The lipolytic degradation of highly structured cubic micellar nanoparticles of soy phosphatidylcholine and glycerol dioleate by phospholipase A2 and triacylglycerol lipase.

The effects of different lipolytic enzymes on the structure of lipid liquid crystalline nano-particles (LCNP) have been investigated by cryogenic transmission electron microscopy (cryo-TEM) and synchrotron small angle X-ray diffraction (SAXD). Here we used highly structured cubic micellar (Fd3m) nanoparticles of 50/50 (wt%/wt%) soy phosphatidyl choline (SPC)/glycerol dioleate (GDO) as substrate. Two types of lipolytic enzymes were used, phospholipase A2 (PLA2) that catalyses degradation of the phospholipid component, SPC, and porcine pancreatic triacylglycerol lipase (TGL) that facilitate the hydrolysis of the diglyceride, GDO. Evolution of the structure was found to be very different and linked to specificity of the two types of enzymes. PLA2, which hydrolyses the lamellar forming component, SPC, induces a reversed micellar lipid phase, while TGL which hydrolysis the reverse phase forming compound, GDO, induces a lamellar phase.

Relationship between Structure and Fluctuations of Lipid Nonlamellar Phases Deposited at the Solid-Liquid Interface.

The structure and dynamics of nanostructure films formed by mixtures of soy phosphatidylcholine and glycerol dioleate at the silicon-aqueous interface were studied by grazing incidence neutron spin echo spectroscopy (GINSES), specular and off-specular neutron reflectometry, and small-angle X-ray diffraction. Reverse hexagonal (HII) and micellar cubic phase (Fd3m) layers at the solid-liquid interface have been identified with neutron reflectometry measurements. A preferred orientation of the liquid crystalline (LC) domains was observed only for the anisotropic HII phase. The size of the LC domains was found to be about 1 micrometer as estimated from the width of the diffraction peaks. GINSES revealed that the cubic phase forms rather rigid films. In comparison, the HII film was more flexible, appearing as a modified undulation spectrum of the cylinders due to the interaction with the substrate.

Interfacial properties of POPC/GDO liquid crystalline nanoparticles deposited on anionic and cationic silica surfaces.

Reversed lipid liquid crystalline nanoparticles (LCNPs) of the cubic micellar (I2) phase have high potential in drug delivery applications due to their ability to encapsulate both hydrophobic and hydrophilic drug molecules. Their interactions with various interfaces, and the consequences for the particle structure and integrity, are essential considerations in their effectiveness as drug delivery vehicles. Here, we have studied LCNPs formed of equal fractions of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and glycerol dioleate in the presence of different fractions of the stabilizer Polysorbate 80. We have used a combination of ellipsometry, quartz crystal microbalance with dissipation monitoring and neutron reflectometry to reveal the structure and composition of the adsorbed layer on both anionic silica and cationic (aminopropyltriethoxysilane) silanized surfaces. For both types of surfaces, there is a spread near-surface layer comprising lipid and polymer as well as a sparse coverage of intact particles. The composition of the near-surface layer is very close to that of the particles, in contrast to the lipid bilayer observed with related systems. The interaction is stronger for cationic than anionic surfaces, which is rationalized in terms of the negative zeta potential of the LCNPs. The work shows that the attachment of and spreading from LCNPs is influenced by the properties of the surface, the internal structure, composition and stability of the particles as well as the nature of the stabilizer.

Non-lamellar lipid liquid crystalline structures at interfaces.

The self-assembly of lipids leads to the formation of a rich variety of nano-structures, not only restricted to lipid bilayers, but also encompassing non-lamellar liquid crystalline structures, such as cubic, hexagonal, and sponge phases. These non-lamellar phases have been increasingly recognized as important for living systems, both in terms of providing compartmentalization and as regulators of biological activity. Consequently, they are of great interest for their potential as delivery systems in pharmaceutical, food and cosmetic applications. The compartmentalizing nature of these phases features mono- or bicontinuous networks of both hydrophilic and hydrophobic domains. To utilize these non-lamellar liquid crystalline structures in biomedical devices for analyses and drug delivery, it is crucial to understand how they interact with and respond to different types of interfaces. Such non-lamellar interfacial layers can be used to entrap functional biomolecules that respond to lipid curvature as well as the confinement. It is also important to understand the structural changes of deposited lipid in relation to the corresponding bulk dispersions. They can be controlled by changing the lipid composition or by introducing components that can alter the curvature or by deposition on nano-structured surface, e.g. vertical nano-wire arrays. Progress in the area of liquid crystalline lipid based nanoparticles opens up new possibilities for the preparation of well-defined surface films with well-defined nano-structures. This review will focus on recent progress in the formation of non-lamellar dispersions and their interfacial properties at the solid/liquid and biologically relevant interfaces.

Structural effects of the dispersing agent polysorbate 80 on liquid crystalline nanoparticles of soy phosphatidylcholine and glycerol dioleate.

Well-defined, stable and highly structured I2 (Fd3̅m) liquid crystalline nanoparticles (LCNP) of 50/50 (wt/wt) soy phosphatidylcholine (SPC)/glycerol dioleate (GDO), can be formed by using a low fraction (5-10 wt%) of the dispersing polymeric surfactant polyoxyethylene (20) sorbitan monooleate (polysorbate 80 or P80). In the present study we used small angle neutron scattering (SANS) and deuterated P80 (d-P80) to determine the location and concentration of P80 within the LCNP and small angle X-ray scattering (SAXS) to reveal the internal structure. SANS data suggests that some d-P80 already penetrates the particle core at 5%. However, the content of d-P80 is still low enough not to significantly change the internal Fd3̅m structure of the LCNP. At higher fractions of P80 a phase separation occurs, in which a SPC and P80 rich phase is formed at the particle surface. The surface layer becomes gradually richer in both solvent and d-P80 when the surfactant concentration is increased from 5 to 15%, while the core of the particle is enriched by GDO, resulting in loss of internal structure and reduced hydration. We have used neutron reflectometry to reveal the location of the stabiliser within the adsorbed layer on an anionic silica and cationic (aminopropyltriethoxysilane (APTES) silanized) surface. d-P80 is enriched closest to the supporting surface and slightly more so for the cationic APTES surface. The results are relevant not only for the capability of LCNPs as drug delivery vehicles but also as means of preparing functional surface coatings.

Formation of highly structured cubic micellar lipid nanoparticles of soy phosphatidylcholine and glycerol dioleate and their degradation by triacylglycerol lipase.

Lipid nanoparticles of reversed internal phase structures, such as cubic micellar (I2) structure show good drug loading ability of peptides and proteins as well as some small molecules. Due to their controllable small size and inner morphology, such nanoparticles are suitable for drug delivery using several different administration routes, including intravenous, intramuscular, and subcutaneous injection. A very interesting system in this regard, is the two component soy phosphatidylcholine (SPC)/glycerol dioleate (GDO) system, which depending on the ratio of the lipid components form a range of reversed liquid crystalline phases. For a 50/50 (w/w) ratio in excess water, these lipids have been shown to form a reversed cubic micellar (I2) phase of the Fd3m structure. Here, we demonstrate that this SPC/GDO phase, in the presence of small quantities (5-10 wt %) of Polysorbate 80 (P80), can be dispersed into nanoparticles, still with well-defined Fd3m structure. The resulting nanoparticle dispersion has a narrow size distribution and exhibit good long-term stability. In pharmaceutical applications, biodegradation pathways of the drug delivery vehicles and their components are important considerations. In the second part of the study we show how the structure of the particles evolves during exposure to a triacylglycerol lipase (TGL) under physiological-like temperature and pH. TGL catalyzes the lipolytic degradation of acylglycerides, such as GDO, to monoglycerides, glycerol, and free fatty acids. During the degradation, the interior phase of the particles is shown to undergo continuous phase transitions from the reversed I2 structure to structures of less negative curvature (2D hexagonal, bicontinuous cubic, and sponge), ultimately resulting in the formation of multilamellar vesicles.

Bioadhesive lipid compositions: self-assembly structures, functionality, and medical applications.

Lipid-based liquid crystalline compositions of phospholipids and diglycerides have unique bioadhesive properties with several medical applications, as exemplified by a lipid-based medical device indicated for management and relief of intraoral pain. The present paper describes the relation between self-assembly properties of phosphatidyl choline (PC) and glycerol dioleate (GDO) mixtures in the presence of aqueous fluids and functional attributes of the system, including: film formation and bioadhesion, intraoral coverage, acceptance by patients, and potential as a drug delivery system. The phase behavior of PC/GDO was characterized using synchrotron small-angle X-ray scattering. Functional properties, including the presence of study formulations at intraoral surfaces, ease of attachment, taste, and degree of and intraoral pain, were assessed in a crossover clinical pilot study in head and neck cancer patients. An optimum in functional properties was indicated for formulations with a PC/GDO weight ratio of about 35/65, where the lipids form a reversed cubic liquid crystalline micellar phase structure (Fd3m space group) over the relevant temperature range (25-40 °C).

Effect of phospholipid composition and phase on nanodisc films at the solid-liquid interface as studied by neutron reflectivity.

Nanodiscs are disc-like self-assembled structures formed by phospholipids and amphipatic proteins. The proteins wrap like a belt around the hydrophobic part of the lipids, basically producing nanometer-sized patches of lipid bilayers. The bilayer in the nanodisc constitutes a native-like model of the cell membrane and can act as a nanometer-sized container for functional single membrane proteins. In this study, we present a general nanodisc-based system, intended for structural and functional studies of membrane proteins. In this method, the nanodiscs are aligned at a solid surface, providing the ability to determine the average structure of the film along an axis perpendicular to the interface as measured by neutron reflectivity. The nanodisc film was optimized in terms of nanodisc coverage, reduced film roughness, and stability for time-consuming studies. This was achieved by a systematic variation of the lipid phase, charge, and length of lipid tails. Herein, we show that, although all studied nanodiscs align with their lipid bilayer parallel to the interface, gel-phase DMPC nanodiscs form the most suitable film for future membrane protein studies since they yield a dense irreversibly adsorbed film with low roughness and high stability over time. This may be explained by the appropriate matching between the thickness of the hydrophobic lipid core of gel phase DMPC and the height of the belt protein. Moreover, once formed the gel-phase DMPC nanodiscs film can be heated up to melt the lipid bilayer, thus providing a more biologically friendly environment for membrane proteins.

Monitoring shifts in the conformation equilibrium of the membrane protein cytochrome P450 reductase (POR) in nanodiscs.

Nanodiscs are self-assembled ∼50-nm(2) patches of lipid bilayers stabilized by amphipathic belt proteins. We demonstrate that a well ordered dense film of nanodiscs serves for non-destructive, label-free studies of isolated membrane proteins in a native like environment using neutron reflectometry (NR). This method exceeds studies of membrane proteins in vesicle or supported lipid bilayer because membrane proteins can be selectively adsorbed with controlled orientation. As a proof of concept, the mechanism of action of the membrane-anchored cytochrome P450 reductase (POR) is studied here. This enzyme is responsible for catalyzing the transfer of electrons from NADPH to cytochrome P450s and thus is a key enzyme in the biosynthesis of numerous primary and secondary metabolites in plants. Neutron reflectometry shows a coexistence of two different POR conformations, a compact and an extended form with a thickness of 44 and 79 Å, respectively. Upon complete reduction by NADPH, the conformational equilibrium shifts toward the compact form protecting the reduced FMN cofactor from engaging in unspecific electron transfer reaction.

Aligning nanodiscs at the air-water interface, a neutron reflectivity study.

Nanodiscs are self-assembled nanostructures composed of a belt protein and a small patch of lipid bilayer, which can solubilize membrane proteins in a lipid bilayer environment. We present a method for the alignment of a well-defined two-dimensional layer of nanodiscs at the air-water interface by careful design of an insoluble surfactant monolayer at the surface. We used neutron reflectivity to demonstrate the feasibility of this approach and to elucidate the structure of the nanodisc layer. The proof of concept is hereby presented with the use of nanodiscs composed of a mixture of two different lipid (DMPC and DMPG) types to obtain a net overall negative charge of the nanodiscs. We find that the nanodisc layer has a thickness or 40.9 ± 2.6 Å with a surface coverage of 66 ± 4%. This layer is located about 15 Å below a cationic surfactant layer at the air-water interface. The high level of organization within the nanodiscs layer is reflected by a low interfacial roughness (~4.5 Å) found. The use of the nanodisc as a biomimetic model of the cell membrane allows for studies of single membrane proteins isolated in a confined lipid environment. The 2D alignment of nanodiscs could therefore enable studies of high-density layers containing membrane proteins that, in contrast to membrane proteins reconstituted in a continuous lipid bilayer, remain isolated from influences of neighboring membrane proteins within the layer.