A novel approach for reversal of autophagic defects using lysosome-targeted nanoparticles

使用溶酶体靶向纳米颗粒逆转自噬缺陷的新方法

• 批准号: 9752911
• 负责人: MARK W. GRINSTAFF
• 金额: $ 25.48万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-04-15 至 2021-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9752911
• 关键词: Acidity; Acids; Acute; Address; Autophagocytosis; Autophagosome; Beta Cell; Butylene Glycols; Caliber; Cell Line; Cell physiology; Cells; Cellular biology; Charge; Chronic; Comorbidity; Cultured Cells; Cytosol; Data; Defect; Development; Diabetes Mellitus; Digestion; Disease; Dyes; Electron Microscopy; Endocytosis; Endosomes; Environment; Exposure to; Failure; Fluorescent Dyes; Formulation; Functional disorder; Glycols; Goals; Heart failure; Hepatocyte; Impairment; Intercellular Fluid; Investigation; Journals; Label; Lipids; Lysosomes; Mitochondria; Modeling; Monitor; Names; Obesity; Organelles; Patients; Pharmacology; Plasma; Polyesters; Proteins; Public Health; Publishing; Quality Control; Recycling; Role; Succinic Acids; Technology; Testing; Translating; design; ethylene glycol; in vivo; inhibitor/antagonist; insulin secretion; insulin sensitivity; late endosome; liver function; nanoparticle; non-alcoholic fatty liver disease; novel; novel strategies; restoration; stem; uptake

ABSTRACT Ample data support a role for impaired lysosomal acidification and reduced autophagic flux in comorbidities related to obesity, including non-alcoholic fatty liver disease (NAFLD), heart failure, and impaired insulin secretion in diabetes. Investigation of the role of impaired lysosomal acidification in the pathophysiology of NAFLD, heart failure, and diabetes has been hampered by the lack of a technology to acidify lysosomes. To facilitate mechanistic studies of acutely restoring lysosomal pH, we recently published that photoactivated release of acid from lysosome-targeted nanoparticles (NPs) provides immediate short-term restoration of optimal pH and autophagic flux. However, to translate such technology for long-term acidification and for in vivo use, there is a need to develop NPs that release acid independent of an external trigger. This proposal stems from our development of novel formulations that allow lysosome-targeted NPs to sense their entry into the lysosomes and then trigger the release of acid. To this end, we capitalized on the observation that impaired acidification, documented in various diseases, only elevates the pH by 0.6-1 units. Therefore, the pH of the dysfunctional lysosome is still significantly more acidic than the cytosol and plasma. Consequently, we designed a new NP formulation that releases acid at pH 6 and below to assure that acid release will only occur in the lysosome. We hypothesize that acid-activated-acid-release NPs (acNPs) will allow for continuous treatment and long-term restoration of lysosomal acidity and autophagic flux. In this study we will develop, test, and validate acNP in cultured cells. Our long term goal is to develop the acNPs to be used in vivo. Preliminary data show that the acNPs efficiently target lysosomes. Furthermore, preliminary data in a hepatocyte cell line demonstrate the capacity of the acNPs to restore pH and autophagic flux under conditions of impaired lysosomal function induced by chronic exposure to excess lipids. To address our hypothesis we propose the following two aims: Aim1: Synthesize and characterize lysosome targeted acNPs that activate at pH 6 where we will test the hypothesis that lysosome targeted acNPs enter the cells through endocytosis and follow them through their maturation into the lysosome, where at pH ≤6 they will trigger acid release and restore lysosomal pH and autophagic flux. And Aim 2: Determine the capacity of acNPs to enable long-term restoration of autophagic flux under conditions that impair lysosomal acidification where we will test the hypothesis that in cells treated with excess lipid environment, re-acidification of lysosomes by acNP will restore autophagic flux, mitochondrial turnover, and cellular functions including insulin sensitivity in the hepatocyte and insulin secretion in the beta cell.

抽象的 充足的数据支持溶酶体酸化受损和自噬通量减少在合并症中的作用 与肥胖相关，包括非酒精性脂肪肝病 (NAFLD)、心力衰竭和胰岛素受损 糖尿病中的分泌。溶酶体酸化受损在病理生理学中的作用的研究 由于缺乏酸化溶酶体的技术，NAFLD、心力衰竭和糖尿病一直受到阻碍。到 为了促进快速恢复溶酶体 pH 值的机制研究，我们最近发表了光激活 溶酶体靶向纳米颗粒 (NP) 释放酸可立即短期恢复最佳状态 pH 值和自噬通量。然而，为了将这种技术转化为长期酸化和体内使用， 需要开发独立于外部触发而释放酸的纳米颗粒。这项提议源于 来自我们开发的新颖配方，使靶向溶酶体的纳米粒子能够感知它们进入 溶酶体，然后触发酸的释放。为此，我们利用了损害 据记载，在各种疾病中，酸化只会使 pH 值升高 0.6-1 个单位。因此，pH值 功能失调的溶酶体的酸性仍然明显高于细胞质和血浆。因此，我们设计了 一种新的 NP 配方，可在 pH 值 6 及以下时释放酸，以确保酸释放仅发生在 溶酶体。 我们假设酸激活酸释放纳米颗粒（acNP）将允许持续治疗和 溶酶体酸度和自噬通量的长期恢复。在本研究中，我们将开发、测试和 验证培养细胞中的 acNP。我们的长期目标是开发可在体内使用的 acNP。初步数据 表明 acNP 有效地靶向溶酶体。此外，肝细胞系的初步数据 证明 acNP 在溶酶体受损的条件下恢复 pH 值和自噬通量的能力 长期接触过量脂质引起的功能。为了解决我们的假设，我们提出以下两个 目标：目标 1：合成并表征溶酶体靶向 acNP，这些 acNP 在 pH 6 时激活，我们将在该值进行测试 溶酶体靶向 acNP 通过内吞作用进入细胞并跟随它们完成其代谢过程的假设 成熟进入溶酶体，在 pH ≤ 6 时，它们将触发酸释放并恢复溶酶体 pH 值 自噬通量。目标 2：确定 acNP 实现自噬长期恢复的能力 在损害溶酶体酸化的条件下的通量，我们将测试细胞中的假设 在过量的脂质环境下，acNP对溶酶体的重新酸化将恢复自噬通量， 线粒体周转和细胞功能，包括肝细胞中的胰岛素敏感性和胰岛素分泌 在β细胞中。