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

基本信息

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
项目类别：
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

项目摘要

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有关，包括非酒精性脂肪肝病 (NAFLD)、心力衰竭和胰岛素受损研究溶酶体酸化受损在糖尿病病理生理学中的作用。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。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粒子 (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。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，我们利用了受损的观察结果。据记载，在各种疾病中，酸化只会使 pH 值升高 0.6-1 个单位。我们设计的功能失调的溶酶体的酸性明显高于所测试的细胞质和血浆。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。我们追求酸激活酸释放纳米颗粒 (acNP) 能够实现持续治疗和在这项研究中，我们将开发、测试和研究溶酶体酸度和自噬通量的长期恢复。我们的长期目标是开发用于体内的 acNP 的初步数据。此外，肝细胞系中的初步数据表明 acNP 能有效靶向溶酶体。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为了解决我们的假设，我们提出以下两个假设。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。