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

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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.