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

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使许多以前无法治愈的疾病得到治疗。在这里，PI建议创建包含毛孔的天然小脂质囊泡。这将允许大分子被直接运送到细胞中。为了使这些小脂质囊泡装载治疗分子，将设计由核酸组成的生物材料。为了确保脂质囊泡只针对特定的细胞，囊泡将被修饰成可以检测病变细胞的分子。这项研究预计将在几个科学学科中产生广泛的影响，包括生物材料、核酸和药物输送、纳米技术和细胞模拟材料。从该提案中获得的基本知识将显著改变膜不渗透化合物输送到细胞的方式，特别是对于大分子。这对人类健康具有重要意义，因为这些携带者可以使目前无法治疗的疾病得到更安全、更有效的治疗。该项目还将为研究生开发新的生物材料课程，并向高中提供互动教学模块，从而加强国家的教育基础设施。此外，该项目寻求建立一个指导密集型计划，以促进少数民族和女性本科生的生物材料研究。它还将建立一个支持生物材料研究生、本科生和高中生的网络。在这份职业计划书中，PI计划开发一种新型的细胞模拟载体，称为多孔体。这些孔体绕过核内体，将大分子传递到细胞质。尽管纳米科学取得了巨大的进步，但细胞膜不渗透化合物（如带负电荷的RNA）的细胞质递送仍然具有挑战性。这是因为递送的RNA中有很大一部分被困在核内体中，最终被降解。本CAREER提案的目标是通过设计细胞模拟装载rna的脂质囊泡来克服这些挑战，这些脂质囊泡配备由连接蛋白膜通道组成的孔隙。假设microRNA（miRNA）负载的孔体将连接到受体细胞的连接蛋白膜通道，形成间隙连接。通过这种方式，mirna将直接传递到细胞质中，而绕过降解的内体和溶酶体环境（目的1）。此外，为了有效地将治疗货物装载到孔体中，PI建议合成外显子异构体，这是一种基于rna的序列，能够利用细胞运输机制，将大分子积极地分类到孔体中。将进行结构活性分析以开发高效的序列（目标2）。第三，针对非内化受体的多功能孔体将被设计成介导细胞锚定并促进miRNA通过间隙连接运输（目的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代表性不足的学生的参与；（ii）改善研究生、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质应该提高研究生和本科生的沟通技巧。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

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.