Abstract
The discovery of new membrane-active peptides (MAPs) is an area of considerable interest in modern biotechnology considering their ample applicability in several fields ranging from the development of novel delivery vehicles (via cell-penetrating peptides) to responding to the latent threat of antibiotic resistance (via antimicrobial peptides). Different strategies have been devised for such discovery process, however, most of them involve costly, tedious, and low-efficiency methods. We have recently proposed an alternative route based on constructing a non-rationally designed library recombinantly expressed on the yeasts’ surfaces. However, a major challenge is to conduct a robust and high-throughput screening of possible candidates with membrane activity. Here, we addressed this issue by putting forward low-cost microfluidic platforms for both the synthesis of Giant Unilamellar Vesicles (GUVs) as mimicking entities of cell membranes and for providing intimate contact between GUVs and homologues of yeasts expressing MAPs. The homologues were chitosan microparticles functionalized with the membrane translocating peptide Buforin II, while intimate contact was through passive micromixers with different channel geometries. Both microfluidic platforms were evaluated both in silico (via Multiphysics simulations) and in vitro with a high agreement between the two approaches. Large and stable GUVs (5–100 µm) were synthesized effectively, and the mixing processes were comprehensively studied leading to finding the best operating parameters. A serpentine micromixer equipped with circular features showed the highest average encapsulation efficiencies, which was explained by the unique mixing patterns achieved within the device. The microfluidic devices developed here demonstrate high potential as platforms for the discovery of novel MAPs as well as for other applications in the biomedical field such as the encapsulation and controlled delivery of bioactive compounds.
Highlights
The screening of membrane-active peptides (MAPs) can be accelerated by a low-cost and robust methodology based on the encapsulation of yeast surface-displayed MAPs homologues (i.e., the membraneactive peptide Buforin II (BUF-II) conjugated to chitosan microparticles (CSMPs)) by the controlled and direct interaction with Giant Unilamellar Vesicles (GUVs) that mimic the composition of cell membranes
Synthesis of large and stable GUVs was conducted by forming double emulsions within low-cost microfluidic devices by adapting the octanol-assisted, on-chip de-wetting method, which avoids the use of sophisticated equipment, or specialized facilities such as clean rooms
The device performance was explored in silico aided by Multiphysics simulations, which allowed us to identify operating conditions leading to droplet sizes in the range of 5–150 μm
Summary
Micromachines 2021, 12, 1377 the transport and release of both hydrophilic and hydrophobic molecules intracellularly. Some of these penetrating molecules include different types of proteins and peptides [2]. Penetration, some peptides interact with cell membranes strongly, leading to substantial disorganization (and generally exhibiting antimicrobial activity), intercalation, or even fusion. All these peptide sequences have been group into a big family called membrane-active peptides (MAPs) [3]. There are two major classes of MAPs; antimicrobial peptides (AMPs), which kill cells, and cell-penetrating peptides (CPPs), which can carry cargoes through lipid bilayers [3]
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