NER: Rational Design of Biodegradable Nanoparticles for Gene Delivery

millicent O sullivan特拉华大学：合理设计用于基因传递的可生物降解纳米颗粒基因治疗具有彻底改变疾病治疗的潜力，但这取决于成功的DNA转运和细胞内DNA传递。纳米颗粒（NP） DNA配方原则上是基因治疗的理想选择：纳米颗粒足够小，可以被细胞吸收并进入分子尺度的运输机制，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：它们经常导致在生理条件下聚集的有毒配方。NP解包装和细胞内DNA释放也非常低效。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、环境敏感的NPs，用于位点定向基因传递。智力优势：基因传递途径具有内在的等级性。例如，一旦NP到达目标细胞，它必须依次穿过质膜，离开核内体，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成模仿这种层次结构：(i) NP保护/靶向模块将在使用地点/时间逐步引入；（ii）模块将在使用后移除，以避免阻碍NP的进一步运输和DNA的释放。这种合理的装配策略具有重要的意义。例如，在提议的设计中，功能模块的引入和移除很容易改变。这使得系统地分析每个主动运输步骤的整体递送有效性：阐明通过每个基因递送屏障控制NP运输的参数（例如，物理，化学或生物）的关键功能。为了平衡可行性和新颖性，将采用成熟的化学和生物模块。具体内容：NPs将被构建为一系列可脱落的、功能性的“壳”，围绕着一个可逆包装的质粒DNA （pDNA）核心。每个外壳将通过可生物降解的肽连接物结合在一起，以响应目标降解位点的环境线索进行降解。该项目的目标是：1)制定“最小”NPs，并描述其物理、化学和生物特性作为设计参数的函数。NPs的性质对它们与细胞的相互作用和运输效率有很大的影响。本目标将探讨设计参数和NP属性之间的关系。例如，NP大小将部分由pDNA缩聚程度控制；NP大小又会影响细胞摄取和细胞内运输。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)证明NPs具有分层、定向降解的能力。该平台的新颖性取决于NPs在响应特定位点线索时降解的能力。将配制含有单一可降解“壳”的NPs。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：本提案提供了一个特殊的机会，通过应用化学工程基础知识来解决前沿的生物学问题，来参与和鼓励参与工程。以下策略是有针对性的。外展：与特拉华大学的同事一起，将开发一个曝光/招聘计划，以增加对K - 12级别工程的理解/兴趣。课程设置：开设一门新的选修课程，教育本科生/研究生如何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项目将被用作积极招募代表性不足的群体（如妇女）进入工程领域的框架。本提案将讨论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.