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将聚合物合成技能转化为在细胞生物学和核酸输送方面的价值。这一研究项目的复杂性将汇集从单体设计、受控聚合物合成、材料测试、自组装、细胞转染和基因治疗等领域的研究专业知识。这项研究项目将开发一种工具，作为开发有效的基因疗法的关键的独立技术，并为未来的应用项目奠定基础。目标-使用聚合物诱导自组装(PISA)从染料功能引发剂生产一系列具有各种功能、大小和组成的聚合物。-使用核磁共振和凝胶渗透色谱表征合成的聚合物，包括聚合物纳米颗粒(PNPs)，然后装载编码GFP表达的核酸-使用荧光成像和FACS来鉴定和定量最有效的基因传递PNPs方法-染料-功能引发剂-化学合成和表征技术-高通量显微成像和数据分析(FACS和荧光显微镜)潜在成果-确定有效的基因治疗递送聚合物纳米颗粒-改进的基因治疗转染法-巨噬细胞靶向核酸转染(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，包括作为一系列潜在的干预和治疗一系列人类疾病。聚合物可以在这种情况下使用，在可伸缩性和精确度控制方面提供优势。对于基因传递，多胺通常是首选的：聚合物的正电荷性质允许与核酸静电结合，另外通过在内吞过程中缓冲pH来保护核酸。然而，这些多胺的降解谱控制不佳，有时毒性很高。在基因治疗递送系统中实现阶梯变化需要自下而上地重新考虑聚合物纳米颗粒框架。因此，我们建议开发可生物降解的聚合物，以允许高效和有效地将核酸导入细胞。这些聚合物将被设计成与核酸结合，并在内吞过程中提供保护。聚合物诱导自组装(PISA)将用于从一系列单体生产这些聚合物。利用聚乙二醇化的染料分子(琼斯实验室生产)作为起始物种，将生产一种染料标记的聚合物递送系统，这将允许进行体外示踪和成像。这些将与编码绿色荧光蛋白表达的核酸(基因编辑单元，UOM)组装成聚合物纳米颗粒(菲尔丁实验室)。荧光显微镜和流式细胞仪将用来评估每个PNP的转染率，以确定最有效的转染性PNP。影响驱动的最终项目阶段将利用特定的核酸序列传递给巨噬细胞(体外)，通过合作努力在体内进行研究。

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.