Liposomes caught drunk-dancing in solution

How neutrons help us understand the industrial production of therapeutic molecules

Liposomes - spherical assemblies of amphiphilic molecules called phospholipids - are of great importance for cosmetic and pharmaceutical products. In industrial settings, liposomes are typically produced in the presence of alcohols. A detailed understanding of liposome-alcohol interactions is therefore critical to be able to optimise the industrial processes involved.

Using different neutron techniques, a team of researchers has revealed that liposome membranes become softer in the presence of certain alcohols. The reported softening has important implications for liposome size, which, in turn, affects their properties such as their drug targeting activity. This study underlines the unique strength of neutron techniques for the fundamental investigation of structural and dynamic properties of biomolecules.

Liposomes are hollow spherical particles made of amphiphilic phospholipid molecules. Since their discovery in the 1960s, liposomes have risen to fame due to their outstanding capacity to encapsulate biological molecules for targeted delivery. Today, they are frequently used in cosmetics such as facial creams and also play crucial roles in pharmaceutical applications. Examples include liposome-delivered gene therapy and certain antiparasite treatments. In fundamental research, liposomes are important reductionistic model systems of living cells.

In industrial settings, the first step of liposome production consists in dissolving phospholipids in an organic solvent such as ethanol. After removing some of the solvent, liposomes are produced by applying mechanical forces in a step known as "extrusion". The resulting solutions in which liposomes and solvent molecules coexist are then stored until the final product is manufactured.

Despite the primordial role of alcohol in this process, its molecular interactions with the phospholipids have not been elucidated in much detail to this day. Since a good understanding of these interactions is crucial for the optimisation of large-scale industrial processes, an interdisciplinary team, including two ILL scientists and an ILL PhD student, performed a systematic study of liposomes in the presence of different alcohols.

"Working at the ILL allowed us to combine a variety of cutting-edge neutron and laboratory techniques. This is absolutely essential to obtaining detailed insights of both structural and dynamical properties of our liposome samples", says Fernanda Alvarado-Galindo, the first author of the study and PhD student at the ILL and Technische Universität Berlin in the framework of InnovaXN, a EU-funded doctoral training programme bringing together large-scale research infrastructures and industry.

In a first approach, the team established that increasing concentrations of ethanol during extrusion influences liposome size. Interestingly, this effect is not observed if ethanol is added once extrusion has been completed. "Using neutron spin echo (NSE) - a unique method which allows for tracking molecular motions on time scales that are difficult to access otherwise - we observed that ethanol and butanol soften the liposome membranes", explains Ingo Hoffmann, one of the ILL scientists involved in the study. "The softening depends on the type of alcohol used and was not observed with methanol or glycerol, which is great news since the latter is used purely as a viscosity modifier and is expected to be inert with respect to the liposomes."

The dynamical information obtained by NSE was corroborated by small-angle neutron scattering (SANS). The authors observed a progressive thinning of the liposome membrane in the presence of ethanol and butanol, but not methanol. A possible explanation of the outsider role of methanol could be its reduced interactions with the liposome components due to its small size. Modeling of the SANS data provided even more precise structural information: in the presence of a water-alcohol mixture, both water and alcohol penetrate the liposome membrane. As the alcohol content increases, alcohol molecules gradually replace water molecules, effectively dehydrating the liposome membranes.

"Overall, our neutron experiments have allowed us to obtain very precise insights into the interactions between alcohols and liposomes in solution", says Fernanda Alvarado-Galindo. "This information is highly valuable for the industrial manufacturing of liposomes for medical purposes and showcases the unique strength of neutron techniques for such fundamental studies."

Text: Olga Matsarskaia

ILL Instruments : D11 - Lowest momentum transfer & lowest background small-angle neutron scattering instrument and IN15 - High Resolution Spin-echo spectrometer

Reference : Fernanda Alvarado Galindo, Joachim Venzmer, Sylvain Prévost, Ingo Hoffmann, and Michael Gradzielski.
Incorporation of short-chain alcohols into fluid bilayers and its effect on membrane dynamic properties as seen by neutron scattering.
Colloids and Surfaces A: Physicochemical and Engineering Aspects 702 (2024): 135014.
doi.org/10.1016/j.colsurfa.2024.135014

ILL Contacts : Ingo Hoffmann, Sylvain Prévost

Industrial partner : Evonik Operations GmbH, Germany