Synthesis and identification of highly efficient polymeric nanoparticles for transfection of cells

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This inherently multidisciplinary project will require the student to translate polymer synthesis skills to create value in cell biology and nucleic acid delivery. The complex nature of this research project will bring together research expertise ranging from monomer design, controlled polymer synthesis, material testing, self-assembly, cell transfection and gene therapy. This research project will develop both a tool that can serve as a stand-alone technology crucial for developing effective gene therapies as well as a foundation for future applied projects.Objectives-Use polymer induced self-assembly (PISA) to produce a range of polymers with a variety of functionalities, sizes and compositions from a dye functional initiator.-Characterise the synthesised polymers using NMR and GPC including the polymeric nanoparticles (PNPs) followed by loading with nucleic acids that code for GFP expression-Use fluorescence imaging and FACS to identify and quantify the most efficient PNPs for gene deliveryMethods-Dye-functional initiator - wet chemistry synthesis and characterisation techniques (NMR, Mass Spectroscopy)-PISA - PNP synthesis, purification and characterization (DLS, TEM).-High-throughput microscopy imaging and data analysis (FACS and Fluorescence Microscopy)Potential Outcomes-Identification of potent gene therapy delivery polymeric nanoparticles-Improved transfection for gene therapies-Macrophage transfection with targeted nucleic acids (in-vitro) leading to further collaborations for in-vivo studiesGene therapy transfers genetic material into cells to provide new functions, including as potential interventions and treatments for a wide range of human diseases. Polymers can be used in this instance, providing advantages in scalability and precision control. For gene delivery, polyamines are often preferred: the positively charged nature of the polymer allows for electrostatic binding to nucleic acids, additionally providing protection to the nucleic acids by buffering pH during endocytosis. These polyamines however, have poorly controlled degradation profiles and are sometimes highly toxic. Accessing a step-change in gene-therapy delivery systems requires a bottom-up rethink of the polymer nanoparticle framework.Thus we propose to develop biodegradable polymers that will allow for efficient and effective transfection of nucleic acids to cells. These polymers will be designed for binding to nucleic acids and to provide protection during endocytosis. Polymer induced self-assembly (PISA) will be used to produce these polymers from a range of monomers. Using poly(ethylene glycol) (PEG) functionalised dye molecules (produced in the Jones Lab) as the initiating species a dye labeled polymeric delivery system will be produced, which will allow for in-vitro tracking and imaging. These will be assembled into polymeric nanoparticles (Fielding Lab) with nucleic acids encoding for GFP expression (Gene Editing Unit, UoM). Fluorescence microscopy and FACS will be used to assess the transfection efficiency of each PNP in order to identify the most potent transfection PNP. Impact-driven final project stages will exploit specific nucleic acid sequences for delivery to macrophages (in-vitro), to be investigated in-vivo through collaborative efforts.