NER: Rational Design of Biodegradable Nanoparticles for Gene Delivery

CBET-0707583 Millicent O Sullivan特拉华大学：用于基因递送的可生物降解纳米颗粒的合理设计基因治疗具有彻底改变疾病治疗的潜力，但取决于成功的DNA转运和细胞内DNA递送。纳米颗粒（NP）DNA制剂原则上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NP组装技术是有问题的：通常，它们导致在生理条件下聚集的有毒制剂。NP解包装和细胞内DNA释放也是非常低效的。该项目的目标是设计和展示一种新的组装策略，用于生理稳定，环境响应的NPs定点基因递送。智力优点：基因传递途径本质上是分层的。例如，一旦NP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。所提出的递送系统将被设计为模拟该层级：（i）用于NP保护/靶向的模块将在使用部位/时间逐步引入;（ii）模块将在使用后被移除，以避免阻碍进一步的NP运输和DNA释放。这种合理的装配策略具有重要的意义。例如，在所提出的设计中，功能模块的引入和移除很容易改变。这使得能够系统地分析每个主动转运步骤对递送的总体有效性的影响：阐明参数的关键功能（例如，物理的、化学的或生物的），其控制NP通过每个基因递送屏障的转运。为了平衡可行性和新奇，将采用成熟的化学和生物模块。具体说明：NP将被构建为围绕可逆包装质粒DNA（pDNA）核心的一系列可脱落的功能性“壳”。每个壳将通过可生物降解的肽接头并入，所述肽接头被工程化以响应于靶降解位点处的环境线索而降解。该项目的目标：1）制定“最小”NP，并表征其物理，化学和生物特性作为设计参数的函数。纳米颗粒的性质对其与细胞的相互作用及其运输效率有很大影响。这个目标将探讨设计参数和NP属性之间的关系。例如，NP大小将部分地由pDNA缩合的程度控制; NP大小将进而影响细胞摄取和细胞内转运。将确定NP尺寸、机械性能和相互作用（与蛋白质和细胞）的控制参数。2)证明NP分级靶向降解的能力。该平台的新奇取决于NP响应于位点特异性线索而降解的能力。将配制含有单一可降解“壳”的NP。将验证用于监测生物降解的荧光共振能量转移（FRET）系统，并用于确定生物降解的速率和（细胞）定位。更广泛的影响：该提案提供了一个特殊的机会，通过将化学工程基本原理应用于尖端生物问题来参与和鼓励参与工程。有针对性的战略如下。外联：与特拉华州大学的同事一起，将制定一项曝光/招聘计划，以提高K 12水平对工程的理解/兴趣。课程设置：将开发一门新的选修课，以教育本科生/研究生将化学工程概念应用于药物输送和组织工程中的问题。调研：特拉华州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代表性不足的群体（如妇女）的框架。该提案将涉及主动纳米结构的主题。

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.