NER: Rational Design of Biodegradable Nanoparticles for Gene Delivery

CBET-0707583 Millicent O Sullivan DelawareNER：用于基因传递的生物可降解纳米颗粒的合理设计基因治疗具有革命性疾病治疗的潜力，但依赖于成功的DNA转运和细胞内DNA输送。纳米粒(NP)DNA制剂原则上是基因治疗的理想选择：纳米粒小到足以被细胞摄取并进入分子尺度的转运机制，但大到足以包含全长基因以及细胞和细胞内靶向部分。不幸的是，常见的静电NP组装技术是有问题的：它们经常导致在生理条件下聚集的有毒制剂。NP的拆解和细胞内DNA的释放也是非常低效的。这个项目的目标是设计和展示一种新的组装策略，用于定点基因传递的生理稳定的、环境响应的NPs。智力优势：基因传递途径具有内在的层次性。例如，一旦NP到达其目标细胞，它必须顺序地穿过质膜，离开内体，穿过细胞质，进入细胞核，然后解包。拟议的递送系统将被设计为模仿这一层次：(1)将在使用地点/时间分步引入NP保护/靶向模块；(2)使用后将移除模块，以避免阻碍NP的进一步传输和DNA的释放。这种理性的集结策略产生了重要的后果。例如，功能模块的引入和移除很容易在拟议的设计中进行更改。这使得能够系统地分析每一个活跃的运输步骤，以了解传递的整体有效性：这是阐明控制NP通过每个基因传递屏障的参数(例如，物理、化学或生物)的关键功能。为了平衡可行性和新颖性，将使用成熟的化学和生物模块。具体内容：NPs将被构建成一系列可剥离的、有功能的“壳”，围绕着可逆包装的质粒DNA(PDNA)的核心。每个壳层将通过可生物降解的多肽连接物结合在一起，这些多肽连接物被设计成根据目标降解部位的环境线索进行降解。本项目的目标是：1)制定“最低限度的”核动力源，并根据设计参数表征它们的物理、化学和生物特性。纳米粒子的性质对其与细胞的相互作用和运输效率有很大的影响。这一目标将探索设计参数与NP性质之间的关系。例如，NP的大小将部分受PDNA缩合程度的控制；NP的大小反过来会影响细胞的摄取和细胞内的运输。将确定NP大小、机械性能和(与蛋白质和细胞)相互作用的控制参数。2)展示核动力源的分级、定向降级的能力。这个平台的新颖性取决于NP对特定地点线索的反应能力。含有单一可降解“壳”的NPS将被配制。用于监测生物降解的荧光共振能量转移(FRET)系统将被验证并用于确定生物降解的速率和(细胞)定位。更广泛的影响：这项建议提供了一个特殊的机会，通过将化学工程基础应用于前沿生物学问题来参与和鼓励参与工程。以下是有针对性的战略。外展：与特拉华大学的同事一起，将开发一个暴露/招聘计划，以增加对K12级工程的了解/兴趣。课程：将开发一门新的选修课，教育本科生/研究生如何将化学工程概念应用于药物输送和组织工程中的问题。研究：特拉华州生物技术研究所的多学科互动以及与该大学生物技术IGERT计划的互动将被用作积极招募女性等代表性不足的群体进入工程学的框架。这项提案将涉及活性纳米结构的主题。

CBET-0707583Millicent O SullivanUniversity of DelawareNER: Rational Design of Biodegradable Nanoparticles for GeneDeliveryGene therapy holds the potential to revolutionize disease treatment, but depends upon successful DNA transport and intracellular DNA delivery. Nanoparticle (NP) DNA formulations are in principle ideal for gene therapy: NPs are small enough to be ingested by cells and to access molecular-scale transport mechanisms, but large enough to contain full-length genes as well as cellular and intracellular targeting moieties. Unfortunately, common electrostatic NP assembly techniques are problematic: frequently, they result in toxic formulations that aggregate under physiological conditions. NP unpackaging and intracellular DNA release are also highly inefficient. The goal of this project is to design and demonstrate a novel assembly strategy for physiologically stable, environmentally responsive NPs for site-directed gene delivery. Intellectual merit: The gene delivery pathway is inherently hierarchical. For example, once an NP has reached its target cell, it must sequentially cross the plasma membrane, exit the endosome, traverse the cytoplasm, enter the nucleus, and unpackage. The proposed delivery system will be designed to mimic this hierarchy: (i) modules for NP protection/targeting will be introduced step-wise, at the site/time of use; (ii) modules will be removed following use to avoid hindering both further NP transport and DNA release. This rational assembly strategy has important consequences. For example, the introduction and removal of functional modules is easily altered in the proposed design. This enables the systematic analysis of each active transport step on the overall effectiveness of delivery: a critical function for elucidating the parameters (e.g., physical, chemical, or biological) that govern NP transport through each gene delivery barrier. To balance feasibility with novelty, well-established chemistries and biomodules will be employed. Specifics: The NPs will be constructed as a series of sheddable, functional "shells" surrounding a core of reversibly-packaged plasmid DNA (pDNA). Each shell will be incorporated via biodegradable peptide linkers engineered to degrade in response to environmental cues at the target degradation site. The objectives of this Project: 1) To formulate "minimal" NPs and characterize their physical, chemical, and biological properties as a function of the design parameters. The properties of NPs have a strong influence on their interactions with cells and the efficiency of their transport. This objective will explore the relationship between design parameters and NP properties. For example, NP size will be controlled in part by the degree of pDNA condensation; NP size will in turn affect cellular uptake and intracellular transport. The controlling parameters for NP size, mechanical properties, and interactions (with proteins and cells) will be determined. 2) To demonstrate the capacity of the NPs for hierarchical, targeted degradation. The novelty of this platform depends upon the capacity of the NPs to degrade in response to site-specific cues. NPs containing a single degradable "shell" will be formulated. A fluorescence resonance energy transfer (FRET) system for monitoring biodegradation will be validated and used to determine the rate and (cellular) localization of biodegradation. Broader impacts: This proposal provides an exceptional opportunity to engage and encourage participation in engineering by the application of chemical engineering fundamentals to a cutting-edge biological problem. The following strategies are targeted. Outreach: With colleagues at the University of Delaware, an exposure/recruitment program will be developed to increase understanding/interest in engineering at the K 12 level. Curriculum: A new elective course will be developed to educate undergraduate/graduate students on the application of chemical engineering concepts to problems in drug delivery and tissue engineering. Research: Multidisciplinary interactions at the Delaware Biotechnology Institute and with the university's biotechnology IGERT program will be used as a framework for the active recruitment of underrepresented groups such as women to engineering. This proposal will address the theme of active nanostructures.