Lipid digestion, a crucial process for mammal survival, remains surprisingly understudied. The lipid-water interface is critical, where reactions solubilize lipids and nutrients. Studies have shown that unilamellar vesicles form at emulsion-water interfaces during lipid hydrolysis, and nanostructures play a significant role in lipid absorption. Understanding the formation of these nanostructures, particularly the self-assembly of fatty acids, is crucial for regulating fat digestion and absorption. Lipid digestion is an enzyme-catalyzed hydrolysis reaction with unique characteristics: (1) Lipase acts at the oil-water interface, influenced by droplet size, interfacial area, surface tension, and viscoelasticity; and (2) hydrolysis produces amphiphilic fatty acids that form nanostructured self-assemblies along with other amphiphiles like phospholipids and cholates. The evolution of these aggregates affects the adsorption of surface-active materials and the kinetics of lipid digestion. Creating a simplified model to study the relationship between nanostructured self-assemblies and lipid hydrolysis efficiency is a key challenge. Previous research suggested that the autocatalytic hydrolysis of fatty acid anhydrides in crowded media could serve as a model, encompassing fatty acid production, vesicle catalysis, and the mimicking of crowded biological networks. Fatty acid vesicles have also been used in transdermal delivery. However, the association of reaction models involving fatty acid production, vesicle microstructure evolution, and interfacial catalysis in lipid digestion has not been thoroughly explored. Macromolecular crowding, the influence of volume exclusion on macromolecule properties, is significant in the gut, influencing the self-assembly of nanostructures and lipid hydrolysis. While studies have examined the effects of crowding from polysaccharides on lipase activity, a quantitative study on how crowding degree affects fatty acid self-assembly and lipid digestion is lacking. This paper aims to address this gap using fatty acid anhydrides and PEG to create a model with adjustable crowding degrees, investigating the nanostructure changes and hydrolysis reactions in confined spaces.

Existing literature highlights the importance of the lipid-water interface in lipid digestion, noting the formation of unilamellar vesicles and organized nanostructures during this process. Studies have shown the influence of droplet size, interfacial area, surface tension, and viscoelasticity on digestion efficiency. The self-assembly of amphiphilic molecules, including fatty acids, phospholipids, and cholates, significantly impacts lipid digestion kinetics. Previous research explored the use of autocatalytic hydrolysis of fatty acid anhydrides in crowded media as a model system for studying lipid digestion, while acknowledging the role of macromolecular crowding in influencing enzyme activity and the overall digestive process. However, a comprehensive understanding of how the degree of macromolecular crowding specifically affects the self-assembly of fatty acids and the kinetics of lipid hydrolysis remains absent from the literature.

Decanoic and oleic anhydrides were used as model fatty acid anhydrides. Polyethylene glycols (PEGs) with molecular weights of 200, 2000, and 20000 were used to create crowded media with varying crowding degrees. The autocatalytic production of fatty acids was monitored over time in both dilute solutions and crowded media. The size, polydispersity, and morphology of the resulting fatty acid vesicles were characterized using dynamic light scattering (DLS) and transmission electron microscopy (TEM). Rheological measurements, using a TA DHR-2 rheometer, were performed to investigate the viscoelastic properties of the fatty acid vesicles in the presence and absence of PEG. The concentration of fatty acids was determined using Fourier transform infrared (FT-IR) spectroscopy. Preformed vesicles were also used in some experiments to investigate their catalytic effect. The buffers used were Tricine (0.3 mol/L, pH 8.25) for decanoic anhydride and Bicine (0.2 mol/L, pH 8.50) for oleic anhydride hydrolysis.

The study revealed a significant dependence of fatty acid production rates on both the chain length of the fatty acid and the degree of macromolecular crowding. Higher crowding degrees significantly slowed the production rate of decanoic acid (C10), while conversely accelerating the production rate of oleic acid (C18). DLS and TEM analyses showed that increased crowding led to larger decanoic acid vesicles and smaller oleic acid vesicles. Rheological studies indicated that decanoic acid vesicles displayed viscous behavior in crowded media, whereas oleic acid vesicles exhibited elastic behavior. Experiments using preformed vesicles confirmed the catalytic role of vesicles in the hydrolysis reaction, although the effects of crowding were different for C10 and C18 vesicles. Specifically, the addition of preformed vesicles increased the C10 production rate, but decreased the C18 production rate in a crowded environment, suggesting different underlying mechanisms at play.

The contrasting effects of macromolecular crowding on decanoic and oleic acid production highlight the complexity of lipid digestion. The observed changes in vesicle size and viscoelastic properties are central to understanding these differences. The larger size of decanoic acid vesicles under crowded conditions likely hinders diffusion and reduces catalytic efficiency, thus slowing down the reaction. Conversely, the smaller size of oleic acid vesicles in crowded environments likely enhances diffusion and catalytic activity, accelerating the reaction. The observed viscous and elastic behaviors further support these findings, as viscosity can limit diffusion, while elasticity can facilitate it. The study provides a valuable model for understanding the influence of macromolecular crowding on lipid digestion. The differences observed between the short-chain (C10) and long-chain (C18) fatty acids suggest that the effect of crowding is not simply a matter of bulk viscosity, but also involves interactions at the vesicle interface.

This study demonstrates the significant influence of macromolecular crowding on the self-assembly of fatty acids and the kinetics of lipid hydrolysis. The different responses of decanoic and oleic acids to crowding highlight the complexity of this process and the importance of considering macromolecular effects in biological systems. Future research could explore the effects of other types of crowding agents and the role of specific interactions between crowding agents and fatty acid molecules. Investigating the in vivo relevance of these findings through further experiments using biological systems would also be valuable.

The study utilized a simplified model system using PEG to mimic macromolecular crowding. While PEG is a commonly used crowding agent, it might not perfectly represent the complex environment of the gastrointestinal tract. Further studies using more biologically relevant crowding agents would be beneficial. The focus on only two fatty acids with different chain lengths limits the generalizability of the findings. Future studies could explore a broader range of fatty acids to determine whether the observed trends apply more generally. The in vitro nature of the study limits its direct translatability to in vivo systems. Further in vivo research is necessary to validate these findings in a physiological context.