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

生物制药(又名生物制品)正越来越多地应用于临床。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，而且由于它们是由多个链组成的，通过二硫键连接在一起，很难获得，因此作为治疗药物非常昂贵。我们一直在研究一种替代的蛋白质支架，即人类载铁蛋白，它可以在大肠杆菌中作为成像和药物输送载体而高产生产。当pH高于5.0时，载脂蛋白从24个亚基自我组装成蛋白质胶囊，其天然的氧化铁可被成像分子取代，包括荧光量子点、MRI和PET成像剂或药物分子。载脂蛋白被认为优先被过度表达转铁蛋白或TIM2受体的细胞所摄取，其中包括许多类型的癌细胞。通过将特定的多肽融合到载铁蛋白的N端，使其出现在蛋白质包膜的表面，应该可以将载脂蛋白重定向到不同的细胞类型。Apoferritins通常是内吞的，被认为是晚期内体或溶酶体中的蛋白质被解体，实现其内容，Apoferritin蛋白要么被蛋白酶体降解，要么被回收并从细胞中释放。在第一次旋转中(Thomas实验室)，将产生新的载脂蛋白-多肽融合并优化它们的生产。这些新的融合蛋白的细胞靶向和摄取能力将通过共聚焦显微镜在不同类型的细胞中进行研究。在第二次连锁旋转(Bradshaw Lab)中，将确定修饰后的Apoferritin对细胞活力和整个细胞周期的影响，并将利用荧光显微镜和HR-TEM研究Apoferritin在内体-溶酶体中的定位和分解。在第三次旋转中(Grabowska实验室)，将使用荧光/发光技术研究有无原位肿瘤的小鼠体内载脂蛋白多肽融合的定位。

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.