Nanoplasmonically-Induced Defects in Lipid Membrane Monitored by

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Nanoplasmonically-Induced Defects in Lipid Membrane Monitored by Ion Current: Transient Nanopores versus Membrane Rupture Raghavendra Palankar, Bat-El Shani Pinchasik, Boris N. Khlebtsov, Tatiana A. Kolesnikova, Helmuth Möhwald, Mathias Winterhalter, and Andre G. Skirtach Nano Lett., Just Accepted Manuscript • DOI: 10.1021/nl500907k • Publication Date (Web): 25 Jun 2014 Downloaded from http://pubs.acs.org on July 8, 2014

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Nanoplasmonically-Induced Defects in Lipid Membrane Monitored by Ion Current: Transient Nanopores versus Membrane Rupture

Raghavendra Palankar,a,b,# Bat-El Pinchasik,c,# Boris N. Khlebtsov,d Tatiana A. Kolesnikova,b,c Helmuth Möhwald,c Mathias Winterhalter,b Andre G. Skirtach,c,e,f,*

a

ZIK HIKE, Nanostructure Group, Ernst-Moritz-Arndt-Universität Greifswald, 17489 Greifswald, Germany b Biophysics, School of Engineering and Sciences, Jacobs University of Bremen, 28759 Bremen, Germany c Max Planck Institute of Colloids and Interfaces, Potsdam, D-14424 Potsdam, Germany d Russian Acad Sci., Inst. Biochem & Physiol Plants & Microorganisms, Saratov 410049, Russia e Department of Molecular Biotechnology, Ghent University, 9000 Ghent, Belgium, and f NB (NanoBio)-Photonics, Ghent University, 9000 Ghent, Belgium

# These authors contributed equally to this work * Corresponding Author, E-mail: [email protected]

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ABSTRACT: We have developed a nanoplasmonic-based approach to induce nanometer-sized local defects in the phospholipid membranes. Here, gold nanorods and nanoparticles having plasmon resonances in the near-infrared (NIR) spectral range are used as optical absorption centers in the lipid membrane. Defects optically induced by NIR-laser irradiation of gold nanoparticles are continuously monitored by high-precision ion conductance measurement. Localized laser-mediated heating of nanorods and nanoparticle aggregates cause either a) transient nanopores in lipid membranes or b) irreversible rupture of the membrane. To monitor transient opening and closing, an electrophysiological setup is assembled, wherein a giant liposome is spread over a micrometer hole in a glass slide forming a single bilayer of high Ohmic resistance (so-called gigaseal), while a laser light is coupled in and focused on the membrane. The energy associated with the localized heating is discussed and compared with typical elastic parameters in the lipid membranes. The method presented here provides a novel methodology for better understanding of transport across artificial or natural biological membranes.

KEYWORDS: nanopore, liposomes, ion current, nanoparticles, electrophysiology, lipids


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A fundamental principle of life is the compartmentalization into well-defined “in” and “out” volumes separated by a cell envelope. To control the permeability of ions, nutrients, metabolic intermediates, proteins or drugs, nature developed numerous protein channels as molecular gates. Malfunction of the permeability control may cause severe diseases.1 Most of these channels allow selective passive diffusion of small molecules, and the underlying principles are the basis for molecular filtering. In addition to molecular selectivity channels sense transmembrane voltages or pH gradients and may act by reversible opening or closing. Switchable devices are interesting for nanobiotechnological applications in particular in the area of controlled delivery.2,

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During recent years

efforts have been made to utilize membrane proteins in switchable devices for drug release and uptake. As an exciting example, the otherwise mechanical stress induced gating of the MscL channel has been transformed into a light switch.2 In the last decade, a novel principle based on laser-mediated photo-/optoporation of phospholipid membranes with gold4 nanoparticles (so called optical injection) has been developed and employed for the delivery of hydrophilic substances through the biological membranes.5-7 Application of a single laser source allowing for both activation and injection of nanoparticles in a single step experiment is the main advantage of this approach. However, to introduce gold nanoparticles inside the lipid vesicles or cells, UV or green laser light was commonly used, increasing the risk of photochemical damage of cells due to the lack of biocompatibility of UV-light and/or generation of reactive oxygen species (ROS) upon irradiation.8, 9 In contrast the low absorption of biomolecules and water in the infra-red (IR) frequency range renders IR-laser irradiation particularly attractive.10 Using IR-light reduces side-effects to the individual cells and biological tissue, and allows development of applications ranging from cancer treatment to gene therapy.11-15 Nanoplasmonics covers in general all electromagnetic field effects of light with nanoparticles. Intensive research revealed that the shape of individual or aggregates of nanoparticles provides strong


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absorption in the near-infrared range of the spectrum. Localized heating around gold nanoparticles has been used for release of biomolecules from liposomes [see review3] or microcapsules functionalized with nanoparticles.16, 17 In this work we develop a novel approach to induce nanometer-sized local defects in the phospholipid membranes by NIR-laser irradiation of gold nanoparticles. We demonstrate that nanoplasmonic absorption centers: gold rods (referred to as nanorods or AuNR) can be used as active absorption centers for transient laser-triggered opening of the phospholipid membrane of giant unilamellar vesicles (GUVs) with subsequent sealing of the membrane, whereas gold nanoparticles forming larger aggregates (referred to as nanoparticles or AuNP) cause membrane rupture. The action of laser light on nanoplasmonic absorption centers is ascribed to localized resonant optical heating around these absorbing centers causing nanopore formation. Throughout the whole text, AuNR and AuNP are also referred to as absorbing nanocenters. This is conducted by assembling a novel setup in which a laser beam is focused on a micrometer sized hole over which a giant liposome is spread (due to pressure difference) (Figure 1). Further experiments are conducted by monitoring ion current after adding absorbing centers on the membrane and exposing it to the laser. Along with other fluorescent methods of temperature measurement,18 electrophysiology has been used for monitoring the temperature rise and profiling the intensity distributions.19, 20 Compared to the standard fluorescence microscopy-based approaches electrophysiology

allows detection of single

nanosized defects (nanopores) induced by localized plasmonic heating of absorbing centers in the lipid membrane with high temporal resolution. Therefore, it is particularly attractive for studying the mechanism of membrane permeability upon laser irradiation. Furthermore, the method presented here provides a novel methodology for optimization of controlled transport of molecules through the artificial or biological membranes.


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Figure 1, I schematically presents our experimental setup assembled to record the ionic current across the lipid membrane. Giant liposomes were spread on top of a micrometer sized hole in a glass slide. Subsequently, the liposomes were aspirated from below (external pump is not shown) to form lipid bilayers across the hole in the glass slide (for materials and Methods see Supporting Information and Ref.

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for details). Prior each experiment, formation of a bilayer was verified by a capacitance

measurement as a standard routine. As the capacitance depends on the thickness, a multilayer corresponds to substantially reduced capacitance value (more than half as also the inter-bilayer water has to be accounted for) comparing to a planar bilayer. Though the bilayer capacitance varies slightly from one experiment to another, formation of multilayer or other non-bilayer structures can be certainly excluded. Conductance measurements performed on this planar lipid membrane revealed a so-called gigaseal, an electrically dense membrane reducing the ion currents substantially below < 1 pA even at applied trans-membrane voltages up to 100-200 mV. Neither illumination of the lipid membrane by laser nor sole addition of gold nanorods without laser did influence the conductance. Figure 1, II shows the main steps in alignment of the laser exactly on the top of the hole in the glass slide. By careful positioning the laser beam on top of the hole, first without and then with buffer solution, we assured that the laser always stayed focused on the hole (II). Figure 2 presents typical recordings for AuNR. In our set-up the membrane permeability is followed by the onset of conductance distributed around 10.6±1.8 pA, 25.4±1.6 pA, 50.5±3.1, 80.2±0.9 pA and 139.6±5.8 pA at 1 M KCl. It is essential to note that our experiments were performed only after the absorbing nanocenters were “settled” on the lipid membrane, while monitoring has been continuously performed starting with the initial gigaseal formation. During these measurements the AuNR alone do not influence the stability of the membrane (which was electrically sealed before and after AuNR absorption, (Figure S1, Supporting Information). Salt concentration22 and surface charges23 influence the stability of the lipid membrane only weakly. In a control experiment, irradiation of the


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lipid membrane with near-IR laser in absence of absorbing nanocenters did not cause any conductivity (Figure S2, Supporting Information). This is qualitatively expected since only high power (and higher absorption) affects the lipid membrane.7,

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A more detailed discussion is provided in thermal

distribution and nanorod location analysis (Figures 3 and 4). To investigate the second case causing the membrane rupture, we conducted experiments using gold nanoparticles (AuNP) instead of gold nanorods (AuNR). The essential difference between these two cases was that in our experiment the AuNP were smaller in size (up to 10 nm) and present at much higher concentration (> 1014 NP/mL, at least two orders of magnitude higher number than that for AuNR). In addition, AuNP tend to cluster in a physiological buffer forming large aggregates.25 The transient current changes detected in case of AuNR (Figure 3 (a), red arrows) show current versus time traces. The onset of transient current change is observed without a large initial current change and no essential dip has been registered upon addition of AuNR. This is consistent with optical images of a giant liposome (Figure S3 (a), Supporting Information), where no aggregates were observed. A very different phenomenon is observed upon addition of AuNP. Figure 3 (b) shows a very distinct current change. The initial current dip marked by the light blue arrow in Figure 3 (b). One possibility for the occurrence of this dip might be the formation of AuNP aggregates consistent with previous studies of nanoparticle interaction with red blood cells, when interaction of AuNP with lipids and oligosaccharides was confirmed by Raman microscopy.25 Another distinct phenomenon observed in our studies is the rupture of the lipid membrane upon laser illumination. After the initial interaction of AuNP aggregates with the membrane, some ion current changes were observed, but differ substantially from the ion current changes observed for the case of AuNR (the area between the light and dark blue arrows in Figure 3 (b)). Most significantly is that the effect was not completely reversible and the ion current (opening of the channel) did not return to the same level and eventually the membrane was observed to break, as is pointed at by the dark blue arrow in Figure 3 (b).


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To investigate the behavior of nanoparticles in the salt buffer solution, we have conducted DLS (dynamics light scattering) and UV-Vis (ultraviolet-visible) absorption studies of both AuNP and AuNR in the solution with the same ionic strength. It can be seen from Figure 4 that AuNP, although stable in water, form aggregates (Figure 3 (c), blue line). A very different behavior is exhibited by AuNR, which do not show any aggregation (Figure 3 (c), red line). This difference is attributed to the presence of different

stabilizing

surfactants,

4-dimethylaminopyridine

(DMAP)

for

AuNP

and

centrylmethylammonium bromide (CTAB) in the case of AuNR. The latter stabilizer is responsible for the shape anisotropy during the nanoparticle synthesis, yielding rod-shaped particles. We have further confirmed this behavior by UV-Vis spectroscopy. At the beginning of the experiment both AuNP and AuNR exhibited their characteristic features (a surface plasmon peak around 520 nm for AuNP, and two peaks at 520 and 800 nm for AuNR (Figure 3 (d), dashed curves). 1600 seconds after adding nanoparticles or nanorods to the salt solution, the spectra of nanorods showed no aggregation behavior (Figure 3 (d), solid red curve), while AuNP exhibited typical aggregation behavior with the surface plasmon resonance peak shifting to the red part of the spectrum (Figure 3 (d), solid blue curve). This data correlates well with the results of ionic current monitoring upon interaction of AuNP and AuNR with the lipid membrane: light blue arrow on Figure 3 (b) presumably points to a moment of nanoparticle aggregate interaction with the membrane (which also takes place within tens of seconds), while no interaction of nanorods or their aggregates was registered. Another interesting system is gold nanocages (AuNC).26,

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AuNC behaved in the salt solution similar to AuNP: they are both stable in

water, but they both form aggregates (Figure 3 (c), blue and black lines, respectively). The synthesized AuNC, were stabilized with PVP poly(vinyl pyrrolidone) of molecular weight of 10000. Under these conditions, AuNC exhibited very strong aggregation, Figure 3 (c). Stabilization of AuNR with 4dimethylaminopyridine (DMAP) for AuNP, poly(vinylpyrrolidone) made a difference in regard with


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aggregation. AuNCs exhibited also a very different behavior of the lipid membrane (Supporting Information). Measuring conductance of such defects allows a very rough estimate of an effective hole size.28 Neglecting the access resistance and assuming in first approximation a cylindrical hole spanning the lipid membrane give a simple relation between conductance G and effective pore radius a: G = κ πa2/l,

(1)

where κ is the bulk electrolyte conductivity (about 12 S/m for 1 M KCl) and l ~ 4 nm is the approximate lipid membrane thickness.28, 29 Subsequently, an ion current of 10.6 pA under applied potential of 100 mV corresponds to a small defect size of about 1-2 Angstroms. It should be mentioned that pores of such small sizes depend on the exact pore structure and pore surface charges; in absence of detailed structural information this value seems to be reasonable for lipid defects. In general, the membrane stability is described by two parameters.28 First, the membrane is set under a mechanical tension increasing the area per lipid and leading to rupture above a critical tension. Here we need to point out that a planar lipid membrane under a constant tension σ is strongly influenced by the interaction of the lipid membrane with the support. Formation of defects necessitates release of a stress, and this is proportional to the pore area, but one has to form an edge which is proportional to the pore radius. A combination of both opposing energy contributions gives: Epore= 2πaγ − πa2σ,

(2)

where a is the pore radius, σ is the membrane tension which favors the creation of pores, and γ is the line tension which opposes pore formation (in other words this is the energy of the channel rim which depends on elasticity).29, 30 Minimization leads to a critical radius a* = γ/σ and an energy barrier E(a*), above which irreversible rupture will occur. It should be noted that the pore formation occurs likely in the lipid membrane spanning the glass hole, whereas the remaining part of the liposome is supported on the glass slide.31,

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nserting typical values for lipid membranes (γ = 10−11 J/m and σ = 2·10-3 J/m2), ACS Paragon Plus Environment

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yields a critical radius above which the pore will cause irreversible rupture of about a ~ 2-5 nm. In the case of nanoplasmonically induced pore formation we expect local heating which may considerably increase the membrane tension through increasing the area of the lipid. This, in turn, reduces the energy barrier for pore formation. Two other curves are further analyzed in comparison to energy associated with localized heating. Figure 4 (a) presents calculated pore energy versus pore radius with a maximum at its critical pore radii. The green line in Figure 4 (a) represents the thermal energy (the energy due to heating around AuNR). It can be noted that the σ values of 2·10-3 J/m2 were used in our calculations (the solid black line); a larger value of σ would position the energy curve somewhat lower as shown by the dashed orange line in Figure 4 (a); under these conditions lower temperatures can be used for the regime corresponding to the sustainable pore openings or membrane breakage. The temperature rise of 90 °C (Supporting Information) for the polarization of the laser beam parallel to the larger nanorod axis is consistent with the previously reported data.33,34 Since temperature rise is proportional to absorption coefficient of nanoparticles and laser power,18,34 the energy of the laser-nanoparticle interaction for nanoparticle aggregates is substantially larger, which would permit reversible and transient current modulation.. In the case (for gold nanoparticle aggregates), the energy associated with a higher temperature rise is larger.25 The irreversible membrane rupture (Figure 3 (b)) could be reached if the temperature rise was high enough and no adjacent lipids would be able to get in proximity for resealing. On the energy scale, this temperature range (temperature rise) would correspond to the dashed blue line (Figure 4 (a)). The formation of large enough aggregates (more than ten and twenty nanoparticles) is possible, and can be incurred for the aggregation process even at lower salt concentrations.35 In addition, we have also modeled the temperature distribution around nanorods and nanoparticles with parameters similar to those employed in our experiments (see Supporting Information for more details). The heat conductivity of gold (318 W/mK) is much larger than the heat conductivity


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of an surrounding environment (0.54 W/mK for water), so one would expect that a nanorod acts on the membrane as one local point, and not as two “hot-spots” generated at both ends of each rod if the longitudinal plasmon is excited by the polarized laser light. Different positions/orientations of nanorods would correspond to different behavior observed in our experiment. Polarization plays an important role; if the polarization of the electromagnetic field is parallel to the longest axis of the rod, the highest absorption is achieved (Figure 4 (b)). It was shown that the radiation pressure pushes larger (in comparison to the focus of the beam) anisotropic objects along the optical beam,36 while smaller objects can be trapped and aligned along the electric field.37, 38 Diffusion and Brownian motion are set to place the nanorod out of the position in which their longest axis is parallel to the polarization of the laser beam. Two cases of temperature rise are modeled in Figure 4 (b) and (c), where larger temperature rise is achieved when one nanorod is situated parallel to the polarization of the electric field of the optical beam (Figure 4 (b)). Figure 4 (c) corresponds to the situation when two nanorods are elongated parallel to the polarization of light, while Figure 4 (d) depicts the case where no nanorods are aligned parallel to the polarization. Thermal equilibration processes are proportional to the ratio of square of the size of nanoparticle to the thermal diffusivity, which in our case corresponds to sub-picosecond time scale (Supporting Information); that does not contradict to our analysis. Interplay between these effects can open and close the current through the membrane. To summarize the above mentioned results, we demonstrated that thermal energy may suffice to generate the threshold energy necessary for the pore formation. Thermal heating could enhance fluctuations of the membrane causing the initial defect;39 which, in turn, favors membrane rupture. An electric field may also facilitate the opening for longer times.29 Phase transitions due to the plasmonic heating effect could be excluded; due to the bulky side chains DPhPC in the fully hydrated state does not have a particular phase transition up to 120°C.40 It is possible however that several nanorods can be located close to each other, thus forming an aggregate; then higher temperature can be reached. Under


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such conditions, formation of gas bubbles upon laser irradiation of gold nanorods would be conceivable.41 It is worth to mention previous reports on pushing nanoparticles through lipid vesicles, where the authors speculate on large pore openings during an extended period of time.7 Other reports included utilization of nanoplasmonic nanopores for release of molecules from red blood cells or liposomes,3, 25, 42-44

and current monitoring across pores45 as well as using nanorods for monitoring the phase transition

of the lipid membrane.5, 33 Unlike planar lipid bilayers, vesicles after poration will eject their content relaxing the stress.30 In case that the water release is fast enough, the pore in the liposome will not cause irreversible rupture (as reported in this study for nanoparticle aggregates), but reseal (as reported for nanorods).28 It can be noted that in our considerations an aqueous environment was taken as an ambient medium around nanorods,46 and no broadening of the absorption bands of nanorods was observed.47 We also note that our data of the interaction of an aggregate with the lipid membrane are consistent with recently reported interaction of nanoparticles also monitored via the ionic current.48 In conclusion, we have presented results on: a) monitoring the transient ionic current change through a lipid membrane, and b) irreversible membrane rupture by means of the interaction of a near-IR laser (830 nm) with light-absorbing nanocenters (AuNR and AuNP aggregate, respectively). In the case of transient current changes (AuNR), a series of openings (or generation of the current) in the lipid membrane was observed. By measuring conductance of such events it is possible to estimate an effective size of defects in the lipid membrane. Thus, the current through the opening ~ 10 pA corresponds to ~ 0.1-0.2 nm holes in the membrane. The membrane was resealed after each opening event demonstrating its otherwise good stability and transient character of nanopore generation. The extreme case of irreversible rupture of the membrane is achieved upon addition of AuNP present at larger concentration and forming aggregates. The energy associated with the localized heating is discussed and compared with typical elastic parameters in the lipid membranes. To the best of our knowledge, an


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electrophysiology study of nanoplasmonically-induced transient opening and rupture of lipid membranes via continuous monitoring of ion current through the membrane upon NIR-laser irradiation of gold nanoparticles is reported here for the first time. There is now a growing interest in remotely controlled transport through the phospholipid membranes at the nanoscale. Obviously an understanding of the mechanism of nanopore formation upon laser irradiation of gold nanoparticles in the lipid membrane is required (e.g. by varying parameters of laser light (regime (pulsed vs. continuous), power, pulse duration, etc.). In this study we have shown that parameters like shape, size, material and density of optical absorption centers in the membrane are of high importance and have a direct impact on the type of induced nanopore (transient vs. irreversible). We believe that the results of this work will help to understand the basic principles of biological membrane permeability control, applicability of nanoplasmonics,49 and will be of interest for broad application areas ranging from analysis of molecules in nanopores,50-53 interface of cells with environment,54, 55 membranes,56, 57 intracellular delivery,58-60 release61-63,18 to drug development.


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Supporting Information Available: Materials and methods, synthesis of nanorods, nanoparticles and nanocages, synthesis of giant liposomes, monitoring of stability of the lipid membrane, details of the setup and its alignment, visualization of nanoparticles, temperature simulations. This material is available free of charge via the Internet at http://pubs.acs.org.

Acknowledgments We thank Prof. Peter Fratzl for valuable discussions; technical assistance of Rona Pitschke, Annegret Praast and assistance of Mohamed Kreir with data treatment are also acknowledged. We gratefully acknowledge the support of BOF (Bijzondere Onderzoek Fonds) of the University of Ghent (Belgium), FWO (Fonds Wetenschappelijk Onderzoek, Belgium) and the DFG Wi 2278/18-1 (Germany). AGS acknowledges also the support of the NB-Photonics multidisciplinary platform at Ghent University, Belgium. BNK was supported by the Government of the Russian Federation (grant 14.Z50.31.0004 to support scientific research projects implemented under the supervision of leading scientists at Russian institutions and Russian institutions of higher education) and by a grant from the Russian Scientific Foundation (project No. 14-13-01167).


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(28) Winterhalter, M. Adv. Colloid Interface Sci. 2014, 208,121-128. (29) Winterhalter, M. Curr. Opin. Colloid Interface Sci. 2000, 5, 250-255. (30) Zhelev, D. V.; Needham, D. Biochim. Biophys. Acta 1993, 1147, 89-104. (31) Tanaka, M.; Sackmann, E. Nature 2005, 437, 656-663. (32) Reimhult, E.; Kasemo, B.; Hook, F. Int. J. Mol. Sci. 2009, 10, 1683-1696. (33) Ma, H. Y.; Bendix, P. M.; Oddershede, L. B. Nano Lett. 2012, 12, 3954-3960. (34) Baffou, G.; Rigneault, H. Phys. Rev. B 2011, 84, 035415. (35) Bedard, M. F.; Braun, D.; Sukhorukov, G. B.; Skirtach, A. G. ACS Nano 2008, 2, 1807-1816. (36) Grover, S. C.; Gauthier, R. C.; Skirtach, A. G. Opt. Express 2000, 7, 533-539. (37) Yan, Z. J.; Pelton, M.; Vigderman, L.; Zubarev, E. R.; Scherer, N. F. ACS Nano 2013, 7, 87948800. (38) Selhuber-Unkel, C.; Zins, I.; Schubert, O.; Sonnichsen, C.; Oddershede, L. B. Nano Lett. 2008, 8, 2998-3003. (39) Blicher, A.; Wodzinska, K.; Fidorra, M.; Winterhalter, M.; Heimburg, T. Biophys. J. 2009, 96, 4581-4591. (40) Lindsey, H.; Petersen, N. O.; Chan, S. I. Biochim. Biophys. Acta 1979, 555, 147-67. (41) Lukianova-Hleb, E. Y.; Mutonga, M. B. G.; Lapotko, D. O. ACS Nano 2012, 6, 10973-10981. (42) Paasonen, L.; Laaksonen, T.; Johans, C.; Yliperttula, M.; Kontturi, K.; Urth, A. J. Control. Release 2007, 122, 86-93. (43) Volodkin, D. V.; Skirtach, A. G.; Moehwald, H. Angew. Chem. Int. Ed. 2009, 48, 1807-1809. (44) Troutman, T. S.; Leung, S. J.; Romanowski, M. Adv. Mater. 2009, 21, 2334-2338. (45) Leontiadou, H.; Mark, A. E.; Marrink, S. J. Biophys. J. 2007, 92, 4209-4215. (46) Huang, J. Y.; Park, J.; Wang, W.; Murphy, C. J.; Cahill, D. G. ACS Nano 2013, 7, 589-597. (47) Juve, V.; Cardinal, M. F.; Lombardi, A.; Crut, A.; Maioli, P.; Perez-Juste, J.; Liz-Marzan, L. M.; Del Fatti, N.; Vallee, F. Nano Lett. 2013, 13, 2234-2240. (48) Carney, R. P.; Astier, Y.; Carney, T. M.; Voitchovsky, K.; Silva, P. H. J.; Stellacci, F. ACS Nano 2013, 7, 932-942. (49) Alessandri, I.; Depero, L. E. Chem. Commun. 2009, 2359-2361. (50) Wanunu, M.; Bhattacharya, S.; Xie, Y.; Tor, Y.; Aksimentiev, A.; Drndic, M. ACS Nano 2011, 5, 9345-9353. (51) Belkin, M.; Maffeo, C.; Wells, D. B.; Aksimentiev, A. ACS Nano 2013, 7, 6816-6824. (52) Howorka, S.; Siwy, Z. Chem. Soc. Rev. 2009, 38, 2360-2384. (53) Soskine, M.; Biesemans, A.; Moeyaert, B.; Cheley, S.; Bayley, H.; Maglia, G. Nano Lett. 2012, 12, 4895-4900. (54) Maus, L.; Dick, O.; Bading, H.; Spatz, J. P.; Fiammengo, R. ACS Nano 2010, 4, 6617-6628. (55) Kolesnikova, T. A.; Kohler, D.; Skirtach, A. G.; Mohwaldt, H. ACS Nano 2012, 6, 9585-9595. (56) Balazs, A. C.; Kuksenok, O.; Alexeev, A. Macromol. Theory Simul. 2009, 18, 11-24. (57) Jiang, C. Y.; Markutsya, S.; Pikus, Y.; Tsukruk, V. V. Nat. Mater. 2004, 3, 721-728. (58) Skirtach, A. G.; Javier, A. M.; Kreft, O.; Koehler, K.; Alberola, A. P.; Moehwald, H.; Parak, W. J.; Sukhorukov, G. B. Angew. Chem. Int. Ed. 2006, 45, 4612-4617. (59) Huschka, R.; Neumann, O.; Barhoumi, A.; Halas, N. J. Nano Lett. 2010, 10, 4117-4122. (60) He, J.; Wei, Z. J.; Wang, L.; Tomova, Z.; Babu, T.; Wang, C. Y.; Han, X. J.; Fourkas, J. T.; Nie, Z. H. Angew. Chem. Int. Ed. 2013, 52, 2463-2468. (61) Skirtach, A. G.; Antipov, A. A.; Shchukin, D. G.; Sukhorukov, G. B. Langmuir 2004, 20, 69886992. (62) Radt, B.; Smith, T. A.; Caruso, F. Adv. Mater. 2004, 16, 2184-2189. (63) Angelatos, A. S.; Radt, B.; Caruso, F. J. Phys. Chem. B 2005, 109, 3071-3076.


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FIGURE CAPTIONS

Figure 1. I) Side view of the scheme of the experiment. II) Images (top view) demonstrating the laser alignment on the chip with the following steps: a) chip in air (the inset shows the side view schematics); b) chip with water filling the lower chamber (the inset shows water in blue filling the lower chamber); c) chip with water filling the lower chamber with laser focused at the top; d) chip with water filling both chambers, the location of the opening is marked by the dashed yellow cross (the inset shows water filling both chambers and indicates presence of the laser is illuminating the top). The yellow dashed lines are drawn to highlight the location of the hole. Figure 2. Examples of ion current traces versus time upon laser-AuNR interaction cut out from a 5 minute long record and -100mV applied voltage: a) the upper trace shows small openings corresponding to the c-level (10.6 pA); b) the middle trace corresponds to one opening at b-level (25.4 pA); c) the short spikes corresponding to large short openings at a-level (80.2 pA); d) open pore statistics of the 5 minute trace. Figure 3. Typical ion current at a lipid membrane: a) with adsorbed AuNR showing the ion current versus time trace. The red arrows point at transient current changes. b) Adsorbed AuNP aggregates: rupture of the membrane. The light blue arrow presumably points to the current associated with the addition of AuNP, the dark blue points to the membrane break-up. (c) DLS (Dynamic Light Scattering) of AuNP (blue) and AuNR (red) and AuNC (black) upon adding the same buffer in which liposomes are placed (at similar volume ratios); the time point of addition is indicated by green arrow. The size is monitored versus time (normalized to the size at the beginning of the experiments (t = 0 seconds). (d) UV-vis spectra of AuNP (blue cures) and AuNR (red cures), and AuNC (black curves); spectra were taken when salt was added (dashed lines in (d), the time interval is marked by the green arrow in (c)) and after 1600 seconds (solid lines). (e), (f), (g) TEM images of nanoparticles, nanorods and nanocages, respectively. Scale bars in (e-g) correspond to 200 nm. Figure 4. a) Energy for creation of a pore in a lipid membrane versus the radius of the pore. The maximum temperature rise corresponds to a temperature difference of about ∆T = 90 K for nanorods being oriented parallel to the incident field (Supporting Information). Thermal energy (the green line) on


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AuNR, while that for AuNP (the blue dashed line) are given in units of kT; the energy of the pore was calculated using equation 2, where γ =10−11 J/m and two different σ values of 2·10-3 J/m2 (solid squares), 5·10-3 J/m2 (the orange dashed line). Temperature distribution around several nanorods with one (b) and two (c) nanorods aligned parallel to the polarization (blue double headed arrow) of the laser beam; (d) shows the case when alignments of all nanorods does not coincide with the alignment of the polarization of the laser light. Blue arrow in (d) points to the nanorod similar to that in (b) but with a different orientation now; for diversity zoom in image (d) is shown higher than those in (b) and (c). Insets show enlarged middle areas in all temperature distribution images (b through d), in (a) a dashed square points to the enlarged area.


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Figure 1.


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Figure 2.


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Figure 3.


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Figure 4.


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For TOC only


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Nanoplasmonically-Induced Defects in Lipid Membrane Monitored by

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