Novel Self Assembly of siRNA for Efficient and Safe Delivery

[0933966]本提案描述了纳米技术和生物医学工程跨学科研究、教育和推广的综合方法。该研究的目的是开发一种创新且无毒的递送平台，使小干扰rna （sirna）的细胞特异性递送能够在体外和体内沉默其靶向癌基因。本研究的另一个目的是寻求对siRNA纳米颗粒及其生物性能的基本物理化学特性，特别是纳米力学特性的广泛理解。为了达到这些目标，该提案有三个相关的具体目标：(1)开发一种新的方法来组装和传递siRNA的无毒性，使用一些低代树突修饰的不稳定的金纳米颗粒。(2)对siRNA纳米颗粒的表面进行工程化处理，以实现目标递送，并研究单个siRNA纳米颗粒的力学性能（包括包裹和不包裹金纳米颗粒，以及工程化和非工程化）。(3)确定siRNA纳米颗粒在体外和体内的结构/性质-生物分布、生物效力和毒性关系。知识优势：一种新的siRNA组装方法，其中Au纳米颗粒（Au NPs）将用于显着增强无毒，低代树状大分子，以有效地将siRNA凝聚成离散的纳米颗粒。然而，Au NPs可以被控制在最终siRNA复合物的“内”或“外”，这是与之前报道的关键区别。通过在siRNA复合物递送之前选择性去除Au NPs，可以解决Au NPs伴随的潜在毒性问题。此外，为了满足siRNA通过全身途径在体内靶向递送的要求，形成的siRNA纳米颗粒将通过一层一层的模块化方法进行设计，使其能够在特定的亚细胞区室中进行空间和时间控制的释放。这些特性将增加额外的治疗活性，并进一步减少基于rnai的治疗的副作用。除了物理化学性质外，不同配方的siRNA纳米颗粒的纳米力学性质将通过单力显微镜进行研究。通过结合这些siRNA纳米颗粒的生物学研究，本研究将首次将siRNA纳米颗粒的物理化学性质和纳米力学性质与其细胞内化、循环、生物分布，以及它们在体外和体内全身给药过程中的靶向性和治疗效果联系起来。对这些关系的更好理解将导致未来设计和开发新的材料和策略，以有效和安全地递送siRNA，从而有助于实现其全部治疗潜力。这项研究的成功完成也将为我们如何利用由工程无机纳米载体（相对硬）构建的各种多功能纳米医学平台获得的信息来指导高效有机纳米载体（相对软）的开发提供关键的理解，反之亦然。更广泛的影响：拟议的研究对有效和安全的siRNA递送具有根本和实际的重要意义。该提案的重点是设计多功能siRNA纳米颗粒，能够细胞特异性递送和沉默EZH2基因的基因表达，用于乳腺癌治疗。鉴于siRNA在许多基础和治疗应用中的广泛应用，从该项目中获得的知识将对制药和生物技术产业以及卫生保健产生深远的科学和经济影响。这项教育计划将把纳米科学的工具和概念带给全国最多样化的罗格斯大学两个校区的广大学生。本研究固有的跨学科性质将培养出在纳米技术、生物医学工程、分子生物学和药物输送方面受过特殊训练的学生。为本科生和高中生设计的研究活动将促进更多有天赋的少数民族学生进入纳米科学行列。在少数民族占主导地位的纽瓦克地区进行广泛的推广，将提高公众对纳米科学和纳米技术影响的认识。

0933966HeThis proposal describes an integrated approach for interdisciplinary research, education, and outreach in nanotechnology and biomedical engineering. The objective of the research is to develop an innovative and nontoxic delivery platform that will enable cell specific delivery of small interference RNAs (siRNAs) to silence their targeted oncogenes both in vitro and in vivo. The other objective of this research is to seek an extensive understanding of the fundamental physicochemical characteristics, especially nanomechanical properties, of siRNA nanoparticles with their biological performance. To reach these goals, the proposal has three associated specific aims: (1) To develop a novel approach for assembly and delivery of siRNA without toxicity using labile Au nanoparticles modified with several low generation dendrimers. (2) To engineer the surface of siRNA nanoparticles for target delivery, and to study the mechanical properties of the individual siRNA nanoparticles (both with and without Au nanoparticles encapsulated, and both engineered and non-engineered). (3) To determine structure/property-biodistribution, biological potency, and toxicity relationships of the siRNA nanoparticles in vitro and in vivo. Intellectual Merit: A novel siRNA assembly approach where Au nanopartices (Au NPs) will be used to dramatically enhance non toxic, low-generation dendrimers to efficiently condense siRNA to discrete nanoparticles. However, the Au NPs can be controlled "in" or "out" of the final siRNA complexes, which is the key difference from previous reports. The potential toxic problem accompanied with the Au NPs will be solved by selectively removing the Au NPs before the siRNA complexes are delivered. In addition, to satisfy the requirements for in vivo targeted delivery of siRNA through a systemic route, the formed siRNA nanoparticles will be engineered by a layer-by-layer modular approach to enable them for spatially- and temporally- controlled release in specific sub-cellular compartments. These properties will add additional therapeutic activities and further decrease the side effects of RNAi-based therapy.In addition to the physicochemical properties, the nanomechanical properties of the individual siRNA nanoparticles from various formulations will be studied by single force microscopy. By combining the biological investigation of these siRNA nanoparticles, this proposal will link, for the first time, the physicochemical properties and the nanomechanical properties of the siRNA nanoparticles with their cellular internalization, circulation, biodistribution, and therefore, their targeting and therapeutic efficacy during in vitro and in vivo systemic delivery. The improved understanding of these relationships will lead to future design and development of new materials and strategies for efficient and safe delivery of siRNA, and therefore help to realizing its full therapeutic potentials. Successful completion of this research will also provide critical understanding how we can utilize information obtained from various multifunctional nanomedicine platforms which are constructed by engineered inorganic nanocarriers (relatively hard) to guide the development of efficient organic nanocarriers (relatively soft) and vice versa. Broader Impacts: The proposed investigations are fundamentally and practically important for efficient and safe siRNA delivery. The proposal focuses on design multifunctional siRNA nanoparticles capable of cell specific delivery and silencing of gene expression of EZH2 genes for breast cancer therapy. Given the widespread applications of siRNA in numerous fundamental and therapeutic applications, the knowledge gained from this project will have far reaching scientific and economic impacts on pharmaceutical and biotechnology industry and health care. The educational plan will bring nanoscience tools and concepts to a wide range of students on two campus of Rutgers known as the most diverse in the nation. The inherently interdisciplinary nature of this research will produce students with exceptional training in nanotechnology, biomedical engineering, molecular biology, and drug delivery. Research activities designed for undergraduates and high school students will promote more gifted minority students into the nanoscience ranks. Extensive outreach to the Newark area, a minority-dominated region, will raise the public awareness of the impact of nanoscience and nanotechnology.