Range and magnitude of the steric pressure between bilayers containing phospholipids with covalently attached poly(ethylene glycol).

The interactive properties of liposomes containing phospholipids with covalently attached poly(ethylene glycol) (PEG-lipids) are of interest because such liposomes are being developed as drug delivery vehicles and also are ideal model systems for measuring the properties of surface-grafted polymers. For bilayers containing PEG-lipids with PEG molecular weights of 350, 750, 2000, and 5000, pressure-distance relations have been measured by X-ray diffraction analysis of liposomes subjected to known applied osmotic pressures. The distance between apposing bilayers decreased monotonically with increasing applied pressure for each concentration of a given PEG-lipid. Although for bilayers containing PEG-350 and PEG-750 the contribution of electrostatic repulsion to interbilayer interactions was significant, for bilayers containing PEG-2000 and PEG-5000 the major repulsive pressure between bilayers was a steric pressure due to the attached PEG. The range and magnitude of this steric pressure increased both with increasing PEG-lipid concentration and PEG size, and the extension length of the PEG from the bilayer surface at maximum PEG-lipid concentration depended strongly on the size of the PEG, being less than 35 A for PEG-750, and about 65 A for PEG-2000 and 115 A for PEG-5000. The measured pressure-distance relations have been modeled in terms of current theories (deGennes, 1987; Milner et al., 1988b) for the steric pressure produced by surface-grafted polymers, as modified by us to take into account the effects of polymer polydispersity and the possibility that, at low grafting densities, polymers from apposing bilayers surfaces can interpenetrate or interdigitate. No one theoretical scheme is sufficient to account for all the experimental results. However, for a given pressure regime, PEG-lipid size, and PEG-lipid surface density, the appropriately modified theoretical treatment gives a reasonable fit to the pressure-distance data.     

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.     

Poly(ethylene glycol)-poly(ester-carbonate) block copolymers carrying PEG-peptidyl-doxorubicin pendant side chains: synthesis and evaluation as anticancer conjugates

Water soluble polymer anticancer conjugates can improve the pharmacokinetics of covalently bound drugs by limiting cellular uptake to the endocytic route, thus prolonging plasma circulation time and consequently facilitating tumor targeting by the enhanced permeability and retention (EPR) effect. Many of the first generation antitumor polymer conjugates used nonbiodegradable polymeric carriers which limits the molecular weight that can be safely used to <40,000 g/mol. The aim of this ambitious study was to synthesize and evaluate a novel, prototype biodegradable polymeric system based on high molecular weight, water-soluble functionalized polyesters. The main polymeric platform was prepared from bis(4-hydroxy)butyl maleate (DBM) and poly(ethylene glycol) (PEG4000) blocks to give the polymer DBM2-PEG4000 containing biodegradable carbonate bonds and having a M(w) of 100,000-190,000 g/mol; M(n) of 37,000-53,000 g/mol, and M(w)/M(n) of 3.0-3.7. Using thioether linkages, this polymer was then grafted with HS-PEG3000-Gly-Phe-Lue-Gly doxorubicin (HS-PEG3000-GFLG-Dox) pendant side chains ( approximately 30 per DBM2-PEG chain). The final construct, DBM2-PEG4000-S-PEG3000-GFLG-Dox had a total Dox content of 3-4 wt % and a free Dox content of < or = 0.7% total Dox. During incubation with isolated lysosomal enzymes, the rate of Dox release from the polymer backbone was relatively slow (<5% release over 5 h) compared to that seen for PEG5000-GFLG-Dox alone (>20% over 5 h). The in vitro cytotoxicity was assessed using B16F10 murine melanoma (MTT assay). DBM2-PEG4000-S-PEG3000-GFLG-Dox was 10-20-fold less toxic than free Dox. In vivo antitumor activity of the DBM2-PEG4000-S-PEG3000-GFLG-Dox conjugates was assessed using a subcutaneous (s.c.) B16F10 murine melanoma model, and an intraperitoneal (i.p.) L1210 leukaemia model. The increased toxicity (attributed to poor solubility) and low antitumor activity of DBM2-PEG4000-S-PEG3000-GFLG-Dox conjugates compared to PEG5000-GFLG-Dox and HPMA copolymer-Dox conjugates was attributed to the slow rate of Dox release. The DBM2-PEG4000-S-PEG3000-GFLG-Dox conjugates were considered unfavorable as candidates for further development. However, the successful scale-up synthesis of DBM2-PEG4000-S-PEG3000 constructs suggest that they are worthy of further investigation as carriers for controlled release and targeting of less hydrophobic agents.     

Quantitative experimental assessment of macromolecular crowding effects at membrane surfaces

We examined how crowding of the surfaces of lipid vesicles with either grafted polyethyleneglycol (PEG) chains or bilayer-anchored protein molecules affects the binding of soluble proteins to the vesicle surface. Escherichia coli dihydrofolate reductase (DHFR, 18 kDa) or a larger fusion protein, NusA-DHFR (72 kDa), binds reversibly but with high affinity to a methotrexate-modified lipid (MTX-PE) incorporated into large unilamellar vesicles. Incorporation of phosphatidylethanolamine-PEG5000 into the vesicles strongly decreases the affinity of binding of both proteins, to a degree that varies roughly exponentially with the lateral density of the PEG chains. Covalently coupling maltose-binding protein (MBP) to the vesicle surfaces also strongly decreases the affinity of binding of NusDHFR or DHFR, to a degree that likewise varies roughly exponentially with the surface density of anchored MBP. Surface-coupled MBP strongly decreases the rate of binding of NusDHFR to MTX-PE-incorporating vesicles but does not affect the rate of NusDHFR dissociation. The large magnitudes of these effects (easily exceeding an order of magnitude for moderate degrees of surface crowding) support previous theoretical analyses and suggest that surface-crowding effects can markedly influence a variety of important aspects of protein behavior in membranes.     

Measurements of interbilayer forces and protein adsorption on uncharged lipid bilayers displaying poly(ethylene glycol) chains

Poly(ethylene glycol) (PEG)-stabilized liposomes were recently shown to exhibit differences in cell uptake that were linked to the liposome charge. To determine the differences and similarities between charged and uncharged PEG-decorated liposomes, we directly measured the forces between two supported, neutral bilayers with terminally grafted PEG chains. The measurements were performed with the surface force apparatus. The force profiles were similar to those measured with negatively charged PEG conjugates of 1, 2-distearoyl-sn-glycero-3-phosphatidyl ethanolamine (DSPE), except that they lacked the longer ranged electrostatic repulsion observed with the charged compound. Theories for simple polymers describe the forces between end-grafted polymer chains on neutral bilayers. The force measurements were complemented by surface plasmon resonance studies of protein adsorption onto these layers. The lack of electrostatic forces reduced the adsorption of positively charged proteins and enhanced the adsorption of negatively charged ones. The absence of charge also allowed us to determine how membrane charge and the polymer grafting density independently affect protein adsorption on the coated membranes. Such studies suggest the physical basis of the different interactions of charged and uncharged liposomes with proteins and cells.     

Dispersion and bilayer interaction of single-walled carbon nanotubes modulated by covalent and noncovalent PEGylation

Single-walled carbon nanotubes (SWNTs) covalently functionalised with polyethylene glycol (PEG) or noncovalently coated with PEGylated lipids were simulated in water and in lipid bilayers at different PEG sizes and grafting densities using coarse-grained force fields. Starting with the random position of three SWNT–PEG complexes in water, larger PEGs at higher grafting densities more significantly inhibit the aggregation of SWNTs because of larger radii of gyration and hydrodynamic radii of the SWNT–PEG complex, which influence the thickness and the wrapping extent of PEG layer. In particular, PEG-functionalised SWNTs, where PEGs are evenly grafted along the SWNT, disperse, while PEG-coated SWNTs aggregate because SWNTs are less covered by randomly adsorbed PEGylated lipids. Simulations of SWNT–PEGs in lipid bilayers show that PEG (M_w = 550 and 2000)-functionalised SWNTs bind to the bilayer surface but do not insert into the bilayer, while PEG-coated SWNTs insert into the bilayer because PEGylated lipids detach from SWNTs and mix with bilayer lipids. These findings support recent experiments at the same PEG size and density, which suggested that PEG-coated SWNTs may form bundles and thus cannot be easily excreted through the renal route, while PEG-functionalised SWNTs may remain individual and thus show more renal excretion.     

Assessment of the roles of ordered lipid microdomains in post-endocytic trafficking of glycosyl-phosphatidylinositol-anchored proteins in mammalian fibroblasts

We have used artificial phosphatidylethanolamine-polyethylene glycol (PE-PEG)-anchored proteins, incorporated into living mammalian cells, to evaluate previously proposed roles for ordered lipid 'raft' domains in the post-endocytic trafficking of glycosylphosphatidylinositol (GPI)-anchored proteins in CHO and BHK cells. In CHO cells, endocytosed PE-PEG protein conjugates colocalized strongly with the internalized GPI-anchored folate receptor, concentrating in the endosomal recycling compartment, regardless of the structure of the hydrocarbon chains of the PE-PEG 'anchor'. However, internalized PE-PEG protein conjugates with long-chain saturated anchors recycled to the plasma membrane at a slow rate comparable to that measured for the GPI-anchored folate receptor, whereas conjugates with short-chain or unsaturated anchors recycled at a faster rate similar to that observed for the transferrin receptor. These findings support the proposal (Mayor et al. Cholesterol-dependent retention of GPI-anchored proteins in endosomes. EMBO J 1998;17:4628-4638) that the slow recycling of GPI proteins in CHO cells rests on their affinity for ordered lipid domains. In BHK cells, internalized PE-PEG protein conjugates with either saturated or unsaturated 'anchors' colocalized strongly with simultaneously endocytosed folate receptor and, like the folate receptor, gradually accumulated in late endosomes/lysosomes. These latter findings do not support previous suggestions that the sorting of GPI proteins to late endosomes in BHK cells depends on their association with lipid rafts.     

Structure and phase behavior of lipid suspensions containing phospholipids with covalently attached poly(ethylene glycol).

Liposomes containing phospholipids with covalently attached poly(ethylene glycol) (PEG-lipids) are being developed for in vivo drug delivery. In this paper we determine the structure and phase behavior of fully hydrated distearoylphosphatidylcholine (DSPC) suspensions containing PEG-lipids composed of distearoylphosphatidylethanolamine with attached PEGs of molecular weights ranging from 350 to 5000. For DSPC:PEG-lipid suspensions containing 0-60 mol % PEG-lipid, differential scanning calorimetry shows main endothermic transitions ranging from 55 to 64 degrees C, depending on the size of the PEG and concentration of PEG-lipid. The enthalpy of this main transition remains constant for all PEG-350 concentrations but decreases with increasing amounts of PEG-750, PEG-2000, or PEG-5000, ultimately disappearing at PEG-lipid concentrations greater than about 60 mol %. Low-angle and wide-angle x-ray diffraction show that tilted gel (L beta') phase bilayers are formed for all PEG-lipid molecular weights at concentrations of about 10 mol % or less, with the distance between bilayers depending on PEG molecular weight and PEG-lipid concentration. At PEG-lipid concentrations greater than 10 mol %, the lipid structure depends on the size of the PEG moiety. X-ray diffraction analysis shows that untilted interdigitated (L beta I) gel phase bilayers form with the incorporation of 40-100 mol % PEG-350 or 20-70 mol % PEG-750, and untilted gel (L beta) phase bilayers are formed in the presence of about 20-60 mol % PEG-2000 and PEG-5000. Light microscopy, turbidity measurements, x-ray diffraction, and 1H-NMR indicate that a pure micellar phase forms in the presence of greater than about 60% PEG-750, PEG-2000, or PEG-5000.     

Hydration of polyethylene glycol-grafted liposomes.

This study aimed to characterize the effect of polyethylene glycol of 2000 molecular weight (PEG2000) attached to a dialkylphosphatidic acid (dihexadecylphosphatidyl (DHP)-PEG2000) on the hydration and thermodynamic stability of lipid assemblies. Differential scanning calorimetry, densitometry, and ultrasound velocity and absorption measurements were used for thermodynamic and hydrational characterization. Using a differential scanning calorimetry technique we showed that each molecule of PEG2000 binds 136 +/- 4 molecules of water. For PEG2000 covalently attached to the lipid molecules organized in micelles, the water binding increases to 210 +/- 6 water molecules. This demonstrates that the two different structural configurations of the PEG2000, a random coil in the case of the free PEG and a brush in the case of DHP-PEG2000 micelles, differ in their hydration level. Ultrasound absorption changes in liposomes reflect mainly the heterophase fluctuations and packing defects in the lipid bilayer. The PEG-induced excess ultrasound absorption of the lipid bilayer at 7.7 MHz for PEG-lipid concentrations over 5 mol % indicates the increase in the relaxation time of the headgroup rotation due to PEG-PEG interactions. The adiabatic compressibility (calculated from ultrasound velocity and density) of the lipid bilayer of the liposome increases monotonically with PEG-lipid concentration up to approximately 7 mol %, reflecting release of water from the lipid headgroup region. Elimination of this water, induced by grafted PEG, leads to a decrease in bilayer defects and enhanced lateral packing of the phospholipid acyl chains. We assume that the dehydration of the lipid headgroup region in conjunction with the increase of the hydration of the outer layer by grafting PEG in brush configuration are responsible for increasing thermodynamic stability of the liposomes at 5-7 mol % of PEG-lipid. At higher PEG-lipid concentrations, compressibility and partial volume of the lipid phase of the samples decrease. This reflects the increase in hydration of the lipid headgroup region (up to five additional water molecules per lipid molecule for 12 mol % PEG-lipid) and the weakening of the bilayer packing due to the lateral repulsion of PEG chains.     

Using hydrated reversed micelles to evaluate the dimensions of polymer-protein adducts.

Hydrolysis of N-trans-cynnamoylimidazole catalyzed by conjugates and complexes of alpha-chymotrypsin (ChT) with poly(ethylene glycol) (PEG) of different molecular mass (from 300 to 5000 daltons) was studied in the system of the hydrated reversed micelles of aerosol OT (AOT) in octane at 25 degrees C. The plot of the deacylation constant k3 for PEG--ChT conjugates and complexes versus the degree of hydration of reversed micelles (w0 = [H2O]/[AOT]) was studied. These plots are bell-shaped with maxima shifted to higher degrees of micelle hydration compared to the corresponding value of the shift for ChT. As for PEG--ChT conjugates, the value of the shift of w0 increases with increasing of molecular mass of the attached PEG and/or with the number of polymer chains per ChT molecule. Another picture was observed for PEG--ChT complexes for which the position of the maximum on k3 versusw0 curves was practically the same for all compounds. The values of the thickness of the polymer layer for PEG--ChT conjugates and complexes were calculated. Thus, polymer chains in conjugates placed in hydrated micelles are highly packed, whereas in the case of complexes they form a flat layer on the surface of the protein.     

Prolonged circulating lives of single-chain Fv proteins conjugated with polyethylene glycol: a comparison of conjugation chemistries and compounds

The utility of single-chain Fv proteins as therapeutic agents would be substantially broadened if the circulating lives of these minimal antigen-binding polypeptides were both prolonged and adjustable. Poly(ethylene glycol) (PEG) bioconjugate derivatives of the model single-chain Fv, CC49/218 sFv, were constructed using six different linker chemistries that selectively conjugate either primary amines or carboxylic acid groups. Activated PEG polymers with molecular weights of 2000, 5000, 10 000, 12 000, and 20 000 were included in the sFv bioconjugate evaluation. Additionally, the influence of PEG conjugate geometry in branched PEG strands (U-PEG) and the effect of multimeric PEG-sFv bioconjugates on circulating life and affinity were examined. Although random and extensive PEG polymer conjugations have been achievable in highly active derivatives of the prototypical PEG-enzymes, PEGylation of CC49/218 sFv required stringent adjustment of reaction conditions in order to preserve antigen-binding affinity as measured in either mucin-specific or whole cell immunoassays. Purified bioconjugates with PEG:sFv ratios of 1:1 through 2:1 were identified as promising candidates which exhibit sFv affinity (K(d)) values within 2-fold of the unmodified sFv protein. Interestingly, PEG conjugation to carboxylic acid moieties, using a PEG-hydrazide chemistry, achieved significant activity retention in bioconjugates at a higher PEG:sFv ratio (5:1) than with any of the amine-reactive activated PEG polymers. Prolonged circulating life in mice was demonstrated for each of the PEG conjugates. An increase in PEG polymer length was found to be more effective for serum half-life extension than a corresponding increase in total PEG mass. For example, CC49/218 sFv conjugated to either one strand of PEG-20000, or four strands of PEG-5000, displayed about 20- or 14-fold increased serum half-life, respectively, relative to the unmodified sFv. The demonstrated suitability of established random conjugation chemistries for PEGylation of sFv proteins, in conjunction with innovative site-specific conjugation methods, indicates that production of a panoply of sFv proteins with both engineered affinity and tailored circulating life may now be achievable.     

Ion-exchange macroporous hydrophilic gel monolith with grafted polymer brushes

The grafting of functional polymers to the pore surface of macroporous monolithic polyacrylamide cryogels was found to be an efficient and convenient method for the preparation of macroporous polyacrylamide gels, so-called cryogels (pAAm cryogels), with both controlled extent of functional group incorporated and with tailored surface chemistries. Anion-exchange polymer chains of poly(2-(dimethylamino)ethyl methacrylate) (pDMAEMA) and poly([2-(methacryloyloxy)ethyl]-trimethylammonium chloride) (pMETA), and cation-exchange polymer chains of polyacrylate have been grafted onto pAAm cryogels using potassium diperiodatocuprate as initiator. It was possible to achieve the ion-exchange capacity up to 0.2-0.5 mmol/ml. The graft polymerization did not alter the macroporous structure of the pAAm cryogel, however the flow rate of solutes through the cryogel matrix decreased with increase in the density of polymer grafted. The sorption of low-molecular-weight (metal ion, dye) and high-molecular-weight (protein) substances on the grafted monolithic pAAm column has been studied. The results indicate that a 'tentacle'-type binding of protein to grafted polymer depended on the architecture of the grafted polymer layer and took place after a certain degree of grafting has been reached. The binding of proteins by tentacle-like polymer chains allowed for increasing the binding capacity for proteins on the grafted pAAm cryogels up to 6-12 mg/ml.     

PEG-doxorubicin conjugates: influence of polymer structure on drug release, in vitro cytotoxicity, biodistribution, and antitumor activity

Polymer-drug conjugates (polymer therapeutics) are finding increasing use as novel anticancer agents. Here a series of poly(ethylene glycol) PEG-doxorubicin (Dox) conjugates were synthesized using polymers of linear or branched architecture (molecular weight 5000-20000 g/mol) and with different peptidyl linkers (GFLG, GLFG, GLG, GGRR, and RGLG). The resultant conjugates had a drug loading of 2.7-8.0 wt % Dox and contained <2.0% free drug (% total drug). All conjugates containing a GFLG linker showed approximately 30% release of Dox at 5 h irrespective of PEG molecular weight or architecture. The GLFG linker was degraded more quickly (approximately 57% Dox release at 5 h), and the other linkers more slowly (<16% release at 5 h), by lysosomal enzymes in vitro. In vitro there was no clear relationship between cytotoxicity toward B16F10 cells and the observed Dox release rate. All PEG conjugates were more than 10-fold less toxic (IC50 values > 2 microg/mL) than free Dox (IC50 value = 0.24 microg/mL). Biodistribution in mice bearing sc B16F10 tumors was assessed after administration of PEGs (5000, 10000, or 20000 g/mol) radioiodinated using the Bolton and Hunter reagent or PEG-Dox conjugates by HPLC. The 125I-labeled PEGs showed a clear relationship between Mw and blood clearance and tumor accumulation. The highest Mw PEG had the longest plasma residence time and consequently the greatest tumor targeting. The PEG-Dox conjugates showed a markedly prolonged plasma clearance and greater tumor targeting compared to free Dox, but there was no clear molecular weight-dependence on biodistribution. This was consistent with the observation that the PEG-Dox conjugates formed micelles in aqueous solution comprising 2-20 PEG-Dox molecules depending on polymer Mw and architecture. Although PEG-Dox showed greater tumor targeting than free Dox, PEG conjugation led to significantly lower anthracycline levels in heart. Preliminary experiments to assess antitumor activity against sc B16F10 in vivo showed the PEG5000linear (L)-GFLG-Dox and PEG10000branched (B)-GLFG-Dox (both 5 mg/kg Dox-equiv) to be the most active with T/C values of 146 and 143%, respectively. Free Dox did not show significant activity in this model (T/C = 121%). Dose escalation of PEG5000(L)-GFLG-Dox to 10 mg/kg Dox-equiv prolonged further animal survival (T/C = 161%). Using the Dox-sensitive model ip L1210 (where Dox displayed a T/C = 150% after single ip dose), the PEG5000(L)-GFLG-Dox displayed a maximum T/C of 141% (10 mg/kg Dox-equiv) using a once a day (x3) schedule. Further studies are warranted with PEG5000(L)-GFLG-Dox to determine its spectrum of antitumor activity and also the optimum dosing schedule before clinical testing.     

Bilayer interfacial properties modulate the binding of amphipathic peptides

The free energy of transfer (DeltaG degrees ) from water to lipid bilayers was measured for two amphipathic peptides, the presequence of the mitochondrial peptide rhodanese (MPR) and melittin. Experiments were designed to determine the effects on peptide partitioning of the addition of lipids that produce structural modifications to the bilayer/water interface. In particular, the addition of cholesterol or the cholesterol analog 6-ketocholestanol increases the bilayer area compressibility modulus, indicating that these molecules modify lipid-lipid interactions in the plane of the bilayer. The addition of 6-ketocholestanol or lipids with attached polyethylene glycol chains (PEG-lipids) modify the effective thickness of the interfacial region; 6-ketocholestanol increases the width of hydrophilic headgroup region in the direction of the acyl chains whereas the protruding PEG chains of PEG-lipids increase the structural width of the headgroup region into the surrounding aqueous phase. The incorporation of PEG-lipids with PEG molecular weights of 2000 or 5000 had no appreciable effect on peptide partitioning that could not be accounted for by the presence of surface charge. However, for both MPR and melittin DeltaG degrees decreased linearly with increasing bilayer compressibility modulus, demonstrating the importance of bilayer mechanical properties in the binding of amphipathic peptides.     

The presence of PEG-lipids in liposomes does not reduce melittin binding but decreases melittin-induced leakage

Poly(ethyleneglycol) (PEG), anchored at the surface of liposomes via the conjugation to a lipid, is commonly used for increasing the liposome stability in the blood stream. In order to gain a better understanding of the protective properties of interfacial polymers, we have studied the binding of melittin to PEG-lipid-containing membranes as well as the melittin-induced efflux of a fluorescent marker from liposomes containing PEG-lipids. We examined the effect of the polymer size by using PEG with molecular weights of 2000 and 5000. In addition, we studied the role of the anchoring lipid by comparing PEG conjugated to phosphatidylethanolamine (PE) which results in a negatively charged PEG-PE, with PEG conjugated to ceramide (Cer) which provides the neutral PEG-Cer. Our results show that interfacial PEG does not prevent melittin adsorption onto the interface. In fact, PEG-PE promotes melittin binding, most likely because of attractive electrostatic interactions with the negative interfacial charge density of the PEG-PE-containing liposomes. However, PEG-lipids limit the lytic potential of melittin. The phenomenon is proposed to be associated with the change in the polymorphic tendencies of the liposome bilayers. The present findings reveal that the protective effect associated with interfacial hydrophilic polymers is not universal. Molecules like melittin can sense surface charges borne by PEG-lipids, and the influence of PEG-lipids on liposomal properties such as the polymorphic propensities may be involved in the so-called protective effect.     

The presence of PEG-lipids in liposomes does not reduce melittin binding but decreases melittin-induced leakage.

Poly(ethyleneglycol) (PEG), anchored at the surface of liposomes via the conjugation to a lipid, is commonly used for increasing the liposome stability in the blood stream. In order to gain a better understanding of the protective properties of interfacial polymers, we have studied the binding of melittin to PEG-lipid-containing membranes as well as the melittin-induced efflux of a fluorescent marker from liposomes containing PEG-lipids. We examined the effect of the polymer size by using PEG with molecular weights of 2000 and 5000. In addition, we studied the role of the anchoring lipid by comparing PEG conjugated to phosphatidylethanolamine (PE) which results in a negatively charged PEG-PE, with PEG conjugated to ceramide (Cer) which provides the neutral PEG-Cer. Our results show that interfacial PEG does not prevent melittin adsorption onto the interface. In fact, PEG-PE promotes melittin binding, most likely because of attractive electrostatic interactions with the negative interfacial charge density of the PEG-PE-containing liposomes. However, PEG-lipids limit the lytic potential of melittin. The phenomenon is proposed to be associated with the change in the polymorphic tendencies of the liposome bilayers. The present findings reveal that the protective effect associated with interfacial hydrophilic polymers is not universal. Molecules like melittin can sense surface charges borne by PEG-lipids, and the influence of PEG-lipids on liposomal properties such as the polymorphic propensities may be involved in the so-called protective effect.     

Effects of acylation on the structure,lipid binding,and transfer activity of wheat lipid transfer protein

Study of the effect of protein chemical acylation on their functional properties or activity often brings valuable information regarding structure-function relationships. We performed such work on wheat lipid transfer protein, LTP1, to investigate the role of grafted acyl chains on the lipid binding and transfer properties. LTP1 was acylated by using anhydride derivatives of various chain lengths from C2 to C6. Only the chemical modifications with hexanoic acid yielded a marked effect on the tertiary structure and a slight change in the secondary structure. The affinity of the modified proteins for myristoyl-lysophosphatidylcholine was similar to that of the native protein accompanied by a slight decrease in stoichiometry. Interestingly, the acylation of LTP1 enhanced the lipid transfer activity by at least a factor of 10 for hexanoic chain length. Finally, the grafting of acyl chains was investigated by means of molecular modelling, and an attempt is made to correlate with our experimental data.     

Particle-by-particle quantification of protein adsorption onto poly(ethylene glycol) grafted surfaces

The use and advantage of flow cytometry as a particle-by-particle, low sampling volume, high-throughput screening technique for quantitatively examining the non-specific adsorption of proteins onto surfaces is presented. The adsorption of three proteins: bovine serum albumin (BSA), immunoglobulin gamma (IgG) and protein G, incubated at room temperature for 2 h onto organosilica particles modified with poly(ethylene glycol) (PEG) of increasing MW (2000, 3400, 6000, 10,000 and 20,000 g mol(-1)) and grafted amounts (0.14-1.4 mg m(-2)) was investigated as a model system. Each protein exhibited Langmuir-like, high affinity monolayer limited adsorption on unmodified particles with the proteins reaching surface saturation at 1.8, 4.0 and 2.5 mg m(-2) for BSA, IgG and protein G, respectively. Protein adsorption on PEG-modified surfaces was found to decrease with increasing amounts of grafted polymer. PEG grafting amounts >0.6 mg m(-2) effectively prevented the adsorption of the larger two proteins (BSA and IgG) while a PEG grafting amount >1.3 mg m(-2) was required to prevent the adsorption of the smaller protein G.     

Interaction of poly(L-lysine)-g-poly(ethylene glycol) with supported phospholipid bilayers

Interactions between the graft copolymer poly(L-lysine)-g-poly(ethylene glycol), PLL-g-PEG, and two kinds of surface-supported lipidic systems (supported phospholipid bilayers and supported vesicular layers) were investigated by a combination of microscopic and spectroscopic techniques. It was found that the application of the copolymer to zwitterionic or negatively charged supported bilayers in a buffer of low ionic strength led to their decomposition, with the resulting formation of free copolymer-lipid complexes. The same copolymer had no destructive effect on a supported vesicular layer made up of vesicles of identical composition. A comparison between poly(L-lysine), which did not induce decomposition of supported bilayers, and PLL-g-PEG copolymers with various amounts of PEG side chains per backbone lysine unit, suggested that steric repulsion between the PEG chains that developed upon adsorption of the polymer to the nearly planar surface of a supported phospholipid bilayer (SPB) was one of the factors responsible for the destruction of the SPBs by the copolymer. Other factors included the ionic strength of the buffer used and the quality of the bilayers, pointing toward the important role defects present in the SPBs play in the decomposition process.     

Kinetic parameters of the interactions of retinol with lipid bilayers

The process of transfer of vitamin A alcohol (retinol) between unilamellar vesicles of phosphatidylcholine was studied. The transfer was found to proceed spontaneously by hydration from the bilayer and diffusion through the aqueous phase. The rate-limiting step for transfer was the dissociation from the bilayer, a step that was characterized in bilayers of egg phosphatidylcholine (PC) by a rate constant koff = 0.64 s-1. The rate constant for association of retinol with bilayers of egg PC was also determined: kon = 2.9 x 10(6) s-1. The relative avidities for retinol of vesicles comprised of PC lipids with the various fatty acyl chains were measured. It was found that the binding affinity was determined by the composition of the lipids, such that PC with symmetric acyl chains had a lower affinity for retinol vs those with mixed chains. To clarify the mechanism underlying this observation, the rates of dissociation and association of retinol bound to vesicles of dioleoyl-PC were determined. The rate of association of retinol with bilayers strongly depended on the composition of the fatty acyl chains of the lipids. The rate of dissociation of retinol from the bilayers of PC was found to be independent of that composition. The implications of the observations for the interactions of hydrophobic ligands with lipid bilayers are discussed.