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

Liposomes are well-established systems for drug delivery and biosensing applications. The design of a liposomal carrier requires careful choice of lipid composition and formulation method. These determine many vesicle properties including lamellarity, which can have a strong effect on both encapsulation efficiency and the efflux rate of encapsulated active compounds. Despite this, a comprehensive study on how the lipid composition and formulation method affect vesicle lamellarity is still lacking. Here, we combine small-angle neutron scattering and cryogenic transmission electron microscopy to study the effect of three different well-established formulation methods followed by extrusion through 100 nm polycarbonate membranes on the resulting vesicle membrane structure. Specifically, we examine vesicles formulated from the commonly used phospholipids 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) via film hydration followed by (i) agitation on a shaker or (ii) freeze–thawing, or (iii) the reverse-phase evaporation vesicle method. After extrusion, up to half of the total lipid content is still assembled into multilamellar structures. However, we achieved unilamellar vesicle populations when as little as 0.1 mol % PEG-modified lipid was included in the vesicle formulation. Interestingly, DPPC with 5 mol % PEGylated lipid produces a combination of cylindrical micelles and vesicles. In conclusion, our results provide important insights into the effect of the formulation method and lipid composition on producing liposomes with a defined membrane structure.

Neutron scattering data were fitted using several models in the SasView v3.1.2 program. It provides the form factor ( ), defined as: Where φ is a scale factor, ℎ is the volume of the shell, is the volume of the core, is the total volume, is the radius of the core, is the outer radius of the shell, is the scattering length density of the solvent (which is the same for the core), is the scattering length density of the shell and J1 = (sinx-x cosx) / x 2 and the = 4 sin( )/ .
Bilamellar Vesicle model. The 1D scattering intensity ( ) is calculated as the sum of the form factors ( ) for the volume fractions 1 and 2 of uni-and bilamellar vesicles and a flat background to account for incoherent and/or inelastic scattering: The form factor ( ) is representative of a vesicle with a number of shells = i and is normalized by the particle volume: For a spherical particle system it is possible to assume that the volume fraction of a particle population is: where is the number of particles and is the volume per particle/shell. From the core radius , the bilayer thickness = − and the water layer thickness , it is possible to calculate the total volume per particle , and the total shell volume per particle , . Therefore, the fraction of bilamellar vesicles as a function of total particle volume, particle number and lipid can be calculated.  (S4) where n = 4 as the sum of the form factors ( ) for the volume fractions 1 , 2 , 3 and 4 of uni-, bi-, tri-and quadrilamellar vesicles and a flat background.

S3
The angle is defined as the orientation of the major axis of the ellipse respect to the vector , the angle is the angle between the axis of the cylinder and the vector. To get the 1D scattering intensity, the form factor 2 ( ) is averaged over all the possible orientations and normalised by particle volume: 2 ( ) = < 2 > (S12).
In a similar way to the multilamellar models, from the core radius rc and the bilayer thickness = − , it is possible to calculate , and , (i.e. the total volume per particle and the total shell volume per particle). Therefore, the proportion of vesicles as a function of total particle volume, particle number and lipid can be calculated.
Broad Peak model. The 1D scattering intensity is calculated as: where ξ is the Lorentzian screening length, m is the Lorentzian exponent (fixed as 2 in the fittings), and are scaling factors for the Porod and the Lorentz terms, respectively, n is the Porod exponent and 0 is the peak position. The peak position can be used to calculate the d-spacing ( = 2 0 ⁄ ), which is the sum of the bilayer thickness and the coordinated water layer.

S4
Neutron scattering data of unextruded DPPC vesicles were fitted with either a Broad Peak model (AS and the FT methods) or a unilamellar vesicle model (REV method). A Gaussian polydispersity function of 20% for the vesicle core radii was used in this latter case. (core radii, rc). Table S2. Fitting parameters and simple estimates of the particle number and lipid volume ratios of the uni (i=1) and bilamellar (i=2) vesicle component for extruded DPPC vesicles prepared via the AS and the FT methods. Lengths and volumes are in Å and Å 3 , respectively.  To fully capture the hump in the scattering profile at Q ~ 0.1 Å, a quadrilamellar vesicle model was used to fit the small-angle neutron scattering data of extruded POPC vesicles prepared via the AS method. A trilamellar vesicle model and a bilamellar vesicle model were instead sufficient in the case of the FT and the REV methods, respectively. A Gaussian polydispersity function of 20% for the vesicle core radii was used in all the cases. We also formulated POPC vesicles via agitation at room temperature for 1 hour and extruded them through a 100 nm membrane for 35 times and we observed no substantial differences with POPC vesicles prepared via the AS method described here. The fitted data for the experiment had errors from 0.22% to 1.14% (ts), 5.16% to 18.65% (φi), 1.67% to 6.82% (tw), and 5.26% to 19.67% (rc). Table S4. Fitting parameters and simple estimates of the particle number and lipid volume ratios of the uni (i=1), bi (i=2), tri (i=3) and quadrilamellar (i=4)   were best fitted with a trilamellar vesicle and a bilamellar vesicle model, respectively. Data of vesicles prepared with 0.5, 1 and 5 mol% of DOPE-PEG2000 were fitted using a simple unilamellar vesicle model. A Gaussian polydispersity function of 20% for the vesicle core radii was used in all the cases, together with a Gaussian polydispersity function of bilayer thickness of 20% for 0.1, 0.5 mol% and 1 mol% and 40% for 5 mol% of DOPE-PEG2000 to account for variation in membrane bilayer thickness due to the presence of the PEGylated lipid. No Gaussian distribution of bilayer thickness was used for vesicles containing 0 mol% DOPE-PEG2000 as it was assumed that they have a uniform thickness.

S8
Neutron scattering data of extruded DOPC vesicles containing 0 mol% of DOPE-PEG2000 were fitted with a trilamellar vesicle model. Scattering data of vesicles prepared with 0.1, 0.5, 1 and 5 mol% of DOPE-PEG2000 were fitted using a simple unilamellar vesicle model. A Gaussian polydispersity function of 20% for the vesicle core radii was used in all the cases, together with a Gaussian polydispersity function of bilayer thickness of 20% for 0.5 mol% and 1 mol% and 40% for 5 mol% of DOPE-PEG2000 to account for variation in membrane bilayer thickness due to the presence of the PEGylated lipid. The fitted data for the experiment had errors from 0.03% to 7.38% (φi), 2.24% to 2.71% (tw), maximum 1.03% (ts), and maximum 8.30% (rc). Small-angle neutron scattering data of extruded DPPC vesicles containing 0 mol% of DOPE-PEG2000 were fitted with a bilamellar vesicle model. Data of vesicles prepared with 0.1, 0.5 and 1 mol% of DOPE-PEG2000 were fitted using a unilamellar vesicle model. A Gaussian polydispersity function of 20% for the vesicle core radii was used in all the cases, together with a Gaussian polydispersity function of bilayer thickness of 20% for 0.5 mol% and 1 mol% of DOPE-PEG2000 to account for variation in membrane bilayer thickness due to the presence of the PEGylated lipid.

S10
A model obtained combining a unilamellar vesicle model and a core shell cylindrical model was used to fit the scattering profile of extruded DPPC ensembles containing 5 mol% DOPE-PEG2000. A Gaussian polydispersity function of 20% was used for the vesicle core radius and bilayer thickness.
The fitted data for the experiment had errors from 0.9% to 11.47%. Table S8. Fitting parameters of small-angle neutron scattering data of DPPC ensembles containing 5 mol% DOPE-PEG2000 prepared via the FT method and extruded through 100 nm pore size membrane. Simple estimates of the particle number and lipid volume ratios of the unilamellar vesicle component are reported below. Lengths and volumes are in Å and Å 3 , respectively.  Figure S1. Cryo-TEM of unextruded DPPC vesicles prepared via the REV method. Scale bar: 100 nm.