Bacteriomimetic invasin-functionalized nanocarriers for intracellular delivery

Abstract

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 ..•

Understanding and improving assays for cytotoxicity of nanoparticles: what really matters?

“…13,14 This includes checking colloidal stability in acellular 15 and cellular conditions, testing NP interference with assay reagents 16,17 and use of appropriate controls. 2,3 This is further explained in the following paragraphs. A schematic showing an overview of the present study is presented in the ESI methods section.…”

“…Liposomes were previously reported to interfere and fuse with the cell membrane lipids. 2,46,47 We could not, however, test that due to liposomal interference with the phospholipidosis assay reagent. On the other hand, toxicity of QDs was observed to be at least partially attributed to the production of reactive oxygen species ( Fig.…”

“…Cell association of NPs was also assessed in terms of the frequency of pixels above a certain uorescence threshold. 2,30 This approach was reported as superior over approaches based on intensity measurements because intensities may vary with the location of the NPs in the imaged 3D specimen owing to light scattering. For pixel measurements, imaging settings could be freely adjusted for each measurement individually according to the signal level required to detect NPs at different depths in the examined cells.…”

“…The latter approach rather determines the "fold decrease or increase from the control experiment", whereas working up the same data to calculate percentage viability based on both negative and positive controls will result in accurate values for the determined response. 2,3 Deciency of appropriate controls can greatly reduce the explanatory power of cell-based toxicity models and potentially lead to overestimation of toxicity.…”

Invasin-functionalized liposome nanocarriers improve the intracellular delivery of anti-infective drugs

“…Covalent coupling of InvA497 (produced as described in the ESI †) to LFH liposomal surfaces was performed as described previously. 20,25 Briey, 2 ml of LFH liposomal suspension was incubated for a minimum of 3 h with a crosslinking reagent solution, consisting of N-(3-dimethylaminopropyl)-N 0 -ethylcarbodiimide hydrochloride (EDC, 48 mM, Sigma Aldrich, Steinheim, Germany) and N-hydroxysuccinimide (NHS, 19 mM, Carbolution Chemicals, Saarbruecken, Germany) in an ice bath with gentle mixing. Liposomes were then washed (described below) to remove excess crosslinking reagent.…”