R21: Acidic Nanoparticles for Restoration of Autophagy in Age-associated NAFLD

摘要 这项探索性的R21提案描述了两种新的纳米技术，用于溶酶体的酸化和 自噬通量的恢复。自噬，蛋白质和细胞器在细胞内的过程 降解，需要酸性溶酶体。不能酸化溶酶体隔室会导致 自噬小体、脂类和相关的未消化内容物。溶酶体酸化受损和减少 自噬通量是非酒精性脂肪性肝病(NAFLD)的核心。非酒精性脂肪肝主要发生在 中老年人发病的风险因素随着年龄的增长而增加。当前 药物和分子工具能够降低溶酶体的酸度(增加pH)；然而，NO 有工具可以增加溶酶体的酸度(降低pH)。我们进步的关键是溶酶体的使用 积累纳米颗粒(NPs)，释放酸来降低当地的PH值。具体地说，我们提出两个新NP 增加溶酶体酸度的技术。第一种是刺激反应的NP，它会使当地环境酸化。 暴露在光下(即，光激活的酸纳米颗粒，光-ACNP)。第二个NP响应于 一种功能失调的溶酶体(5.7-6)，以释放酸，进一步将pH降低到健康/正常值(4-5)。这些 PH活化酸纳米粒(pH-acNPs)是由一种有效的二元酸组成的聚酯基纳米粒， 四氟琥珀酸(TFSA)，以进一步降低NP降解时的pH。我们假设在 在溶酶体中积累，acNPs将通过光激活或pH激活释放酸，并 从而迅速恢复溶酶体的酸性和自噬通量。此外，我们还假设， 修复的程度将取决于光暴露的长度和聚合物中TFSA的量， 分别进行了分析。重要的是，大量的初步数据支持拟议的研究，具有良好的特点 建立了材料和严格的实验设计，以及基本的跨学科合作和 专业知识(纳米技术、聚合物化学、细胞新陈代谢和自噬)已经到位来解决这些问题。 假设。这项为期两年的提案的具体目标是：目标1.评估溶酶体靶向acNPs 用光激活(light-acNPs)；以及，目标2.合成和评估溶酶体靶向acNPs PH 6(pH-acNPs)。该提案的成功完成将为科学家、临床医生和 生物医学工程师研究自噬生物学，便于制剂筛选和作用机理(S)。从… 从纳米技术的角度来看，将确定新的纳米颗粒组合物和功能以及一种手段 通过纳米颗粒控制一个关键的细胞过程。最后，这些结果将支持对 在活体小鼠模型中使用AcNPs，将安全性、PK/PD和疗效研究作为未来R01提案的一部分 重点关注非酒精性脂肪肝。

ABSTRACT This exploratory R21 proposal describes two novel nanotechnologies for acidification of the lysosome and for restoration of autophagic flux. Autophagy, the intracellular process by which proteins and organelles are degraded, requires an acidic lysosome. Failure to acidify the lysosomal compartment affords accumulation of autophagosomes, lipids, and associated undigested content. Impaired lysosomal acidification and reduced autophagic flux are central to non-alcoholic fatty liver disease (NAFLD). NAFLD occurs primarily among the middle-aged and the elderly given that the risk factors for its development increase with advancing age. Current pharmacological and molecular tools are able to reduce lysosomal acidity (increase pH); however, no tools exist to increase lysosomal acidity (decrease pH). Key to our advance is the use of lysosomal accumulating nanoparticles (NPs) that release acid to lower the local pH. Specifically, we propose two new NP technologies to increase lysosomal acidity. The first is a stimuli-responsive NP that acidifies its local environment upon exposure to light (i.e., light activated acid nanoparticle, light-acNP). The second NP responds to the pH of a dysfunctional lysosome (5.7 - 6) to release acid further lowering the pH to a healthy/normal value (4 - 5). These pH-activated acid nanoparticles (pH-acNPs) are polyester-based NPs composed of a potent diacid, tetrafluorosuccinic acid (TFSA), to further lower the pH upon NP degradation. We hypothesize that upon accumulation in lysosomes, the acNPs will release acid through light activation or pH-activation and thereby restore lysosomal acidity and autophagic flux quickly. Further, we hypothesize that the extent of restoration will depend on the length of light exposure and the amount of TFSA in the polymer, respectively. Importantly, substantial preliminary data support the proposed studies, well-characterized materials and rigorous experimental designs are established, and essential cross-disciplinary collaborations and expertise (nanotechnology, polymer chemistry, cell metabolism, and autophagy) are in place to address these hypotheses. The specific aims of this two-year proposal are: Aim 1. Evaluate lysosome-targeted acNPs that activate with light (light-acNPs); and, Aim 2. Synthesize and evaluate lysosome-targeted acNPs that activate at pH 6 (pH-acNPs). Successful completion of the proposal will provide two new tools for scientists, clinicians, and biomedical engineers to study autophagy biology to facilitate agent screening and mechanism(s) of action. From a nanotechnology perspective, new nanoparticle compositions and functions will be identified as well as a means to control a key cellular process via a nanoparticle. Finally, these results would support further evaluation of the acNPs in in vivo murine models to include safety, PK/PD, and efficacy studies as part of a future R01 proposal focused on NAFLD.