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Cite this: Chem. Commun., 2013, 49, 11647 Received 16th September 2013, Accepted 17th October 2013
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Biomimetic artificial ion channels based on beta-cyclodextrin† Yassine El Ghoul,a Ruddy Renia,a Ibrahima Faye,a Soumassoudrane Rassou,a ´ronique Bennevault-Celton,a Ce ´cile Huin*a and Philippe Gue ´ganc Nezha Badi,zb Ve
DOI: 10.1039/c3cc47071g www.rsc.org/chemcomm
Star polymers based on b-cyclodextrin were synthesized by a ‘‘click-chemistry’’ process and characterized by NMR, SEC and BLM. The resulting triazole functional groups create, at a specific pH range, electrostatic hindrance between pores inserted in lipid bilayers, preventing their aggregation, allowing formation of well-defined isolated unitary pores and mimicking biological natural channels.
Natural ion channels are essential for life, mainly because of their ability to regulate the flow of ions and molecules through the cell membrane. Natural macromolecules self-assemble to form isolated nanopores/nanochannels in the lipid bilayer (a-hemolysin, gramicidin A, aerolysin).1,2 Design of biomimetic systems is of prime importance to understand the nature and to develop devices useful for the bio- and nanotechnology fields (antimicrobial agents, nanofluidic devices, tools for translocation and detection, single-molecule experiments).1,3,4 The first requirement for the formation of suitable nanopores is the design of molecules partially soluble in water, such as monomers forming the self-assembled a-hemolysin.2a Numerous examples of synthetic amphiphilic systems forming artificial channels are reported in the literature,4–7 showing the necessity to work under very dilute conditions to avoid aggregation in solution or/and in the membrane. Aggregates are considered detrimental to nanotechnology applications, such as protein unfolding8 or nanopore force spectroscopy:9 the function of the nanopores is not ensured due to pollution from the aggregates. In the case of polymers, different kinds of interactions could exist with lipid a
LAMBE, UMR8587, UEVE-CNRS-CEA, Bld François Mitterrand, 91025 Evry, France. E-mail: [email protected]; Fax: +33 1 69 47 76 65; Tel: +33 1 69 47 77 17 b Institut Charles Sadron, UPR22-CNRS, 23 rue du Loess, BP 84047, 67034 Strasbourg Cedex 2, France c Laboratoire de Chimie des Polyme`res, UMR 7610, UPMC, 3 rue Galile´e, 94200 Ivry sur Seine, France † Electronic supplementary information (ESI) available: Experimental details: protocols used to synthesize the star polymers and physicochemical characterization (NMR, critical aggregation concentration). See DOI: 10.1039/c3cc47071g ‡ Present address: Precision Macromolecular Chemistry Group, Institut Charles Sadron, UPR22-CNRS, 23 rue du Loess, BP 84047, 67034 Strasbourg Cedex 2, France.
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bilayer membranes, involving supramolecular aggregates (amphiphilic block copolymers PEO–PPO–PEO) or formation of transient pores (hydrophilic polymers grafted hydrophobic side chains, polyelectrolytes).7 In order to ascertain the presence of unitary, isolated pores, steric or electrostatic hindrance should be encountered. Badi et al. demonstrated that star polymers based on b-cyclodextrin, bearing heptyl chains on the secondary face and poly(ethylene glycol) PEG arms on the primary face, may form well-defined channels, in the concentration range of 106 mol L1.6c However, a precise tuning of the concentration must be done to avoid aggregation. Here, we report the design of cyclodextrin (CD)-based channels, synthesized by the Huisgen [2+3] cycloaddition reaction, which behave as biomimetic isolated systems. The role of triazole groups, resulting from this reaction, was already mentioned by Fyles et al. for the design of artificial channels,6d–f mainly for the simple and efficient synthetic process. In addition, the triazole functional groups possess an interesting property of pH-sensitivity10 that has never been explored in the application of pore-forming molecules. The ‘‘click-chemistry’’ process allows the synthesis of per(2,3-di-O-heptyl)-6-methoxyPEG-6-(1,2,3-triazole)-b-CDs, named CD-triazole-PEG, by grafting 7 PEG branches on the primary face of a per(2,3-di-O-acetyl)-(6-deoxy-6-azido)-b-CD (MPEG = 550, 1100 or 2000 g mol1).6d–f,11 Deprotection of the acetyl groups and modification by heptyl chains of the secondary face of the cyclodextrin derivatives provide hydrophobicity,12 which is required for a possible insertion of the macromolecules in a lipidic bilayer.6c The high molar masses of star polymers containing 7 PEG arms of 1100 and 2000 g mol1 make the NMR characterization difficult and the evidence of the exact architecture difficult to ascertain. The star polymer with 7 arms of 550 g mol1 and the intermediates are discussed in the ESI† (Fig. S1 and S2 for the final product). 1H and 13 C NMR proves the quantitative modification of the cyclodextrin derivative (absence of the signal corresponding to the CH2–N3 in the 13 C NMR spectrum). DOSY NMR analysis shows only one population. The large difference in diffusion coefficients obtained by comparing a-methoxy-o-propargyl PEG550 (D = 1.7 1010 0.1 m2 s1 – Fig. S4, ESI†) and CD-triazole-PEG550 (D = 5.8 1011 0.2 m2 s1 – Fig. S3, ESI†) allows us to ascertain the star Chem. Commun., 2013, 49, 11647--11649
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polymer architecture and to demonstrate the complete absence of the linear polymer. The purity of the star polymer is also observed by size exclusion chromatography (Fig. S5, ESI†) with one monodisperse population and a molar mass in agreement with the theoretical one. Triazole functional groups have been envisioned as proton transport facilitators.10 Their pKa have been determined to be close to 1.2. However, bitriazole provides a higher proton affinity and its pKa is close to 3.2.10 This protonation property was then encountered by the CD-triazole-PEG derivatives. For these derivatives exhibiting a polyelectrolyte behavior, classical acid–base titration, 1H NMR experiments of CD-triazole-PEG550 in D2O at various pHs and isothermal titration calorimetry did not allow for the determination of pKa, due to the degradation of the PEG arms and the CD at such pHs.13 Dynamic light scattering, using different buffers (KCl 1 mol L1, Hepes 5 mmol L1, pH = 7 and KCl 1 mol L1, sodium citrate 5 mmol L1, pH = 3), shows self-aggregation of the amphiphilic star polymers, the critical aggregation concentration being around 2 102 mol L1, independently from the tested pH (Fig. S6, ESI†). In solution, hydrophobic interactions between heptyl chains seem to be predominant compared to electrostatic repulsions. Badi et al. proved, by the ‘‘black lipid membrane’’ (BLM) technique, that amphiphilic star PEG polymers based on cyclodextrin, without any triazole functional groups, called CD-PEG, form channels in the lipidic model membrane,6c only under very dilute conditions. Indeed, for M n ¼ 5000 g mol1 (MPEG arm = 530 g mol1), at pH = 7 and at a concentration below 4 106 mol L1, discrete jumps are observed and histograms, representing the number of events at each current, show the existence of well-defined unitary pores of mean intensity of 1.2 pA for an applied voltage of 100 mV (Fig. S7, ESI†). The Hill plot represents the open probability P0 of a single ion channel versus the polymer concentration. The obtained profile gives information about the stoichiometry of the assembly and the number of molecules involved in the pore formation, according to eqn (1). log P0 = n log C n log KD
Chem. Commun., 2013, 49, 11647--11649
2000 g mol1, permeabilization of the membrane is observed (Fig. S11, ESI†), which is not desirable for applications of artificial channels.
(1)
where n is the Hill coefficient and KD the dissociation constant. The determination of the Hill plot1,14 demonstrates the requirement of two cyclodextrin derivatives to form a unitary pore and provides direct evidence for the hemi-channel insertion mechanism (Fig. S8, ESI†). However, as soon as the concentration used is above 5 106 mol L1, the current values are much higher than the one obtained for unitary pores (Fig. S9, ESI†), current values not reproducible, depending on the experimental conditions (membrane capacitance for instance). These events correspond to the insertion of rafts, ‘‘aggregates’’ or ‘‘clusters’’ of cyclodextrin derivatives, visible whatever the pH. At this point, it is necessary to recall that the aggregation only occurs in the membrane and does not occur in the solution. The concentration used during the BLM experiments (around 106 mol L1) is indeed below the critical aggregation concentration, as proved by the radius distribution centred around 5 nm, corresponding to a unimer size (Fig. S10, ESI†). In addition, the molar mass of the star polymers is important: when using CD with arms above 11648
Fig. 1 BLM conductance measurements and analysis for CD-triazole-PEG550 at a concentration of 2 107 mol L1 at pH = 7, current intensity versus time for an applied voltage: 100 mV. (A) Complete BLM electrical measurements. (B) Zoom of the BLM electrical measurements on ‘unitary pores’ events. (C) Number of events versus current corresponding to trace B. (D) Zoom of the BLM electrical measurements on ‘clusters and unitary pores’ events. (E) Number of events versus current corresponding to trace D.
Fig. 2 BLM conductance measurements and analysis for CD-triazole-PEG550 at a concentration of 2 107 mol L1 at pH = 3, current intensity versus time for an applied voltage: 100 mV. (A) BLM electrical measurements – example 1. (B) Number of events versus current corresponding to trace A. (C) Current versus pore number of trace A. (D) BLM electrical measurements – example 2. (E) BLM electrical measurements – example 3. (F) Number of events versus current corresponding to trace E. (G) Current versus pore number of trace E. (H) IV curves at pH 3 and 7.
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Scheme 1
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Insertion mechanism for CD-triazole-PEGs, according to the pH.
Fig. 1 presents the electrical measurements obtained with CD-triazole-PEG550 at pH = 7, showing the coexistence of two kinds of events. Important jumps are visible, corresponding to aggregates of cyclodextrins, and, among these populations, discrete single channel conductance events are obtained, corresponding to ‘‘unitary pores’’. The unitary pore current value was deduced to be around 1 pA for an applied voltage of 100 mV, which is similar to the one obtained in the presence of CD-PEG star polymers. The low conductance is explained by the low dielectric constant of the membrane, creating a high energy barrier.6c,15 In some traces, short events of low conductance values could be observed. The addition of one triazole functional group on the CD-PEG increases the length of the hydrophobic part of the molecule, leading to some monomolecular-based signals. The hemi-channel mechanism is supported by an asymmetric experiment consisting in adding the CD derivatives on one side of the chamber as already discussed.6c The pore formation occurred after a long time, suggesting the insertion of the CD derivative on one side of the lipid bilayer, followed by a flip-flop mechanism of the inserted CD derivatives to equilibrate the concentration of the pore-forming molecules on both sides of the lipid bilayer. Then, the occurrence of pores could be detected. As for the CD-PEG, the concentration used during the BLM experiments was below the critical aggregation concentration (Fig. S6, ESI†), the aggregation occurring then in the membrane and not in solution. Interestingly, the triazole functional groups shift the hydrophilic/lipophilic balance (HLB) of the pore-forming molecules towards a higher value, enabling the observation of the insertion of the CD-based pores at lower concentration. However, at pH = 3, only one kind of event was observed, corresponding to unitary pores, whatever the molar mass of the arm (Fig. 2), at the tested concentrations. It has to be recalled that protonation of the CD-triazole-PEG550 may occur from pH = 3.10 Thus, the absence of clusters at pH = 3 in the lipid membrane may be explained by the protonated triazole groups present on the primary face of the cyclodextrin star polymers, which creates an electrostatic hindrance between the pores, preventing cluster formation in the membrane. IV curves were
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obtained at pH 7 and pH 3. Conductances of 0.018 nS at pH 7 and 0.012 nS at pH 3 were obtained. The demonstration of the formation of pores by CD-triazole-PEG550 is achieved by two methods. The first one is based on the opening and closing of the pores (current versus time) and the second one considers the relationship between the number of opened pores and the measured intensity. Considering the hemi-channel insertion mechanism of the CD-PEG550, an insertion mechanism is proposed, as illustrated in Scheme 1, which explains the behavior of the pH-sensitive macromolecules in contact with the lipidic membrane. At pH = 7, the neutral triazole groups belong to the hydrophobic part of the channel, whereas at pH = 3, the slight protonation of the triazole shifts this function to the hydrophilic part of the channel, hindering the formation of clusters in the membrane and allowing the formation of isolated unitary pores, similar to the behavior of biological nanopores.
Notes and references 1 B. Hille, Ionic Channels of Excitable Membranes, Sinauer Associates, Inc., Sunderland, MA, 3rd edn, 2001. 2 (a) L. Song, M. R. Hobaugh, C. Shustak, S. Cheley, H. Bayley and J. E. Gouaux, Science, 1996, 274, 1859; (b) D. W. Urry, Proc. Natl. Acad. Sci. U. S. A., 1971, 68, 672; (c) H. U. Wilmsen, F. Pattus and J. T. Buckley, J. Membr. Biol., 1990, 115, 71. 3 (a) Z. S. Siwy and S. Howorka, Chem. Soc. Rev., 2010, 39, 1115; (b) X. Hou, W. Guo and L. Jiang, Chem. Soc. Rev., 2011, 40, 2385. 4 A. L. Sisson, M. R. Shah, S. Bhosale and S. Matile, Chem. Soc. Rev., 2006, 35, 1269. 5 (a) G. W. Gokel and A. Mukhopadhyay, Chem. Soc. Rev., 2001, 30, 274; (b) M. R. Ghadiri, J. R. Granja, R. A. Milligan, D. E. McRee and N. Khazanovich, Nature, 1993, 366, 324; (c) N. Yoshino, A. Satake and Y. Kokuke, Angew. Chem., Int. Ed., 2001, 40, 457; (d) K. Kim, N. Selvapalam, Y. H. Ko, K. M. Park, D. Kim and J. Kim, Chem. Soc. Rev., 2007, 36, 267. 6 (a) I. Tabushi, Y. Kuroda and K. Yokota, Tetrahedron Lett., 1982, 23, 4601; (b) L. Jullien, T. Lazrak, J. Canceill, L. Lacombe and J.-M. Lehn, J. Chem. Soc., Perkin Trans. 2, 1993, 1011; (c) N. Badi, ´gan, Adv. Mater., 2009, 21, 4054; (d) T. M. Fyles, L. Auvray and P. Gue Chem. Soc. Rev., 2007, 36, 335; (e) J. K. W. Chui and T. M. Fyles, Chem. Commun., 2010, 46, 4169; ( f ) J. K. W. Chui and T. M. Fyles, Chem. Soc. Rev., 2012, 41, 148. 7 W. H. Binder, Angew. Chem., Int. Ed., 2008, 47, 3092. 8 (a) C. Merstorf, B. Cressiot, M. Pastoriza-Gallego, A. G. Oukhaled, J.-M. Betton, L. Auvray and J. Pelta, ACS Chem. Biol., 2012, 7, 652; (b) L. Payet, M. Martinho, M. Pastoriza-Gallego, J.-M. Betton, ´, Anal. Chem., 2012, 84, 4071. L. Auvray, J. Pelta and J. Mathe ´, H. Visram, V. Viasnoff, Y. Rabin and A. Meller, Biophys. J., 9 J. Mathe 2004, 87, 3205. 10 R. Subbaraman, H. Ghassemi and T. Zawodzinski Jr., Solid State Ionics, 2009, 180, 1143. 11 (a) S. Srinivasachari, K. M. Fichter and T. M. Reineke, JACS, 2008, 130, 4618; (b) W. H. Binder and R. Sachsenhofer, Macromol. Rapid Commun., 2007, 28, 15; (c) A. Brik, J. Muldoon, Y. Lin, J. Elder, D. Goodsell, A. Olson, V. Fokin, B. Sharpless and H. Wong, ChemBioChem, 2006, 4, 1246; (d) Z. Li, T. Seo and J. Ju, Tetrahedron Lett., 2004, 45, 3143; (e) V. Rostovtsev, L. Green, V. Fokin and B. Sharpless, Angew. Chem., Int. Ed., 2002, 41, 2596. ´gan, Tetrahedron Lett., 2006, 47, 8925. 12 N. Badi, N. Jarroux and P. Gue 13 C. Huin, Z. Eskandani, N. Badi, A. Farcas, V. Bennevault-Celton and ´gan, Carbohydr. Polym., 2013, 94, 323. P. Gue 14 S. Matile and N. Sakai, The Characterization of Synthetic Ion Channels and Pores, in Analytical Methods in Supramolecular Chemistry, ed. C. Schalley, Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, Germany, 2007, p. 391. 15 A. Parsegian, Nature, 1969, 221, 844.
Chem. Commun., 2013, 49, 11647--11649
11649
Published on 18 October 2013. Downloaded by York University on 22/10/2014 10:54:39.
COMMUNICATION
Cite this: Chem. Commun., 2013, 49, 11647 Received 16th September 2013, Accepted 17th October 2013
View Article Online View Journal | View Issue
Biomimetic artificial ion channels based on beta-cyclodextrin† Yassine El Ghoul,a Ruddy Renia,a Ibrahima Faye,a Soumassoudrane Rassou,a ´ronique Bennevault-Celton,a Ce ´cile Huin*a and Philippe Gue ´ganc Nezha Badi,zb Ve
DOI: 10.1039/c3cc47071g www.rsc.org/chemcomm
Star polymers based on b-cyclodextrin were synthesized by a ‘‘click-chemistry’’ process and characterized by NMR, SEC and BLM. The resulting triazole functional groups create, at a specific pH range, electrostatic hindrance between pores inserted in lipid bilayers, preventing their aggregation, allowing formation of well-defined isolated unitary pores and mimicking biological natural channels.
Natural ion channels are essential for life, mainly because of their ability to regulate the flow of ions and molecules through the cell membrane. Natural macromolecules self-assemble to form isolated nanopores/nanochannels in the lipid bilayer (a-hemolysin, gramicidin A, aerolysin).1,2 Design of biomimetic systems is of prime importance to understand the nature and to develop devices useful for the bio- and nanotechnology fields (antimicrobial agents, nanofluidic devices, tools for translocation and detection, single-molecule experiments).1,3,4 The first requirement for the formation of suitable nanopores is the design of molecules partially soluble in water, such as monomers forming the self-assembled a-hemolysin.2a Numerous examples of synthetic amphiphilic systems forming artificial channels are reported in the literature,4–7 showing the necessity to work under very dilute conditions to avoid aggregation in solution or/and in the membrane. Aggregates are considered detrimental to nanotechnology applications, such as protein unfolding8 or nanopore force spectroscopy:9 the function of the nanopores is not ensured due to pollution from the aggregates. In the case of polymers, different kinds of interactions could exist with lipid a
LAMBE, UMR8587, UEVE-CNRS-CEA, Bld François Mitterrand, 91025 Evry, France. E-mail: [email protected]; Fax: +33 1 69 47 76 65; Tel: +33 1 69 47 77 17 b Institut Charles Sadron, UPR22-CNRS, 23 rue du Loess, BP 84047, 67034 Strasbourg Cedex 2, France c Laboratoire de Chimie des Polyme`res, UMR 7610, UPMC, 3 rue Galile´e, 94200 Ivry sur Seine, France † Electronic supplementary information (ESI) available: Experimental details: protocols used to synthesize the star polymers and physicochemical characterization (NMR, critical aggregation concentration). See DOI: 10.1039/c3cc47071g ‡ Present address: Precision Macromolecular Chemistry Group, Institut Charles Sadron, UPR22-CNRS, 23 rue du Loess, BP 84047, 67034 Strasbourg Cedex 2, France.
This journal is
c
The Royal Society of Chemistry 2013
bilayer membranes, involving supramolecular aggregates (amphiphilic block copolymers PEO–PPO–PEO) or formation of transient pores (hydrophilic polymers grafted hydrophobic side chains, polyelectrolytes).7 In order to ascertain the presence of unitary, isolated pores, steric or electrostatic hindrance should be encountered. Badi et al. demonstrated that star polymers based on b-cyclodextrin, bearing heptyl chains on the secondary face and poly(ethylene glycol) PEG arms on the primary face, may form well-defined channels, in the concentration range of 106 mol L1.6c However, a precise tuning of the concentration must be done to avoid aggregation. Here, we report the design of cyclodextrin (CD)-based channels, synthesized by the Huisgen [2+3] cycloaddition reaction, which behave as biomimetic isolated systems. The role of triazole groups, resulting from this reaction, was already mentioned by Fyles et al. for the design of artificial channels,6d–f mainly for the simple and efficient synthetic process. In addition, the triazole functional groups possess an interesting property of pH-sensitivity10 that has never been explored in the application of pore-forming molecules. The ‘‘click-chemistry’’ process allows the synthesis of per(2,3-di-O-heptyl)-6-methoxyPEG-6-(1,2,3-triazole)-b-CDs, named CD-triazole-PEG, by grafting 7 PEG branches on the primary face of a per(2,3-di-O-acetyl)-(6-deoxy-6-azido)-b-CD (MPEG = 550, 1100 or 2000 g mol1).6d–f,11 Deprotection of the acetyl groups and modification by heptyl chains of the secondary face of the cyclodextrin derivatives provide hydrophobicity,12 which is required for a possible insertion of the macromolecules in a lipidic bilayer.6c The high molar masses of star polymers containing 7 PEG arms of 1100 and 2000 g mol1 make the NMR characterization difficult and the evidence of the exact architecture difficult to ascertain. The star polymer with 7 arms of 550 g mol1 and the intermediates are discussed in the ESI† (Fig. S1 and S2 for the final product). 1H and 13 C NMR proves the quantitative modification of the cyclodextrin derivative (absence of the signal corresponding to the CH2–N3 in the 13 C NMR spectrum). DOSY NMR analysis shows only one population. The large difference in diffusion coefficients obtained by comparing a-methoxy-o-propargyl PEG550 (D = 1.7 1010 0.1 m2 s1 – Fig. S4, ESI†) and CD-triazole-PEG550 (D = 5.8 1011 0.2 m2 s1 – Fig. S3, ESI†) allows us to ascertain the star Chem. Commun., 2013, 49, 11647--11649
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polymer architecture and to demonstrate the complete absence of the linear polymer. The purity of the star polymer is also observed by size exclusion chromatography (Fig. S5, ESI†) with one monodisperse population and a molar mass in agreement with the theoretical one. Triazole functional groups have been envisioned as proton transport facilitators.10 Their pKa have been determined to be close to 1.2. However, bitriazole provides a higher proton affinity and its pKa is close to 3.2.10 This protonation property was then encountered by the CD-triazole-PEG derivatives. For these derivatives exhibiting a polyelectrolyte behavior, classical acid–base titration, 1H NMR experiments of CD-triazole-PEG550 in D2O at various pHs and isothermal titration calorimetry did not allow for the determination of pKa, due to the degradation of the PEG arms and the CD at such pHs.13 Dynamic light scattering, using different buffers (KCl 1 mol L1, Hepes 5 mmol L1, pH = 7 and KCl 1 mol L1, sodium citrate 5 mmol L1, pH = 3), shows self-aggregation of the amphiphilic star polymers, the critical aggregation concentration being around 2 102 mol L1, independently from the tested pH (Fig. S6, ESI†). In solution, hydrophobic interactions between heptyl chains seem to be predominant compared to electrostatic repulsions. Badi et al. proved, by the ‘‘black lipid membrane’’ (BLM) technique, that amphiphilic star PEG polymers based on cyclodextrin, without any triazole functional groups, called CD-PEG, form channels in the lipidic model membrane,6c only under very dilute conditions. Indeed, for M n ¼ 5000 g mol1 (MPEG arm = 530 g mol1), at pH = 7 and at a concentration below 4 106 mol L1, discrete jumps are observed and histograms, representing the number of events at each current, show the existence of well-defined unitary pores of mean intensity of 1.2 pA for an applied voltage of 100 mV (Fig. S7, ESI†). The Hill plot represents the open probability P0 of a single ion channel versus the polymer concentration. The obtained profile gives information about the stoichiometry of the assembly and the number of molecules involved in the pore formation, according to eqn (1). log P0 = n log C n log KD
Chem. Commun., 2013, 49, 11647--11649
2000 g mol1, permeabilization of the membrane is observed (Fig. S11, ESI†), which is not desirable for applications of artificial channels.
(1)
where n is the Hill coefficient and KD the dissociation constant. The determination of the Hill plot1,14 demonstrates the requirement of two cyclodextrin derivatives to form a unitary pore and provides direct evidence for the hemi-channel insertion mechanism (Fig. S8, ESI†). However, as soon as the concentration used is above 5 106 mol L1, the current values are much higher than the one obtained for unitary pores (Fig. S9, ESI†), current values not reproducible, depending on the experimental conditions (membrane capacitance for instance). These events correspond to the insertion of rafts, ‘‘aggregates’’ or ‘‘clusters’’ of cyclodextrin derivatives, visible whatever the pH. At this point, it is necessary to recall that the aggregation only occurs in the membrane and does not occur in the solution. The concentration used during the BLM experiments (around 106 mol L1) is indeed below the critical aggregation concentration, as proved by the radius distribution centred around 5 nm, corresponding to a unimer size (Fig. S10, ESI†). In addition, the molar mass of the star polymers is important: when using CD with arms above 11648
Fig. 1 BLM conductance measurements and analysis for CD-triazole-PEG550 at a concentration of 2 107 mol L1 at pH = 7, current intensity versus time for an applied voltage: 100 mV. (A) Complete BLM electrical measurements. (B) Zoom of the BLM electrical measurements on ‘unitary pores’ events. (C) Number of events versus current corresponding to trace B. (D) Zoom of the BLM electrical measurements on ‘clusters and unitary pores’ events. (E) Number of events versus current corresponding to trace D.
Fig. 2 BLM conductance measurements and analysis for CD-triazole-PEG550 at a concentration of 2 107 mol L1 at pH = 3, current intensity versus time for an applied voltage: 100 mV. (A) BLM electrical measurements – example 1. (B) Number of events versus current corresponding to trace A. (C) Current versus pore number of trace A. (D) BLM electrical measurements – example 2. (E) BLM electrical measurements – example 3. (F) Number of events versus current corresponding to trace E. (G) Current versus pore number of trace E. (H) IV curves at pH 3 and 7.
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Scheme 1
Communication
Insertion mechanism for CD-triazole-PEGs, according to the pH.
Fig. 1 presents the electrical measurements obtained with CD-triazole-PEG550 at pH = 7, showing the coexistence of two kinds of events. Important jumps are visible, corresponding to aggregates of cyclodextrins, and, among these populations, discrete single channel conductance events are obtained, corresponding to ‘‘unitary pores’’. The unitary pore current value was deduced to be around 1 pA for an applied voltage of 100 mV, which is similar to the one obtained in the presence of CD-PEG star polymers. The low conductance is explained by the low dielectric constant of the membrane, creating a high energy barrier.6c,15 In some traces, short events of low conductance values could be observed. The addition of one triazole functional group on the CD-PEG increases the length of the hydrophobic part of the molecule, leading to some monomolecular-based signals. The hemi-channel mechanism is supported by an asymmetric experiment consisting in adding the CD derivatives on one side of the chamber as already discussed.6c The pore formation occurred after a long time, suggesting the insertion of the CD derivative on one side of the lipid bilayer, followed by a flip-flop mechanism of the inserted CD derivatives to equilibrate the concentration of the pore-forming molecules on both sides of the lipid bilayer. Then, the occurrence of pores could be detected. As for the CD-PEG, the concentration used during the BLM experiments was below the critical aggregation concentration (Fig. S6, ESI†), the aggregation occurring then in the membrane and not in solution. Interestingly, the triazole functional groups shift the hydrophilic/lipophilic balance (HLB) of the pore-forming molecules towards a higher value, enabling the observation of the insertion of the CD-based pores at lower concentration. However, at pH = 3, only one kind of event was observed, corresponding to unitary pores, whatever the molar mass of the arm (Fig. 2), at the tested concentrations. It has to be recalled that protonation of the CD-triazole-PEG550 may occur from pH = 3.10 Thus, the absence of clusters at pH = 3 in the lipid membrane may be explained by the protonated triazole groups present on the primary face of the cyclodextrin star polymers, which creates an electrostatic hindrance between the pores, preventing cluster formation in the membrane. IV curves were
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The Royal Society of Chemistry 2013
obtained at pH 7 and pH 3. Conductances of 0.018 nS at pH 7 and 0.012 nS at pH 3 were obtained. The demonstration of the formation of pores by CD-triazole-PEG550 is achieved by two methods. The first one is based on the opening and closing of the pores (current versus time) and the second one considers the relationship between the number of opened pores and the measured intensity. Considering the hemi-channel insertion mechanism of the CD-PEG550, an insertion mechanism is proposed, as illustrated in Scheme 1, which explains the behavior of the pH-sensitive macromolecules in contact with the lipidic membrane. At pH = 7, the neutral triazole groups belong to the hydrophobic part of the channel, whereas at pH = 3, the slight protonation of the triazole shifts this function to the hydrophilic part of the channel, hindering the formation of clusters in the membrane and allowing the formation of isolated unitary pores, similar to the behavior of biological nanopores.
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