NER: Rational Design of Biodegradable Nanoparticles for Gene Delivery

NER：用于基因传递的可生物降解纳米颗粒的合理设计

• 批准号: 0707583
• 负责人: Millicent Sullivan
• 金额: --
• 依托单位: University of Delaware
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-07-15 至 2009-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0707583&HistoricalAwards=false
• 关键词: NER; Rational; Design; Biodegradable; Nanoparticles

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.

CBET-0707583米利森特·O·沙利文特拉华大学NER：用于基因传递的可生物降解纳米粒子的合理设计基因疗法具有彻底改变疾病治疗的潜力，但取决于成功的 DNA 运输和细胞内 DNA 传递。原则上，纳米颗粒 (NP) DNA 配方是基因治疗的理想选择：NP 足够小，可以被细胞摄取并进入分子级运输机制，但又足够大，可以包含全长基因以及细胞和细胞内靶向部分。不幸的是，常见的静电纳米粒子组装技术存在问题：它们常常会产生在生理条件下聚集的有毒制剂。 NP 解包和细胞内 DNA 释放效率也很低。该项目的目标是设计并展示一种新颖的组装策略，用于生理稳定、环境响应的纳米粒子，用于定点基因传递。智力价值：基因传递途径本质上是分层的。例如，一旦纳米粒子到达其靶细胞，它必须依次穿过质膜、离开内体、穿过细胞质、进入细胞核并解包。拟议的交付系统将被设计为模仿这种层次结构：（i）用于 NP 保护/瞄准的模块将在使用地点/时间逐步引入； (ii) 使用后模块将被移除，以避免阻碍进一步的 NP 运输和 DNA 释放。这种合理的组装策略具有重要的影响。例如，在所提出的设计中，功能模块的引入和删除很容易改变。这使得能够系统分析每个主动运输步骤对递送整体有效性的影响：这是阐明控制 NP 通过每个基因递送屏障运输的参数（例如物理、化学或生物）的关键功能。为了平衡可行性与新颖性，将采用成熟的化学物质和生物模块。具体细节：纳米颗粒将被构建为一系列可脱落的功能性“壳”，围绕可逆包装的质粒 DNA (pDNA) 核心。每个壳将通过可生物降解的肽接头结合在一起，这些肽接头被设计为响应目标降解位点的环境线索而降解。该项目的目标： 1) 配制“最小”纳米颗粒，并根据设计参数表征其物理、化学和生物特性。纳米粒子的性质对其与细胞的相互作用及其运输效率有很大影响。该目标将探讨设计参数与 NP 特性之间的关系。例如，NP 尺寸将部分由 pDNA 缩合程度控制；纳米粒子的大小反过来会影响细胞的摄取和细胞内的运输。纳米粒子尺寸、机械性能和相互作用（与蛋白质和细胞）的控制参数将被确定。 2) 展示纳米颗粒分级、定向降解的能力。该平台的新颖性取决于纳米颗粒响应特定位点线索而降解的能力。将配制含有单一可降解“壳”的纳米粒子。用于监测生物降解的荧光共振能量转移（FRET）系统将得到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：该提案提供了一个绝佳的机会，通过将化学工程基础知识应用于前沿的生物问题来参与和鼓励参与工程。以下策略是有针对性的。外展活动：将与特拉华大学的同事一起制定一个接触/招聘计划，以增加 K 12 级别对工程的理解/兴趣。课程：将开发一门新的选修课程，以教育本科生/研究生应用化学工程概念解决药物输送和组织工程问题。研究：特拉华生物技术研究所的多学科互动以及该大学的生物技术 IGERT 计划将被用作积极招募女性等代表性不足的群体从事工程工作的框架。该提案将讨论活性纳米结构的主题。