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

这个固有的多学科项目将要求学生翻译聚合物合成技能，以创造细胞生物学和核酸输送的价值。该研究项目的复杂性将汇集单体设计，受控聚合物合成，材料测试，自组装，细胞转染和基因治疗等研究专业知识。该研究项目将开发一种工具，可以作为一个独立的技术开发有效的基因疗法，以及为未来的应用项目的基础。目标-使用聚合物诱导自组装（比萨），以染料功能引发剂产生一系列具有各种功能，大小和组成的聚合物。使用NMR和GPC表征合成的聚合物，包括聚合物纳米颗粒（PNP），然后加载编码GFP表达的核酸-使用荧光成像和FACS鉴定和定量用于基因递送的最有效的PNP方法-染料官能引发剂-湿化学合成和表征技术（NMR，质谱）-比萨- PNP合成，纯化和表征（DLS，TEM）。高通量显微成像和数据分析（流式细胞仪和荧光显微镜）潜在的结果-有效的基因治疗递送聚合物纳米颗粒的鉴定-基因治疗的改进的转染-用靶向核酸转染巨噬细胞（体外）导致体内研究的进一步合作基因治疗将遗传物质转移到细胞中以提供新的功能，包括作为广泛的人类疾病的潜在干预和治疗。在这种情况下可以使用聚合物，提供可扩展性和精确控制方面的优势。对于基因递送，多胺通常是优选的：聚合物的带正电荷的性质允许与核酸静电结合，另外通过在内吞作用期间缓冲pH来为核酸提供保护。然而，这些多胺具有较差的控制降解特性，并且有时具有高毒性。基因治疗传递系统的一个重大变化需要对聚合物纳米颗粒框架进行自下而上的重新思考。因此，我们建议开发可生物降解的聚合物，使核酸能够高效地转染到细胞中。这些聚合物将被设计用于结合核酸并在内吞作用期间提供保护。聚合物诱导自组装（比萨）将用于从一系列单体生产这些聚合物。使用聚（乙二醇）（PEG）官能化染料分子（在琼斯实验室生产）作为起始物质，将生产染料标记的聚合物递送系统，这将允许体外跟踪和成像。这些将与编码GFP表达的核酸（基因编辑单元，UoM）组装成聚合物纳米颗粒（菲尔丁实验室）。将使用荧光显微镜和FACS来评估每个PNP的转染效率，以鉴定最有效的转染PNP。影响驱动的最终项目阶段将利用特定的核酸序列递送到巨噬细胞（体外），通过合作在体内进行研究。

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.