Gómez Saúl C, Quezada Valentina, Quiroz Isabella, Muñoz-Camargo Carolina, Osma Johann F, Reyes Luis H, Cruz Juan C
Department of Biomedical Engineering, Universidad de los Andes, Cra. 1E No. 19a-40, Bogotá 111711, Colombia.
Department of Electrical and Electronic Engineering, Universidad de los Andes, Cra. 1E No. 19a-40, Bogotá 111711, Colombia.
Micromachines (Basel). 2021 Nov 10;12(11):1377. doi: 10.3390/mi12111377.
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.
新型膜活性肽（MAPs）的发现是现代生物技术中一个备受关注的领域，因为它们在多个领域具有广泛的适用性，从新型递送载体的开发（通过细胞穿透肽）到应对抗生素耐药性的潜在威胁（通过抗菌肽）。针对这一发现过程已经设计了不同的策略，然而，其中大多数都涉及成本高、繁琐且效率低下的方法。我们最近提出了一种基于构建在酵母表面重组表达的非合理设计文库的替代途径。然而，一个主要挑战是对具有膜活性的可能候选物进行强大且高通量的筛选。在这里，我们通过提出低成本的微流控平台来解决这个问题，该平台用于合成作为细胞膜模拟实体的巨型单层囊泡（GUVs），并用于使GUVs与表达MAPs的酵母同系物紧密接触。同系物是用膜转位肽Buforin II功能化的壳聚糖微粒，而紧密接触是通过具有不同通道几何形状配置的被动微混合器实现的。这两个微流控平台都通过计算机模拟（通过多物理场模拟）和体外实验进行了评估，两种方法之间具有高度一致性。有效地合成了大而稳定的GUVs（5 - 100 µm），并对混合过程进行了全面研究，从而找到了最佳操作参数。配备圆形特征的蛇形微混合器显示出最高的平均包封效率，这可以通过该装置内实现的独特混合模式来解释。这里开发的微流控装置作为发现新型MAPs的平台以及在生物医学领域的其他应用，如生物活性化合物的包封和控制递送，具有很高的潜力。
