Published on 10 January 2014. Downloaded by State University of New York at Stony Brook on 22/10/2014 12:49:41.
ChemComm COMMUNICATION
Cite this: Chem. Commun., 2014, 50, 2642 Received 18th December 2013, Accepted 9th January 2014
View Article Online View Journal | View Issue
Reconstitution of OmpF membrane protein on bended lipid bilayers: perforated hexagonal mesophases† Alexandru Zabara,a Renata Negrini,a Patric Baumann,b Ozana Onaca-Fischerb and Raffaele Mezzenga*a
DOI: 10.1039/c3cc49590f www.rsc.org/chemcomm
Membrane proteins have been reconstituted on lipid bilayers with zero mean-curvature (cubic phases or vesicles). Here we show that reconstitution of pore-forming membrane proteins can also occur on highly curved lipidic bilayers of reverse hexagonal mesophases, for which the mean-curvature is significantly different from zero. We further show that the membrane protein provides unique topological interconnectivities between the aqueous nanochannels, significantly enhancing mesophase transport properties.
A central milestone in drug delivery and therapeutics has been set by optimizing the efficacy of the drug, that is, by maintaining the drug concentration within the therapeutic range over a prolonged time period. For this reason, significant efforts have converged towards the design and development of more sophisticated delivery systems that are able to ensure a controlled release of the bioactive compound and protect it from denaturing processes such as enzymatic or acidic hydrolysis.1–5 Lyotropic liquid crystalline phases (LLCs), based on selfassembled lipids in an aqueous environment, present appealing properties for these tasks. These thermodynamically stable mesophases of various architectures are characterized by nanosized hydrophilic and hydrophobic domains separated by a bilayer that offers a native-mimetic environment for membrane protein reconstitution; additionally these heterogeneous nanostructured fluids are ideally suited for encapsulating both hydrophilic and hydrophobic compounds.2–5 This has contributed towards placing emphasis on the use of LLCs as drug hosting and delivery matrices.6–12 a
Food & Soft Material Science, Institute of Food Nutrition and Health, ETH Zurich, Schmelzbergstrasse 9, LFO E23, 8092, Zurich, Switzerland. E-mail: raff[email protected]; Web: http://www.ifnh.ethz.ch/lwm; Tel: +41-446329140, +41-446323284 b Department of Chemistry, University of Basel, Klingelbergstrasse 80, 4056, Basel, Switzerland † Electronic supplementary information (ESI) available: Experimental details, structural parameters calculations, SAXS data after the full drug release and for the samples used in conductivity experiments, caffeine UV calibration curve. See DOI: 10.1039/c3cc49590f
2642 | Chem. Commun., 2014, 50, 2642--2645
Ever since the initial research of Ericsson et al.6 on use of LLCs for oral and transdermal drug administration, several mesophase architectures were considered as hosting matrices but the most significant advances, both in vitro and in vivo, were achieved by means of bicontinuous cubic phases.6–10 The reverse hexagonal phase, however, is equally suited to serve drug delivery purposes, since it exists in thermodynamic equilibrium with excess water; nonetheless, the high temperature at which this becomes typically accessible has long been a hindering factor.14 However, recent studies have clearly shown that addition of a co-surfactant or an oil can counteract this limitation and allow the formation of a stable hexagonal phase at room temperature.11–13 The structural topology of the reverse hexagonal phase consists of densely packed water filled cylinders, each surrounded by a lipidic bilayer, following a 1D anisotropic symmetry. This grants the system the distinct advantage of a slower and prolonged release of target hydrophilic molecules, according to the diffusion coefficient rank: DCUBIC c DLAM Z DHEX.15 Accordingly, these systems were recently proved to be extremely effective transdermal delivery vehicles, allowing for the enhanced permeation of target therapeutic agents (i.e. sodium diclofenac).12 In a recent study we explored a new, unique approach towards the development of advanced LLC-based drug delivery systems and demonstrated that by means of membrane protein-based interconnecting pores, hybrid liquid crystalline cubic phases of Pn3m symmetry can be engineered, capable of modulating their transport properties in response to variations in protein pore density or moderate changes in environmental pH.16 In this communication we expand our previous findings and show that this approach is general in that it can be universally used to control the transport properties of reverse liquid crystalline systems regardless of their three-dimensional symmetry. To this end, we show that we can successfully reconstitute the bacterial pore-forming protein, Outer Membrane Protein F (OmpF)17,18 within the lipidic bilayer of highly curved reverse hexagonal phases, thus opening gating pathways between distinct water nanochannels. This reconstitution of the protein allows significantly faster solute transport rates within the mesophase and opens new possibilities in the use of
This journal is © The Royal Society of Chemistry 2014
View Article Online
Published on 10 January 2014. Downloaded by State University of New York at Stony Brook on 22/10/2014 12:49:41.
Communication
reverse hexagonal phases as delivery tools. From a more fundamental standpoint, this works demonstrates for the first time that it is possible to reconstitute membrane proteins at lipid bilayers with nonzero mean-curvature without losing their functionality, in contrast to the general assumption of highly curved interfaces being detrimental to the functional reconstitution of membrane proteins. In order to develop a functional perforated mesophase it is imperative to understand how the individual components can come together in a cohesive and reproducible manner. To start with, small angle X-ray measurements coupled with geometrical models that account for the packing frustrations in the interstitial spaces allow us to assess the structural parameters of the unperturbed phase and accurately determine the size of its two distinct domains (a water channel diameter of 25.5 Å and a lipidic bilayer length of 27.9 Å – see ESI†). Additionally, high-resolution crystallographic data on the three dimensional structure of the functional form of OmpF revealed that the protein monomer has a pore central elliptical cross-section of 27 Å 38 Å (width length)17,18 and a height of 53 Å, dimensions that correlate perfectly with the structural parameters of the host mesophase. The protein monomers follow a well-established mechanism of folding and membrane insertion.19–21 Initial reconstitution of the protein monomer within the structure of a lipidic membrane initiates the structural conformations required for the assembly of an active dimeric intermediate state. Under favourable conditions the dimeric state undergoes further changes that eventually lead to the final, native, conformation of a functional trimer.19–21 However, reconstitution of a functional trimeric form of OmpF requires a hydrophobic region of 59 Å in width (see ESI,† Fig. S1), which would induce a massive deformation of the mesophase structure, i.e. an energetically unaffordable event. Thus, we assume that a possible reconstitution of OmpF within the highly curved bilayer of a reverse hexagonal phase can proceed only through monomers or dimers. This is not to jeopardize diffusion, though, as active transport of OmpF dimers has been reported before.19–21 The schematic concept behind the design of this new structural type of reverse hexagonal phase is graphically sketched in Fig. 1. Small angle X-ray scattering (SAXS) provided key insights towards detection of any potential mesophase structural changes occurring due to membrane protein reconstitution. Fig. 2 shows the SAXS spectra of scattered intensities plotted versus scattering vector, Q,
Fig. 1 Schematic overview of the perforated reverse hexagonal (HII) phase based on the hybrid monolinolein–tetradecane–OmpF–water system. Due to spatial restraints within the structure of the host mesophase correct reconstitution of OmpF can only be achieved for monomers or dimers.
This journal is © The Royal Society of Chemistry 2014
ChemComm
Fig. 2 Effects of membrane protein reconstitution on the structure of the host mesophase (with or without added OmpF). 1D SAXS spectra and calculated structural parameters (inset) for the systems without (blue), with 0.24 wt% (red) and respectively with 0.5 wt% OmpF (green).
azimuthally averaged into 1D profiles for both the unperturbed (blank) reverse hexagonal phase (blue spectra) and the system containing either 0.24 wt% (red spectra) or 0.5 wt% (green spectra) OmpF. Structural data for the systems with or without added OmpF, are shown within the figure inset. More importantly the corresponding analysis of the structural parameters unveiled no significant topological difference between the OmpF-containing mesophases and the unperturbed phase, confirming that the OmpF reconstitutes within the host mesophase without significant structural deformations. Determination of molecular transport properties was achieved using a combination of two independent techniques: caffeine release studies in conditions of excess water and bulk phase ion conductivity measurements. These assessment tools offer an accurate and reliable way of investigating the specific changes in the mesophase transport behaviour caused by doping the host LLC with different amounts of pore-forming protein. Moreover, it allows us to encompass a broad region of the phase diagram and monitor the induced changes of transport properties of the host mesophase under conditions of both bulk mesophase and mesophase in the presence of excess water. Fig. 3a shows the caffeine release profiles at physiological pH (pH 7.4) plotted as a percentage of released caffeine vs. square root of time for both the unperturbed reverse hexagonal phase (blue) and the two perforated phases containing progressively larger amounts of OmpF: 0.24 wt% (red) and respectively 0.5 wt% (green). In all cases, background subtracted data were normalized by the concentration plateau observed after long periods of time (>72 h). This was done under the assumption of negligible specific interactions (i.e. electrostatic) between the released molecule and the mesophase, compared to the osmotic pressure gradient driving the release process: under these assumptions the cumulative drug released at long times corresponds to 100% release. The scattering
Chem. Commun., 2014, 50, 2642--2645 | 2643
View Article Online
Published on 10 January 2014. Downloaded by State University of New York at Stony Brook on 22/10/2014 12:49:41.
ChemComm
Communication
Fig. 3 Assessment of the effect of OmpF reconstitution on the transport behaviour of the host mesophase. (a) Caffeine release profiles of the systems containing progressively larger protein amounts: 0 wt% (blue); 0.24 wt% OmpF (red); 0.5 wt% OmpF (green). The inset highlights the linear part of the release profile and the difference in calculated diffusion coefficient for the three systems. (b) Bulk phase ion conductivity values for the three LLCs. In all the figures the error bars represent the standard deviation value of four different samples.
experiments clearly showed that all the tested systems maintained their reverse hexagonal symmetry within the time frame of the drug release study (see ESI† for SAXS spectra after release of caffeine), as revealed by the observed SAXS diffraction peaks in the ratio O1 : O3 : O4. Addition of membrane proteins led to a systematic increase in the slope of the cumulated caffeine release outdoing the unperturbed mesophase by 27.4% and 46.8%, respectively. This corresponds to an increase in the diffusion coefficient of 55.9% and 115.5% (see ESI,† eqn (S3)) for the mesophases doped with 0.24 wt% OmpF and 0.5 wt% OmpF (Fig. 3a-inset). Thus, the increase in the transport diffusion coefficient correlates directly with the protein concentration, i.e. the surface density of protein-mediated interconnecting pores. The observed increase in caffeine transport across the reverse hexagonal mesophase was independently validated by bulk phase ion conductivity measurements. Fig. 3b shows the specific conductivity values measured for the hexagonal mesophases of composition identical to those in Fig. 3a: blank (blue), 0.24 wt% (red) and 0.5 wt% (green). Once more, perforation of the lipidic bilayer and the opening of active gating pores between the independent aqueous domains of the hexagonal phase led to a direct increase in ion transport across the mesophase surface by 30.5% and respectively 50.4% for the two OmpF-containing systems. These measured differences in ion conductivity come in close correlation with the observed changes in solute release rates and conclusively demonstrate the improved transport capacity of the perforated mesophase. Fig. 4 rationalizes the enhanced transport properties caused by OmpF by plotting the relative increase in ionic conductivity (C/C0) against the square root of the relative diffusion coefficients (D/D0). Remarkably, a 1 : 1 correlation (slope = 1.07; R2 = 0.999) between the two sets of data is found. The inset of Fig. 4 further emphasizes this agreement by the two nearly overlapping profiles of transport rates, C/C0 and (D/D0)1/2, plotted as a function of OmpF concentration. These findings unambiguously demonstrate the universal effect of protein-mediated perforation of the lipidic bilayer on the transport of either ions
2644 | Chem. Commun., 2014, 50, 2642--2645
Fig. 4 Correlation between the measured relative ionic conductivity plotted as a function of the square root of the relative diffusion coefficient for the three OmpF concentrations studied. The inset gives the relative increase in transport rates (C/C0 and (D/D0)1/2) plotted as a function of OmpF concentration. All error bars represent the standard deviation over four independent samples.
(bulk phase) or target hydrophilic molecules (caffeine) and highlight the possibility of controlling diffusion by varying the surface density of the protein pores. In summary we have shown for the first time that complex membrane proteins can functionally reconstitute within the highly curved bilayer of a reverse hexagonal liquid crystalline phase. Implicitly, integration of these transporters within the three dimensional structure of the mesophase provides a new strategy for the design of drug delivery matrices with unprecedented properties. Using the model system monolinolein–tetradecane– OmpF we have demonstrated that the solute transport can be accurately modulated by the membrane protein surface density (i.e. number of interconnecting pores). These findings may open new perspectives in the field of drug delivery systems exploiting lipidic mesophases.
This journal is © The Royal Society of Chemistry 2014
View Article Online
Communication
Published on 10 January 2014. Downloaded by State University of New York at Stony Brook on 22/10/2014 12:49:41.
Notes and references 1 T. R. S. Kumar, K. Soppimath and S. K. Nachaegari, Curr. Pharm. Biotechnol., 2006, 7, 271–276. 2 E. M. Landau and J. P. Rosenbusch, Proc. Natl. Acad. Sci. U. S. A., 1996, 93, 14532–14535. 3 J. Clogston and M. Caffrey, J. Controlled Release, 2005, 107, 97–111. 4 J. C. Shah, Y. Sadhale and D. M. Chilukuri, Adv. Drug Delivery Rev., 2001, 47, 229–250. 5 S. Z. Mohammady, M. Pouzot and R. Mezzenga, Biophys. J., 2009, 96, 1537–1546. ¨froth and S. Engstro ¨m, ACS Symp. 6 B. Ericsson, P. O. Eriksson, J. E. Lo Ser., 1991, 469, 251–265. 7 A. Feher, E. Urban, I. Eros, P. Szabo-Revesz and E. Csanyi, Int. J. Pharm., 2008, 358, 23–26. 8 L. B. Lopes, J. H. Collett and M. V. L. B. Bentley, Eur. J. Pharm. Biopharm., 2005, 60, 25–30. 9 C. J. Drummond and C. Fong, Curr. Opin. Colloid Interface Sci., 1999, 4, 449–456. 10 R. Negrini and R. Mezzenga, Langmuir, 2011, 27, 5296–5303.
This journal is © The Royal Society of Chemistry 2014
ChemComm 11 D. Libster, A. Aserin, E. Wachtel, G. Shoham and N. Garti, J. Colloid Interface Sci., 2007, 308, 514–524. 12 M. Cohen-Avrahami, A. Aserin and N. Garti, Colloids Surf., B, 2010, 77, 131–138. 13 Y. D. Dong, I. Larson, T. Hanley and B. J. Boyd, Langmuir, 2006, 22, 9512–9518. 14 R. Mezzenga, C. Meyer, C. Servais, A. I. Romoscanu, L. Sagalowicz and R. C. Hayward, Langmuir, 2005, 21, 3322–3333. 15 L. Sagalowicz, R. Mezzenga and M. E. Leser, Curr. Opin. Colloid Interface Sci., 2006, 11, 224–229. 16 A. Zabara, R. Negrini, O. Onaca-Fischer and R. Mezzenga, Small, 2013, 9, 3602–3609. 17 S. W. Cowan, T. Schirmer, G. Rummel, M. Steiert, R. Ghosh, R. A. Pauptit, J. N. Jansonius and J. P. Rosenbusch, Nature, 1992, 358, 727–733. 18 R. G. Efremov and L. A. Sazanov, J. Struct. Biol., 2012, 178, 311–318. ¨hnig, Biochemistry, 1996, 35, 19 T. Surrey, A. Schmid and F. Ja 2283–2288. 20 Y. Watanabe, J. Protein Chem., 2002, 21, 169–175. 21 H. Naveed, D. Jimenez-Morales, J. Tian, V. Pasupuleti, L. J. Kenney and J. Liang, J. Mol. Biol., 2012, 419, 89–101.
Chem. Commun., 2014, 50, 2642--2645 | 2645
ChemComm COMMUNICATION
Cite this: Chem. Commun., 2014, 50, 2642 Received 18th December 2013, Accepted 9th January 2014
View Article Online View Journal | View Issue
Reconstitution of OmpF membrane protein on bended lipid bilayers: perforated hexagonal mesophases† Alexandru Zabara,a Renata Negrini,a Patric Baumann,b Ozana Onaca-Fischerb and Raffaele Mezzenga*a
DOI: 10.1039/c3cc49590f www.rsc.org/chemcomm
Membrane proteins have been reconstituted on lipid bilayers with zero mean-curvature (cubic phases or vesicles). Here we show that reconstitution of pore-forming membrane proteins can also occur on highly curved lipidic bilayers of reverse hexagonal mesophases, for which the mean-curvature is significantly different from zero. We further show that the membrane protein provides unique topological interconnectivities between the aqueous nanochannels, significantly enhancing mesophase transport properties.
A central milestone in drug delivery and therapeutics has been set by optimizing the efficacy of the drug, that is, by maintaining the drug concentration within the therapeutic range over a prolonged time period. For this reason, significant efforts have converged towards the design and development of more sophisticated delivery systems that are able to ensure a controlled release of the bioactive compound and protect it from denaturing processes such as enzymatic or acidic hydrolysis.1–5 Lyotropic liquid crystalline phases (LLCs), based on selfassembled lipids in an aqueous environment, present appealing properties for these tasks. These thermodynamically stable mesophases of various architectures are characterized by nanosized hydrophilic and hydrophobic domains separated by a bilayer that offers a native-mimetic environment for membrane protein reconstitution; additionally these heterogeneous nanostructured fluids are ideally suited for encapsulating both hydrophilic and hydrophobic compounds.2–5 This has contributed towards placing emphasis on the use of LLCs as drug hosting and delivery matrices.6–12 a
Food & Soft Material Science, Institute of Food Nutrition and Health, ETH Zurich, Schmelzbergstrasse 9, LFO E23, 8092, Zurich, Switzerland. E-mail: raff[email protected]; Web: http://www.ifnh.ethz.ch/lwm; Tel: +41-446329140, +41-446323284 b Department of Chemistry, University of Basel, Klingelbergstrasse 80, 4056, Basel, Switzerland † Electronic supplementary information (ESI) available: Experimental details, structural parameters calculations, SAXS data after the full drug release and for the samples used in conductivity experiments, caffeine UV calibration curve. See DOI: 10.1039/c3cc49590f
2642 | Chem. Commun., 2014, 50, 2642--2645
Ever since the initial research of Ericsson et al.6 on use of LLCs for oral and transdermal drug administration, several mesophase architectures were considered as hosting matrices but the most significant advances, both in vitro and in vivo, were achieved by means of bicontinuous cubic phases.6–10 The reverse hexagonal phase, however, is equally suited to serve drug delivery purposes, since it exists in thermodynamic equilibrium with excess water; nonetheless, the high temperature at which this becomes typically accessible has long been a hindering factor.14 However, recent studies have clearly shown that addition of a co-surfactant or an oil can counteract this limitation and allow the formation of a stable hexagonal phase at room temperature.11–13 The structural topology of the reverse hexagonal phase consists of densely packed water filled cylinders, each surrounded by a lipidic bilayer, following a 1D anisotropic symmetry. This grants the system the distinct advantage of a slower and prolonged release of target hydrophilic molecules, according to the diffusion coefficient rank: DCUBIC c DLAM Z DHEX.15 Accordingly, these systems were recently proved to be extremely effective transdermal delivery vehicles, allowing for the enhanced permeation of target therapeutic agents (i.e. sodium diclofenac).12 In a recent study we explored a new, unique approach towards the development of advanced LLC-based drug delivery systems and demonstrated that by means of membrane protein-based interconnecting pores, hybrid liquid crystalline cubic phases of Pn3m symmetry can be engineered, capable of modulating their transport properties in response to variations in protein pore density or moderate changes in environmental pH.16 In this communication we expand our previous findings and show that this approach is general in that it can be universally used to control the transport properties of reverse liquid crystalline systems regardless of their three-dimensional symmetry. To this end, we show that we can successfully reconstitute the bacterial pore-forming protein, Outer Membrane Protein F (OmpF)17,18 within the lipidic bilayer of highly curved reverse hexagonal phases, thus opening gating pathways between distinct water nanochannels. This reconstitution of the protein allows significantly faster solute transport rates within the mesophase and opens new possibilities in the use of
This journal is © The Royal Society of Chemistry 2014
View Article Online
Published on 10 January 2014. Downloaded by State University of New York at Stony Brook on 22/10/2014 12:49:41.
Communication
reverse hexagonal phases as delivery tools. From a more fundamental standpoint, this works demonstrates for the first time that it is possible to reconstitute membrane proteins at lipid bilayers with nonzero mean-curvature without losing their functionality, in contrast to the general assumption of highly curved interfaces being detrimental to the functional reconstitution of membrane proteins. In order to develop a functional perforated mesophase it is imperative to understand how the individual components can come together in a cohesive and reproducible manner. To start with, small angle X-ray measurements coupled with geometrical models that account for the packing frustrations in the interstitial spaces allow us to assess the structural parameters of the unperturbed phase and accurately determine the size of its two distinct domains (a water channel diameter of 25.5 Å and a lipidic bilayer length of 27.9 Å – see ESI†). Additionally, high-resolution crystallographic data on the three dimensional structure of the functional form of OmpF revealed that the protein monomer has a pore central elliptical cross-section of 27 Å 38 Å (width length)17,18 and a height of 53 Å, dimensions that correlate perfectly with the structural parameters of the host mesophase. The protein monomers follow a well-established mechanism of folding and membrane insertion.19–21 Initial reconstitution of the protein monomer within the structure of a lipidic membrane initiates the structural conformations required for the assembly of an active dimeric intermediate state. Under favourable conditions the dimeric state undergoes further changes that eventually lead to the final, native, conformation of a functional trimer.19–21 However, reconstitution of a functional trimeric form of OmpF requires a hydrophobic region of 59 Å in width (see ESI,† Fig. S1), which would induce a massive deformation of the mesophase structure, i.e. an energetically unaffordable event. Thus, we assume that a possible reconstitution of OmpF within the highly curved bilayer of a reverse hexagonal phase can proceed only through monomers or dimers. This is not to jeopardize diffusion, though, as active transport of OmpF dimers has been reported before.19–21 The schematic concept behind the design of this new structural type of reverse hexagonal phase is graphically sketched in Fig. 1. Small angle X-ray scattering (SAXS) provided key insights towards detection of any potential mesophase structural changes occurring due to membrane protein reconstitution. Fig. 2 shows the SAXS spectra of scattered intensities plotted versus scattering vector, Q,
Fig. 1 Schematic overview of the perforated reverse hexagonal (HII) phase based on the hybrid monolinolein–tetradecane–OmpF–water system. Due to spatial restraints within the structure of the host mesophase correct reconstitution of OmpF can only be achieved for monomers or dimers.
This journal is © The Royal Society of Chemistry 2014
ChemComm
Fig. 2 Effects of membrane protein reconstitution on the structure of the host mesophase (with or without added OmpF). 1D SAXS spectra and calculated structural parameters (inset) for the systems without (blue), with 0.24 wt% (red) and respectively with 0.5 wt% OmpF (green).
azimuthally averaged into 1D profiles for both the unperturbed (blank) reverse hexagonal phase (blue spectra) and the system containing either 0.24 wt% (red spectra) or 0.5 wt% (green spectra) OmpF. Structural data for the systems with or without added OmpF, are shown within the figure inset. More importantly the corresponding analysis of the structural parameters unveiled no significant topological difference between the OmpF-containing mesophases and the unperturbed phase, confirming that the OmpF reconstitutes within the host mesophase without significant structural deformations. Determination of molecular transport properties was achieved using a combination of two independent techniques: caffeine release studies in conditions of excess water and bulk phase ion conductivity measurements. These assessment tools offer an accurate and reliable way of investigating the specific changes in the mesophase transport behaviour caused by doping the host LLC with different amounts of pore-forming protein. Moreover, it allows us to encompass a broad region of the phase diagram and monitor the induced changes of transport properties of the host mesophase under conditions of both bulk mesophase and mesophase in the presence of excess water. Fig. 3a shows the caffeine release profiles at physiological pH (pH 7.4) plotted as a percentage of released caffeine vs. square root of time for both the unperturbed reverse hexagonal phase (blue) and the two perforated phases containing progressively larger amounts of OmpF: 0.24 wt% (red) and respectively 0.5 wt% (green). In all cases, background subtracted data were normalized by the concentration plateau observed after long periods of time (>72 h). This was done under the assumption of negligible specific interactions (i.e. electrostatic) between the released molecule and the mesophase, compared to the osmotic pressure gradient driving the release process: under these assumptions the cumulative drug released at long times corresponds to 100% release. The scattering
Chem. Commun., 2014, 50, 2642--2645 | 2643
View Article Online
Published on 10 January 2014. Downloaded by State University of New York at Stony Brook on 22/10/2014 12:49:41.
ChemComm
Communication
Fig. 3 Assessment of the effect of OmpF reconstitution on the transport behaviour of the host mesophase. (a) Caffeine release profiles of the systems containing progressively larger protein amounts: 0 wt% (blue); 0.24 wt% OmpF (red); 0.5 wt% OmpF (green). The inset highlights the linear part of the release profile and the difference in calculated diffusion coefficient for the three systems. (b) Bulk phase ion conductivity values for the three LLCs. In all the figures the error bars represent the standard deviation value of four different samples.
experiments clearly showed that all the tested systems maintained their reverse hexagonal symmetry within the time frame of the drug release study (see ESI† for SAXS spectra after release of caffeine), as revealed by the observed SAXS diffraction peaks in the ratio O1 : O3 : O4. Addition of membrane proteins led to a systematic increase in the slope of the cumulated caffeine release outdoing the unperturbed mesophase by 27.4% and 46.8%, respectively. This corresponds to an increase in the diffusion coefficient of 55.9% and 115.5% (see ESI,† eqn (S3)) for the mesophases doped with 0.24 wt% OmpF and 0.5 wt% OmpF (Fig. 3a-inset). Thus, the increase in the transport diffusion coefficient correlates directly with the protein concentration, i.e. the surface density of protein-mediated interconnecting pores. The observed increase in caffeine transport across the reverse hexagonal mesophase was independently validated by bulk phase ion conductivity measurements. Fig. 3b shows the specific conductivity values measured for the hexagonal mesophases of composition identical to those in Fig. 3a: blank (blue), 0.24 wt% (red) and 0.5 wt% (green). Once more, perforation of the lipidic bilayer and the opening of active gating pores between the independent aqueous domains of the hexagonal phase led to a direct increase in ion transport across the mesophase surface by 30.5% and respectively 50.4% for the two OmpF-containing systems. These measured differences in ion conductivity come in close correlation with the observed changes in solute release rates and conclusively demonstrate the improved transport capacity of the perforated mesophase. Fig. 4 rationalizes the enhanced transport properties caused by OmpF by plotting the relative increase in ionic conductivity (C/C0) against the square root of the relative diffusion coefficients (D/D0). Remarkably, a 1 : 1 correlation (slope = 1.07; R2 = 0.999) between the two sets of data is found. The inset of Fig. 4 further emphasizes this agreement by the two nearly overlapping profiles of transport rates, C/C0 and (D/D0)1/2, plotted as a function of OmpF concentration. These findings unambiguously demonstrate the universal effect of protein-mediated perforation of the lipidic bilayer on the transport of either ions
2644 | Chem. Commun., 2014, 50, 2642--2645
Fig. 4 Correlation between the measured relative ionic conductivity plotted as a function of the square root of the relative diffusion coefficient for the three OmpF concentrations studied. The inset gives the relative increase in transport rates (C/C0 and (D/D0)1/2) plotted as a function of OmpF concentration. All error bars represent the standard deviation over four independent samples.
(bulk phase) or target hydrophilic molecules (caffeine) and highlight the possibility of controlling diffusion by varying the surface density of the protein pores. In summary we have shown for the first time that complex membrane proteins can functionally reconstitute within the highly curved bilayer of a reverse hexagonal liquid crystalline phase. Implicitly, integration of these transporters within the three dimensional structure of the mesophase provides a new strategy for the design of drug delivery matrices with unprecedented properties. Using the model system monolinolein–tetradecane– OmpF we have demonstrated that the solute transport can be accurately modulated by the membrane protein surface density (i.e. number of interconnecting pores). These findings may open new perspectives in the field of drug delivery systems exploiting lipidic mesophases.
This journal is © The Royal Society of Chemistry 2014
View Article Online
Communication
Published on 10 January 2014. Downloaded by State University of New York at Stony Brook on 22/10/2014 12:49:41.
Notes and references 1 T. R. S. Kumar, K. Soppimath and S. K. Nachaegari, Curr. Pharm. Biotechnol., 2006, 7, 271–276. 2 E. M. Landau and J. P. Rosenbusch, Proc. Natl. Acad. Sci. U. S. A., 1996, 93, 14532–14535. 3 J. Clogston and M. Caffrey, J. Controlled Release, 2005, 107, 97–111. 4 J. C. Shah, Y. Sadhale and D. M. Chilukuri, Adv. Drug Delivery Rev., 2001, 47, 229–250. 5 S. Z. Mohammady, M. Pouzot and R. Mezzenga, Biophys. J., 2009, 96, 1537–1546. ¨froth and S. Engstro ¨m, ACS Symp. 6 B. Ericsson, P. O. Eriksson, J. E. Lo Ser., 1991, 469, 251–265. 7 A. Feher, E. Urban, I. Eros, P. Szabo-Revesz and E. Csanyi, Int. J. Pharm., 2008, 358, 23–26. 8 L. B. Lopes, J. H. Collett and M. V. L. B. Bentley, Eur. J. Pharm. Biopharm., 2005, 60, 25–30. 9 C. J. Drummond and C. Fong, Curr. Opin. Colloid Interface Sci., 1999, 4, 449–456. 10 R. Negrini and R. Mezzenga, Langmuir, 2011, 27, 5296–5303.
This journal is © The Royal Society of Chemistry 2014
ChemComm 11 D. Libster, A. Aserin, E. Wachtel, G. Shoham and N. Garti, J. Colloid Interface Sci., 2007, 308, 514–524. 12 M. Cohen-Avrahami, A. Aserin and N. Garti, Colloids Surf., B, 2010, 77, 131–138. 13 Y. D. Dong, I. Larson, T. Hanley and B. J. Boyd, Langmuir, 2006, 22, 9512–9518. 14 R. Mezzenga, C. Meyer, C. Servais, A. I. Romoscanu, L. Sagalowicz and R. C. Hayward, Langmuir, 2005, 21, 3322–3333. 15 L. Sagalowicz, R. Mezzenga and M. E. Leser, Curr. Opin. Colloid Interface Sci., 2006, 11, 224–229. 16 A. Zabara, R. Negrini, O. Onaca-Fischer and R. Mezzenga, Small, 2013, 9, 3602–3609. 17 S. W. Cowan, T. Schirmer, G. Rummel, M. Steiert, R. Ghosh, R. A. Pauptit, J. N. Jansonius and J. P. Rosenbusch, Nature, 1992, 358, 727–733. 18 R. G. Efremov and L. A. Sazanov, J. Struct. Biol., 2012, 178, 311–318. ¨hnig, Biochemistry, 1996, 35, 19 T. Surrey, A. Schmid and F. Ja 2283–2288. 20 Y. Watanabe, J. Protein Chem., 2002, 21, 169–175. 21 H. Naveed, D. Jimenez-Morales, J. Tian, V. Pasupuleti, L. J. Kenney and J. Liang, J. Mol. Biol., 2012, 419, 89–101.
Chem. Commun., 2014, 50, 2642--2645 | 2645
