Membrane Active Peptides Remove Surface Adsorbed Protein Corona From Extracellular Vesicles of Red Blood Cells

Abstract

Besides the outstanding potential in biomedical applications, extracellular vesicles (EVs) are also promising candidates to expand our knowledge on interactions between vesicular surface proteins and small-molecules which exert biomembrane-related functions. Here we provide mechanistic details on interactions between membrane active peptides with antimicrobial effect (MAPs) and red blood cell derived EVs (REVs) and we demonstrate that they have the capacity to remove members of the protein corona from REVs even at lower than 5 µM concentrations. In case of REVs, the Soret-band arising from the membrane associated hemoglobins allowed to follow the detachment process by flow-Linear Dichroism (flow-LD). Further on, the significant change on the vesicle surfaces was confirmed by transmission electron microscopy (TEM). Since membrane active peptides, such as melittin have the affinity to disrupt vesicles, a combination of techniques, fluorescent antibody labeling, microfluidic resistive pulse sensing, and flow-LD were employed to distinguish between membrane destruction and surface protein detachment. The removal of protein corona members is a newly identified role for the investigated peptides, which indicates complexity of their in vivo function, but may also be exploited in synthetic and natural nanoparticle engineering. Furthermore, results also promote that EVs can be used as improved model systems for biophysical studies providing insight to areas with so far limited knowledge.

Membrane Association Modes of Natural Anticancer Peptides: Mechanistic Details on Helicity, Orientation, and Surface Coverage

“…Structural effects upon REV-ACP interactions were also followed by CD and IR spectroscopy. Very similar spectral changes were detected here (Figure S6 and related text in SI) as observed previously on melittin and CM15 [92], which is indicative of a common action exerted by this set of peptides. All these findings suggest that the novel function, e.g., the removal of REV-associated proteins revealed for melittin and its hybrid CM15, can be extended to several cationic membrane-active peptides.…”

“…Distinctive perturbations were also found for the lipid ester and acyl chain vibrations, especially for LL-37 with PC/PS liposomes, showing a strong membrane interaction affecting deeper regions of the bilayer perturbing lipid order and packing as well. The results with REVs suggested that they could efficiently remove the surface-associated protein corona in a concentration-dependent manner, which promotes that their helices covering the vesicle occupy a relatively large space on the EV surface [92] (Figure 7D). In this case, the longer LL-37 was proven to be more effective.…”

“…Previous studies have suggested that red blood-cell derived extracellular vesicles (REVs) can be used as improved model systems to study membrane interactions, as they can be produced on a large scale, are non-toxic, and have a monodispersed size distribution making them suitable for biophysical experiments [90,91]. We have demonstrated that membrane-active peptides target not only the lipid bilayer of extracellular vesicles but also that their unique action can result in the removal of EV-associated protein corona members [92]. To improve our mechanistic understanding, LL-37, FK-16, and Tempo-La were selected, and their interactions with REVs were studied by increasing ACP concentration and monitoring with LD spectroscopy.…”

Blood Nanoparticles – Influence on Extracellular Vesicle Isolation and Characterization

“…Recent studies on EVs and previous studies on nanoparticles of nonbiological origin have suggested that the same sets of proteins can bind physiologically to the surface of EVs in body fluids to form the protein corona, which thus coats the EVs ( Palviainen et al, 2019 ; Priyanka et al, 2020 ). The composition of the protein corona depends on the composition of the biofluid and its protein concentration, on the fluid conditions (i.e., static vs. flowing), and on the temperature and nanoparticle properties ( Nguyen and Lee, 2017 ).…”

Liposomes and Extracellular Vesicles as Drug Delivery Systems: A Comparison of Composition, Pharmacokinetics, and Functionalization

“…[ 150 ] Furthermore, melittin and CM15, two membrane active peptides with antimicrobial effect, drastically affect the protein corona adsorbed to red blood cell‐derived EVs, further suggesting the presence of a protein corona. [ 151 ] Further research investigating the in vivo emergence of a protein corona on the surface of (exogenously) administered EVs and the consequences in regard to EV clearance, is required.…”