Chemotherapy is ineffective against brain metastases (BM), because haematogenous drug delivery fails to achieve therapeutic and evenly distributed intratumoural concentrations. Heterogeneous tumour-brain tissue architecture, abnormal perfusion dynamics, hypoxic regions, and high interstitial pressure are factors limiting drug delivery, compounded by patchy blood-tumour-barrier permeability. Furthermore, BM are usually detected late, after patients became symptomatic. Hypothesis Theranostic nanomedicines will improve diagnostic sensitivity for earlier detection of BM, as well as therapeutic efficacy and side-effect profiles of existing chemotherapeutics through tumour targeting, delayed drug clearance and microenvironment-mediated activation. Aims 1) Develop monoclonal antibody (mAb) fragments (scFvs) against the BM markers HER2 and HER3, including 2) evaluation of independent therapeutic potential. 3) Functionalised polyethylene glycol (PEG)-based nano-drug-carriers with the scFvs, along with imaging agents to facilitate in vitro and in vivo analysis of tissue distribution. 4) Functionalise HER2/HER3-targeted nano-drug-carriers with doxorubicin via an acid-labile linkage for pH-trigger release in hypoxic microenvironment or endosome compartments after internalisation. Results to date HER2- and HER3- scFvs based on epitope-binding sequence of clinically-approved mAbs were synthesized. Binding affinities were found to be higher than parental mAbs (KD 2-8x10E-11 M). The scFvs were cytostatic and moderately cytotoxic in vitro with an IC50 of 0.4-1.0µM. In addition, they exhibited dose-dependent, additive growth inhibition when used in combination, and induced internalisation of their receptor ligands within 4 hours in a HER2+ cell line (SKBr3). Conclusion These scFvs are strong nanocarrier tethering candidates in terms of both intracellular and extracellular payload release. HER2 and HER3 scFvs exhibited dose-dependent, additive growth inhibition when used in combination. ScFv-tentered nanocarrier synthesis is now currently underway and in vivo investigation using a BM mouse model will follow.