Design and development of new Lipid Nano Particle delivery systems for new RNA-based c therapeutics: A rationally designed chemistry and microfluidics

基于信使核糖核酸的疗法通过沉默特定基因或过度表达治疗蛋白来发挥作用，现已被证明是辉瑞和现代新冠肺炎疫苗的基础。由于母体分子的不稳定性和不良的物理化学性质，将寡核苷酸治疗药物输送到患者体内是有限的，因此可以通过将其掺入基于脂质的纳米颗粒(LNP)来实现疗效。LNPs通常直径在50-100 nm之间，由有助于控制颗粒大小的聚乙二醇化脂质、帮助形成纳米颗粒结构的非甾体酰化磷脂酰胆碱和也有助于结构的胆固醇组成。最重要的是，它们还含有一种可电离的阳离子脂类，它包裹着带负电荷的寡核苷酸，有助于增加RNA的稳定性。该项目的方法是将合成脂类化学与微流体相结合，设计定制的可电离阳离子脂类，将其并入用于COVID和/或RNA癌症治疗的最佳LNP递送系统。在一种合理的方法中，将制备一系列修饰的可电离脂质，将其并入LNP并负载特定的寡核苷酸，然后进行物理化学性质研究以确定颗粒大小、包封率和pKA(包括单独的脂类和LNP系统)。在所选的特定分析中的效力评估(将在课程项目中适当地确认)和随后的稳定性研究之后，将生成结构活性关系轮廓，该轮廓将被用来推动迭代合成运动以寻找最佳的LNP体系。作为本博士课程的一部分，学生将获得的具体技能如下：标准和并行合成高官能化的脂分子使用核磁共振、LC MS、IR、MALDI、HRMS、CHN表征和分析脂质分子及其前体，通过闪光柱色谱通过微流体/微混合器制备低聚脂纳米颗粒体系Zeta电位、pKA测定、差示扫描量热法和寡聚包埋/吸附效率。

The potential of messenger ribonucleic acid (mRNA) based therapeutics which exhibit their effect by the silencing of specific genes or overexpression of therapeutic proteins has now been proven as basis of the Pfizer and Moderna COVID-19 vaccines. As delivery of oligonucleotides therapeutics into the patient is limited due to the instability and undesirable physicochemical properties of the parent molecules, efficacy is achieved by incorporation into a lipid based nanoparticle (LNP). LNPs typically, have diameters of between 50-100 nm and are comprised of a PEGylated lipid that helps control particles size, diasteraroylphosphatidylcholine to help form the structure of the nanoparticle and cholesterol which also contributes to the structure. Most importantly, they also contain an ionisable cationic lipid that encapsulates the negatively charged oligonucleotide and helps to increase the stability of the RNA. The approach in this project is to combine synthetic lipid chemistry with microfluidics to design tailor made ionisable cationic lipids to be incorporated into optimum LNP delivery systems for COVID and/or RNA cancer therapeutics. In a rational approach, a series of modified ionisable lipids will be prepared, incorporated into the LNP and loaded with the specific oligonucleotide before undergoing physicochemical property investigations to determine particle size, encapsulation loading and pKa (both individual lipid and LNP system). Following potency evaluation in specific chosen assays (to be confirmed during the course project as appropriate) and subsequent stability studies, a structure activity relationship profile will be generated which will be used to drive forward an iterative synthesis campaign to find the optimum LNP systems.The specific technical skills that the student will gain as part of this PhD are listed below:Standard and parallel synthesis of highly functionalised lipid moleculesCharacterisation and analysis of lipids using NMR, LCMS, IR, MALDI, HRMS, CHNPurification of lipid molecules and their pre-cursors, by flash column chromatographyPreparation of oligo-lipid nano particle systems via microfluids/micromixerAnalysis of physicochemical properties of LNP systems eg Z-average, PDI, zeta-potential, pKa determination, DLS and oligo encapsulation/adsorption efficiency.