Sandra Rocha
email: sandra.rocha@fe.up.pt

Andreas F Thünemann

Maria Do

Carmo Pereira

Manuel Coelho

Helmuth Möhwald

Gerald Brezesinski

Influence of Fluorinated and Hydrogenated Nanoparticles on the Structure and Fibrillogenesis of Amyloid beta-Peptide

Keywords: amyloid-beta peptide, circular dichroism, conformation analysis, electron microscopy, fluorinated complexes, nanoparticles

Peptide aggregation in amyloid fibrils is implicated in the pathogenesis of several diseases such as Alzheimer's disease. There is a strong correlation between amyloid fibril formation and a decrease in conformational stability of the native state. Amyloid-β peptide (Aβ), the aggregating peptide in Alzheimer's disease, is natively unfolded. The deposits found in Alzheimer's disease are composed of Aβ fibrillar aggregates rich in β-sheet structure. The influence of fluorinated complexes on the secondary structure and fibrillogenesis of Aβ peptide was studied by Circular Dichroism (CD) spectroscopy and Transmission Electron Microscopy (TEM). CD spectra show that complexes of polyampholyte and fluorinated dodecanoic acid induce α-helix structure in Aβ, but their hydrogenated analogous lead to β-sheet formation and aggregation. The fluorinated nanoparticles with highly negative zeta potential and hydrophobic fluorinated core have the fundamental characteristics to prevent Aβ fibrillogenesis.

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Introduction

Soluble peptides and proteins convert, under certain conditions, into highly organized fibrillar aggregates described as amyloid fibrils and intracellular inclusions [START_REF] Chiti | Protein Misfolding, Functional Amyloid, and Human Disease[END_REF]. The fibrillar aggregates have a common structure of polypeptide chains organized in β-sheets. Such transition is implicated in the pathogenesis of several diseases including neurodegenerative Alzheimer's disease (AD). The extracellular deposits that characterized AD are composed of fibrils of a small peptide with 39-43 amino acids, the amyloid-β (Aβ) peptide [START_REF] Masters | Amyloid Plaque Core Protein in Alzheimer-Disease and Down Syndrome[END_REF]. Aβ is continuously secreted by normal cells in culture and is detected as a soluble peptide in the plasma and cerebrospinal fluid of healthy individuals [START_REF] Haass | Amyloid Beta-Peptide Is Produced by Cultured-Cells During Normal Metabolism[END_REF][START_REF] Seubert | Isolation and Quantification of Soluble Alzheimers Beta-Peptide from Biological-Fluids[END_REF]. In AD patients, Aβ exists as insoluble fibrillar aggregates [START_REF] Selkoe | Alzheimer's Disease: Genes, Proteins, and Therapy[END_REF]. This process results from a progressive transition from natively unfolded peptide to β-sheet structure. Aggregation of Aβ is preceded by the formation of series of nonfibrillar species, the protofibrils, which are by their turn preceded by other oligomeric species [START_REF] Chiti | Protein Misfolding, Functional Amyloid, and Human Disease[END_REF]. Prefibrillar aggregates are toxic to cells [START_REF] Lambert | Diffusible, Nonfibrillar Ligands Derived from a Beta(1-42) Are Potent Central Nervous System Neurotoxins[END_REF][START_REF] Haass | Soluble Protein Oligomers in Neurodegeneration: Lessons from the Alzheimer's Amyloid Beta-Peptide[END_REF]. The size and structure of the aggregated species appear to be associated with toxicity [START_REF] Wang | Development of a Novel Diffusion-Based Method to Estimate the Size of the Aggregated a Beta Species Responsible for Neurotoxicity[END_REF]. Oligomers are composed of 2 to 6 molecules and circular dichroism measurements show that they are relatively disorganized [START_REF] Huang | Structural Studies of Soluble Oligomers of the Alzheimer Beta-Amyloid Peptide[END_REF][START_REF] Bitan | Amyloid Beta-Protein (a Beta) Assembly: A Beta 40 and a Beta 42 Oligomerize through Distinct Pathways[END_REF]. The lack of regular secondary structure in the small oligomers suggested that the binding of monomeric Aβ to nascent fibrils requires large conformational changes and correspondingly high activation energies [START_REF] Kusumoto | Temperature Dependence of Amyloid Beta-Protein Fibrillization[END_REF]. If peptide conformational change and aggregation represent an early event in the pathogenesis of AD [START_REF] Selkoe | Toward a Comprehensive Theory for Alzheimer's Disease. Hypothesis:Alzheimer's Disease Is Caused by the Cerebral Accumulation and Cytotoxicity of Amyloid Β-Protein[END_REF], a plausible therapeutic approach would be to design molecules that control and stabilize Aβ secondary structure. Conditions such as long incubation times, high concentration and temperature promote β-sheet formation [START_REF] Hilbich | Aggregation and Secondary Structure of Synthetic Amyloid Beta-A4 Peptides of Alzheimers-Disease[END_REF][START_REF] Barrow | Solution Conformations and Aggregational Properties of Synthetic Amyloid Beta-Peptides of Alzheimers-Disease -Analysis of Circular-Dichroism Spectra[END_REF][START_REF] Chu | Temperature-Induced Conformational Changes in Amyloid Βnext Term(1-40) Previous Termpeptidenext Term Investigated by Simultaneous Ft-Ir Microspectroscopy with Thermal System[END_REF]. Aβ forms β-sheet structure on hydrophobic graphite surfaces [START_REF] Kowalewski | In Situ Atomic Force Microscopy Study of Alzheimer's Beta-Amyloid Peptide on Different Substrates: New Insights into Mechanism of Beta-Sheet Formation[END_REF] and at air-water interface [START_REF] Schladitz | Amyloid-Beta-Sheet Formation at the Air-Water Interface[END_REF]. In contrast, the peptide adopts α-helix structure in solution of negatively charged micelles and teflon particles [START_REF] Coles | Solution Structure of Amyloid Beta-Peptide(1-40) in a Water-Micelle Environment. Is the Membrane-Spanning Domain Where We Think It Is?[END_REF][START_REF] Shao | Solution Structures of Micelle-Bound Amyloid Beta-(1-40) and Beta-(1-42) Peptides of Alzheimer's Disease[END_REF][START_REF] Giacomelli | Conformational Changes of the Amyloid Beta-Peptide (1-40) Adsorbed on Solid Surfaces[END_REF][START_REF] Rocha | Adsorption of Amyloid Beta-Peptide at Polymer Surfaces: A Neutron Reflectivity Study[END_REF]. Fluorinated solvents (trifluoroethanol, hexafluoroisopropanol) are described to stabilize the structure of Aβ monomers at low concentrations

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(10%), but ethanol and 2-propanol have exhibited reasonable stabilizing effects only at very high concentrations (75%) [START_REF] Vieira | Change and Stabilization of the Amyloid-Beta(1-40) Secondary Structure by Fluorocompounds[END_REF].

This work focuses on the influence of hydrophilic surfaces, silica particles and hydrogenated and fluorinated nanoparticles on amyloid-β peptide (1-40) (Aβ 40 , Figure 1) to better understand the conditions that stabilize the peptide helical structure. Aβ 40 solution was incubated for 6 days in sealed quartz cuvettes that were previously cleaned by the RCA procedure. The structure of the peptide in contact with hydrophilic quartz cuvettes was characterized by Circular Dichroism (CD) spectroscopy and compared with a solution stored in microcentrifuge or glass test tubes. In addition, the conformation of Aβ 40 was studied in the presence of silica particles and polyampholyte-dressed micelles [START_REF] Thunemann | Polyampholyte-Dressed Micelles of Fluorinated and Hydrogenated Dodecanoic Acid[END_REF]. The silica particles used have a diameter of 86 nm and a zeta potential of -40 mV. Polyampholytes with alternating cationic (N,N'-diallyl-N,N'-dimethylammonium chloride) and anionic charged monomers (maleamic acid or N-phenylmaleamic acid) were complexed with perfluorododecanoic or hydrogenated dodecanoic acid, resulting in nanoparticles with hydrodynamic diameters of 3 to 5 nm (Figure 2) [START_REF] Thunemann | Polyampholyte-Dressed Micelles of Fluorinated and Hydrogenated Dodecanoic Acid[END_REF].

The nanoparticles were incubated with Aβ and their effect on the peptide was studied by CD spectroscopy and Transmission Eelectron Microscopy (TEM). Previous work has demonstrated that the fluorinated complexes induce α-helix structure in B18 peptide, a sequence with tendency to form amyloid fibrils [START_REF] Rocha | The Conformation of B18 Peptide in the Presence of Fluorinated and Alkylated Nanoparticles[END_REF].

Material and Methods

Chemicals

Amyloid-β peptide (1-40) (Aβ 40 ) was purchased from Bachem (Weil am Rhein, Germany). Potassium dihydrogen phosphate, di-potassium hydrogen phosphate, sodium chloride, sodium azide, uranyl acetate, hexafluoroisopropanol, dimethyl sulfoxide and Ham's F-12 medium were purchased from Fluka (Seelze, Germany). Silica particles were prepared according to the method reported by Stöber et al [START_REF] Stober | Controlled Growth of Monodisperse Silica Spheres in Micron Size Range[END_REF].

Preparation of complexes

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The complexes were prepared as previously described. [START_REF] Thunemann | Polyampholyte-Dressed Micelles of Fluorinated and Hydrogenated Dodecanoic Acid[END_REF] Briefly a solution of 0.1 g of polyampholyte,

Poly(N,N'-diallyl-N,N'dimethylammoniumalt-maleamic Carboxylate) (P 1 ) or Poly(N,N'-diallyl-N,N'dimethylammonium-alt-N-phenylmaleamic Carboxylate) (P 2 ), in 100 mL of water was adjusted to pH 8 using sodium hydroxide solution (0.1 mol L -1 ) and heated to 90 °C. A solution (100 mL, 90 °C) of 1.0 equiv of the dodecanoic acid and perfluorododecanoic acid, respectively, was added in droplets to the polyampholyte solution, which was then adjusted to pH 8. The mixture was stirred for 60 min at 90 °C and then cooled to room temperature.

Peptide solution

Aβ 40 was dissolved at a concentration of 1 mg/mL in 100% hexafluorisopropanol (HFIP). The solution was incubated at room temperature for several hours. The peptide/HFIP solution was dried down under a gentle stream of nitrogen gas and the peptide was resuspended in 10 mM potassium phosphate buffer (pH 7.4) with or without NaCl (0.5 M NaCl; final concentration 0.1 M) for CD spectroscopy or 100%

dimethyl sulfoxide for TEM. Sodium azide (0.05 mM) was added to prevent microbial growth.

Cuvette modification

Quartz cuvettes (Hellma GmbH, Germany) were carefully cleaned to obtain hydrophilic surfaces. The cuvettes were treated by the RCA method: immersion in solution of H 2 O 2 (30%) + H 2 O + NH 3 (30%), ratio 1:5:1 by volume, heated to 80 °C for 10 min, followed by rinsing with Milli-Q-water. After RCA the surface is well saturated with silanol groups, conferring very high hydrophilicity. Above pH 2, the oxide surface is negatively charged.

Circular dichroism (CD) spectroscopy

Far UV CD spectra of Aβ 40 solution were recorded in a Jasco J-715 Spectropolarimeter (Jasco Co., Tokyo, Japan), using a cuvette with 0.5 or 1 mm path length at 0.2 nm intervals between 190 and 260 nm. The spectra were taken as the average of 10 scans recorded at a speed of 20 nm/min. The peptide conformation was analyzed during incubation (6 days) in quartz cuvettes and in the presence of

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ACCEPTED MANUSCRIPT particles at room temperature and at 37 ºC. Spectra were corrected by subtracting the buffer or particle solution baseline.

The content of secondary structure motifs was calculated using the CONTIN/LL, a self-consistent method with an incorporated variable selection procedure included in the CDPro software. [START_REF] Sreerama | Estimation of Protein Secondary Structure from Circular Dichroism Spectra: Comparison of Contin, Selcon, and Cdsstr Methods with an Expanded Reference Set[END_REF] Before secondary structure analysis, the CD spectra were smoothed using the noise reduction routines provided with the J-715 spectropolarimeter. In general, CONTIN/LL program with the reference set including unordered structures produced the most reliable results. [START_REF] Sreerama | Estimation of Protein Secondary Structure from Circular Dichroism Spectra: Inclusion of Denatured Proteins with Native Proteins in the Analysis[END_REF] Normalized root mean standard deviation (NRMSD) was used to determine the quality of the fit of the calculated structure to the data and was less than 0.1. Although the program used to calculate the secondary structure elements is meant for proteins, the reference set with unordered structures gave consistent results. These results give an idea on changes in the secondary structure and on the predominant structure motif of Aβ. Other programs such as CDNN/PEPFIT were not suitable for deconvolute the spectra resulting in errors higher than 10%, even with a base spectra of polypeptides [START_REF] Reed | A Set of Constructed Type Spectra for the Practical Estimation of Peptide Secondary Structure from Circular Dichroism[END_REF][START_REF] Poschner | An Automated Application for Deconvolution of Circular Dichroism Spectra of Small Peptides[END_REF]. The problem is probably related to the fact that the base spectra are from homopolymers or binary and trinary copolymers. The intensity and position of the CD bands for a given secondary structure can vary significantly with the side chain. Spectra constructed from an average of the spectra existent in the literature for a number of different model peptides to ameliorate this problem, as performed for CDNN/PEPFIT program, [START_REF] Reed | A Set of Constructed Type Spectra for the Practical Estimation of Peptide Secondary Structure from Circular Dichroism[END_REF] was not sufficient to give consistent results for Aβ spectra.

Dynamic Light Scattering (DLS)

The hydrodynamic radius of the particles was measured by DLS on a Malvern Zetasizer Nano ZS. The samples were filtered with 0.2 µm syringe filter before measurement. All data were averaged from 5 measurements.

Transmission electron microscopy (TEM)

Aβ α-helix, 34% β-sheet and 31% unordered (Table 1) after 6 days incubation time. Similar behavior is observed at 37 ºC, but the conformational transition in test tubes occurs faster when compared to that at room temperature (data not shown).

The measured CD spectrum of Aβ 40 (50 µM) in solution of negatively charged silica particles is shown in Figure 4. It can be seen that the peptide displays the typical spectrum of unordered protein in the presence of silica particles, similar to that observed in the absence of particles.

The conformation of Aβ 40 (50 µM) was additionally characterized in solutions of 0, 2, 4 and 8 g L -1 of complexes, which are polyampholytes of a cationic and anionic monomers (maleamic acid, P 1 or phenylmaleamic acid, P 2 ) complexed with perfluorododecanoic (F) or hydrogenated dodecanoic acid (H) (Figure 2). The particles have a hydrophobic core (formed by the surfactant chains) and a hydrophilic shell (polyampholyte). The hydrodynamic diameters are in the range of 3 to 5 nm (Figure 5 a). The zeta potentials are highly negative for fluorinated complexes, with values of -(47 ± 5) mV when

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R is the hydrogen (P 1 F) and -(48 ± 5) mV when R is the phenyl group (P 2 F) [START_REF] Thunemann | Polyampholyte-Dressed Micelles of Fluorinated and Hydrogenated Dodecanoic Acid[END_REF]. The hydrogenated complexes display zeta potentials of +(25 ± 10) mV (P 1 H) and -(20 ± 4) mV (P 2 H) (Figure 5 b). The concentrations of complexes were chosen to be above their critical aggregation concentration (P 1 , 1 g L - 1 ; P 2 , 10 -2 g L -1 ). The measured CD spectra of Aβ 40 solution with and without complexes are shown in were reminiscent of α-helix structure, particularly at high complex concentration (8 g L -1 ), as observed in Figure 7 (a). In this case the increase in α-helix content was, however, smaller (from 3% to 21%) than the increase observed after addition of 8 g L -1 of complex P 1 F (from 3% to 31%), as shown in Table 2. The unordered fraction decreased to 36%. The titration of Aβ 40 with hydrogenated complexes P 2 H resulted in a decrease of the circular dichroism signal with increasing concentration of the complexes (Figure 7 b), suggesting the occurrence of aggregation. The presence of peptide aggregates is known to reduce the circular dichroism signal intensity due to light scattering and a shadowing effect [START_REF] Duysens | The Flattening of the Absorption Spectrum of Suspensions, as Compared to That of Solutions[END_REF].

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The secondary structure of Aβ 40 in contact with P 1 nanoparticles (8 g L -1 ) was analysed in the presence of salt (0.1 M NaCl) at 37 ºC. Figure 9 shows that P 1 F induces a small change in the CD spectrum of Aβ 40 when salt is present. A higher nanoparticle concentration is needed to have the helical effect observed in the absence of salt. P 1 H nanoparticles induced aggregation and sedimentation and thus CD measurement could not be performed.

Since P 1 F shows higher α-helix inducing effect, Aβ 40 was incubated with P 1 complex (8 g L -1 ) for 6 days at 37 ºC to evaluate its ability to inhibit Aβ aggregation. The incubation was performed at low ionic strength (0.04 M) to accelerate the fibril formation. Aliquots were taken immediately after sample preparation and at 6 days incubation time to be observed by TEM. After sample preparation, TEM

shows already the presence of spherical Aβ aggregates and short fibrils, which may correspond to protofibrils (Figure 10, planes a and c). [START_REF] Walsh | Amyloid Beta-Protein Fibrillogenesis -Detection of a Protofibrillar Intermediate[END_REF] Aβ 40 incubated alone for 6 days at 37 ºC formed amyloidlike fibrils (Figure 10, plane b). However when the peptide was co-incubated from the beginning with fluorinated complexes, long and unbranched fibrils are not any longer visible (Figure 10, plane d). It is not possible to determine unambiguously whether the spherical structures observed are Aβ aggregates or complex aggregates. However, these structures are similar to the ones observed for the complexes incubated alone (Figure 10, panel e and f). In addition spherical Aβ aggregates are more electron lucent than the complexes, indicating that the spherical structure are aggregates of the complexes. The coincubation of Aβ 40 with P 1 H resulted in amyloid fibril formation (Figure 11), demonstrating that these particles are not able of inhibiting amyloid fibril formation.

Discussion

Aβ is detected as a circulating peptide in the plasma and cerebrospinal fluid of healthy individuals [START_REF] Haass | Amyloid Beta-Peptide Is Produced by Cultured-Cells During Normal Metabolism[END_REF][START_REF] Seubert | Isolation and Quantification of Soluble Alzheimers Beta-Peptide from Biological-Fluids[END_REF]. Under physiological conditions, the peptide appears to be unfolded at the beginning of the aggregation. NMR studies have shown that monomers exist predominately as random extended chains, without α-helical or β-sheet structures [START_REF] Kirkitadze | Identification and Characterization of Key Kinetic Intermediates in Amyloid Beta-Protein Fibrillogenesis[END_REF]. However, amyloid deposits isolated from AD brain tissue

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are composed predominantly of Aβ in a β-pleated sheet conformation [START_REF] Kirschner | X-Ray-Diffraction from Intraneuronal Paired Helical Filaments and Extraneuronal Amyloid Fibers in Alzheimer-Disease Indicates Cross-Beta Conformation[END_REF]. Although a causal relationship between peptide deposition and the development of AD is not conclusively proved, it was observed that the fibrillar and the oligomeric forms of the peptide, but not the monomeric form, cause changes in cultured neurons [START_REF] Walsh | Naturally Secreted Oligomers of Amyloid Beta Protein Potently Inhibit Hippocampal Long-Term Potentiation in Vivo[END_REF][START_REF] Pike | Invitro Aging of Beta-Amyloid Protein Causes Peptide Aggregation and Neurotoxicity[END_REF].

This work demonstrates that negatively charged surfaces inhibit the conformational transition of Aβ 40 from unfolded to β-sheet that normally occurs if the peptide is incubated at room temperature or at 37 ºC in test tubes. This might be explained by the fact that negatively charged groups prevent the interaction between peptide molecules since they can interact directly with polar side chains in unfolded peptide, inhibiting conformational transitions. However Aβ 40 does not adsorb irreversibly on negatively charged surfaces, as demonstrated by previous results obtained with neutron reflectivity measurements [START_REF] Rocha | Adsorption of Amyloid Beta-Peptide at Polymer Surfaces: A Neutron Reflectivity Study[END_REF]. This indicates a dynamic interaction where peptide molecules are going to the surface and to the bulk phase. After incubation in test tubes, Aβ 40 undergoes conformational changes. In this case, the peptide is more in contact with air than when incubated in hydrophilic quartz cuvettes (that were hermetically closed) and conformational transitions can not be prevented. The peptide was previously found to adsorb at air-water interface, which acts as a hydrophobic-hydrophilic surface and merely βsheet structure was detected [START_REF] Schladitz | Amyloid-Beta-Sheet Formation at the Air-Water Interface[END_REF].

Negatively charged silica particles do not induce structural changes in the peptide. However when fluorinated complexes are added to Aβ 40 solution an α-helix structure is observed, whereas hydrogenated analogues lead to β-sheet formation and aggregation. Previous studies have shown that hydrophobic teflon particles with a size of 200 nm and a zeta potential of -47 mV are able to induce αhelix structure in Aβ 40 [START_REF] Giacomelli | Conformational Changes of the Amyloid Beta-Peptide (1-40) Adsorbed on Solid Surfaces[END_REF]. Interactions between hydrophobic residues and fluorinated chains must stabilize the peptide intramolecular hydrogen bonds, to the detriment of any intermolecular hydrogen bonding with water. Organofluorine compounds are effective inhibitors of Aβ self-assembly [START_REF] Torok | Organofluorine Inhibitors of Amyloid Fibrillogenesis[END_REF]. The inhibitor activity of these compounds was attributed to the acidic character of a hydroxyl group, that

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increases by introducing the strongly electron withdrawing CF 3 group. Perfluorododecanoic acid is a stronger acid than dodecanoic, suggesting that the acidic character plays as well a key role in α-helix formation. This is support by the observation of a smaller structural change when measurements are performed in the presence of salt (NaCl 0.1 M). Montserret et. al. have shown that while the folding of peptides in sodium dodecy sulphate (SDS) micelles is mostly driven by hydrophobic effects, electrostatic interactions play a significant role in the formation and the stabilization of α-helix structure [START_REF] Montserret | Involvement of Electrostatic Interactions in the Mechanism of Peptide Folding Induced by Sodium Dodecyl Sulfate Binding[END_REF]. Usually, this structure is induced upon dehydrating apolar sites of the peptide and the micelle, directly related to hydrophobic interactions, in line with current thoughts about the formation of local secondary structure [START_REF] Dill | Denatured States of Proteins[END_REF][START_REF] Dyson | Defining Solution Conformations of Small Linear Peptides[END_REF]. However the role of both charged and hydrophobic amino acid side chains is essential in the sequential cooperative process of the SDS-induced mechanism of α-helix formation.

In the absence of electrostatic interactions, the binding of hydrophobic C12 tails is not able to form a stable peptide-detergent complex allowing the formation of a hydrophobic environment required for helix stabilization [START_REF] Montserret | Involvement of Electrostatic Interactions in the Mechanism of Peptide Folding Induced by Sodium Dodecyl Sulfate Binding[END_REF]. The fact that fluorinated complexes show high negative surface charges seems to be determinant to the folding. However, it is not sufficient since silica particles that have a zeta potential in the order of that of fluorinated ones can not induce α-helices.

Hydrogenated complexes are not able of inducing an α-helix structure in Aβ 40 , but rather β-sheets and aggregation. The reason might be that dodecanoic acid is less hydrophobic than perfluorododecanoic.

Taking into consideration that SDS micelles, that are formed by a core of hydrogenated C12 chains, induce α-helix structure in Aβ 40 , [START_REF] Coles | Solution Structure of Amyloid Beta-Peptide(1-40) in a Water-Micelle Environment. Is the Membrane-Spanning Domain Where We Think It Is?[END_REF][START_REF] Shao | Solution Structures of Micelle-Bound Amyloid Beta-(1-40) and Beta-(1-42) Peptides of Alzheimer's Disease[END_REF] the charge contribution has to be important. It has been demonstrated that SDS adsorbed on the surface of activated carbons and carbon blacks gives a zeta potential in the order of -50 mV [START_REF] Gallardo-Moreno | Arrangement of Sds Adsorbed Layer on Carbonaceous Particles by Zeta Potential Determinations[END_REF]. Thus the lower zeta potentials in absolute numbers of hydrogenated complexes contribute to the fact that these particles are not able of preventing the interaction between peptide molecules, resulting in aggregation. The lower efficiency of fluorinated complex with the phenyl group on the polyampholyte (P 2 F) compared with the complex with hydrogen

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(P 1 F) can be understood as a steric effect. The phenyl group sterically hinders the hydrophobic residues on the peptide from interacting with fluorine groups. A C C E P T E D M A N U S C R I P T

  40 /DMSO was added to the control (Ham's F-12) or complexes and Ham's F-12 medium (diluted; final ionic strength 0.04 M) to a peptide final concentration of 100 μM (pH 7.4). The samples were A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT incubated for 6 days at 37 ºC. Aliquots of 5 μl of each sample immediately after preparation and at 6 days incubation time were adsorbed for 2 min to formvar-coated Ni grids. Grids were washed with ultrapure water 3 times and negatively stained with 1% filtered uranyl acetate solution for visualization by TEM (Zeiss microscope operated at 60 kV). Results Aβ 40 (40 µM) was incubated in sealed quartz cuvettes of 1 mm path length previously cleaned by RCA method, for 6 days, at room temperature (20 ± 1 °C) and pH 7.4. CD measurements showed that the peptide is mainly in unordered conformation (minimum at 198 nm) when in contact with hydrophilic quartz surfaces and no structural changes are observed during the incubation period (Figure 3 a). As opposed to that, the CD spectrum of Aβ 40 solution incubated 6 days in microcentrifuge tubes or glass tubes has a broad band at 214 nm and a positive band at 192 nm (Figure 3 b). Secondary structure estimation resulted in a content of 3% α-helix, 25% β-sheet and 59% unordered at initial time and 14%

Figure 6 and 7 .

 7 Figure 6 and 7. In the absence of particles, Aβ 40 displays the typical spectrum (curve 1) of a unordered protein. The secondary structure estimation resulted in a content of 3% α-helix, 27% β-sheet, 14% β-

FluorinatedFigure 1 .

 1 Figure Legend

Figure 2 .Figure 3 .

 23 Figure 2. Complexes of polyampholytes and the sodium salt of dodecanoic acid (X = H) and

Figure 4 .

 4 Figure 4. CD data of Aβ 40 (50 µM) in the absence (1) and presence (2) of silica particles.

Figure 5 .

 5 Figure 5. Particle size distribution of the hydrogenated complex P 2 H as determined by DLS

Figure 6 .

 6 Figure 6. CD data of Aβ 40 (50 µM) in the presence of different concentrations of fluorinated (a) and hydrogenated (b) P 1 complexes: 0 g L -1 (curve 1), 2 g L -1 (curve 2), 4 g L -1 (curve 3) and 8 g L -1 (curve

Figure 7 .

 7 Figure 7. CD data of Aβ 40 (50 µM) in the presence of different concentrations of fluorinated (a) and hydrogenated (b) P 2 complexes: 0 g L -1 (curve 1), 2 g L -1 (curve 2), 4 g L -1 (curve 3) and 8 g L -1 (curve

Figure 8 .

 8 Figure 8. Percentage of secondary structure motifs of Aβ 40 in the presence of different concentrations of fluorinated (a) and hydrogenated (b) P 1 complexes: α-helix (•), β-sheet (■), β-turn (▼) and

Figure 9 .

 9 Figure 9. CD spectra of Aβ 40 (50 µM) in phosphate buffer containing 0.1 M NaCl in the absence (curve 1) and presence of 8 g L -1 of fluorinated P 1 complex.

Figure 10 .

 10 Figure 10. Electron microscopy analysis of the effect of fluorinated complexes on fibril formation. Aβ 40

Figure 11 .FiguresFigure 1 DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV

 111 Figure 11. Electron microscopy analysis of Aβ 40 (100 µM) incubated with hydrogenated complexes (8 g L -1 ) at 37 ºC. The scale bar is 200 nm.

Figure 2 Figure 3 190 5 RFigure 4 190

 2354 Figure 2

Figure 7 190

 7 Figure 7

Table 1

 1 Percentage of secondary structure motifs (±3%) of Aβ 40 incubated in microcentrifuge or glass tubes.

	Time (days) Helix Strand Turn Unordered
	0	3	25	13	59
	6	14	34	21	31

Table 2

 2 Percentage of secondary structure motifs (±3%) of Aβ 40 in the presence of fluorinated complexes P 2 F at different concentrations.

	Conc. (g L -1 ) Helix Strand Turn Unordered
	0	3	27	14	56
	2	4	29	15	52
	4	8	33	21	38
	8	21	24	19	36

Acknowledgment

S. Rocha thanks the FCT for a Fellowship (BPD/25641/2005). These investigations were supported by FCT research project PTDC/BIO/69359/2006, Max Planck Society, Fraunhofer Society and Federal Institute of Material Research and Testing. Isabel Cardoso (IBMC) is gratefully acknowledged for scientific discussions. We thank Heidi Zastrow (MPI) and Rui Fernandes for assistance in DLS and TEM measurements, respectively.


Mariusz Gagoś

Wiesław I Gruszecki
email: wieslaw.gruszecki@umcs.pl

Organization of polyene antibiotic amphotericin B at the argon-water interface

Keywords: polyene antibiotics, amphotericin B, membrane channels, molecular aggregates, excitonic interactions, monomolecular films, linear dichroism

Amphotericin B (AmB) is a life-saving antibiotic, used to treat deep-seated mycotic infections. Both the therapeutic and toxic side effects of AmB are directly dependent on its molecular organization. Organization of AmB was studied in monocomponent monomolecular layers formed at the argon-water interface, by means of polarized and nonpolarized electronic absorption spectroscopy and analyzed in terms of the exciton splitting theory. The results provide direct indication that AmB forms spontaneously dimers that can be assembled into molecular structures characterized by homogeneous orientational distribution in the monolayer, interpreted as cylindrical pores. The structures are not stable at surface pressures higher than 20 mN/m and therefore dimers are concluded as abundant molecular organization forms of AmB in biomembranes. Possibility of stabilization of the cylindrical structures, at higher surface pressures, by other molecules, eg. sterols, is also discussed.

Introduction

Amphotericin B (AmB) is a macrolide poliene antibiotic (Fig. 1) popular in treatment of deepseated mycotic infections [START_REF] Baginski | Interaction of Macrolide Antibiotics with Lipid Membranes[END_REF][START_REF] Hartsel | Amphotericin B: new life for an old drug[END_REF][START_REF] Martinez | An update on the use of antifungal agents[END_REF]. AmB has been in use for almost fifty years and despite its strong side effects is still popular as a life saving antibiotic. It is generally accepted that the biological effect of AmB is based upon interaction to cellular membranes but detailed molecular mechanisms responsible for both pharmacological as well as toxic side effects are still not fully understood. The most popular concept regarding the effect of AmB on biomembranes is directly associated with formation of transmembrane pores that are able to affect severely the physiological ion transport [START_REF] Kruijff | Polyene antibiotic-sterol interaction in membranes of Acholeplasma Laidlawii cells and lecithin liposomes; III Molecular structure of the polyene antibiotic-cholesterol complex[END_REF]. The ability of AmB molecules to form cylindrical structures that can play possibly the role of membrane pores, gained support from the theoretical studies carried out with application of the electrostatic interaction analysis [START_REF] Bonilla-Marin | A microscopic electrostatic model for the amphotericin B channel[END_REF], molecular dynamics approach [START_REF] Baginski | Interaction of amphotericin B and its selected derivatives with membranes: molecular modeling studies[END_REF][START_REF] Baginski | Comparative molecular dynamics simulations of amphotericin B-cholesterol/ergosterol membrane channels[END_REF][START_REF] Baginski | Molecular properties of amphotericin B membrane channel: a molecular dynamics simulation[END_REF] and from the experimental studies on ion permeability [START_REF] Cotero | On the role of sterol in the formation of the amphotericin B channel[END_REF]. The ability of AmB to form ring-shaped molecular aggregates has been demonstrated by the scanning force microscopic analysis of monomolecular layers of the drug [START_REF] Gruszecki | Polyene antibiotic amphotericin B in monomolecular layers: spectrophotometric and scanning force microscopic analysis[END_REF]. On the other hand, the results of recent experiments carried out with application of the 1 H-NMR [START_REF] Gabrielska | Binding of antibiotic amphotericin B to lipid membranes: a 1H NMR study[END_REF] and FTIR [START_REF] Gagos | Binding of antibiotic amphotericin B to lipid membranes: Monomolecular layer technique and linear dichroism-FTIR studies[END_REF] techniques demonstrated that the principal site of localization of AmB with respect to the lipid membrane is the polar headgroup region. The results of the linear dichroism analysis of orientation of AmB molecules in the lipid membranes were interpreted in terms of existence of two pools of AmB, one parallel and one perpendicular with respect to the plane of the lipid bilayer [START_REF] Gruszecki | Dimers of polyene antibiotic amphotericin B detected by means of fluorescence spectroscopy: molecular organization in solution and in lipid membranes[END_REF][START_REF] Gruszecki | Organization of antibiotic amphotericin B in model lipid membranes. A mini review[END_REF]. Interestingly, it was documented that low molar fractions of AmB (below 1 mol% with respect to lipid) increased the barrier for the transmembrane proton transport and the transport was facilitated only at higher concentrations of the drug, that promote formation of aggregated structures [START_REF] Herec | An effect of antibiotic amphotericin B on ion transport across model lipid membranes and tonoplast membranes[END_REF]. Such a finding correlates with the results reported recently, according to which AmB present in the lipid membrane system, at the concentrations higher than 1 mol%, aggregates and is able to penetrate the

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M A N U S C R I P T hydrophobic membrane core [START_REF] Herec | Effect of antibiotic amphotericin B on structural and dynamic properties of lipid membranes formed with egg yolk phosphatidylcholine[END_REF]. Despite those recent reports the question still remains open whether AmB molecules which penetrate the hydrophobic core of the lipid membrane, form cylindrical aggregates (pores) or remain in the form of small molecular aggregates e.g. dimers. Very efficient formation of dimeric structures by AmB has been predicted on the basis of the theoretical analysis of molecular interactions [START_REF] Caillet | Theoretical study of the self-association of amphotericin B[END_REF] and has been demonstrated with application of electronic absorption and fluorescence spectroscopic analyses [START_REF] Gruszecki | Dimers of polyene antibiotic amphotericin B detected by means of fluorescence spectroscopy: molecular organization in solution and in lipid membranes[END_REF][START_REF] Gruszecki | Dimers of polyene antibiotic amphotericin B[END_REF][START_REF] Stoodley | Fluorescence of amphotericin B-deoxycholate (fungizone) monomers and aggregates and the effect of heat-treatment[END_REF][START_REF] Gagos | Anomalously high aggregation level of the polyene antibiotic amphotericin B in acidic medium: Implications for the biological action[END_REF]. The formation of AmB dimers has been also concluded on the basis of monomolecular layer studies [START_REF] Saint-Pierre-Chazalet | Amphotericin B-Sterol Complex formation and competition with egg phosphatidylcholine: A Monolayer Study[END_REF]. In the present work we address the problem of molecular organization of AmB using electronic absorption spectroscopy with non-polarized and polarized light and monomolecular layer technique. Our results show efficient formation of dimers of AmB which, under certain conditions, may be assembled into the symmetrical molecular forms interpreted as cylindrical pores. Such structures of AmB, formed in the environment of biomembranes are able to disturb severely physiological ion transport and are believed to be responsible for pharmaceutical activity of the drug. The analysis of the spectral shifts have been very frequently applied to study molecular organization of AmB [START_REF] Milhaud | Interactions of the drug amphotericin B with phospholipid membranes containing or not ergosterol: new insight into the role of ergosterol[END_REF][START_REF] Rinnert | Structural studies on aqueous and hydroalcoholic solutions of a polyene antibiotic: Amphotericin B[END_REF][START_REF] Barwicz | Spontaneous organization of amphotericin B in aqueous medium[END_REF][START_REF] Gagos | Spectrophotometric analysis of organisation of dipalmitoylphosphatylcholine bilayers containing the polyene antibiotic amphotericin B[END_REF][START_REF] Gaboriau | Physico-chemical properties of the heatinduced 'superaggregates' of amphotericin B[END_REF][START_REF] Hargreaves | Spectroscopic studies of amphotericin B solubilized in nanoscale bilayer membranes[END_REF][START_REF] Hemenger | Structure of Amphotericin B Aggregates Based on Calculations of Optical Spectra[END_REF][START_REF] Mazerski | Effect of the modifications of ionizable groups of amphotericin B on its ability to form complexes with sterols in hydroalcoholic media[END_REF][START_REF] Mazerski | Influence of net charge on the aggregation and solubility behaviour of amphotericin B and its derivatives in aqueous media[END_REF][START_REF] Barwicz | A kinetic study of the oxidation effects of amphotericin B on human low-density lipoproteins[END_REF][START_REF] Bolard | One-sided action of amphotericin B on cholesterol-containing membranes is determined by its self-association in the medium[END_REF][START_REF] Charbonneau | The interactions of amphotericin B with various sterols in relation to its possible use in anticancer therapy[END_REF][START_REF] Tancrede | The effect of surfactants on the aggregation state of amphotericin B[END_REF][START_REF] Blanc | Oligonucleotide delivery by a cationic derivative of the polyene antibiotic amphotericin B. I: interaction oligonucleotide/vector as studied by optical spectroscopy and electron microscopy[END_REF][START_REF] Barwicz | Effects of the aggregation state of amphotericin B on its toxicity to mice[END_REF]. The approach applied in the present work enables to link the spectral changes in the electronic absorption spectra with defined molecular organization patterns.

Materials and methods

Amphotericin B (AmB) in a crystalline form was purchased from Sigma Chem. Co. AmB was dissolved in 40% 2-propanol and then centrifuged for 15 min at 15 000 x g in order to remove micro crystals of the drug still remaining in the sample. Glan-Taylor polarizer was used in the polarization experiments (Fig. 2). Spectral analysis was performed with Grams/AI software from ThermoGalactic (USA). During compression of AmB monolayers, desorption of molecules from the interface into the subphase was not observed before the monolayer collapse, as concluded on the basis of absorbance measurements of the subphase medium. To assure that AmB desorbed into the aqueous phase do not interfere with the spectroscopic measurements of monomolecular layers each experiment was performed with the freshly prepared subphase.

Results and discussion

Fig. 3 presents the isotherm of compression of the monomolecular film formed with AmB at the argon-water interface. The shape of the isotherm represents a two phase process, as can be deduced from two distinct linear portions of the isotherm of compression, corresponding to the specific molecular areas of 148 Å 2 /molecule and 36 Å 2 /molecule [START_REF] Gruszecki | Polyene Antibiotic Amphotericin B in Monomolecular Layers: Spectrophotometric and Scanning Force Microscopic Analysis[END_REF]. These values of the specific area correspond to different orientation of AmB at the argon-water interface, most

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probably the horizontal at low surface pressure values and vertical with AmB anchored in the subphase with mycosamine polar head [START_REF] Gagos | Binding of antibiotic amphotericin B to lipid membranes: Monomolecular layer technique and linear dichroism-FTIR studies[END_REF][START_REF] Gruszecki | Organization of antibiotic amphotericin B in model lipid membranes. A mini review[END_REF][START_REF] Wojtowicz | Effect of amphotericin B on dipalmitoylphosphatidylcholine membranes: calorimetry, ultrasound absorption and monolayer technique studies[END_REF][START_REF] Minones | Orientational Changes of Amphotericin B in Langmuir Monolayers Observed by Brewster Angle Microscopy[END_REF] (see inset to Fig. 3). The specific molecular area of 36 Å 2 /molecule is close to the theoretical cross-section of AmB (approximately 6 Å x 7 Å) [START_REF] Bonilla-Marin | A microscopic electrostatic model for the amphotericin B channel[END_REF] and therefore indicates tight molecular packing of the monolayer at high surface pressures, despite the amphiphilic character of the AmB molecule.

The relatively high pressure of collapse, close to 45 mNm -1 , is an indication that such structures are relatively stable and can be also stable in lipid biomembranes (30-35 mN/m [START_REF] Minones | Orientational Changes of Amphotericin B in Langmuir Monolayers Observed by Brewster Angle Microscopy[END_REF][START_REF] Feng | Interpretation of mechanochemical properties of lipid bilayer vesicles from the equation of state or pressure-area measurement of the monolayer at the air-water or oil-water interface[END_REF][START_REF] Nagle | Theory of lipid monolayer and bilayer phase transitions: Effect of headgroup interactions[END_REF]). Characteristic plateau phase in the molecular area range 100 Å 2 to 40 Å 2 corresponds to the reorientation of AmB molecules at the interface [START_REF] Gruszecki | Polyene antibiotic amphotericin B in monomolecular layers: spectrophotometric and scanning force microscopic analysis[END_REF][START_REF] Gagos | Binding of antibiotic amphotericin B to lipid membranes: Monomolecular layer technique and linear dichroism-FTIR studies[END_REF][START_REF] Wojtowicz | Effect of amphotericin B on dipalmitoylphosphatidylcholine membranes: calorimetry, ultrasound absorption and monolayer technique studies[END_REF]. Fig. 4 presents the electronic absorption spectra of the AmB monolayer recorded during compression, at different surface pressures: 0, 25 and 32 mN/m. AmB molecules deposited at the argon-water interface display typical light absorption spectrum, characteristic of the antibiotic in monomeric form [START_REF] Gruszecki | Dimers of polyene antibiotic amphotericin B detected by means of fluorescence spectroscopy: molecular organization in solution and in lipid membranes[END_REF], with characteristic vibrational bands peaking at 409, 385 and 364 nm (see Tab. I). Gaussian analysis of the absorption spectrum shows that even in the beginning of compression, the molecules of AmB deposited at the argon-water interface, interact one to each other, as can be deduced from the spectral broadening represented by the minor Gaussian components visible in the red edge of the spectrum as well as in the short wavelength spectral region. This effect can be particularly observed at higher surface pressure values. The absorption spectrum of the AmB monolayer compressed to the surface pressure 32 mN/m demonstrates pronounced hypsochromic shift, typical of the card-pack aggregates of AmB [START_REF] Gruszecki | Dimers of polyene antibiotic amphotericin B detected by means of fluorescence spectroscopy: molecular organization in solution and in lipid membranes[END_REF][START_REF] Barwicz | Spontaneous organization of amphotericin B in aqueous medium[END_REF][START_REF] Tancrede | The effect of surfactants on the aggregation state of amphotericin B[END_REF], formed also in the lipid phase [START_REF] Gabrielska | Binding of antibiotic amphotericin B to lipid membranes: a 1H NMR study[END_REF][START_REF] Gruszecki | Dimers of polyene antibiotic amphotericin B detected by means of fluorescence spectroscopy: molecular organization in solution and in lipid membranes[END_REF][START_REF] Gruszecki | Organization of antibiotic amphotericin B in model lipid membranes. A mini review[END_REF]. Both the short-as well as the long-wavelength spectral shifts of the absorption spectrum of AmB can be observed in the absorption spectrum recorded at the surface pressure 25 mN/m. Such a spectral feature, along with a distinct vibrational substructure of the spectrum, suggests that excitonic interactions which give rise to the spectral shifts observed, are associated with formation of small

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molecular aggregates, most probably dimers, with oblique orientation of the dipole moments of the neighboring molecules. The excitonic interactions accompanying formation of molecular aggregates [START_REF] Kasha | The exciton model in molecular spectroscopy[END_REF] are manifested by spectral shifts that can be described, within the framework of the exciton splitting theory, by the following dependency [START_REF] Parkash | Depolarized Resonance Light Scattering by Porphyrin and Chlorophyll a Aggregates[END_REF]:

         1 cos 2 N m mon m     (1)
where  m is the position of the m-th excitation state in the spectrum of the aggregated form,  mon is the position of the electronic transition in the spectrum of monomeric chromophores, N is a number of all exciton states and m is the number of the exciton state considered. In the case of a maximum hypsochromic shift m=1.  is a dipole-dipole coupling matrix element [START_REF] Parkash | Depolarized Resonance Light Scattering by Porphyrin and Chlorophyll a Aggregates[END_REF] and is expressed as:

       2 3 2 2 cos 3 cos 4   R n o mon (2)
In the formula  mon is a dipole transition moment of a monomer,  o is the dielectric permittivity of vacuum, n is the refractive index of the medium and R is the distance between the centers of the transition dipoles of the nearest neighbors in the aggregate. The angle between monomeric transition moment vectors,  , and the angle between the transition moment vector and the axis connecting the centers of the transition moment vectors of neighboring molecules,  , were assumed to be 0 o and 90 o , respectively, in the case of Haggregates of AmB (neighboring molecules are parallel one to each other and perpendicular to the plane of aggregation). A value of 11.3 Debye for the transition dipole of monomeric AmB of the absorption bands corresponding to the monomeric form of AmB (409 nm) and to the dimeric spectral form (353 nm [START_REF] Gruszecki | Dimers of polyene antibiotic amphotericin B detected by means of fluorescence spectroscopy: molecular organization in solution and in lipid membranes[END_REF][START_REF] Gagos | Anomalously high aggregation level of the polyene antibiotic amphotericin B in acidic medium: Implications for the biological action[END_REF]) enables an analysis of formation of molecular dimers in course of the monolayer compression (Fig. 6). The surface pressure dependency of the absorbance ratio A 409/353 is presented in Fig. 6C, as superimposed on the isotherm of compression. Interestingly, the comparison shows that dimerization of AmB, represented as the dimer to monomer ratio, does not follow exactly the compression of the layer, represented

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by the mean area per molecule. In particular, the compression-induced reorientation of molecules, from the horizontal to the vertical position, observed at the surface pressures between 5 and 15 mN/m, is not associated with pronounced change in the monomer to dimer ratio. This means that molecular dimers of AmB are formed spontaneously at 0 and very low surface pressure values and that dimers, like monomers, are reoriented in the course of compression and take part in formation of higher molecular aggregates. A pronounced, surface pressure-induced dimerization of AmB, begins in the monomolecular layer when practically all molecules are oriented vertically and are located relatively close one to each other (mean molecular area below 90 Å 2 ).

The absorption spectra of AmB in the monomolecular layers subjected to the compression were also recorded with polarized light. Fig. 7 presents the surface pressure dependency of the absorbance level at 409 nm (where all the spectral forms absorb, including monomers, dimers and large aggregates) recorded with polarized light, with the electric vector of radiation polarized parallel (A ║ ) and perpendicular (A  ) with respect to the direction of the compression (see Fig. 2B for the geometry of the measurements). The dichroic ratio R can be represented by the average orientation angle (Φ) of the projection of the transition dipole  on the plane of the surface of the monolayer and the compression direction: Interestingly, the first pronounced phase of molecular reorganization of AmB begins directly after the beginning of the monolayer compression, at relatively low surface pressures.

1 cos 1 cos sin 2 2 2            R (3)

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10 Essentially, no average horizontal orientation changes during molecules reorientation at the interface were observed (surface pressures 5-15 mN/m). As can be seen from Fig. 7B, very pronounced phase of ordering of AmB molecules in the monolayer (between 16-19 mN/m) corresponds to the massive dimerization of the molecules, as deduced from the analysis of Fig. 6. Very interestingly, such an organization phase leads to the <cos 2 Φ> value of 0.5, which is diagnostic of totally homogenous horizontal distribution of the AmB transition dipoles and may represent formation of symmetrical cylindrical structures interpreted as molecular pores (Fig. 7B, inset a). The formation of such structures in the monomolecular layers has been observed in the monomolecular layers, by means of scanning force microscopic analysis [START_REF] Gruszecki | Polyene antibiotic amphotericin B in monomolecular layers: spectrophotometric and scanning force microscopic analysis[END_REF].

The structures formed during compression become decomposed ("broken", Fig. 7B, inset b) upon further compression of the monolayer (value of <cos 2 Φ> drops down). The fact that symmetrically-ordered structures of AmB are not stable at the surface pressures above 20 mN/m implies that dimer is the most abundant form of organization of the drug in natural biomembranes and suggests possible role of sterols in stabilization of molecular porous aggregated structures at higher surface pressures [START_REF] Charbonneau | The interactions of amphotericin B with various sterols in relation to its possible use in anticancer therapy[END_REF][START_REF] Kruijff | Polyene antibiotic-sterol interactions in membranes of Acholeplasma laidlawii cells and lecithin liposomes. I. Specificity of the membrane permeability changes induced by the polyene antibiotics[END_REF].

The approach applied in the present work to record absorption spectra of AmB in the monomolecular layers in situ, under the conditions of controlled molecular distance (compression to selected surface pressure values) enabled, for the first time, to analyze the effect of the molecular organization of the drug on shape of the spectrum. The analyses of the spectral shifts have been very frequently applied to study molecular organization of AmB [START_REF] Milhaud | Interactions of the drug amphotericin B with phospholipid membranes containing or not ergosterol: new insight into the role of ergosterol[END_REF][START_REF] Rinnert | Structural studies on aqueous and hydroalcoholic solutions of a polyene antibiotic: Amphotericin B[END_REF][START_REF] Barwicz | Spontaneous organization of amphotericin B in aqueous medium[END_REF][START_REF] Gagos | Spectrophotometric analysis of organisation of dipalmitoylphosphatylcholine bilayers containing the polyene antibiotic amphotericin B[END_REF][START_REF] Gaboriau | Physico-chemical properties of the heatinduced 'superaggregates' of amphotericin B[END_REF][START_REF] Hargreaves | Spectroscopic studies of amphotericin B solubilized in nanoscale bilayer membranes[END_REF][START_REF] Hemenger | Structure of Amphotericin B Aggregates Based on Calculations of Optical Spectra[END_REF][START_REF] Mazerski | Effect of the modifications of ionizable groups of amphotericin B on its ability to form complexes with sterols in hydroalcoholic media[END_REF][START_REF] Mazerski | Influence of net charge on the aggregation and solubility behaviour of amphotericin B and its derivatives in aqueous media[END_REF][START_REF] Barwicz | A kinetic study of the oxidation effects of amphotericin B on human low-density lipoproteins[END_REF][START_REF] Bolard | One-sided action of amphotericin B on cholesterol-containing membranes is determined by its self-association in the medium[END_REF][START_REF] Charbonneau | The interactions of amphotericin B with various sterols in relation to its possible use in anticancer therapy[END_REF][START_REF] Tancrede | The effect of surfactants on the aggregation state of amphotericin B[END_REF][START_REF] Blanc | Oligonucleotide delivery by a cationic derivative of the polyene antibiotic amphotericin B. I: interaction oligonucleotide/vector as studied by optical spectroscopy and electron microscopy[END_REF][START_REF] Barwicz | Effects of the aggregation state of amphotericin B on its toxicity to mice[END_REF] but the present work links directly numerous spectral bands observed with the organization patterns of chromophores (dimers, large aggregates). Such elaboration will be helpful to carry future spectroscopic analyses of molecular organization of antibiotic amphotericin B in model lipid membranes and natural biomembranes and also to reanalyze (vibrational levels 0-0, 0-1, 0-2) and dimeric form, based on the results of the Gaussian deconvolution of the absorption spectra (Fig. 4). Individual energy levels correspond to the spectral bands as assigned in Tab. I. The observed splitting of the energy levels 2 1 and 2 2 enabled determination of the dipole-dipole interaction energies  1 =890 cm -1 and  2 =3741 cm -1 and distances between the centers of chromophores of neighboring molecules R 1 and R 2 as schematically depicted in the inset. Grey circles represent projections of the chromophores of amphotericin B on the plane of the monolayer. Each vibrational energy level of AmB in the monomeric form is shifted toward lower and higher energies by the values corresponding to the dipole-dipole interaction energies  1 and  2 .

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Table I

Parameters of Gaussian deconvolution of absorption spectra recorded from amphotericin B monomolecular layers at different surface pressures (Fig. 4). The assignment of the bands corresponds to the energy level diagram presented in Fig. 5.

Gaussian component

  AmB was further purified by means of HPLC on YMC C-30 coated phase reversed column (length 250 mm, internal diameter 4.6 mm) with 40% 2-propanol in H 2 O as a mobile phase. The final concentration of AmB was A C C E P T E D M A N U S C R I P T calculated from the absorption spectra on the basis of the molar extinction coefficient 1.3 x 10 5 M -1 cm -1 in the 0-0 absorption maximum at 408 nm. Monomolecular layers of the AmB were formed at the argon-water interface. As a first step, AmB solution was deposited at many places of the interface. Monolayers were formed on mQ water. Monomolecular layers were formed in a Teflon trough 40 cm x 4 cm and were compressed along the long side with the speed of 4.8 cm 2 /min. Surface pressure was monitored by NIMA Technology tensiometer, model PS3 (Coventry, England). Monolayer compression, surface pressure measurements and data acquisition were controlled by our own software. Monolayers compression was carried out at 25  1 o C. Detailed analysis of formation of monomolecular layers of AmB was presented by us previously [10, 12, 37]. Light absorption measurements and linear dichroism measurements were carried out with a Diode array UV-Vis spectrophotometer HP 8453.

  on the integration of the absorption spectra. Analysis of the spectral components of AmB monolayer compressed to 25 mN/m (see Tab. I) indicates that the principal absorption bands are symmetrically shifted both towards the lower and the higher energies by two different values of  termed  1 (890 cm -1 ) and  2 (3741 cm -1 ). Despite very low level of absorbance of a single monomolecular layer of AmB and some differences in proportions of intensity of the spectral bands recorded, both the bathochromic and hypsochromic spectral shifts observed in course of film compression were highly reproducible in all repetition of the experiment. The analysis of possible molecular arrangements based on Eq. 2 let us to conclude that in the case of  1 the chromophores (dipole transitions) of neighboring molecules are separated by less than 7.4 Å and in the case of  2 by less than 4.4 Å. It is therefore possible that both interaction energy values correspond to one molecular organization pattern in which the hydrophobic portions of neighboring AmB molecules interact one to each other by van der Waals forces (a short distance between the chromophores, R 2 ) and polar sides of the neighboring AmB molecules are linked by the hydrogen bonding (a long distance between the chromophores, R 1 ). The energy level diagram corresponding to the spectral shifts observed and possible molecular organization of AmB leading to the observed excitonic interaction is presented in Fig.5. The analysis of the absorption spectrum of AmB in the monomolecular layer compressed to 25 mN/m fits exceptionally well to the dimeric organization of the molecules. Comparison of the intensity

Fig. 7B presents

  Fig. 7B presents the surface pressure dependency of the average cos 2 Φ. A change of this

AFigure captions Fig. 1 Fig. 2 (

 captions12 Figure captions

Fig. 3

 3 Fig. 3 Isotherm of compression of amphotericin B monomolecular layer deposited at the

Fig. 4 Fig. 5

 45 Fig. 4 Light absorption spectra of monomolecular layer of amphotericin B deposited at the

Fig. 6

 6 Fig. 6 Surface pressure dependencies of absorbance level of monomolecular layer of

Fig. 7 (

 7 Fig. 7 (A) Surface pressure dependencies of absorbance level of monomolecular layer of

18 Fig. 2 19 Fig. 3 20 Fig. 4 21 Fig. 5 22 Fig. 6 23 Fig. 7

 182193204215226237 Fig. 1 Gagos and Gruszecki

Acknowledgements

This research was financed by the Ministry of Education and Science of Poland from the budget founds for science in the years 2004-2007 within the research project 2P05F04327.

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Comment on: Construction of simplified models to simulate estrogenic disruptions by esters of 4-hydroxybenzoic acid (parabens).

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Fax. 01992 825566 e-mail: dgodfrey@sblack.com I wish to point out several serious failings in the conclusions of this recent paper [START_REF] Guadarrama | Construction of simplified models to simulate estrogenic disruptions by esters of 4-hydroxy benzoic acid (parabens)[END_REF].

Both claims -that methylparaben is the most potent oestrogen mimic and the most active antibacterial compound amongst the parabens -are in direct contravention with all the published evidence to date. Several groups have studied the oestrogenic activities of some of the parabens using the rat uterotrophic assay, including

Routledge [START_REF] Routledge | Some alkyl hydroxybenzoate preservatives (parabens) are oestrogenic[END_REF] and Houssani [START_REF] Hossaini | Lack of oestrogenic effects of food preservatives (parabens) in uterotrophic assays[END_REF] and, in all cases methylparaben was concluded to be orders of magnitude weaker than butylparaben. For example, Routledge determined that methylparaben was 2,500,000 times weaker than oestradiol, whereas butylparaben was only 10,000 times weaker than oestradiol in their in vitro study. It may be concluded, therefore, that methylparaben is 250 times weaker than butylparaben. Indeed, in Routledge's in vivo study [START_REF] Routledge | Some alkyl hydroxybenzoate preservatives (parabens) are oestrogenic[END_REF], methylparaben demonstrated no detectable oestrogenic activity, compared with butylparaben being determined as 100,000 times weaker than oestradiol. From these data it is difficult to accept that the model proposed by the authors of [START_REF] Guadarrama | Construction of simplified models to simulate estrogenic disruptions by esters of 4-hydroxy benzoic acid (parabens)[END_REF] has any semblance of accuracy.

The authors' claim that the results obtained are in agreement with experimental studies of extraction and quantification of parabens in tumours is totally illogical, since the study to which the authors refer, by Darbre et al [START_REF] Darbre | Concentrations of parabens in human breast tumours[END_REF] contains no determination of oestrogenic activity, and only claims to have detected the presence of parabens in the tumours. There is no connection between the presence of parabens and their relative potency as oestrogen mimics as neither the presence, nor the relative concentrations present can be indicative of relative oestrogenic activity. It is difficult, therefore, to comprehend how the authors concluded that methylparaben is more

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oestrogenic than butylparaben simply because it appeared to be present in the tissues tested at a higher concentration. Indeed, the Darbre study fails to prove conclusively the presence of parabens in tumour tissue as the blank samples were also found to contain parabens, and at concentrations statistically indistinguishable from those found in the tumours themselves.

The claim that methylparaben is the most potent antibacterial amongst the parabens is not borne out by the wealth of data available from any manufacturer or supplier of these materials. Table 1 lists the minimum inhibitory concentrations of methylparaben against a range of bacteria and, for comparison, against some fungi.

Table 1. Minimum inhibitory concentrations (mic) of parabens against various microbial species [5]

The data in Table 1 are typical of the values quoted by all parabens manufacturers.

The lower the minimum inhibitory concentration, the more powerful the activity and it is very clear that the order of activity is methylparaben < ethylparaben < propylparaben < butylparaben for all organisms. This is entirely the opposite of what is claimed by the authors for their model and, therefore, it must surely be concluded that this model is not suitable for purpose.

Declaration of interest:

The author is employed by S. Black Ltd., a distributor of raw materials to the personal care industry. The company supplies parabens and most other preservatives commonly used in this industry.

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Comment on: Construction of simplified models to simulate estrogenic disruptions by esters of 4 -

 4 hydroxybenzoic acid (parabens). Patricia Guadarrama, Serguei Fomine, Roberto Salcedo, Ana Martínez. Biophys. Chem. 137 (2008) 1-6. doi:10,1016/j.bpc2008.06.001 Dene Godfrey To cite this version: Dene Godfrey. Comment on: Construction of simplified models to simulate estrogenic disruptions by esters of 4-hydroxybenzoic acid (parabens). Patricia Guadarrama, Serguei Fomine, Roberto Salcedo, Ana Martínez. Biophys. Chem. 137 (2008) 1-6. doi:10,1016/j.bpc2008.06.001. Biophysical Chemistry, 2008, 138 (1-2), pp.63. 10.1016/j.bpc.2008.09.009. hal-00501719

Table 1 .

 1 Minimum inhibitory concentrations (mic) of parabens against various

	microbial species [5]
	Organism	Paraben	mic (%)
	Pseudomonas aeruginosa (bacterium)	methylparaben	0.200
	Pseudomonas aeruginosa	ethylparaben	0.120
	Pseudomonas aeruginosa	propylparaben	0.080
	Pseudomonas aeruginosa	butylparaben	0.015
	Staphylococcus aureus (bacterium)	methylparaben	0.160
	Staphylococcus aureus	ethylparaben	0.120
	Staphylococcus aureus	propylparaben	0.045
	Staphylococcus aureus	butylparaben	0.015
	Candida albicans (fungus)	methylparaben	0.100
	Candida albicans	ethylparaben	0.060
	Candida albicans	propylparaben	0.025
	Candida albicans	butylparaben	0.015
	Aspergillus niger (fungus)	methylparaben	0.085
	Aspergillus niger	ethylparaben	0.045
	Aspergillus niger	propylparaben	0.035
	Aspergillus niger	butylparaben	0.025