Engineering and study of a new multifunctional sensing nanoprobe for direct and rapid delivery of cargo to cytosol, reporting on the delivery event, and quantification of delivered

1159500 GesquireGoal.拟议的研究计划的目标是设计一种OFF/ON生物传感探针，该探针将专门设计用于直接细胞摄取到细胞质，将报告细胞内环境中货物的递送和相关的递送位置，并将提供有关细胞内环境中递送的货物量的定量数据。该生物传感器将这些多功能集成在一个一体化设计中。纳米生物传感器和多功能纳米颗粒货物输送系统的发展导致了最近的重大进展，在生物医学研究。然而，关于这些发展，许多问题仍未解决，包括（i）缺乏从这些纳米生物传感器获得的定量数据，（ii）缺乏对细胞毒性/生物相容性的研究，（iii）缺乏对细胞摄取机制的理解，以及（iv）缺乏对这些纳米生物传感器在使用后的命运（例如，崩解、在胞质溶胶中聚集）的研究和理解。特别是对于具有瞄准或/和感测功能的货物递送系统，通常不清楚递送是否成功。在本文中，将开发新的多功能纳米生物传感平台，其将解决（i）设计用于直接摄取和递送到胞质溶胶的生物传感器颗粒的开发，（ii）感测细胞内递送事件，即报告成功递送，和（iii）定量感测递送的货物的量，同时研究（iv）生物传感器的细胞毒性，和（v）作为细胞摄取机制的函数的生物传感器的命运。所提出的方案将产生基本的洞察力，从这样一个系统的设计规则的功能/应用程序和命运的建议的生物传感器颗粒在体外细胞水平。本提案中提出的方法在以下几个方面是独特和变革性的：（i）感测和量化能力不限于特定的染料-目标对（例如，Ca2+比率成像需要使用专门设计的染料），因为报告功能被NAC-货物构建体覆盖，其中NAC（N-乙酰基-L-半胱氨酸）充当QDot猝灭剂，即该概念广泛适用于报告可连接至NAC的不同货物类型的递送和定量，（ii）所有功能都内置于生物传感器颗粒中，因此无需额外的染色来报告递送或定量递送的货物（一体化设计）。将把研究、教育和外联结合起来，以便以包容的方式为提高对STEM的认识和发展学生、教师和广大社区的STEM技能作出重大贡献。将参与这项研究的学生将在化学，材料，细胞生物学和纳米技术的跨学科领域进行培训。与拟议的研究项目相关的主题将被整合到课程和实验室中。研究生和本科生的招聘和培训将通过REU项目和NanoFlorida研讨会（www.example.com）进行。NanoFlorida是一个跨佛罗里达州的学生组织的会议。来自代表性不足的群体的学生已经占了这项提议的调查人员实验室的一半以上。通过NSF进一步招募少数民族？s佛罗里达格鲁吉亚路易斯斯托克斯少数民族参与工程联盟和纳米佛罗里达会议的组织。一个主要的更广泛的影响组成部分是拟议的社区外展计划与奥兰多科学中心（OSC）合作。OSC？的扫描电子显微镜（SEM）位于展厅的一个房间将提供给UCF研究生参与教育公众对我们的研究活动。通过在他们的工作中使用这个设施，研究生将学习与人合作的重要性，以及如何与人沟通的外行，他们不能在大学环境中轻松体验的东西。将开发实验室活动和在线学习模块，儿童和家长可以互动，共同学习纳米科学和生物技术。这些活动将面向广大受众。这些举措将作为一系列长期计划的开端，这些计划将导致在OSC创建大量纳米科学和生物学/生物技术相关的展览和教育材料。成功后，OSC将启用PI？s进入中央佛罗里达学校系统，以便将科学教育带到教室。考虑到中佛罗里达？的人口统计，学校的目标往往符合？多数少数？历史上的黑人院所

1159500GesquiereGoal. The goal of the proposed research program is to engineer an OFF/ON biosensing probe that will be specifically designed for direct cellular uptake to the cytoplasm, will report on the delivery of cargo in the intracellular environment and the associated location of delivery, and will provide quantitative data on the amount of cargo delivered in the intracellular environment. The biosensor will integrate these multiple functionalities in an all-in-one design.Intellectual Merit. The development of nanobiosensors and multifunctional nanoparticle cargo delivery systems has lead to recent significant advances in biomedical research. However, many issues have remained unresolved with respect to these developments, including (i) a lack of quantitative data obtained from these nanobiosensors, (ii) a lack of study of the cytotoxicity/biocompatibility, (iii) a lack of understanding of the cellular uptake mechanism, and (iv) a lack of study and understanding of the fate (e.g. disintegration, aggregation in cytosol) of these nanobiosensors after use. Specifically for cargo delivery systems with targeting or/and sensing functionality, it is often not clear if the delivery was successful. Herein, a new multifunctional nanobiosensing platform will be developed that will address (i) the development of a biosensor particle designed for direct uptake and delivery to cytosol (ii) sensing the intracellular delivery event i.e. report on successful delivery, and (iii) quantitative sensing of amount of delivered cargo, while investigating the (iv) cytotoxicity of biosensor, and (v) fate of biosensor as a function of cellular uptake mechanism. The proposed program will yield fundamental insight from the design rules of such a system to the function/application and fate of the proposed biosensor particle in vitro at the cellular level. The approach presented in this proposal is unique and transformational in several ways: (i) the sensing and quantification capabilities are not limited to specific dye-target pairs (e.g. Ca2+ ratiometric imaging requires use of specifically designed dyes) since reporting functions are covered by a NAC-cargo construct where NAC (N-Acetyl-L-Cysteine) acts as the QDot quencher, i.e.this concept is broadly applicable to reporting on delivery and quantification of different cargo types that can be attached to NAC, (ii) all functionalities are built into the biosensor particle, so no additional staining is necessary to report on delivery or for quantification of delivered cargo (integrated all-in-one design).Broader Impact. Research, education and outreach will be integrated in order to make significantcontributions to increasing the awareness for STEM and development of STEM skills of students,teachers and the community at large in an inclusive manner. The students that will be involved in thisproposed research will be trained in the interdisciplinary field of chemistry, materials, cell biology andnanotechnology. Topics that are related to the proposed research project will be integrated in courses and labs. Recruitment and Training of graduate and undergraduate students will occur through REU projects and the NanoFlorida symposium (www.nanoflorida.org). NanoFlorida is a trans-Florida student-organized conference. Students from underrepresented groups already make up more than half of the labs of the investigators on this proposal. Further recruiting of minorities will occur through NSF?s Florida Georgia Louis Stokes Alliance for Minorities Participation in Engineering and the organization of the NanoFlorida conference. A major broader impact component is the proposed community outreach program in collaboration with the Orlando Science Center (OSC). OSC?s Scanning Electron Microscope (SEM) located in a room in the exhibit halls will be made available to UCF graduate students to participate in educating the general public about our research activities. By using this facility in their work graduate students will learn about the importance of working with people and how to communicate with people in layman terms, something they can not experience easily in a college setting. Lab activities and online learning modules will be developed where children and parents can interact and learn together about nanoscience and biotechnology. The activities will be designed to be accessible to a broad audience. These initiatives will serve as a kick-off for a series of longer term plans that will lead to the creation of a critical mass of nanoscience and biology/biotechnology related exhibits and educational materials at the OSC. When successful the OSC will enable the PI?s to enter the Central Florida school system in order to take science education to classrooms. Given Central Florida?s demographic, the schools that are targeted often qualify as ?majority minority? and historically black? institutes.