Binary Lipid Mixtures Research Articles

Several common methods of making vesicles (except an emulsion method) capture intended lipid ratios

Researchers choose different methods of making giant unilamellar vesicles to satisfy different constraints of their experimental designs. A challenge that arises when researchers use a variety of methods is that each method may produce vesicles with a different average lipid ratio, even if all experiments use lipids from a common stock mixture. Here, we use mass spectrometry to investigate ratios of lipids in vesicle solutions made by five common methods: electroformation on indium tin oxide slides, electroformation on platinum wires, gentle hydration, emulsion transfer, and extrusion. We made vesicles from either five-component or binary mixtures of lipids chosen to span a wide range of physical properties: di(18:1)PC, di(16:0)PC, di(18:1)PG, di(12:0)PE, and cholesterol. For a mixture of all five of these lipids, ITO electroformation, Pt electroformation, gentle hydration, and extrusion methods result in only minor shifts in lipid ratios (≤5 mol %) relative to a common stock solution. In contrast, emulsion transfer results in ∼80% less cholesterol than expected from the stock solution, which is counterbalanced by a surprising overabundance of saturated PC-lipid relative to all other phospholipids. Experiments using binary mixtures of saturated and unsaturated PC-lipids and cholesterol largely support results from the five-component mixture. In general, our results imply that experiments that increment lipid ratios in small steps will produce data that are highly sensitive to the technique used and to sample-to-sample variations. For example, sample-to-sample variations are ∼±2 mol % for five-component vesicles produced by a single technique. In contrast, experiments that explore larger increments in lipid ratio or that seek to explain general trends and new phenomena will be less sensitive to sample-to-sample variation and the method used.

Lipid-related ion suppression on the herbicide atrazine in earthworm samples in ToF-SIMS and matrix-assisted laser desorption ionization mass spectrometry imaging and the role of gas-phase basicity.

In mass spectrometry imaging (MSI), ion suppression can lead to a misinterpretation of results. Particularly phospholipids, most of which exhibit high gas-phase basicity (GB), are known to suppress the detection of metabolites and drugs. This study was initiated by the observation that the signal of an herbicide, i.e., atrazine, was suppressed in MSI investigations of earthworm tissue sections. Herbicide accumulation in earthworms was investigated by time-of-flight secondary ion mass spectrometry and matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). Additionally, earthworm tissue sections without accumulation of atrazine but with a homogeneous spray deposition of the herbicide were analyzed to highlight region-specific ion suppression. Furthermore, the relationship of signal intensity and GB in binary mixtures of lipids, amino acids, and atrazine was investigated in both MSI techniques. The GB of atrazine was determined experimentally through a linear plot of the obtained intensity ratios of the binary amino acid mixtures, as well as theoretically. The GBs values for atrazine of 896 and 906 kJ/mol in ToF-SIMS and 933 and 987 kJ/mol in MALDI-MSI were determined experimentally and that of 913 kJ/mol by quantum mechanical calculations. Compared with the GB of a major lipid component, phosphatidylcholine (GBPC = 1044.7 kJ/mol), atrazine's experimentally and computationally determined GBs in this work are significantly lower, making it prone to ion suppression in biological samples containing polar lipids.

Predicting lipid sorting in curved membranes.

Most biological membranes are curved, and both lipids and proteins play a role in generating curvature. For any given membrane shape and composition, it is not trivial to determine whether lipids are laterally distributed in a homogeneous or inhomogeneous way, and whether the inter-leaflet distribution is symmetric or not. Here we present a simple computational tool that allows to predict the preference of any lipid type for membranes with positive vs. negative curvature, for any given value of curvature. The tool is based on molecular dynamics simulations of tubular membranes with hydrophilic pores. The pores allow spontaneous, barrierless flip-flop of most lipids, while also preventing differences in pressure between the inner and outer water compartments and minimizing membrane asymmetric stresses. Specifically, we provide scripts to build and analyze the simulations. We test the tool by performing simulations on simple binary lipid mixtures, and we show that, as expected, lipids with negative intrinsic curvature distribute to the tubule inner leaflet, the more so when the radius of the tubular membrane is small. Compared to other existing computational methods, relying on membrane buckles and tethers, our method is based on spontaneous inter-leaflet transport of lipids, and therefore allows to explore lipid distribution in asymmetric membranes. The method can easily be adapted to work with any molecular dynamics code and any force field.

The Development of Lipid-Based Sorafenib Granules to Enhance the Oral Absorption of Sorafenib.

Sorafenib (SFN) is an anticancer multi-kinase inhibitor with great therapeutic potential. However, SFN has low aqueous solubility, which limits its oral absorption. Lipids and surfactants have the potential to improve the solubility of water-insoluble drugs. The aim of this study is thus to develop novel lipid-based SFN granules that can improve the oral absorption of SFN. SFN powder was coated with a stable binary lipid mixture and then absorbed on Aeroperl 300 to form dry SFN granules with 10% drug loading. SFN granules were stable at room temperature for at least three months. Compared to SFN powder, SFN granules significantly increased SFN release in simulated gastric fluid and simulated intestinal fluid with pancreatin. Pharmacokinetics and tissue distribution of SFN granules and SFN powder were measured following oral administration to Sprague Dawley rats. SFN granules significantly increased SFN absorption compared to SFN powder. Overall, the lipid-based SFN granules provide a promising approach to enhancing the oral absorption of SFN.

Origin of the nonlinear structural and mechanical properties in oppositely curved lipid mixtures.

Structural and mechanical properties of membranes such as thickness, tail order, bending modulus and curvature energetics play crucial role in controlling various cellular functions that depend on the local lipid organization and membrane reshaping. While behavior of these biophysical properties are well understood in single component membranes, very little is known about how do they change in the mixed lipid membranes. Often various properties of the mixed lipid bilayers are assumed to change linearly with the mole fractions of the constituent lipids which, however, is true for "ideal" mixing only. In this study, using molecular dynamics simulations, we show that structural and mechanical properties of binary lipid mixture change nonlinearly with the lipid mole fractions, and the strength of the nonlinearity depends on two factors - spontaneous curvature difference and locally inhomogeneous interactions between the lipid components.

Food-grade hexosomes as efficient vehicles for delivery of fish-purified antioxidant peptide

Herein, we describe the potential use of food-grade hexosomes (HEXs) for delivering fish-purified antioxidant peptide (PF10). Using a binary lipid mixture of Dimodan U/citrem, the nanocarriers were produced with a size range of 202.7–569.8 nm and peptide encapsulation efficiency of 64.6–89.3%. These HEXs were also characterized by SAXS and cryo-TEM, and were able to sustain the release of PF10, where only 32.2% released in PBS after 24 h. SAXS findings verified that PF10 modulate the internal structure of HEXs in a pH-dependent manner. Antioxidant assays proved the efficacy of such nano-self-assemblies in maintaining the bioactivity of the loaded peptide. Moreover, the in vitro gastrointestinal stability test indicated that the antioxidant capacity of the free- and PF10-loaded HEXs decreased under SGF/SIF conditions with the reduction in activity being greater for the free PF10. The present findings may provide a useful basis for development of pH-responsive nano-self-assemblies for delivery of antioxidant peptides.

Small-Angle Neutron Scattering Study of a Phosphatidylcholine-Phosphatidylethanolamine Mixture.

The properties of single-component phospholipid lipid bilayers have been extensively characterized. Natural cell membranes are not so simple, consisting of a diverse mixture of lipids and proteins. While having detailed structural information on complex membranes would be useful for understanding their structure and function, experimentally characterizing such membranes at a level of detail applied to model phospholipid bilayers is challenging. Here, small-angle neutron scattering with selective deuteration was used to characterize a binary lipid mixture composed of 1,2-dimyristoyl-3-sn-glycero-phosphatidylcholine and 1,2-dimyristoyl-3-sn-glycero-phosphatidylethanolamine. The data analysis provided the area per lipid in each leaflet as well as the asymmetry of the composition of the inner and outer leaflets of the bilayer. The results provide new insight into the structure of the lipid bilayer when this lipid mixture is used to prepare vesicles.

Quantitative Comparison against Experiments Reveals Imperfections in Force Fields' Descriptions of POPC-Cholesterol Interactions.

Cholesterol is a central building block in biomembranes, where it induces orientational order, slows diffusion, renders the membrane stiffer, and drives domain formation. Molecular dynamics (MD) simulations have played a crucial role in resolving these effects at the molecular level; yet, it has recently become evident that different MD force fields predict quantitatively different behavior. Although easily neglected, identifying such limitations is increasingly important as the field rapidly progresses toward simulations of complex membranes mimicking the in vivo conditions: pertinent multicomponent simulations must capture accurately the interactions between their fundamental building blocks, such as phospholipids and cholesterol. Here, we define quantitative quality measures for simulations of binary lipid mixtures in membranes against the C-H bond order parameters and lateral diffusion coefficients from NMR spectroscopy as well as the form factors from X-ray scattering. Based on these measures, we perform a systematic evaluation of the ability of commonly used force fields to describe the structure and dynamics of binary mixtures of palmitoyloleoylphosphatidylcholine (POPC) and cholesterol. None of the tested force fields clearly outperforms the others across the tested properties and conditions. Still, the Slipids parameters provide the best overall performance in our tests, especially when dynamic properties are included in the evaluation. The quality evaluation metrics introduced in this work will, particularly, foster future force field development and refinement for multicomponent membranes using automated approaches.

Tunable biomimetic bacterial membranes from binary and ternary lipid mixtures and their application in antimicrobial testing

The reconstruction of accurate yet simplified mimetic models of cell membranes is a very challenging goal of synthetic biology. To date, most of the research focuses on the development of eukaryotic cell membranes, while reconstitution of their prokaryotic counterparts has not been fully addressed, and the proposed models do not reflect well the complexity of bacterial cell envelopes. Here, we describe the reconstitution of biomimetic bacterial membranes with an increasing level of complexity, developed from binary and ternary lipid mixtures. Giant unilamellar vesicles composed of phosphatidylcholine (PC) and phosphatidylethanolamine (PE); PC and phosphatidylglycerol (PG); PE and PG; PE, PG and cardiolipin (CA) at varying molar ratios were successfully prepared by the electroformation method. Each of the proposed mimetic models focuses on reproducing specific membrane features such as membrane charge, curvature, leaflets asymmetry, or the presence of phase separation. GUVs were characterized in terms of size distribution, surface charge, and lateral organization. Finally, the developed models were tested against the lipopeptide antibiotic daptomycin. The obtained results showed a clear dependency of daptomycin binding efficiency on the amount of negatively charged lipid species present in the membrane. We anticipate that the models proposed here can be applied not only in antimicrobial testing but also serve as platforms for studying fundamental biological processes in bacteria as well as their interaction with physiologically relevant biomolecules.

Kinetic relaxation of giant vesicles validates diffusional softening in a binary lipid mixture.

The stiffness of biological membranes determines the work required by cellular machinery to form and dismantle vesicles and other lipidic shapes. Model membrane stiffness can be determined from the equilibrium distribution of giant unilamellar vesicle surface undulations observable by phase contrast microscopy. With two or more components, lateral fluctuations of composition will couple to surface undulations depending on the curvature sensitivity of the constituent lipids. The result is a broader distribution of undulations whose complete relaxation is partially determined by lipid diffusion. In this work, kinetic analysis of the undulations of giant unilamellar vesicles made of phosphatidylcholine-phosphatidylethanolamine mixtures validates the molecular mechanism by which the membrane is made 25% softer than a single-component one. The mechanism is relevant to biological membranes, which have diverse and curvature-sensitive lipids.

A Small-Angle Neutron Scattering, Calorimetry and Densitometry Study to Detect Phase Boundaries and Nanoscale Domain Structure in a Binary Lipid Mixture

Techniques that can probe nanometer length scales, such as small-angle neutron scattering (SANS), have become increasingly popular to detect phase separation in membranes. But to extract the phase composition and domain structure from the SANS traces, complementary information is needed. Here, we present a SANS, calorimetry and densitometry study of a mixture of two saturated lipids that exhibits solidus-liquidus phase coexistence: 1,2-dipalmitoyl-d62-sn-glycero-3-phosphocholine (dDPPC, tail-deuterated DPPC) and 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC). With calorimetry, we investigated the phase diagram for this system and found that the boundary traces for both multilamellar vesicles (MLVs) as well as 50 nm unilamellar vesicles overlap. Because the solidus boundary was mostly inaccessible by calorimetry, we investigated it by both SANS and molecular volume measurements for a 1:1 dDPPC:DLPC lipid mixture. From the temperature behavior of the molecular volume for the 1:1 dDPPC:DLPC mixture, as well as the individual molecular volume of each lipid species, we inferred that the liquidus phase consists of only fluid-state lipids while the solidus phase consists of lipids that are in gel-like states. Using this solidus-liquidus phase model, the SANS data were analyzed with an unrestricted shape model analysis software: MONSA. The resulting fits show irregular domains with dendrite-like features as those previously observed on giant unilamellar vesicles (GUVs). The surface pair correlation function describes a characteristic domain size for the minority phase that decreases with temperature, a behavior found to be consistent with a concomitant decrease in membrane mismatch between the liquidus and solidus phases.

Copolymeric nanodiscs do not retain the native membrane environment, but do maintain protein-lipid interactions.

Polymer stabilized lipid-protein nanoparticles have become invaluable tools in membrane biology since they can isolate individual proteins with their surrounding lipids. These nanoparticles, termed nanodiscs, have historically relied on membrane scaffolding proteins, but can now be formed using amphipathic copolymers that directly intercalate into membranes. Since copolymeric nanodiscs are formed directly from membranes without requiring detergent solubilization, they have been presumed to retain the native membrane environment, but this assumption has not been thoroughly tested. To test if native membrane lipid packing is retained in styrene-maleic acid (SMA) and diisobutylene-maleic acid (DIBMA) stabilized nanodiscs, lipid packing was analyzed using solvatochromic dyes Laurdan, C-Laurdan, and Pro12A. SMA and DIBMA copolymeric nanodiscs were formed from membranes increasing in complexity from single and binary lipid mixture, to biomimetic ternary mixtures composed of palmitoyl-sphingomyelin (PSM), 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), and cholesterol, and ultimately to mammalian cellular membranes. For all synthetic model membranes, lipid packing of fluid-disordered membranes was significantly increased in both SMA and DIBMA nanodiscs compared to intact membranes, with DIBMA being the least perturbing. In contrast, lipid packing in ordered membranes was unaffected. For cellular membranes, lipid packing was also higher inside nanodiscs than in native membranes. Our data challenges the assumption that native membrane properties are retained inside nanodiscs. While not maintaining native membrane properties, nanodiscs can be useful models for studying protein-lipid interactions, as they are composed of membrane lipids. Interestingly, nanodiscs can exchange lipids through collisions, allowing tuning of their lipid compositions. We used this capability to analyze the preferred surrounding lipidome of multi-pass transmembrane proteins. Altogether, our studies demonstrate that copolymeric nanodiscs have different properties to those of intact membranes, but they are useful models in studying the paralipidomes of membrane proteins.

Asymmetric phase diagram of DPPC/DOPC to investigate inter-leaflet coupling.

Cell membranes are asymmetric, meaning that the bilayer is composed of leaflets with different lipid composition. Simplified models for the exoplasmic leaflet typically form coexisting Ld (liquid disordered) + Lo (liquid ordered) phases in symmetric bilayers. In contrast, the cytoplasmic leaflet lacks high-melting lipids, and models for this leaflet always form a single fluid phase in symmetric bilayers. Thus, among the major open questions is how the phase behavior of these two leaflets is coupled in an asymmetric bilayer. Here, we report the first experimentally determined asymmetric phase diagram for a binary lipid mixture, DPPC/DOPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine/1,2-dioleoyl-sn-glycero-3-phosphocholine) at room temperature. Different from cholesterol-containing model membranes that separate into coexisting liquid phases, this simpler mixture exhibits gel + fluid coexistence over a range of composition and temperature. We used calcium-induced hemifusion to engineer asymmetric GUVs (aGUVs), and we measured the extent of outer leaflet exchange through quantitative analysis of fluorescent probe intensities. Our experiments can be directly compared to theoretical phase diagrams calculated from mean field theory, where phase boundaries and tieline orientations depend on the relative strength of in-plane and out-of-plane lipid interaction energies. The experimental DPPC/DOPC asymmetric phase diagram is consistent with an intermediate coupling strength whereby anti-registered domains coexist with registered domains at lower levels of asymmetry, but phase separation is completely suppressed at higher levels of asymmetry. Our results allow us to estimate a mismatch free energy of 0.2-0.3 kT per square nanometer for a gel domain directly opposite a fluid domain.

Intrinsic lipolysis rate for systematic design of lipid-based formulations

Lipid-based formulations (LBFs) are used by the pharmaceutical industry in oral delivery systems for both poorly water-soluble drugs and biologics. Digestibility is key for the performance of LBFs and in vitro lipolysis is commonly used to compare the digestibility of LBFs. Results from in vitro lipolysis experiments depend highly on the experimental conditions and formulation characteristics, such as droplet size (which defines the surface area available for digestion) and interfacial structure. This study introduced the intrinsic lipolysis rate (ILR) as a surface area-independent approach to compare lipid digestibility. Pure acylglycerol nanoemulsions, stabilized with polysorbate 80 at low concentration, were formulated and digested according to a standardized pH–stat lipolysis protocol. A methodology originally developed to calculate the intrinsic dissolution rate of poorly water-soluble drugs was adapted for the rapid calculation of ILR from lipolysis data. The impact of surfactant concentration on the apparent lipolysis rate and lipid structure on ILR was systematically investigated. The surfactant polysorbate 80 inhibited lipolysis of tricaprylin nanoemulsions in a concentration-dependent manner. Coarse-grained molecular dynamics simulations supported these experimental observations. In the absence of bile and phospholipids, tricaprylin was shielded from lipase at 0.25% polysorbate 80. In contrast, the inclusion of bile salt and phospholipid increased the surfactant-free area and improved the colloidal presentation of the lipids to the enzyme, especially at 0.125% polysorbate 80. At a constant and low surfactant content, acylglycerol digestibility increased with decreasing acyl chain length, decreased esterification, and increasing unsaturation. The calculated ILR of pure acylglycerols was successfully used to accurately predict the IRL of binary lipid mixtures. The ILR measurements hold great promise as an efficient method supporting pharmaceutical formulation scientists in the design of LBFs with specific digestion profiles.Graphical abstract

The Structures of Heterogeneous Membranes and Their Interactions with an Anticancer Peptide: A Molecular Dynamics Study.

We conduct molecular dynamics simulations of model heterogeneous membranes and their interactions with a 24-amino acid peptide—NAF-144–67. NAF-144–67 is an anticancer peptide that selectively permeates and kills malignant cells; it does not permeate normal cells. We examine three membranes with different binary mixtures of lipids, DOPC–DOPA, DOPC–DOPS, and DOPC–DOPE, with a single peptide embedded in each as models for the diversity of biological membranes. We illustrate that the peptide organization in the membrane depends on the types of nearby phospholipids and is influenced by the charge and size of the head groups. The present study sheds light on early events of permeation and the mechanisms by which an amphiphilic peptide crosses from an aqueous solution to a hydrophobic membrane. Understanding the translocation mechanism is likely to help the design of new permeants.

Investigation of diclofenac release and dynamic structural behavior of non-lamellar liquid crystal formulations during in situ formation by UV–Vis imaging and SAXS

In situ formation of high viscous inverse lyotropic non-lamellar liquid crystalline phases is a promising approach for sustained drug delivery in the joint. The in situ forming process on exposure of two diclofenac-loaded preformulations to aqueous media was characterized with respect to depot size and shape, initial release and structural transitions using UV–Vis imaging and spatially and time-resolved synchrotron small-angle X-ray scattering (SAXS). The preformulations consisted of 10 % (w/w) ethanol, 10 % (w/w) water and a binary lipid mixture of glycerol monooleate (GMO):1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) (DOPG) or GMO:medium chain triglycerides (MCT). Upon injection of preformulations into an employed injection-cell containing excess of bio-relevant medium, rapid generation of liquid crystalline depots was observed. UV–Vis images and constructed 2D SAXS maps of the injection-cell showed depots with different shapes and sizes, and features with high nanostructural heterogeneity. More extensive swelling of the GMO:DOPG-based preformulation was observed compared to the GMO:MCT-based preformulation. The UV image analysis found that a higher amount of diclofenac was released in the image area after 20 h from the GMO:MCT depot compared to the GMO:DOPG depot. The injection-cell setup employing UV–Vis imaging and synchrotron SAXS constitutes an attractive approach for evaluating the in situ forming processes of liquid crystalline depots.

Interaction of Cyanine-D112 with Binary Lipid Mixtures: Molecular Dynamics Simulation and Differential Scanning Calorimetry Study.

This comprehensive molecular dynamics (MD) simulation and experimental study investigates the lipid bilayer interactions of dye D112 for potential photodynamic therapy (PDT) applications. PDT involves formation of a reactive oxidant species in the presence of a light sensitive molecule and light, interrupting cellular functions. D112 was developed as a photographic emulsifier, and we hypothesized that its combined cationic and lipophilic nature can render a superior photosensitizing property–crucial in various light therapies. The focus of this study is to elucidate the binding and insertion mechanisms of D112 with mixed lipid bilayers of anionic dipalmitoyl-phosphatidylserine (DPPS) and zwitterionic dipalmitoyl-phosphatidylcholine (DPPC) lipids to resemble cancer cell membranes. Our studies confirm initial electrostatic binding between the positively charged moieties of D112 and negatively charged lipid headgroups. Additionally, MD simulations combined with differential scanning calorimetry (DSC) studies confirm that D112-lipid interactions are governed by enthalpy-driven nonclassical hydrophobic effects in the membrane interior. It was further noted that despite the electrostatic preference of D112 toward the anionic lipids, D112 molecules colocalized on DPPC-rich domains after insertion. Atomistic level MD studies point toward two possible insertion mechanisms for D112: harpoon and flip. Further insights from the simulation showcase the interactions of low and high aggregates of D112 with the bilayer as the concentration of D112 increases in solution. The size of aggregates modulates the orientation and degree of insertion, providing important information for future studies on membrane permeation mechanisms.

Asymmetric phase diagram of a binary lipid mixture

The plasma membranes of eukaryotic cells are asymmetric with respect to their lipid composition. Model membrane studies consistently find that simplified outer leaflet mixtures tend to separate into coexisting liquid phases in symmetric bilayers, while inner leaflet mixtures do not. Among the major unanswered questions is how the phase behavior of the two leaflets is coupled in an asymmetric bilayer. Mean field theory has been used to calculate asymmetric phase diagrams, where phase boundaries and tieline orientations depend on the relative strength of in-plane and out-of-plane lipid interaction energies.

Nanostructured lipid carriers based suppository for enhanced rectal absorption of ondansetron: In vitro and in vivo evaluations

The current research aimed to fabricate ondansetron nanostructured lipid carriers (OND-NLCs) and incorporate them into a suppository base to manage chemotherapy-induced vomiting and nausea, which offer the advantage of both rapid onset and prolonged release. NLCs were fabricated by adopting the solvent diffusion method. The binary lipid mixture of oleic acid (liquid lipid) and lauric acid (solid lipid) were prepared in distinct ratios. The NLCs were characterized concerning the surface charge, size, drug encapsulation efficiency, and surface morphology. In addition, the influence of surfactant, co-surfactant, and lipid on entrapment efficiency and particle size was investigated. Phosphate buffer having pH 7.4 is used for evaluating in vitro drug release by utilizing a dialysis membrane. Various kinetics models were used to estimate the drug release kinetics of fabricated nanostructured lipid carriers. The particle size of the NLCs was calculated between 101 and 378 nm with negative zeta potential on the NLC’s surface. The entrapment efficiency was found between 68 and 87%. Scanning Electron Microscopic analysis showed the spherical shape of nanostructured lipid carriers. The dissolution profile of the ondansetron-loaded NLC suppository depicts biphasic behavior of firstly burst release then slow release was observed. The diffusion controlled release was evident from kinetic modeling. The succeeding step comprehended the fabrication and characterization of NLC-based suppositories utilizing NLC formulations that demonstrated the combined advantage of rapid onset, prolonged release, and better in vivo bioavailability as compared to control suppository.

Dynamic structure factors of fluid membranes exhibiting lateral and flip-flop transitions: Effects of inclusions and the presence of an interacting wall

In this paper, we consider a fluid membrane formed by a mixture of two chemically incompatible lipid molecules A and B. The variation of an adequate parameter (temperature, PH, lateral pressure, etc.) may lead the amphiphile molecules of the bilayer to undergo lateral or flip-flop separations. The physical system is modeled by a two-order parameter field theory. The composition fluctuations relative to the sheets are denoted φ and ψ, standing in fact for the two fields. We first investigate the effect of inclusions like flippase proteins on the dynamics of the lipid mixture, assumed to be suddenly cooled by passing from a starting temperature Ti, towards a temperature Tf nearby to the consolute point Tc. To compute exactly the structure factors use is made of the extended Van Hove approach written as two coupled Langevin equations (with noise). We show that the dynamics is entirely ruled by two kinds of relaxation rates τL and τS. The first is the pertinent time, which is related to long-wavelength fluctuations leading to the formation of macrophase domains. Whereas the local dynamics is characterized by the second one τS, which is a short scale time. We show, notably, that the presence of attractive inclusions tends to lower the relaxation time τL, and consequently the flip-flop transition is accelerated. The existence of two relaxation times is in good agreement with a certain experimental study on the kinetics of binary lipid mixtures phase transitions, namely DMPC-DSPC and DMPC-DPPC. We investigate, in the second part of this work, the effect of the presence of an interacting wall, assumed to adsorb strongly one or both species, on the parallel structure factors S(z,z′). These latter are exactly calculated in all cases: below Tc, above Tc and at T=Tc, as well as their associated local compressibilities S(z). Scaling laws, for these latter, are also derived.