Interaction of pH-Sensitive Liposomes With Blood Components

Abstract

Avoidance of lysosomal degradation of drugs entrapped in liposomes has been one of the major efforts in liposome research. The achievement of high drug deliver}' efficiency using pH-sensitive liposomes over the pH-insensitive liposomes has greatly influenced our strategies in liposome drug delivery. The success of pH-sensitive liposomes in delivering compounds such as fluorescence dye, anti-cancer reagents, toxins and DNA to target cells with high efficiency in vitro shows a great potential to apply the same strategy to in vivo systems. Using human plasma as a simplified model for blood, we have systematically examined the interaction of pH-sensitive liposomes composed of dioleoylphosphatidyl-ethanolamine (DOPE) and oleic acid (OA) with plasma components. Our results show that the bilayer structure of liposomes in plasma depends on their sizes. Small liposomes (d<200nm) were stabilized by plasma components while the larger ones (d>600nm) were rapidly lysed upon the exposure to plasma. Such differences in their stability in plasma may derive from their differences in lipid packing which determines the surface pressure of the membrane. Using purified serum proteins, we found that albumin such as bovine serum albumin (BSA) lyse liposomes by extracting OA from the bilayer. However, BSA induced lysis could be blocked by lipoproteins including HDL, LDL and VLDL, but not by immunoglobulins. Further studies with purified components of HDL demonstrated that apoAl, not the lipids of the HDL, contains the stabilization activity. The extraction of OA from liposomes and the insertion of plasma components into the bilayer modified the bilayer properties such that plasma stabilized liposomes were no longer pH sensitive. Using dipalmitoylsuccinylglycerol (DPSG), a double-chain pH senser for DOPE liposomes, we could preserve 50% pH sensitivity after plasma treatment. The potential application of such liposomes and other essential properties of pH-sensitive liposomes for drug delivery in vivo are also discussed.     

Stabilization of large multilamellar liposomes by human serum in vitro

The leakage of 5,6-carboxyfluorescein from large multilamellar liposomes prepared from dipalmitoylphosphatidylcholine (without or with cholesterol) was investigated in vitro in the presence of human serum. Below the phospholipid phase transition temperature, the rate of dye release is retarted 3–8-fold in the presence of up to 25% human serum in the incubation medium, as compared to the release in isotonic phosphate-buffered saline. This effect is significantly augmented by incorporation of 50 mol% cholesterol into the lipid bilayer. At and above the phase transition temperature, the initial rapid dye leakage in the presence of serum is followed by a slow long-term release. Incubation of the liposomes with serum is assumed to result in the association of serum proteins with the outermost lipid bilayer which in turn will lead to their stabilization, while the inner lamellae are not immediately accessible to the serum proteins. The permeability of the outer protein-rich lipid bilayer appears to be restricted, as concluded from the decreased dye release in the presence of serum. Massive leakage from multilamellar liposomes appears to be primarily due to bilayer defects occurring in the lipid transition region rather than being caused by protein-lipid interactions. The results of our in vitro experiments are discussed in terms of the potential usefulness of multilamellar liposomes as drug carriers in vivo for local and topical applications.     

Separation of large unilamellar liposomes from blood components by a spin column procedure: towards identifying plasma proteins which mediate liposome clearance in vivo.

In order to facilitate the isolation of liposomes from blood components, we have developed a simple and rapid procedure combining chromatographic and centrifugal methods. This 'spin column' procedure was used to isolate liposomes from incubation mixtures with human serum or from the blood of CD1 mice after intravenous administration of liposomes. An advantage of this procedure is that processing times are fast (typically minutes) such that the isolation procedure can be done in the absence of chelators or other coagulation inhibitors which may affect protein/liposome interactions. Furthermore, several samples can be analyzed together and small sample volumes can be processed. In addition, we show that this spin column procedure can be employed to isolate large unilamellar vesicles averaging 100 nm in diameter from lipoproteins and plasma proteins. The applicability of this spin column procedure in studying protein/liposome interactions is demonstrated by quantitating the amount of human complement component C3 bound per liposome using a C3 competitive ELISA assay after incubation with human serum. The proteins associated with the recovered liposomes were further analyzed by conventional SDS-polyacrylamide gel electrophoresis. We show that egg phosphatidylcholine/cholesterol (55:45, mol/mol) or egg phosphatidylcholine/cholesterol/dioleoylphosphatidylserine (35:45:20, mol/mol) liposomes isolated from the circulation of CD1 mice within minutes of administration have distinct, complex profiles of associated proteins. By isolating circulating large unilamellar liposomes using the spin column method and characterizing the proteins associated with their membranes, this protein fingerprinting approach will expedite identifying protein interactions which affect liposome stability and clearance in vivo.     

Interactions of serum proteins with small unilamellar liposomes composed of dioleoylphosphatidylethanolamine and oleic acid: high-density lipoprotein, apolipoprotein A1, and amphipathic peptides stabilize liposomes

Small unilamellar liposomes composed to dioleoylphosphatidylethanolamine (DOPE) and oleic acid (OA) are stabilized by incubation with normal human serum or plasma [Liu, D., & Huang, L. (1989) Biochemistry 28, 7700-7707]. The present report describes a systematic study of interactions of purified serum proteins and lipoproteins with these liposomes. Albumin destabilized liposomes by extracting OA from the liposomes, whereas immunoglobulins and lipoproteins (HDL, LDL, and VLDL) had no effect. However, HDL and, to some extent, VLDL showed a rapid stabilization activity against the lytic effect of albumin. HDL added together with or shortly after the addition of albumin completely abolished the liposome leakage and aggregation effects induced by albumin. SDS-PAGE analysis of the HDL-stabilized liposomes revealed that apolipoprotein A1 was associated with liposomes. Purified apolipoprotein A1, but not a lipid mixture resembling the lipid composition of HDL, showed comparable liposome stabilization activity as HDL. Furthermore, synthetic peptides resembling the amphipathic helices found in apolipoprotein A1 also showed strong liposome stabilization activity. Peptides which were able to form amphipathic helixes of a wedge shape were more effective stabilizers than those which could not. These data indicate that HDL plays a major role in human serum or plasma for the liposome stabilization activity. HDL exerts its activity probably by the interactions of the amphipathic helices of apolipoprotein A1 with the hydrophobic voids found on the outer surface of the highly curved, small liposomes.     

Free diC14-amidine liposomes inhibit the TNF-α secretion induced by CpG sequences and lipopolysaccharides: role of lipoproteins

It has been shown that a preinjection of diC14-amidine cationic liposomes decreased TNF-α secretion induced by lipoplexes intravenous injection. We showed here that free cationic liposomes inhibit CpG sequences- or lipopolysaccharides- induced TNF-α secretion by macrophages. Surprisingly, this effect was strictly dependent on serum. Free cationic liposomes alone did not reveal any anti-inflammatory activity. Low-density lipoproteins and triglyceride-rich lipoproteins were identified as the serum components that confer to the liposomes an anti-inflammatory activity. Lipid fractions of these lipoproteins were able to reproduce the effect of the total lipoproteins and could inhibit, in association with diC14-amidine liposomes, the CpG-induced TNF-α secretion. Serum components confer to cationic liposomes new properties that can be used to modulate the inflammatory response directed against CpG sequences and lipopolysaccharides.     

Free diC14-amidine liposomes inhibit the TNF-alpha secretion induced by CpG sequences and lipopolysaccharides: role of lipoproteins

It has been shown that a preinjection of diC14-amidine cationic liposomes decreased TNF-alpha secretion induced by lipoplexes intravenous injection. We showed here that free cationic liposomes inhibit CpG sequences- or lipopolysaccharides-induced TNF-alpha secretion by macrophages. Surprisingly, this effect was strictly dependent on serum. Free cationic liposomes alone did not reveal any anti-inflammatory activity. Low-density lipoproteins and triglyceride-rich lipoproteins were identified as the serum components that confer to the liposomes an anti-inflammatory activity. Lipid fractions of these lipoproteins were able to reproduce the effect of the total lipoproteins and could inhibit, in association with diC14-amidine liposomes, the CpG-induced TNF-alpha secretion. Serum components confer to cationic liposomes new properties that can be used to modulate the inflammatory response directed against CpG sequences and lipopolysaccharides.     

Effects of phosphatidylserine on membrane incorporation and surface protection properties of exchangeable poly(ethylene glycol)-conjugated lipids

Liposomes containing the acidic phospholipid phosphatidylserine (PS) have been shown to avidly interact with proteins involved in blood coagulation and complement activation. Membranes with PS were therefore used to assess the shielding properties of poly(ethylene glycol 2000)-derivatized phosphatidylethanolamine (PE-PEG(2000)) with various acyl chain lengths on membranes containing reactive lipids. The desorption of PE-PEG(2000) from PS containing liposomes was studied using an in vitro assay which involved the transfer of PE-PEG(2000) into multilamellar vesicles, and the reactivity of PS containing liposomes was monitored by quantifying interactions with blood coagulation proteins. The percent inhibition of clotting activity of PS liposomes was dependent on the PE-PEG(2000) content. 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine (DSPE)-PEG(2000) which transferred out slowly from PS liposomes was able to abolish >80% of clotting activity of PS liposomes at 15 mol%. This level of DSPE-PEG(2000) was also able to extend the mean residence time of PS liposomes from 0.2 h to 14 h. However, PE-PEG(2000) with shorter acyl chains such as 1,2-dimyristyl-sn-glycero-3-phosphoethanolamine-PEG(2000) were rapidly transferred out from PS liposomes, which resulted in a 73% decrease in clotting activity inhibition and 45% of administered intravenously liposomes were removed from the blood within 15 min after injection. Thus, PS facilitates the desorption of PE-PEG(2000) from PS containing liposomes, thereby providing additional control of PEG release rates from membrane surfaces. These results suggest that membrane reactivity can be selectively regulated by surface grafted PEGs coupled to phosphatidylethanolamine of an appropriate acyl chain length.     

Interactions of polymerizable phosphatidylcholine vesicles with blood components: relevance to biocompatibility

We have studied the biocompatibility properties of polymerizable phosphatidylcholine bilayer membranes, in the form of liposomes, with a view toward the eventual utilization of such polymerized lipid assemblies in drug carrier systems or as surface coatings for biomaterials. The SH-based polymerizable lipid 1,2-bis[1,2-(lipoyl)dodecanoyl]-sn-glycero-3-phosphocholine (dilipoyl lipid, DLL) and the methacryl-based lipid 1,2-bis[(methacryloyloxy)dodecanoyl]-sn-glycero-3-phosphocholine (dipolymerizable lipid, DPL) were studied in comparison to ‘conventional’ zwitterionic or charged phospholipids. We examined binding of serum proteins to liposomes and effects of liposomes on fibrin clot formation and on platelet aggregation. All types of liposomes tested bound complex mixtures of serum proteins with IgG being the most abundant bound component. DPL vesicles and anionic vesicles bound substantially more protein than other vesicle types. Polymerized DPL vesicles uniquely bound a protein of about 53 kDa which was not bound to other types of phosphatidylcholine liposomes. Likewise polymerized DPL vesicles, but not other types of phosphatidylcholine vesicles, caused a marked alteration in coagulation as measured by activated partial thromboplastin time (APTT) and prothrombin time (PT) tests; this effect was shown to be due to binding and depletion of clothing factor V by the DPL polymerized vesicles. Polymerized DPL liposomes and DLL liposomes in polymerized or nonpolymerized form, were without substantial effect on platelet aggregation. However, DPL nonpolymerized vesicles, while not causing aggregation, did impair ADP-induced aggregation of platelets. These studies suggest that SH based polymerizable lipids of the DLL type may be very suitable for in vivo use in the contexts of drug delivery systems or biomaterials development. Methacryloyl-based lipids of the DPL type seem to display interactions with the hemostatic process which militate against their in vivo utilization.     

Interaction of positively-charged liposomes with blood: implications for their application in vivo.

Liposomes with positively-charged lipid components have previously demonstrated efficacy in animal models for human diseases, and are currently being evaluated in human clinical studies. Cationic lipids can improve entrapment efficiency of drugs and other substances which are negatively charged, and facilitate penetration of biological membranes in vitro, e.g. in transfection. However, toxic effects have also been reported for positively-charged liposomes containing stearylamine. In this report we have examined gross interactions between plasma components or erythrocytes with cholesterol-rich SUV composed of PC or DPPC and having 0-50 mol% of phospholipid replaced with positively-charged stearylamine, DOTMA, or BisHOP. Plasma interactions observed included increased turbidity of the usually clear stroma and/or formation of a clot-like mass. At plasma concentrations of 0.25 mumol/ml or more, the extent of plasma interactions depended upon the concentration of positive charge, the charge density of cationic lipid initially present in the liposomes, and to a lesser degree, the nature of the lipid providing the positive charge. At liposomal positive charge concentrations of greater than 0.5 mumol/ml plasma, stearylamine provoked a strong increase in plasma turbidity, whereas liposomes incorporating DOTMA or BisHOP provoked a strong clotting response. Some hemolysis of erythrocytes in vitro occurred on interaction with cationic liposomes where positive charge was contributed by DOTMA or stearylamine, but not BisHOP. Implications for the clinical use of liposomes containing cationic lipids, is discussed.     

Studies on the methodology of the carboxyfluorescein assay and on the mechanism of liposome stabilization by red blood cells in vitro

The stability of small unilamellar liposomes was investigated in human blood, in vitro. Using the carboxyfluorescein technique, interaction between the dye, the detergent Triton X-100, and an as yet unidentified component of human serum grossly interferes with the experiment and necessitates the use of other detergents, preferably sodium deoxycholate. Separation of liposomes and blood cells by centrifugation induces a small leakage from the liposomes and can lead to an underestimation of the real liposome stability. Upon incubation with whole blood, intact liposomes are absorbed nonspecifically to erythrocytes and internalized by leukocytes, the extent and kinetics of the former process being insenstive to the presence of metabolic inhibitors. The stability of liposomes is significantly enhanced in whole blood or in serum containing washed erythrocytes. Similarly, liposome stability in serum could be augmented be presaturating the serum lipoproteins with excess phospholipid. Our work adds support to previous notions that stable liposomes with high affinities for certain blood-cell components might be developed as suitable carrier systems for drug targetting in pathological disorders within the blood stream.     

Cryomicroscopy of liposome sytems as simple models to study cellular freezing response

Liposomes represent an attractive model system to study the freeze/thaw-induced alterations in biological membranes primarily because liposomes may be custom-manufactured for a particular investigation.This paper has reviewed briefly the degree of flexibility possible in terms of manufacturing liposomes with desired membrane composition and intraliposomal markers.Earlier research using liposomes as a model to study the freezing response of biological cells demonstrated their usefulness in this respect. Combining these earlier results with new results obtained in our laboratory, many analogies between the responses of biological cells and liposomes have been demonstrated. These analogies are summarized in Table 3.It is significant to note that in virtually every category thus far examined the relevant cryobiological behavior of biological cells can be mimicked qualitatively (and in some cases quantitatively) by a pure lipid membrane system. This is not to say that protein components of the membrane are insignificant. They are likely to be quite important in some cases. The liposome model system offers some interesting possibilities of examining the relative importance of lipids and proteins in model systems.Cryomicroscopic observation of liposome systems represents a promising approach to an improved understanding of membrane-related phenomena which occur during freezing and thawing. Especially interesting are the observations of membrane instabilities (“popping”) which appear to be induced osmotically.Real-time observation of well-defined membrane bilayer systems subjected to computer-controlled freeze/thaw protocols should lead to valuable insights into the nature of membrane freezing injury in the future.     

Correlation between fluidising effects on phospholipid membranes and mitochondrial respiration of propofol and p-nitrosophenol homologues

Nitrosopropofol (2-6-diisopropyl-4-nitrosophenol) has dramatic consequences for respiration, ATP synthesis and the transmembrane potential of isolated rat liver mitochondria at concentrations at which propofol (2-6-diisopropylphenol) does not cause any apparent effects. These results correlate well with the observation that nitrosopropofol is also a stronger perturbing agent of phospholipid membranes. In this paper we verify the possible biological activity of different phenols and nitrosophenols on mitochondrial respiration. We then discuss their interactions with phospholipid liposomes, studied with differential scanning calorimetry, spin labelling techniques and UV-Vis spectrophotometry, in order to obtain information on drug distribution and the modifications they impose on lipid bilayer. The results of the experiments performed on mitochondria and model membranes prove an interesting correlation between the effects of the molecules on both systems.     

Adsorption of non-membrane proteins on the surface of model phospholipid membranes

1. Two new methods are proposed for enhancement of the binding of hydrophilic proteins by liposomes. 2. An alkylating derivative of phosphatidic acid has been obtained by its reaction with N,N,N′-tris(2-chloroethyl)-N′- (p-formylphenyl)propylene-1,3-diamine. The alkylating activity of this derivative is very low due to the electron-acceptor effect of the formyl residue. Phosphatidylcholine liposomes which contain this alkylating derivative in the lipid bilayer may be obtained. The compound residing in the outer monolayer may be reduced by NaBH₄. Upon reduction, the formyl residue is transformed into a hydroxymethyl residue. Therefore, the alkylating group of the compound is activated, and proteins may be attached covalently to the outer monolayer by alkylation with such chemically reactive liposomes. 3. Reaction of alkylating liposomes with myoglobin results in covalent binding of this hydrophilic protein. Complement-mediated leakage of such myoglobin-carrying liposomes may be induced by antibodies against myoglobin. 4. Modification of hydrophilic proteins with dansyl chloride results, even at small extents of modification, in a dramatic increase of the affinity of such proteins to phosphatidylcholine liposomes.     

Inhibitory effect of cholesterol on interaction between cytoplasmic actin and liposomes, and restorative effect of high osmotic pressure

Although cholesterol is one of the major components of plasma membranes in eukaryotic cells, very little is known about its role in biological membranes. We reported previously (Okimasu et al., Cell Struct. Funct. 11, 273-283, 1986) that introduction of cholesterol into the liposomal membrane caused a decrease in membrane permeability, especially by the binding of cytoplasmic proteins to the liposomal membrane. The present study was carried out to further clarify the biochemical function of cholesterol in the membrane-protein interactions, especially under high osmotic pressure. The association of membranes with cytoplasmic proteins and their permeability were decreased by the introduction of cholesterol, but its effects were diminished in a hypertonic medium. The protein species associated with cholesterol-containing liposomes vary depending on the sort of hypertonic condition. It was suggested that since the degree of lipid packing by the cholesterol was reduced by the locally increased curvature in the lipid bilayer under high osmotic pressure, some cytoplasmic proteins can penetrate into the liposomal membrane.     

Oxysterols: biological activities and physicochemical studies.

To improve the understanding of the various biological activities of oxysterols (oxygenated derivatives of cholesterol), studies of their physicochemical properties have been undertaken. Oxysterols modify membrane dynamic properties which consequently trigger several biological effects. Despite the presence of at least one oxygenated group in addition to the C3 beta-hydroxyl, oxysterols insert perfectly into the lipidic bilayer of the membrane inducing a condensing effect similar to, but less potent than, that of cholesterol. In biological membranes oxysterols probably interact with membrane components as they are not easily exchanged after their incorporation into the cell membrane. These lipid-protein interactions are probably crucial for the expression of the biological activities of the oxysterols.     

Giant vesicles as models to study the interactions between membranes and proteins

The interaction between polypeptides and membranes is a fundamental aspect of cell biochemistry. Liposomes have been used in this context as in vitro systems to study such interactions. We present here the case of giant vesicles (GVs), which, due to their size (radius larger than 10 microns), mimic more closely the situation observed in cell membranes and furthermore permit to study protein-membrane interactions by direct optical monitoring. It is shown that GVs formed from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine by electroformation are permeable to certain low molecular weight molecules such as the nucleic acid dye YO-PRO-1 and fluorescein diphosphate whereas conventional liposomes (large or small unilamellar liposomes) are not. In addition, it is shown that non-membrane proteins, such as DNases or RNases, added to the selected GVs from the outside, are able to convert their substrate, which is strictly localized on the internal side of the membrane. This effect is only seen in GVs (also when they are removed from the original electroformation environment) and is absent in conventional liposomes. The fact that these effects are only present in GVs obtained by electroformation and not in conventional small liposomes is taken as an indication that certain physico-chemical properties of the bilayer are affected by the membrane curvature, although the mechanism underlying such differences could not be established as yet.     

Biophysical view of the role of interfaces in biomolecular recognition

Molecular recognition plays a key role in life. Macromolecular interactions at and with interfaces are of paramount importance in this respect. It is therefore crucial to understand and quantify the forces near the surfaces of biological interest in sufficient detail. Specific binding of large molecules, such as antibodies, is affected by the proximity of polar surfaces, for example. On the one hand, the presence of the net surface charges may raise or lower the local macromolecular concentration depending on the relative sign of the charges involved. On the other hand, the ligands attached to strongly polar surfaces always attract and bind their corresponding antibodies less efficiently than the corresponding dissolved molecules. The reason for this is the non-Coulombic repulsion between the ligand-presenting polar surface and the approaching macromolecule. This force is promoted by the surface hydrophilicity and the width of the interfacial region. A simple, direct hydration force is seldom, if ever, seen in such systems. (This is owing to the very short range (Lambda (h ) reverse similar 0.1 nm ) of pure hydration force.) The non-specific adsorption of proteins to the lipid bilayer is also little affected by the overall repulsion between the macromolecule and the bilayer surface; such an adsorption is governed more by the number of defects and/or by the availability of the hydrophobic binding sites in the interfacial region. Artificial lipid membranes typically offer numerous such binding sites to the surrounding macromolecules. Multiple non-specific protein adsorption, which results in partial macromolecular denaturation or complement activation, is therefore one of the main reasons for the rapid elimination of lipid vesicles from the blood stream in vivo. To promote the circulation time of an intravenously injected lipid suspension it is therefore necessary to modify the surfaces of their constituent lipid bilayers. Increasing the surface net charge density and/or increasing the bilayer surface hydrophilicity is of little use in this respect. In order to affect the non-specific bilayer-protein interactions significantly, an optimal number of water-soluble, short and sufficiently mobile polymers must be attached to the lipid head-groups. These polymers then increase the repulsive barrier of the membrane surface dramatically, due to the generation of a thick and mobile as well as strongly hydrated interface. Owing to this, the affinity for proteins of the resulting surface is lowered and the surface-induced protein denaturation or complement insertion is hampered. Polymer-coated liposomes, consequently, are not attractive for the phagocytic cells. Such liposomes, consequently, remain in the blood circulation much longer than simple lipid vesicles; the former, consequently, may spontaneously accumulate in tumors.     

Role of membrane organization and membrane domains in endocytic lipid trafficking

Lipid compositions vary greatly among organelles, and specific sorting mechanisms are required to establish and maintain these distinct compositions. In this review, we discuss how the biophysical properties of the membrane bilayer and the chemistry of individual lipid molecules play a role in the intracellular trafficking of the lipids themselves, as well as influencing the trafficking of transmembrane proteins. The large diversity of lipid head groups and acyl chains lead to a variety of weak interactions, such as ionic and hydrogen bonding at the lipid/water interfacial region, hydrophobic interactions, and van-der-Waals interactions based on packing density. In simple model bilayers, these weak interactions can lead to large-scale phase separations, but in more complex mixtures, which mimic cell membranes, such phase separations are not observed. Nevertheless, there is growing evidence that domains (i.e., localized regions with non-random lipid compositions) exist in biological membranes, and it is likely that the formation of these domains are based on interactions similar to those that lead to phase separations in model systems. Sorting of lipids appears to be based in part on the inclusion or exclusion of certain types of lipids in vesicles or tubules as they bud from membrane organelles.     

Evaluation of temperature and guanidine hydrochloride-induced protein–liposome interactions by using immobilized liposome chromatography

Hydrophobic interactions between nine model proteins and net-neutral lipid bilayer membranes (liposomes) under stress conditions were quantitatively examined by using immobilized liposome chromatography (ILC). Small or large unilamellar liposomes were composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and immobilized in a gel matrix by utilizing covalent coupling between amino-containing lipids and activated gel beads or avidin–biotin biospecific binding. Retardation of bovine carbonic anhydrase (CAB) in ILC was pronounced at particular temperatures (50 and 60 °C) where the local hydrophobicity of theses protein molecules becomes sufficiently large. Protein-induced leakage of a hydrophilic dye (calcein) from immobilized liposomes interior was also drastically enhanced at particular temperatures where large retardation was observed. For other proteins examined, similar results were also observed. The specific capacity factor of the proteins characteristic for the ILC and the amount of calcein released from immobilized liposomes were successfully expressed as a function of the product of the local hydrophobicities of proteins and liposomes, regardless of protein species and the type of the stress conditions applied (denaturant and heating). These findings indicate that lipid membranes have an ability to non-specifically recognize local hydrophobicities of proteins to form stress-mediated supramolecular assemblies with proteins, which may have potential applications in bioprocesses such as protein refolding and separation. ILC was thus found to be a very useful method for the quantitative detection of dynamic protein–liposome interactions triggered by stress conditions.     

Applications of phospholipid bilayer nanodiscs in the study of membranes and membrane proteins

Phospholipid bilayer Nanodiscs are novel model membranes derived from high-density lipoprotein particles and have proven to be useful in studies of membrane proteins. Membrane protein enzymology has been hampered by the inherent insolubility of membrane proteins in aqueous environments and has necessitated the use of model membranes such as liposomes and detergent-stabilized micelles. Current model membranes display a polydisperse particle size distribution and can suffer from problems of inconsistency and instability. It is also unclear how well they mimic biological lipid bilayers. In contrast, Nanodiscs, the particle size of which is constrained by a coat of scaffold proteins, are relatively monodisperse, stable model membranes with a "nativelike" lipid bilayer. Nanodiscs have already been used to study a variety of membrane proteins, including cytochrome P450s, seven-transmembrane proteins, and bacterial chemoreceptors. These proteins are simultaneously monomerized, solubilized, and incorporated into the well-defined membrane environment provided by Nanodiscs. Nanodiscs may also provide useful insights into the thermodynamics and biophysics of biological membranes and binding of small molecules to membranes.