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

摘要 大量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，包括非酒精性脂肪性肝病(NAFLD)、心力衰竭和胰岛素受损 糖尿病患者的分泌物。溶酶体酸化受损在高血压病病理生理机制中作用的研究 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。至 促进急性恢复溶酶体pH的机制研究，我们最近发表了光激活的 从溶酶体靶向纳米粒(NPs)中释放酸可立即在短期内修复最佳 PH和自噬通量。然而，为了将这种技术转化为长期酸化和活体使用， 需要开发与外部触发无关地释放酸的NPs。这项建议源于 从我们开发的允许溶酶体靶向NPs感觉到它们进入 溶酶体，然后触发酸的释放。为此，我们利用了观察到的损害 酸化在各种疾病中都有记载，它只会使PH值升高0.6-1个单位。因此，水中的pH值 功能障碍的溶酶体比胞浆和胞浆的酸性要强得多。因此，我们设计了 一种新的NP配方，在pH 6及以下释放酸，以确保酸只会在 溶酶体。 我们假设酸激活的酸释放NPs(AcNPs)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 在培养的细胞中验证ACNP。我们的长期目标是开发用于活体的acNPs。初步数据 表明acNPs有效地靶向溶酶体。此外，肝细胞系的初步数据 证明在溶酶体受损的条件下，acNPs恢复pH和自噬通量的能力 因长期接触过多的脂类而引起的功能障碍。为了解决我们的假设，我们提出了以下两个假设 目的：Aim1：合成和表征溶酶体靶向acNPs，这些溶酶体靶向acnps在pH值为6时激活，我们将在那里进行测试 假设溶酶体靶向acNPs通过内吞作用进入细胞，并跟随细胞通过其 成熟到溶酶体，在pH≤6时，它们会触发酸释放，恢复溶酶体的pH和 自噬通量。和目标2：确定acNPs的能力，以实现自噬的长期恢复 在损害溶酶体酸化的条件下的通量，我们将在那里测试细胞中的假设 在高脂环境下，ACNP对溶酶体的重新酸化将恢复自噬通量， 线粒体周转率和细胞功能，包括肝细胞中的胰岛素敏感性和胰岛素分泌 在贝塔细胞中。

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.