Intracellular bacteria invade mammalian cells to establish an infectious niche. The current work models adhesion and subsequent internalization strategy of pathogenic bacteria into mammalian cells to design a bacteriomimetic bioinvasive delivery system. We report on the surface functionalization of liposomes with a C-terminal fragment of invasin (InvA497), an invasion factor in the outer membrane of Yersinia pseudotuberculosis. InvA497-functionalized liposomes adhere to mammalian epithelial HEp-2 cell line at different infection stages with a significantly higher efficiency than liposomes functionalized with bovine serum albumin. Covalent attachment of InvA497 results in higher cellular adhesion than liposomes with physically adsorbed InvA497 with non-specific surface protein alignment. Uptake studies in HEp-2 cells indicate active internalization of InvA497-functionalized liposomes via β1-integrin receptor-mediated uptake mechanism mimicking the natural invasion strategy of Yersinia pseudotuberculosis. Uptake studies in Caco-2 cells at different polarization states demonstrate specific targeting of the InvA497-functionalized liposomes to less polarized cells reflecting the status of inflamed cells. Moreover, when loaded with the anti-infective agent gentamicin and applied to HEp-2 cells infected with Yersinia pseudotuberculosis, InvA497-functionalized liposomes are able to significantly reduce the infection load relative to nonfunctionalized drug-loaded liposomes. This indicates a promising application of such a bacteriomimetic system for drug delivery to intracellular compartments. *Graphical AbstractBacterial invas on into a human cellvia outer ..•

Bacterial invasion into eukaryotic cells and the establishment of intracellular infection has proven to be an effective means of resisting antibiotic action, as anti‐infective agents commonly exhibit a poor permeability across the host cell membrane. Encapsulation of anti‐infectives into nanoscaled delivery systems, such as liposomes, is shown to result in an enhancement of intracellular delivery. The aim of the current work is, therefore, to formulate colistin, a poorly permeable anti‐infective, into liposomes suitable for oral delivery, and to functionalize these carriers with a bacteria‐derived invasive moiety to enhance their intracellular delivery. Different combinations of phospholipids and cholesterol are explored to optimize liposomal drug encapsulation and stability in biorelevant media. These liposomes are then surface‐functionalized with extracellular adherence protein (Eap), derived from Staphylococcus aureus. Treatment of HEp‐2 and Caco‐2 cells infected with Salmonella enterica using colistin‐containing, Eap‐functionalized liposomes resulted in a significant reduction of intracellular bacteria, in comparison to treatment with nonfunctionalized liposomes as well as colistin alone. This indicates that such bio‐invasive carriers are able to facilitate intracellular delivery of colistin, as necessary for intracellular anti‐infective activity. The developed Eap‐functionalized liposomes, therefore, present a promising strategy for improving the therapy of intracellular infections.

Intracellular infections caused by invasive pathogens continue to prove difficult to combat, due in part to the commonly poor membrane permeability of anti-infective drugs. The aim of this study was to improve the intracellular delivery of one such poorly permeable (but broad-spectrum) anti-infective, gentamicin. Gentamicin was encapsulated into liposomal nanocarriers which were then surface functionalized with InvA497, a bacteria-derived invasion protein. Treatment of HEp-2 cells infected with the enteroinvasive bacteria Yersinia pseudotuberculosis or Salmonella enterica with gentamicincontaining, InvA497-functionalized liposomes resulted in a significantly greater reduction in infection load than treatment with non-functionalized liposomes, indicating that such a bacteriomimetic nanocarrier was not only able to promote successful cellular uptake of gentamicin but was also able to mediate anti-infective drug delivery to both cell cytoplasm and intracellular compartments. The developed InvA497-functionalized liposomal nanocarrier therefore holds great promise as a strategy for improving the therapy of intracellular infections.

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