Investigating apoferritin-encapsulated antitumour theranostics` delivery - to overcome drug-resistance mechanisms.

对癌症治疗新方法的研究吸引了大量的研究努力。特别具有挑战性的是：1)脑癌治疗的发展；Ii)克服耐药性。通过开发生物相容性蛋白质纳米结构，有可能提供更好的治疗方案，能够靶向特定位点，克服耐药性并降低全身毒性。该方法已将具有治疗潜力但溶解度有限、靶向性弱的抗癌药物引入临床。然而，需要了解：(1)包封剂与细胞/组织之间的相互作用；Ii)如何绕过残留的耐药性。载铁蛋白（AFt）是一种蛋白质纳米笼（直径12纳米，内腔8纳米），自然用于储存和运输铁离子（作为铁蛋白），将被用作运载工具。该蛋白胶囊由24条重链和轻链组成，可用于蛋白质工程和合成生物学操作。我们已经证明，使用马脾和最近重组的人类AFt，我们可以将包括近红外PbS量子点（QDs）、抗癌剂（例如EGFR酪氨酸激酶抑制剂吉非替尼、咪唑四嗪和苯并噻唑类似物）在内的产品输送到一系列癌细胞系。通过利用i)癌细胞中转铁蛋白受体（TfR1）的增强表达和ii) AFt固有的结合特性，AFt包封具有显著的癌症选择性。为了证实这一观点，我们最近证明可以通过诱变TfR1结合识别位点的AFt来消除TfR1的识别（图1）。我们的假设是，AFt包裹的货物被内吞到核内体中，当核内体晚期和酸性溶酶体的pH值下降时，AFt纳米笼解体，释放其货物，然后能够发挥其治疗作用。我们还证实，在二维细胞培养中，替莫唑胺（TMZ）的aft包封能够克服MGMT表达、dna错配修复（MMR）缺陷或p-糖蛋白（p-gp）表达对这种甲基化剂的抗性。在这个项目中，我们将通过研究aft的摄取、通过肿瘤细胞的转运和货物运输来确定aft递送克服耐药性的机制。

Investigation of novel approaches to cancer treatment has attracted considerable research efforts. Particularly challenging are i) development of brain cancer therapies; ii) overcoming drug-resistance. Through development of biocompatible protein-based nanostructures, it is possible to deliver better treatment options able to target specific sites, overcome drug resistance and offer reduced systemic toxicity. Promising anticancer agents with therapeutic potential but limited solubility and weak targeting have been brought to clinic by this approach. However, the need to understand i) interactions between encapsulated agents and cells/tissue; ii) how drug-resistance can be bypassed remain. Apoferritin (AFt), a protein nanocage (12 nm diameter and 8 nm internal cavity), used naturally to store and transport iron ions (as ferritin), will be used as a delivery vehicle. This protein capsule, comprising 24 heavy and light chains is amenable to protein engineering and manipulation by synthetic biology. We have demonstrated using horse spleen and recently recombinant human AFt that we can deliver cargoes including near-infrared PbS quantum dots (QDs), anti-cancer agents (e.g. EGFR tyrosine kinase inhibitor gefitinib, imidazotetrazine- and benzothiazole analogues) to a range of carcinoma cell lines. By exploiting i) cancer cells` enhanced expression of transferrin receptor (TfR1) and ii) the intrinsic binding properties of AFt, AFt-encapsulation confers a significant degree of cancer-selectivity. Corroborating this thesis, we recently demonstrated that TfR1-recognition can be abolished through mutagenesis of AFt at the TfR1 binding recognition site (Figure 1). Our hypothesis is that AFt-encapsulated cargo is endocytosed into the endosome and as pH falls in the late endosome and acidic lysosome, the AFt nanocage disassembles releasing its cargo which is then able to exert its therapeutic effect. We have also established that in 2D cell culture, AFt-encapsulation of temozolomide (TMZ) is able to overcome resistance to this methylating agent conferred by MGMT expression, DNA-mismatch repair (MMR) deficiency or p-glycoprotein (p-gp) expression. In this project we will define mechanisms by which AFt-delivery overcomes drug-resistance by studying AFt-uptake, -trafficking though tumour cells and cargo delivery.