Liposome fractionation and size analysis by asymmetrical flow field-flow fractionation/multi-angle light scattering: influence of ionic strength and osmotic pressure of the carrier liquid

doi:10.1016/j.chemphyslip.2009.10.009

Chemistry and Physics of Lipids

Volume 163, Issue 2, February 2010, Pages 141-147

https://doi.org/10.1016/j.chemphyslip.2009.10.009 Get rights and content

Abstract

Asymmetrical flow field-flow fractionation (AsFlFFF)/multi-angle light scattering (MALS) was employed for studying filter-extruded liposomes in carrier solutions with different ionic strength and osmolarity. By dilution of preformed liposome suspensions with different media, only the ionic strength in the external free aqueous phase was changed. Under such conditions the liposomes were found to elute at almost identical elution times, which is in contrast to earlier studies. This may be explained by two opposing effects: (a) modulation of inter-particulate and particle-wall-repulsion effects and (b) osmotic stress-induced changes in vesicle size. The latter effect was demonstrated when analysing liposomes upon dilution in media of constant ionic strength, but varying osmotic pressure (with or without 150 mmol L⁻¹ sucrose supplement). The osmotic stress-induced change in liposome size was found to be size dependent. Larger liposomes appeared to both shrink and swell when exposed to hyper- or hypoosmotic media, respectively. Smaller liposomes appeared to shrink but not to swell. The potential causes of this effect are discussed.

Introduction

Liposomes or phospholipid vesicles are composed of one or several concentric bilayers encapsulating an aqueous core. Their application as models for biomembranes and carriers for drugs, has continuously broadened during the past 40 years. Particle size and lamellarity are among the most important physicochemical characteristics of liposomes. Size distribution has been demonstrated to govern biodistribution and targeting of liposomal drug carriers upon intravenous application (Liu and Huang, 1992). Therefore considerable effort has been put into optimization and refinement of techniques for size distribution analysis. One of the most versatile sub-categories among the family of field-flow fractionation (FFF) is asymmetrical flow field-flow fractionation (AsFlFFF), where a flow field is used to fractionate the analyte according to diffusivity. It has been used for the characterization of diverse analytes such as proteins (Wahlund and Litzen, 1989), polymers (Benincasa and Giddings, 1992), virus-like particles (Chuan et al., 2008), viruses (Giddings et al., 1977) and cells (Lee et al., 2003). Online coupling of AsFlFFF with multi-angle light scattering (MALS) has rendered the method easily applicable for routine purposes. One specific strength of this technique is, that the analyte is fractionated according to size before passing the MALS detector, which facilitates data analysis of broad and/or complex size distributions (Hupfeld et al., 2006). Simultaneous concentration detection (Hupfeld et al., 2009a) permits construction of quantitative size distribution plots. However, FFF of nanoparticles is governed by several interdependent parameters, which renders method-development a rather complex task. A recent study has demonstrated that fractionation of liposomes by AsFlFFF depends on cross flow rate, focussing conditions, sample load and salt concentration of the carrier liquid. (Hupfeld et al., 2009b). The influence of ionic strength on liposome fractionation is explained by the varying thickness of the electrical double layer, which may prevent the particles from reaching their equilibrium height over the accumulation wall (Moon, 1995, Moon et al., 1998). For successful size fractionation of liposomes, it is recommended to use a moderate salt concentration of the carrier liquid in the magnitude of 10 mmol L⁻¹ (Hupfeld et al., 2009b, Moon et al., 1998). However, for changes of the salt concentration, effects of osmotic stress and ionic strength on the liposome characteristics may have to be taken into account (Alonso et al., 1995, Mui et al., 1993).

The aim of the current study was, to investigate the impact of varying ionic strengths and osmotic pressures, on both AsFlFFF behaviour, and MALS-derived particle sizes of liposomes.

Section snippets

Relationship between retention time and particle size of the analyte

In FFF a field of force is exposed perpendicular onto the flow carrying the analyte in the separation channel (Giddings, 1966). In case of AsFlFFF the separation channel consists of an impermeable top block and a bottom block holding a semipermeable membrane laying on a porous frit constituting the accumulation wall. The perpendicular flow field is caused by restricting the main flow at the channel outlet. This restriction will force portions of the carrier liquid to leave the channel through

Chemicals

Egg-phosphatidylcholine (egg-PC) (Lipoid E80) was a kind gift of Lipoid (Lipoid GmbH, Ludwigshafen, Germany). Lissamine rhodamine B sulfonyl labeled DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-n-(lissamine rhodamine B sulfonyl)) (Rh-PE) was purchased from Avanti Polar Lipids (Avanti Polar Lipids, Alabaster, AL, USA). Sodium nitrate (NaNO₃, 99.5%) and sucrose (C₁₂H₂₂O₁₁, p.a.) were purchased from Merck (Merck KGaA, Darmstadt, Germany). Nanosphere size standards were purchased from Duke

Variation of salt concentration of the carrier liquid

We investigated the elution behaviour of Rh-PE liposomes prepared in 1 mmol L⁻¹ sodium nitrate solution with carrier solutions of increasing salt concentrations. Fractionations were performed in water as well as sodium nitrate solutions of increasing concentration (5–50 mmol L⁻¹). Injection was performed in triplicates and the elugrams resulting from the fractionations with different carrier liquids are displayed in Fig. 1. In water the liposomes began to elute at 11 min, immediately after the void

Conclusion

The combination of AsFlFFF with MALS and concentration detection allowed to study independently electrostatic and osmotic effects of solutes on the retention behaviour and the particle size distributions of filter-extruded liposomes. A change of the actual size of liposomes and consequential change in elution time, is for the first time reported here. We could demonstrate that the change of retention time with varying ionic strength of the carrier liquid is caused by two different effects.

Acknowledgments

We acknowledge support for financing the AsFlFFF instrument from the Norwegian Research Council (AVIT). We also would like to thank Roy Lysaa for designing the swelling-/shrinking-plot, the teams of both Wyatt Technology Europe GmbH, Germany and the Wyatt Technology Corporation for technical support and fruitful discussions, the Pharmaceutical Institute, University of Oslo, for giving us access to zeta potential measurements and Ursula Fattler (Medigene AG, Munich) for providing Rh-PE liposomes

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Liposome fractionation and size analysis by asymmetrical flow field-flow fractionation/multi-angle light scattering: influence of ionic strength and osmotic pressure of the carrier liquid

Abstract

Introduction

Section snippets

Relationship between retention time and particle size of the analyte

Chemicals

Variation of salt concentration of the carrier liquid

Conclusion

Acknowledgments

Colloids Surf. A

Colloids Surf. A

J. Chromatogr. A

Chem. Phys. Lipids

Int. J. Pharm.

Biophys. J.

Biophys. J.

Biochim. Biophys. Acta Biomembr.

J. Chromatogr.

J. Chromatogr. A

Biophys. J.

Biochim. Biophys. Acta

Chem. Phys. Lipids

Int. J. Pharm.

J. Colloid Interf. Sci.

J. Chromatogr.