Asymmetricity in the lipid bilayer is one of the major topics in the study of cell biology. The artificial supported lipid bilayers provide a very good insight into this topic. The self-organization of phospholipids under physiological conditions leads to the formation of Supported Lipid Bilayers (SLBs) [1,2], which mimic native biological membranes. The phase change behavior due to mechanical change in the bilayer is always one of the interesting topics among biophysicists [3,4]. The paper thoroughly studies this effect by using dioleoylphosphatidylcholine (DOPC) in the bilayer. The study particularly focuses on cholesterol’s effect as it is crucial in maintaining the membrane integrity and affects various cellular processes, also its influence on mechanical properties is significant, particularly in the context of interleaflet coupling. While its general role in membrane stability is well understood, this research aims on how cholesterol distributes itself within a simple lipid bilayer and whether it influences the balance between the two layers. Understanding this could help explain many biological phenomena, such as how cholesterol effects cell function and contributes to diseases linked to membrane organization. The experiments were carried out with different concentrations of lipid and cholesterol and later analyzed and visualized using the state-of-the-art technique i.e. Atomic Force Microscopy (AFM) force spectroscopy.

Initially Small unilamellar vesicles (SUVs) were created using a chloroform/methanol solvent mixture and hydrated with HEPES buffer. Then artificial lipid membranes were used i.e., dioleoylphosphatidylcholine (DOPC), and later different concentrations of cholesterol were introduced starting from 10% to 50% (0%, 10%, 20%, 35%, and 50%). These membranes were then studied using Atomic Force Microscopy (AFM), a technique that allows researchers to visualize nanometer-scale structures and measure mechanical properties like stiffness and rupture strength. The AFM images revealed that adding cholesterol initially made the bilayer smoother and more tightly packed, a well-known effect of cholesterol in a bilayer, pulling lipid molecules closer together. However, when cholesterol exceeded 20%, the membrane started to become more irregular, suggesting that too much cholesterol might disrupt the natural arrangement of lipids.

To understand how cholesterol affected the bilayer’s mechanical properties, the researchers used AFM force spectroscopy, a method where a tiny probe applies pressure to the membrane until it punctures through. This allowed them to measure the "breakthrough force" (F_B), or the amount of pressure needed to rupture the bilayer. In a pure DOPC membrane, this force was about 8 nN. When 10% cholesterol was added, the membrane became stronger, requiring 11 nN to puncture. However, at cholesterol levels of 20% or higher, the force curves showed two distinct rupture events instead of one. This meant that instead of breaking through the bilayer in a single step, the probe first pierced the outer leaflet at a lower force (FB1) and then the inner leaflet at a higher force (FB2). This suggested that cholesterol was making the inner leaflet mechanically stronger than the outer one, effectively creating an asymmetric membrane. Further analysis of bilayer thickness supported this idea. Initially pure DOPC bilayer is about 5.5 nm thick. At 10% cholesterol, this thickness reduced slightly due to the lipids being pulled closer together. But at 20% cholesterol and more concentration, two distinct thickness measurements appeared, one for the outer leaflet (~4.1 nm) and one for the inner leaflet (~2.08 nm). This confirmed that cholesterol was not distributed evenly but was instead accumulating more in the inner leaflet, showing that it creates mechanical asymmetry in the membrane.

The author discusses how cholesterol influences the structure and stability of lipid bilayers. This might be because one is chain interdigitation, where lipid tails from one leaflet extend into the other, affecting how the two layers interact. The second is random translocation, where cholesterol molecules move between leaflets, causing an imbalance in lipid organization. In a typical membrane, the two leaflets are closer to each other, meaning they behave as a single unit. But, when cholesterol reaches high concentrations (above 20%), this assembly deteriorates, and the bilayer starts behaving more like two separated layers, with the inner one becoming stronger than the outer one. When cholesterol levels exceeded 50%, the bilayer became unstable, and its structure started to break down. This suggests, at low cholesterol levels, the bilayer remains stable and symmetric, and at high concentration, it disrupts this balance by redistributing between the leaflets, weakening their connection and making the bilayer mechanically unstable. This happens because the bilayer naturally adjusts itself to minimize surface energy and maintain structural integrity. When cholesterol levels become too high (around 70%), the bilayer completely loses stability. The study also points out that this cholesterol influenced asymmetry is more noticeable in simple, fluid-phase bilayers compared to complex lipid mixtures, as observed through AFM experiments. Also, the asymmetric behavior of lipids is studied with different combinations of PC lipids. The author’s previous work also demonstrated the combination of two different phases of lipids show this behavior (DLPC and DSPC) [5]. They also investigated the cholesterol effect on DOPC, where they thoroughly stated the effect on phase change behavior [6]. These results can be confirmed with spectroscopic ellipsometry study along with AFM and MD simulations [7]. These findings show that cells introduce cholesterol into their bilayer to maintain membrane physical function, and increase or decrease in cholesterol may have been an important cause for diseases such as neurodegenerative disorders.

In conclusion, this research article shows a significant contribution to the understanding of cholesterol's role in lipid bilayers. It highlights the importance of using single-component bilayers to study cholesterol effects, revealing how asymmetry arises in bilayers due to cholesterol's presence. Using AFM force spectroscopy, here they demonstrated that cholesterol found more in the inner leaflet at higher concentrations. The use of AFM force spectroscopy proved effective in observing these interactions, helping in the future studies in membrane biophysics.