Effect of Formulation Method, Lipid Composition, and PEGylation on Vesicle Lamellarity: A Small-Angle Neutron Scattering Study


Liposomes are vesicles comprising a lipid membrane and aqueous core, and have found myriad applications in the biomedical field as therapeutics and in diagnostic assays. Liposome design requires careful choice of lipid composition and formulation method in order to produce populations with a defined unilamellar membrane structure. This is crucial in the development of liposome formulations for such drug delivery and biosensing applications, since lamellarity influences encapsulation efficiency and release kinetics. Despite this, a comprehensive study on how the lipid composition and formulation method affect vesicle lamellarity is still lacking.

There are many methods for producing liposomes, beginning typically from either hydrating a dried lipid film in aqueous buffer or mixing lipid dissolved in organic solvent with an aqueous phase. Most formulation processes include a step where the liposomes are extruded over a membrane of specific pore size in the range of 50 to 400 nm. While some residual multilamellarity is expected at larger extrusion pore sizes, it is generally assumed that extruding over membranes with 100 nm pore size or smaller leads to a predominantly unilamellar population with homogeneous size. However, we have observed that for specific lipid compositions and formulation methods, this is not necessarily the case.

Here, we combine small-angle neutron scattering and cryogenic transmission electron microscopy (cryo-TEM) to study the effect of three different well-established formulation methods on the resulting vesicle membrane structure. The ability to characterize and tune the extent of multi-lamellarity of liposome formulations has great significance for their application in diagnostic assays and drug delivery vectors, which often presume a unilamellar membrane bilayer.


Quantifying the extent of residual lamellarity in bulk suspensions is not trivial. While techniques such as cryo-TEM can provide sample snapshots, SANS is the ideal technique to quantify residual multilamellarity in bulk samples at low concentrations in their native environment. Using SANS, we quantified the lamellarity of liposome suspensions with fixed lipid compositions, formulated by either freeze thaw, agitation or reverse phase evaporation techniques, before and after sample extrusion, and corroborated our findings with cryo-TEM. In order to draw conclusions from the SANS data about the lamellarity of extruded vesicle samples, we constructed models in SasView that accommodated populations of uni-, bi-, tri- and quadri-lamellar vesicles and used the extracted parameters to calculate the percentage of the total particle volume, particle number and lipid for each sub-population.

For extruded liposome populations that had been formulated using film hydration-based methods,  a substantial sub-population of multilamellar liposomes remained. However, formulation via the reverse phase evaporation method yielded liposomes with a significantly lower percentage of multilamellar vesicles, and in the case of DPPC formulations the liposomes were unilamellar even before extrusion. We also observed that the addition of PEG at as little as around 0.1 to 0.5 mol% led to entirely unilamellar liposomes in all formulations after extrusion. In DPPC formulations, we also observed the formation of a sub-population of cylindrical micelles at 5 mol% PEG.


The work was carried out by Valeria Neleb, Margaret N. Holmea,b, Ulrike Kauscherb, Michael R. Thomasb, James J. Doutchc, and Molly M. Stevensa,b based at the Department of Medical Biochemistry and Biophysics, Karolinska Instituteta, the Department of Materials, Department of Bioengineering, and Institute of Biomedical Engineering, Imperial College Londonb, and ISIS Neutron and Muon Sourcec. ISIS Neutron and Muon Source (RB number: RB1610368 and RB1710255) is acknowledged for beamtime allocation from the Science and Technology Facilities Council. Financial support was provided by the Ermenegildo Zegna Founder’s Scholarship program, the Rosetrees Trust, the FP7 Marie Curie Intra-European Fellowship “SMase LIPOSOME” (626766), the Swiss National Science Foundation (P300PA_171540 and 406240_147493), the i-sense Engineering and Physical Sciences Research Council (EPSRC) 1610368IRC in Early Warning Sensing Systems for Infectious Diseases (EP/K031953/1; www.i-sense.org.uk), the Medical Research Council (MRC) grant “m-Africa” (MR/P024378/1), the Deutsche Forschungsgemeinschaft (KA4370/1-1), and a Wellcome Trust Senior Investigator Award (098411/Z/12/Z). This work benefited from the use of the SasView application, originally developed under NSF award DMR-0520547. SasView contains code developed with funding from the European Union’s Horizon 2020 research and innovation programme under the SINE2020 project, grant agreement 654000. We also acknowledge use of microscopy facilities within the Harvey Flower Electron Microscopy Suite, Department of Materials, Imperial College London, and would like to thank Dr. Richard K. Heenan for insightful discussion of the SANS analysis.

"Effect of Formulation Method, Lipid Composition, and PEGylation on Vesicle Lamellarity: A Small-Angle Neutron Scattering Study"

Langmuir 2019, 35, 18, 6064–6074

The full paper can be found at DOI: 10.1021/acs.langmuir.8b04256

What´s next?

Through SANS measurements corroborated with cryo-TEM, we have observed that factors such as formulation method, lipid composition and the inclusion of PEGylated lipid lead to vastly varying degrees of multi-lamellarity in extruded liposome formulations. In some cases, almost 50% of the total lipid is not formulated into unilamellar vesicles. These findings highlight important considerations affecting vesicle yield, encapsulation efficiency and release profiles when designing liposome formulations designated for drug delivery vectors and diagnostic assays, where a unilamellar membrane bilayer is often presumed.

Our current work focuses on expanding this study to characterize the effect of formulation method on lamellarity of liposomes formulated from a variety of lipid mixtures relevant to the biomedical field. Our overall aim is to build up a roadmap detailing which formulation methods are most appropriate to obtain unilamellar liposomes with a wide range of lipid compositions.

Figure 1. Using SANS, we quantified residual populations of multilamellar liposomes in extruded large unilamellar vesicle populations.


Dr. Margaret Holme, Karolinska Institutet