Lipid membranes exposed to dispersions of phytantriol and monoolein cubosomes: Langmuir monolayer and HeLa cell membrane studies

https://doi.org/10.1016/j.bbagen.2020.129738Get rights and content

Highlights

  • Incorporation of cubosomes is influenced by the hydration of lipid polar headgroups.

  • Langmuir isotherms and BAM show highest partition of PT cubosomes into DMPS layers.

  • Smaller doses of PT and GMO cubosomes did not affect the viability of HeLa cells.

  • Large concentrations of PT cubosomes destroyed cell membranes and altered viability.

Abstract

The interactions of liquid-crystalline nanoparticles based on lipid-like surfactants, glyceryl monooleate, monoolein (GMO) and 1,2,3-trihydroxy-3,7,11,15-tetramethylhexadecane, phytantriol (PT) with selected model lipid membranes prepared by Langmuir technique were compared. Monolayers of DPPC, DMPS and their mixture DPPC:DMPS 87:13 mol% were used as simple models of one leaflet of a cell membrane. The incorporation of cubosomes into the lipid layers spread at the air-water interface was followed by surface-pressure measurements and Brewster angle microscopy. The cubosome - membrane interactions lead to the fluidization of the model membranes but this effect depended on the composition of the model membrane and on the type of cubosomes. The interactions of PT cubosomes with lipid layers, especially DMPS-based monolayer were stronger compared with those of GMO-based nanoparticles. The kinetics of incorporation of cubosomal material into the lipid layer was influenced by the extent of hydration of the polar headgroups of the lipid: faster in the case of smaller, less hydrated polar groups of DMPS than for strongly hydrated uncharged choline of DPPC. The membrane disrupting effect of cubosomes increased at longer times of the lipid membrane exposure to the cubosome solution and at larger carrier concentrations. Langmuir monolayer observations correspond well to results of studies of HeLa cells exposed to cubosomes. The larger toxicity of PT cubosomes was confirmed by MTS. Their ability to disrupt lipid membranes was imaged by confocal microscopy. On the other hand, PT cubosomes easily penetrated cellular membranes and released cargo into various cellular compartments more effectively than GMO-based nanocarriers. Therefore, at low concentrations, they may be further investigated as a promising drug delivery tool.

Introduction

Lipidic liquid-crystalline nanoparticles (LCNP), cubosomes are studied extensively as therapeutic and diagnostic agents and delivery systems due to their biocompatibility and the ease of preparation, drug encapsulation, and targeting [1]. The cubosome structure consists of two interpenetrating, non-contacting, aqueous channels, which are surrounded by the lipid bilayer arranged in a thermodynamically favorable periodic 3D structure [[2], [3], [4]]. The cubosome structures, which are observed experimentally in the lipid–water systems, are double diamond (Pn3m), primitive (Im3m), and gyroid (Ia3d). They contain 3-, 4-, 6- way network junctions for Ia3d, Pn3m and Im3m phase, respectively. Amphiphilic lipids such as monoolein (GMO) and phytantriol (PT) are most often used to prepare cubosomes. GMO-based cubosomes possess lower stability than PT cubosomes because of the presence of the ester linkage and an unsaturated bond in the hydrocarbon chain, while phytantriol is more resistant to enzymatic degradation or hydrolysis due to the absence of these two moieties. Cubosomes have shown their potential as drug delivery systems for anticancer drugs [5,6], and for application in drug delivery across the blood-brain barrier (BBB), since they are able to transverse the BBB [7]. Cubosomes were also used in MRI and to form dual-modality lipid nanoparticles for fluorescence-MR imaging [8,9].

The mechanism of interaction between lipid - based nanoparticles and cell membranes and their effect on cell tolerability is the subject of intensive investigations. A few mechanisms of cubosome-cell interaction can be considered including: i) adsorption of cubosomes to cells, ii) lipid exchange between cubosome formulated lipids and the cell membrane, iii) endocytosis [10]. The latter can be classified into two main groups: receptor-mediated endocytosis or actin-driven endocytic non-specific uptake process called micropinocytosis [11]. The literature data on the interactions between lipid - based liquid crystalline (LLC) particles of different internal nanostructures and biological systems: soluble plasma constituents, blood cells, and isolated tissue cell lines have been recently reviewed by Tan et al. [11]. Critical analysis of factors affecting cell – nanoparticle tolerability such as type of lipids, type of steric stabilizers, nanoparticle surface charges, and internal nanostructures is provided in this review and allows for a better understanding, which of them can be of importance in selecting the entity optimal for internal applications. Shen et al. suggested that interactions of multicomponent dispersions of lyotropic liquid crystalline particles with cell membranes could involve a multi-step process comprising attachment and fusion as well as lipid mixing [12]. Internalization of the lipid nanoparticles with the cell membranes may depend on the elastic moduli of the lipid nanoparticles [13]. The highest uptake was observed for the nanoparticles with highest stiffness [14, 15]. Cubosomes being more rigid had higher cellular uptake than the softer hexosomes [16]. Nanoparticles with less negatively curved surface exhibit higher in vitro toxicity towards cells than nanoparticles with higher negative curvature [11]. Zou et al. reported that monoolein cubosomes interact more strongly with lipid membrane interface than hexosomes [17]. Different cytotoxicity between cubosomes and hexosomes may be related with the fact that the amount of stabilizer associated with cubosomes is likely to be higher than with hexosomes. The difference in stabilizer density on the surface of lipid nanoparticles contribute to the difference in the mechanism of interaction between nanoparticles and cells [18]. PT cubosomes exhibit a higher degree of membrane disruption than GMO analogue [19]. Cubosomes formed from PT are significantly more toxic and aggressive towards disrupting the cell nuclei membrane than GMO-based ones. Confocal microscopy images revealed that GMO treated cells showed fluorescent probe uptake through endocytosis, while in PT treated cells membrane disruption took place, allowing diffusion of dye-containing extracellular fluid across the membrane of PT treated cells [19]. The safety of using monoolein or phytantriol based cubosomes can be increased by incorporating certain endogenous phospholipids to mimic components of the mammalian cell membranes [20]. Recently, Strachan et al. hypothesized that toxicity of lipid nanoparticles appeared to be primarily determined by the chemical structure of the lipid [21]. Fusion of lipid cubosome nanocarriers with supported lipid bilayer under flow conditions allowed to visualize the fusion even which was sensitive to the lipid composition and rationalized by lipid diffusion. Dyett and co-workers used supported lipid bilayers to study fusion of lipid cubosomes under flow conditions using TIRF and microfluidics to elucidate the interaction of cubosomes with lipid layers [22].

Langmuir monolayers of different composition have been successfully employed as simple models to study the interactions with drugs, peptides or pollutants [[23], [24], [25]]. As a component of a model membrane a 1,2- dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) is used in the present paper. This phospholipid is a representative of phosphatidylcholines, which are the most abundant type of lipids present in cellular membranes [26]. As a second component a 1,2-dimyristoyl-sn-glycero-3-phospho-l-serine (DMPS) has been chosen, since PS lipids are externalized into the outer leaflet of cancerous cell membranes [27,28]. Despite the difference in the lipid acyl chain length the monolayers of these two phospholipids exhibit similar surface properties such as fluidity, which allows for an easier and more reliable comparison of the influence of the two types of cubosomes with respect to the different composition of the polar head of the lipid. Recently, we have demonstrated the effect of cubosomes on the structure and lipid orientation of DMPC bilayers transferred by the Langmuir-Schaefer method onto Au (111) surfaces [29]. We have shown that the effect of cubosomes on the lipid membrane depends on the initial state and organization of the bilayer. In the case of well-organized bilayers, spreading of the cubosomal material over their hydrophilic external surface is favored. On the other hand, less organized layers facilitate incorporation of the cubosomal phytantriol into the lipid matrix enhancing the porosity of the lipid membrane and explaining the observed decrease of the film resistance.

In the present paper, we aim at finding correlation of the effect of GMO and PT cubosomes on the selected cells with their ability to restructure the lipid membrane (Scheme 1). Monolayers of DPPC, DMPS and DPPC:DMPS 87:13 mol% – simple models of one leaflet of a lipid bilayer, and HeLa cell membranes served to study the incorporation of the cubic phase nanoparticles. The penetration of cubosomes into a lipid layer was followed by surface-pressure measurements and Brewster angle microscopy. The observed differences in the interactions of the two types of cubosomes with model membranes of varied composition are explained by the degree of the polar head hydration, which for the first time allowed us to determine the influence of this factor on the speed of cubosome incorporation into the lipidic layer. Differences in toxicity evaluated by MTS studies and the ability to disrupt the lipid membrane demonstrated by confocal microscopy confirmed monolayer study results and were strictly dependent on the nature of the lipidic material forming the cubosome but especially on the concentration of these carriers in the solution, to which the lipid membranes were exposed.

Section snippets

Chemicals

Monoolein (1-oleoyl-rac-glycerol) purity ≥99% (GMO) and Pluronic F108 (PF108) used for the synthesis of the mesophases were purchased from Sigma-Aldrich (USA). Octadecyl rhodamine B was from Thermofisher Sci. Phytantriol (PT) was purchased from Tokyo Chemical Industry (TCI). The model membranes were composed of 1,2-dimyristoyl-sn-glycero-3-phospho-l-serine (sodium salt) (DMPS) and 1,2- dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) of high purity (≥99%), which were purchased from Avanti Polar

Cubosome characterization

The internal structure of all cubosome formulations was verified using small-angle X-ray scattering (SAXS). Fig. S1 shows the diffraction pattern obtained from the formulations prepared using PT and GMO derived cubosomes, which were either loaded with the fluorescent dye or empty. Pluronic F108 has been selected to provide colloidal stability of formulations and to avoid particle aggregation. 1D diffraction patterns for all lipid formulations exhibit a sequence of diffraction peaks with

Conclusions

Interactions of GMO and PT derived cubosomes with model and biological cell membranes were studied. Incorporation of monoolein and phytantriol cubosomes into negatively charged DMPS and neutral DPPC monolayers leads to major changes of the monolayer phase from solid to liquid condensed or more liquid, respectively. It is consistent with the results of our earlier PM-IRRAS studies of supported DMPC bilayers showing a decreasing tilt angle of lipid molecules in the presence of PT cubosomal

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

The study was financed by Polish National Science Centre No. 2016/23/B/ST4/03295. Dr. Piotr Szwedziak from ReMedy-International Research Agenda Unit, Centre of New Technologies, University of Warsaw, 02-097 Warsaw, Poland is acknowledged for the Cryo TEM images of cubosomes.

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