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

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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.