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相当多的研究努力。特别具有挑战性的是i)脑癌疗法的开发；ii)克服耐药性。通过开发生物相容的基于蛋白质的纳米结构，有可能提供更好的治疗选择，能够针对特定部位，克服耐药性，并提供减少的全身毒性。通过这种方法已经将具有治疗潜力但溶解度有限、靶向性弱的抗癌药物带入临床。然而，仍然需要了解i)包埋剂和细胞/组织之间的相互作用；ii)如何绕过耐药性。载脂蛋白(Apoferritin，AFT)是一种蛋白质纳米笼(直径12 nm，内腔8 nm)，天然用于储存和运输铁离子(作为铁蛋白)，将被用作递送载体。这种蛋白质胶囊由24个重链和轻链组成，可以通过合成生物学进行蛋白质工程和操纵。我们已经利用马的脾和最近重组的人AFT证明了我们可以将包括近红外PBS量子点(Qds)、抗癌药物(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)的货物运送到一系列癌细胞系。通过利用1)癌细胞转铁蛋白受体(TfR1)的增强表达和2)AFT的内在结合特性，AFT包埋赋予了显著的癌症选择性。为了证实这一观点，我们最近证明了通过在TfR1结合识别位点突变AFT可以取消TfR1的识别(图1)。我们的假设是，AFT包裹的货物被内吞到内体中，随着晚期内体和酸性溶酶体的pH下降，AFT纳米笼解离释放其货物，从而能够发挥其治疗作用。我们还证实，在2D细胞培养中，包裹替莫唑胺(TMZ)后能够克服由于MGMT表达、DNA错配修复(MMR)缺陷或P-糖蛋白(p-gp)表达而产生的对该甲基化试剂的耐药性。在这个项目中，我们将通过研究AFT摄取、通过肿瘤细胞的贩运和货物递送来确定AFT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.