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

摘要 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（NAFLD）、心力衰竭和胰岛素受损 糖尿病的分泌。溶酶体酸化受损在肝硬化病理生理学中作用的研究 NAFLD、心力衰竭和糖尿病由于缺乏酸化溶酶体的技术而受到阻碍。到 促进急性恢复溶酶体pH的机制研究，我们最近发表了光活化 从溶酶体靶向纳米颗粒（NPs）释放酸提供了最佳的 pH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， 需要开发不依赖于外部触发而释放酸的NP。这一提议源于 从我们开发的新制剂，允许溶酶体靶向的纳米颗粒，以感觉到他们进入 然后触发酸的释放。为此，我们利用了观察结果， 在各种疾病中记录的酸化仅使pH升高0.6-1个单位。因此， 功能障碍的溶酶体仍然明显比胞质溶胶和血浆酸性更强。因此，我们设计了 一种新的NP制剂，其在pH 6及以下释放酸，以确保酸释放仅发生在 溶酶体 我们假设酸活化酸释放纳米粒（acNP）将允许连续治疗， 溶酶体酸性和自噬通量的长期恢复。在这项研究中，我们将开发，测试， 在培养的细胞中验证acNP。我们的长期目标是开发用于体内的acNP。初步数据 表明acNP有效靶向溶酶体。此外，肝细胞系的初步数据 证明acNP在受损的溶酶体损伤条件下恢复pH和自噬通量的能力。 由于长期暴露于过量的脂质而引起的功能。为了解决我们的假设，我们提出以下两个 目的：Aim 1：合成和表征在pH 6下活化的溶酶体靶向acNP，我们将在pH 6下测试 假设溶酶体靶向acNP通过内吞作用进入细胞并跟随它们通过它们的内吞作用， 在pH ≤6时，它们将触发酸释放并恢复溶酶体pH， 自噬通量目标2：确定acNP能够长期恢复自噬的能力。 在损害溶酶体酸化的条件下，我们将测试细胞中的假设， 用过量脂质环境处理，acNP对溶酶体的再酸化将恢复自噬通量， 线粒体更新和细胞功能，包括肝细胞中的胰岛素敏感性和胰岛素分泌 在β细胞中。

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.