The influence of dipalmitoyl phosphatidylserine on phase behaviour of and cellular response to lyotropic liquid crystalline dispersions
Introduction
During the course of the last decade a number of surfactant and lipid based particle dispersions including micelles, liposomes and cubosomes have been suggested for use in biomedical applications such as medical imaging, bioseparations and drug delivery [1], [2], [3]. Safe and effective utilisation of such systems requires that they do not result in an adverse biological response. Factors such as particle size [4], dose [5] and surface characteristics, such as charge and coating [6], [7] are believed to play key roles in the biological response (e.g. cytotoxicity) to these materials. However, little work has been reported regarding the role of lipid/surfactant phase behaviour (polymorphism) [8]. Given that cell membrane fusogenicity is known to be affected by lipid polymorphism [9], it is apparent that a detailed understanding of the role of phase behaviour in determining biological response to lipid/surfactant based particles is important in order to ensure their safe and effective formulation and administration.
The vast majority of studies investigating the biological response to lipid-based particles have focussed on lamellar phase materials such as unilamellar/multilamellar vesicles or liposomes. Of particular note has been the development of the PEG coated ‘Stealth’ liposomes which have been shown to exhibit extended circulatory lifetimes in a number of studies [10], [11]. The ability of amphiphilic lipids and surfactants to form non-lamellar phases, however, offers many exciting opportunities for the formulation of lipid based particles for biomedical applications, as well as for the study of membrane lipid organisation and membrane dynamics in, for example, membrane fusion. Lyotropic liquid crystalline nanoparticles (cubosomes™) based on the cubic phase of the surfactant glyceryl monooleate (monoolein) have received attention by virtue of their ability to act as nanocarriers for delivering bioactive materials, such as drugs [12], peptides [13], and proteins [14], as well as functional foods [15]. However, there are, to our knowledge, no phase behaviour based studies of in vitro cellular response to these systems.
In this work, we have studied in vitro the role of lipid particle phase behaviour and composition in modulating cellular uptake and cytotoxicity of lyotropic liquid crystalline nanoparticles based on the lipid like surfactant: 1,2,3-trihydroxy-3,7,11,15-tetramethylhexadecane (phytantriol). Phytantriol is a commonly used surfactant in the cosmetics and haircare industries [16]. Phytantriol displays phase transitions with increasing water composition and temperature which result in a bicontinuous liquid crystalline phase comprising 15% water in physiological conditions and a reversed hexagonal phase at higher temperatures [17]. Most importantly, an equilibrium is found between the cubic phase and excess water, which provides the necessary conditions required for the dispersion of the cubic phase into colloidal (cubosome) formulations. In terms of its chemical structure, phytantriol comprises a tri-hydroxy headgroup and a branched phytanyl tail, without the presence of labile (e.g. ester) functionalities which affords additional chemical/biological stability relative to monoolein [18]. Hence, it has been suggested that phytantriol can be utilised as an alternative lipid to monoolein for the preparation of dispersed liquid crystalline systems (cubosomes) [19].
In the present study, we have co-formulated phytantriol cubic phases with the naturally occurring charged membrane lipid, DPPS in an effort to modulate the cubic phase nanostructure whilst maintaining biocompatibility. We report on the preparation of DPPS-containing phytantriol dispersions and their nanostructural characterisation using Cryogenic-transmission Electron Microscopy (Cryo-TEM) and Small Angle X-ray Scattering (SAXS).
It has been proposed that cubosomes can fuse with the cellular lipid membranes [20]. To investigate this hypothesis, we have employed SAXS to monitor the phase changes which occur when ‘membrane like’ lipid vesicles interact with cubosome systems. With a view to understanding the cubosome–membrane interaction in greater detail, we have developed a supported lipid membrane system based on 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) as a simplified cell membrane mimic. This has been used to quantify cubosome uptake and lipid/surfactant exchange reactions using Quartz Crystal Microbalance with Dissipation (QCM-D). Finally, we have studied the effect of cubosome nanostructural transitions, which result from liquid crystalline phase changes on cellular uptake and cytotoxicity using microscopic techniques and biological assays. By means of different characterization techniques, we can understand the effect of different formulations on the cellular response and uptake which may have implications for the future design and formulation of lipidic dispersions for therapeutic applications.
Section snippets
Materials
Commercial grade 1,2,3-trihydroxy-3,7,11,15-tetramethylhexadecane (98.0%) was obtained from DSM Nutritional Products. Pluronic® F127 (Poly(ethylene oxide)-poly(propyleneoxide)-poly(ethylene oxide) (PEO-PPO-PEO)) was purchased from Sigma. Dipalmitoyl phosphatidylserine was purchased from Genzyme Pharmaceuticals. 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine was purchased from Avanti Polar Lipids, Inc. Octadecyl rhodamine B chloride (R18) was purchased from Invitrogen. HEPES
Characterisation of phytantriol dispersions
Phytantriol-pluronic® F127 (Phy) dispersions were prepared containing various concentrations of DPPS (0, 2.5, 4.0, and 8.0 wt %) as described above. The surface electrostatic potential of the dispersed particles was confirmed by zeta potential measurements (shown in Table 1), which showed that the pure phytantriol particles possessed a slight negative charge of −36.4 mV in water. The incorporation of the negatively charged lipid DPPS, resulted in an increase in this negative zeta potential as
Conclusions
The interaction of multicomponent dispersions of lyotropic liquid-crystalline particles with cell membranes presumably involves a multi-step process involving attachment, and fusion as well as lipid mixing. These interactions must, in turn, be related to cellular uptake and, hence, cytotoxicity of the particles and their payloads. Whilst there are a plethora of studies which address cytotoxic response to such dispersions at the molecular level in, for instance, drug delivery applications,
Acknowledgements
SAXS/WAXS research was undertaken at the Australian Synchrotron, Victoria, Australia and we thank Dr Nigel Kirby for his assistance. The authors would like to thank Lynne Waddington and Veronica Glattauer for their help with the Cryogenic-Transmission Electron Microscopy studies and in cell culturing, respectively. The authors acknowledge the facilities, scientific and technical assistance of Monash Micro Imaging, Monash University, Victoria, Australia.
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