Developing apoferritin into an imaging and drug delivery platform; Optimisation of production and efficiency of cell uptake

将脱铁铁蛋白开发成成像和药物输送平台；

• 批准号: 1803018
• 金额: --
• 依托单位: University of Nottingham
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2016
• 资助国家: 英国
• 起止时间: 2016 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-1803018
• 关键词: Developing; apoferritin; into; imaging; drug

Biopharmaceutics (aka biologics) are increasingly being used in the clinic. The majority of these compounds are currently antibodies or antibody derived structures which have the disadvantage of needing to be produced in eukaryotic expression systems and because they are made of multiple chains held together by disulphide bonds are difficult to obtain and hence very expensive as therapeutics. We have been investigating an alternative protein scaffold, that of human apoferritin, which can be produced in high yield in E. coli as an imaging and drug delivery vehicle. Apoferritin self assembles into a protein capsule from 24 subunits when above pH 5.0 and its natural cargo of iron(III)oxide can be replaced by imaging molecules including fluorescent quantum dots, MRI and PET imaging agents or drug molecules.Apoferritin is thought to be preferentially taken up by cells that over express the transferrin or TIM2 receptors which includes a number of cancer cell types. By fusing specific peptides to the N-terminal of apoferritin so that they appear on the surface of the protein capsule, it should be possible to redirect the apoferritin to different cell types. Apoferritins are normally endocytosed and it is thought that the protein capsule disassembles in the late endosome or lysosome realising its contents with the apoferritin proteins either being degraded by the proteasome or being recycled and released from the cell. In the first rotation (Thomas lab), new apoferritin-peptide fusions will be produced and their production optimised. The cellular targeting and uptake abilities of these new fusion proteins will be investigated with different cell types using confocal microscopy.. In the second linked rotation (Bradshaw lab) the effect of the modified apoferritins on cell viability and progression through the cell cycle will be determined and the location and disassembly of the apoferritin in the endosome-lysosome will be studied by fluorescence microscopy and HR-TEM. In the third rotation (Grabowska lab) the localisation of the apoferritin peptide fusion in vivo in mice with and without orthotopic tumours will be investigated using fluorescence/luminescence techniques.

生物制药（又名生物制剂）越来越多地用于临床。目前这些化合物中的大多数是抗体或抗体衍生结构，其缺点是需要在真核表达系统中产生，并且由于它们是由二硫键连接在一起的多条链组成，因此难以获得，因此作为治疗药物非常昂贵。我们一直在研究一种替代蛋白支架，即人载铁蛋白，它可以在大肠杆菌中高产地生产，作为成像和药物递送载体。当pH高于5.0时，载铁蛋白自组装成由24个亚基组成的蛋白质胶囊，其天然货物氧化铁可以被成像分子取代，包括荧光量子点，MRI和PET显像剂或药物分子。载铁蛋白被认为优先被过度表达转铁蛋白或TIM2受体的细胞吸收，包括许多癌症细胞类型。通过将特定的肽融合到载铁蛋白的n端，使它们出现在蛋白胶囊的表面，应该有可能将载铁蛋白定向到不同的细胞类型。载铁蛋白通常被内吞，据认为，蛋白质胶囊在内核体或溶酶体晚期解体，载铁蛋白要么被蛋白酶体降解，要么被回收并从细胞中释放出来。在第一次轮换（托马斯实验室），新的载铁蛋白-肽融合将产生并优化其生产。这些新的融合蛋白的细胞靶向和摄取能力将在不同的细胞类型中使用共聚焦显微镜进行研究。在第二次连锁旋转（Bradshaw实验室）中，将确定修饰的载铁蛋白对细胞活力和细胞周期进展的影响，并通过荧光显微镜和HR-TEM研究载铁蛋白在内体溶酶体中的位置和拆卸。在第三轮（Grabowska实验室）中，将使用荧光/发光技术研究携带和不携带原位肿瘤的小鼠体内载铁蛋白肽融合的定位。