Spontaneous Formation of Multiarchitecture Vesicles of [C8mim]Br +

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Spontaneous Formation of Multi-Architecture Vesicles of [C8mim]Br + [Na]DBS in Aqueous Medium: Synergic Interplay of Electrostatic, Hydrophobic and #-# Stacking Interactions Praveen Singh Gehlot, K. Srinivasa Rao, Pankaj Bharmoria, Krishnaiah Damarla, Hariom Gupta, Markus Drechsler, and Arvind Kumar J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.5b09850 • Publication Date (Web): 17 Nov 2015 Downloaded from http://pubs.acs.org on November 18, 2015

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The Journal of Physical Chemistry

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Spontaneous Formation of Multi-Architecture Vesicles of [C8mim]Br + [Na]DBS in Aqueous Medium: Synergic Interplay of Electrostatic, Hydrophobic and π-π Stacking Interactions

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Praveen Singh Gehlot,1 K. Srinivasa Rao, 1Pankaj Bharmoria, 1Krishnaiah Damarla,1 Hariom

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Gupta,2 Markus Drechsler,3 and Arvind Kumar*,1,2

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1

Academy of Scientific and Innovative Research (AcSIR)-Central Salt and Marine Chemicals

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Research Institute, Council of Scientific and Industrial Research (CSIR), G. B. Marg,

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Bhavnagar-364002, Gujarat, India.

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2

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Industrial Research (CSIR), G. B. Marg, Bhavnagar-364002, Gujarat, India.

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3

CSIR-Central Salt and Marine Chemicals Research Institute, Council of Scientific and

Bayreuth Institute of Macromolecular Research (BIMF)–Soft Matter Electron Microscopy

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1 ACS Paragon Plus Environment


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ABSTRACT

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Mixture of a cationic surface active ionic liquid, [C8mim]Br and anionic surfactant, [Na]DBS

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has been shown to form unilamellar vesicles in water over an exceptionally wide mole

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fraction range of [C8mim]Br (x1= 0.2 to 0.8). Formation of vesicles has been evidenced from

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TEM, Cryo-TEM and AFM imaging. Cryo-TEM imaging of an equimolar mixture showed

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multi-architectural unilamellar vesicles (spherical, tubular and ribbon). Such complex

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architectures were earlier reported for Janus dendrimers of different structures (Science,

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2010, 328, 1014). The synergism between oppositely charged single chain surfactants to form

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bilayer structures has been explained based on the evidences of π-π stacking interaction from

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2D NOESY measurements, coulombic interactions from zeta potential measurements and

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magnitude of interaction parameter from the critical aggregation concentration. The

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aggregation concentrations were measured from tensiometry and fluorescence using pyrene

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as polarity probe. The phase behaviour at different mixture compositions has been revealed

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from turbidity measurements and visual inspection. Hydrodynamic radii of self-assembled

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structures in the bulk solution phase were measured from dynamic light scattering. Vesicles

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formed have been explored as delivery vehicles for proteins using bovine serum albumin

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(BSA) as model.

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1. INTRODUCTION

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Biological vesicles formed by intrusion/extrusion of the biomembrane are the chief in vivo

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transporter of biomolecules via endo/exocytosis process.1 These systems have been mimicked

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with the vesicles of synthetic lipids (cationic or anionic) for drug delivery and transfection of

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biomolecules.2-12 But these systems have drawbacks pertaining to bulk phase kinetic

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stabilization as vesicular dispersions undergoes aggregation and reform the lamellar and

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mesophases from which they are made of.13-16 Pure ionic surfactants have limitations

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pertaining to denaturation of

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applications.17 The vesicles formed by oppositely charged surfactants, frequently called as

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cat-anionic mixtures have gained success to circumvent these limitations to certain extent.18-24

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The equimolar mixtures of cat-anionic surfactants exhibits phase behaviour very similar to

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that of swelling lipids,25,26 and are potential colloidal stable alternatives to charged lipid

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vesicles. Thermodynamically stable cat-anionic colloidal vesicular dispersions can be

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achieved by choice of surfactants having head group which favour packing with reduced area

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per head group, can induce hydrogen bonding and π-π stacking interactions between the head

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groups.

biomolecules and hence are not suitable for “in vivo”

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The rise of surface active ionic liquids (SAILs) exhibiting structure tuneable

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property,27-33offers opportunity to utilize them in conjunction with conventional surfactants

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for the preparation of synthetic vesicles exhibiting of good thermodynamic and kinetic

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stability. First attempt in this direction was made by Yuan et al. who have investigated the

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phase behaviour of a mixture of 1-dodecyl-3-methylimidazolium bromide, [C12mim]Br and

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SDS and reported the vesicle formation which were further utilized for the preparation of

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hollow sphere silica.34 Our own group have reported the formation of vesicles in a specific

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composition range of a mixture of ILs, [C4mim]C8OSO3 and [C8mim]Cl which remained

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stable in the bulk solution phase for a very long period due to anion-π and π-π stacking



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interactions between the head groups.35 Ghosh et al. have reported the micelle-vesicle-micelle

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transition in mixture of cationic surfactant, CTAB and anionic SAIL, [C4mim]C8OSO3in

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various volume fraction ranges and observed precipitates and unilamellar/multilamellar

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vesicles in the 0.65-0.70 volume fraction range.36 The biamphiphilic ionic liquid surfactants

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or cat-anionic ionic liquid surfactants which form vesicles in aqueous media can be

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considered as advanced development in the field of cat-anionic surfactant mixtures.37-

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Among these reports the 4 to 25 mmol.L-1 aqueous vesicular solution of [C8mim][C12OSO3]

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reported by us remained very stable for a long time similar to that of [C4mim]C8OSO3 and

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[C8mim]Cl mixture, thus citing the importance of π-π stacking interactions in stabilizing the

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vesicles.38Advancing these studies, recently Chabba et al. have reported the unilamellar

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vesicle formation in a mixture of cationic SAILs, [Cnmim]Cl (n=8,10,12) and [Na]DBS using

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SANS measurements.41 Since the Br- ions are shown to be more effective than Cl- and I- in

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enhancing the surfactant adsorption by adopting a vertical orientation at the air-water

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interface,42 herein we have investigated the system [C8mim]Br + [Na]DBS. In fact we have

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observed the formation multi-architectural vesicular structures which have not yet reported in

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cat-anionic surfactant mixtures. The propensity of such vesicular structures as a potential

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carrier of biomolecules has been checked by investigating their binding behaviour with

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protein, bovine serum albumin (BSA) along with the kinetic conformational stability of a

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BSA in vesicle solution. The propensities of cat-anionic vesicles as delivery vehicles using

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BSA and lysozyme as a model protein were earlier reported by Mesa group.43,44a Authors

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controlled the protein-vesicles electrostatic interactions by manipulating the mixtures

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concentration and solution pH.43,44a An understanding of the such kind of interaction of

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protein or any biomacromolecule with vesicles, such as, DNA–cationic vesicle complex

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formation will allow the production of more homogeneous, efficient delivery systems in


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pharmaceutically acceptable forms.44b Protein-vesicles interactions also serve as good models

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for studies on more complicated biomacromolecule-liposome systems.44c

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The aggregation concentration at different mixture compositions has been

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measured from tensiometry and pyrene fluorescence. Phase behaviour at different mixing

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ratios has been studied from visual inspection of solutions and turbidity measurements. The

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size and morphology of aggregated structures in the different composition range have been

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revealed from DLS,TEM, Cryo-TEM and AFM imaging. The evidences of electrostatic, π-π

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stacking and hydrophobic interactions in the system were collected from the zeta potential,

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2DNOESY and interaction parameters. Interaction of BSA with vesicles was measured from

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isothermal titration calorimetry (ITC), zeta potential and DLS, whereas changes in the

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conformation BSA with time have been investigated using CD spectroscopy.

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2. EXPERIMENTAL SECTION

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2.1. Materials.1-Methyl imidazole and 1-Bromo octane of >98% was purchased from

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Spectrochem and SRL, India respectively whereas the sodium dodecylbenzenesulfonate

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(>98% purity) and sodium dodecylsulfate (>95.0% purity) were procured from TCI Chemical

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(India) Pvt. Ltd. AR grade solvents, n-hexane, dichloromethane, ethyl acetate and diethyl

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ether were obtained from SD-fine chem. Ltd., India. All the chemicals procured were used as

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received. Millipore grade water with specific conductivity 3 µS.cm-1 and surface tension 71

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mN.m-1 was used for preparation of solutions.

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2.2. Synthesis of 1-methyl-3-octylimidazoliumbromide.n-octyl bromide and 1-methyl

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imidazole (molar ratio of 1.2:1) were dissolved and refluxed for 5h in acetonitrile.

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Acetonitrile was removed under reduced pressure after the reaction was completed. Excess 1-

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bromo octane was removed from the reaction mixture by washing with ethyl acetate/n-

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hexane. Coloured impurities were removed by using activated charcoal in DCM. IL thus

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obtained was dried under vacuum oven for 72h at 80oC and characterized with LCMS and 5 ACS Paragon Plus Environment


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H-NMR techniques. 1H-NMR spectra (Figure S1) and ESI data are given in Supporting

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Information.

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2.3. Methods. 2.3.1. Tensiometry. Tensiometry was employed to measure the critical

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aggregation concentration (CAC) and adsorption behaviour of cat-anionic mixtures. The

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surface tension measurements were done on Data Physics DCAT-II automated tensiometer

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by adopting the procedure similar to that reported in our earlier paper.31 Uncertainty in

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measurements was found to be ±0.1 mN.m-1.

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2.3.2. Steady-State Fluorescence Measurements. Fluoremetry was employed to measure

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the CAC, of the cat-anionic mixtures using pyrene as polarity probe. The fluorescence

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measurements of pyrene at different concentration cat-anionic mixtures were carried out

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using Fluorolog (Horiba JobinYvon) spectrometer using a quartz cuvette of 1 cm path length

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at 298.15K. In a typical experiment pyrene,1×10-6 mol L-1 was dissolved in water and changes

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in the fluorescence emission intensity of vibronic bands at I1 (373 nm) and I3 (383 nm) were

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measured at an excitation wavelength of 334 nm at different concentration of cat-anionic

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mixture. I1/I3 ratio is used to measure the polar/nonpolar domains in the solution and gives

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information about CAC when it senses homogenous non-polar environment inside the

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aggregates (micelle, vesicles).45

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2.3.3. Conductometry. This technique was used to determine the CAC and degree of

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counterion dissociation of aggregates (micelle, vesicles) in the solution. A digital

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conductivity meter (Eutech PC 2700) at 298.15 K was used for conductivity measurements

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with an uncertainty of less than ±0.1%.The temperature was controlled with a Julabo

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thermostat to within ±0.1 K.

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2.3.4. Dynamic Light Scattering (DLS). The technique was used to measure the size of

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aggregates in situ. NaBiTec Spectro-Size300 light scattering apparatus (NaBiTec, Germany)


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with a He−Ne laser (633 nm, 4 mW) was used for DLS measurements following the reported

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procedure.31

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2.3.5. UV-Visible Spectroscopy.The variation inturbidity of cat-anionic mixtures at different

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mole fraction were measured from absorption spectra at 660nm at 298.15 K from Schimadzu

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UV-2700 UV-Vis spectrophotometer.

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2.3.6. 1HNMR and 2D NOESY measurements. The

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[C8mim]Br is recorded using Brüker 200 MHz spectrometer in deuterated DMSO. 2D

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NOESY experiment of equimolar cat-anionic mixture was recorded using Brüker 500 MHz

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spectrometer in D2O.31 Phase sensitive 1H homonuclear 2D NOESY NMR experiment was

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acquired with experimental parameters: fid size 2048:F2 & 256:F1, relaxation delay of 4s, 48

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numbers of scan, mixing time of 720 ms and processed using Topspin NMR software. The

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mixing time used for NOESY experiment was optimized through inversion recovery method.

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The total concentration of solution was 10 mM in D2O.

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2.3.7. Atomic Force Microscopy (AFM). AFM imaging of self-assembled structures was

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carried out on an Ntegra Aura atomic force microscope (NT-MDT, Russia) in semi-contact

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mode using an NSG 01 silicon probe. Samples were prepared by putting a drop of the

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solution of cationic mixture on a thin mica sheet and air drying.35

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2.3.8. Determination of Zeta Potential (ζ). ζ is the potential difference between the

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dispersion medium and the stationary layer of fluid attached to the dispersed aggregates.36 It

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gives an idea about the stability of systems. ζ of aqueous solutions [C8mim]Br, [Na]DBS, and

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[C8mim]Br + [Na]DBS mixtures above CAC and vesicle-BSA mixture was measured using a

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Zetasizer Nano ZS light scattering apparatus (Malvern Instruments, U.K.) with a He-Ne laser

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(633 nm, 4mW) at 298.15 K following the reported procedure.35

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2.3.9. Transmission Electron Microscopy (TEM). Sample was prepared by putting a drop

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(2µL) of solutions of catanionic mixture on the carbon-coated copper grid (300 mesh).

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HNMR spectra of synthesised



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Residual liquid was blotted immediately. Samples were imaged under a JEOL JEM-2100

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electron microscope at a working voltage of 80 kV.38

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2.3.10. Cryogenic Transmission Electron Microscopy (Cryo-TEM). Cryo-TEM imaging

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was done on Zeiss EM922 EF-TEM instrument using the procedure reported in our earlier

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paper.31

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2.3.11. Isothermal Titration Calorimetry (ITC). Enthalpy changes (dH) due to the

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interaction of BSA in successive injections with vesicles (2 mmol.L-1) in buffer solution were

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measured using MicroCal ITC200 microcalorimeter, with an instrument controlled

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Hamiltonian syringe having volume capacity of 40 µL. We did both forward and reverse

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titrations. In forward titration we added 2µL aliquots of BSA (1%) stock solution into the

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sample cell containing 200 µL of vesicle or buffer solution with continuous stirring (500

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rpm). In the reverse titration 2µL aliquots of vesicular solution (30 mmol.L-1) was titrated in

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to 200 µL of 0.1% BSA or buffer solution The parameters like time of addition and duration

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between each addition were controlled by software provided with the instrument. The

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enthalpy change at each injection was measured and plotted against concentration by using

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origin software provided with the instrument. The BSA-vesicles binding isotherm was

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determined by subtracting the enthalpy changes due the titration of BSA or vesicles in buffer

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solution from that of BSA in vesicles or vesicles in BSA solution.

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2.3.12. Far-UV Circular Dichroism Spectroscopy. Far-UV circular dichroism (CD) spectra

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of 0.1% BSA in buffer or vesicle in solution were recorded in the wavelength range 200-250

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nm on a Jasco J-815 CD spectrometer at 298.15 K. Experiments were carried out in a quartz

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cuvette having path length of 1 mm. The spectra were collected at a scan rate 100 nm/min.

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The response time and the bandwidth were 2 s and 0.2 nm, respectively.

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3. RESULTS AND DISCUSSION

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3.1. Defining the Critical Aggregation Concentration. The adsorption isotherms of

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[C8mim]Br + [Na]DBS mixtures determined from surface tension ( γ ) measurements at

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298.15 K are shown in Figure 1.The γ decreased polynomially with the increase in

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concentration for all the mixtures before attaining a plateau above CAC due to interfacial

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saturation. The CAC of all the mixtures were defined from the break point in adsorption

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isotherms and fall in the range of 0.396-0.523 mmol.L-1. Such a low CAC compared to parent

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ionic constituents; sodium dodecybenzenesulfonate (2.4 mmol.L-1)46 and 1-octyl-3-methyl

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imidazolium bromide (150 mmol.L-1)47 showed synergic interactions between surface active

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ions of the mixtures in the bulk causing early aggregation. The various thermodynamic

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parameters of aggregation such as adsorption efficiency ( pC20 ), effectiveness of surface

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tot tension reduction ( π CAC ), relative surface excess ( Γmax ) and area occupied by surfactant

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monomers at the interface have been calculated using relevant equations (Annexure 1,

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Supporting Information) and are tabulated in Table 1. The mixtures at all the composition

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decreased the γ to around 26.8 mN.m-1 due to the good interfacial adsorption per unit area due

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to significant reduction in electrostatic repulsions between head groups possessing opposite

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charges. The low pC20 and high π CMC of the mixtures indicated the better adsorption

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efficiency and effectiveness of reducing the γ compared to their parent surafcatnts.46,47

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tot Highest Γmax and lowest Amin was observed at x1 = 0.5 indicating synergic packing of

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adsorbing surfactant moieties at the air-solution interface at equimolar composition .

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tot Comparative analysis of Γmax and Amin of mixtures with parent ionic surfactants indicated

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better adsorption of surfactants in the mixtures with a more dense packing at air-solution

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interface compared to the parent surfactant moieties.46,47



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The CAC obtained from adsorption isotherm were further validated by pyrene fluorescence

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method (Figure S2, Supporting Information).30,35 The intensity ratio of the first and third

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vibronic bands (I373/I383) of pyrene fluorescence indicates variation in polarity around pyrene

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which gives information about arrival of CAC.30,35 The ratio I373/I383 decreased in a sigmoidal

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fashion and attained plateau when CAC was reached. The decrease in I373/I383 occurs because

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I373 band of pyrene senses increasingly nonpolar environmentcausing decrease in its

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fluorescence intensity.30,35,45 The CAC was marked by taking first derivatives of I373/I383 vs

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concentration profiles (Table 1).

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Conductivity ( κ ) behaviour of surfactant mixtures was then investigated to record CAC and

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o to evaluate the degree of couterion binding ( β ), standard free energy of aggregation ( ∆Gagg )

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o and standard free energy of adsorption ( ∆Gad ). The plots are shown in Figure S3A and B,

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Supporting Information. The conductivity increased initially with a higher slope due to the

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increase in number of free ions in the solution. The decrease in slope in the conductivity

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profiles indicated the arrival of CAC due to lower mobility of aggregates (micelles, vesicles)

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as compared to free ions. The CAC was obtained from the intersection point of lines fitting

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the pre and post CAC region of κ vs concentration profiles and is listed in Table 1. The

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feasibility of aggregation depends upon the binding of conterions to the surfactants head

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group in the stern layer of aggregates.30-33 The counterion binding ( β ) was calculated using

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equation, β = 1 − α , where α is degree of counterion dissociation which was calculated from

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the ratio (S2/S1) of post (S2) to pre (S1) aggregation slopes. The standard Gibb’s energy of

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o aggregation ( ∆Gagg ), for all the mixtures at 298.15 K was derived according to pseudo phase

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model of micellization using equation (1).30,35,48

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o ∆Gagg = (1 + β )RT ln xCAC .......................( 1 )


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where xCAC is mole fraction of surfactant mixtures at CAC, R is gas constant and T is

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o temperature of the solution. The standard free energy of interfacial adsorption ( ∆Gad ) of

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surfactant mixture was evaluated from equation (2) 30,35,48

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o o ∆Gad = ∆Gagg −

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o o The calculated values of thermodynamic parameters from conductivity, β , ∆Gagg and ∆Gad

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o for all the mixtures are listed in Table 2. Significantly high negative values of ∆Gagg and

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o indicated the spontaneity of adsorption and aggregation of mixed surfactants in ∆Gad

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o o aqueous solution. The higher negative magnitude of ∆Gad compared to ∆Gagg , showed that

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adsorption of surfactant mixture at the interface is more favourable than aggregation in the

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o bulk. Also the decrease in magnitude of ∆Gagg with the increase in [C8mim]Br content of

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mixtures showed that incorporation of [C8mim]Br in [Na]DBS leads to the decrease in the

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spontaneity of aggregation.

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3.2 . Characterization of Aggregated Structures

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Once the CAC of mixtures were defined, we investigated the phase behaviour, size and

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morphology of aggregates from turbidity, DLS, TEM, Cryo-TEM and AFM imaging.

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3.2.1. Phase Behaviour. The phase behaviour of [C8mim]Br (10 mmol.L-1) and [Na]DBS (10

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mmol.L-1] mixture at different mole fractions in aqueous solution was observed from visual

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inspection (Figure 2).35,36 Transparent solutions were observed at x1< 0.2 and x1>0.8 which

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indicates the micellar phase wherein individual or mixed micelles of [C8mim][Br] and

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[Na]DBS prevails. Turbidity was observed with naked eyes in the mole fraction range of

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0.1>x10.1 was also detected by a sharp increase in turbidity

π CAC tot Γmax

..............................( 2 )



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observed from UV-Vis measurements (Figure S4 Supporting Information). Maximum in

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turbidity was observed at x1=0.4 beyond which the turbidity decreased. The decrease in

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turbidity can be accounted to the change in the size of vesicles. To reveal the role played by

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synergic interactions between the aromatic rings of cations and anion in vesicle formation, we

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compared the phase behaviour of [C8mim]Br + SDS (Figure S5) and [C8mim]Br + [Na]DBS

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mixtures. In [C8mim]Br + SDS mixtures, only a small turbidity was observed in the mole

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fraction range of x1=0.4 to 0.7 which is due to the mixed micelles formation. This observation

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indicated the role played by possible π-π stacking interactions between imidazolium head

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group of cation and benzene head group of anion for the vesicle formation in [C8mim]Br +

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[Na]DBS mixtures.

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3.2.2. Size and Morphology of the Aggregated Structures. The hydrodynamic radii (Rh) of

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aggregated structures formed at different mole fractions was measured from DLS (Figure

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3).30,31,35 The corresponding intensity autocorrelation functions are provided in Figure S6.

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Small Rh of 7.4 and 15 nm were observed at x1= 0.1 and 0.9 which must be mixed micelles

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formed in these regions. Sudden rise in Rh was observed at x1= 0.3 to 0.8 which can be

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accounted to the formation of vesicular structures in the bulk solution phase. The Rh observed

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in this phase varied from 75 to 90 nm. Higher turbidity of solution in this phase also

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vindicated the formation of bigger aggregated structures.

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Morphology of the vesicles was revealed from TEM and Cryo-TEM, and that of micelles was

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observed from AFM imaging. AFM images of the mixture solution above CAC at x1 = 0.1

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and 0.9 showed micellar structures (Figure S7) with heights of 10-15 nm and 10 nm

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respectively. The TEM images of the samples at x1= 0.5 showed vesicles of the diameter

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ranging from 200 to 500 nm (Figure 4A). But when we imaged the sample from Cryo-TEM,

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multi-architecture unilamellar vesicles; sphere, tubular and ribbons were observed (Figure

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4B-D), Figure S8). Such multi-architectural vesicles were earlier reported for Janus


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dendrimer by facile coupling of tailored hydrophilic and hydrophobic branched segments.49

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Preparation of such diverse vesicular morphologies by simple mixing of two different

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surfactants is a unique phenomenon. The diameter of spheres varied from 100 nm to 1µm

4

showing formation of small to giant vesicles. The length of the ribbons varied from 1.0 to 2.5

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µm whereas the diameter varied from 100 to 200 nm. The ends of the ribbons protruded to

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form vesicles which are similar to the formation of vesicles in vivo by plasma membrane

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during exo/endocytosis.

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3.3. Forces Driving the Formation Multi-architectural Vesicles. To understand the

9

synergism between the surfactant moieties to drive vesicles formation we compared

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experimental CAC with theoretical CAC calculated from Clint’s equation (3).50

11

(1 − x1 ) .....................( 3 ) 1 x = 1 + CACmix CAC1 CAC2

12

Where CACmix is experimentally determined CAC of mixtures in aqueous medium, CAC1

13

and CAC2 are the CAC of [C8mim]Br and [Na]DBS respectively in aqueous solution and x1 is

14

mole fraction of [C8mim]Br in the mixtures. The plot showing difference in experimental and

15

theoretically calculated CAC is shown in Figure 5. The experimental values of CAC have

16

been found to be much lower than that of CACmix, reflecting non-ideality in the system and

17

hence the synergism in the surfactant mixture. Generally the large difference between

18

theoretical and experimental CAC values indicates high synergy between two surfactants.51

19

Therefore, the negative deviation of CAC values from CACmix indicated that formation of

20

mixed aggregates is driven by synergic mixing in the solution. The non-ideality of the mixed

21

cat-anionic system has been estimated through the regular solution approximation of

22

Rubingh.52 According to this theory the critical aggregate concentration (CAC) of surfactant

23

mixtures (C12) are given by equation (4).52

24

x1m . f 1C 1m = α1.C12m .......... .......... .....( 4)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

where C12m and C1m are the CAC’s of the cat-anionic mixture and surfactant 1 ([C8mim]Br),

2

respectively, α 1 is the mole fraction of surfactant 1 in solution, x1m is the mole fraction of

3

surfactant 1 in the mixed aggregates, and f1 is the activity coefficient which is given by

4

equation (5).52

5

ln f1 = β M (1 − x1m ) 2 ....................(5)

6

Where β M is the constant which signifies the net pair wise interactions in the mixed

7

aggregates and has the forms as shown in equation (6).52

8

βM =

9

where, W11, W12, and W22 are pair wise interaction energies between surfactant molecules in

10

the aggregates, L is Avogadro’s Number. Synergism of different surfactant moieties leading

11

to the formation of mixed aggregate exists when C12m of the mixture is less than that of

12

individual surfactants moieties. The defined conditions for synergism in the mixture are as

13

follows: (a) β M must be negative and (b) β M > C1m C 2m .53 The x1m and β M were calculated

14

using equation (7) and (8).54,55

15

 α Cm  x12m ln 1 12m   x1mC1  1= ....................(7)  (1 − α1 )C12m  2 (1 − x1m ) ln  m  (1 − x1m )C2 

16

βM =

17

Equation 7 was solved iteratively to obtain the value of x1m . Then substitution of the value of

18

x1m into equation (8) gave the value of β M .54,55 The values of β M for the [C8mim]Br +

19

[Na]DBS mixed systems calculated using above equations are given in Table 2. The negative

L(W11 + W22 − 2W12 ) ..............(6) RT

[

]

ln α1C12m x1mC1m ............................(8) (1− x1m )2


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1

values of β M observed in the whole mole fraction range indicated that interactions between

2

[C8mim]Br and [Na]DBS in the mixture are more attractive than between individual ionic

3

moieties.41,53 Since, in the system studied the chain lengths of surfactant used are different,

4

β M can only explain the interaction between head groups of the two surfactants.55 The

5

presence of aromatic head group containing π electrons, hydrogen bond donor and acceptor

6

sites in both the surfactants gives opportunity for surfactant head group to interact via non-

7

covalent interactions such as π-π stacking and H-bonding along with electrostatic

8

interactions. The effective overlap of head groups in a proper geometry along with inevitable

9

hydrophobic interactions between alkyl tails must be driving the formations of such multi-

10

architectural vesicles.

11

We further validated the electrostatic interactions in the system from zeta potential ( ζ )

12

measurements (Figure 6). Zeta potential of colloidal solution indicates the potential

13

difference between the dispersion medium and the stationary layer of fluid attached to the

14

dispersed micellar aggregates.36 The ζ of negatively charged micelles of [Na]DBS (10

15

mmol.L-1) was found to be -79.2 mV. The ζ decreased to -128mV at x1=0.1 which may be

16

due to the inclusion of Br- anion in the stern layer of [Na]DBS micelles. Further addition of

17

[C8mim]Br at higher mole fractions led to the increase in ζ up to x1=0.3 which showed the

18

prevalence of electrostatic interactions between oppositely charged head group of surfactants

19

A small dip in ζ at x1=0.4 was followed by sudden increase in ζ up to x1=0.9 which further

20

indicated the electrostatic interactions in the system. The value of ζ for most part of the plot

21

was found to > ±30mV which showed stability colloidal dispersions in the bulk solution.56

22

The evidence of π−π interactions between the DBS anion and [C8mim] cation in the

23

mixture were obtained from 2D NOESY NMR spectra of mixture at x1=0.5 (Figure 7). The

24

π−π in the present system is expected on account of the presence of (i) electron rich aromatic

25

π system in both the ions, (ii) low desolvation penalty in aqueous system and (iii) low degree 15 ACS Paragon Plus Environment


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1

of hydration.57,58,59 2D NOESY results shows that NOEs originating from intermolecular

2

interactions between the protons of aromatic head groups (imidazolium-benzene), aliphatic-

3

aromatic and aliphatic-aliphatic of region of [C8mim]Br and [Na]DBS. In Figure 7A, the

4

NOE cross peak corresponds to the alkyl chain of octyl group and dodecyl group with

5

aromatic protons. In Figure 7B aromatic protons of the imidazolium head groups (1H, 2H,

6

3H) show NOE with aromatic protons of the benzene head group of the DBS anion (4H, 5H)

7

indicating that imidazolium cations are present in the palisade layer of vesicles.31 Other cross-

8

peaks which show strong interactions between (i) the N−CH2 (8H) and N−CH3 (6H) group

9

with alkyl chain, and (ii) N−CH2 (8H),N-CH3 (6H) and aromatic protons (1−5H) (iii) cross

10

peak corresponding to the alkyl chain of octyl group and dodecyl groups collectively

11

suggested that there is a compact and efficient packing between the cation and anion in

12

vesicular arrangement. The expanded form of these cross-peaks is shown in Figure S9. The

13

perfect orientation of various groups showing cross peaks in NOE spectra is due to π-π

14

stacking interaction of aromatic head group of oppositely charged surfactants. The significant

15

role of aromatic counterions in the construction of higher self-assembled structures such as

16

vesicles via π-π interactions has been already reported.60 The schematic showing various

17

interactions in [C8mim]Br and [Na]DBS mixture is depicted in Figure 8.

18

3.4. [C8mim]Br + [Na]DBS Vesicles as Protein Delivery Vehicle. We explored the

19

application of these stable cat-anionic vesicles as protein delivery vehicles by investigating

20

their binding to protein, bovine serum albumin (BSA) and consequent kinetic stability of

21

BSA in vesicular dispersion. The interactional behaviour of BSA with vesicles was

22

investigated from ITC, DLS and zeta potential ( ζ ) measurements. The binding isotherm of

23

BSA to vesicles (Figure 9A) has been found to be entirely endothermic. The endothermic

24

enthalpy changes due to the interaction with proteins arises when molecules binds to proteins

25

via hydrophobic association.61,62 It is possible when BSA molecules interacts directly with the


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1

hydrophobic bilayers of vesicles. Considering the charged nature of both BSA and vesicles

2

we also looked into the role of electrostatic interactions between BSA and vesicle from zeta

3

potential ( ζ ) measurements.43,44 The ζ of native vesicle at equimolar composition in

4

phosphate buffer solution was found to be -80±5 mV, which increased to -42±2 mV upon

5

addition of 15µM BSA. An increase in ζ indicated the charge neutralization due to

6

electrostatic binding of BSA to the vesicles. To further confirm the electrostatic interactions

7

we again moved back to ITC and performed reverse titrations of vesicles into 0.1% BSA

8

solution (Figure. 9B). The initial exothermic enthalpy changes and small endothermic

9

enthalpy changes at higher vesicular concentration confirmed the prevalence of electrostatic

10

interactions in the system.61,62 It is though surprising why purely endothermic enthalpy

11

changes were observed upon titration of BSA into vesicle solution (Figure 9A). Therefore, it

12

can be concluded that BSA interacts with vesicles largely via hydrophobic interactions which

13

are also accompanied by electrostatic interactions. DLS measurements further confirmed the

14

association of BSA with vesicles (Figure S 10). The hydrodynamic radii (Rh) of BSA at the

15

studied concentration were found to be 3.52±0.2 nm. It was observed that upon interaction

16

with BSA the size of vesicles (62.2±5 nm) decreased to 40±5 nm, thus indicating the

17

contraction of vesicles upon association with BSA. Similar behaviour of vesicle contraction

18

of biamphiphillic IL vesicles of [C8mim][C12OSO3] was observed upon binding to BSA.63

19

TEM micrographs also showed BSA adsorbed on [C8mim]Br + [Na]DBS vesicles (Figure

20

10A). Efficiency of protein delivery depends upon their stability in the vesicular solution,

21

therefore, we studied the changes in secondary structure of BSA as a function of time in

22

vesicular dispersion (Figure 10B) and buffer solution as control (Figure S11) from CD

23

measurements. No alteration in the secondary structure of BSA was observed upon binding to

24

vesicles as evident from the retention of all-α CD spectrum with signatory bands at 208 (n-

25

π*) and 222 nm (π-π*). Moreover, the structure remained intact for 7 days in vesicular



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

dispersions. On the other hand, though the all-α CD spectrum of BSA is retained in buffer

2

solution, the ellipsity decreased with time (Figure S11) which usually occurs due to the

3

aggregation of protein. The possible reasons for the stability of BSA in vesicle must be the

4

lesser interactions with vesicles as charged interactions between surfactant moieties in

5

vesicles are more dominant compared to vesicle-BSA interactions.63 Therefore, these results

6

shows the potential utility of [C8mim]Br + [Na]DBS vesicles as efficient protein delivery

7

vehicle.43 Schematic of vesicle formation to BSA-vesicle interaction is provided in Figure 11.

8 9

4. CONCLUSION

10

Based on the evidences obtained from various physicochemical and imaging characterization

11

of [C8mim]Br + [Na]DBS mixtures in aqueous solution we conclude this paper based on the

12

following points: (1) The aqueous cat-anionic mixture of [C8mim]Br with [Na]DBS formed

13

unilamellar vesicles spontaneously in an exceptionally large mole fraction (x1 = 0.2 to 0.8)

14

range; (2) Cryo-TEM imaging at equimolar composition showed multi-architectural vesicles

15

(sphere, tubes and ribbons) which is unusual to earlier reported cat-anionic mixtures which

16

form either mixed micelles or spherical vesicles;18-24 (3) Negative value of interaction

17

parameter and lower experimental CMC of mixtures compared to the theoretically

18

determined values indicated high synergy between [C8mim]+ and DBS- ions; (4) Major forces

19

governing the synergic interactions were found to be electrostatic, hydrophobic and π-π

20

stackingbetween aromatic rings; (5) Br- as counter ion in palisade layer assisted in compact

21

packing of ions leading to the formation of vesicles; and (6) Stability of protein BSA in the

22

vesicles indicated their suitability as protein delivery vehicles.

23

This work gives useful insights of controlled transformation of organized assemblies in

24

mixed SAIL-conventional surfactant colloidal systems and promotes its application in

25

delivery of biomolecules.43


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1

■ ASSOCIATED CONTENT

2

Supporting Information

3

Annexure1, NMR spectra of synthesized [C8mim]Br, fluorescence spectra of pyrene,

4

conductivity plots, turbidity plot, digital images, intensity autocorrelation functions, 3-D

5

AFM images, Cryo-TEM images and NOESY spectra in expanded form for [C8mim]Br +

6

[Na]DBS mixtures, DLS plots of BSA and BSA-vesicle mixture, CD spectra of 0.1% BSA in

7

buffer.

8

■ AUTHOR INFORMATION

9

Corresponding Author

10

E-mail: [email protected]; Phone: +91-278-2567039; Fax: +91-278-2567562

11

■ ACKNOWLEDGEMENTS

12

Department of Science and Technology (DST), Government of India for the financial support

13

for this work (No. SB/S1/PC-104/2012). P. S. Gehlot is thankful to UGC for SRF fellowship

14

and K. S. Rao is thankful to CSIR for SRF fellowship. The analytical division & central

15

instrumentation facility of CSMCRI is acknowledged for helping with sample

16

characterization. Authors are thankful to Shruti Chabba, and Dr. Tejwant Singh Kang for

17

discussion on interaction parameters.

18

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48. Phillips, J. N. The Energetics of Micelle Formation. Trans. Faraday Soc. 1955, 51, 561569.


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1

49. Percec, V.; Wilson, D. A.; Leowanawat, P.; Wilson, C. J.; Hughes, A. D.; Kaucher, M. S.;

2

Hammer, D. A.; Levine, D. H.; Kim, A. J.; Bates, F. S.; Davis, K. P. Self-Assembly of

3

Janus Dendrimers Into Uniform Dendrimersomes and other Complex Architectures

4

Science. 2010, 328, 1009-1014.

5 6 7

50. Clint, J. H. Micellization of Mixed Non-ionic Surface Active Agents. J. Chem. Soc., Faraday Trans. 1975, 71, 1327-1334. 51. Holgate, C.; Glenn, K.; Palepu, R. M.; Ray, G. B.; Chakraborty, I.; Ghosh, S.; Moulik, S.

8

P.

9

Tetradecyltrimethylammonium Bromide (C14TAB), Tetradecyltriphenylphosphonium

10

Bromide (C14TPB), and Tetradecylpyridinium Bromide (C14PB). A Critical Analysis of

11

Their Interfacial and Bulk Behaviors. J. Phys. Chem. B 2007, 111, 9828-9837.

12 13 14 15

Studies

on

Binary

and

Ternary

Amphiphile

Combinations

of

52. Holland, P. M.; Rubingh, D. N. Non-ideal Multicomponent Mixed Micelle Model J. Phys. Chem. 1983, 87, 1984-1990. 53. Rosen, M. J. Surfactants and Interfacial Phenomena, second ed., Wiley, New York,

1989.

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54. Hao, L.; Deng,Y.; Zhou, L.; Ye, H.; Nan, Y.; Hu,P.; Mixed Micellization and the

17

Dissociated Margules Model for Cationic/Anionic Surfactant Systems. J. Phys. Chem. B

18

2012, 116, 5213-5225.

19 20

55. Ghosh, S.; Khatua, D.; Dey, J.; Interaction between Zwitterionic and Anionic Surfactants: Spontaneous Formation of Zwitanionic Vesicles. Langmuir 2011, 27, 5184-5192.

21

56. Bharmoria, P.; Singh, T.; Kumar, A. Complexation of Chitosan with Surfactant like Ionic

22

Liquids: Molecular Interactions and Preparation of Chitosan Nanoparticles. J Colloids

23

Interface Sci. 2013, 407, 361-369.

24 25

57. Meyer, E. A.; Castellano, R. K.; Diederich, F. Interactions with Aromatic Rings in Chemical and Biological Recognition. Angew. Chem., Int. Ed. 2003, 42, 1211-1239.



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58. Gallivan, J. P.; Dougherty, D. A. A Computational Study of Cation-π Interactions vs Salt

2

Bridges in Aqueous Media: Implications for Protein Engineering. J. Am. Chem. Soc.

3

2000, 122, 870-874.

4 5

59. Lu, Q.; Oh, D. X.; Lee, Y.; Jho, Y.; Hwang, D. S.; Zeng, H. Nanomechanics of Cation−π Interactions in Aqueous Solution. Angew. Chem., Int. Ed. 2013, 52, 3944-3948.

6

60. Xu, W.; Wang, T.; Cheng, Ni; Hu, Q.; Bi, Y. ;Gong, Y. ;Yu, L. Experimental and DFT

7

Studies on the Aggregation Behavior of Imidazolium-Based Surface-Active Ionic Liquids

8

with Aromatic Counterions in Aqueous Solution. Langmuir 2015, 31, 1272-1282.

9 10

61. Ross, P. D.; Subramanian, S. Thermodynamics of Protein Association Reactions: Forces Contributing to Stability?. Biochemistry 1981, 20, 3096-3102.

11

62. Bharmoria, P.; Kumar, A. Thermodynamic Investigations of Protein’s Behaviour with

12

Ionic Liquids in Aqueous Medium Studied by Isothermal Titration Calorimetry, Biochim.

13

Biophys. Acta. 2015, 10.1016/j.bbagen.2015.08.022.

14

63. Bharmoria, P.; Rao, K. S.; Trivedi, T. J.; Kumar, A. Biamphiphilic Ionic Liquid Induced

15

Folding Alterations in The Structure of Bovine Serum Albumin in Aqueous Medium. J.

16

Phys. Chem. B 2014, 118, 115-124.

17 18 19 20 21 22 23 24 25


Page 26 of 34

Page 27 of 34

1 2

0.01

3 4 5 6 7

γ /m N .m -1

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


8 9 10 11 12 13

14

0.1

1

65 52 39 26 75 60 45 30 75 60 45 30 75 60 45 30 75 60 45 30

x1=0.9

x1=0.7

x1=0.5

x1=0.3

x1=0.1 CAC

1E-3

0.01

0.1

-1

1

logCtot./mmol.L 15

Figure 1. Adsorption isotherms of [C8mim]Br + [Na]DBS mixture at various mole fractions

16

in aqueous solution at 298.15 K. 17

18

x1= 0.1

x1= 0.2 – 0.8

x1= 0.9

Vesicles

Micelle

19

20

Micelle

Figure 2. Digital photographs of [C8mim]Br + [Na]DBS 21

mixtures at different mole fractions.

22 23



1

x1=0.9

0.65

2

0.00

N o r m a liz e d I n t e n s it y

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

3 4 5 6 7 8

0.65

x1=0.7

0.00 0.7

x1=0.5

0.0 0.7

x1=0.3

0.0

x1=0.1

0.65 0.00 0

9

50

100 150 200 250 300 350 400 450 500

Rh(nm)

10

Figure 3. Hydrodynamic radii distribution of aggregated structures

11

of [C8mim]Br + [Na]DBS mixtures at different mole fractions.

12

(A)

(B)

(C)

(D)

13 14 15 16 17 18 19 20

500 nm

21 22 23 24 25 26 27

Figure 4. (A) TEM and (B-D) Cryo-TEM images of [C8mim]Br + [Na]DBS mixture at x1 = 0.5, showing unilamellar vesicles of different architectures viz. sphere and tubules and ribbons. 28 ACS Paragon Plus Environment

Page 28 of 34

Page 29 of 34

1 160

2 150

Theoritical values Experimental values

140

3

12

5

CAC

4

10 8 6 4

6

2 0

7

0.00 0.15 0.30 0.45 0.60 0.75 0.90 1.05

x1

8 9

Figure 5. Comparison of ideal CAC values obtained

10

from the Clint equation with the experimental CAC

11

(average) values measured from various techniques.

12 13 0

14 15 16 17

-25

ζ / mV

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


-50 -75

-100

M I C E L L E

VESICLES

-125 -150

18 19

M I C E L L E

-175 0.00 0.15 0.30 0.45 0.60 0.75 0.90 1.05

x1

20

Figure 6. Variation of zeta potential (ζ) of (x1)

21

[C8mim]Br + [Na]DBS solution at different

22

mole fractions.

23 24 25 26



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 34

1 2 9 3 4 5

1-5

1-5

A

6,8

7

A

9

6 7

1-5

B

8 9

1-5

B

10 11 12 13

Figure 7. 2D NOESY spectrum of [C8mim]Br + [Na]DBS at x1 = 0.5 in aqueous medium. NOEs originate from (A) aromatic protons (imidazole

14

and benzene ring) interactions with alkyl chains protons and (B) aromatic

15

protons of imidazolium cation and benzene ring of DBS anion.

16 17 18 19 20 21 22 23 24 25

Figure 8. Scheme representing peak positions and possible interactions in vesicular structures of [C8mim]Br + [Na]DBS at x1 = 0.5

26 27 30 ACS Paragon Plus Environment

1

12

6

Time / min

30

36

42

Time / min 48

6

60 14

(A)

1.4

6

50

4

48

2

46

0

44

-2

42

-1

BSA Binding to Vesicles (2 mmol.L )

0.005

0.010

0.015

-1

0.020

-1

dH / kJ.mol

8

52

9

24

30

36

42

48 1.2 0.6

0.0

0.0

-0.7

-0.6

-1.4

-1.2

-2.1

-1.8

-2.8

-2.4

-4

Vesicles binding to 0.1% BSA

-3.5

-6 0.025

0.0

0.7

1.4

2.1

2.8

3.5

Vesicle / mmol.L

Figure 9. ITC thermograms of (A) BSA binding to vesicles (2 mmol.L-1) with corresponding differential power (dP) plot as inset and (B) vesicles

10

binding to BSA with dP plot as inset. 11 12 13 (A)

Native BSA BSA+Vesicles Day 1 Day 7 Day 14

(B)

-1

20

-2

15

[θ]MRE/10 xdeg.cm dmol

14

16

10

0

6

17 18 500 nm

19

-10

-20 200

210

220

230

λ / nm

240

250

20

Figure 10. (A) TEM image of BSA adsorbed on [C8mim]Br + [Na]DBS 21 22 23

vesicles,

(B) CD spectra of BSA in [C8mim]Br + [Na]DBS vesicular

dispersions at different time (days) intervals.

24 25 26


-3.0

4.2

-1

BSA / mmol.L

8

18

0.7

10

54

12

(B)

12

40

7

54

56 -1

5

24

58

3 4

18

dP / µ Watts

60

2

dH / kJ.mol

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


dP / µ Watts

Page 31 of 34


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 34

1 2 3 4 5 6 7 8 9

Figure 11. Schematic showing multi-architectural self-assembled structures

10

of [C8mim]Br + [Na]DBS and protein BSA associated with vesicles in

11

aqueous medium. 12 13 14

Table 1. Critical aggregation concentration (CAC), surface tension at CAC with ±0.1mN.m-1 ), effective surface tension reduction with ±0.1mN.m-1 accuracy (

accuracy (

adsorption efficiency (

), maximum surface excess concentration (

),

), and minimum

) of [C8mim]Br + [Na]DBS

area occupied by a single molecule at the air-water interface ( mixtures in aqueous at 298.15 K

γ CAC

CAC (mmolL-1) x1

π CAC -1

-1

(mN.m )

(mN.m )

tot pC 20 Γmax

Amin

(µmol.m-2)

(Å)

S.T.

Cond.

Flr.

Avg.

0.1

0.32

0.53

0.35

0.40

26.66

44.91

4.56

2.6379

63.18

0.3

0.23

0.26

0.30

0.26

26.62

43.89

4.63

2.9983

55.59

0.5

0.21

0.22

0.29

0.24

27.29

43.75

4.56

3.9567

42.12

0.7

0.25

0.45

0.33

0.34

26.79

44.85

4.51

3.5655

46.74

0.9

0.46

0.56

0.52

0.51

27.12

43.82

4.40

2.5166

66.22

15 16


Page 33 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1 2

Table 2. Critical aggregation concentration (CAC), Gibbs free energy of

3

aggregation (

4

), Gibbs free energy of adsorption (

) at different mole

fraction for the mixtures [C8mim]Br + [Na]DBS in aqueous medium at 298.15 K.

5 x1

CAC experimental (mmol.L-1)

CAC theoretical (mmol.L-1)

βM

8

0.1

0.40

1.55

9

0.3

0.26

10

0.5

6

o ∆Gagg

o ∆Gad

(kJ.mol-1)

(kJ.mol-1)

-13.43

-33.97

-51.00

1.99

-14.84

-34.54

-49.17

0.24

2.78

-15.44

-35.99

-47.05

0.7

0.34

4.57

-14.78

-31.14

-43.62

0.9

0.51

12.91

-15.14

-30.73

-48.14

7

11 12 13 14 15 16

TOC 600dpi, 5.1×5.1 cm

17 18 19 20 21 22 23 24 25 26



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

54x54mm (300 x 300 DPI)

ACS Paragon Plus Environment

Page 34 of 34

Spontaneous Formation of Multiarchitecture Vesicles of [C8mim]Br +

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