in vitro and in vivo is essential. Currently, EVs are detected by analyzing the endogenous marker proteins such as trans-membrane cluster of differentiation (CD) proteins (CD81, CD63, and CD9), and tumor susceptibility gene 101 protein (TSG101). However, these markers are present in nearly almost all cells at different level which results in analysis heterogeneity. Therefore, the exogenous tag is very important to study realistic cellular interaction and tracking of EVs. Within this background, the major goal of this proposal is to define the importance of reconstruction of EVs with exogenous fluorescent and metal tags as an analytical handle to elucidate its quantitative uptake in vital organs. Recently, the PI demonstrated delivery of water-soluble doxorubicin (DOX) to breast tumor xenografts using natural killer (NK) cell-derived EVs reconstructed liposome. Results showed the therapeutic advantage of reconstructed EVs with a tumor inhibition rate of 80% (free DOX = 65%). Similarly when macrophage-derived EVs reconstructed with liposome, a higher order of colloidal stability and drug loading were observed. Engineered EVs showed the differential targeting and uptake against normal and cancerous cells thereby putting itself in the group of potential tumor-targeted drug delivery candidates. These outstanding findings from re- engineered EVs validated our hypothesis that acquired properties from parent cells navigate to target recipient cell. Lesson learned from re-engineered system, our overarching hypothesis is to incorporate exogenous components such as near-infrared (NIR) dye, macrocyclic metal, and/or radiopharmaceuticals as a tag in EVs for downstream quantitative analysis. The related but independent aims are Aim 1. To re-engineer EVs with exogenous tags and study its physicochemical properties. Aim 2. To elucidate the cellular specificity and biocompatibility of re-engineered EVs. Aim 3. To evaluate the biodistribution of EVs using breast cancer models. We will incorporate exogenous tags into the EVs derived from human breast cancer cells (MCF-7 and MDA-MB- 231) to study its interaction and distribution in human breast cancer xenograft developed in humanized mice. These tags will be detected using state-of-art analytical techniques established in the PI’s lab. The research team anticipates the results will assist EVs system designs in accelerating the diagnosis and therapy of cancer.

细胞外囊泡（EV）是由细胞持续分泌的磷脂和蛋白质构建体，并携带亲本细胞特异性的遗传物质、蛋白质和脂质，它们可以被邻近的或远离释放的靶细胞。来源于具有异常状况（如病毒感染）的细胞的EV，神经退行性疾病和癌症具有特定的物质，可以用作潜在的生物标志物针对各自的疾病状况。此外，报告表明，肿瘤EV作为转移位点发挥作用，是一种重要的生物学机制，对于为转移性细胞创造有利的微环境或生态位至关重要。因此在为了阐明EV及其组分的作用模式，体外和体内追踪EV是必要的。目前，EV的检测是通过分析内源性标记蛋白，如跨膜簇，分化（CD）蛋白（CD 81、CD 63和CD 9）和肿瘤易感基因101蛋白（TSG 101）。然而，这些标记物以不同的水平存在于几乎所有的细胞中，这导致分析异质性因此，外源性标签对于研究真实的细胞相互作用和跟踪EV。在此背景下，本提案的主要目标是确定以下方面的重要性：用外源性荧光和金属标签作为分析手柄重建EV，以阐明其重要器官中的定量摄取。最近，PI证明了水溶性阿霉素（DOX）对乳腺肿瘤异种移植物的递送使用自然杀伤（NK）细胞衍生的EV重构脂质体。结果显示，肿瘤抑制率为80%（游离DOX = 65%）。类似地，当巨噬细胞衍生的脂质体修饰的EV具有较高的胶体稳定性和载药量。工程EV显示出针对正常细胞和癌细胞的差异靶向和摄取，从而将本身在一组潜在的肿瘤靶向药物输送的候选人。这些杰出的发现来自于重新- 工程EV验证了我们的假设，即从亲本细胞获得的特性导航到靶受体 cell.从重新设计的系统中吸取的教训，我们的首要假设是将外源性组分，例如近红外（NIR）染料、大环金属和/或放射性药物作为EV中的标签用于下游定量分析。相关但独立的目标是目标1。重新设计电动汽车，外源性标签，并研究其理化性质。目标2.为了阐明细胞特异性，重新设计的电动汽车的生物相容性。目标3.使用乳腺癌模型评价EV的生物分布。我们将外源性标签掺入来源于人乳腺癌细胞（MCF-7和MDA-MB-1）的EV中。 231)研究其在人源化小鼠中的人乳腺癌异种移植物中的相互作用和分布。将使用PI实验室中建立的最新分析技术检测这些标签。研究研究小组预计，这些结果将有助于电动汽车系统设计加速癌症的诊断和治疗。

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）

Indocyanine-type Infrared-820 Encapsulated Polymeric Nanoparticle-Assisted Photothermal Therapy of Cancer.

DOI：
10.1021/acsomega.2c00306
发表时间：
2022-04-12
期刊：
ACS OMEGA
影响因子：
4.1
作者：
Marasini, Ramesh;Aryal, Santosh
通讯作者：
Aryal, Santosh

Extracellular vesicles production and proteomic cargo varies with incubation time and temperature.

DOI：
10.1016/j.yexcr.2022.113454
发表时间：
2022-12
期刊：
Experimental cell research
影响因子：
3.7
作者：
Sagar Rayamajhi;S. Sulthana;Colin Ferrel;Tej B. Shrestha;S. Aryal
通讯作者：
Sagar Rayamajhi;S. Sulthana;Colin Ferrel;Tej B. Shrestha;S. Aryal

Effect of Nanoparticle Weight on the Cellular Uptake and Drug Delivery Potential of PLGA Nanoparticles.

DOI：
10.1021/acsomega.3c02273
发表时间：
2023-08-01
期刊：
ACS OMEGA
影响因子：
4.1
作者：
Kattel, Prabhat;Sulthana, Shoukath;Trousil, Jiri;Shrestha, Dinesh;Pearson, David;Aryal, Santosh
通讯作者：
Aryal, Santosh

Re-engineered imaging agent using biomimetic approaches.