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Interactions between an amyloid beta peptide, responsible for Alzheimer’s disease, and biomembranes

Andreia Cuco, Benilde Saramago (supervisor) and Ana Paula Serro (co-supervisor)

Técnico Lisboa – Universidade de Lisboa – Lisboa, Portugal

Abstract. Alzheimer’s disease is characterized by plaque formation, in brain, composed by amyloid-β peptide (Aβ) aggregates. The Aβ

neurotoxicity seems to be related to its interaction with the cell membrane which can be influenced by the peptide conformation or the lipid membrane composition. Lipid rafts are microdomains rich in sphingomyelin (SM) and cholesterol (Chol), which have been associated with specific interactions with Aβ peptide. Used an equimolar mixture of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocoline (POPC), SM and Chol as a membrane model for studying lipid raft interaction with Aβ25-35 peptide fragment, at different concentrations. Investigated the effect of cholesterol studying the interaction between Aβ25-35 and POPC/SM (1:1) mixture, and the pH effect by pH changes between 4 and 7. The experimental techniques used were: Nanoparticle Tracking Analysis (NTA) and Circular Dichroism, to Aβ25-35 characterization, and Quartz Crystal Microbalance with Dissipation (QCM-D), Differential Scanning Calorimetry (DSC) and Langmuir Monolayers, to characterize the lipid-peptide interaction. At low Aβ25-35 peptide concentrations, the peptide is adsorbed in the polar region of the monolayers, slightly affecting the organization of lipids. The increase of Aβ25-35 concentration leads to the formation of peptide rich domains in both lipid mixtures, being stronger in the presence of Chol and at pH=4 (peptide aggregates smaller). In supported liposomes, the interaction with Aβ25-35 is favored POPC/SM (1: 1) and increasing concentrations of peptide did not affect the liposomes. In suspended liposomes, at low concentrations the peptide stabilizes the liposomes while increasing the Aβ25-35 concentration leads to their destruction. Keywords: Lipid rafts, Amyloid β-peptide, Liposome, Langmuir Monolayers, QCM-D, DSC

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I. INTRODUCTION

Alzheimer’s disease is characterized by a neuronal loss and a formation of plaques in the brain composed by aggregates of amyloid-β peptide (Aβ), exhibiting a β-sheet structure. According to other authors, the neurotoxicity of Aβ peptides may be related to the interaction of these peptides with the cell membrane, and therefore it is important to understand this interaction phenomenon [1, 2]. Thus, the inhibition of this aggregation process is a potential therapeutic approach for the Alzheimer’s disease treatment [3, 4].

Many mechanisms are suggested as being the cause for neurotoxicity of the interaction between Aβ peptides with cell membrane [5]. The neurotoxicity of these interactions may be associated with increased permeability of phospholipid bilayer, ion channels formation due to peptide aggregation, Aβ electrostatic bonds to phospholipids polar group and lipid oxidation in cell membranes, with consequent loss of integrity and increase in cell membrane fluidity and neuronal apoptosis [1, 2, 6-10]. The Aβ peptide-cell membrane interactions are influenced by the peptide conformation, amount of peptide in interaction with cell membrane, physic-chemical properties and lipid composition of cell membrane [2, 11-17].

Exist microdomains in cell membrane, called lipid rafts, which are rich in cholesterol and sphingomyelin. These microdomains are a preferential place for lipid-peptide interactions and its main functions are signal transduction and mediate the membrane transport, and are an important pathway for proliferation and apoptosis process [18-25]. Lipid rafts are a liquid-ordered phase (Lo), which coexists with domains rich in unsaturated glycerophospholipids in a liquid-disordered phase (Ld) [26-28]. Changes in microdomains structure and activity may be associated with neurodegenerative diseases, like Alzheimer’s disease, since lipid rafts play an important role in peptide aggregation [19, 24].

The amyloid-β peptide have a high tendency to aggregate, which are mostly insoluble aggregates and composed mainly for β-sheet. This tendency to aggregate due to H-bonding, involving CO and NH groups of Aβ peptide [29-31]. Aβ25-35 is located in the hydrophobic domain of Aβ1-42 and has neurotoxicity and aggregation properties identical to the full peptide fragment. It’s an

amphiphilic peptide fragment and less complex than Aβ1-42. This fragment shows mostly the β sheet conformation at pH 7 and random coil at pH 4. Thus, Aβ25-35 is a very attractive model to study the peptide aggregation and the interaction between the peptide and biomembranares models [14, 29, 32-44]. Fluorinated solvents such as hexafluoroisopropanol (HFIP) is used to stabilize the monomeric structure of the Aβ peptide, favouring the monomeric state and solubility of the peptide. This pre-treatment also keeps the α-helix structure of the peptide Aβ, however is not fully effective [42, 45-48].

In this work, it was studied the interaction between lipid rafts and Aβ25-35

peptide. An equimolar lipid mixture of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), sphingomyelin (SM) and cholesterol (Chol), as a model for lipid rafts, was prepared. There was also prepared an equimolar lipid mixture of POPC and SM, in order to study the effect of cholesterol in peptide-model biomembranar interactions. As biomembranar model, it was used supported or suspended liposomes and Langmuir monolayer.

Liposomes are one of the most adequate model to study cell membrane, since it is very similar to the phospholipid bilayer of native cell [26, 49-51]. Langmuir monolayers are an organized film of active molecules, with a thickness equivalent to the molecule thickness, at air/liquid interface. The polar groups are in contact with the subphase and nonpolar hydrocarbon chains pointing to the air. Monolayers are somewhat realistic biomembranar model, but are a very useful platform to study, in detail, the molecular interactions that occur at hydrophobic / hydrophilic interfaces [52, 53].

The experimental techniques used to characterize the Aβ25-35 peptide and the interactions between model systems and Aβ25-35 were: nanoparticle tracking analysis (NTA), circular dichroism, quartz crystal microbalance with dissipation (QCM-D) Langmuir monolayers and differential scanning calorimetry (DSC). NTA allows viewing and measuring, in real time, the size of nanoparticles present in the sample, and information about refractive index, fluorescence and zeta potential of nanoparticles [54, 55]. Circular dichroism is based on the differential absorption of the components of the circularly polarized light and can determine and analyse the secondary structure of peptides in

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solution [56-58]. QCM-D is the measurement of the shifts of the resonance frequency and dissipation and its working principle is based on the fact that piezoelectric quartz crystals are leading to a mechanical deformation when exposed to an oscillating electric field and a wave shear that propagates through the sample. The variation of the frequency and dissipation may be due to absorption/desorption of molecules to the crystal surface coated with gold or changes in the viscoelastic properties of the layer adsorbed to the crystal [51, 59, 60]. Langmuir monolayers is based on the formation of a monolayer at the air/liquid interface and provides information about variation of the surface pressure which causes compression of the monolayer, indicating the molecules phase and the concentration effect on lipid-peptide interactions [61-63]. DSC allows a thermal characterization of the samples and provides information about the reversibility of the thermal behavior of the sample, and the temperature associated with the phase transition of the sample within a temperature range [65-66].

II. MATERIALS AND METHODS

Materials. Sphingomyelin (brain SM) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocoline (POPC) were purchased from Avanti Polar Lipids, Inc. Cholesterol (Chol), hexafluoroisopropanol (HFIP), N-(2-Hydroxyethyl) piperazine-N’-(2-ethanesulfonic acid) (HEPES), dimethyl sulfoxide (DMSO), sodium dodecyl sulphate (SDS), chloroform and sodium acetate were purchased from Sigma Aldrich, Co. Amyloid-β peptide (25-35) (95% purity) was purchased from Intavis. Sodium chloride (NaCl) was supplied by Panreac, Hellmanex ® II was supplied by Hellma Gmbh & Co KG, and acetic acid was purchased from Glacial Reagent Grade. Chromafil ® Xtra filters, with 25mm diameter, for filter the buffers, was supplied by Macherey Nagel. Polycarbonate filters of 600, 200 and 100 nm of diameter from Nuclepore, Whatman were used for liposomes extrusion. The gold-coated quartz crystals (AT–cut, 4.95 MHz, 14mm diameter), for QCM-D experiments, were supplied by Q-Sense AB.

Preparation of liposomes. The protocol supplied by Avanti Polar Lipids, Inc. was used as a support for the preparation of liposomes [67]. The amount of lipids necessary for a final concentration of 3.195 mM in lipids were solubilized on a chloroform solution and put on a round bottom flask. After drying the lipid mixture in the flash with nitrogen, the resulting lipid film was kept under vacuum, overnight, to remove all traces of organic solvent. Thereafter, the lipid film was hydrated with HEPES buffer (10mM, 0.1M NaCl, pH=7) and kept on a thermostatized water bath at 65ºC, for 1 hour, interchanging heating with manual and vortex agitation for a complete dissolution of the lipid film. After this process, there was obtained a suspension of large multilamellar vesicle (LMV) and this suspension was then subjected to freeze-thaw cycles, using liquid nitrogen. Large unilamellar vesicles (LUV) were obtained from extrusion of the solution, passing sequentially the samples 20 times, through polycarbonate filters with pore diameter of 600, 200 and 100nm (5, 5 and 10 times, respectively). Finally, the liposome solution was diluted with HEPES buffer to a final concentration of 1.12mM and was stored at 4/5ºC and used within a maximum period of 7days after the preparation.

Preparation of peptide solution. The dry film of Aβ25-35 peptide was prepared with a final concentration of 0.849mM. 10mg of peptide was dissolved in HFIP, to prevent aggregation, with a buffer volume of 10mL. The solution is incubated at room temperature, for 30 minutes and then placed in a glass flask. The solution is subjected to vortex and sonication for peptide solubilisation. The stock solution was distributed by Eppendorf tubes and these tubes were left open, in the fume hood, overnight, to evaporate the HFIP. The Eppendorf tubes are then placed in a vacuum, for 1hour, to remove all traces of HFIP. Finally, the dried

peptide film formed on each Eppendorf was stored at -20 ° C. For each experiment, there was prepared a peptide solution with a desire final concentration. It was removed the Eppendorf tube from the freezer and allowed to cool, at room temperature, for 30minutes. Next, was added 400μL of buffer, in order to dissolve the peptide film, placing it in ultrasound, for 10seconds, and in vortex, for 20minutes. The peptide solution is transferred to a polypropylene tube, previously washed with buffer solution. It is added again to the Eppendorf 1000μL of buffer and repeat the agitation cycle, twice. Finally, add the volume of buffer required to obtain the desired peptide concentration for the different techniques.

Preparation of lipid solution. Solutions of pure lipids (POPC, SM and Chol) with a final concentration of 0.5mM were prepared. The amount of peptide required was weighed in a glass flask and, after, was added 5mL of chloroform and methanol solution (4:1), to dissolve the lipid. The lipid solution was stored at 4/5ºC.

NTA. The experiments were performed using a microscope and a

Capture software, to visualize the peptide solution with a final concentration of 1, 4 and 25µM, at pH=4 and pH=7. The buffers were centrifuged on Hermille Z323K, with Centrifugal Filters Units for 40minutes, at 20ºC and 8000rpm and after were filtered twice using filters with 0.20µm of pore size. The chamber was filled with Milli-Q water, to confirm the absence of residues of other solutions. Subsequently, the peptide solution was injected into the chamber and 15 measurements were performed, for 90seconds, with a contrast level camera of 8.

Circular dichroism. The experiments were perform in Applied Photophysics π*-180. First, the HEPES buffer baseline, at pH=7 and sodium acetate/acetic acid, at pH=4 were performed. After, the measurements were done at 21ºC with a wavelength of 185 to 250 nm, for pH=4, and 195 to 250 nm, for pH=7, and with 0.25seconds time per point, 2.5nm of step, 5 nm of bandwidth. The spectrum was obtained using Pro-Data PiStar software. The peptide solution was prepared with a final concentration of 80µM.

QCM-D. Q-Sense E4 (Q-Sense AB), fitted with 4-sensor chamber and connected to a flow pump (Imastec IPC-N 4) was used in this work. Before each experiment, the gold-coated quartz crystal were submitted to 2 cycles of UV-Ozone exposition on PSD Series UVOzone Cleaning & Sterilization device (Novascan), for 10minutes, intercalated by washing with Milli-Q water and blow-drying with nitrogen. All experiments were performed at 37ºC, pH=7 and a rate of 0.100ml/min. For each experiments, the frequency (Δf) and dissipation (ΔD) shifts were monitored for the fundamental and up to 9th overtone. In order to analyse the results, the mean ± standard deviation of Δf and ΔD for each overtone were calculated and the viscoelastic modelling of the results was performed on the QTools 3 software, using Voigt model. Thus, it is considered a film with a single layer of adsorbed liposomes and used 3rd to 9th overtone. The boundary conditions for viscoelastic modelling are show in Table1.

Table 1. Boundary conditions to estimate the viscosity, shear modulus and thickness film, in QTools 3 software.

Parameter Min. Max. Steps

ɳ (Pa.s) 0.0005 0.01 50

G (Pa) 1000 1x109 50

d (m) 1x10-10 1x10-6 50

-A Isotherms. All experiments were performed at 25°C, in a

Teflon micro trough (total area of 8400mm2 and volume of 50mL), equipped with a movable Teflon barrier. The experiments were performed at a speed rate of 10mm/min. At the beginning of the

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test, the micro trough is filled with the subphase (HEPES buffer to pH=7 or sodium acetate/acetic acid buffer to pH=4). Before and after each test, the monolayers are washed with DMSO, ethanol, hot Milli-Q water (about 50°C) and Milli-Q water, at room

temperature. Note that were performed at least 3 -A isotherms for

each lipid and lipid mixture, with a deviation less than 6%.

-A lipid isotherms: Lipids were carefully spread on the subphase,

and the compression was performed after 15minutes.

-A lipid-peptide mixture isotherms: Peptide with a final

concentration of 0.25 to 4µM was injected on the subphase and 15minutes then, the lipid mixtures were spread on the subphase. After 60minute, the compression was performed.

-A Aβ25-35 isotherms: Peptide with a final concentration of 1 to 4

µM was injected into the subphase. After 75minutes, the compression was performed.

DSC. The experiments were performed in a VP-DSC MicroCalorimeter (MicroCal®), at pH=7. The six heating/cooling cycles were performed at a temperature range from 10 to 50ºC, at a scanning rate of 60ºC/hour. Before each cycle, the samples pass through a thermostating period of 15 minutes. The reference cell was filled with HEPES buffer. Before the experiments, reference scans were performed with the sample cell filled with HEPES. The mixtures of liposomes with Aβ25-35 peptide were prepared immediately before injection in cell, with a final lipid concentration of 1.12mM and Aβ25-35 between 10 to 80 µM. The HEPES buffer was subjected to degassing through Thermovac (MicroCal®), to

minimize the presence of air bubbles in the sample. Since liposomes are very sensitive and their disruption may occur, samples containing liposomes not subjected to this process. At the end of experiments, the thermograms obtained were analysed using Origin 7.0 software (OriginLab Corporation). Reference scans were performed with the sample cell filled with buffer and its thermogram was subtracted from all thermograms.

III. RESULTS AND DISCUSSION

A. NTA

The pH has a direct influence on the size of the Aβ25-35 peptide aggregation. Independently of peptide concentration, at pH=7 the peptide aggregates are considerably higher, i.e. at pH=4 Aβ25-35

peptide has a lower aggregation tendency, which is consistent with Loew [68].

For lower peptide concentrations (1 and 4μM), the size distribution of peptide aggregates ranges between 50 to 800 nm and at higher concentrations (25μM), the aggregate size distribution ranges from 50 to 500 nm (Fig.1). In both pHs, there was an average concentration of 0.03x106 particles/ml for 1µM and 0.05x106 particles/ml for 4µm of Aβ25-35. For 25µM, there is an increased of average concentration 0.65x106 particles/ml at pH=4 and 0.5x106 particles/ml at pH=7. This increase was expected in relation to the lower concentrations. For higher concentrations, a large number of particles in solution, allowing the formation of many aggregates with different sizes (Table 2). At concentrations of 1 and 4µm the number of particles in solution is significantly smaller, forming fewer but larger aggregates.

Table 2. Amount of aggregate Aβ25-35 peptide particles, in solution, at pH=7.

1µM 4µM 25µM

9x10-7 2x10-6 4x10-5

B. Circular dichroism

The circular dichroism spectrum, at pH = 4, shows a minimum peak at 198nm (Fig.2), demonstrating that Aβ25-35 peptide has a predominantly random coil conformation [69, 70]. At pH 7, there is a peak at 203nm and a minimum peak at 228nm (Fig. 2). According to Labbé et al, at physiological pH, Aβ25-35 peptide has a predominant β-turn secondary structure at low concentrations and β-sheet at high concentration, like in present case [29, 69, 71].

The differences in spectrum intensities are justified by the fact that peptide aggregation influence the spectrum, as an increase of aggregates leads to a decrease of the spectrum intensity [72]. As pH=4 there are fewer aggregates compared to pH=7, this justifying that the intensity of the spectrum at pH=4 is lower.

C. QCM-D

The mean frequency and dissipation values for POPC/SM (1:1) and POPC/SM/Chol (1:1:1) liposomes (Table 3), at 37ºC, observed is consistent with the liposome film formation on the quartz crystal coated with gold [60, 73-77]. The HEPES buffer does not affect the lipid mixtures, which means that this liposomes layers are properly adsorbed to the crystal surface. The dissipation shifts of raftlike mixture was always higher, which is consistent with the fact that this mixture is more viscoelastic and dissipative layer. According to Viitala et al., liposomes doesn’t have a rigid behavior and adopted a flattened shape after adsorption onto the crystal surface and this liposomes are less rigid, compared to the raftlike liposomes [78]. The POPC/SM (1:1) liposome layer, at 37ºC, is in liquid-disordered lamellar phase (Ld), with greater deformation of the liposomes. Thus, the area occupied by these liposomes is larger and the adsorbed mass is smaller, compared to the raftlike

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Figure 2. Dicrhroism circular spectrum of Aβ25-35 peptide 80µM, at pH=4 (right) and pH=7 (left).

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Figure 1. pH effect in peptide aggregation at different peptide concentration. pH=4: dashed line; pH=7: solid line.

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liposomes. The presence of cholesterol in liposomes leads to the formation of an adsorbed layer with more viscosity, on the crystal surface. Raftlike liposomes showed a more rigid behavior, compared to POPC/SM liposomes, due to the cholesterol molecular conformation and its condensing effect [79, 80].

Table 3. Mean ± S.D. of frequency and dissipation shifts for 3rd overtone of adsorption experiments of POPC/SM and POPC/SM/Chol liposomes, at 37ºC.

Liposomes POPC/SM (1:1) POPC/SM/Chol (1:1:1)

Δf3 (Hz) -200 ± 3 -271 ± 3

ΔD3 (1x10-6) 28 ± 1 42 ± 1

The introduction of the Aβ25-35 peptide causes no significant disturbances in the stability of the liposome adsorbed layer to the quartz crystal (Fig. 3). However, differences exist between the behavior of the lipid mixture POPC/SM and POPC/SM/Chol when the interaction with the peptide Aβ25-35 occurs. The Aβ25-35 peptide influences the behavior of the layer of liposomes POPC/SM, being more pronounced with increasing peptide concentration. The dissipation remains almost constant, independently of peptide concentration. The variations in frequency and dissipation shifts of raftlike liposomes are negligible, independently of peptide concentration. It is important to note that for both mixtures, the variation in frequency of several harmonics is similar, meaning that the change in the adsorbed layer is constant throughout the film thickness.

In Fig. 4 can be seen that when the Aβ25-35 interacts with the liposomes leading to a -10Hz decrease in POPC/SM mixture, but in raftlike mixture this variation is almost zero. Thus, it appears that Aβ25-35 peptide-adsorbed liposomes interaction is more evident in the absence of cholesterol, i.e., the Aβ25-35 peptide destabilizes more POPC/SM liposome layer. The frequency shifts after interaction with Aβ25-35 is in agreement with the study of other authors who found that, according to the type and concentration peptide, the frequency can increase due to breakage of the adsorbed liposomes leading to decreasing of adsorbed mass or destabilization of adsorbed layer due to the interactions of

liposomes with peptides [81-83]. However, in this case, the decrease in frequency shifts was negligible, suggesting a slight fluidization of the bilayer may cause a deformation of the adsorbed liposomes with the release of interstitial water.

The dissipation shifts after Aβ25-35 peptide-liposomes interactions, was 1x10-6 for POPC/SM mixture and 0.5x10-6 for raftlike mixture. Independently of peptide concentration, the shifts dissipation is always higher, in absolute value, in POPC/SM/Chol mixture maintaining its viscoelastic and dissipative properties in the presence of Aβ25-35 peptide.

The frequency and dissipation shifts resulting from the rinsing process is almost zero for both lipid mixtures, meaning that most liposomes are irreversibly adsorbed to the crystal surface [83]. The slight shifts, which occurs occasionally after rinsing, results from the removal of weakly adsorbed liposomes, which are swept away by buffer solution or, if after addition of the Aβ25-35 peptide, may result in desorption of peptides. The frequency and dissipation shifts after rinsing can also be due to partial rupture of the smaller liposome, with water release from liposomes to the surroundings.

Table 4. Mean ± S.D. values of viscoelastic properties of POPC/SM liposomes 80µM, at 37ºC, obtained from QTools 3 modeling.

Parameters Liposomes HEPES Aβ25-35 HEPES

ɳ (mPa.s) 2.0 ± 0.03 1.9 ± 0.04 1.9 ± 0.04 1.9 ± 0.04

G (kPa) N.E. N.E. N.E. N.E.

d (nm) 106 ± 2 107 ± 3 110 ± 4 112 ± 4

Table 5. Mean ± S.D. values of viscoelastic properties of POPC/SM/Chol liposomes 80µM, at 37ºC, obtained from QTools 3 modeling.

Parameters Liposomes HEPES Aβ25-35 HEPES

ɳ (mPa.s) 2.6 ± 0.2 2.5 ± 0.2 2.5 ± 0.2 2.5 ± 0.2

G (kPa) 6 ± 2 5 ± 1 5 ± 2 5 ± 2

d (nm) 106 ± 4 106 ± 4 107 ± 5 107 ± 5

The layer thickness of POPC/SM liposomes increases slightly as a result of interaction with the Aβ25-35, at high peptide

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Figure 3. Example of QCM-D experiment of interaction of POPC/SM liposomes

(above) and POPC/SM/Chol liposomes (bottom) with Aβ25-35 peptide 80µM at 37ºC and pH=7. Left: frequency shifts. Right: dissipation shifts. (1) liposome introduction; (2) 10min rising; (3) Aβ25-35 injection; (4) 10min rising.

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Figure 4. Frequency (left) and dissipation (right) shifts, for 3rd overtone, after injection of POPC/SM (1:1) (above) and POPC/SM/Chol (bottom), rising, injection of 10, 25, 50 and 80µM of Aβ25-35 peptide and, again, rising. The grey intensity correspond to injections listed in the box.

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concentrations, but the viscosity does not change (Table 3). It was not possible to obtain reasonable shear modulus values, it is not possible to assess any changes in liposomes rigidity. In raftlike mixture, no significant change in viscosity, shear modulus and thickness in peptide-liposome interaction was observed (Table 4). The small increase in the layer thickness of liposomes POPC/SM may be explained by the disruption of liposomes layer caused by the interaction with the peptide. This disruption causes electrostatic repulsion in lipids and the overall volume increases, leading to increased thickness and decreased viscosity [83]. However, it is important to note that the variation observed in this case is very small, and these very significant effects.

The preferential interaction of the Aβ25-35 peptide at high concentrations, with POPC/SM liposomes can be explained by the fact that this mixture, at 37ºC, be in a disordered liquid phase, while the raftlike system has SM/Chol ordered domains.

D. -A Isotherms

The -A isotherm of Aβ25-35 is directly influenced by peptide concentration (Fig.5) and occurs the Gibbs monolayer formation, since the peptide has a soluble and amphiphilic behavior. Independently of peptide concentration, there is no monolayers collapse, which means may be occurring, along the compression, an increase in thickness of the adsorbed monolayer at the interface, forming multiple layers on top of each other. At 1µM, surface

pressure is not affected significantly (≈0) because the peptide surface concentration is very low. The increase in peptide concentration leads to an increase in monolayer surface concentration at the interface. Thus, for a molecular area of 100Å2, only for 4µM, or higher concentrations, there is enough peptide in the interface which leads to an increase of surface pressure. A lower molecular area, as 40Å2, monolayer surface concentration is enough to cause changes in surface pressure, independently of peptide concentration.

During the compression process, lipid molecules tend to orient themselves perpendicular to interface, in order to achieve a more

dense conformation [84]. The value of mean molecular area in =0 is called lift-off (A0) and corresponds to the state in which the surface pressure of the monolayer increases when it is subjected

to compression. The SM and POPC -A isotherm indicate the formation of a monolayer on liquid-expanded state (LE), at low

surface pressure, and Chol -A isotherm appears very abrupt, which corresponds to the formation of a dense and incompressible monolayer (Fig.6A). The area occupied by a POPC molecule is greater than the area occupied by a SM and Chol molecules, for the same surface pressures. POPC has an acidic unsaturated chain inhibits the formation of a dense monolayer [85]. Chol easily form a dense and compact monolayer, due to their molecular conformation and its condensing effect. The final zone of isotherm corresponds to monolayer collapse, i.e., occurs a lipid monolayer disruption or a collapse of the arranged state of molecules. The

POPC collapse, at lower surface pressures than the SM collapse due to phase transition from LE state to liquid-condensed state

(LC) (=23mN/m), where SM molecules adopt a more compact form.

The interaction between Chol and POPC/SM leads to a deviation from the isotherm to smaller areas, for same surface pressure (Fig.6B). This behavior is due to the condensing effect of Chol, making raftlike monolayer more dense and compact. The A0 of POPC/SM and POPC/SM/Chol monolayer is 103Å2 and 75Å2, respectively, and corresponds to the LE monolayer formation. These monolayers do not shows a LC state, since the double bond of POPC prevents of a compact monolayer organization, inhibiting the SM phase transition. The collapse of both monolayers occurs at 44mN/m.

Fig. 7 (-●- line) shows the theoretical isotherms at pH=7, calculated from the linear combination of pure lipid isotherms, taking into account equimolar proportion of mixtures. In POPC/SM monolayer there is an equivalent of both lipids contribute, since there is an almost complete overlap between theoretical and experimental isotherm. The slight deviation that is observed at higher surface pressures is a consequence of POPC inhibition in SM transition phase. In raftlike mixture, there was observed a highly significant deviation from experimental isotherm due to the fact that the raftlike isotherm be calculated assuming ideal behavior of lipids, not taking into account Chol condensing effect.

pH effect (Fig.6, solid line) is opposite to the behavior of POPC and SM monolayers, since the SM isotherm deviates to larger molecular area, while POPC isotherm deviates to smaller molecular area. Chol isotherm is not influenced by pH variation. At pH 4, SM reaches the LE state at higher molecular areas, but the LE to LC transition occurs at the same surface pressure

(=23mN/m). POPC reaches LE state at lower molecular areas, at pH=4. Monolayers collapse does not change with pH variation, meaning that the pH does not influence the organization molecular state just before the monolayer rupture (Table6). In lipid monolayers, there wasn’t any effect of pH variation, since it was detected an almost complete overlap isotherms (Fig.6). Therefore,

Figure 5. -A isotherm of Aβ25-35, at 25ºC and pH=7, at different peptide

concentration (1, 2 and 4µM).

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50

60

0 50 100 150

Surf

ace p

ressure

(m

N/m

)

Mean molecular area(Å2)

5

4

B.

Figure 6. -A isotherm of POPC (1), SM (2), Chol (3), POPC/SM (4) and

POPC/SM/Chol (5), at pH=4 (dashed lines) and pH=7 (solid lines), ate 25ºC.

0

10

20

30

40

50

60

0 20 40 60 80 100 120 140 160

Surf

ace p

ressure

(m

N/m

)

Mean molecular area (Å2)

1

23

B

0

10

20

30

40

50

60

0 20 40 60 80 100 120 140 160

Surf

ace p

ressure

(m

N/m

)

Mean molecular area(Å2)

1

2

A

Figure 7. Experimental vs. Theoretical -A isotherm experimental, at 25ºC and

pH=7. (1) POPC, (2) SM, (3) Chol. (A) POPC/SM and (B) POPC/SM/Chol.

Experimental POPC/SM and POPC/SM/Chol -A isotherm: -●-.

6

the pH doesn’t have a significant influence on POPC/SM and POPC/SM/Chol isotherms, as had been verified by other authors [85-87].

Table 6. A0 and collapse values of lipid monolayers (POPC, SM and Chol), at pH=4

and pH=7 (25ºC).

A0 (Å2/molec) Collapse (mN/m)

pH=4 pH=7 pH=4 pH=7

POPC 116 127 44 45

SM 93 85 54 55

Chol 44 44 42 43

While compression is performed, the available molecular area is becoming smaller and changes occurring at the interface, due to reduced surface tension and consequently increase in surface pressure. The increase in peptide concentration leads to a decrease in the available molecular area, leading to isotherms deviation for smaller areas.

The increase in peptide concentration leads to an isotherm deviation to higher areas (Fig.8). In POPC/SM, there is a progressive isotherm deviation with increasing peptide concentration. In raftlike monolayer, at lower peptide concentrations (0.25 to 1µM), peptide has slight effect on lipid monolayer, due to the Chol condensing effect. This is due to the peptide being adsorbed to polar region of the lipid monolayer, slightly affecting its organization, i.e., exists a peptide adsorbed layer on lipid monolayers basis. Isotherms variations are more significant in 2 and 4µM, in both monolayer: peptide rich domains are formed in air/liquid interface, contributing to the increase of monolayer area. The increase in concentration leads to an increase of initial surface pressure, due to a sufficient peptide surface concentration already exists to affect initial surface pressure. From equation 1:

(1)

where Acp is the area occupied by Aβ25-35-lipid mixture monolayer lipid and AC0 is the area occupied by lipid mixture monolayer, were calculated the area deviation of lipid monolayer interacting with Aβ25-35, at pH=7 (Fig.9).

Fig.9 shows that the increase of peptide concentration induces an increase in molecular area, especially for peptide concentrations higher than 1µM, (slope is greater). This behavior was also observed for pH=4. The area deviation of Aβ25-35-raflike monolayer interaction is greater than that observed for the POPC/SM monolayer, verifies a direct effect of Chol presence.

For lower peptide concentrations, the amount of adsorbed peptide on the monolayer corresponds to a lower lipids fraction at the interface, slightly affecting the monolayer, explaining the slight

area deviation to 0.25 to 1µM. Increasing the peptide concentration to 2 and 4µM leads to an increase of peptide fraction higher of lipid fraction, forming peptide rich domains which causes an increase in area deviation. Chol induces a higher area deviation, since the presence of Chol rich domains facilitates interaction between Aβ25-35 and lipid monolayer. Raftlike monolayer presents more condensed domains due to condensing effect that Chol induced in monolayer. Thus, peptide interaction is stronger with condensate domains than expanded domains, since this domains have a higher polar groups density to interact with peptide. Area deviation is higher at 10mN/m because molecules are in LE state, i.e., more disorganized. At 30mN/m, molecules are already a more organized state and closer to achieve the monolayer collapse. This behavior is verified independently of peptide concentration.

At pH=4, area deviation occurs for higher areas in both Aβ25-35-lipid monolayer interactions (Fig.10). Thus, subphase pH influences area deviation of isotherms for both lipid mixtures. Fig.10 shows that the increase in monolayer area is higher at pH=4, for all peptide concentrations tested in interaction with lipid mixtures. Other authors have confirmed that variations pH induces a monolayer area deviation [88, 89]. For this observation, it was chosen a surface pressure of 30 mN/m, since at this stage the monolayer is compressed almost completely, being quite stable, compact and organized. In Fig.1 it has been verified that exist a larger number of smaller aggregates at pH=4, but with smaller dimensions. Thus, it was found that the interaction between the smaller peptide aggregates with monolayer is more efficient.

0

10

20

30

40

50

60

0 50 100 150

Surf

ace p

ressure

(m

N/m

)

Mean molecular area (Å2)

cA

0

10

20

30

40

50

60

0 50 100 150

Surf

ace p

ressure

(m

N/m

)

Mean molecular area (Å2)

cA

Figure 8. -A isotherm of POPC/SM (left) and POPC/SM/Chol (right) (solid line) and

Aβ25-35 peptide, at 25ºC and pH=7. Red arrow indicate the increase of peptide concentration.

100)(

0

0

A

AAAverage Ccp

0

20

40

60

80

100

0 1 2 3 4

Are

a d

evia

tion (

%)

Aβ25-35 concentration (µM)

Figure 9. Chol effect in lipid monolayer in the presence of different peptide

concentration, at 25ºC and pH=7, for 10 and 30mN/m

0

20

40

60

80

100

0,25 µM 0,5 µM 1 µM 2 µM 4 µM

Are

a d

eiv

iation (

%)

pH=4

pH=7

0

20

40

60

80

100

0,25 µM 0,5 µM 1 µM 2 µM 4 µM

Are

a d

evia

tion (

%)

Figure 10. pH effect in area deviation of POPC/SM (left) and POPC/SM/Chol (right) monolayer in interaction with 0.25, 0.5, 1, 2 and 4µM of Aβ25-35, at 25ºC and 30mN/m.

7

E. DSC

In the presence of Chol was not possible to observe a phase transition of raftlike mixture, in the temperature range studied (10 to 50°C). This lipid stabilizes liquid-ordered domains, since there is a preferential interaction with acyl chains of SM. When exposed to high temperatures, Chol interferes with fatty acid chains movement of phospholipids, become less fluid lipid membrane [90-95]. Thus, was not studied the thermal behavior of raftlike liposomes with Aβ25-35. There was, within the studied temperatures, a peptide phase transition. Horng-Lun Chu et al [96] have shown that temperature variation (25 to 120°C) leads to an irreversible change in peptide conformation to β-sheet.

POPC/SM mixture shows a broad peak that starts at temperatures below 10°C, i.e. outside the temperature range that the device allows to analyse, and ends close to 37°C, occurring a phase transition at 23,75ºC (Fig.11, dashed line). POPC is an unsaturated lipid and presents a melting temperature (Tm) of -3ºC and destabilizes the acyl chain of SM molecules. SM undergoes a phase transition from gel to liquid crystalline phase with a Tm of 38°C. The mixture of these lipids in an equimolar ratio, leads to an enlargement of lipid mixture phase transition [92]. POPC/SM mixture presents a system with rigid SM-rich domain and fluid POPC-rich domain.

Aβ25-35+POPC/SM mixture was subjected to 6cycles of heating/cooling and, with exception of first cycle, all cycles presented overlapping. The peptide addition leads to an increase of phase transition of lipid mixture, due to stabilize the Aβ25-35 cause in liposomes, making them more rigid, since its phase transition occurs at higher temperatures compared to pure liposomes (Fig.11). At 10 and 25µM there is an increase in peak width due to the decrease in the liposomes cooperativity. At 50µM, Aβ25-35-liposome mixture peak 50um has a width similar to pure liposomes, but the area under the peak is lower, meaning that there is a decrease in enthalpy associated with phase transition. In thermogram of peptide 80µM is not possible to identify a maximum corresponding to the temperature of phase transition and there is a decrease in heat capacity, suggesting the liposomes destruction. The thermograms shape obtained after peptide addition, particularly at a concentration of 50µm, suggests the existence of several overlapping peaks, which can be associated with the existence of phase separation in the mixture.

Fig.12 shows that the phase transition temperature is maximum at 10µM, decreasing to higher concentrations, but always remains above the phase transition temperature of pure liposomes. Other authors have reported that the peptide-lipid interaction leads to a change in the transition temperature of the

lipid phase, but that is dependent upon the lipid and peptide type in interaction [97-99].

Figure 12. Phase transition temperature of POPC/SM (1:1) mixture as a function of Aβ25-35 concentration, at pH=7.

IV. CONCLUSIONS

Aggregation tendency is a major characteristic of Aβ25-35 peptide, and seems to have a direct consequence on Alzheimer's disease. With the aim to characterize the Aβ25-35, was investigated its aggregation state and structure, through NTA and Circular Dichroism, respectively. Aβ25-35 peptide has a greater aggregation tendency, at physiological pH. At low concentrations, Aβ25-35 aggregates have a wider size distribution (50 to 800nm) as compared to high concentrations (50 to 500nm). However, there is a much larger number of aggregates, at high concentrations. Regarding peptide conformation, it was verified that pH influences directly the conformation adopted. Thus, at pH=4 peptide has mainly a random coil secondary structure and a β-sheet secondary structure at pH=7. The highest level of β-sheet clustering has led to association of this secondary structure with peptide aggregates formation.

Interaction between Aβ25-35 and biomembranes was studied

using Langmuir monolayers (-A isotherms), in the case of lipid

monolayers, and using QCM-D and DSC to liposomes. The most interesting results were obtained using the Langmuir monolayers.

In studies with QCM-D, it has been verified that the interaction between Aβ25-35 peptide and POPC/SM liposomes resulted in a slight increase in layer thickness, may be related to a weak fluidizing liposomes layer. In raftlike liposomes, the interaction with Aβ25-35 peptide did not cause any significant change in the adsorbed layer properties.

For suspended liposomes, it was only possible to study the Aβ25-35 effect on POPC/SM/ liposomes, since the absence of a phase transition in liposomes raftlike was observed within the temperature range studied (10 to 50°C). The Aβ25-35 effect on POPC/SM liposomes depends on the peptide concentration. The addition of low peptide concentrations (10 and 25µM) led to an increase of phase transition temperature of lipid mixture, suggesting that the peptide stabilizes suspended liposomes, making it more rigid. However, at higher peptide concentrations (80µm) there was no phase transition, indicating suspended liposomes destruction. These results do not coincide with those obtained with adsorbed liposomes onto QCM-D, where only verified a fluid action of POPC/SM liposomes after 80µM peptide addition. Thus, it seems reasonable to conclude that the adsorbed liposome resist to high peptide concentration addition, while suspended liposomes undergo breakage.

Once the modifications induced by peptide in adsorbed liposomes are small and it was not possible to study the Aβ25-35

20

21

22

23

24

25

26

27

28

0 10 25 50

Tem

pera

ture

(ºC

)

Aβ25-35 peptide concentration (µM)

-0,4

-0,3

-0,2

-0,1

0

10 15 20 25 30 35 40 45 50 55

Cp

(kC

al/ºC

/mo

l)

Temperature (ºC)

POPC/SM (1:1)

POPC/SM (1:1) + 10µM Aβ25-35

POPC/SM (1:1) + 25µM Aβ25-35

POPC/SM (1:1) + 50µM Aβ25-35

POPC/SM (1:1) + 80µM Aβ25-35

Figure 11. POPC/SM 1.12mM thermogram with different Aβ25-35 concentration, at pH=7.

8

effect in suspended raftlike, the pH effect has not been studied in this techniques.

At 25ºC, POPC/SM and POPC/SM/Chol monolayers organized in a LE state, although Chol becomes raftlike monolayer denser and compact, due to it condensing effect. At 25ºC and pure state, pH variation causes no effect on lipid monolayers. Therefore, it appears that the Chol effect is identical in monolayers and adsorbed or suspended liposomes.

Interaction between Aβ25-35 and both lipid monolayers, at 25ºC, leads to an increase of molecular area more significantly at higher peptide concentration. At low concentrations, Aβ25-35 adsorbed on polar groups of monolayer, affecting little the lipid organization. The increase of peptide concentration leads to the formation Aβ25-

35-rich domains, due to the increased proportion of peptide relatively to the lipids. The lower peptide concentrations produce a more pronounced effect on POPC/SM monolayers, since it is less dense and compact. For higher peptide concentrations, both monolayers undergo significant changes in its organization, being more evident in POPC/SM/Chol monolayer once the Aβ25-35-monolayer interaction is stronger with condensed domains, due to the higher polar groups density available to interact with Aβ25-35. This observation is contrary to the results with supported liposomes, in the Chol absence leads to a preferential interaction with Aβ25-35.

At pH=4 and 25°C, Aβ25-35 leads to greater changes in both lipid monolayers organization, compared to that observed at physiological pH. This organization change is more evident in raftlike monolayer, being denser. It was verified that interaction between smaller Aβ25-35 aggregates and lipid monolayer is more efficient, since there is a greater amount of these small aggregates at pH=4.

Experimental techniques did not allow making direct comparisons, since experimental conditions were different, but allowed to obtain some conclusions regarding differences between Aβ peptide-POPC/SM and POPC/SM/Chol mixtures interactions.

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