Department of Chemistry and Biochemistry, University of California, Merced, Merced, California 95343, United States.
Department of Bioengineering, University of California, Merced, Merced, California 95343, United States.
Langmuir. 2023 Apr 18;39(15):5579-5590. doi: 10.1021/acs.langmuir.3c00457. Epub 2023 Apr 6.
Giant unilamellar vesicles (GUVs) are micrometer-scale minimal cellular mimics that are useful for bottom-up synthetic biology and drug delivery. Unlike assembly in low-salt solutions, assembly of GUVs in solutions with ionic concentrations of 100-150 mM Na/KCl (salty solutions) is challenging. Chemical compounds deposited on the substrate or incorporated into the lipid mixture could assist in the assembly of GUVs. Here, we investigate quantitatively the effects of temperature and chemical identity of six polymeric compounds and one small molecule compound on the molar yields of GUVs composed of three different lipid mixtures using high-resolution confocal microscopy and large data set image analysis. All the polymers moderately increased the yields of GUVs either at 22 or 37 °C, whereas the small molecule compound was ineffective. Low-gelling temperature agarose is the singular compound that consistently produces yields of GUVs of greater than 10%. We propose a free energy model of budding to explain the effects of polymers in assisting the assembly of GUVs. The osmotic pressure exerted on the membranes by the dissolved polymer balances the increased adhesion between the membranes, thus reducing the free energy for bud formation. Data obtained by modulating the ionic strength and ion valency of the solution shows that the evolution of the yield of GUVs supports our model's prediction. In addition, polymer-specific interactions with the substrate and the lipid mixture affects yields. The uncovered mechanistic insights provide a quantitative experimental and theoretical framework to guide future studies. Additionally, this work shows a facile means for obtaining GUVs in solutions of physiological ionic strengths.
巨大的单室囊泡 (GUV) 是微米级的最小细胞模拟物，对于自下而上的合成生物学和药物输送非常有用。与低盐溶液中的组装不同，在离子浓度为 100-150mM Na/KCl（咸溶液）的溶液中组装 GUV 具有挑战性。沉积在基底上或掺入脂质混合物中的化学化合物可以协助 GUV 的组装。在这里，我们使用高分辨率共焦显微镜和大数据集图像分析，定量研究了六种聚合物和一种小分子化合物的温度和化学性质对由三种不同脂质混合物组成的 GUV 的摩尔产率的影响。所有聚合物都适度提高了 22°C 或 37°C 下 GUV 的产率，而小分子化合物则无效。低胶凝温度琼脂糖是唯一一种始终能产生大于 10%GUV 产率的化合物。我们提出了一个出芽的自由能模型来解释聚合物在协助 GUV 组装中的作用。溶解聚合物对膜施加的渗透压平衡了膜之间增加的粘附力，从而降低了芽形成的自由能。通过调节溶液的离子强度和离子价来获得的数据表明，GUV 产率的演变支持我们模型的预测。此外，聚合物与基底和脂质混合物的特异性相互作用会影响产率。所揭示的机制见解为指导未来的研究提供了定量的实验和理论框架。此外，这项工作展示了一种在生理离子强度溶液中获得 GUV 的简便方法。
