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There is a growing interest in designing a nanocarrier containing an EGFR targeting affibody to direct toward cancer cells. Here, cytolysin A was cloned at the N-terminus of Z EGFR:1907 affibody to guarantee its surface presentation on the OMVs while targeting the epidermal growth factor receptors (EGFRs). A separate construct including a fusogenic peptide (GALA) was also designed for the endosomal escape of the nanocarrier. Binding of the two constructs ClyA-affi EGFR and ClyA-affi EGFR -GALA to domain III of EGFR was investigated using molecular docking and molecular dynamic simulations. The higher stability of the ClyA-affi EGFR -GALA/EGFR as compared to the ClyA-affi EGFR /EGFR complex was evident. The ClyA-affi EGFR -GALA structure showed a higher RMSD during the first half of the simulation time implying a much less stable behavior. Plateau state of the radius of gyration plot of ClyA-affi EGFR -GALA confirmed a well-folded structure in the presence of the GALA sequence. Solvent accessible surface area for both proteins was in the same range. The data obtained from hydrogen bond analysis revealed a more equilibrated and stable form of the ClyA-affi EGFR -GALA structure upon interaction with EGFR. The data provided here was a requisite for our biological evaluation of the synthesized constructs as a component of a novel drug delivery system., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Bacterial outer membrane vesicles (OMVs) have recently drawn a great deal of attention due to their therapeutic efficiency and ability to target specific cells. In the present study, we sought to probe engineered OMVs as novel and promising carriers to target breast cancer cells. Following the fusion of the affi EGFR -GALA structure to the C-terminal of ClyA as an anchor protein, the ClyA-affi EGFR -GALA construct was successfully expressed on the surface of ∆msbB/∆pagP E. coli W3110-derived OMVs. Morphological features of the engineered and wild-type OMVs were identical. The engineered OMVs induced no endotoxicity, cytotoxicity, or immunogenicity, indicating the safety of their application. These OMVs could specifically bind to EGF receptors of MDA-MB-468 cells expressing high levels of EGFR and not to those with low levels of EGFR (HEK293T cells). Interestingly, despite a lower binding affinity of the engineered OMVs relative to the positive control Cetuximab, it was strong enough to identify these cells. Moreover, confocal microscopy revealed no uptake of the modified OMVs by the EGFR-overexpressing cells in the presence of EGFR competitors. These results suggest that OMVs might internalize into the cells with EGF receptors, as no OMVs entered the cells with any EGFR expression or those pretreated with EGF or Cetuximab. Regarding the EGFR-binding affinity of the engineered OMVs and their cellular uptake, they are presented here as a potential carrier for cell-specific drug delivery to treat a wide variety of cancer cells. Interestingly, the engineered OMVs are capable of reaching the cytoplasm while escaping the endosome due to the incorporation of a fusogenic GALA peptide in the construct., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Sepahdar, Miroliaei, Bouzari, Khalaj and Salimi.)