Design of Lipid-Based Nanocarriers via Cation Modulation of Ethanol-Interdigitated Lipid Membranes


The phase behavior of phospholipids dictates various membrane properties, such as permeability, shape, and stiffness. Some phospholipids exhibit a polymorphic gel phase and are capable of undergoing membrane interdigitation under specific conditions, such as in the presence of short-chain alcohols (e.g., ethanol). Ethanol-induced interdigitation of diacyl–phosphatidylcholine bilayers has been leveraged to formulate large unilamellar vesicles with high entrapped aqueous volumes via the so called interdigitation-fusion vesicle method. In this case, ethanol is added to a small unilamellar vesicle (SUV) suspension below the lipid transition temperature (Tm), which induces vesicle fusion and rupture, together with bilayer interdigitation. A sol–gel transition of the vesicle suspension typically occurs as a result. Upon a temperature increase above the lipid Tm, the interdigitated bilayers refold into large unilamellar vesicles.

In order to induce bilayer interdigitation, water molecules at the lipid–water interface need to be replaced by ethanol, which alters the magnitude of hydration forces responsible for interbilayer repulsion. Binding of cations to the polar headgroups of phospholipid molecules can also modify the water layer coordinated to the lipid bilayer. However, most of the studies on the interactions between ethanol and PC bilayers in the context of bilayer interdigitation have been carried out in water and little is known on the role of cations, in particular Ca2+ and Na+. Here, we employed a combination of small- and wide-angle X-ray scattering (SAXS and WAXS), small-angle neutron scattering (SANS), and all-atom MD simulations to elucidate the nature of complex interactions between calcium and sodium ions, ethanol, and PC membranes. While bulk phase DPPC exhibited membrane interdigitation at all of the NaCl and CaCl2 concentrations explored, DPPC vesicles underwent membrane interdigitation and vesicle fusion in interdigitated lamellae only for a specific CaCl2 concentration. Large, multilamellar vesicles formed in the other conditions. These findings highlight the important role of cations in ethanol-induced membrane interdigitation and have implications for cell biology and biochemistry, as well as being directly applicable for, i.e., the fabrication of interdigitation-fusion vesicles with high entrapped volumes.


Neutron scattering studies allowed us to understand the morphology of DPPC vesicles formulated in the presence of either NaCl or CaCl2 at varying concentrations following ethanol addition. We configured sample-detector distances to give a scattering vector Q = 4π/λsin(θ/2) range of 0.004–0.722 Å–1, where θ is the scattering angle. Neutrons of wavelengths λ of 1.75–16.5 Å were used simultaneously by time of flight. The data was reduced by using MantidPlot, while SasView v4.1.0 was used to fit the SANS curves. In order to ensure good neutron contrast, partially deuterated ethanol (CH3CH2OD) was added to bulk and vesicular DPPC; the solvent scattering density was calculated with the scattering length density calculator plug-in of SasView.

We were able to estimate the bilayer thickness of the DPPC vesicles upon ethanol addition and we found bilayer thicknesses of 37 Å for the majority of salt concentrations explored, which may be due to the coexistence of interdigitated and gel phases within the sample. For DPPC vesicles containing 0.15 M CaCl2 we observed the formation of lamellar stacks with bilayer thicknesses of 25 Å upon ethanol addition, which was also confirmed by the sol-gel transition of the vesicle suspension. This observation was particularly relevant and has implications for the production of interdigitation-fusion vesicles. Ethanol could induce vesicle fusion in interdigitated lamellae only in the case of 0.15 M CaCl2; for the other conditions, we observed aggregation in large, multilamellar ensembles.

Figure 1. The addition of ethanol (showed in green in the right panel) to small unilamellar vesicles below the lipid Tm leads to the formation of bilayer stacks with interdigitated membranes.

What´s next?

In this work, calcium ions at low concentrations promoted vesicle fusion upon ethanol addition due to their fusogenic properties; we are interested in investigating the effect of other fusogenic molecules on ethanol-induced interdigitation and vesicle fusion. Future studies will also aim at elucidating the behavior of mixtures of saturated PCs with different chain lengths and the role of other divalent cations such as magnesium.


This work stemmed from an international collaboration between researchers based at Imperial College London (United Kingdom), Karolinska Insitutet (Sweden), RMIT (Australia), and the ISIS Neutron and Muon Source (United Kingdom). Funding was obtained by the Australian Research Council, the Ermenegildo Zegna Foundation, the Rosetrees Trust, the FP7 Marie Curie Intra-European Fellowship Program, the Swiss National Science Foundation, and Horizon 2020 Individual Marie Skłodowska-Curie Fellowship Program. Neutron scattering experiments at the ISIS Neutron and Muon Source were supported by beamtime allocations from the Science and Technology Facilities Council, while the SasView application was originally developed under an NSF award. SasView also contains code developed with funding from the European Union’s Horizon 2020 research and innovation programme under the SINE2020 project. X-ray scattering beamtime was provided by Diamond Light Source (SM 18658).


Cite: Langmuir 2021, 37, 40, 11909–11921

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Prof.  Molly Stevens

Department of Materials, Department of Bioengineering and Institute of Biomedical Engineering, Imperial College London, London SW7 2AZ, U.K. and Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-171 77 Stockholm, Sweden.


Prof. Irene Yarovsky

School of Engineering, RMIT University, Melbourne, Victoria 3001, Australia.