Endosomal escape of lipid-based nanoparticles comprising Gaussian curvature lipids

包含高斯曲率脂质的基于脂质的纳米粒子的内体逃逸

• 批准号: 10798629
• 负责人: Cecilia Leal
• 金额: $ 16.79万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-06-10 至 2026-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10798629
• 关键词: Acoustics; COVID-19 pandemic; COVID-19 vaccine; Cell Therapy; Cells; Cholesterol; Clinical Trials; Computer software; Cytosol; Development; Disease; Drug Delivery Systems; Endosomes; Ensure; Equipment; Event; FDA approved; Flow Cytometry; Formulation; Gene Delivery; Genetic Diseases; Goals; Grant; Health; Human; Image; Image Enhancement; Individual; Intake; Lipids; Location; Malignant Neoplasms; Membrane; Messenger RNA; Morphology; Nanostructures; Organism; Pathway interactions; Phospholipids; Population; Property; RNA; RNA delivery; Records; Research; Research Support; Resolution; Speed; System; Testing; Therapeutic Studies; Virus; Work; chronic infection; delivery vehicle; design; endosome membrane; experimental study; gene therapy; insight; instrument; lipid nanoparticle; monoolein; novel; particle; success; therapeutic RNA

PROJECT SUMMARY We propose the acquisition of a new image-enhanced flow cytometer (Attune CytPix) to support the research of 1R01GM143723-01A1: Endosomal escape of lipid-based nanoparticles comprising Gaussian curvature lipids. RNA-based therapies offer significant potential for treating a variety of diseases that have a major impact on human health. These include chronic infections, genetic disorders, specific cancers, and the current COVID-19 pandemic. Non-viral lipid-based nanoparticles (LNPs) are the primary RNA delivery vehicles approved by the FDA and are also being evaluated in numerous clinical trials. LNPs are composed of standard phospholipids, cholesterol, and ionizable lipids (ILs) that become protonated in acidic conditions. Similar to enveloped viruses, LNPs exploit the endocytic pathway to gain entry into cells. The success of RNA delivery depends on the ability of LNPs to fuse with the endosomal membrane and escape the endosome. However, the mechanisms that govern LNP-endosome fusion remain largely unknown. The central goal of 1R01GM143723-01A1 is to test the hypothesis that the inclusion of a new class of structural lipids with single chains and small headgroups like glycerol monooleate onto state-of-the art LNP formulations allows us to prescribe well-defined internal nanostructures of LNPs directly impacting their ability to fuse with endosomal membranes and releasing RNA cargo into the cytosol. Acquiring the new Attune CytPix equipment is vital to the progression of our project for two main reasons. Firstly, the only flow cytometry instrument in close proximity to our primary research location is currently non-operational due to frequent breakdowns and high user demand, resulting in prolonged periods of downtime. Secondly, the Attune CytPix offers simultaneous high throughput flow cytometry and high resolution brightfield imaging through acoustic focusing, which is not available with our current equipment. This feature is particularly important for quantifying LNP cargo loading and delivery efficiency. The high-speed brightfield camera records individual events as they pass through the flow cell, and the Attune Cytometric Software ensures that analyzed events originate from single cells and particles rather than doublets, clumps, or debris. This capability is critical in cell and gene therapy research, as well as other flow cytometry experiments that aim to understand the morphology of each cell population. Through our research, we aim to uncover novel physical insights into the endosomal escape of LNPs and determine the optimal membrane properties of LNPs to enhance fusion in living systems. This will lead to the development of RNA delivery vehicles that are significantly more effective in delivering their cargo.

项目摘要 我们建议购买一种新的图像增强流式细胞仪（Attune CytPix），以支持以下研究： 1 R 01 GM 143723 - 01 A1：包含高斯曲率脂质的基于脂质的纳米颗粒的内体逃逸。 基于RNA的疗法为治疗多种疾病提供了巨大的潜力， 人体健康其中包括慢性感染、遗传性疾病、特定癌症以及目前的COVID-19 流行病基于非病毒脂质的纳米颗粒（LNP）是由FDA批准的主要RNA递送载体。 FDA和也正在许多临床试验中进行评估。LNP由标准磷脂组成， 胆固醇和在酸性条件下质子化的可电离脂质（IL）。与包膜病毒类似， LNP利用内吞途径进入细胞。RNA递送的成功取决于 的LNP与内体膜融合并逃离内体。然而， 控制LNP-内体融合的基因在很大程度上仍然未知。 1 R 01 GM 143723 - 01 A1的中心目标是检验以下假设： 将具有单链和小头部基团的脂质（如甘油单油酸酯）添加到最先进的LNP制剂中 使我们能够规定明确的内部纳米结构的LNP直接影响他们的能力，融合 内体膜和释放RNA货物进入胞质溶胶。 获得新的Attune CytPix设备对我们项目的进展至关重要，主要有两个原因。第一、 在我们主要研究地点附近唯一的流式细胞仪目前是不能使用的 由于频繁的故障和高用户需求，导致停机时间延长。二是 Attune CytPix提供同步高通量流式细胞术和高分辨率明场成像， 声聚焦，我们目前的设备不具备这一功能。这一特点对于 量化利比里亚国家警察货物装载和交付效率。高速明场摄像机记录下 Attune Cytometric软件可确保分析的事件 起源于单个细胞和颗粒，而不是双联体、团块或碎片。这种能力在细胞中至关重要 和基因治疗研究，以及其他旨在了解形态学的流式细胞术实验， 每个细胞群体。 通过我们的研究，我们的目标是揭示新的物理见解内体逃逸的LNP和 确定LNP的最佳膜特性，以增强生命系统中的融合。这会导致 开发RNA递送载体，其在递送其货物方面显著更有效。