The formation of giant unilamellar vesicles (GUVs) is crucial for various applications, including drug delivery, artificial cells, and studying membrane dynamics. However, achieving high yields of GUVs, particularly in solutions mimicking physiological ionic strength, remains a challenge. Traditional methods often result in low yields and a heterogeneous size distribution. This study addresses this challenge by exploring the role of osmotic pressure in enhancing GUV formation. The central hypothesis is that manipulating osmotic pressure, particularly through the controlled dissolution of polymers, can counteract the increased membrane adhesion observed in high-salt solutions, thereby promoting a higher yield of GUVs. The importance of this research stems from the need for efficient and reliable methods for producing GUVs for various biomimetic and biomedical applications. High-yield production in physiologically relevant conditions is particularly important for studying membrane processes and developing drug delivery systems that accurately reflect cellular environments.

The authors reference several key studies. Tsumoto et al. (2009) demonstrated efficient GUV formation using fructose-doped phosphatidylcholine films. Pazzi and Subramaniam (2020) explored the use of nanocellulose paper for high-yield GUV formation. Israelachvili's work (2011) on intermolecular and surface forces provides a theoretical framework for understanding membrane interactions. Wang et al. (2002) investigated the second virial coefficients of polyethylene glycol, relevant to understanding polymer osmotic pressure. Several other studies (Sun et al., 2011; Marra & Israelachvili, 1985; Leckband et al., 1993; Mahnke et al., 1999; Kucerka et al., 2021; Kurakin et al., 2021) are cited to provide background on lipid bilayer adhesion, the influence of ions on membrane interactions, and the effects of dissolved gases and cations on surface interactions.

The researchers employed a gentle hydration technique on various substrates to form GUVs. They investigated different assisting compounds, including various types of agarose (ULGT, LGT, MGT, HGT) and polyvinyl alcohol (PVA), as well as fructose-doped lipids. GUV formation was examined in solutions with varying ionic strengths, including low-salt solutions and those containing PBS, MgCl2, KCl, and CaCl2. The yield of GUVs was quantified by microscopy, and the size distribution was analyzed. They used several equations to model the osmotic pressure, membrane adhesion energy, and Debye screening length to understand the influence of polymers and ions on vesicle formation. Sedimentation experiments were conducted to assess the effect of dissolved polymers on GUV behavior. Statistical analysis, including t-tests and ANOVA, was performed to compare the yields obtained under different conditions. The maximum concentration of dissolved polymers was calculated considering the partial solubility of the polymers used. The Debye screening length was calculated to estimate the range of electrostatic repulsion between membranes.

The study found significantly higher GUV yields using the fructose-doped technique in physiological salt solutions compared to previous reports. The use of LGT agarose as an assisting compound resulted in high GUV yields in PBS, comparable to yields obtained in low-salt conditions without assisting compounds. Analysis of the osmotic pressure contribution of dissolved polymers suggested that even low concentrations can effectively balance the increased membrane adhesion in high-salt solutions. The Debye screening length calculations showed a drastic reduction in the range of electrostatic repulsion in the presence of high concentrations of ions, promoting membrane adhesion. Sedimentation experiments showed that low concentrations of dissolved polymers do not significantly affect the sedimentation behavior of GUVs. Statistical analysis using t-tests and ANOVA confirmed significant differences in GUV yields between various experimental conditions, demonstrating that the optimized method yields significantly more GUVs than existing methods.

The findings support the hypothesis that osmotic pressure plays a critical role in enhancing GUV formation in physiological ionic strengths. By using polymers to control osmotic pressure, the increased membrane adhesion caused by high salt concentrations can be effectively counteracted, leading to higher yields. The results highlight the importance of both the polymer type and the ionic composition of the buffer. The low impact of the polymer on GUV sedimentation indicates that the effect of osmotic pressure is a primary factor in GUV formation. These results have significant implications for the development of more efficient and reliable methods for producing GUVs for various biomedical and biomimetic applications. The ability to create high yields of GUVs under physiological conditions opens doors for further investigation of membrane properties and the design of improved drug delivery systems.

This study successfully demonstrates a high-yield method for assembling GUVs in physiological ionic strength solutions. The use of osmotic pressure, controlled by the addition of polymers, is identified as a key factor in achieving this result. Future research could explore a wider range of polymers and ionic conditions to further optimize the process. Investigating the impact of different lipid compositions on the efficiency of this method would also be beneficial.

The study assumes ideal behavior for polymers and lipids in their calculations, which might not fully reflect the complex interactions in the system. The partial solubility of the polymers might lead to variations in the actual osmotic pressure compared to the calculated values. Further investigation is needed to explore the long-term stability of the GUVs produced by this method.