Cavity Orientation Regulated by Mixture Composition and Clustering

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Cavity Orientation-Regulated by Mixture Composition and Clustering of Amphiphilic Cyclodextrins in Phospholipid Monolayers John Jairo Pinzón Barrantes, Bruno Maggio, Rita Hoyos de Rossi, and Raquel Viviana Vico J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.7b01247 • Publication Date (Web): 11 Apr 2017 Downloaded from http://pubs.acs.org on April 16, 2017

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The Journal of Physical Chemistry

Cavity Orientation-Regulated by Mixture Composition and Clustering of Amphiphilic Cyclodextrins in Phospholipid Monolayers John J. Pinzón Barrantes,a Bruno Maggio,b Rita H. de Rossia and Raquel V. Vicoa* a

Instituto de Investigaciones en Fisicoquímica de Córdoba (INFIQC-UNC-CONICET), Departamento

de Química Orgánica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba. Haya de la Torre y Medina Allende, Ciudad Universitaria, X5000HUA, Córdoba, Argentina b

Centro de Investigaciones en Química Biológica de Córdoba (CIQUIBIC-UNC-CONICET),

Departamento de Química Biológica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba. Haya de la Torre y Medina Allende, Ciudad Universitaria, X5000HUA, Córdoba, Argentina

AUTHOR EMAIL ADDRESS AND TELEPHONE NUMBER [email protected]

TE: +54-0351-5353867 ext. 53318

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ABSTRACT: Artificial supramolecular-hierarchical structures that emulated nature represent an overcoming alternative for the design of new drug delivery systems. Thermodynamic and topographic properties of films formed by a monoacylated amphiphilic β-cyclodextrin (βCD-C16) with the phospholipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) at the air/water interface were studied. βCD-C16 formed stable mixed films with POPC at several proportions when spread together at the air/water interface. The orientation of βCD-C16 cavity at the interface depends on its mole fraction in the film as reveled by the analysis of partial mean molecular areas as function of composition. Furthermore, βCD-C16 was able to penetrate POPC pre-formed films in a broad range of initial surface pressures, including that near the collapse pressure of the phospholipid. These results demonstrated the strong tendency of βCD-C16 to be inserted into this lipid matrix commonly used in liposome formulations. Topography studies show that βCD-C16 segregate from POPC forming clusters enriched in βCD-C16. Segregation of βCD-C16 was especially noticeable when βCD-C16 were incorporated by themselves into a pre-formed POPC matrix leading to ordered and highly birefringent structures that suggest the formation of hierarchical stacked βCD-C16 arrangement at the interface.


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INTRODUCTION Artificial supramolecular-hierarchical structures that emulated nature represent an overcoming alternative for the development of new functional drug delivery systems that could surmount many disadvantages of the currently available systems with these purposes. The design of these systems demands a deep knowledge about the interactions among molecules or supramolecules, miscibility, segregation, clustering and spatial distribution of the components. Cyclodextrins (CDs) are versatile compounds that currently find numerous applications in the design of novel smart materials with a special interest in the field of nanomedicine1–6. Native cyclodextrins have been employed for years in the pharmaceutical industry for improving the physicochemical properties of bioactive compounds7. However, the need of biocompatible functional systems for the transport, delivery and targeting of drugs still represent a challenge8. Cyclodextrin derivatives offer a wide range of possibilities to fulfill these purposes that seem to be limited only by the chemist imagination. To accomplish these purposes cyclodextrins have been incorporated or grafted onto the surface of nanoparticles9–11, mesoporous materials12,13 and polymers4,14 among others. Another important strategy for the design of CD-based smart drug delivery systems is the use of amphiphilic cyclodextrins15,16. Whatever the design was, the cyclodextrin cavity plays an important and ubiquitous role in reaching the objectives because can be involved in loading the active compound to be delivered, or in forming a complex with a moiety useful for targeting or having another active role4,5,17. Amphiphilic cyclodextrins can be obtained by joining acyl chains or other hydrophobic counterparts to the native cyclodextrin moiety15,18–20; amphiphilic cyclodextrins are able to form hierarchical supramolecular structures such us micelles, liposomes, or monolayers whose properties and structure depend mainly on the geometry and hydrophilic/hydrophobic balance of the cyclodextrin amphiphile17–21. Depending on the synthetic procedure, it is possible to obtain persubstituted or monosubstituted compounds with a wide variety of hydrophobic counterpart20. In general, persubstituted amphiphilic cyclodextrins are molecules with scarce conformational flexibility, and due


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to their molecular geometry, are able to form liposomes where the cyclodextrin cavity is located into the surface and exposed to the aqueous media22,23. On the contrary, monosubstituted cyclodextrins with acyl chains are compounds possessing highly conformational flexibility15,18,20,21 that allows regulating the orientation of the cavity under appropriated conditions, the last property offers the possibility to manipulate the accessibility of cyclodextrin cavity toward a compound, adding to the system an extra regulating point to a suitable external stimulus. Langmuir and Gibbs monolayers of phospholipids and other amphiphiles have been widely employed as models for studying the surface behavior of bioactive amphipathic molecules. The organization of monolayer components in two dimensions makes such systems amenable for comprehensive physicochemical investigation and structural manipulation of surface properties under controlled molecular conditions. In previous work, we studied the properties and structural orientation adopted by monoacylated β-cyclodextrin βCD-C16 (Figure 1) along compression-decompression Langmuir isotherms at the air/water interface18,21. We concluded that the oligosaccharide ring of βCD-C16 is capable of adopting a large number of different orientations, relative to the interfacial plane. This was interpreted by analyzing the surface pressure-mean molecular area isotherms (π-MMA), the elasticity of the films (Cs-1), the surface potential-mean molecular areas isotherms (∆V-MMA), the perpendicular dipole moment per unit area (µ⊥), the surface topography and the intermolecular interaction and orientation at the interface18,21. Our analysis revealed that the cyclic oligosaccharide modifies its position beneath compression from one in which the wider side of the cavity is located almost parallel to the interface to another in which the plane of the wider side of the cavity is perpendicular to the interface. The reorientation of the cyclodextrin moiety is favored because among the oligosaccharides is established an intermolecular net of hydrogen bonds that plays an important role in imparting the dynamic properties of the film. The hydrogen bonding network becomes more important with the increase of the packing


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degree, up to a molecular packing limit, while the surface pressure rise, and it determines the surface properties of the film for future compression-decompression cycles21. The aim of this work was to access the thermodynamic properties and topographic characteristics of films formed by mixtures of the monoacylated amphiphilic β-cyclodextrin βCD-C16 with the phospholipid POPC at the air/water interface. Relevant information was obtained that will allow the design of hierarchical supramolecular systems that could be used as novel drug delivery platforms as well as for targeting to a specific site of action. To these purposes, we studied the Langmuir monolayers obtained by deposition at air/water interface of pre-formed mixtures of βCD-C16 and POPC at different mole fractions. Besides, in order to know the ability of βCD-C16 to absorb to the bare air/water interface Gibbs monolayers were studied, and to evaluate the capability of βCD-C16 to penetrate into a pre-formed phospholipid matrix of POPC penetration experiments were performed. From these results, we conclude that βCD-C16 was able of forming stable mixed films with POPC at several proportions and have a strong tendency to penetrate into POPC model biomembranes giving gripping organized supramolecular structures.

EXPERIMENTAL Amphiphilic β-cyclodextrin βCD-C16 was synthesized and characterized as previously reported18. The average degree of substitution was equal to 1.5 determined by 1H NMR. The phospholipid 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) was purchased to Avanti Polar Lipids, Inc (Alabama, USA). The organic solvents employed for preparing βCD-C16, POPC, or βCDC16/POPC stock solutions were dimethyl sulfoxide-methanol-chloroform (1:1:1) of the highest purity available (HPLC, Merck or J.T Baker). For preparing the βCD-C16/POPC stock solutions adequate volumes of known concentration solutions of pure βCD-C16 and POPC were mixed. The concentration of POPC and βCD-C16 stock solutions were routinely determined by using their Langmuir isotherm. Also, for determining βCD-C16 concentration the anthrone method, that allows quantifying the total ACS Paragon Plus Environment

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amount of carbohydrate, was employed18,24. Water was purified by a Milli-Q system (Millipore, Billerica, MA) to yield a product with a resistivity of ~18.5 MΩ. NaCl used for preparing the subphase was roasted to 400°C for 5 hours. The subphase employed in all experiments consist in a 145 mM NaCl solution. All experiments were performed at 23 ± 2 °C. Compression-decompression Langmuir monolayers were prepared by seeding the stock solutions of βCD-C16, POPC, or βCD-C16/POPC at the air/aqueous (NaCl 145 mM) interface of a KSV minithrough equipment (KSV, Helsinki, Finland) at a compression-decompression rate of 10 mm/min. Typically 10-20 µl of the stock solution was spread on the surface of 273 cm2 trough. The compression started 15 minutes after spreading. The absence of impurities with surface activity in the aqueous subphase or spreading solvents was daily checked. Compression-decompression isotherms are reported as surface pressure (π) vs. mean molecular area (MMA) plots. The π was taken as shown in Equation 1 where γ and γo represent the surface tension of the air/water interface in the presence or in the absence of an amphiphile film, respectively. = −

(1)

The mean molecular area (MMA) was calculated as the total monolayer area (trough area) divided by the number of molecules present at the interface. Surface pressure (π) was measured using the Wilhelmy method with a Pt plate. Triplicate monolayer isotherms were obtained and averaged. Reproducibility was within a maximum standard error of the mean (SEM) of ±1 mN/m for π and ±0.5 Å2 for MMA. The ideal isotherms were calculated accordingly to Equation 2, where A represent the area and X the molar fraction of each compound. = [ β β + ]

(2)

The interfacial elastic modulus of area compressibility (Cs-1), that reflect variations of the film in-plane elasticity, was calculated as Equation 3 from the experimental isotherms, where Cs is the compressibility, π is the surface pressure at the corresponding area A. ACS Paragon Plus Environment

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C = −A

(3)

The in-plane elasticity of the mixtures was compared with the calculated mean elasticity of ideally mixed films, taking into account the Cs-1of the pure components, the mole fraction β and

!"! , and the mean molecular area β and !"! in the mixture at the corresponding π as is shown in Equation 4 25,26 *+

##### C = $ β % & β()*(+, . + & /0/1 -β()*(+,

*+

2 3 β β + !"! !"! 4

-/0/1

(4)

The area in excess was calculated as shown in Equation 5 56 = (859:; − )

(5)

The free energy of compression (∆Gcomp) and expansion (∆Gexpan) was calculated as follow in Equation 6 and 7. -

(6)

-

(7)

∆>6?8@ = − A- C B D

∆> 5@ E = − A- C B D

The area (A) was taken as the MMA of the mixed film and ∆Gcomp or ∆Gexpan was calculated as the area beneath the compression curve between certain limits of surface pressure, here we have taken A0 and Ai as the MMA at 1 and 43 mN/m, respectively. The free energy of hysteresis (∆Ghys) was calculated according Equation 8. ∆>FGH = ∆> 5@ E − ∆>6?8@

(8)

I ) was calculated by using the method of intercepts27,28. The The partial mean molecular area ( plots shown in the supporting information (Figure S2) for surface pressures equal to 5 mN/m and 20 mN/m were fitted to a polynomial function by using the Table Curve 2D v5.01 software and then I for βCD-C16 and POPC as function of composition according to derived in order to obtain the Equations 9 and 10. ̅β = + KL

β()*(+,

M . 31 − β 4

(9)


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̅!"! = − K

-

Lβ()*(+,

M . β

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(10)

When to the aqueous subphase of a Langmuir trough is added a concentrated solution of βCDC16, the adsorption of βCD-C16 molecules from the bulk subphase to the bare air/aqueous interface start off and lead the formation of a Gibbs monolayer. The adsorption experiments were typically done by adding into the aqueous subphase of a circular homemade Teflon trough 400 µL of βCD-C16 stock solution (about 9 mM) under continuous stirring (trough volume: 17 mL, area: 28 cm2). The injection of up to 400 µL of dimethyl sulfoxide-methanol-chloroform (1:1:1) into the subphase (control experiment) did not produce an increase of π. The final concentration of βCD-C16 into the subphase was kept above the critical aggregation concentration (CAC) determined as explained bellow. While the Gibbs monolayer was formed at the interface, the changes in π as a function of time, and constant area were registered. After adsorption of βCD-C16 to the interface, the subphase concentration may diminish by less than 5%. For penetration experiments, previous to the injection of βCD-C16 into the aqueous subphase, a POPC stock solution was spread at the air/water interface until achieving the desired surface pressure (πo) in this way, a phospholipid monolayer composed by pure POPC was formed at the interface. After 10 minutes of stabilization of the POPC monolayer, at the chosen πo, the injection of βCD-C16 into the subphase was performed under continuous stirring (trough volume: 17 mL, area: 28 cm2). For quantification of the βCD-C16 that had penetrated in the POPC film the anthrone method was used18,24. For this, once the βCD-C16 had penetrated into the POPC monolayer (at 5 mN/m or 35 mN/m) and reached a stable surface pressure (πf), the film present at air/aqueous interface was swept to an adjacent trough by a movable barrier29. The film had been swept at about 80-90 minutes from the injection of βCD-C16 into the subfase. The film was transferred to the surface of clean water (17 mL), present in an adjacent trough with the same size than the trough used for the penetration experiment. After the film was transferred, both the water and the film forming molecules were placed in a vial and


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the water content was reduced to 5 ml under vacuum. A volume of 250 µL of this solution was used for βCD-C16 quantification by using the anthrone method18,24. The film swept was performed twice at each surface pressure (5 and 35 mN/m) and from each experiment the concentration of βCD-C16 were measured by duplicate. The calibration curve was done by using a β-cyclodextrin standard solution (between 1-18 µM). POPC (80 mM) was added to the calibration curve and to the blank; the absorbance was determined at 620 nm. The critical aggregation concentration (CAC) of βCD-C16 was determined by using a Fisher Surface Model 21 tensiometer (Fisher Scientific, Iowa, USA). Solutions of βCD-C16 in Milli-Q water and 2% DMSO in the concentration range of µM to mM were prepared and left to stabilize 24 h at 4 ºC previous surface tension measurement at 25.0 ± 0.1 °C. Triplicate determinations of each solution were performed. The CAC value for βCD-C16 is 63 µM (plot shown in supporting information). For topography studies, Brewster angle visualization was performed by using a Brewster angle microscope (BAM) that allows the observation of mesoscale structures in monolayers

30,31

. Under the

Brewster angle of incidence, on a bare interface, a minimum of reflectance is achieved but when a film is present, the film acts as a third optical medium reflecting light. The amount of reflected light depends on the local thickness and refractive index of the film. Langmuir monolayers (compression experiments) of βCD-C16, POPC and of selected molar fractions of βCD-C16:POPC were studied by BAM and prepared as described above, using a KSV Minitrough apparatus (KSV NIMA-Biolin Scientific, Finland). To study Gibbs monolayers of the adsorbed βCD-C16 at the air/aqueous interface and the films resulting of penetration of βCD-C16 into pre-formed monolayers of POPC a circular homemade Teflon through was used (volume: 28 mL); the system was under stirring along the whole experiment and before acquiring the BAM images the stirring was stopped. The Langmuir equipment or the circular Teflon through were mounted on the stage of a Nanofilm EP3 Imaging Ellipsometer (Accurion, Goettingen, Germany), which was employed in the Brewster Angle Microscopy (BAM) mode. Minimum reflection was set with a polarized laser (532 λ) incident on the bare aqueous surface at the ACS Paragon Plus Environment

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Brewster angle (53.1º). During monolayer compression, adsorption or penetration, the reflected light was collected through a 20x objective and an analyzer-polarized lens to a CCD camera.

RESULTS AND DISCUSSION Mixed Langmuir Monolayers of βCD-C16:POPC. We previously studied the Langmuir films formed by βCD-C16 (Figure 1), the organization adopted by this amphiphilic molecule and the intermolecular interactions established along the compression isotherm reveals a rich variety of packing states which involve profound reorganizations of the hydrophobic and hydrophilic moieties of this molecule at the air/water interface depending on the lateral surface pressure18,21. The topography of these films also present features that depended on the packing degree18. On the other hand, the isotherm corresponding to POPC shows a full liquid-expanded (LE) phase along the isotherm at 25 °C with homogeneous topography32,33. To investigate the properties of mixed films formed by βCD-C16 and POPC, Langmuir isotherm obtained from pre-formed mixed solutions containing different mole fractions were studied.

A

B

16

O HO O

OH O

O

OH OH

O O

O

H

6 OH

OH HO

1

O

II O O

Figure 1. (A) Structure of βCD-C16, the acyl chain is linked to the position 6 of one glucose unit of βcyclodextrin. Isomers I and II are present. (B) Schematic representation of βCD-C16.


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When studying mixed monolayers an important question that arises is whether the components are miscible or immiscible in the film. This point can be answered by evaluating different parameters such as the deviation from the ideal behavior, by comparing the experimental mixed isotherm with the ideal one, by inspecting the collapse surface pressure of the mixed films, the surface potential fluctuations over a range of film composition and by inspecting the surface topography27,33. The surface pressure-mean molecular area compression isotherms (π-MMA) and the compression modulus (Cs-1) for different βCD-C16:POPC mixtures are shown in Figure 2 and S1 and compared with the ideal ones; these parameters were calculated as indicated in the experimental section Equations 2-4. It can be observed that for all mixtures positive deviation of the isotherms (area expansion) from the ideal one had occurred. In Figure S2 the areas obtained for the mixed films were compared with the ideal one and Figure S3 shows the calculated excess area per molecule (Aexcess, Equation 5) as function of composition at different surface pressures. The Aexcess when the mole fractions of βCD-C16 (XβCD-C16) were higher than 0.30 do not exceed the 10-12 Å2, but a major positive deviation is observed for the mixture containing less amount of βCD-C16 (XβCD-C16= 0.10). Regarding miscibility in monolayers, two components are considered to be immiscible in the surface film when the properties of the film reflect those of the separated individual components, so the area occupied by the combined film will be the weighted sum of the area of the individual components (Equation 2)33 Also, it should be noted that miscibility-immiscibility can be ambiguous concepts if not clearly focused on the particular scale range of the systems to which it is applied27. At the nm scale (molecular level), intermolecular immiscibility in binary monolayers can be judged by additive variation of the properties as stated above that can be accompanied with or without macroscopic phase separation. In binary systems, lateral immiscibility can be observed as segregated domains of one component from the other on the µm scale; sometimes the domains can be very tiny, containing only a few molecules, and can be difficult to detect them by microscopy methods on the mesoscale. For more complex systems regarding composition or surface topography, the cooperativity at the molecular level (nm) can be


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abolished by the emergence of lateral thermodynamic tensions and interfacial energy. This has the consequence that the mixed film can exhibit additive behavior and smooth compression isotherms but also a richly patterned surface topography revealing immiscible domain coexistence34. The presence of surface heterogeneity on the µm scale range implies local interactions leading to favorable or unfavorable intermolecular mixing of the different components along the lateral plane on the nm scale range27.

Figure 2. Surface pressure vs. MMA (A, C, E) and compression modulus vs. MMA (B, D, F) for films containing βCD-C16:POPC with mole fractions of 0.90:0.10 (A, B), 0.45:0.55 (C, D) and 0.10:0.90 (E, F). Pure βCD-C16: blue; pure POPC: black; ideal mixtures: gray dashed-line; experimental βCDC16:POPC: 0.90:0.10 red, 0.45:0.55 green, 0.10:0.90 pink. NaCl 145 mM aqueous solution was used as subphase (T= 23 ± 2 ºC). ACS Paragon Plus Environment

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The topography of mixed βCD-C16:POPC films was inspected by Brewster angle microscopy (BAM) along the compression isotherm, the images obtained for representatives mixtures at different surface pressures are shown in Figures 3, S4 and S5. These experiments show surface microheterogeneity in the 2D dimension with phase segregation of the amphiphilic β-cyclodextrin and the phospholipid at all surface pressures. Inspection of Figure 3 clearly shows the components immiscibility in the mesoscale range that was evident from the gaseous region (π < 1 mN/m) and kept along the entire isotherm. The appearance of highly birefringent domains upon compression can be attributed to βCD-C16 clusters that were also observed in the films of pure βCD-C1618.

Figure 3. Brewster angle microscopy images of a mixed film containing βCD-C16:POPC in mole fractions 0.10:0.90. The micrographs were obtained during the compression of the film at different surface pressures and are representative of 5-10 images from two independent experiments. NaCl 145 mM aqueous solution was used as subphase (T= 23 ± 2 ºC).

The two-dimensional work involved in bringing together the film-forming molecules from a loosely packed state (π∼ 1 mN/m) to a certain intermolecular packing can be accessed by the free ACS Paragon Plus Environment

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energy of compression (∆Gcomp), see Equation 6 in the experimental section. The ∆Gcomp is a complex quantity that reflects the energy balance between intermolecular interactions and the loss of entropy upon compression of a film. Table 1 shows the free energy of compression for different mixtures of βCD-C16:POPC as well as that of the pure βCD-C16. All mixed films have comparable ∆Gcomp values that are also similar to that found for pure βCD-C16. More noteworthy conclusions arise from the analysis of hysteresis in the mixed films. For ideally fluid films, the energy given to the system during the compression process (∆Gcomp) is equal to the energy returned by the system in the expansion process (∆Gexpan). When these energies are not equal (and ∆Gcomp > ∆Gexpan), it means that a certain amount of energy is stored by the system that is not restored after expansion. This is represented by the free energy of hysteresis (∆Ghys), which can be assigned to the compression-expansion process and is calculated as shown in Equation 8 (Experimental section). Table 1 and Figure S6 show that the films containing higher amounts of βCD-C16 (mole fraction of βCD-C16 above 0.45) show larger ∆Ghys than that with a smaller amount of the amphiphilic β-cyclodextrin moreover, the films with mole fractions of βCD-C16 above 0.45 possess almost the same ∆Ghys than that found in the pure βCD-C16 film. All these results indicate that the amphiphilic cyclodextrins seem to be a major responsible in determining properties of the mixed films such as segregation and clustering of the components, and the presence of hysteresis. It appears that high proportion of βCD-C16 induce a notable hysteretic behavior. This is not surprising considering the relevant intermolecular interactions that can be established among the oligosaccharides units of βCD-C16, mainly given by the hydrogen bonding network, whose magnitude can be modulated by the packing state controlled with compression21. On the other hand, the major deviation from the ideality observed for the mixed film containing a mole fraction of βCD-C16 equal to 0.10 can be attributed to the difficulty of βCD-C16 to establish efficiently the hydrogen bonds in the phospholipid matrix along the surface due to a minor statistical possibility of encounter another


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βCD-C16 molecule. These results reflect the vital roles that possess carbohydrates in life, and how they participate in the mesoscale level in transmitting information on bio-interfaces35,36.

Table 1. Compression Work and Compression-Decompression Energy Gaps for βCD-C16:POPC Mixed Films mole fraction ∆Gcomp ∆Gexpan ∆Ghys (Kcal/mol) (Kcal/mol) (Kcal/mol) βCD-C16:POPC 0.10 : 0.90 1.44 1.35 -0.09 0.45 : 0.55 1.28 0.67 -0.61 0.90 : 0.10 1.26 0.71 -0.55 a 1.0: 0.00 1.34 0.72 -0.62 a Data taken from Ref.18

The divergence of the mean molecular area of a binary film with respect to the ideal mixture in a monolayer can be attributed to modification of the molecular parameters of one or both components. To evaluate if βCD-C16 or POPC contributes mostly to such deviations from the ideal behavior, the I ) with composition was calculated as explained in the variation of the partial mean molecular area ( I at mole experimental section, Equations 9-10. In Table 2 are summarized the values obtained for

fractions of βCD-C16 equal to 0.90, 0.45, and 0.10 at 5 mN/m and 20 mN/m. From the analysis of these data it can be inferred that for both low or high surface pressures, and mole fractions of βCD-C16 equal to 0.90 and 0.45 the area contribution to the mixture of βCD-C16 is close to the area occupied by this pure amphiphile. Contrary, for the lowest mole fraction of βCD-C16 studied, the area contribution of βCD-C16 to the mixture is almost double than that expected for the pure compound for both surface pressures. These findings are in line with the idea that a small amount of the βCD-C16, immersed into the phospholipid matrix, prevent to establish the hydrogen bonding network between the oligosaccharides units of the amphiphile which is also reflected by the smaller ∆Ghys observed for this mixture upon a compression-decompression cycle. This has an important consequence because by controlling the amount of βCD-C16 it is possible to regulate the orientation of the cavity respect to the interfacial plane. At low mole fraction of βCD-C16 it seems that the oligosaccharide lies with the cavity more parallel to the interfacial plane, exposing a major area, situation that could be favorable to allow ACS Paragon Plus Environment

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the formation of inclusion complexes with a suitable guest, Figure 4A. Conversely, at higher mole fractions of βCD-C16 most of the oligosaccharide units are involved in forming the hydrogen bonding network, and as consequence the cavity is orientated more perpendicular to the interfacial plane21, with less chances to be accessible to a guest compound, Figure 4B. Similar observations regarding phase separation of amphiphilic cyclodextrins anchored to cholesterol or esterified by two acyl chains in phospholipid bilayers were reported by Djedaíni-Pilard & col. In that work, it was also proposed that the amphiphilic cyclodextrins organized in a 2D network are stabilized by intermolecular hydrogen bonds between the oligosaccharide at the bilayer surface15,20.

I ) as Function of Composition Table 2. Partial Mean Molecular Area (P for βCD-C16:POPC Mixed Films I βCD-C16 I POPC A ideala P P XβCD-C16 2 2 2 (Å /molecule) (Å /molecule) (Å /molecule) 5 mN/m 1.0 51.8b 0.90 56.5 48.2 130.7 0.45 78.4 53.5 98.8 0.10 93.9 94.3 93.4 0 98.7c 20 mN/m 1.0 32.7d 0.90 36.8 29.5 102.8 0.45 55.8 32.0 75.4 0.10 69.3 74.1 68.7 0 73.3e a

Obtained from the ideal isotherm at the indicated mole fraction and surface pressure Correspond to the mean molecular area of pure βCD-C16 at 5 mN/m c Correspond to the mean molecular area of pure POPC at 5 mN/m d Correspond to the mean molecular area of pure βCD-C16 at 20 mN/m e Correspond to the mean molecular area of pure POPC at 20 mN/m b


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βCD-C16

POPC

A

B

Figure 4. Proposed molecular orientation adopted by βCD-C16 relative to the interfacial plane at low (A) and high (B) mole fraction in the mixed films with POPC.

Adsorption of βCD-C16 to the Bare Interface and Penetration into POPC Monolayers. When a concentrated solution of βCD-C16 was dissolved into the bulk of a NaCl 145 mM solution, the adsorption of the amphiphiles into the bare air/aqueous interface resulted in the formation of Gibbs monolayer that is evidenced by an increase of π (or decrease in the surface tension). A Gibbs monolayer arises from the equilibrium between the amphiphile in the bulk solution and the monolayer formed at the interface37,38. Additionally, the βCD-C16 aggregates would provide a reservoir for replacement of the monomers integrated to the surface and thus keep the monomer concentration in the subphase nearly constant. The critical aggregation concentration (CAC) of βCD-C16 was determined as 63 µM, Figure S7. When βCD-C16 is present in the bulk subphase, in a concentration well above the CAC, the surface pressure increases with time, and reached an equilibrium value (πeq) equal to 33 ± 1 mN/m. By assuming a similar lateral organization respect to that adopted in the Langmuir monolayers, we estimated that the mean area occupied by a βCD-C16 molecule in the Gibbs monolayer at the πeq was about 22 Å2/molecule which implies that the amphiphilic β-cyclodextrin has the plane of the cavity perpendicular to the interface as proposed before18.


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The surface topography of the adsorbed βCD-C16 into the bare air/NaCl 145 mM aqueous solution interface was inspected by BAM, Figure 5. The topography acquired by self-incorporation of βCD-C16 at the bare interface shows highly refringent ordered structures with preferential anisotropic grown along a line. The adsorption process of βCD-C16 led to topographical features that considerably differs from that observed from their compression in Langmuir isotherm.

Figure 5. Brewster angle microscopy visualization of adsorbed βCD-C16 at the bare air/NaCl 145 mM interface (T= 23±2 ºC). Panel A: NaCl 145 mM, panels B-G: adsorbed βCD-C16 at different times, images C-G correspond to βCD-C16 at πeq.

For studying the βCD-C16 penetration into POPC monolayers, phospholipid monolayers initially packet at different surface pressures (πo) were pre-formed at the air/aqueous interface. Then a βCD-C16 stock solution was injected into the NaCl 145 mM subphase reaching a concentration above the CAC. The changes of π as function of time were recorded (∆π=πf-πo) until the π value was invariant (πf). Any increase in the π of the films reflects the penetration of βCD-C16 amphiphile into the POPC monolayer. As shown in Figure 6 A, the increase in surface pressure (∆π) observed after βCD-C16 addition depends on the initial surface pressure of POPC and in all cases the πf reached was higher than that achieved for βCD-C16 when adsorbed to the bare interface (33 ± 1 mN/m). The cut off plot shown in Figure 6 B allow determining the maximum π at which βCD-C16 would be able to penetrate (cut off point) the POPC monolayer, this value ranged the 46-47 mN/m. It was proposed that the average surface pressure of bilayers in biomembranes fall between 30-35 mN/m39. It is worth noting that the cut off value for βCD-C16 into POPC monolayers are far higher than the average surface pressure assigned to


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bilayers membranes. These results suggest that βCD-C16 would have a great ability to penetrate POPC bilayers and other more densely packed assemblies having this biological, pharmaceutical and medical relevance. The slope of ∆π vs. πoPOPC plot accounts for the type of interactions between βCD-C16 and POPC40. Regressions lines with a slope equal to -1 would represent an ideal situation with no interaction among the components. A steeper slope with absolute values larger than 1 indicates repulsive interactions while a slope with absolute vale lower than 1 attractive interactions40. The slope value for the system βCDC16 / POPC is -0.83±0.06 accounting thus for attractive interactions among the components.

40

50

A

B

35

40

30

30

∆π (mN/m)

Surface pressure (mN/m)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


βCD-C16 πo POPC: 5 mN/m πo POPC: 10 mN/m πo POPC: 15 mN/m

20 10

25 20 15

πo POPC: 20 mN/m πo POPC: 30 mN/m

10

πo POPC: 35 mN/m πo POPC: 40 mN/m

5

Cut off point

0

0 0

20

40

60

Time (min)

80

0

10

20

30

40

50

πo POPC (mN/m)

Figure 6. A) Time curves for the adsorption of βCD-C16 to the bare air/NaCl 145 mM interface (black) and penetration of βCD-C16 into POPC monolayers initially packed at different surface pressure πoPOPC: 5 mN/m (red), 10 mN/m (green), 15 mN/m (yellow), 20 mN/m (blue), 30 mN/m (magenta), 35 mN/m (dark red), 40 mN/m (violet). Representative experiments are shown that vary less than 2 mN/m from duplicated. B) Cut off curve for the penetration of βCD-C16 into pre-formed POPC films at different initial surface pressures. The final subphase concentration of βCD-C16 was above the CAC in all cases.


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The topography of the resulting mixed films due to the penetration of βCD-C16 into POPC monolayer at πo equal to 5 and 35 mN/m were accessed by BAM and the results are shown in Figure 7. The surface texture resulting from the penetration of βCD-C16 into the POPC monolayers closely resembles that acquired for βCD-C16 when absorbed to the bare interface, see Figure 5. Nevertheless, the wire-like structures formed by the penetration of βCD-C16 into the phospholipid film are more refringent than that observed for the adsorption of βCD-C16 to the bare interface, especially when POPC was packed at 35 mN/m. Note that images E-F in Figure 7 II were acquired with lower gain in order to avoid image bleaching. In BAM images, the relative reflectance is proportional to the relative optical thickness of the film region observed. The high reflectance observed lead to suppose that multistaked cyclodextrin structures are formed at the interface region. In order to estimate the amount of amphiphilic β-cyclodextrin that had penetrated into the POPC film, once reached πf, βCD-C16 was quantified after being transferred into a clean water subphase (for details see Experimental section). The amount of βCD-C16 present in the transferred film, coming from the POPC monolayer at 5 mN/m and 35 mN/m, exceed by 6 and 9 times the amount of β-cyclodextrin that could be present if we considered that all the surface was covered only by βCD-C16 with an orientation relative to the interface such that the amphiphilic β-cyclodextrin occupied 22 Å2/molecule (minimum area that can be occupied by a βCD-C16 before the collapse in a Langmuir monolayer of βCD-C16). This amount of βCD-C16 together with the BAM images support the idea that multi-staked structures of βCD-C16 are formed probably by insertion of the hydrocarbon chain into the cavity of another βCD-C16 that was previously inserted. These multi-staked structures become more important especially when POPC is highly packed; under this condition it is most likely that βCD-C16 cannot be inserted so deeply into the POPC film remaining the cyclodextrin cavity more exposed or dislocated from the interfacial plane favoring then the self-inclusion of other amphiphilic cyclodextrin and leading to structures with a larger thickness than that observed for a single βCD-C16 molecule. Once more, the topographical features observed in Figure 7 are dissimilar from that observed in the compressed mixed films formed by the co-spreading of βCD-C16:POPC as shown Figures 3, S4 ACS Paragon Plus Environment

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and S5. Nevertheless, both the mixed Langmuir monolayers and the penetrated βCD-C16:POPC films showed self-segregation and clustering of βCD-C16 in the phospholipid matrix. The major difference resides in the degree of order reached associated to the self-insertion of βCD-C16 into the POPC film. In order to check if the compression rate used for obtaining the films by co-spreading of βCDC16:POPC modified the topography, the rate of compression was lowered 5 times but no differences were observed on the surface topography, slower rate compression cannot be achieved with our experimental set up. Also, the compressed monolayer was left to evolve on time (about 1 hour) upon compression until 30 mN/m but the wire-like ordered structures were not formed.

I)

πoPOPC: 5 mN/m

II)

πoPOPC: 35 mN/m

Figure 7. BAM images obtained for the mixed films formed after penetration of βCD-C16 into preformed POPC monolayers at the air/NaCl 145 mM interface as function of time, images D-G were acquired at a surface pressure equal to πf. I) πoPOPC: 5 mN/m, II) πoPOPC: 35 mN/m. Panels A shown the surface observed for NaCl 145 mM and panels B for POPC at 5 mN/m (I) and 35 mN/m (II). All images were obtained using a microscope gain of 100% except images E-G in II that were collected using a gain of 40% in order to avoid high reflectance.


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CONCLUSIONS The Langmuir films of βCD-C16:POPC with different composition showed immiscibility and area expansion respect to the ideal behavior. The excess of the area is noticeably greater when the mole fraction of βCD-C16 is the smallest, moreover little hysteretic behavior was present in this mixture. The analysis of the partial mean molecular area as function of composition for βCD-C16:POPC mixed films revealed that at high or low surface pressures, and high mole fractions of βCD-C16 (XβCD-C16 > 0.30) the area contribution to the mixture of βCD-C16 is comparable to the area occupied by the pure amphiphile. Contrary, when the mole fraction of βCD-C16 is small (XβCD-C16 = 0.10) the area contribution to the mixture of βCD-C16 almost double that expected for the pure compound. These results suggest that a small amount of the βCD-C16, immersed into the phospholipid matrix, is prevented to form efficiently the hydrogen bonding network between the oligosaccharides units of the amphiphile which is also reflected by the smaller ∆Ghys observed for this mixture upon a compression-decompression cycle. This has an important consequence because by controlling the amount of βCD-C16 it is possible to regulate the orientation of the cavity respect to the interfacial plane. Our studies also revealed that βCD-C16 have a strong tendency to self-organize in ordered assemblies when adsorbed to a bare interface or to a lipid matrix such POPC. In the last case βCD-C16 is able to form domains or clusters enriched on it probably guided by successive intermolecular interactions mainly present in the oligosaccharide units; the brighter wire-like regions observed by BAM on the surface could be attributed to being composed by βCD-C16 and probably these structures are out of the plane (dislocated from the interfacial plane) and formed by successive stacking of amphiphilic cyclodextrin with some volume phase based on the high reflectance observed. All these events can be favored by the formation of hydrogen bonding and complexation of the acyl chain from one βCD-C16 into the cavity of another βCD-C16 as was previously observed in micelles of a similar amphiphilic cyclodextrin with shorter chain41.


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SUPPORTING INFORMATION βCD-C16:POPC Langmuir isotherms, excess area plot, BAM images, βCD-C16:POPC compressiondecompression isotherms, βCD-C16 critical aggregation concentration. This material is available free of charge.

ACKNOWLEDGMENT This work was supported by SECyT-UNC, FONCYT PICT 2013-1175, Argentina. J.P.B. is a doctoral fellow from SECyT-UNC. B.M. and R.H.de R, and R.V.V. are Career Investigators of CONICET.

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