Bilayer Membrane Systems Research Articles

HIV-1 Gag specificity for PIP2 is regulated by macromolecular electric properties of both protein and membrane local environments

HIV-1 assembly occurs at the plasma membrane, with the Gag polyprotein playing a crucial role. Gag association with the membrane is directed by the matrix domain (MA), which is myristoylated and has a highly basic region that interacts with anionic lipids. Several pieces of evidence suggest that the presence of phosphatidylinositol-(4,5)-bisphosphate (PIP2) highly influences this binding. Furthermore, MA also interacts with nucleic acids, which is proposed to be important for the specificity of GAG for PIP2-containing membranes. It is hypothesized that RNA has a chaperone function by interacting with the MA domain, preventing Gag from associating with unspecific lipid interfaces. Here, we study the interaction of MA with monolayer and bilayer membrane systems, focusing on the specificity for PIP2 and on the possible effects of a Gag N-terminal peptide on impairing the binding for either RNA or membrane. We found that RNA decreases the kinetics of the protein association with lipid monolayers but has no effect on the selectivity for PIP2. Interestingly, for bilayer systems, this selectivity increases in presence of both the peptide and RNA, even for highly negatively charged compositions, where MA alone does not discriminate between membranes with or without PIP2. Therefore, we propose that the specificity of MA for PIP2-containing membranes might be related to the electrostatic properties of both membrane and protein local environments, rather than a simple difference in molecular affinities. This scenario provides a new understanding of the regulation mechanism, with a macromolecular view, rather than considering molecular interactions within a ligand-receptor model.

Structure and stability of polydiacetylene membrane systems: Molecular dynamics simulation studies

We have performed full atomistic molecular dynamics (MD) simulations to investigate structure and stability of bilayer membrane systems consisting of monomeric or polymeric 10,12-pentacosadiynoic acid (PCDA) units connected with lysine groups by amide bonds. The PCDA monomer molecules show a twisted three-rod-domain structure with two kinks but upon polymerization, they possess more elongated conformation. The resulting polydiacetylene (PDA) membrane systems have stable membrane structures at room temperature, which is similar to biological lipid bilayer membranes and maintain their gel-like membrane integrity even up to as high as 370 K. Structural properties such as area per monomer, membrane thickness, density profile, 2D pair distribution function, and orientational correlation function are also calculated to understand the membrane structure and check its stability upon thermal fluctuation with atomistic resolution. This study is expected to provide the understanding about PDA membrane systems in atomistic details as well as significant insights into designing new novel PDA sensors.

Dynamics and mechanism of a deswelling transition of the sponge phase in a bilayer membrane system

In surfactant solutions, membranes are self-organized and divide water into two domains, an ``inside'' and an ``outside.'' When the membrane phase transforms into a dilute sponge (${L}_{1}$) phase, the ${L}_{1}$ phase is also partitioned into inside and outside domains. Such systems are critical not only for material exchange mechanisms in biology but also for the formation of boundaries between topologically distinct regions. Nevertheless, the mechanism and associated behavior of the system at the transition remain unclear. Here, we use both experimental observations and numerical simulations to investigate the phase transition dynamics. It is found that domains of inside and outside are formed which appear connected to each other and that they nucleate as droplets despite the ${L}_{1}$ phase being the majority phase. Although such behavior is significantly different from what is seen in ordinary phase separation, we successfully reproduced all typical behavior seen in the experiments with numerical simulations. For the formation of the connected domains, it is found that the correlation length associated with the concentration needs to be larger than that of the inside/outside patterns. This offers a means to control pattern formation in systems with a parity mechanism, such as chirality.

Conductive nerve guide conduits based on wet-adhesive hydrogel to accelerate peripheral nerve repair

Peripheral nerve injury is a serious traumatic condition, and primary repair or tension-free anastomosis remains an unresolved challenge in nerve restoration. Currently, surgical treatment has critical limitations, such as granulomatous inflammation and numbness of the end limbs. Nerve guidance conduits (NGCs), which are tissue-adaptable and conductive, have shown promise for effective nerve repair. Inspired by the adhesion mechanism (cation–π interactions) of bilayer membrane systems and mussel foot proteins, we developed a promising wireless suture using wet-adhesive hydrogel NGCs. The prepared hydrogel conduits had good electrical conductivity, flexibility, mechanical stability, and wet tissue adhesion and exhibited excellent cytocompatibility with neuronal and Schwann cells. An in vivo rat sciatic nerve transection injury model confirmed that the hydrogel NGCs not only firmly adhere to wet nerve tissue and corkingly connect the proximal to distal ends, but also improve electroconduction velocity and sciatic nerve function index, which significantly enhance peripheral nerve repair. Histological analysis and immunostaining confirmed robust and orderly nerve regeneration through the conductive hydrogel NGCs. In summary, wet-adhesive hydrogel NGCs may serve as effective wireless suture candidates for promoting neural repair.

Bioinorganic Platforms for Sensing, Biomimicry, and Energy Catalysis

Bioinorganic chemistry is a broad field with multiple branches. In this Highlight Review, the newest excitements in the branch of Bioinorganic Platforms are reviewed. Artificial bioinorganic platforms come in mesmerizing flavors, in particular the following 5 types are the major focus of this Highlight Review. First, single-stranded and double-stranded DNA, RNA, PNA, and their hybrids can be attached to Au electrodes for in vitro biosensing purposes as well as real-time monitoring of DNA repair processes via magnetoelectrochemistry. Second, Pseudomonas aeruginosa biofilms can be grown into interdigitated array (IDA) electrodes to investigate electron transport mechanisms in vivo. Third, protein films can be formed on glassy carbon (GC) electrodes to examine the effect of electrostatics on the redox behavior of metalloenzymes. Fourth, lipid-covered hybrid bilayer membrane (HBM) systems can be constructed to control the thermodynamics and kinetics of proton-coupled electron transfer (PCET) steps important to renewable energy schemes. Fifth, metal complexes can be physisorbed onto highly porous carbon powder as facile oxygen reduction reaction (ORR) catalysts.

In Silico Design of a Single Protein Molecular Photoreaction Center

Background: Research on harvesting solar energy through an artificial photosynthetic device is gaining momentum in the present era. The device is a promising solution to the energy crisis by overcoming the crunch in fossil fuel and low efficiency of heat engine. Reports are available on design of isolated parts of the photosynthetic device, for example, only sensitizer or only redox unit containing metallo-peptides. Objective: An attempt has been made to design an in silico photoreaction center in a single chain protein matrix containing all the three basic units: sensitizer, electron donor, and acceptor mimicking the photosynthetic reaction center II. Methods: A single chain of a protein containing a closely packed transmembrane four-helix bundle (PDB ID 2bl2 A) is selected for the purpose. The protein is suitably mutated in silico to accommodate the basic elements of a reaction center: Mn-Ca binding site as water oxidizing moiety, Fe-binding site as quinine reducing moiety, and MgDPP as photosensitizer to achieve the desired function of photoredox reaction. Results: A photoelectron transport protein has been designed, which may incorporate into the bilayer membrane system. It has the potential to photo-oxidize water to oxygen on one side and reduce quinone on the other side of the membrane. The stability and transmembrane orientation of the molecular device in an artificial membrane system has been validated theoretically by molecular dynamics study. Conclusion: An attempt to incorporate in silico all the elements essential for a photoelectron transport device into a single chain transmembrane protein model is the first of its kind. Donor and acceptor moieties are separated on the inner and outer side of a membrane bilayer. These features make the model unique and novel. The design of the model is the first step towards the study of experimental viability of the model, which remains to be validated in future.

Ultrastructural modeling of small angle scattering from photosynthetic membranes

The last decade has seen a range of studies using non-invasive neutron and X-ray techniques to probe the ultrastructure of a variety of photosynthetic membrane systems. A common denominator in this work is the lack of an explicitly formulated underlying structural model, ultimately leading to ambiguity in the data interpretation. Here we formulate and implement a full mathematical model of the scattering from a stacked double bilayer membrane system taking instrumental resolution and polydispersity into account. We validate our model by direct simulation of scattering patterns from 3D structural models. Most importantly, we demonstrate that the full scattering curves from three structurally typical cyanobacterial thylakoid membrane systems measured in vivo can all be described within this framework. The model provides realistic estimates of key structural parameters in the thylakoid membrane, in particular the overall stacking distance and how this is divided between membranes, lumen and cytoplasmic liquid. Finally, from fitted scattering length densities it becomes clear that the protein content in the inner lumen has to be lower than in the outer cytoplasmic liquid and we extract the first quantitative measure of the luminal protein content in a living cyanobacteria.

Plasma‐induced Water Pore Formation in Model Cell Membranes: Molecular Dynamics Simulation

We have investigated the mechanism of plasma‐induced water pore formation in model 1,2‐dioleoyl‐sn‐glycero‐3‐phosphocholine (DOPC) bilayer membrane systems using atomistic molecular dynamics (MD) simulations. Oxidized by reactive oxygen species generated upon the plasma treatment, unsaturated hydrocarbon tails of DOPC lipids are converted into shortened hydrocarbon tails with terminal groups such as peroxide or aldehyde. Among them, the lipids with both hydrocarbon tails oxidized into aldehyde groups are particularly susceptible to the stable water pore formation. By analyzing the water pore formation dynamics, lipid escape, and lipid clustering for the plasma‐damaged DOPC membrane systems, we have found that a stable water pore is formed in the membrane region where the plasma‐damaged lipids are highly concentrated or locally clustered. In the plasma‐damaged lipid‐rich region, a continuous water channel through the membrane is easily established with the help of the terminal aldehyde groups in the tails of damaged lipids, and it continuously grows with time to form a stable water pore. The rapid local clustering or domain formation of the plasma‐damaged lipids is due to both the hydrophobic mismatch between normal and oxidized DOPC lipids and enhanced lateral diffusion of the oxidized lipids in the membrane. We have also observed that the onset concentration of oxidized lipids for the stable water pore formation is approximately 30% in the model DOPC membrane systems.

The Use of Integra® Dermal Regeneration Template for the Orbital Exenteration Socket: A Novel Technique.

Integra® dermal regeneration template is a bilayer membrane system that acts as a scaffold for regenerating dermal skin cells. It is used for wound reconstruction following burns, extensive injuries, and a large tumor excision in multiple parts of the body. The dermal layer is made of porous matrix of bovine tendon collagen and glycosaminoglycan. The epidermal layer is made of polysiloxane layer. In this study, the authors evaluated the use of Integra® dermal regeneration template for the immediate reconstruction of the orbital exenteration socket. Five patients who underwent exenteration and immediate reconstruction of the socket with Integra® dermal regeneration template were included in this study. Demographic and clinical features, healing time, complications, and follow-up time were recorded. The study included 4 male patients and 1 female patient with a mean age of 74 years (range, 49-87 years). The primary diagnoses were orbital extension of conjunctival melanoma in 2 patients, squamous cell carcinoma in 1 patient, and uveal melanoma in 1 patient, and aggressive orbital Wegener granulomatosis in 1 patient. There was no postoperative infection, necrosis, hematoma, or fluid accumulation in any patients. The mean postoperative follow-up period was 20 months (range, 11-42 months). The sockets were completely granulated by 4 weeks, and epithelized, getting ready for the prosthesis in 8 weeks. Integra® dermal regeneration template can be used for the immediate reconstruction of the socket following exenteration. It is easy to use, and provides a short healing time without any need for any additional reconstructive procedures.

Structure, Dynamics, and Phase Behavior of DOPC/DSPC Mixture Membrane Systems: Molecular Dynamics Simulation Studies

Full atomistic molecular dynamics simulations have been performed for model mixture bilayer membrane systems consisting of 1,2‐dioleoyl‐sn‐glycero‐3‐phosphocholine (DOPC) and 1,2‐distearoyl‐sn‐glycero‐3‐phosphocholine (DSPC) phospholipids to understand the effects of two essential parameters such as lipid composition and temperature on the structural, dynamical, and phase behavior of mixture membrane systems. Although pure DSPC membranes are in the gel‐like (L β ′ or P β ′) phase at 323 K, raising the temperature by only 10 K or replacing 20% of DSPC lipids by DOPC lipids can change the gel‐like phase into the completely liquid‐crystalline phase (L α ). This phase change is accompanied by dramatic change in both structural properties such as area per lipid, membrane thickness, deuterium order parameter, and tail angle distribution, and dynamics properties such as mobility map. We also observe that the full width at half‐maximum (FWHM) data of tail angle distribution as well as area per lipid (or membrane thickness) can be used as order parameters for the membrane phase transition.

Cholesterol-based tethers and markers for model membranes investigation

A series of new bifunctional cholesterol compounds for tethered bilayer membrane (tBLM) systems were synthesized and tested. The compounds containing cyclic disulfide group may be used as molecular anchors for phospholipid bilayers. Anchoring occurs through the insertion of the cholesterol moiety into the hydrophobic slab of the phospholipid layer, while the surface density of anchor molecules may be adjusted using disulfides terminated spacers. Five ethylene oxide segments between the disulfide group and the cholesteryl provide hydration of the layer separating solid support and model membrane. Another group of cholesterol derivatives described in this work contains either fluorescence probe or electroactive functional groups. We demonstrated the practical utility of these compounds for visualization of cholesterol extraction from and loading to tBLMs. We demonstrated that electroactive group containing cholesterol derivatives can be reconstituted either into vesicles or tBLMs. In both cases an electrochemical signal can be generated on electrodes from these probes. In general, the newly synthesized compound may be utilized in a variety of applications involving tethered bilayer systems and vesicles.

Contact bubble bilayers with flush drainage.

Planar lipid bilayers have been used to form stable bilayers into which membrane proteins are reconstituted for measurements of their function under an applied membrane potential. Recently, a lipid bilayer membrane is formed by the apposition of two monolayers that line an oil-electrolyte interface. Here, a bilayer membrane system is developed with picoliter bubbles under mechanically and chemically manipulable conditions. A water bubble lined with a phospholipid monolayer is blown from a glass pipette into an oil phase. Two blowing pipettes are manipulated, and bubbles (each with a diameter of ~ 50 μm) are held side by side to form a bilayer, which is termed a contact bubble bilayer. With the electrode implemented in the blowing pipette, currents through the bilayer are readily measured. The intra-bubble pressure is varied with the pressure-controller, leading to various sizes of the bubble and the membrane area. A rapid solution exchange system is developed by introducing additional pressure-driven injection pipettes, and the blowing pipette works as a drain. The solution is exchanged within 20 ms. Also, an asymmetric membrane with different lipid composition of each leaflet is readily formed. Example applications of this versatile method are presented to characterize the function of ion channels.

Stepping between Membrane Microdomains

Lateral membrane microdomains are postulated to be involved in a number of important functions in mammalian cells, such as membrane trafficking, exocytosis, endocytosis, signal transduction, and protein activity (1,2). These putative cell microdomains have been the subject of extensive research using a variety of biophysical and biochemical techniques, including detergent extraction (2), fluorescence microscopy (3), electron microscopy (4), fluorescence resonance energy transfer microscopy (5), fluorescence correlation spectroscopy (6), fluorescence recovery after photobleaching (7), and single-molecule tracking (8).

Effect of Asymmetry on Bilayer Membrane Systems

Many biological processes, such as membrane fusion and the action of microbial proteins, involve asymmetric uptake of proteins. In addition, it has been demonstrated that large mechaosensitive channels open by the asymmetric inclusion of lyso-lipids in the bilayer in the absence of external pressure. These phenomena can be interpreted in terms of the asymmetric local pressure profile in the bilayer, which lacks the quantitative understanding. By noting that such asymmetric pressure profile can be induced in bilayer membranes with asymmetric number of lipids between leaflets, to provide quantitative understanding, we performed molecular dynamics simulations of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) bilayer membranes for a set of systems with different level of asymmetry with and without gramicidin A (gA) or WALP23. We observed that even highly asymmetric lipid bilayers with and without gA or WALP23 were stable during the whole simulation time, which is consistent with the previous study of dipalmitoyl-phosphatidylcholine (DPPC) bilayers [J. Am. Chem. Soc. 131, 15194 (2009)]. As the number of lipids in the lower leaflet becomes smaller, the lipids are more tightly packed in the top leaflet and less ordered in the bottom leaflet, which are quantified in terms the lateral pressure profile, the order parameter, the area per lipid, etc. However, the hydrophobic thickness for pure lipid bilayers does not show meaningful dependence on the asymmetry, which results from the stretched and less ordered lipids in the upper and lower leaflets, respectively. We will also discuss the influence of the asymmetry on the properties of protein-lipid interactions, such as the distance dependence of the lipid properties and the interaction patterns around gA or WALP23.

Development of a CCK1R-membrane nanoparticle as a fish-out tool for bioactive peptides

The cholecystokinin receptor type 1 (CCK1R) is a G protein-coupled receptor (GPCR) that is involved in several biological processes including the regulation of the secretion of digestive enzymes. The peptide hormone cholecystokinin (CCK) binds to CCK1R, which is an important pharmacological target for several diseases, including obesity. Interestingly, nutritional dietary peptides also appear to activate CCK1R, and may play a role in CCK1R signaling in the gut. In this study, a novel technique to screen for CCK1R ligands based on affinity-selection is described. Functional expressed CCK1R is reconstituted into membrane nanoparticles called NABBs (nanoscale apo-lipoprotein bound bilayers). NABBs are native-like bilayer membrane systems for incorporation of GPCRs. CCK1R-NABBs were characterized using a fluorescently labeled CCK analog and can be used as a cutting-edge technology to screen for CCK1R ligands using affinity-selection mass spectrometry.

Localization of trehalose in partially hydrated DOPC bilayers: insights into cryoprotective mechanisms

Trehalose, a natural disaccharide with bioprotective properties, is widely recognized for its ability to preserve biological membranes during freezing and dehydration events. Despite debate over the molecular mechanisms by which this is achieved, and that different mechanisms imply quite different distributions of trehalose molecules with respect to the bilayer, there are no direct experimental data describing the location of trehalose within lipid bilayer membrane systems during dehydration. Here, we use neutron membrane diffraction to conclusively show that the trehalose distribution in a dioleoylphosphatidylcholine (DOPC) system follows a Gaussian profile centred in the water layer between bilayers. The absence of any preference for localizing near the lipid headgroups of the bilayers indicates that the bioprotective effects of trehalose at physiologically relevant concentrations are the result of non-specific mechanisms that do not rely on direct interactions with the lipid headgroups.

Proton switch for modulating oxygen reduction by a copper electrocatalyst embedded in a hybrid bilayer membrane.

Molecular switches gate many fundamental processes in natural and artificial systems. Here, we report the development of an electrochemical platform in which a proton carrier switches the activity of a catalyst. By incorporating an alkyl phosphate in the lipid layer of a hybrid bilayer membrane, we regulate proton transport to a Cu-based molecular oxygen reduction reaction catalyst. To construct this hybrid bilayer membrane system, we prepare an example of a synthetic Cu oxygen reduction reaction catalyst that forms a self-assembled monolayer on Au surfaces. We then embed this Cu catalyst inside a hybrid bilayer membrane by depositing a monolayer of lipid on the self-assembled monolayer. We envisage that this electrochemical system can give a unique mechanistic insight not only into the oxygen reduction reaction, but into proton-coupled electron transfer in general.

Improved membrane fluidity of ionic polysaccharide bead-supported phospholipid bilayer membrane systems

Supported phospholipid bilayer membranes on polysaccharide-based cationic polymer beads (cationic group: –[OCH2CH(OH)CH2]2N+(CH3)3·X−, 45–165μm in diameter) were prepared using small unilamellar vesicles from mixtures of phosphatidylserine (PS) and phosphatidylcholine (PC). Confocal fluorescence microscopic observations with a fluorescent membrane probe (N-4-nitrobenzo-2-oxa-1,3-diazole-phosphatidylethanolamine) revealed that the phospholipid molecules in the phospholipid-bead complexes were along the outer surface of the beads. The fluidity of the phospholipid bilayer membranes in the PS/PC-bead complexes was investigated by the fluorescence recovery after photobleaching (FRAP) technique. The lateral diffusion coefficients (D) for the PS/PC-bead complexes were lower than that for the 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine giant unilamellar vesicles without solid supports. Such less fluid membranes in the complexes appeared to be due to the immobilization of the phospholipid bilayer membranes by electrostatic attractive forces between PS and the bead. The D values for the PS/PC-bead complexes were dependent on the phospholipid composition; the PS(100mol%)/PC(0mol%)-bead complex had the least fluid membranes among the PS/PC-bead complexes tested in this study. The phospholipid bilayer membranes formed on the polysaccharide-based cationic polymer beads were much more fluid than those on a polystyrene-based one. Furthermore, such fluid phospholipid bilayer membranes formed on the polysaccharide-based cationic polymer bead were maintained for 10 days, even though the complex sample was stood in plain buffer (pH 8.5) at ambient temperature.

Structural Characterization of the Voltage-Sensor Domain and Voltage-Gated K+-Channel Proteins Vectorially Oriented within a Single Bilayer Membrane at the Solid/Vapor and Solid/Liquid Interfaces via Neutron Interferometry

The voltage-sensor domain (VSD) is a modular four-helix bundle component that confers voltage sensitivity to voltage-gated cation channels in biological membranes. Despite extensive biophysical studies and the recent availability of X-ray crystal structures for a few voltage-gated potassium (Kv) channels and a voltage-gate sodium (Nav) channel, a complete understanding of the cooperative mechanism of electromechanical coupling, interconverting the closed-to-open states (i.e., nonconducting to cation conducting) remains undetermined. Moreover, the function of these domains is highly dependent on the physical-chemical properties of the surrounding lipid membrane environment. The basis for this work was provided by a recent structural study of the VSD from a prokaryotic Kv-channel vectorially oriented within a single phospholipid (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)) membrane investigated by X-ray interferometry at the solid/moist He (or solid/vapor) and solid/liquid interfaces, thus achieving partial to full hydration, respectively (Gupta et al. Phys. Rev. E2011, 84, 031911-1-15). Here, we utilize neutron interferometry to characterize this system in substantially greater structural detail at the submolecular level, due to its inherent advantages arising from solvent contrast variation coupled with the deuteration of selected submolecular membrane components, especially important for the membrane at the solid/liquid interface. We demonstrate the unique vectorial orientation of the VSD and the retention of its molecular conformation manifest in the asymmetric profile structure of the protein within the profile structure of this single bilayer membrane system. We definitively characterize the asymmetric phospholipid bilayer solvating the lateral surfaces of the VSD protein within the membrane. The profile structures of both the VSD protein and phospholipid bilayer depend upon the hydration state of the membrane. We also determine the distribution of water and exchangeable hydrogen throughout the profile structure of both the VSD itself and the VSD:POPC membrane. These two experimentally determined water and exchangeable hydrogen distribution profiles are in good agreement with molecular dynamics simulations of the VSD protein vectorially oriented within a fully hydrated POPC bilayer membrane, supporting the existence of the VSD's water pore. This approach was extended to the full-length Kv-channel (KvAP) at a solid/liquid interface, providing the separate profile structures of the KvAP protein and the POPC bilayer within the reconstituted KvAP:POPC membrane.

Fluorescence microscopic characterization of ionic polymer bead-supported phospholipid bilayer membrane systems

Supported phospholipid membrane structures on cationic organic polymer beads were prepared using mixtures of dioleoylphosphatidylserine (PS) and egg yolk phosphatidylcholine (PC). Confocal fluorescence microscopic observations using a fluorescent membrane probe (N-4-nitrobenzo-2-oxa-1,3-diazole-phosphatidylethanolamine) revealed that the phospholipid molecules in the PS/PC-bead complexes were along the outer surface of the beads, but not inside the beads. The anionic PS on the most outer surface of the PS/PC-bead complexes was responsible for the binding of a positively charged macromolecule, rhodamine isothiocyanate dextran (Mw 70,000) by electrostatic attractive forces. The fluidity of the membranes in the PS/PC-bead complexes was investigated by the fluorescence recovery after a photobleaching technique. The lateral diffusion coefficients (D) for the PS/PC-bead complexes were one-half or less than that for 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine giant unilamellar vesicles without solid supporting materials. Such a constrain of the phospholipid bilayer membrane in the complexes appeared to be due to its immobilization on the cationic polymer bead by electrostatic attractive forces between the PS and ammonium group on the surface of the bead. The D values for the complexes were dependent on the phospholipid composition; the PS(25mol%)/PC(75mol%)-bead complex produced a more fluid membrane than the PS(50mol%)/PC(50mol%)-bead one. Thus, the fluidity of the phospholipid bilayer membranes formed on the cationic polymer beads was significantly affected by the anionic phospholipid fraction used for the preparation of the complexes.