DOI: 10.1039/b000000x. We report the production of a robust hybrid polyene/artificial solid-state membrane. This membrane is based on the confinement of ...
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Structure and ionic selectivity of hybrid polyene/artificial polymer solid state membrane Daniela Thiele,a Sebastian Kraszewski,b Sébastien Balme,*a,c Fabien Picaud,b Jean-Marc Janota and Philippe Déjardina 5
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Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX DOI: 10.1039/b000000x We report the production of a robust hybrid polyene/artificial solid-state membrane. This membrane is based on the confinement of Amphotericin B inside the cylindrical nanopores of a polymeric membrane. The experiments supported by molecular dynamic simulations reveal new filtration properties never seen in biological membranes. Indeed this biomimetic membrane exhibits anionic permeation better than for both monovalent and divalent cations one. This work opens a new route of separation in the domain of nanobiofiltration, especially for tunable nanodevices based on differential ion conduction, using a fundamental understanding of the confinement mechanism.
Introduction 15
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Polymer solid-state membrane technologies are commonly used in many industrial, health or environment applications. Since many years an important research topic focuses on the development of membrane which exhibits both high ion permeability and selectivity without energy intake. Nevertheless, these features are often considered as antagonist. For example, the specific separation of different monovalent ions is commonly performed by electro-dialysis methods which require electric energy.1 With the recent advances in nanoporous materials and biological technology, the development of biomimetic systems is emerging at the nanoscale.2-4 These systems mimic biological formations at molecular level to obtain functionalized solid state membrane, exhibiting the same ionic selectivity properties than the biological one. Today, different approaches are developed to obtain such biomimetic membranes. The first one consists in the construction of synthetic pore (or channel) at nanoscale using polymers brushed onto solid state nanopore,5-7 or asymmetric shape of single pore track-etched membrane.8,9 The second approach is based on direct insertion of biological transmembrane ionic channels into nanopore leading to hybrid biological/artificial membrane. Recently the feasibility of this method has been demonstrated by the confinement of gramicidine A into polycarbonate track-etched membrane.10 This novel way presents the strong advantage of combining both biological selectivity and solid-state material properties, such as robustness, and sustainability. However, their realization is still based on two major challenges: (i) the production of well-characterized solidstate nanopore and (ii) the understanding of the confinement mechanisms of ionic channel.2 Except the specific case of gramicidin A, where the ionic channel is formed inside a β-helix in most cases, all ionic channels are This journal is © The Royal Society of Chemistry [year]
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formed by the self-assembling protein subunits. Among them, the polyene antibiotics, being a large class of antifungals produced by bacteria, have the ability to increase the ion permeability of lipid membranes. In this work, a specific polyene, Amphotericin B (noted AmB), has been chosen.11 In general overview, this molecule is composed by a polar head, a hydrophilic polyhydroxyl chain and a hydrophobic chain (Fig. 1a). In biological membrane AmB forms a barrel throughout selfassembly of 8 molecules, where hydrophobic chains form the external part of the barrel (permitting its intercalation inside lipid membrane) and the hydrophilic chains form the inner channel permeable to ions.12 The properties of AmB channels have been extensively studied and we only attract the attention of the reader to the fact that so formed channel is only permeable to monovalent ions (cations and anions).13
Fig. 1 (a) Ball and sticks molecular representation of Amphotericin B in stretched conformation found after optimization and relaxation in the solvent. White, cyan, blue and red balls corresponds to hydrogen, carbon, nitrogen and oxygen atoms, respectively. (b) MEB imaging of MbPC with 770x1020 nm² field of view, 250 nm scale bar.
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In this study we investigate the feasibility of hybrid polyene/artificial polymer solid state membrane based on the AmB confinement inside track-etched polycarbonate membrane and its relationship with ionic transport properties. First the experimental characterization of confined AmB into nanopore is described and confirmed by molecular dynamic (MD) simulations. Then, ionic transport of Na+, K+, Ca2+ and Cl- and selectivity of hybrid membrane is studied experimentally and supported by simulations, with comparation to biological system. Finally, we conclude about hybrid AmB/artificial membrane properties and their potentialities.
Materials and methods
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Ion selectivity measurement 60
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Polyene intercalation
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Amphotericin B (Sigma A4888) has been confined inside pore of polycarbonate track-etched membrane (GE water and process technologies ref. KNISH02500, and noted MbPC). Firstly, polycarbonate membranes have been treated by ethanol percolation (15 ml). Secondly, AmB confinement has been performed by percolation of 15 ml of polyene solution (7 µM AmB and 0.7 µM cholesterol) under 8 µL.min-1 flow rate. Thus obtained membrane is referred as MbPCAmB.
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The recordings of intensity/voltage (I/V) curve and voltage at I = 0 A (E0) have been performed on potentiostat EG&E princeton applied research model 236A. These measurements have been performed with two Ag/AgCl electrodes (Tacussel), which have been prepared to have a dissymmetry potential less than 1 mV. The measurement cell composed of two compartments (“in” and “out”) has been separated by a membrane surface area of 38.5 mm2. Membrane conductivity Membrane
conductance
Gmem
has
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been
obtained
by
where E0 is the measured voltage, Eelect is the voltage caused by the difference of salt concentration between both compartments of the measurement cell, and E inv is the voltage of current inversion. The relative contribution of ions to the conductance is given by the ratio of their permeability coefficient P ion. To obtain these relative permeations, data analyses have been performed using generalizations of Goldman-Hodgin-Katz (Eq. 2).16
Einv
RT S1 T1 ln F
S1 T1 2 16T1S2 T2 S1 4T2 S2 2S1 4T2
(2)
S1 i Pi aoi i Pi aii
(3)
T1 i Pi aii i Pi aoi
(4)
S 2 i P i aoi i P i aii
(5)
T2 i Pi aii i Pi aoi
(6)
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where i±± (respectively i±) represents divalent (respectively monovalent) cation, Pi is the permeation of cation and anion, i i and ai and ao are the ion activities inside the “in” and the “out” compartments, respectively. The measurement of relative permeation between cation and chloride ( Pcat PCl ) is determined from Einv when the membrane is placed between two compartments filled with a given electrolyte of the variable ion concentration in the “in” compartment [ion]i (between 6 mM to 720 mM) and of the constant salt concentration in the “out” compartment [salt]o (0.6 mM), respectively. The relative permeation PK PNa has been obtained for symmetrical concentrations of solutions in both compartments, where compartment “in” is filled with KCl solution and compartment “out” with NaCl solution (concentrations between 6 mM to 720 mM).
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Current and voltage measurement
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(1)
where R is the gaz constant, T is the temperature, F is the Faraday constant, and S1, T1, S2 and T2 are given as following equations:
Optical characterization UV-Vis absorbance spectra have been performed on Jasco V-570. Membranes have been analyzed with integrating sphere INS-470. All spectra have been recorded for a wavelength between 300 to 450 nm with 1 nm step. Confined polyenes and membrane infrared fourier transform (IRTF) spectrometry analyzes have been recorded on IRTF-Nexus with ATR mode. All samples have been recorded for wavenumber ranged between 600 cm-1 to 4500 cm-1 with 128 scans. The fluorescence spectra of the membrane with the labeled AmB have been obtained by means of a high resolution fluorescence confocal spectrometer whose the setup is described elsewhere.14,15 It allowed us to measure the fluorescence through the thickness of the membrane with steps of 0.1 μm. Observed area is about 2.83×10−14 m2, which corresponds statistically to 2 nanopores according to the density of pores.
Ion selectivity has been obtained by the voltage (E0) collected at I = 0 A. These measurements have been performed with the same cell, where compartments have been filled with same salt solution at different concentrations or different salt solutions at same concentration. In case of dissymmetric concentration between both compartments the AgCl electrode contribution has been subtracted according to the Eq. (1). E E E 0 elect inv
Fluorescent polyene labeling The experimental protocol of labeling consists in the addition of Amphotericin B in methanol solution (6 µM) to Alexa-fluor 594 succinimidyl-ester (purchased from Invitrogen®) with a ratio of 3:1. The mixture was stirred for one hour.
current/voltage measurements, since it is directly given by the slope of I/V curve. The both compartments of the measurement cell has been filled with 100 ml of saline solution at the same concentration. The current has been recorded for a voltage ramp between -1 V and 1 V with a step of 50 mV.s-1.
Molecular dynamics simulations The model for Amphotericin B was built using the 3D structure of DB00681 model in drug bank database. For the missing This journal is © The Royal Society of Chemistry [year]
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potential parameters of the AmB, we have followed the general procedure described by Norrby and Brandt17 basing on construction of the Hessian matrix (the second derivatives matrix of the energy with respect to geometry) for further use in the force field parameterization. This matrix has been obtained via ab-initio quantum calculations using Gaussian 09 package software.18 Due to a huge number of atoms in AmB molecule, several bases were used to gradually optimize the geometry of the molecule with the DFT approach using B3LYP hybrid functional. The latest quantum calculations were performed using 6-31++G medium-sized basis set. The Mulliken partial charges were applied to the molecular model. The cholesterol parameters were taken from Ref. [19]. The water molecules were treated using TIP3P model,20 and all-atom AMBER force field (param94)21 was used for ions and carbon atoms of the rigid nanotube. Before a complete building of the system, each polyene was first equilibrated during 1 ns in a water box of 64 nm3 in order to check its stability. Then, polyene molecules were transferred to the solution containing: the nanopore modelled by a carbon nanotube (diameter of 7.5 nm and denoted NT) that presents hydrophobic properties, the cholesterol molecules, 12266 water molecules and 0.1 M dissociated KCl – uniformly distributed 23 chloride and potassium counter ions ensuring the neutrality of the system. The complete system analyzed hereafter contains thus 48 Amphotericin B and 48 cholesterol molecules in a simulation box of 936 nm3 (for a total of 51860 atoms). All MD simulations were carried out in the NPT ensemble using the NAMD 2.7b2 suite of program.22 Langevin dynamics and Langevin piston methods were applied to keep constant temperature (300 K) and stable pressure (1 atm) of the system, respectively. Long-range electrostatic forces were taken into account using the particle-mesh Ewald approach,23 and the integration timestep was equal to 2 fs. For a statistical overview of the problem, 4 initial configurations were used to build theoretically the MbPCAmB. All these runs lead to the total of 1.62 s simulation time, but we focalize here on results of only one, the most representative configuration. We have also performed an additional 50 ns run with applied a low external field corresponding to 300 mV. The equilibrium polyene structures were used to study the small voltage influence on the ionic current, as also performed during the experimental measurements. 60
Results and discussion Polyene confinement 45
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Experimental analysis AmB in water solution (Fig. 1a) has been confined inside the commercial polycarbonate membrane (MbPC) of 5 µm thickness. The pore size distribution and polydispersity of Mb PC have been obtained by MEB analysis. The pictures of both membrane sides reveal a mean pore diameter of 24±6 nm and a pore density 9×108 pores.cm-2 (one side shown in Fig. 1b). The analysis of confined AmB inside the membrane pore (noted as MbPCAmB) by confocal fluorescence spectroscopy allows obtaining information about their spatial localization inside the nanopores.10,15 Nevertheless, these investigations need to be carried out with polyene labeled by a fluorescent label. The fluorescence signal measured at different spots of the membrane This journal is © The Royal Society of Chemistry [year]
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Fig. 2 Membrane characterization: (a) confocal fluorescence spectra of MbPCAmB (fluorescence intensity - red line, scatter light diffusion - black line). (b) IRTF spectra of MbPC and MbPCAmB, (c) UV-Vis absorbance of AmB in water solution at low concentration (dashed line) and the differential absorbance of AmB in MbPC (solid line) presented as MbPCAmB absorbance after raw membrane spectrum substraction.
surface reveals that the protein concentration is homogeneous throughout the sample. Confocal fluorescence spectra (Fig. 2a) of MbPCAmB show a superposition of scatter light diffusion peak and fluorescence emission. According to previous studies,6 these results confirm that AmB molecules are located inside the membrane pore without any adsorption on the membrane surface. AmB confinement inside the nanopore has also been characterized by IRTF (Fig. 2b) spectrometry and UV-Vis absorbance (Fig. 2c) without use of the supplementary fluorescence labeling. IRTF spectra of MbPCAmB exhibit two absorption bands at 2963 cm-1 and 2930 cm-1. The first one originates from polycarbonate membrane, and the second one is Journal Name, [year], [vol], 00–00 | 3
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Fig. 3 Subsequent confinement of the AmB molecules inside the hydrophobic nanopore. At t = 338ns, the hole at the center of the pore begins stable. Red arrow indicates the total dipolar moment coming from all the polyene molecules. Circular nanopore is indicated as cyan balls, and each AmB molecule is represented using different colors. All atoms of given AmB molecule are shown as balls. 5
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the most likely due to the presence of AmB inside membrane, since this strong absorption band can be attributed to C-N stretching vibration in the NH3+ groups of the AmB.24 Different photophysical studies have shown that the AmB structure modifies the absorbance properties. The two absorbance peaks in the UV-Vis spectra (λ=390 nm and λ=416 nm) can be clearly attributed to AmB, even if slightly shifted compared to those obtained in methanol solution (λ=383 nm and λ=406 nm, respectively). Moreover, the same peak shift, than observed here in polycarbonate membrane, is induced by the self-assembly of AmB in aqueous media at high concentration, and in lipid membrane.25-27 This result clearly suggests an organization of AmB inside the membrane pore. MD simulations Experimental characterizations reveal that polyenes are confined inside nanopore exhibiting some structural arrangement. Nevertheless, these experiments cannot give information about polyene stability and interaction within the nanopore. In order to answer to these questions, MD simulations have been conducted according to experimental setup. AmB insertion inside the hydrophobic nanopore (NT), mimicking the experimental membrane, has been performed. We started the MD simulations with 48 AmB molecules in front of the NT entrance and in 0.1 M KCl solution. Following the experimental conditions, we added also cholesterol in the same amount as AmB molecules, in form of cholesterol-polyene couples. However, their presence being necessary to reproduce the selfassembly mechanism of AmB channels, decreases the AmB hydrophobicity. As a consequence, the AmB insertion time into the NT is considerably extended. The dynamics of the polyene and the solvent necessary to disorganize the cholesterol-polyene couples takes about 80 ns. Then, the AmB molecules penetrate spontaneaously the NT. A total of 310 ns run has been necessary to observe the complete insertion of AmB. During their diffusion
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inside the NT, their root mean square deviation (RMSD) varied around 3.8ű1.0 Å (average value for each of 48 AmB). This confirms the geometric stability of the AmB skeletons. Only translational displacements are thus in play affirming the supposed self-assembly mechanism during the AmB confinement inside the nanopore. We would attract the reader attention to the fact that several MD simulations were performed using different initial configurations of cholesterol/polyene couples. At the light of these simulations, conclusions on the insertion time, RMSD value and self-assembly process can be generalized. After their complete incorporation, we have followed the dynamics of the polyene inside the NT. Fig. 3 presents different snapshots of successive molecular arrangements during the simulation until the formation of a large canal in the nanopore. From these snapshots, it is rather clear that AmB try to organize themselves in an appearing channel form, as it occurs in hydrophobic core of cell membrane. However, we have observed several occurences of polyene lacks inside the NT, where unselective ionic diffusion may largely take place. These shorttime defects seem to be the results of constant AmB diffusion alongthe internal NT surface. Finally, after a total simulation time of 338 ns inside the membrane, the AmB molecules are completely organized around the inner surface of the tube leaving the formation of a big hole near the membrane center. The approximate cross-section of this hole being of about 8.5 nm² is significantly smaller than those of the nanopore one (44 nm²). This system is likely stabilized and able to accommodate much more AmB molecules, which stays for now beyond of the actual computational power. Our data, although not sufficient to directly support the total self-assembly of AmB molecules inside the hydrophobic nanopore, are consistent with such an interpretation. These conclusions are also valid for all the tested initial configurations across the different simulation runs.
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Ionic conductivity membrane
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through
hybrid
polyene/artificial
Since AmB channel is well known to be permeable only to monovalent cation and anion, we decided to verify, if this particular property is still maintained after the AmB confinement inside the nanopore. To this end, membrane conductance G mem has been obtained for different concentrations of KCl and NaCl solutions varying from 6 mM to 720 mM (Fig. 4a), and between 6 mM to 340 mM for CaCl2 (Fig. 4b). The very first view on the raw membrane conductance at 10 mM KCl reveals 724 µS in contrast to 9.62 µS for MbPCAmB. This decrease of conductance can be directly recognized as the signature of AmB confinement inside the membrane pore. However, the conductance of MbPCAmB measured at same concentrations of NaCl or KCl is similar. Thus it can be supposed that the channel formed by AmB does not exhibit any particular selectivity between NaCl and KCl. For these monovalent salts, the linear increase of the membrane conductance with salt concentration is only valid at concentrations smaller than 400 mM. In biological membranes the same behavior has also been observed.10,28 In the case of the CaCl2 transport through MbPCAmB, we should expect the much lower conductance, if the primary monovalent ions selectivity is preserved. However, data shown in Fig. 4b presents higher conductance than for monovalent salts, already in the low CaCl2 concentrations range. As obtained for monovalent salts solution, measured membrane conductance increases again linearly for CaCl2 concentrations lower than 400 mM. This evident lack of monovalent ions selectivity is even more expressed when the membrane conductances are drawn as a function of the solution conductivities (Fig. 4c). Ionic selectivity of hybrid polyene/artificial membrane
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Experiments In biological conditions AmB channel permeability for K+ is larger than for Na+.12 Moreover, the anion vs cation selectivity is depending on pH,29 anion and salt concentrations.13 According to Eq. (2) the selectivity of MbPCAmB given by the Pcat PCl ratio has been determined by the measurement of current inversion (Einv). The permeation ratios obtained on MbPCAmB reported in Fig. 5a, as a function of the “in” compartment concentration, show an anionic behavior for all salts studied. In other words, the chlorine diffusion is favored compared to the cation one. The Pcat PCl ratio decreases when the “in” concentration increases until an asymptotic value of 0.16 for both KCl and NaCl, while it takes a much lower asymptotic value for CaCl2 of 0.047. This corroborates the anionic behavior, amplified for Ca2+ when compared to K+ or Na+. Moreover, the similar conductance obtained for NaCl and KCl are in very good accordance with the similar Pcat PCl ratios. On the contrary, higher conductance for CaCl2 than for KCl seems to be antagonist with such a low value of Pcat PCl ratio for Ca2+, when compared to monovalent ions. Two assumptions could be proposed to explain this apparent paradox. The first one is that the electric field, applied during the measurements, induces a reorganization of the polyenes inside the pore. However, it was not observed during the MD run expressly performed with applied external electric field. The second one is that the anionic behavior of membrane induces an increase of Cl- concentration inside the pore. The enhancement of This journal is © The Royal Society of Chemistry [year]
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Fig. 4 (a) MbPCAmB conductance as a function of monovalent salt concentration: NaCl (red squares) and KCl (black circles). The linear dependency is only valid up to 0.4 M of NaCl (red dashed line) and KCl (black solid line) concentrations, respectively. (b) MbPCAmB conductance as a function of CaCl2 salt concentration. The linear dependency can be also drawn (blue dashed line). (c) Membrane conductance as a function of solution conductivity. Red squares, black circles and blue triangles correspond to NaCl, KCl and CaCl2, respectively.
CaCl2 conductivity can thus mainly be attributed to the faster transfer of Cl- over Ca2+ cations. To compare the monovalent cationic selectivity of Mb PCAmB, we Journal Name, [year], [vol], 00–00 | 5
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Fig. 5 (a) Permeation ratio of cations over chlorine anions. (Na+ - red squares, K+ - black circles,Ca2+ - blue triangles). Please note the ordinate logarytmic scale. (b) Permeation ratio of K+ vs Na+ as a function of salt concentration.
reported in Fig. 5b the PK PNa ratio obtained for concentrations ranging from 6 mM to 720 mM. It is clearly shown that MbPCAmB exhibits a higher permeability through membrane for K+ than for Na+, since PK PNa 1 for all concentrations >100 mM. The behavior is quite different at low salt concentration (
