Enhancement of Lateral Diffusion in Catanionic Vesicles during

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Enhancement of Lateral Diffusion in Catanionic Vesicles during Multilamellar-to-Unilamellar Transition Subhankur Mitra, Veerendra Kumar Sharma, Victoria Garcia-Sakai, Andrea Orecchini, Tilo Seydel, Mark Johnson, and Ramaprosad Mukhopadhyay J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.6b02997 • Publication Date (Web): 31 Mar 2016 Downloaded from http://pubs.acs.org on April 4, 2016

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The Journal of Physical Chemistry

Enhancement of Lateral Diffusion in Catanionic Vesicles during Multilamellar-to-Unilamellar Transition 1

1$

S. Mitra , V.K. Sharma , V. Garcia Sakai2, A. Orecchini3, T. Seydel4, M. Johnson4, and R. Mukhopadhyay1* 1 2 3

Solid State Physics Division, Bhabha Atomic Research Centre, Mumbai,40085, India

Science and Technology Facilities Council, Rutherford Appleton Laboratory, Didcot, UK

Dipartimento di Fisica e Geologia, Università di Perugia, Via Pascoli, I-06123 Perugia, Italy 4

Institut Laue-Langevin, BP 156, 6, rue Jules Horowitz, 38042 Grenoble Cedex 9, France

Abstract Catanionic vesicles are formed spontaneously by mixing cationic and anionic dispersions in aqueous solution in suitable conditions. Due to spontaneity in formation, long-term stability, and easy modulation of size and charge, they have numerous advantages over conventional lipid-based vesicles. The dynamics of such vesicles is of interest in the field of biomedicine, as they can be used to deliver drug molecules into the cell membrane. Dynamics of catanionic vesicles based on Sodium Dodecyl Sulfate (SDS) and Cetyltrimethylammonium Bromide (CTAB) have been studied using incoherent elastic and quasielastic neutron scattering (QENS) techniques. Neutron scattering experiments have been carried out on two backscattering spectrometers, IRIS and IN16B, which have different energy resolutions and energy transfer windows. An elastic fixed-window scan carried out using IN16B shows a phase transition at ~307 K during the heating cycle, whereas on cooling the transition occurred at ~294 K. DSC results are found to be in close agreement with the elastic scan data. This transition is ascribed to a structural rearrangement from a multilamellar to a unilamellar phase [P. Andreozzi, et al., J. Phys. Chem. B, 2010, 114, 8056–8060]. It is found that a model in which the surfactant molecules undergo both lateral and internal motions can describe the QENS data quite well. While the data from IRIS have contributions from both dynamical processes, the data from IN16B probe only lateral motions, as the internal motions are too fast for the energy window of the spectrometer. It is found that, through the transition, the fraction of surfactant molecules undergoing lateral motion increases of a factor of two from the multilamellar to the unilamellar phase, indicating an enhanced fluidity of the latter. The lateral motion is found to be Fickian in nature, while the internal motion has been described by a localized translational diffusion model. The results reported here could have direct interest for a number of applications, such as molecular transport, and the effect of specific drug molecules or hormones through the membrane.

*Corresponding Author: [email protected]; Tel. +91-22-25594667; $

Present Address: Biology and Soft Matter Division, Neutron Science Directorate. ORNL, Oak Ridge, Tennessee 37831, US ACS Paragon Plus Environment


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INTRODUCTION Lipids are amphiphilic molecules, where one part of the molecule is hydrophilic and the other part is hydrophobic. The amphiphilic nature of lipid molecules is what drives their self-assembly into different structures such as micelles, vesicles, bilayers, etc., in aqueous environments. Liposomes are colloidal, concentric bilayered vesicles where the aqueous compartment is entirely enclosed by a bilayer membrane, mainly composed of natural or synthetic lipids. If there is only one lipid bilayer, these vesicles are called unilamellar liposome vesicles; otherwise they are called multilamellar liposome vesicles. Vesicles have numerous practical applications such as in the delivery of drugs and genetic material in the human body.1 In the last decade, vesicular systems have raised increasing interest because of possible further applications, spanning from biomedicine to biophysics and, from pharmacology (vaccine production) to cosmetics (skin penetration). An additional advantage of vesicular systems derives from their relatively limited cytoxicity at low concentration. Recent studies evaluated the cytotoxic effects of some catanionic vesicles on different cell types, to ascertain the extent and nature of damage at a cellular and molecular level, and finally left open the possibility to use these supramolecular aggregates in nano-biotechnology applications without significant toxic effects.2 Natural lipid-based vesicle systems present some shortcomings, such as non-spontaneous structures with limited colloidal stability. In addition, they can easily undergo chemical degradation by hydrolysis and peroxidation and therefore are less stable chemically.3 Consequently, recent efforts are aimed at finding artificial vesicular systems giving the same performance as natural lipid-based ones. Synthetic surfactant species were found to be useful for this purpose.4 It was found that mixtures of oppositely charged surfactants can be used, since they exhibit a phase behavior very similar to that occurring in lipids.5 Catanionic vesicles are colloidal aggregates formed by mixing cationic and anionic surfactants in non-stoichiometric ratios.6,7 They form spontaneously and thus have many advantages over those prepared by other means. Non-stoichiometric mixtures composed by such surfactants show a rich polymorphic behaviour at room temperature with formation of micelles, vesicles, solids and lyotropic mesophases. The thermal stability and the presence of temperature-induced phase transitions are important in these vesicles in order to use them for biomedical applications. As the temperature increases, the bilayers of catanionic vesicles become more fluid which may change the curvature of these vesicles and, in turn, produce a new phase of the vesicle. Non-stoichiometric ratios of Sodium Dodecyl Sulfate (SDS), an anionic surfactant, and Cetyltrimethylammonium Bromide (CTAB), a cationic surfactant, were found to form catanionic vesicles. The phase behavior of these vesicles, as a function of both temperature and composition, was investigated in details with different experimental techniques.7 It has been shown that initially

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the size of the vesicles increases on heating and, at a critical temperature, the size reduces abruptly with a transition from multi to unilamellar state. Surfactant molecules in this type of catanionic vesicles exhibit a complex dynamical behavior in the bilayer system. The local dynamics of such assemblies is very important since the dynamics of surfactant molecules could influence the enzymatic activity of the encapsulated proteins or the release of drugs for drug delivery applications. Various other phenomena, such as receptor clustering8, ligand-receptor interactions9 and conventional chemical reactions,10 are also known to be rate-limited by the two-dimensional lateral diffusion at the cell surface. The lateral diffusion coefficients of lipid molecules in phospholipid bilayers have been estimated to be ~10-8 -10-6 cm2/s as probed by fluorescence photobleaching, pulsed field gradient NMR, EPR imaging11 and neutron scattering techniques.12,13,14 A molecular dynamics simulation study15 on catanionic bilayers, composed of ion pair amphiphiles with double-tailed cationic surfactant, showed that the deuterium order parameter along the hydrocarbon chain is less in head and tail. Therefore, it is of interest to study the various dynamical behaviors of these vesicles, such as internal dynamics of the surfactant or lateral diffusion of surfactants along the surface as a function of temperature. Despite many studies about the local dynamics in lipid bilayers, there are only few aimed at understanding the dynamics in surfactant-based vesicular systems. Our aim in this paper is to probe the local dynamics of surfactant bilayers in the different phases of SDS/CTAB based catanionic vesicles using quasielastic neutron scattering (QENS). QENS is suitable for observing the local motions on a picoto nanosecond timescale and on a length scale from angstroms to few nanometers.16-22

EXPERIMENTAL DETAILS Sodium Dodecyl Sulfate (SDS), an anionic surfactant,Cetyltrimethylammonium Bromide (CTAB), a cationic surfactant, and D2O (99.9% atom D purity) were obtained from Aldrich. Catanionic vesicles are colloidal aggregates formed by mixing equimolar cationic and anionic micellar solution in non-stoichiometric ratios. 0.16M catanionic vesicles based on SDS and CTAB were prepared, inside a glove box, by mixing 0.16M SDS and CTAB micellar solution with molar ratio R= [SDS]/[CTAB]=1.6. DSC experiments were performed using a NANO DSC Series III System with Platinum Capillary Cell (TA Instruments). The sample cell was filled with about 300 µl of 0.16M catanionic vesicles solution, and an equal volume of D2O was used as a reference. Measurements were carried out in the range of 280–330 K at a scan rate of 0.5 K /min in both the heating and cooling cycles. In neutron spectroscopy, the elastic fixed-window scan (EFWS) technique is an established tool for detecting the onset of motions in glass, melting or other kinds of transitions. In an EFWS, the sample scattering intensity at zero energy transfer is recorded as a function of ACS Paragon Plus Environment


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temperature. Our EFWS experiments were carried out on 0.16M catanionic vesicles in D2O using the high-resolution backscattering spectrometer IN16B23 at ILL, Grenoble, France. IN16B operates in backscattering configuration and achieves an energy resolution of ~0.9 µeV (full width at half-maximum). The elastic intensity scans were carried out in the temperature range from 270 to 330 K with a scan rate of 0.5 K/min, to map out the multilamellar to unilamellar transition both on the heating and cooling cycles. LAMP software24 was used to carry out standard data reduction, including background subtraction and detector efficiency corrections. QENS experiments were carried out using the backscattering spectrometer IN16B23 at ILL, Grenoble, and the IRIS spectrometer25 at the ISIS facility, UK. Quasi-elastic spectra were recorded in the Q range of 0.5 -1.8 Å-1 on IRIS and 0.2-1.9 Å-1 on IN16B spectrometers. To complement the slow dynamics recorded on IN16B, IRIS was used with an energy resolution of 17.5 µeV (FWHM). IRIS is an inverted geometry near backscattering spectrometer which uses (002) reflection of pyrolytic graphite analyser. The energy transfer range for the measurements was between -0.3 and 1.1 meV. In IN16B, the energy transfer range is ± 30 µeV. The sample was placed in an annular aluminum can with an internal spacing of 0.5 mm chosen to achieve no more than 10% scattering and to minimize multiple scattering. In order to remove the contribution of D2O from the vesicles solution, the QENS data were recorded in the same cell for D2O alone at different temperatures. The data analysis package, MANTID was used to carry out background subtraction and detector efficiency corrections.26

Results and Discussion In neutron scattering data from hydrogenous systems, the incoherent contribution from hydrogen atoms dominates the signal because of their high incoherent scattering cross-sections relative to coherent or incoherent scattering cross-sections of any other element. The measured scattering intensity then is proportional to the incoherent scattering law, Sinc(Q,ω), which is the double Fourier transform of the space-time self-correlation function that describes single-particle motions. Since the system of interest here is vesicles, deuterated water was used as solvent to minimize its scattering contribution. Elastic Fixed Window Scan (EFWS) The elastic intensity in an EFWS is sensitive to any dynamical process that might occur at timescales roughly shorter than ~ℏ/ࢾࡱ, with ࢾࡱbeing the elastic energy resolution of the instrument and ℏ the reduced Planck constant. A step-like loss/gain of intensity in an elastic scan experiment is an indication of a transition related to a change in dynamical behaviour at that temperature. Fig. 1a ACS Paragon Plus Environment

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shows the elastic intensity as obtained from IN16B, at ILL, Grenoble, France, for the catanionic vesicle, 0.16M SDS/CTAB with mole ratio R=1.6, averaged over all accessible scattering angles upto 1.0 Å-1 in heating as well as in cooling cycles. As evident from the figure, upon heating, the elastic intensity decreases monotonically with increasing in temperature and shows a sharper fall between 307 and 315K. In the cooling cycle the reverse transition is found to occur at ~294 K, indicating the presence of a hysteresis effect.

Heating Cooling

Heat Flow (mW)

800 400 0 -400 -800

(b)

0.015

Elastic Intensity

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Heating Cooling

0.010

0.005

(a) 0.000 280

290

300

310

320

330

T(K) Fig. 1 a)Temperature dependence of the Q-averaged (up to 1 Å -1) elastic intensity as observed for 0.16M SDS/CTAB catanionic vesicles with molar ratio R=1.6, in heating and cooling cycles as obtained using IN16B, at ILL, Grenoble. A transition at ~307 K in the heating cycle and at 294 K in the cooling cycle is evident. b) DSC scans in the heating and cooling cycles. A hysteresis effect on the transition is evident. DSC measurements on the same sample also show a sharp peak at 307 K on heating and at 294 K in the cooling cycle (Fig. 1b). These results are consistent with each other and also confirm that this is not a time effect. It is not clear at this stage whether the hump-like extra features observed at 309 K in the heating cycle and 292 K in the cooling cycle are related to another transition or not. It may be



noted that the elastic scan also indicates small steps at 300 K during heating and at 288 K in the cooling cycle. DSC measurements in similar systems have been reported for different SDS/CTAB ratios (R) and showing that thermograms depend sensitively on the ratio.27 Quasielastic Neutron Scattering (QENS) Andreozzi et al.7 have shown that 6mM catanionic vesicles (R=[SDS/CTAB]= 1.7) undergo a transition from multilamellar (MLV) to unilamellar (ULV) phase at 320 K. Elastic scan (Fig. 1a) and DSC data (Fig. 1b), for the present sample 0.16 M [R=1.6], show the transition to occur at 307 K. The difference between the transition temperatures in Ref. 7 and here was confirmed by DSC and is caused by the difference in concentration. This could be due to electrostatic interactions which might influence the chain packing and thus the transition temperature. Small angle neutron scattering (SANS) experiment performed on samples at 300 K before heating showed that the radius of the vesicles is more than 1000 Å and for sample at 300 K after heating it is around 260 Å. This is consistent with the earlier study7 which indicates that with heating the sample undergoes a transition from MLV to ULV phase. To investigate the details of the dynamical behavior in the different phases of the vesicles, QENS data were recorded at different temperatures, before and after the transition. First, QENS data were recorded at 300 K where vesicles are in MLV phase and then heated at 340 K (well above the transition temperature). As seen from Fig. 1, the transition is initiated at 307 K and completes at around 313 K - QENS data were thus recorded at 315 K where the system should be in the ULV phase. After that, the sample was cooled back to 300 K. As the reverse transition occurs at 294 K, the sample remains in the ULV phase at 300 K. QENS spectra were then recorded at 300 K in ULV phase.

S(Q,ω) in arb unit

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-1

1

Q=1.01 Å

0.1

300K MLV 300K ULV Vanadium

0.01

1E-3 -0.3

0.0

0.3

0.6

0.9

E (meV) Fig. 2 QENS spectra as obtained from IRIS spectrometer, at ISIS Facility, UK, for 0.16M catanionic vesicles (SDS/CTAB R=1.6) at 300 K before and after heating to 340K at a typical Q value of 1.01 ÅACS Paragon Plus Environment

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1

. The contribution from D2O is subtracted. The spectra are normalized to the peak intensities. The

instrument resolution is shown by a dashed line. In order to proceed with the data analysis, the spectra of the pure solvent (D2O) were subtracted from that of the vesicles solution. Both spectra were normalized to the monitor counts, and the final scattered intensity from the vesicles was obtained using the following relation:

φ SVesicles (Q , ω ) = S solution (Q , ω ) − (1 − φ ) S D O (Q , ω )

(1)

2

where φ is the volume fraction of surfactants (SDS/CTAB) in the D2O. The factor φ accounts for the fact that the amount of D2O in the vesicles solution is less than that in the pure D2O sample. The scattering law for the vesicles, as obtained by Eq. 1 is shown in Fig. 2 in two different phases at a typical value of Q=1.01 Å-1. The instrumental resolution, as measured using a standard vanadium sample, is also shown in the figure. For comparison, each spectrum is normalized to the peak amplitudes. Significant quasielastic broadening over the instrumental resolution is evident for both MLV and ULV phases, and, at first sight, the mobility in the MLV phase appears slower than in the ULV phase. Surfactant molecules in vesicles have the freedom to undergo two kinds of motion, namely lateral diffusion and fast internal motions, both of which are expected to contribute within the time scales of the QENS technique (10-9 - 10-12 sec)12,13,14,28,29. To interpret the data, we assume that the different dynamical processes are independent of each other, and the scattering law Svesicles(Q,ω) can be expressed as,

Svesicles (Q,ω) = [ Slat (Q,ω) ⊗ Sint (Q,ω)]

(2)

where Slat(Q,ω) and Sint(Q,ω) correspond to the scattering functions due to the lateral and internal motions of the surfactant molecules, respectively. Concerning lateral motions, it is not necessary that all the surfactant molecules participate in the dynamical process. To take this in to account we introduce the factor Px as the fraction of the total number being immobile at any given temperature. The scattering law for lateral motions in this situation can be written as

Slat = Pxδ (ω) + (1 − Px ) Llat (Γlat , ω)

(3)

The first term in the RHS of the above equation represents the contribution to the elastic scattering from to the surfactant molecules which are not undergoing lateral diffusion. The second term corresponds to the quasielastic broadening due to the lateral motion of the surfactant molecules. We



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model this contribution with a Lorentzian function Llat (Γlat, ω),withΓlat the half width at half maximum (HWHM). The internal motions are locally confined as defined by the chemical structure of the surfactant molecules and there remains a finite probability of finding the scatterer within the chain volume after a relatively long relaxation time. This gives rise to an elastic contribution to the scattering law. Therefore, the scattering law for the internal motion, Sint(Q,ω) can be expressed as Sint ( Q, ω ) = A( Q) δ (ω ) + (1 − A( Q) ) Lint (Γint , ω)

(4)

where the elastic part arises from either immobile particles or motions slower than the longest observable time of the spectrometer. The second term in Eq. 5 represents the quasielastic component, which is approximated by a single Lorentzian function Lint (Γint, ω) with half width at half maximum (HWHM) Γint,. Combining Eqs. 2, 3 and 4, the generalized scattering law for vesicles can be written as,

Svesicles ( Q, ω ) =  Pxδ (ω ) + (1 − Px ) LLat ( Γ Lat , ω)  ⊗  A ( Q ) δ (ω ) + (1 − A (Q ) ) Lint ( Γint , ω )  = Px A ( Q ) δ (ω ) + (1 − Px ) A (Q ) LLat ( Γ Lat , ω ) + (1 − A (Q ) ) Px Lint ( Γint , ω ) + (1 − Px ) (1 − A ( Q ) ) LLat ( Γ Lat , ω ) ⊗ Lint ( Γint , ω )

(5)

It is known that the diffusivity corresponding to lateral motion30 is typically anorder of magnitude smaller than that of internal motion.17-20 Therefore, Γlat

Enhancement of Lateral Diffusion in Catanionic Vesicles during

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