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，细胞内的过程，蛋白质和细胞器， 降解需要酸性溶酶体未能酸化溶酶体隔室提供积累 自噬体、脂质和相关的未消化内容物。溶酶体酸化受损， 自噬流是非酒精性脂肪肝（NAFLD）的核心。NAFLD主要发生在 中年和老年人，因为其发展的危险因素随着年龄的增长而增加。电流 药理学和分子工具能够降低溶酶体酸度（增加pH值）;然而， 存在增加溶酶体酸度（降低pH）的工具。我们取得进展的关键是利用溶酶体 积累纳米颗粒（NP），释放酸，以降低局部pH值。具体而言，我们提出了两个新的NP 增加溶酶体酸度的技术。第一种是刺激反应NP，它酸化了它的局部环境 在暴露于光时（即，光活化酸纳米颗粒，光-acNP）。第二个NP响应于 功能失调的溶酶体（5.7 - 6）释放酸，进一步降低pH至健康/正常值（4 - 5）。这些 pH活化的酸纳米颗粒（pH-acNP）是由有效的二酸组成的基于聚酯的NP， 在NP降解时，通过使用聚丁二酸（TFSA）来进一步降低pH。我们假设， 当acNP在溶酶体中积累时，acNP将通过光活化或pH活化释放酸， 从而迅速恢复溶酶体酸性和自噬通量。此外，我们假设， 修复的效果将取决于光暴露的长度和聚合物中TFSA的量， 分别重要的是，大量的初步数据支持拟议的研究， 材料和严格的实验设计建立，必要的跨学科合作， 专业知识（纳米技术，聚合物化学，细胞代谢和自噬）可以解决这些问题 假设这项两年期提案的具体目标是：目标1。评估溶酶体靶向acNP， 用光活化（light-acNP）;以及，目标2。合成并评估在100 ℃活化的溶酶体靶向acNP pH 6（pH-acNP）。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，以促进药物筛选和作用机制。从 从纳米技术的角度来看，新的纳米粒子组合物和功能将被确定，以及一种手段， 通过纳米粒子来控制关键的细胞过程。最后，这些结果将支持进一步评价 acNP在体内鼠模型中的应用，包括安全性、PK/PD和疗效研究，作为未来R 01提案的一部分 专注于NAFLD

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.