Binding of the antimicrobial peptide temporin L to liposomes assessed by Trp fluorescence

The structure and membrane topology of the antimicrobial peptide temporin L (FVQWFSKFLGRIL- NH(2)) were studied using liposomes as model bilayers. Circular dichroic spectra revealed temporin L to adopt an alpha-helical conformation when bound to liposomes. Binding of temporin L to liposomes induced significant blue shifts of the emission spectra of the single Trp residue (Trp(4)) and also changed its quantum yield. The observed changes in the characteristics of the Trp(4) fluorescence are in keeping with the insertion of this residue into the hydrophobic region of the liposomal bilayers. Access of the aqueous quencher acrylamide to Trp(4) decreased in the sequence 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC)/cholesterol (X(chol) = 0.1) > SOPC > SOPC/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG, X(POPG) = 0.1) > SOPC/POPG (X(POPG) = 0.2) approximately SOPC/POPG (X(POPG) = 0.4), where X represents molar fraction of the indicated lipid. Whereas quenching of Trp(4) by brominated phospholipids was significant in SOPC liposomes, the quenching efficiency was enhanced when the vesicles contained POPG. The depth of insertion of Trp(4) into lipid bilayers was calculated by both the parallax method and distribution analysis and revealed this residue to reside at an average distance of d approximately 8.0 +/- 0.5 A from the center of both SOPC and SOPC/POPG bilayers. However, in the presence of cholesterol, d was increased to 9.5 +/- 0.5 A, thus revealing Trp(4) to become accommodated more superficially in the bilayer. The above data suggest the presence of two populations of temporin L in SOPC- and POPG-containing membranes with parallel and perpendicular orientation with respect to the plane of the membrane surface.     

The spectroscopic analysis for binding of amphipathic and antimicrobial model peptides containing pyrenylalanine and tryptophan to lipid bilayer

The binding of basic amphipathic fluorescent peptides to lipid bilayers was studied in relation to their antimicrobial activity. Four fluorescent peptides containing pyrenylalanine or tryptophan in an amphipathic basic peptide (4(4] consisting of four repeated units of tetrapeptide, -L-Leu-L-Ala-L-Arg-L-Leu-, were found to have antimicrobial activities against Gram-positive bacteria and to take conformations with fairly high alpha-helical content both in aqueous solutions and liposomes. The fluorescence spectroscopic data suggested that the pyrenylalanine-peptide existed as a monomer in methanol or liposomes but as an oligomer in aqueous solutions to form an excimer between pyrenylalanyl residues. Upon binding with liposomes, the fluorescence spectra of the tryptophan-containing peptide shifted to a shorter wavelength, indicating the change in the state of tryptophan from hydrophilic environment to hydrophobic one. The analytical data for the quenching of tryptophan fluorescence by I- anion suggest that the tryptophan residue in the peptide is not deeply buried in the hydrophobic core of the bilayers. Based on these findings, it is suggested that the peptides may interact with liposomes in such a manner that they lie parallel to the surface of the lipid bilayers with their hydrophobic regions shallowly in the amphipathic moiety of the bilayers.     

Bilirubin diffusion through lipid membranes

The possibility that bilirubin can diffuse through lipid bilayers is investigated with liposomes prepared from dipalmitoylphosphatidylcholine (DPPC), egg phosphatidylcholine (egg PC) with 22 mole percent cholesterol, and a lipid extract preparation from N115 neuroblastoma cells. Liposomes were prepared with internalized bilirubin and bovine or human serum albumin, and bilirubin efflux into an exogenous solution of human serum albumin was measured. Efflux from DPPC liposomes was significantly higher above the phase transition temperature than below it. This change was dependent on the lipid undergoing a phase transition and could not be accounted for by 6 K change in temperature. Maximum bilirubin efflux from egg PC-cholesterol liposomes was found to depend on the relative internal and external albumin pools, suggesting an equilibrium distribution of bilirubin between them. These observations demonstrate that bilirubin can diffuse freely through these lipid membranes.     

Membrane-active properties of alpha-MSH analogs: aggregation and fusion of liposomes triggered by surface-conjugated peptides.

Reaction of the melanotropin hormone analogs [Nle(4),D-Phe(7)]-alpha-MSH and [Nle(4),D-Phe(7)]-alpha-MSH(4-10), which were extended at their N-terminus by a thiol-functionalized spacer arm, with preformed liposomes containing thiol-reactive (phospho)lipid derivatives resulted in the aggregation of the vesicles and in a partial leakage of their inner contents. This aggregation/leakage effect, which was only observed when the peptides were covalently conjugated to the surface of the liposomes, was correlated with the fusion of the vesicles as demonstrated by the observed decrease in resonance energy transfer between probes in a membrane lipid mixing assay. A limited fusion was confirmed by monitoring the mixing of the liposome inner contents (formation of 1-aminonaphthalene-3,6,8-trisulfonic acid/p-xylene bis(pyridinium bromide) complex). The membrane-active properties of the peptides could be correlated with changes in the fluorescence emission spectra of their tryptophan residue, which suggested that after their covalent binding to the outer surface of the liposomes they can partition within the core of the bilayers. A blue shift of 10 nm was observed for [Nle(4),D-Phe(7)]-alpha-MSH which was correlated with an increase in fluorescence anisotropy and with changes in the accessibility of the coupled peptide as assessed by the quenching of fluorescence of its tryptophan residue by iodide (Stern-Volmer plots). These results should be related to the previously described capacity of alpha-MSH, and analogs, to interact with membranes and with the favored conformation of these peptides which, via a beta-turn, segregate their central hydrophobic residues into a domain that could insert into membranes and, as shown here, trigger their destabilization.     

Cationic carbosilane dendrimers-lipid membrane interactions

The aim of this work was to study interactions between cationic carbosilane dendrimers (CBS) and lipid bilayers or monolayers. Two kinds of second generation carbosilane dendrimers were used: NN16 with Si-O bonds and BDBR0011 with Si-C bonds. The results show that cationic carbosilane dendrimers interact both with liposomes and lipid monolayers. Interactions were stronger for negatively charged membranes and high concentration of dendrimers. In liposomes interactions were studied by measuring fluorescence anisotropy changes of fluorescent labels incorporated into the bilayer. An increase in fluorescence anisotropy was observed for both fluorescent probes when dendrimers were added to lipids that means the decreased membrane fluidity. Both the hydrophobic and hydrophilic parts of liposome bilayers became more rigid. This may be due to dendrimers' incorporation into liposome bilayer. For higher concentrations of both dendrimers precipitation occurred in negatively charged liposomes. NN16 dendrimer interacted stronger with hydrophilic part of bilayers whereas BDBR0011 greatly modified the hydrophobic area. Monolayers method brought similar results. Both dendrimers influenced lipid monolayers and changed surface pressure. For negatively charged lipids the monitored parameter changed stronger than for uncharged DMPC lipids. Moreover, NN16 dendrimer interacted stronger than the BDBR0011.     

Design of a novel membrane-destabilizing peptide selectively acting on acidic liposomes

The design of amphipathic peptides resulted in a novel peptide with a selective ability to destabilize lipid bilayers of acidic liposomes. The newly synthesized peptide, termed mast 21, is a 21-residue long amino acid chain and can only act effectively on acidic liposomes lacking cholesterol. Moreover, mast 21 killed gram-positive and gram-negative bacteria, and it had no hemolytic activity. The antimicrobial and hemolytic activities paralleled the results of membrane destabilizing activity using liposomes. Circular dichroism and Trp-fluorescence emission spectra showed changes in the peptide conformation and circumstances around the peptide during interaction with liposomes. These changes were consistent with an increased alpha-helical content and a less polar environment for the tryptophan residue of the peptide. Mast 21 was observed under dark-field microscopy in real time attacking liposomes. Acidic liposomes were attacked, which resulted in peeling of the lipid bilayer with its subsequent destruction.     

Synthesis of an amphiphilic tetraantennary mannosyl conjugate and incorporation into liposome carriers

We have synthesized a novel conjugate (Man(4)K(3)DOG) composed of a tetramannosyl head group connected, via a polyethylene glycol spacer, to a lipid moiety. This amphiphilic molecule was easily incorporated into the bilayers of liposomes. As expected from the clustering effect, such multivalent mannose residues when exposed on the surface of the vesicles showed much higher binding affinity for Concanavalin A than their monomannosyl analogue. Mannosylated liposomes prepared with the tetravalent antenna could be promising carriers for e.g., loading dendritic cells with antigens for vaccination purposes.     

Fusion of liposomes with planar lipid bilayers

The fusion of liposomes with planar lipid bilayers was monitored by two different methods. (a) Liposomes consisting of phospholipids and cholesterol were added to the aqueous phase bathing the cholesterol-deficient planar lipid bilayers in the presence of nystatin. The resulting increase in the planar lipid bilayer's electrical conductance was considered indicative of fusion. (b) Transplanar lipid bilayer injection of ³⁵SO₄^2? trapped inside the liposomes.It is shown by both methods that fusion is specifically dependent on the presence of negatively charged phospholipids both in the liposomes and the planar lipid bilayers and on Ca²⁺ in the aqueous phase of the fusion system.     

Kinetics of stearic acid transfer between human serum albumin and sterically stabilized liposomes

The kinetics of the transfer of stearic acids between human serum albumin (HSA) and long circulating sterically stabilised liposomes (SSL) composed of dipalmitoylphosphatidylcholine (DPPC) and of submicellar content of the polymer-lipid poly(ethylene glycol:2000)-dipalmitoylphosphatidylethanolamine (PEG:2000-DPPE) have been studied by fluorescence spectroscopy. The study exploits the fact that HSA has a single tryptophan (Trp) residue and that the intrinsic Trp-emission intensity is quenched by the presence of doxyl spin-labelled stearic acids (SASL). Protein/lipid dispersions are considered in which SASL molecules are inserted either in the protein or in the SSL, and the transfer of SASL between the protein and SSL is conveniently monitored by the time variation of the inherent Trp-fluorescence intensity of HSA. It was found that the transfer of fatty acids between HSA and SSL depends on the type of donor and acceptor matrix, on the temperature (i.e., on the physical state of the lipid bilayers) and on the grafting density of the PEG-lipids at the lipid/protein interface. In the absence of polymer-lipids, the rate of transfer increases with temperature in both directions of transfer, and it is higher for the passage from DPPC bilayers to HSA. The presence of polymer-lipids reduces the rate of transfer both in the mushroom and in the brush regime of the polymer chains, especially at low grafting density and for lipid membranes in the fluid phase.     

Effect of variations in the structure of a polyleucine-based alpha-helical transmembrane peptide on its interaction with phosphatidylglycerol bilayers

High-sensitivity differential scanning calorimetry and Fourier transform infrared spectroscopy were used to study the interaction of a cationic alpha-helical transmembrane peptide, acetyl-Lys(2)-Leu(24)-Lys(2)-amide (L(24)), and members of the homologous series of anionic n-saturated diacyl phosphatidylglycerols (PGs). Analogues of L(24), in which the lysine residues were replaced by 2,3-diaminopropionic acid (L(24)DAP), or in which a leucine residue at each end of the polyleucine sequence was replaced by a tryptophan (WL(22)W), were also studied to investigate the roles of lysine side-chain snorkeling and aromatic side-chain interactions with the interfacial region of phospholipid bilayers. The gel/liquid-crystalline phase transition temperature of the host PG bilayers is altered by these peptides in a hydrophobic mismatch-dependent manner, as previously found with zwitterionic phosphatidylcholine (PC) bilayers. However, all three peptides reduce the phase transition temperature and enthalpy to a greater extent in anionic PG bilayers than in zwitterionic PC bilayers, with WL(22)W having the largest effect. All three peptides form very stable alpha-helices in PG bilayers, but small conformational changes are induced in response to a mismatch between peptide hydrophobic length and gel-state lipid bilayer hydrophobic thickness. Moreover, electrostatic and hydrogen-bonding interactions occur between the terminal lysine residues of L(24) and L(24)DAP and the polar headgroups of PG bilayers. However, such interactions were not observed in PG/WL(22)W bilayers, suggesting that the cation-pi interactions between the tryptophan and lysine residues predominate. These results indicate that the lipid-peptide interactions are affected not only by the hydrophobic mismatch between these peptides and the host lipid bilayer, but also by the tryptophan-modulated electrostatic and hydrogen-bonding interactions between the positively charged lysine residues at the termini of these peptides and the negatively charged polar headgroups of the PG bilayers.     

磷脂组成对脱脂蛋白模型多肽与脂质体相互作用的影响

根据脱脂蛋白的脂结合序列合成了两个两亲性多肽Ａｍｐ１和Ａｍｐ２,在Ａｍｐ２在缬氨酸残基取代了Ａｍｐ１第４位的赖氨酸残基。用内源荧光谱发射峰的蓝移,包埋的钙氯黄素在脂质体中的渗漏,丙烯酰胺对多肽色氨酸残基的淬灭等手段比较了Ａｍｐ１与Ａｍｐ２与具有不同磷脂组成的脂质体的相互作用,并研究了温度的影响。     

Liposome-assisted selective polycondensation of alpha-amino acids and peptides

The main question of this paper is whether and to what extend lipid bilayers can aid in the polycondensation of amino acids and peptides. This means in particular how such bilayers can favor the selection of certain sequences out of a large number of theoretical possible ones. In a first series of experiments we started from a library of Trp-containing dipeptides of the type Trp-X where X is an amino acid residue; and we could show that, when adding this mixture to the POPC liposomes containing a hydrophobic quinoline condensing agent (EEDQ), only the hydrophobic Trp-Trp dipeptide is selected out by the liposomes and transformed into a longer oligomer. Trp-oligomers up to 29 monomers long (water insoluble) could be obtained by using the matrix support of liposomes. Mixed POPC/DDAB liposomes (positive charge) were used to produce co-oligopeptides that contain Trp and Glu residues in the same sequence. Arg/Trp and His/Trp containing sequences were obtained in presence of negatively charged liposomes (mixed POPC/DOPA-liposomes). The polycondensation of racemic NCA-amino acids has been studied to clarify if homochiral sequences are produced preferentially in presence or absence of liposomes. LC-MS and isotope labeling of the L-amino acid, participating in the polymerization reaction achieved this on the level of a direct product analysis. So the individual stereoisomer distribution up to a polymerization degree of 10 (in the case of Trp) could be determined. The data for Trp and other amino acids (Leu, Ile) and amino acid mixtures (Trp/Leu, Trp/Ile, Leu/Ile and Trp/Leu/Ile) show that homochiral sequences are produced preferentially if compared with a random (Bernoulli) distribution.     

Pressure dependence of pyrene excimer fluorescence in human erythrocyte membranes

The intensity of pyrene excimer fluorescence in human erythrocyte membranes and in sonicated dispersions of the membrane lipid (liposomes) was examined as a function of pressure (1–2080 bar) and temperature (5–40°C). Higher pressure or lower temperature decreased the excimer/monomer intensity ratios. A thermotropic transition was detected in both membranes and liposomes by plots of the logarithm of the excimer/monomer intensity ratio versus 1/K. The transition temperature of the membranes was 19–21°C at 1 bar and 28–31°C at 450 bar, a shift with pressure of approx. 20–22 K per kbar. Corresponding transition temperatures of the liposomes were 21°C at 1 bar and 33°C at 450 bar, a shift of approx. 27 K per kbar. The observed pressure dependence of the thermotropic transition temperature is similar to that reported for phospholipid bilayers and greatly exceeds that of protein conformation changes. In concert with the liposome studies the results provide direct evidence for a lipid transition in the erythrocyte membrane.     

Reconstitution of sodium channels in large liposomes formed by the addition of acidic phospholipids and freeze-thaw sonication

Phosphatidylcholine (PC) alone or with phosphatidylethanolamine (PE) are sufficient for the reconstitution of Na+ channels in planar lipid bilayers. However, when Na+ channels were first reconstituted into liposomes using the freeze-thaw-sonication method, addition of acidic phospholipids, such as phosphatidylserine (PS), to the neutral phospholipids was necessary to obtain a significant toxin-modulated 22Na uptake. To further investigate the acidic phospholipid effect on reconstitution into liposomes, Na+ channels purified from Electrophorus electricus electrocytes were reconstituted into liposomes of different composition by freeze-thaw sonication and the effect of batrachotoxin and tetrodotoxin on the 22Na flux was measured. The results revealed that, under our experimental conditions, the presence of an acidic phospholipid was also necessary to obtain a significant neurotoxin-modulated 22Na influx. Though neurotoxin-modulated 22Na fluxes have been reported in proteoliposomes made with purified Na+ channels and PC alone, the 22Na fluxes were smaller than those found using lipid mixtures containing acidic phospholipids. Electron microscopy of negatively stained proteoliposomes prepared with PC, PC/PS (1:1 molar ratio), and PS revealed that the acidic phospholipid increases the size of the reconstituted proteoliposomes. The increment in size caused by the acidic phospholipid, due to the associated increase in internal volume for 22Na uptake and in area for Na+ channel incorporation, appears to be responsible for the large neurotoxin-modulated 22Na fluxes observed.     

Membrane lysis by the antibacterial peptides cecropins B1 and B3: A spin-label electron spin resonance study on phospholipid bilayers

Custom antibacterial peptides, cecropins B1 (CB1) and B3 (CB3), were synthesized. These peptides have particular sequence characteristics, with CB1 having two amphipathic alpha-helical segments and CB3 having two hydrophobic alpha-helical segments. These differences were exploited for a study of their efficacy in breaking up liposomes, which had different combinations of phosphatidic acid (PA) and phosphatidylcholine (PC), and a study of their lipid binding ability. Binding and nonbinding lysis actions of CB1 and CB3 on liposomes were examined further by electron spin resonance (ESR). The spin-labeled lipids 5'SL-PC, 7'SL-PC, 10'SL-PC, 12'SL-PC, and 16'SL-PC were used as probes. The ESR spectra revealed larger outer hyperfine splittings (2A(max)) for CB1 when the interactions of CB1 and CB3 with liposomes were compared. These observations indicate a larger restriction of the motion of the spin-labeled chains in the presence of CB1. Plots of the effective order parameter at the various probe positions (chain flexibility gradient) versus the peptide-lipid ratio further suggested that the lysis action of CB1 is related to its capacity to bind to the lipid bilayers. In contrast, there is no evidence of binding for CB3. To augment these findings, four spin-labeled peptides, C8SL-CB1, C32SL-CB1, C5SL-CB3, and C30SL-CB3, were also examined for their binding to and their state of aggregation within the lipid bilayers. Association isotherms of the peptides were measured for liposomes containing two molar fractions of PA (0.25 and 0.75). The membrane binding of the CB1 peptides exhibited a cooperative behavior, whereas the association isotherm of CB3 revealed binding to the lipid only for beta = 0.75 liposomes. To further identify the location of CB1 in the lipid bilayers, measurements of the collision rate with chromium oxalate in solution were conducted. Results from ESR power saturation measurements suggested that the NH(2)-terminal alpha-helix of CB1 is located on the surface of the lipid bilayers, whereas the COOH-terminal alpha-helix of CB1 is embedded below the surface of the lipid bilayers. These conclusions were further supported by the observed relationship between the partition distribution of peptides bound to liposomes at different PA/PC ratios and the amounts of free peptides. Based on the above observations, possible mechanisms of the bilayer lysis induced by CB1 and CB3 on liposomes of different composition are discussed.     

Effect of Membrane Composition on Antimicrobial Peptides Aurein 2.2 and 2.3 From Australian Southern Bell Frogs

The effects of hydrophobic thickness and the molar phosphatidylglycerol (PG) content of lipid bilayers on the structure and membrane interaction of three cationic antimicrobial peptides were examined: aurein 2.2, aurein 2.3 (almost identical to aurein 2.2, except for a point mutation at residue 13), and a carboxy C-terminal analog of aurein 2.3. Circular dichroism results indicated that all three peptides adopt an α-helical structure in the presence of a 3:1 molar mixture of 1,2-dimyristoyl-sn-glycero-3-phosphocholine/1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DMPC/DMPG), and 1:1 and 3:1 molar mixtures of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPC/POPG). Oriented circular dichroism data for three different lipid compositions showed that all three peptides were surface-adsorbed at low peptide concentrations, but were inserted into the membrane at higher peptide concentrations. The ³¹P solid-state NMR data of the three peptides in the DMPC/DMPG and POPC/POPG bilayers showed that all three peptides significantly perturbed lipid headgroups, in a peptide or lipid composition-dependent manner. Differential scanning calorimetry results demonstrated that both amidated aurein peptides perturbed the overall phase structure of DMPC/DMPG bilayers, but perturbed the POPC/POPG chains less. The nature of the perturbation of DMPC/DMPG bilayers was most likely micellization, and for the POPC/POPG bilayers, distorted toroidal pores or localized membrane aggregate formation. Calcein release assay results showed that aurein peptide-induced membrane leakage was more severe in DMPC/DMPG liposomes than in POPC/POPG liposomes, and that aurein 2.2 induced higher calcein release than aurein 2.3 and aurein 2.3-COOH from 1:1 and 3:1 POPC/POPG liposomes. Finally, DiSC₃5 assay data further delineated aurein 2.2 from the others by showing that it perturbed the lipid membranes of intact S. aureus C622 most efficiently, whereas aurein 2.3 had the same efficiency as gramicidin S, and aurein 2.3-COOH was the least efficient. Taken together, these data show that the membrane interactions of aurein peptides are affected by the hydrophobic thickness of the lipid bilayers and the PG content.     

Interaction of taxifolin (dihydroquercetin) with dimyristoylphosphatidylcholine multilamellar liposomes

Differential scanning calorimetry was used to study the influence of the flavonoid taxifolin (dihydroquercetin) on the temperature-dependent phase transition of dimyristoylphosphatidylcholine multilamellar liposomes. Taxifolin was added to organic solution of the lipid during the procedure of liposomes preparation (addition from-within) or to a suspension of prepared liposomes (addition from-without). In the first case, liposomes contained from 2 to 50 mol% of taxifolin added from-within; in the second case, lyposomes were treated with 0.001% or 0.01% taxifolin. In both cases, the effect was similar. When the concentration of taxifolin increased, the temperature of lipid melting decreased while the width of transition considerably enlarged. Freeze-fracture electron microscopy revealed that taxifolin did not rupture multilamellar liposomes, while the formation of ripple-phase was retarded in all bilayers even when the liposomes were treated from without. This suggested the ability of taxifolin to penetrate through numerous bilayers of multilamellar liposomes.     

Stabilization of liposome bilayers to freezing and thawing: Effects of cryoprotective agents and membrane proteins

Lipid bilayer vesicles (liposomes) with and without glycoprotein incorporated into the membranes were tested for stability during freezing and thawing, in presence and absence of the cryoprotective agents (CPA) glycerol and dimethyl sulfoxide. Changes in turbidity and loss of energy transfer between fluorescent probes present in the bilayers were used to estimate membrane integrity.Freezing caused a 30 to 40% destruction of protein-free liposomes, in absence of CPA. CPA at 10 to 20% concentration prevented such losses, but at higher concentrations destabilized liposomes even without freezing. Protein-containing liposomes suffered no loss on freezing in absence or presence of CPA at moderate concentrations.Lowering of the storage temperature of frozen samples within the range of ?5 to ?27 °C increased the freeze damage. Slower rates of cooling and warming caused a slightly greater loss.The results are interpreted in terms of the liquid mosaic model for lipid bilayers. CPA at higher concentrations destabilize bilayers by dissolving phospholipids. At moderate concentrations, however, they prevent the damaging effect of dehydration of the lipid on freezing. Proteins appear to stabilize bilayers by providing increased hydration at the membrane surface, and by additional hydrophobic binding in the membrane interior.     

Association of ethanol with lipid membranes containing cholesterol, sphingomyelin and ganglioside: a titration calorimetry study.

The association of ethanol at physiologically relevant concentrations with lipid bilayers of different lipid composition has been investigated by use of isothermal titration calorimetry (ITC). The liposomes examined were composed of combinations of lipids commonly found in neural cell membranes: dimyristoyl phosphatidylcholine (DMPC), ganglioside (GM(1)), sphingomyelin and cholesterol. The calorimetric results show that the interaction of ethanol with fluid lipid bilayers is endothermic and strongly dependent on the lipid composition of the liposomes. The data have been used to estimate partitioning coefficients for ethanol into the fluid lipid bilayer phase and the results are discussed in terms of the thermodynamics of partitioning. The presence of 10 mol% sphingomyelin or ganglioside in DMPC liposomes enhances the partitioning coefficient by a factor of 3. Correspondingly, cholesterol (30 mol%) reduces the partitioning coefficient by a factor of 3. This connection between lipid composition and partitioning coefficient correlates with in vivo observations. Comparison of the data with the molecular structure of the lipid molecules suggests that ethanol partitioning is highly sensitive to changes in the lipid backbone (glycerol or ceramide) while it appears much less sensitive to the nature of the head group.     

Adhesion of Positively Charged Liposomes to Mucosal Tissues

Abstract

A series of positively charged phospholipid and cholesterol derivatives was synthesized and evaluated as membrane components for liposomes. Small unilamellar liposomes containing up to 40 mole% of the synthetic lipids were prepared by sonication. Selected liposome preparations containing these synthetic lipid materials were found to be noncytotoxic in vitro by using a cell growth inhibition assay, whereas liposomes containing more classic positively charged components (stearylamine and cetyltrimethylammonium bromide) showed considerable cytotoxicity. Using an unanesthetized rabbit eye model, we have found that inclusion of the positively charged lipid derivatives into the liposomes significantly enhanced the ocular retention compared to neutral or negatively charged liposomes, presumably by molecular association with poly anionic corneal and conjunctival surface mucoglycoproteins. the increased retention was dependent on charge density and rigidity of the lipid bilayer. An assay for primary amino groups in these liposomes suggested that the distribution of the charged molecules between the inner and outer leaflets of the bilayer could be manipulated by lipid composition. Studies of liposomes containing cholesteryl esters of amino acids of various carbon chain lengths indicated that the charged amino groups need to extend from the surface of the lipid bilayers for better adhesion and retention. the ocular surface was saturable with respect to applied liposomes, which were cleared slowly from the eye with a half-time of clearance of about 2 hr. these data suggest a specific adhesion of the cationic liposomes to the surface of mucosal tissues.