Understanding Aß Peptide Binding to Lipid Membranes: A Biophysical Perspective.

Aß peptides are known to bind neural plasma membranes in a process leading to the deposit of Aß-enriched plaques. These extracellular structures are characteristic of Alzheimer's disease, the major cause of late-age dementia. The mechanisms of Aß plaque formation and deposition are far from being understood. A vast number of studies in the literature describe the efforts to analyze those mechanisms using a variety of tools. The present review focuses on biophysical studies mostly carried out with model membranes or with computational tools. This review starts by describing basic physical aspects of lipid phases and commonly used model membranes (monolayers and bilayers). This is followed by a discussion of the biophysical techniques applied to these systems, mainly but not exclusively Langmuir monolayers, isothermal calorimetry, density-gradient ultracentrifugation, and molecular dynamics. The Methodological Section is followed by the core of the review, which includes a summary of important results obtained with each technique. The last section is devoted to an overall reflection and an effort to understand Aß-bilayer binding. Concepts such as Aß peptide membrane binding, adsorption, and insertion are defined and differentiated. The roles of membrane lipid order, nanodomain formation, and electrostatic forces in Aß-membrane interaction are separately identified and discussed.

The Binding of Aß42 Peptide Monomers to Sphingomyelin/Cholesterol/Ganglioside Bilayers Assayed by Density Gradient Ultracentrifugation.

The binding of Aß42 peptide monomers to sphingomyelin/cholesterol (1:1 mol ratio) bilayers containing 5 mol% gangliosides (either GM1, or GT1b, or a mixture of brain gangliosides) has been assayed by density gradient ultracentrifugation. This procedure provides a direct method for measuring vesicle-bound peptides after non-bound fraction separation. This centrifugation technique has rarely been used in this context previously. The results show that gangliosides increase by about two-fold the amount of Aß42 bound to sphingomyelin/cholesterol vesicles. Complementary studies of the same systems using thioflavin T fluorescence, Langmuir monolayers or infrared spectroscopy confirm the ganglioside-dependent increased binding. Furthermore these studies reveal that gangliosides facilitate the aggregation of Aß42 giving rise to more extended ß-sheets. Thus, gangliosides have both a quantitative and a qualitative effect on the binding of Aß42 to sphingomyelin/cholesterol bilayers.

Detection and Characterization of Different Brain-Derived Subpopulations of Plasma Exosomes by Surface Plasmon Resonance Imaging.

The use of exosomes for diagnostic and disease monitoring purposes is becoming particularly appealing in biomedical research because of the possibility to study directly in biological fluids some of the features related to the organs from which exosomes originate. A paradigmatic example are brain-derived exosomes that can be found in plasma and used as a direct read-out of the status of the central nervous system (CNS). Inspired by recent remarkable development of plasmonic biosensors, we have designed a surface plasmon resonance imaging (SPRi) assay that, taking advantage of the fact that exosome size perfectly fits within the surface plasmon wave depth, allows the detection of multiple exosome subpopulations of neural origin directly in blood. By use of an array of antibodies, exosomes derived from neurons and oligodendrocytes were isolated and detected with good sensitivity. Subsequently, by injecting a second antibody on the immobilized vesicles, we were able to quantify the amount of CD81 and GM1, membrane components of exosomes, on each subpopulation. In this way, we have been able to demonstrate that they are not homogeneously expressed but exhibit a variable abundance according to the exosome cellular origin. These results confirm the extreme variability of exosome composition and demonstrate how SPRi can provide an effective tool for their characterization. Besides, our work paves the road toward more precise clinical studies on the use of exosomes as potential biomarkers of neurodegenerative diseases.

Antibody-functionalized polymer nanoparticle leading to memory recovery in Alzheimer's disease-like transgenic mouse model.

Alzheimer's disease (AD) is a neurodegenerative disorder related, in part, to the accumulation of amyloid-ß peptide (Aß) and especially the Aß peptide 1-42 (Aß1-42). The aim of this study was to design nanocarriers able to: (i) interact with the Aß1-42 in the blood and promote its elimination through the "sink effect" and (ii) correct the memory defect observed in AD-like transgenic mice. To do so, biodegradable, PEGylated nanoparticles were surface-functionalized with an antibody directed against Aß1-42. Treatment of AD-like transgenic mice with anti-Aß1-42-functionalized nanoparticles led to: (i) complete correction of the memory defect; (ii) significant reduction of the Aß soluble peptide and its oligomer level in the brain and (iii) significant increase of the Aß levels in plasma. This study represents the first example of Aß1-42 monoclonal antibody-decorated nanoparticle-based therapy against AD leading to complete correction of the memory defect in an experimental model of AD.

Retro-inverso peptide inhibitor nanoparticles as potent inhibitors of aggregation of the Alzheimer's Aß peptide.

Aggregation of amyloid-ß peptide (Aß) is a key event in the pathogenesis of Alzheimer's disease (AD). We investigated the effects of nanoliposomes decorated with the retro-inverso peptide RI-OR2-TAT (Ac-rGffvlkGrrrrqrrkkrGy-NH2) on the aggregation and toxicity of Aß. Remarkably low concentrations of these peptide inhibitor nanoparticles (PINPs) were required to inhibit the formation of Aß oligomers and fibrils in vitro, with 50% inhibition occurring at a molar ratio of ~1:2000 of liposome-bound RI-OR2-TAT to Aß. PINPs also bound to Aß with high affinity (Kd=13.2-50 nM), rescued SHSY-5Y cells from the toxic effect of pre-aggregated Aß, crossed an in vitro blood-brain barrier model (hCMEC/D3 cell monolayer), entered the brains of C57 BL/6 mice, and protected against memory loss in APPSWE transgenic mice in a novel object recognition test. As the most potent aggregation inhibitor that we have tested so far, we propose to develop PINPs as a potential disease-modifying treatment for AD.

Multifunctional liposomes reduce brain ß-amyloid burden and ameliorate memory impairment in Alzheimer's disease mouse models.

Alzheimer's disease is characterized by the accumulation and deposition of plaques of ß-amyloid (Aß) peptide in the brain. Given its pivotal role, new therapies targeting Aß are in demand. We rationally designed liposomes targeting the brain and promoting the disaggregation of Aß assemblies and evaluated their efficiency in reducing the Aß burden in Alzheimer's disease mouse models. Liposomes were bifunctionalized with a peptide derived from the apolipoprotein-E receptor-binding domain for blood-brain barrier targeting and with phosphatidic acid for Aß binding. Bifunctionalized liposomes display the unique ability to hinder the formation of, and disaggregate, Aß assemblies in vitro (EM experiments). Administration of bifunctionalized liposomes to APP/presenilin 1 transgenic mice (aged 10 months) for 3 weeks (three injections per week) decreased total brain-insoluble Aß1-42 (-33%), assessed by ELISA, and the number and total area of plaques (-34%) detected histologically. Also, brain Aß oligomers were reduced (-70.5%), as assessed by SDS-PAGE. Plaque reduction was confirmed in APP23 transgenic mice (aged 15 months) either histologically or by PET imaging with [(11)C]Pittsburgh compound B (PIB). The reduction of brain Aß was associated with its increase in liver (+18%) and spleen (+20%). Notably, the novel-object recognition test showed that the treatment ameliorated mouse impaired memory. Finally, liposomes reached the brain in an intact form, as determined by confocal microscopy experiments with fluorescently labeled liposomes. These data suggest that bifunctionalized liposomes destabilize brain Aß aggregates and promote peptide removal across the blood-brain barrier and its peripheral clearance. This all-in-one multitask therapeutic device can be considered as a candidate for the treatment of Alzheimer's disease.

Repeated intraperitoneal injections of liposomes containing phosphatidic acid and cardiolipin reduce amyloid-ß levels in APP/PS1 transgenic mice.

The accumulation of extracellular amyloid-beta (Aß) peptide and intracellular neurofibrillary tangles in the brain are two major neuropathological hallmarks of Alzheimer's disease (AD). It is thought that an equilibrium exists between Aß in the brain and in the peripheral blood and thus, it was hypothesized that shifting this equilibrium towards the blood by enhancing peripheral clearance might reduce Aß levels in the brain: the 'sink effect'. We tested this hypothesis by intraperitoneally injecting APP/PS1 transgenic mice with small unilamellar vesicles containing either phosphatidic acid or cardiolipin over 3weeks. This treatment reduced significantly the amount of Aß in the plasma and the brain levels of Aß were lighter affected. Nevertheless, this dosing regimen did modulate tau phosphorylation and glycogen synthase kinase 3 activities in the brain, suggesting that the targeting of circulating Aß may be therapeutically relevant in AD. FROM THE CLINICAL EDITOR: Intraperitoneal injection of small unilamellar vesicles containing phosphatidic acid or cardiolipin significantly reduced the amount of amyloid-beta (Aß) peptide in the plasma in a rodent model. Brain levels of Aß were also affected - although to a lesser extent - suggesting that targeting of circulating Aß may be therapeutically relevant of Alzheimer's disease.

Ceramides and sphingomyelinases in senile plaques.

The senile plaque is a hallmark lesion of Alzheimer disease (AD). We compared, without a priori, the lipidome of the senile plaques and of the adjacent plaque-free neuropil. The analysis by liquid chromatography coupled with electrospray ionization mass spectrometry revealed that laser microdissected senile plaques were enriched in saturated ceramides Cer(d18:1/18:0) and Cer(d18:1/20:0) by 33 and 78% respectively with respect to the surrounding neuropil. This accumulation of ceramides was not explained by their affinity for Aß deposits: no interaction between ceramide-liposomes and Aß fibrils was observed in vitro by surface plasmon resonance and fluorescent ceramide-liposomes showed no affinity for the senile plaques in AD brain tissue. Accumulation of ceramides could be, at least partially, the result of a local production by acid and neutral sphingomyelinases that we found to be present in the corona of the senile plaques.

Liposomes bi-functionalized with phosphatidic acid and an ApoE-derived peptide affect Aß aggregation features and cross the blood-brain-barrier: implications for therapy of Alzheimer disease.

Targeting amyloid-ß peptide (Aß) within the brain is a strategy actively sought for therapy of Alzheimer's disease (AD). We investigated the ability of liposomes bi-functionalized with phosphatidic acid and with a modified ApoE-derived peptide (mApoE-PA-LIP) to affect Aß aggregation/disaggregation features and to cross in vitro and in vivo the blood-brain barrier (BBB). Surface plasmon resonance showed that bi-functionalized liposomes strongly bind Aß (kD=0.6 µM), while Thioflavin-T and SDS-PAGE/WB assays show that liposomes inhibit peptide aggregation (70% inhibition after 72 h) and trigger the disaggregation of preformed aggregates (60% decrease after 120 h incubation). Moreover, experiments with dually radiolabelled LIP suggest that bi-functionalization enhances the passage of radioactivity across the BBB either in vitro (permeability=2.5×10(-5) cm/min, 5-fold higher with respect to mono-functionalized liposomes) or in vivo in healthy mice. Taken together, our results suggest that mApoE-PA-LIP are valuable nanodevices with a potential applicability in vivo for the treatment of AD. From the clinical editor: Bi-functionalized liposomes with phosphatidic acid and a modified ApoE-derived peptide were demonstrated to influence Aß aggregation/disaggregation as a potential treatment in an Alzheimer's model. The liposomes were able to cross the blood-brain barrier in vitro and in vivo. Similar liposomes may become clinically valuable nanodevices with a potential applicability for the treatment of Alzheimer's disease.

The Influence of Lipid Electric Charge on the Binding of Aß(1-42) Amyloid Peptide to Bilayers in the Liquid-Ordered State.

The amyloidogenic Aß peptides are widely considered as a pathogenic agent in Alzheimer's disease. Aß(1-42) would form aggregates of amyloid fibrils on the neuron plasma membranes, thus perturbing neuronal functionality. Conflicting data are available on the influence of bilayer order on Aß(1-42) binding to membranes. In the present study, a biophysical approach was used in which isothermal calorimetry and surface pressure measurements were applied to explore the interaction of Aß(1-42) in either monomeric, oligomeric, or fibrillar form with model membranes (bilayers or monolayers) in the liquid-ordered state that were either electrically neutral or negatively charged. In the latter case, this contained phosphatidic acid, cardiolipin, or ganglioside. The calorimetric studies showed that Aß(1-42) fibrils, oligomers, and monomers could bind and/or be inserted into bilayers, irrespective of electric charge, in the liquid-ordered state, except that monomers could not interact with electrically neutral bilayers. The monolayer studies in the Langmuir balance demonstrated that Aß(1-42) aggregation hindered peptide insertion into the monolayer, hindered insertion in the decreasing order of monomer > oligomer > fibril, and that lipid composition did not cause large differences in insertion, apart from a slight facilitation of monomer and oligomer insertion by gangliosides.

Abnormal cross-talk between mutant presenilin 1 (I143T, G384A) and glycosphingolipid biosynthesis.

Mutations in the presenilin 1 (PS1) gene are associated with early onset familial Alzheimer's disease (FAD). In this study, we found that the expression of mutant-PS1 in stable transfectants of SH-SY5Y neuroblastoma cells results in a reduction of the biosynthesis and steady-state levels of glucosylceramide. As an in vivo corroboration of these data, there was a significant reduction of brain glucosylceramide and gangliosides in an animal model of FAD. In mutant-PS1-transfectants (I143T, G384A), immunocytochemistry disclosed a remarkable reduction of glucosylceramide synthase (GlcT-1)-like immunoreactivity in the cells when compared with those of mock- and wild-PS1 transfectants. Immunoprecipitation of GlcT-1 protein from mutant-PS1 transfectants demonstrated a marked reduction in GlcT-1 protein, but there was no reduction in the levels of GlcT-1 mRNA. Both coprecipitation and γ-secretase inhibition experiments suggest that mutant-PS1 seems to form a complex with GlcT-1 protein and to be involved in GlcT-1 degradation, which was never found in other cell types. Thus, mutations in the PS1 gene result in profound glycosphingolipids abnormalities by abnormal molecular interaction with GlcT-1.

Aß42 production in brain capillary endothelial cells after oxygen and glucose deprivation.

Although the diverse triggers of AD are still under debate, the hypothesis of the contribution of cerebrovascular deficiencies has emerged in recent years. Cerebrovascular dysfunction may precede cognitive decline and onset of neurodegeneration. Indeed, the toxic Aß(42) aggregates constituting senile plaques, one of AD hallmarks, is often detected as amorphous material or fine fibrils in the brain capillary of AD patients. Aß(42) causing cerebral microangiopathy might originate either from the circulating blood, the vessel wall itself or the brain parenchyma. In the present investigation we show, for the first time, that in rat brain capillary endothelial cells (RBE4), in vitro oxygen glucose deprivation treatment elicits 250% of Aß(42) peptide production increase through a mechanism that involves the hypoxia inducible factor-1-mediated ß-secretase (BACE1) up-regulation. Furthermore, we observed a time dependent increase of amyloid protein precursor (AßPP) gene and protein expression, confirming previous reports which established the existence of AßPP in the cerebrovascular domain. Our experimental evidences point out that ischemic events may directly contribute in brain capillary endothelial cells to the enhancement of the amyloidogenic metabolism, leading to intracellular deposition of Aß(42). This events may contribute to the impairment of Aß brain clearance and AD related blood brain barrier dysfunctions.

Amyposomes, a nanotechnological chaperone with anti-amyloidogenic activity.

AIM: The effect of liposomes bi-functionalized with phosphatidic acid and with a synthetic peptide derived from human apolipoprotein E has been evaluated on the aggregation features of different amyloidogenic proteins: human Amyloid ß1-40 (Aß1-40), transthyretin (TTR) variant S52P, human ß2microglobulin (ß2m) variants ΔN6 and D76N, Serum Amyloid A (SAA). METHODS: The formation of fibrillar aggregates of the proteins was investigated by ThioflavinT fluorescence assay and validated by Atomic Force Microscopy. RESULTS: The results show that liposomes are preventing the transition of non-aggregated forms to the fibrillar state, with stronger effects on Aß1-40, ß2m ΔN6 and SAA. Liposomes also induce disaggregation of the amyloid aggregates of all the proteins investigated, with stronger effects on Aß1-40, ß2 D76N and TTR.SPR assays show that liposomes bind Aß1-40 and SAA aggregates with high affinity (KD in the nanomolar range) whereas binding to TTR aggregates showed a lower affinity (KD in the micromolar range). Aggregates of ß2m variants showed both high and low affinity binding sites. Computed Structural analysis of protein fibrillar aggregates and considerations on the multidentate features of liposomes allow to speculate a common mechanism of action, based on binding the ß-stranded peptide regions responsible for the amyloid formation. CONCLUSION: Thus, multifunctional liposomes perform as pharmacological chaperones with anti-amyloidogenic activity, with a promising potential for the treatment of a number of protein-misfolding diseases.Key messageAmyloidosis is a group of diseases, each due to a specific protein misfolding.Anti-amyloidogenic nanoparticles have been gaining the utmost importance as a potential treatment for protein misfolding disorders.Liposomes bi-functionalized with phosphatidic acid and with a synthetic peptide derived from human apolipoprotein E showed anti-amyloidogenic activity.

Binding of ß-amyloid (1-42) peptide to negatively charged phospholipid membranes in the liquid-ordered state: modeling and experimental studies.

To explore the initial stages of amyloid ß peptide (Aß42) deposition on membranes, we have studied the interaction of Aß42 in the monomeric form with lipid monolayers and with bilayers in either the liquid-disordered or the liquid-ordered (L(o)) state, containing negatively charged phospholipids. Molecular dynamics (MD) simulations of the system have been performed, as well as experimental measurements. For bilayers in the L(o) state, in the absence of the negatively charged lipids, interaction is weak and it cannot be detected by isothermal calorimetry. However, in the presence of phosphatidic acid, or of cardiolipin, interaction is detected by different methods and in all cases interaction is strongest with lower (2.5-5 mol%) than higher (10-20 mol%) proportions of negatively charged phospholipids. Liquid-disordered bilayers consistently allowed a higher Aß42 binding than L(o) ones. Thioflavin T assays and infrared spectroscopy confirmed a higher proportion of ß-sheet formation under conditions when higher peptide binding was measured. The experimental results were supported by MD simulations. We used 100 ns MD to examine interactions between Aß42 and three different 512 lipid bilayers consisting of palmitoylsphingomyelin, dimyristoyl phosphatidic acid, and cholesterol in three different proportions. MD pictures are different for the low- and high-charge bilayers, in the former case the peptide is bound through many contact points to the bilayer, whereas for the bilayer containing 20 mol% anionic phospholipid only a small fragment of the peptide appears to be bound. The MD results indicate that the binding and fibril formation on the membrane surface depends on the composition of the bilayer, and is the result of a subtle balance of many inter- and intramolecular interactions between the Aß42 and membrane.

Nanotechnology for neurodegenerative disorders.

The efficacy, cellular uptake and specific transport of drugs and/or imaging agents to target organs, tissues and cells are common issues in the diagnosis and treatment of different disorders. In the case of neurodegenerative diseases, they represent complex problems, since brain targeting remains a still unsolved challenge in pharmacology, due to the presence of the blood-brain barrier, a tightly packed layer of endothelial cells that prevents unwanted substances to enter the brain. Engineered nanomaterials, objects with dimensions of 1-100 nm, are providing interesting biomedical tools potentially able to solve these problems, thanks to their physico-chemical features and to the possibility of multi-functionalization, allowing to confer them different features at the same time, including the ability to cross the blood-brain barrier. This review focuses on the state-of-the-art of nanomaterials suitable for therapy and diagnostic imaging of the most common neurodegenerative disorders, as well as for neuroprotection and neuronal tissue regeneration. Finally, their potential neurotoxicity is discussed, and future nanotechnological approaches are described.

Abeta peptide toxicity is reduced after treatments decreasing phosphatidylethanolamine content in differentiated neuroblastoma cells.

We investigated whether the toxicity of oligomeric amyloid-beta peptide (Abeta1-42) upon differentiated human neuroblastoma SH-SY5Y cells, can be affected by changes of membrane lipid composition. An immunostaining technique, using lipids extracted from the cells and separated by thin layer chromatography, suggested that Abeta preferentially binds to phosphatidylethanolamine (PE), one of the major lipids in the cell extract. For this reason, we utilized treatments with putative inhibitors of phosphatidylethanolamine biosynthesis (choline, phosphocholine, R59949) to decrease its proportion in the cell membrane; choline treatment (2.5 mM, 24 h) showed the best performance, reducing phosphatidylethanolamine content from 5.7 to 3.3 µg phosphorous/mg protein. Either the extent of Abeta binding or its toxicity decreased onto choline-treated cells. These data may open the possibility to develop future strategies aiming to reduce Abeta toxicity in Alzheimer disease.

Liposomes functionalized with acidic lipids rescue Aß-induced toxicity in murine neuroblastoma cells.

The loss of synapses and neurons in Alzheimer's disease (AD) is thought to be at least partly induced by toxic species formed by the amyloid beta (Aß) peptide; therefore, therapeutics aimed at reducing Aß toxicity could be of clinical use for treatment of AD. Liposomes are suitable vehicles for therapeutic agents and imaging probes, and a promising way of targeting the various Aß forms. We tested liposomes functionalized with phosphatidic acid, cardiolipin, or GM1 ganglioside, previously shown to have high Aß-binding capacity. Mimicking Aß-induced toxicity in mouse neuroblastoma cell lines, combined with administration of cell viability-modulating agents, we observed that functionalized liposomes rescued cell viability to different extents. We also detected rescue of the imbalance of GSK-3ß and PP2A activity, and reduction in tau phosphorylation. Thus, these liposomes appear particularly suitable for implementing further therapeutic strategies for AD.

Functionalization of liposomes with ApoE-derived peptides at different density affects cellular uptake and drug transport across a blood-brain barrier model.

A promising strategy to enhance blood-brain barrier penetration by drugs is the functionalization of nanocarriers with uptake-facilitating ligands. We studied the cellular uptake, by cultured RBE4 brain capillary endothelial cells, of nanoliposomes (NLs) covalently coupled with monomer or tandem dimer of apolipoprotein E (ApoE)-derived peptides (residues 141-150), at various densities. NLs without functionalization did not show either relevant membrane accumulation or cellular uptake, as monitored by confocal microscopy and quantified by fluorescence-activated cell sorting. Functionalization with peptides mediated an efficient NLs uptake that increased with peptide density; NLs carrying monomeric peptide performed the best. Moreover, we studied the ability of ApoE-NLs to enhance the transport of a drug payload through a RBE4 cell monolayer. The permeability of a tritiated curcumin derivative was enhanced after its entrapment into ApoE-NLs, in particular those functionalized with the dimer (+83% with respect to free drug, P < 0.01). Thus, these NLs appear particularly suitable for implementing further strategies for drug brain targeting.

Effect of curcumin-associated and lipid ligand-functionalized nanoliposomes on aggregation of the Alzheimer's Aß peptide.

The effect of various types of nanoliposomes (associated with curcumin, phosphatidic acid, cardiolipin, or GM1 ganglioside) on the aggregation of the amyloid-ß(1-42) (Aß(1-42)) peptide was investigated. Nanoliposomes incorporating curcumin (curcumin-liposomes) were prepared by adding curcumin in the lipid phase during liposome preparation, whereas curcumin surface-decorated liposomes were prepared by using a curcumin-lipid conjugate (lipid-S-curcumin liposomes) or by attaching a curcumin derivative on preformed liposomes by click chemistry (click-curcumin liposomes). The lipid ligands (phosphatidic acid, cardiolipin, or GM1) were also incorporated into nanoliposomes during their formation. All nanoliposomes with curcumin, or the curcumin derivative, were able to inhibit the formation of fibrillar and/or oligomeric Aß in vitro. Of the three forms of curcumin liposomes tested, the click-curcumin type was by far the most effective. Liposomes with lipid ligands only inhibited Aß fibril and oligomer formation at a very high ratio of liposome to peptide. Curcumin-based liposomes could be further developed as a novel treatment for Alzheimer's disease.

The interaction of Aß42 peptide in monomer, oligomer or fibril forms with sphingomyelin/cholesterol/ganglioside bilayers.

Aß42 peptide binds neuronal membranes and aggregates into plaques that are characteristic of Alzheimer's disease. Aß42 peptide has been proposed to be generated in membrane (nano) domains in the liquid-ordered phase, ganglioside GM1 being a major facilitator of peptide binding to the membrane. The peptide exists in solution in various degrees of aggregation, either monomers, oligomers or fibrils, of which oligomers appear to be particularly toxic. The present study reports on the binding of Aß42 peptide, in monomer, oligomer or fibril form, to model membranes (lipid vesicles or monolayers), composed of sphingomyelin and cholesterol in equimolar ratios, to which 1-5 mol% of different gangliosides were incorporated. Thermodynamic binding parameters obtained from calorimetric data indicate a strong tendency to bind the membrane (ΔG ≈ 7 kcal/mol peptide), in a process dominated in most cases by the increase in entropy. ΔG was virtually invariant with the ganglioside species and the aggregation state of the peptide. The Langmuir balance demonstrated the capacity of all peptide preparations to become inserted in lipid monolayers of any composition and initial π in the range 10-30 mN/m, although fibrils were less capable to do so than oligomers or monomers, their maximum initial π being ≈25 mN/m.