Biomimetic Nanotubes Based on Cyclodextrins for Ion-Channel

Letter pubs.acs.org/NanoLett

Biomimetic Nanotubes Based on Cyclodextrins for Ion-Channel Applications Hajar Mamad-Hemouch,† Hassen Ramoul,† Mohammad Abou Taha,† Laurent Bacri,† Cécile Huin,† Cédric Przybylski,† Abdelghani Oukhaled,‡ Bénédicte Thiébot,‡ Gilles Patriarche,§ Nathalie Jarroux,*,† and Juan Pelta*,† †

Université d’Evry-Val-d’Essonne, LAMBE, UMR CNRS 8587, bd F. Mitterrand, 91025 Evry, France Université de Cergy Pontoise, LAMBE, UMR CNRS 8587, 2 avenue A. Chauvin, 95302 Cergy Pontoise, France § LPN CNRS, UPR20, 91460 Marcoussis, France ‡

S Supporting Information *

ABSTRACT: Biomimetic membrane channels offer a great potential for fundamental studies and applications. Here, we report the fabrication and characterization of short cyclodextrin nanotubes, their insertion into membranes, and cytotoxicity assay. Mass spectrometry and high-resolution transmission electron microscopy were used to confirm the synthesis pathway leading to the formation of short nanotubes and to describe their structural parameters in terms of length, diameter, and number of cyclodextrins. Our results show the control of the number of cyclodextrins threaded on the polyrotaxane leading to nanotube synthesis. Structural parameters obtained by electron microscopy are consistent with the distribution of the number of cyclodextrins evaluated by mass spectrometry from the initial polymer distribution. An electrophysiological study at single molecule level demonstrates the ion channel formation into lipid bilayers, and the energy penalty for the entry of ions into the confined nanotube. In the presence of nanotubes, the cell physiology is not altered. KEYWORDS: Cyclodextrin nanotubes, biomimetic ion channels, single molecule, lipid membrane, cytotoxicity assays, ion transport

B

iomimetic membrane channels,1−6 synthetic, protein, solid-state nanopores and nanotubes coupled with an electric detection, are powerful tools to address fundamental questions at the single-molecule level and to develop medical, biotechnological, and energy applications. Potential applications include protein folding7,8 dynamics of ions, liquid, or molecules in confined medium,6,9−11 and molecule−ligand interactions.12−14 Several applications concern ultrafast sequencing of DNA/RNA,15 single-molecule mass spectrometry,16,17 disease or pathogenic agents detection,2,18,19 and energy conversion.6 Upcoming applications are dependent on the design of new biomimetic channels for biological imaging, medical therapy, biotechnology, and the renewal industrial. Synthetic channels either based on macrocycles1 or nanotubes comprising carbon,5,20 DNA,4,21,22 or peptides1,23 have been designed to form ion conducting channels into membranes. Few translocation studies have been performed with these channels and concern only DNA4 and carbon5,20 nanotubes and generally these nanotubes remain cytotoxic.21,24 However, several challenges should be addressed for the development of future applications: versatile synthesis, in terms of pore diameter, length, short monodisperse nanotubes manufactured to optimize the formation of membrane channels and to obtain sensors with a high resolution and sensitivity; the absence of cytotoxicity for potential medical applications; the ability to © 2015 American Chemical Society

produce a huge quantity of monodisperse nanotubes for industrial applications. To address these challenges, we used natural cyclic oligosaccharides, cyclodextrins, obtained by enzymatic conversion of starch. Modified cyclodextrins are well-known to form ion-conducting channels25 and for their industrial applications.26,27 We have manufactured new short α-cyclodextrin nanotubes from modified Harada’s synthesis method28 (Figure 1). In this chemical reaction the number of cyclodextrins threaded on the polyrotaxane is not controlled. The general pathway consists of four steps: cyclodextrins threaded on a polymer chain named pseudopolyrotaxane (1), addition of bulky groups at each end-chain leading to a polyrotaxane (2), formation of the nanotubes by creating covalent bonds (3), and hydrolysis of the blocking groups leading to the nanotube (4). We have optimized the synthesis at step 2 by using a radical coupling preventing side reactions and allowed for faster kinetics than the aromatic nucleophilic reaction initially used by Harada. As a consequence, the number of unthreaded cyclodextrins resulting from polyrotaxane formation can be controlled as well as the nanotube length. Received: September 28, 2015 Revised: October 14, 2015 Published: October 16, 2015 7748

DOI: 10.1021/acs.nanolett.5b03938 Nano Lett. 2015, 15, 7748−7754

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Figure 1. Synthesis route and characterization by MALDI-TOF MS of CDNT1000. (a) Illustration of the reaction steps leading to CDNT1000. The synthesis consists of the formation of a pseudopolyrotaxane PPR based on α-cyclodextrins (in red) and α−ω dimethacrylate PEO (in green), formation of the PR structure by addition of pyrene based blocking groups (in blue) using a radical coupling reaction, and formation of nanotubes by creating covalent diether bonds (in gray). Nanotubes are finally isolated after hydrolysis of the bulky blocking groups. (b) Characterization of the nanotubes by MALDI-TOF MS analysis. The spectrum highlights the presence of three CDNTs populations. The first one matches with a dimer of CDs, the second one is consistent with a trimer of CDs, and the third one corresponds to four CDs linked by several diether bridges. A detailed analysis proved presence of one sulfate modification on each CD of the CDNT and enabled to attribute each peak a number of CDs linked with a precise amount of diether bridges (Supplementary Data 1).

The reliability of the modified synthesis pathway has been verified by MALDI-TOF mass spectrometry (MS) and a structural analysis was conceivable with a resolution around the nanometer due to the precision supplied by high-resolution transmission electronic microscopy (HRTEM). In order to optimize nanotube insertion into membranes, channel length is adjusted to the lipid membrane thickness. We have performed covalent assembly between cyclodextrins to favor channel stability into the membrane by comparison with hemichannels based on cyclodextrin.25 Electrical measurements were used to probe the capability of these nanotubes to form membrane channels into lipid bilayer and examine the channels’ sensitivity as a sensor. The future potential for pharmaceutical applications was examined by cytotoxicity assays. To demonstrate the nanotube length control, two types of polymer chains of various number-average molar mass (Mn), a dimethacrylated 1000 g·mol−1 PEO chains (PEO1000) and a dihydroxylated 1500 g·mol−1 PEO chains (PEO1500), were used. In both cases, the oligosaccharide used is the αcyclodextrin, leading to two kinds of nanotubes, CDNT1000 and CDNT1500, respectively. The nanotubes synthesis is explained Figure 1. The four-step chemical pathway used to obtain the CDNTs was confirmed by MALDI-TOF MS analysis (Figure 1, Supplementary Data 1). Here, the crucial improvement of the nanotube synthesis is during the polyrotaxane (PR) formation from the pseudopolyrotaxane (PPR), by capping the edges of the polymer chain by pyrene based blocking groups, whose steric properties prevent the

unthreading of the CDs. These groups are added by a radical coupling reaction performed between the methacrylate functions of the polymer chain and 1-pyrene butyric acid Nhydroxysuccinimid ester in a solvent mixture of water/DMSO. This solvent mixture, and particularly the proportion of water, has a major role in the control of the number of CDs threaded on the PR.29 The MALDI-TOF MS spectrum analysis confirms the expected structure and proves linkage between CDs by a consistent number of diether bridges. The low molecular masses desorb better than the higher molecular masses, then the MALDI-TOF MS analysis supports the chemical route but cannot be interpreted as a direct quantitative analysis. In order to obtain quantitative structural information, we have performed HRTEM experiments with CDNT1000 and CDNT1500 nanotubes (Figures 2 and S2). We can observe the alignment of the nanotubes (Figure 2a) and the cyclodextrins along the nanotube (Figure 2a,b). We have made a statistical analysis of each structural parameters (Figure 2d−i) from several HRTEM pictures (20 pictures, 194 counts). We have obtained the distributions of nanotube length (Figure 2d), diameter (Figure 2e), cyclodextrin length (Figure 2f), number of CDs (Figure 2i), distance between two CDs (Figure 2g), and two nanotubes (Figure 2h). We have compared the experimental nanotube length to the expected length according to the polymer molecular weight (Supplementary Data 3). The control of the synthetic pathway shows that experimental results determined by HRTEM are consistent with the 7749


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Figure 2. HRTEM image of CDNT1000. (a) Picture of CDNT1000 obtained with conventional HRTEM. (b) Zoom on the area delineated by the red frame. (c) Fourier transform pattern obtained from CDNT1000s of the area delineated by the red frame and enabling to obtain the distance between two CDs of the same CDNT1000. (d) Length distribution of CDNT1000 measured from HRTEM images (lNT = 2.7 ± 1.1 nm). (e) Diameter distribution of CDNT1000 measured from HRTEM images (Ø = 4.6 ± 0.5 Å). (f) Cyclodextrin length distribution of CDNT1000 measured from HRTEM images (lCD = 5.3 ± 0.9 Å). (g) Distribution of the distance between two CDs of the same CDNT1000 measured from HRTEM images (dCD = 3.1 ± 0.3 Å). (h) Distribution of the distance between two CDNT1000 measured from HRTEM images (dNT = 1.1 ± 0.2 Å). (i) Distribution of the number of CDs composing a CDNT1000 counted on the HRTEM images (nCD = 6 ± 3).

change the structural parameters of the individual nanotubes within the standard deviation. For NT1500, the average length and the number of CDs increase (Table 1). The mean experimental length increased from 2.7 ± 1 to 4.5 ± 1 nm, demonstrating the dependence of the chain molecular weight on the synthesized nanotube (Supplementary Data 3). The average values are obtained from Gaussian fits realized on each distribution (Table 1). To determine whether the small nanotubes based on αcyclodextrins could form ion-channels we performed electrical measurements with a nanopore setup (Figure 4) after membrane lipid bilayer formation. Without CDNT, we do not observe any current fluctuation (data not shown). After addition of the same amount of CDNT in both compartments

distribution of the CD number evaluated by MALDI-TOF MS from initial PEO distribution. To avoid aggregates or supramolecular organization of the concentrated nanotube solution due to hydrogen bonding, we have manufactured silylated CDNTs based on PEO1000 (S1) and observed these nanotubes by CryoTEM (Figure 3). The pictures show only individual nanotubes without any aggregate. As expected, the silylation modification made on the nanotubes prevents either nanotubes association or supramolecular organization. We have compared the structural parameters of individual nanotubes in terms of length (Figure 3d), diameter (Figure 3e), cyclodextrin length, (Figure 3f), and number of CDs (Figure 3g) to those obtained with the native nanotube solution (Table 1). The supramolecular organization does not 7750


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Figure 3. CryoTEM image of CDNT1000Si. (a) Isolated CDNT1000Si obtained by CryoTEM. (b,c) Zoom on isolated CDNT1000Si (red frames) of the previous TEM image. (d) Length distribution of CDNT1000Si measured from TEM images (lNT = 2.8 ± 1.1 nm). (e) Diameter distribution of CDNT1000Si measured from TEM images (Ø = 3.9 ± 0.8 Å). (f) Cyclodextrin length distribution of CDNT1000Si measured from TEM images (lCD = 5.6 ± 1.4 Å). (g) Distribution of the number of CDs composing a CDNT1000Si counted on the TEM images (nCD = 6 ± 2).

Table 1. Average Structural Parameters of CDNT1000, CDNT1000Si and CDNT15000 Obtained after Statistical Measurements on the HRTEM Image Analysis CDNT1000 CDNT1000Si CDNT1500

lNT (nm)

ØNT (Å)

lCD (Å)

nCD (counted)

dNT (Å)

dCD (Å)

2.7 ± 1.1 2.8 ± 1.1 4.5 ± 1.0

4.6 ± 0.5 3.9 ± 0.8 3.8 ± 0.7

5.3 ± 0.9 5.6 ± 1.4 5.9 ± 1.0

6±3 6±2 10 ± 2

1.1 ± 0.2

3.1 ± 0.3

1.1 ± 0.2

3.1 ± 0.4

membrane. By using a numeric method commonly used in blockade detection,30 we are able to locate each nanotube insertion, characterized by its current magnitude and time duration. Then, the measurement of the current insertion magnitude of one CDNT is more accurate than the usual method based on the current distribution of the entire current trace: the peaks are twice as narrow with a location error of 6.0 ± 0.1 pA (Figure 4d). Both current histogram and insertion detection methods result in the same current level measure-

(Figure 4a), the magnitude of ionic current through the lipid bilayer fluctuates with high amplitude (Figure 4b,i). If we increase the time resolution on a part of this current trace, these fluctuations show several well-characterized jumps of current (Figure 4b,ii). The difference of position between two successive peaks is steady, allowing a quantification of each current level (Figure 4c). Each insertion is characterized by a specific current gap, called “unitary current” (Figure 4e). This result shows that we probe the insertion of nanotube inside the 7751


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Figure 4. Cyclodextrin short nanotubes (CDNT) insertion into lipid membrane and ionic transport. (a) Set-up for current and conductance recording though CDNT1000. Two compartments (cis and trans) filled with salt and nanotube solution are separated by a lipid bilayer. Voltage is applied by two Ag/AgCl electrodes. (b) Ionic current trace of insertions of 1, 2, 3, and 4 individual nanotubes (i,ii). (c) Distribution of ionic current through CDNT (n = 37 × 103). Each peak is fitted by a Gaussian distribution characterized by its position and standard deviation. (d) Opening current of each CDNT1000 insertion separated by 7.8 ± 1.0 pA. (e) Opening current according to the inserted CDNT1000 channel number. The data are determined from current distribution (b,c). The applied voltage is ΔV = 100 mV, and the buffer is composed of 1 M KCl and 5 mM HEPES pH 7.5. (f) Distribution of CDNT conductance (n = 37 × 103). Each peak is fitted by a Gaussian distribution characterized by its position and standard deviation separated by 0.078 ± 0.01 nS. (g) Distribution of the conductance of each CDNT insertion. (h) IV curve of CDNT channels. The unitary currents (red dots) are calculated from the current traces. The blue IV curve is normalized from the insertion of 25 channels, 1 M KCl, 5 mM HEPES, pH 7.5. 7752


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Figure 5. Effect of CDNT1000 on growth and morphology of MDA-MB231 and MCF7. (A) Initial cell number seeded was 104 cells per well in 96 well plates. Cell proliferation for 72 h in the presence of FCS. (B) No cytotoxic effect of CDNT1000 until 1 mM for MDA-MB231 (circle) and MCF7 (square) with FCS. (C) No change in cell morphology treated with 0.1 or 1 mM of CDNT1000, in the presence of FCS. Cell morphology was observed with light microscopy (200×).

magnitude is equal to δE = 2.2 ± 0.2 kBT. As the free energy of the membrane deformations is estimated to 3−5 kBT,32 the magnitude of the thermal fluctuations is large enough to distort the lipid bilayer and to facilitate nanotube insertion. Then, we assume that the real channel length is 2.7 < l (nm) < 5 and that the average energy barrier magnitude is δE = 2.5 ± 0.4 kBT. Now, we can consider the gramicidin-A channel31 where both radius (about 0.2 nm) and length (about 2.5 nm) are similar to the biomimetic nanotube ion-channel. However, gramicidin conductance is smaller (∼41 pS in 1 M KCl)33 than the nanotube. This result suggests that the membrane deformation curvature is not the same for both channels, and therefore, the apparent length of the gramicidin is larger than the CDNT one. If we examine the energy barrier for gramicidin, this one is ∼1.8 kBT, which is not very different from the CDNT one. This fact could be explained by the strong confinement of the molecules inside the gramicidin channel, which corresponds to seven water molecules and one ion.34 Then, channel length deviation does not influence the entrance energy barrier δE, but the ion mobility into the channel is certainly modified by interactions between the ions and the inner channel. The next step is to examine cytotoxicity of the nanotubes. We use a sensitive cytotoxicity test enzyme-based method to detect mitochondrial dehydrogenase activity in the living cells (Figure 5a). The optimum cell concentration, as determined by the growth profile of MDA-MB231 and MCF7, was 104 cells per well. In the presence of fetal calf serum (FCS), cells

ment of 7.8 ± 1.0 pA (Figure 4e). The CDNT conductance found is 0.077 ± 0.005 nS (Figure 4h). To discuss these results, we consider the nanotube as a cylinder characterized by its radius r and its length l. Then, from the resolution of Nersnt− Planck equation,31 we obtain the theoretical conductance G0 of the channel:

G0 = 2πr 2cλ /l

(1)

where λ is the KCl equivalent conductance in water and c the KCl concentration. From the nanotube length (2.7 ± 1.1 nm) and diameter (4.6 ± 0.5 Å) measured by HRTEM (see Table 1), we estimate this theoretical conductance G0 ≈ 1.3 ± 1 nS. This value is 17 times larger than the conductance measured in our experiments. In order to explain this difference, we take into account the energy cost or energy barrier (δE) for the entry of ions in confined medium into the nanotubes. Then, the relation 1 must be modified as G = G0 exp( −δE /kBT )

(2)

where T is the absolute temperature and kB the Boltzmann constant. From this expression, we deduce the magnitude of the energy barrier per kBT: δE = −ln(G/G0). We found δE = 2.8 ± 0.7 kBT. In this calculation, we assume that the apparent channel length is equal to the CDNT length. If we consider that the largest value of this apparent length is equal to the lipid bilayer thickness (∼5 nm), the calculated conductance decreases to G0 ≈ 0.7 ± 0.15 nS, and the new energy barrier 7753


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(5) Geng, J.; Kim, K.; Zhang, J.; Escalada, A.; Tunuguntla, R.; Comolli, L. R.; Allen, F. I.; Shnyrova, A. V.; Cho, K. R.; Munoz, D.; Wang, Y. M.; Grigoropoulos, C. P.; Ajo-Franklin, C. M.; Frolov, V. A.; Noy, A. Nature 2014, 514, 612−615. (6) Siria, A.; Poncharal, P.; Biance, A.-L.; Fulcrand, R.; Blase, X.; Purcell, S. T.; Bocquet, L. Nature 2013, 494, 455−458. (7) Oukhaled, G.; Mathé, J.; Biance, A.-L.; Bacri, L.; Betton, J.-M.; Lairez, D.; Pelta, J.; Auvray, L. Phys. Rev. Lett. 2007, 98, 158101. (8) Nivala, J.; Marks, D. B.; Akeson, M. Nat. Biotechnol. 2013, 31, 247−250. (9) Bezrukov, S. M.; Vodyanoy, I.; Parsegian, V. A. Nature 1994, 370, 279−281. (10) Song, W.; Pang, P.; He, J.; Lindsay, S. ACS Nano 2013, 7, 689− 694. (11) Biesemans, A.; Soskine, M.; Maglia, G. Nano Lett. 2015, 15, 6076−6081. (12) Wei, R.; Gatterdam, V.; Wieneke, R.; Tampé, R.; Rant, U. Nat. Nanotechnol. 2012, 7, 257−263. (13) Mohammad, M. M.; Iyer, R.; Howard, K. R.; McPike, M. P.; Borer, P. N.; Movileanu, L. J. Am. Chem. Soc. 2012, 134, 9521. (14) Plesa, C.; Ruitenberg, J. W.; Witteveen, M. J.; Dekker, C. Nano Lett. 2015, 15, 3153−3158. PMID: 25928590. (15) Jain, M.; Fiddes, I. T.; Miga, K. H.; Olsen, H. E.; Paten, B.; Akeson, M. Nat. Methods 2015, 12, 351−356. (16) Robertson, J. W. F.; Rodrigues, C. G.; Stanford, V. M.; Rubinson, K. A.; Krasilnikov, O. V.; Kasianowicz, J. J. Proc. Natl. Acad. Sci. U. S. A. 2007, 104, 8207−8211. (17) Baaken, G.; Halimeh, I.; Bacri, L.; Pelta, J.; Oukhaled, A.; Behrends, J. C. ACS Nano 2015, 9, 6443−6449. (18) Yusko, E. C.; Johnson, J. M.; Majd, S.; Prangkio, P.; Rollings, R. C.; Li, J.; Yang, J.; Mayer, M. Nat. Nanotechnol. 2011, 6, 253−260. (19) Pagès, J.-M.; James, C. E.; Winterhalter, M. Nat. Rev. Microbiol. 2008, 6, 893−903. (20) Liu, L.; Yang, C.; Zhao, K.; Li, J.; Wu, H.-C. Nat. Commun. 2013, 4, 2989. (21) Burns, J. R.; Al-Juffali, N.; Janes, S. M.; Howorka, S. Angew. Chem. 2014, 126, 12854−12854. (22) Seifert, A.; Göpfrich, K.; Burns, J. R.; Fertig, N.; Keyser, U. F.; Howorka, S. ACS Nano 2015, 9, 1117−1126. (23) Ghadiri, M. R.; Granja, J. R.; Buehler, L. K. Nature 1994, 369, 301−304. (24) Hong, G.; Diao, S.; Antaris, A. L.; Dai, H. Chem. Rev. 2015, 115, 10816. (25) Bacri, L.; Benkhaled, A.; Guegan, P.; Auvray, L. Langmuir 2005, 21, 5842−5846. (26) Del Valle, E. M. Process Biochem. 2004, 39, 1033−1046. (27) Davis, M. E.; Brewster, M. E. Nat. Rev. Drug Discovery 2004, 3, 1023−1035. (28) Harada, A.; Li, J.; Kamachi, M. Nature 1993, 364, 516−518. (29) Jarroux, N.; Guégan, P.; Cheradame, H.; Auvray, L. J. Phys. Chem. B 2005, 109, 23816−23822. (30) Oukhaled, A.; Bacri, L.; Pastoriza-Gallego, M.; Betton, J.-M.; Pelta, J. ACS Chem. Biol. 2012, 7, 1935−1949. (31) Jordan, P. C. Biophys. Chem. 1981, 13, 203−212. (32) Helfrich, P.; Jakobsson, E. Biophys. J. 1990, 57, 1075−1084. (33) Hladky, S.; Haydon, D. Biochim. Biophys. Acta, Biomembr. 1972, 274, 294−312. (34) Allen, T. W.; Andersen, O. S.; Roux, B. Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 117−122.

underwent an exponential growth until a cell culture period of 72 h (data not shown). Adherent cells were preincubated for 24 h in DMEM in the presence of FCS, without nanotubes, to get back to their exponential growth phase. Then, cells were incubated with CDNT1000 from 0 to 1 mM for 48 h in the media previously indicated. The fluorescence intensity remains constant as a function of nanotubes concentration for the two types of cell (Figure 5b) and demonstrate no alteration in cell proliferation. We have also observed cell morphology (Figure 5c). The shape of the living cells remained the same with or without presence of cyclodextrin nanotubes. These results suggest no alteration in cell physiology. To conclude, we have demonstrated the synthesis and the control of the cyclodextrin nanotube’s structural parameters length, diameter, and cyclodextrin number. The experimental nanotube length fits the expected length according to the molecular weight of the polymer. The number of cyclodextrins determined highresolution transmission electron microscopy is consistent with the distribution of the number of cyclodextrins evaluated by mass spectrometry from the initial polymer distribution. These small noncytotoxic nanotubes form well-defined biomimetic ion-conducting channels. The dynamics of ion transport and sensitivity are similar to biological channels. The energy penalty, few kBT, for the entry of ions into the confined nanotube is similar to the gramicidin channel, but the ions mobility is different inside this channel. These synthetic nanotubes are able to be mass produced easily making them a suitable material for biotechnology and industrial applications.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.nanolett.5b03938. Additional materials related to the fabrication process, experimental methods as well as supporting figures (PDF)

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AUTHOR INFORMATION

Corresponding Authors

* E-mail: [email protected]. * E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Agence Nationale de la Recherche (ANR 12-NANO-0012-03) and the Region Ile-deFrance in the Framework of DIM Nano-K (No. 094251). We thank Reagan Meredith for kindly correcting the language of the manuscript.

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REFERENCES

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Biomimetic Nanotubes Based on Cyclodextrins for Ion-Channel

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