CAREER: Active-Loadable Poresomes for the Cytoplasmic Delivery of Membrane-Impermeable Compounds

职业：用于膜不可渗透化合物的细胞质递送的活性可负载孔体

• 批准号: 2000256
• 负责人: Juliane Nguyen
• 金额: $ 36.42万
• 依托单位: University of North Carolina at Chapel Hill
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2000256&HistoricalAwards=false
• 关键词: CAREER; Active; Loadable; Poresomes; Cytoplasmic

NON-TECHNICAL Unlike small molecules, large molecules including proteins and nucleic acids are unable to penetrate the cellular membrane. However, effectively delivering proteins and nucleic acids into cells could enable the treatment of a number of previously incurable diseases. Here, the PI proposes to create natural small lipid vesicles that contain pores. These will allow large molecules to be transported directly into cells. To load these small lipid vesicles with therapeutic molecules, biomaterials composed of nucleic acids will be engineered. To ensure that the lipid vesicles will only target specific cells, the vesicles will be decorated with molecules that can detect diseased cells. This research is expected to have broad impact across several scientific disciplines including biomaterials, nucleic acid and drug delivery, nanotechnology, and cell-mimicking materials. The fundamental knowledge gained from the proposal will significantly change the way that membrane-impermeable compounds are delivered to cells, especially for large molecules. This has important implications for human health because these carriers should allow diseases that are currently undruggable to be treated more safely and efficiently. The project will also enhance the nation's education infrastructure by developing a new biomaterials curriculum for graduate students and by delivering interactive teaching modules to high schools. Further, this project seeks to establish a mentoring-intensive program that will foster biomaterials research for minority and female undergraduate students. It will also establish a network of support for biomaterials graduate, undergraduate, and high school students.TECHNICAL In this CAREER proposal, the PI plans to develop a novel class of cell-mimicking carriers, termed poresomes. These poresomes will be used to deliver macromolecules to the cytoplasm while bypassing endosomes. Despite tremendous progress in nanoscience, the cytoplasmic delivery of membrane-impermeable compounds such as negatively charged RNA remains challenging. This is because a significant fraction of the RNA that is delivered remains trapped in the endosomes, where it ultimately degrades. The objective of this CAREER proposal is to overcome these challenges by engineering cell-mimicking RNA-loaded lipid vesicles that are equipped with pores composed of connexin membrane channels. It is hypothesized that microRNA(miRNA)-loaded poresomes will connect to the connexin membrane channels of recipient cells to form gap junctions. In this way, miRNAs will be directly delivered into the cytoplasm whilst bypassing the degradative endosomal and lysosomal environments (Objective 1). Further, to effectively load poresomes with therapeutic cargo, the PI proposes to synthesize EXOmers, RNA-based sequences that are capable of exploiting the cellular trafficking machinery used to actively sort macromolecules into poresomes. Structure activity analyses will be performed to develop highly efficient sequences (Objective 2). Third, multifunctional poresomes that target non-internalizing receptors will be engineered to mediate cellular anchoring and facilitate miRNA transport through gap junctions (Objective 3). The educational goals are to (i) increase participation of underrepresented students through more understandable, relatable, and accessible science and (ii) improve biomaterials and nanoscience education for graduate, undergraduate, and high school students. We will harness the learning advantages provided by visualization and animation to teach complex scientific subject matter. The integrative nature of our education outreach program should improve the communication skills of both graduate and undergraduate students.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

与小分子不同，包括蛋白质和核酸在内的大分子不能穿透细胞膜。然而，有效地将蛋白质和核酸输送到细胞中可以治疗一些以前无法治愈的疾病。在这里，PI建议创造包含毛孔的天然小脂泡。这将允许大分子直接输送到细胞内。为了将治疗分子装载到这些小的脂泡中，将会设计出由核酸组成的生物材料。为了确保脂泡只针对特定的细胞，这些囊泡将被装饰上可以检测到病变细胞的分子。这项研究预计将在生物材料、核酸和药物输送、纳米技术和细胞模拟材料等几个科学学科产生广泛影响。从这项提议中获得的基本知识将显著改变不透膜化合物输送到细胞的方式，特别是对大分子。这对人类健康具有重要影响，因为这些携带者应该允许更安全和有效地治疗目前无法用药治疗的疾病。该项目还将通过为研究生开发新的生物材料课程和向高中提供互动教学模块来加强国家的教育基础设施。此外，该项目寻求建立一个导师密集的计划，将促进少数族裔和女性本科生的生物材料研究。它还将建立一个为生物材料研究生、本科生和高中生提供支持的网络。技术在这份职业提案中，PI计划开发一种新型的模仿细胞的载体，称为孔体。这些孔小体将被用来将大分子运送到细胞质，同时绕过内小体。尽管纳米科学取得了巨大的进步，但带负电荷的RNA等膜不透性化合物的细胞质递送仍然具有挑战性。这是因为交付的RNA中有很大一部分仍然被困在内体中，在那里它最终会降解。这项职业计划的目标是通过设计模拟细胞的RNA负载脂泡来克服这些挑战，这些脂泡配备了由连接蛋白膜通道组成的毛孔。有人推测，携带miRNA(MiRNA)的微孔体将与受体细胞的连接蛋白膜通道连接，形成缝隙连接。通过这种方式，miRNAs将被直接输送到细胞质中，同时绕过降解的内体和溶酶体环境(目标1)。此外，为了有效地将治疗货物装载到孔体中，PI建议合成EXOmer，这是一种基于RNA的序列，能够利用细胞运输机制将大分子主动分类到孔体中。将进行结构活性分析，以开发高效序列(目标2)。第三，以非内化受体为靶标的多功能孔体将被设计成介导细胞锚定并促进miRNA通过缝隙连接的运输(目标3)。教育目标是(I)通过更容易理解、更相关和更容易获得的科学来增加未被充分代表的学生的参与，以及(Ii)改善对研究生、本科生和高中生的生物材料和纳米科学教育。我们将利用可视化和动画提供的学习优势来教授复杂的科学主题。我们的教育推广计划的综合性应该提高研究生和本科生的沟通技能。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

项目成果

MiR-101a loaded extracellular nanovesicles as bioactive carriers for cardiac repair.

• DOI: 10.1016/j.nano.2020.102201
• 发表时间: 2020-07
• 期刊: Nanomedicine : nanotechnology, biology, and medicine
• 作者: Wang J;Lee CJ;Deci MB;Jasiewicz N;Verma A;Canty JM;Nguyen J
• 通讯作者: Nguyen J

Boosting the Biogenesis and Secretion of Mesenchymal Stem Cell-Derived Exosomes

• DOI: 10.3390/cells9030660
• 发表时间: 2020-03-01
• 期刊: CELLS
• 影响因子: 6
• 作者: Wang, Jinli;Bonacquisti, Emily E.;Nguyen, Juliane
• 通讯作者: Nguyen, Juliane