Small-Angle X-ray Scattering for the characterization of liposomes in pharmaceutical applications

Liposomes, first described in the early 60s, are spherical vesicles with lipid bilayer walls mainly consisting of phospholipids. Their size varies from tens of nanometers to over 200 micrometers. There are two types of liposomes: unilamellar and multilamellar. Multilamellar vesicles are further categorized into multilamellar, oligolamellar and multivesicular liposomes (see the following figure). The relative ease of manufacturing and their biocompatibility renders them an ideal carrier for active pharmaceutical ingredients (APIs) as well as in cosmetic [1] and food formulations. [2]

To safeguard the health of consumers and to maintain a constant quality, the production of liposomes for these applications is regulated under Good Manufacturing Practice (GMP) guidelines. These guidelines state that manufacturing processes must be clearly defined and controlled, and that any changes in the process must be evaluated.[ii] Morphological analysis is a crucial part of the quality control and Small-Angle X-ray Scattering is a well-suited tool for this.

Lamellarity, i.e. the amount of lamellar bilayers, is one of the parameters the FDA (Food and Drug Administration) marks as important. The lamellarity is a predominant factor in the ability of the liposome to contain and retain the drug substance.[iii] The number of lipid bilayers and their exact conformation can easily be accessed with SAXS. Coupling the measurement with Wide Angle X-ray Scattering (WAXS), information about Alkyl chain spacing can be obtained. This allows the determination of gel, lipid or ripple phases.

As an example, in a recent publication from scientists from Universita degli Studi dell Aquila and University of La Sapienza in Italy, SAXS was used together with other characterization methods to study the loading of Quinoline in liposomes composed of different lipids [3]. Quinoline is a well-known, and versatile compound, however with low solubility limiting its impact for pharmacological applications which can be overcome with its inclusion into liposomes. The figure below shows SAXS and WAXS measurements of quinoline-loaded multilamellar vesicles with different quinoline formulations and for liposomes made of different phospholipids (DOPC, DPPC). Well defined, periodic scattering peaks confirm the presence of vesicles with a desired degree of lamellarity. The DPPC liposome loaded with quinoline7 forms an exception. Here, broad scattering peaks suggest the presence of liposomes with a much lower number of stacked bilayers.

Characterizing lamellarity and lipid bilayer thickness with SAXS

Apart from studies of the bilayer morphology, SAXS can be used for the size characterization of liposomes in the range from a few nanometers to a few hundred nanometers, depending on the type of liposome and instrument. In a recent publication from the Karlsruhe Institute of Technology (KIT) [10], SAXS was used to investigate the use of liposomes as a means of stabilizing fluorocarbons-based emulsions. In this foundational research, phospholipids with various head sizes, chain lengths and different concentrations of the phospholipids were investigated. Firstly, SAXS unambiguously confirms the formation of liposomes, whereas this was not clear for freshly prepared samples from other measurement techniques. Secondly, SAXS is insensitive to gas bubbles in the samples that are introduced during production. Therefore, SAXS can provide a more reliable measure for the size of the liposomes than the intensity weighted mean hydrodynamic size as measured by photon correlated spectroscopy (PCS). In this particular case, PCS measurements are further hindered by the small difference in the refractive indices of both phases, this is not a problem for SAXS as the contrast relies on differences in electron density rather than refractive index.

As can be seen in the images below, the radially averaged scattering patterns of the liposomes with different phospholipids (left) and at different concentrations (right) can be modeled with high quality fits. The results indicate that the size of the liposomes increases with increasing length of the phospholipid tail from 48 nm for DMPC to 59 nm for DSPC and 61 nm for DPPC. Varying the concentration of DPPC from 25 to 300 mM results in the formation of liposomes from 61 nm to well over the detection limit of the instrument. Furthermore, the increase in scattering intensity leads to the conclusion that the more phospholipids are present, the more liposomes can be formed.