Van de Cauter Lori, Jawale Yash K, Tam Daniel, Baldauf Lucia, van Buren Lennard, Koenderink Gijsje H, Dogterom Marileen, Ganzinger Kristina A
Autonomous Matter Department, AMOLF, Amsterdam 1098 XG, The Netherlands.
Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, Delft 2629 HZ, The Netherlands.
ACS Omega. 2024 Sep 25;9(41):42278-42288. doi: 10.1021/acsomega.4c04825. eCollection 2024 Oct 15.
Giant unilamellar vesicles (GUVs) are widely used as in vitro model membranes in biophysics and as cell-sized containers in synthetic biology. Despite their ubiquitous use, there is no one-size-fits-all method for their production. Numerous methods have been developed to meet the demanding requirements of reproducibility, reliability, and high yield while simultaneously achieving robust encapsulation. Emulsion-based methods are often praised for their apparent simplicity and good yields; hence, methods like continuous droplet interface crossing encapsulation (cDICE), which make use of this principle, have gained popularity. However, the underlying physical principles governing the formation of GUVs in cDICE and related methods remain poorly understood. To this end, we have developed a high-speed microscopy setup that allows us to visualize GUV formation in real time. Our experiments reveal a complex droplet formation process occurring at the capillary orifice, generating >30 μm-sized droplets and only in some cases GUV-sized (∼15 μm) satellite droplets. According to existing theoretical models, the oil-water interface should allow for the crossing of all droplets, but based on our observations and scaling arguments on the fluid dynamics within the system, we find a size-selective crossing of GUV-sized droplets only. The origin of these droplets remains partly unclear; we hypothesize that some small GUVs might be formed from large droplets sitting at the second interface. Finally, we demonstrate that proteins in the inner solution affect GUV formation by increasing the viscosity and altering the lipid adsorption kinetics. These results will not only contribute to a better understanding of GUV formation processes in cDICE but ultimately also aid in the development of more reliable and efficient methods for GUV production.
巨型单层囊泡（GUVs）在生物物理学中被广泛用作体外模型膜，在合成生物学中则被用作细胞大小的容器。尽管它们被广泛使用，但目前还没有一种适用于所有情况的生产方法。为了满足对可重复性、可靠性和高产率的严格要求，同时实现强大的包封效果，人们已经开发了许多方法。基于乳液的方法因其明显的简单性和良好的产率而经常受到称赞；因此，利用这一原理的方法，如连续液滴界面交叉包封（cDICE），已经受到欢迎。然而，在cDICE及相关方法中，控制GUV形成的潜在物理原理仍然知之甚少。为此，我们开发了一种高速显微镜装置，使我们能够实时观察GUV的形成过程。我们的实验揭示了在毛细管孔口处发生的复杂液滴形成过程，产生了大于30μm大小的液滴，并且仅在某些情况下产生了GUV大小（约15μm）的卫星液滴。根据现有的理论模型，油水界面应该允许所有液滴通过，但基于我们的观察以及对系统内流体动力学的尺度论证，我们发现只有GUV大小的液滴会发生尺寸选择性通过。这些液滴的起源仍部分不明；我们推测一些小的GUV可能由位于第二界面的大液滴形成。最后，我们证明内部溶液中的蛋白质通过增加粘度和改变脂质吸附动力学来影响GUV的形成。这些结果不仅将有助于更好地理解cDICE中GUV的形成过程，最终也将有助于开发更可靠、更高效的GUV生产方法。
