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Sodium Chloride Triggered the Fusion of Vesicle Composed of Fatty Acid Modified Protic Ionic Liquid: A New Insight Into the Membrane Fusion Monitored Through Fluorescence Lifetime Imaging Microscopy Niloy Kundu, Pavel Banerjee, Sangita Kundu, Rupam Dutta, and Nilmoni Sarkar J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.6b09298 • Publication Date (Web): 13 Dec 2016 Downloaded from http://pubs.acs.org on December 16, 2016

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The Journal of Physical Chemistry

Sodium Chloride Triggered the Fusion of Vesicle Composed of Fatty Acid Modified Protic Ionic Liquid: A New Insight into the Membrane Fusion Monitored through Fluorescence Lifetime Imaging Microscopy Niloy Kundu, Pavel Banerjee, Sangita Kundu, Rupam Dutta and Nilmoni Sarkar* Department of Chemistry, Indian Institute of Technology, Kharagpur 721302, WB, India E-mail: [email protected] Fax: 91-3222-255303

Abstract The development of stable vesicular assemblies and understanding their interaction and dynamics in aqueous solution is a long standing topic in the research of chemistry and biology. Fatty acids are known to form vesicle structure in aqueous solution depending on the pH of the medium. Protic ionic liquid of fatty acid with ethyl amine (Oleate ethyl amine, OEA) as a component spontaneously form vesicle in aqueous solution. The general comparison of dynamics and interaction of these two vesicles have been drawn using fluorescence correlation spectroscopy (FCS) and fluorescence lifetime imaging microscopy (FLIM) measurements. Further, FLIM images of a single vesicle are taken in multiple wavelengths and the solvation of the probe molecules has been observed from the multiwavelength FLIM images. The lifetime of the probe molecule in OEA vesicle is higher than that in simple fatty acid vesicles. Therefore, it suggests that the membrane of the OEA vesicle is more dehydrated compared to that of fatty acid vesicles and it facilitates OEA vesicles to fuse themselves in presence of electrolyte, sodium chloride (NaCl). However, under same condition only fatty acid vesicles do not fuse. The fusion of OEA vesicles is successfully demonstrated by the time scan FLIM measurements. The different events in fusion process are analyzed in the light of reported model of vesicle fusion. Finally, the local viscosity of the water pool of the vesicle is determined using kiton red, as a molecular rotor. With addition of NaCl, the fluidity in the interior of the vesicle is increased which lead to disassembly of vesicle. The rich dynamic properties of this vesicular assembly and the FLIM based approach of vesicle fusion will provide better insight into the growth of protocell membrane.

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1. Introduction. In recent years, the field of ionic liquid (IL) have received significant attention in the field of chemistry and biology. Different physical as well as chemical properties of ILs, such as nonvolatility, non-flammability, high thermal stability improve its applicability in specific applications along with fundamental research.1-4 Because of the rising necessity of sustainable green chemistry, ILs are becoming increasingly popular. Protic ionic liquids (PILs) are the subset of ILs which are prepared through the proton transfer reaction between the Brǿnsted acid and Brǿnsted base.5,6 Thus, simultaneously hydrogen bond donor and acceptor sites are created. As they can be prepared very easily PILs are used in different industrial applications which involve design of electrolyte fuel cells, biocatalytic reactions, separation and self assembly processes etc.7-10The first synthesized PIL, ethylammonium nitrate (EAN) was made by the proton transfer reaction between ethyl amine and nitric acid. However, recently, a wide range of PILs based on fatty acids or their derivatives have been used as a surfactant or other purposes.11,12Due to the long carbon chain and low toxicity, fatty acid based PILs can be an appropriate choice with properties tailored by an appropriate proton acceptor. Early cell membranes are thought to comprise of fatty acid or other single chain amphiphiles instead of phospholipids and the reason of the hypothesis came from the fact that fatty acid spontaneously form bilayer vesicles.13Now, fatty acid vesicles, an alternative to phospholipids vesicles have been extensively used in a wide variety of applications and it is considered as one of the important biomimetic model membrane systems.14-17The use of giant fatty acid vesicles incorporated with drugs, proteins and various other biological macromolecules has gained significant interest in recent years.18,19 However, the most important feature of the fatty acid vesicle is the self replication.20-22 Therefore, it has a great relevance to understand the emergence of cellular life in real biological system. Thus, the self assembly of fatty acid vesicles is a great paradigm of origin of life and corresponding protocell membrane.17,18 Extensive studies have been performed to understand the small molecule diffusion, membrane mediated pH gradient and competitive growth and division.15,20 It has been known for a long time that fatty acid spontaneously form vesicles in aqueous solution when the pH of the solution is close to the pKa of fatty acid.21,23Oleic acid, an important fatty acid forms oleate micelle at basic pH (pH ~11) and they are converted into vesicles when the pH of the solution is adjusted to 8.5. At acidic pH, oleic acid become fully protonated and forms an oil droplet. Thus, the vesicle is only stable when the pH of the 2 ACS Paragon Plus Environment

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solution is adjusted between 9 and 7.8.13 This pH dependent self aggregation of fatty acid has been extensively studied by molecular dynamics simulation as well as NMR (nuclear magnetic resonance), electron spin resonance (ESR) and X-ray diffraction (XRD) etc.2225

However, the major disadvantage of pure fatty acid vesicle is that they are highly permeable

to protons and therefore, incapable of maintaining pH gradients.15Thus, after addition of small amount of fatty acid into phospholipids vesicles the pre-stabilized pH gradient dissipates immediately. Moreover, the constituting components of fatty acid vesicles remain in dynamic equilibrium with the solution phase and also with each other.26The utility of vesicles solely of pure fatty acids is therefore limited because of their poor stability with respect to the variation of pH, temperature and concentration of the medium.27However, such stability can be enhanced to a wider pH range by mixing with fatty alcohols, fatty acid glycerol esters or other amphiphilic molecules.28-30 In this manuscript, we have synthesized fatty acid based protic ionic liquid (oleate ethyl amine, OEA) where oleic acid act as Brǿnsted acid and ethyl amine act as Brǿnsted base. In aqueous solution, the formation of large vesicle is characterized by fluorescence lifetime imaging microscopy (FLIM), dynamic light scattering (DLS) and Transmission Electron Microscopy (TEM) measurements. The major advantage of this vesicular system over fatty acid vesicles (oleic acid/oleate vesicle) is that they spontaneously form vesicle in aqueous solution and the structure of the vesicle is maintained when the pH of the medium is adjusted between 6.5 and 11. A general comparison of structure, interaction and dynamic properties between OEA vesicles and oleic acid/oleate vesicles has been drawn using FCS and FLIM experiments. Heterogeneity in the size of the vesicles is revealed by the FCS measurements. Moreover, FLIM images of a single vesicle are taken in different emission wavelengths using multi-wavelength detector and the solvation in the single vesicle is observed from the FLIM images. FLIM analysis indicates that the membrane of the OEA vesicles are more dehydrated compared to that of oleic acid/oleate vesicles. The dehydration of membrane surface of OEA vesicles facilitates to fuse themselves in presence of sodium chloride (NaCl). However, oleic acid/oleate vesicle does not fuse under the same condition. The fusion process of different liposomes in presence of monovalent as well as divalent salts is well reported in the literature.31-34In the present system, the different events in vesicle fusions are characterized by time dependent FLIM measurements. The changes in lifetime of the probe molecule associated with each FLIM image are discussed in the light of different reported models of vesicles fusion. The local viscosity in the interior of the vesicle in presence of NaCl is also 3 ACS Paragon Plus Environment


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calculated using kiton red, a popular molecular rotor. The FLIM measurements in different stages of vesicle fusion make this study unique and the rich dynamic properties of these vesicles provide interesting model of fusion and growth of the protocell membrane which might have occurred in response to different physical or chemical forces. 2. Experimental Section. 2.1. Materials and Synthesis of Protic Ionic Liquid. Oleic acid is purchased from Loba Chemie ltd. (India) and ethyl amine is obtained from SRL (India). The protic ionic liquid (oleate ethyl amine, OEA) is synthesized by the reaction of an equimolar amount of oleic acid and ethyl amine following the literature procedure.11The mixture is magnetically stirred at low temperature (273K-278K) and slightly yellow coloured viscous liquid is obtained as a product. Water is removed by rotary evaporation followed by lyophilisation and the purity of the compound is checked by proton (1H) NMR and

13

C NMR and they are given in

Supporting Information (figure S1 and S2 of Supporting Information). Sodium Chloride (NaCl) and sodium hydroxide (NaOH) are obtained from SRL (India). 4-(dicyanomethylene)-2-methyl-6-(4-dimethylaminostyryl)-4H-pyran (DCM) is used as a probe for FLIM and FCS measurements and it is obtained from Exciton. Kiton red or Sulforhodamine b (SRh-B) is used to determine the local viscosity in the interior of the OEA vesicle and received from Lambda Physik. All the chemicals are used without further purification and the structure of all the chemicals are shown in Scheme-1. 2.2. Preparation of Vesicle solution. For the preparation of oleic acid/oleate vesicle we have followed the literature procedure.13 At first, 0.1 (M) oleic acid and 50 mM NaOH solution are prepared and then, oleate micelle is prepared by adding appropriate amount of 50 mM NaOH solution and pH of the solution is maintained greater than 10.5. The stable oleic acid/oleate vesicle is obtained by adding appropriate amount of 1(M) HCl into the oleate micelle and throughout the experiments, the pH of the solution is maintained as 8.4. However, 0.1(M) oleate ethyl amine (OEA) vesicle solution is prepared in aqueous solution and the solution was sonicated for 1-2 minute. The stock solution of DCM (1 mM) is prepared in methanol. At first, small aliquot of the dye solution from the stock is taken in the volumetric flask and methanol is evaporated in hot air flow. Further, requisite amount of 0.1 (M) vesicular solution is added to adjust the concentration of dye.

The dye concentration is maintained as ~1nM for the FCS 4 ACS Paragon Plus Environment

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measurements and 0.1 for the FLIM measurements,. The dye loaded solution is kept for few hours for the proper encapsulation of the dye in the vesicular solution. 2.3. Instrumentation. The formation of OEA vesicle is characterized by dynamic light scattering (DLS) and transmission electron microscopy (TEM) measurements. Further, the dynamic properties of the vesicles are characterized by fluorescence correlation spectroscopy (FCS) and fluorescence lifetime imaging microscopy (FLIM). The detailed descriptions of the instruments are discussed in the supporting information. 3. Results and Discussion. The synthesized protic ionic liquid, OEA represents an amphiphilic and asymmetric molecular structure which contains a hydrophilic (ethyl ammonium cation and carboxylate anion) and hydrophobic moiety. The self aggregation of these kinds of protic ionic liquid is well reported in the literature.11These self assembled structures are transformed from micelle to more complex structures in aqueous solution with increasing concentration of IL. In aqueous solution, 0.1 (M) OEA spontaneously form vesicle which can be identified visually by observing an opalescent solution. The formation of large vesicle is confirmed by the TEM images (figure S3 (a,b) of Supporting Information) and FLIM images using DCM as a fluorophore (figure 1(a,b)). In the FLIM images, vesicles with very broad distribution in size are observed (figure 1(c)). DLS measurement also confirms the formation of vesicles with broad size distribution (figure 1(d)). The lifetime associated with each FLIM image is shown in figure 1(a,b) and we have observed that the DCM lifetime is not uniform which indicates the complex organization of PILs in water. The images of the vesicles are collected in the xy plane (z being vertical) at different z values. The z stack images of OEA vesicles and oleic acid/oleate vesicles are shown in figure 2. The 3D views of the vesicles are obtained by moving the z axis. A general comparison of structure, interaction and dynamic property is required for the OEA vesicles with oleic acid/oleate vesicles in order to understand the kinetic and thermodynamic aspects of different vesicular assemblies.35 From the DLS measurement and the FLIM images it is very much clear that the size of vesicles vary significantly. Now, the changes in the diffusion behaviour of the DCM dye in two different vesicular assemblies are studied by FCS. The change in the diffusion coefficient in different self-assemblies depends on the location of the probe molecule in that system.3638

DCM is a neutral and hydrophobic molecule and it resides at the hydrophobic bilayer region

of the vesicle. In aqueous solution, the translational diffusion coefficient of DCM was found 5 ACS Paragon Plus Environment


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to be 302 and it is supported by earlier literature reports.36 Figure 3(a) shows the FCS traces of DCM in two different vesicular assemblies. Due to the higher extent of polydispersity in the size of the vesicles, multiple species fitting equation is used in order to fit the FCS traces. The fitted diffusion coefficients ( ) are tabulated in table 1. In the bulk solution, DCM exhibits two diffusion coefficients; the faster component of 302 corresponds to the diffusion of DCM in the bulk water and the slower component contributes to the diffusion of the vesicle as a whole. In both the vesicles, relative contribution of in bulk water is very small. However, the change in the diffusion coefficient for OEA vesicle is much higher compared to that of oleic acid/oleate vesicle. The experiments are performed more than 10 times and a distribution in the diffusion coefficient is plotted in figure 3(b). The distribution of is fitted to a Gaussian function and the mean value is taken as an average. For OEA vesicle and oleic acid/oleate vesicle the slow diffusion coefficients are obtained as 0.55±0.05 and 1.67±0.06 respectively. The much slower diffusion coefficient for OEA vesicle can be attributed to the larger size of OEA vesicle compared to that of oleic acid/oleate vesicle. Now, the size of the vesicles can also be estimated from the diffusion coefficient following the Stokes-Einstein equation, =

(1)

In the above equation, is the hydrodynamic radius of the vesicle and is the viscosity of the medium. Thus, following the equation, diffusion coefficient of 0.5 corresponds to vesicle of diameter roughly 1 which is much smaller than the size of the vesicles obtained from the FLIM images. The observed value of 0.5 suggests that a significant contribution is coming from the other freely diffusing vesicles which have much smaller size.37 This is also applicable for the oleic acid/oleate vesicle. Following the StokesEinstein equation, the diffusion coefficient of 1.67 corresponds to the vesicle of approximately 300 nm size and the size of the vesicles obtained from FLIM image are much higher than that. Due to significant polydispersity in the size of the vesicles, the size obtained from the FCS measurements and DLS measurements may not be same. Sen et al have recently performed FCS in different microemulsion droplets and compared the result with the DLS measurements.39They showed that both the measurements have certain disadvantages. Now, in case of FCS, Gapinski et al recently have shown that if the size of the spherical particle is higher than the size of the confocal volume (~365 nm), the autocorrelation function can also be fitted well with the standard formula which is used for infinite small 6 ACS Paragon Plus Environment

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particles.40However, the effective confocal volume is increased in that case. Thus, the FCS measurements indicate an ensemble average measurement over many differently sized vesicles. In case of vesicles in bulk water, we get an average of many vesicles of different sizes which are freely moving in bulk water. Hence, we attempt to study the dynamics of a single vesicle using FLIM. For this, 50 vesicle solution is placed on the slide and the solution is allowed to equilibrate for 15 minutes. They show same position for the individual solution which confirms the proper immobilization of vesicle on the glass surface and they remain stationary during the data acquisition. We have used multi-wavelength FLIM (MW-FLIM) to collect the FLIM images of DCM in two different vesicular assemblies in different emission wavelengths. MW-FLIM analysis is important in order to understand the different physical parameters of biomolecules which include the solvation aspects of probe molecules or autofluorescence of tissue cells etc. The detailed description of the MW-FLIM is described in the supporting information. Briefly, MW-FLIM uses the capability of time correlated single photon counting (TCSPC) modules to record in several detector channels and the result can be interpreted as the number of FLIM images collected in different emission wavelengths.41 Thus, we are able to collect the FLIM images of a single vesicle in different emission wavelengths from the blue end to the red end of the emission spectrum. The MW-FLIM images of single OEA vesicle of size ~5.5 and oleic acid/oleate vesicle of size ~ 2.5 are shown in figure 4 (i) and 4(ii) respectively and the images are collected in six different wavelength regions. The lifetime decays of DCM in OEA vesicle at two different wavelengths are shown in figure 5. Decays are collected at a particular position (pixel) of the vesicle (figure 5). Solvent relaxation takes place in two different vesicular systems resulting in the fast decay at the blue end and slow rise at the red end of the emission spectrum. The lifetime decays of DCM are fitted with a biexponential function and the lifetime values at two different wavelengths in two different vesicular assemblies are summarized in table 2. The average lifetime of DCM in OEA vesicle is increased to 4.51 ns at 731-744 nm from 2.17 ns at 606-620 nm. However, in case of oleic acid/oleate vesicle, the average lifetime of DCM is increased to 2.77 ns at 731-744 nm form 0.93 ns at 606-620 nm. Thus, from table 2, it is obtained that the average lifetime of DCM in OEA vesicle is higher than that in oleic acid/oleate vesicle and it indicates that the OEA vesicular membrane is less hydrated and more rigid compared to the oleic acid/oleate vesicular bilayer. Recently, Bhattacharyya et al have performed the solvation dynamics of C-153 (Coumarin-153) in a single DLPC (1,27 ACS Paragon Plus Environment


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dilauroyl-sn-glycero-3-phosphocholine) vesicle of diameter 20 42. They have shown that the average solvation time and diffusion behaviour of C-153 are not same everywhere in the single vesicle and it depends on the position as well as the microenvironment of the probe molecule. For this reason, we have also emphasized on the lifetime distribution of DCM in the two vesicular assemblies (figure 6) and we have obtained a broad lifetime distribution of DCM in different emission wavelengths which clearly indicates the microenvironments around the DCM molecules is highly heterogeneous in a single vesicle. Similar to earlier measurements, we have also observed the average lifetime of DCM is gradually increased with increasing the emission wavelengths from the blue end to the red end of the emission spectrum. This result is very unique compared to other ensemble average measurements in bulk solution. Thus, the wide distribution in the lifetime of DCM in a single vesicle suggest the spatial variation in polarity, dynamics and reactivity in a single vesicle and it is markedly different from the other ensemble average measurements reported earlier in the literature.43-45 Vesicle fusion process in lipid based systems or polymeric vesicle are explored in the literature in great detail.31-34,46 Moreover, in cell biology, membrane fusion is a fundamentally important process because of its involvement in different biological processes which includes cell fusion, cell division, exo and endo-cytosis etc.47,48 In real biological system, vesicle fusion involves membrane specific proteins.49,50 However, the fusion processes in biological systems are highly specific and it requires different models for proper establishment of the process.51,52 For this reason, to understand such processes several attempts have been made to investigate the fusion process involved in synthetic lipid vesicles under various physicochemical conditions.51,52 The fusion of different vesicles in presence of monovalent and divalent salts and their reversible aggregation are well documented in the literature.31-34 Recently, Paxton et al have demonstrated the fusion of large unilamelar vesicle into giant vesicle in presence of dilute aqueous sodium chloride (NaCl) solution under agitation.32 The fusion process of fatty acid containing vesicle can be correlated with the fusion process in natural biological system due to its existence in prebiotic world. In our study, we have observed the growth of the OEA vesicle in presence of low concentration of NaCl. However, under same condition we have not observed any growth of the oleic acid/oleate vesicle. The preliminary indication of the vesicle fusion is observed visually; after addition of low concentration of aqueous NaCl solution to the vesicle, the solution becomes turbid. For the turbidity measurement, the large OEA vesicles are converted into the small unilamelar vesicle by probe sonication for 15 minutes using ultrasonic sonicator bath45and the formation 8 ACS Paragon Plus Environment

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of vesicle of 100 nm size is confirmed by the TEM image (figure S3e of Supporting Information). With increasing concentration of NaCl, the turbidity of the solution is increased gradually which is shown in figure S4(a) of Supporting Information. (the turbidity measurement is performed by measuring the absorbance of the solution at a particular wavelength where there is no contribution of the individual components.). The size of the fused vesicles is measured with the dynamic light scattering (DLS) measurements. Addition of 0.1 (M) NaCl induces a change in the intensity distribution of the OEA vesicles. For the small unilamelar OEA vesicle, the size obtained from DLS measurement is about 100 nm and addition of 0.1 (M) NaCl results in a broadening of the intensity distribution which may be due to the presence of unfused and fused vesicles in the solution (figure 7(a)). However, we have not observed any significant change in the intensity distribution of oleic acid/oleate vesicle in presence of 0.1 (M) NaCl (figure 7(b)). The formation of large vesicles after the fusion is also demonstrated by the TEM images (figure S3(c,d) of supporting information). Now, DLS measurements cannot distinguish between the aggregated state and fused state. The fusion of OEA vesicles is further confirmed by the FLIM measurement which is discussed later. Earlier, Chaimovich et al showed the NaCl induced aggregation and fusion of dioctadecyldimethylammonium chloride (DODAC) and sodium dihexadecylphosphate (DHP) vesicles53 and they have showed that the DLVO (Derjaguin-Landau-VerweyOverbeek) theory for colloid stability which is based on the long range electrostatic interaction or electrodynamic forces cannot explain the relative stability of small and large vesicle in presence of salts. Thus, the short range hydration repulsion may be the possible reason for the energy barrier of the vesicle fusion. Now, it is well established that the fatty acid vesicles show higher membrane fluidity than the vesicle containing cholesterol or phospholipids and the membrane surface of oleic acid/oleate vesicles are hydrated.30 Suga et al. have showed that modification of fatty acid membrane surface with cationic surfactant results in a slight dehydration of the membrane surface.30 From the FLIM measurements we have also observed that the membrane of the OEA vesicles is more dehydrated compared to the oleic acid/oleate vesicles. Thus, hydration repulsive force at close approach due to the water structure is lower for OEA vesicle than oleic acid/oleate vesicle and it accounts for the experimentally established stability of oleic acid/oleate vesicle in presence of electrolytes. It is important to mention here that besides the hydration repulsive force osmolarity is also a potential factor for the vesicle fusion. To determine the osmotic properties, the change in the absorbance of 0.1 (M) OEA vesicles in 20 mM NaCl is recorded in different time (figure S5, supporting information). The change of turbidity due to the osmotic gradient is the 9 ACS Paragon Plus Environment


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consequence of swelling or shrinking of vesicles. Earlier, Chaimovich et al. showed the osmotic gradient induced time dependent absorbance change of fused vesicles46and the change in the absorption due to fusion was interpreted with a model based on light scattering laws and geometric consideration of vesicles. If the size of the spherical vesicle is comparable to the wavelength of light source (), the absorbance at a particular time depends on the number of aggregated particle and the ratio between the size of the original particle and that of aggregated particle. An initial jump in absorbance is observed due to the fusion of vesicles forming a larger aggregate. The decrease in absorbance corresponds to the swelling of the vesicles. Now, the decrease rate of absorbance for larger OEA vesicle is more prominent compared to small sonicated OEA vesicles and this fact is well justified by the analysis proposed by Chaimovich et al.46 Thus, it is suggested that Na+ promotes the close association of the vesicles which increases the fusion rate and due to the osmotic gradient the water molecules flow across the membrane and into the vesicles results in vesicle swelling and subsequent fusion. We have also measured the zeta potential of OEA vesicle in presence of different concentration of NaCl (figure S4(b) in Supporting Information) and the zeta potential is slightly increased in presence of NaCl which indicates the decreased electrostatic repulsion between the vesicles. Therefore, addition of NaCl into the solution allows the vesicles to come close to each other. The vesicle fusion is visually evidenced by the FLIM experiments undertaken on OEA vesicles in presence of 60 mM NaCl and stained with DCM dye. We have preferred to perform the time series FLIM analysis to understand different stages of vesicle fusion and they are shown in figure 8. The collection time of each image is 3 seconds and the collected images are assembled together and the images are converted into movie frame using software (movie 1 and movie 2). They are very much helpful to visualize the motion of vesicles and their fusion in solution. The time frame of vesicle fusion is varied from milliseconds to seconds. In movie 1, we have observed that the fusion between two vesicles is random in nature. In movie 2, we observe that the vesicles with small size fuse more readily compared to other giant vesicles or they move much faster than the other larger sized vesicles. Small sized vesicles have higher curvature. So, their tails are more exposed to water and have much more freedom in movement54. The different events in vesicle fusion are extensively studied by computer simulation.5557

However, there are limited experimental evidences in literature about the different stages in

vesicle fusion.58 Vesicle bilayer fusion proceeds through several distinct steps. Firstly, two 10 ACS Paragon Plus Environment

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vesicles come close and an initial contact is formed which involve lipid mixing of contacting monolayers. This stage is known as hemifusion.51After this step, full fusion is reached. In literature, there are valid evidences that fusion of lipid bilayers proceed through hemifusion rather than direct fusion.51,52Direct fusion which involves pore edges formation requires much higher energy in lipid vesicle fusion. However, in hemifusion, an earlier intermediate which is known as fusion stalk cost much lower energy. The different pathways in fusion process in our case can be understood by measuring the lifetime associated with each image. We have measured the lifetime as well as lifetime distribution of DCM in different events of vesicle fusion processes and they are summarized in figure S6, Supporting Information. To avoid the complexity, lifetime images are taken in a selected area focusing only two vesicles which will be fused and we have observed that the lifetime value of the DCM dye is decreased after the fusion (figure S6, Supporting Information) and we have repeated it for multiple fusion processes in different vesicles and in every cases, we have ended up with same observation. Besides the lifetime distribution, lifetime values are taken at a single point of the vesicle. DCM is mainly located at the bilayer of the vesicle. Lifetime values are measured at the junction point where two vesicles are fused and with increasing time, lifetime values are steadily decreased at that point (figure S7, Supporting Information). Now, in case of hemifusion, only the lipid exchange between the outer membranes is allowed. After the hemifusion, the inner membranes of two vesicles are merged and form a nascent fusion pore.51At this stage, the inner content (water) of the vesicles are mixed. As DCM is a hydrophobic dye, during the exchange of the water molecules between vesicles the lifetime of the DCM is decreased. Now to determine the local viscosity of the interior of the OEA vesicle under varying salt concentration vesicles are treated with kiton red (figure 9). The detailed description of calculation of local viscosity from the lifetime images are shown in the Supporting Information. Oleic acid/oleate vesicle does not produce any change in the fluorescence lifetime in presence of different concentration of NaCl.14 With increasing concentration of NaCl, the local viscosity in the interior content of OEA vesicle (i.e. the water pool inside the vesicle) is gradually decreased from approximately 7.45 cp in absence of NaCl to 0.95 cp in presence of 80 mM NaCl (figure 9(c)). This is consistent with progressive disassembly of the interior contain of the OEA vesicle in presence of NaCl. In order to mix the inner content of the vesicle, the fluidity of the water molecules need to be increased. Mann et al have also determined the local viscosity of coacervate microdroplets of fatty acid in presence of NaCl 11 ACS Paragon Plus Environment


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and they have ended up with the same observation.14 Decrease of the local viscosity in the water pool of the vesicle clearly indicates the increase in the fluidity of the system and facilitates to fuse the vesicles. 4. Conclusion. In summary, we have successfully demonstrated the formation of large OEA vesicle by TEM and FLIM measurements. The major advantage of this vesicle is that they are stable in aqueous media compared to the simple fatty acid vesicles which are stable in between a certain pH range. FCS measurements have been performed to explore the dynamics and interaction of these vesicles and the heterogeneity in size is obtained from the FCS measurements for both the vesicles. Due to the wide variation in size of the vesicles a wide distribution in the diffusion coefficient of the dye is observed. Besides the FCS, FLIM images are taken of a single vesicle in multiple wavelengths. Wide distribution of lifetime signifies that the microenvironment of the probe molecule in a single vesicle is heterogeneous which is very uncommon from the reported ensemble average measurements. However, the higher lifetime in OEA vesicle compared to that in oleic acid/oleate vesicle signifies that the membrane of OEA vesicle is more dehydrated which facilitates to fuse themselves in presence of NaCl. The fusion of OEA vesicle is established by time scan FLIM measurements and different events in vesicle fusion can also be demonstrated by the change in lifetime of the probe molecule during fusion process and the local viscosity of the interior of the vesicle is decreased with increasing concentration of NaCl which indicates the progressive disassembly of the interior of the vesicle. Additionally, the modified fatty acid vesicle may have the important implication for the drug delivery vesicles because of its existence in prebiotic world and stability in physiological condition. More importantly, although exploration of design, function and potential of model protocells is in its infancy, this approach should provide better insight into the mechanism of growth of the protocell membrane. Acknowledgment: N.S. gratefully acknowledges SERB (Grant No: IR/S1/LU-001/2013 dated 24/03/2015), Department of Science and Technology (DST) and Council of Scientific Industrial Research (CSIR), Government of India for providing generous research grants. N.K is thankful to IIT Kharagpur and P.B., S.K., R.D acknowledge CSIR for their research fellowships. Supporting Information: Instrumentation section, determination of local viscosity using FLIM, Proton NMR and 13C NMR of the synthesized IL, TEM images of the vesicles, 12 ACS Paragon Plus Environment

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turbidity and zeta potential measurement in presence of NaCl, Movie 1 and 2 regarding the vesicle fusion, FLIM images and lifetime distribution of OEA vesicles in different times are included in the Supporting Information. This information is available free of charge via the Internet at http://pubs.acs.org. Reference. (1) Seddon, K.R. Ionic liquids: A Taste of the Future. Nature 2003, 2, 363–365. (2) Anderson, J.L.; Ding, J.; Welton, T.; Armstrong, D.W. Characterizing Ionic Liquids On the Basis of Multiple Solvation Interactions. J. Am. Chem. Soc. 2002, 124, 14247–14254. (3) Anderson, J.L.; Armstrong, D.W.; Wei, G.T. Ionic Liquids in Analytical Chemistry. Anal. Chem. 2006, 78, 2892–2902. (4) Hayes, R.; Warr, G.G.; Atkin, R. Structure and Nanostructure in Ionic Liquids. Chem. Rev .2015, 115, 6357−6426. (5) Noda, A.; Susan, M. A. B. H.; Kudo, K.; Mitsushima, S.; Hayamizu, K.; Watanabe, M. Brønsted Acid−Base Ionic Liquids as Proton-Conducting Nonaqueous Electrolytes. J. Phys. Chem. B 2003, 107, 4024−4033. (6) Miran, M. S.; Yasuda, T.; Susan, M. A. B. H.; Dokko, K.; Watanabe, M. Binary Protic Ionic Liquid Mixtures as a Proton Conductor: High Fuel Cell Reaction Activity and Facile Proton Transport. J. Phys. Chem. C 2014, 118, 27631−27639. (7) Greaves, T.L.; Drummond, C.J. Protic Ionic Liquids: Properties and Applications. Chem. Rev. 2008, 108, 206–237. (8) Greaves, T.L.; Drummond, C.J. Protic Ionic Liquids: Evolving Structure–Property Relationships and Expanding Applications. Chem. Rev. 2015, 115, 11379–11448. (9) Mann, J. P.; McCluskey, A.; Atkin, R. Activity and Thermal Stability of Lysozyme in Alkylammonium Formate Ionic Liquids-Influence of Cation Modification. Green Chem. 2009, 11, 785−792. (10) Yoshizawa, M.; Xu, W.; Angell, C. A. Ionic Liquids by Proton Transfer: Vapor Pressure, Conductivity, and the Relevance of pKa from Aqueous Solutions. J. Am. Chem. Soc. 2003, 125, 15411−15419. (11) Maximo, G.J.; Santos, R.J.B.N.; Lopes-da-Silva, J.A.; Costa, M.C.; Meirelles, A.J.A.; Coutinho, J.A.P. Lipidic Protic Ionic Liquid Crystals. ACS Sustainable Chem. Eng. 2014, 2, 672−682. (12) Mu, L.; Shi, Y.; Ji, T.; Chen, L.; Yuan, R.; Wang, H.; Zhu, J. Ionic Grease Lubricants: Protic [Triethanolamine][Oleic Acid] and Aprotic [Choline][Oleic Acid]. ACS Appl. Mater. Interfaces 2016, 8, 4977−4984. (13) Morigaki, K.; Walde, P. Fatty Acid Vesicles. Curr.Opin. Colloid Interface Sci. 2007, 12, 75–80.



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(14) Tang, T.-Y. D.; Hak, C. R. C.; Thompson, A. J.; Kuimova, M. K.; Williams, D. S.; Perriman, A. W.; Mann, S. Fatty acid Membrane Assembly on Coacervate Microdroplets as a Step Towards a Hybrid Protocell Model Nat. Chem .2014, 6, 527−533. (15) Chen, I. A.; Szostak, J. W. Membrane Growth can Generate a Transmembrane pH Gradient in Fatty Acid Vesicles. Proc. Natl Acad. Sci. USA 2004, 101, 7965–7970. (16) Stano, P.; Luisi, P. L. Achievements and Open Questions in the Self-Reproduction of Vesicles and Synthetic Minimal Cells, Chem. Commun. 2010, 46, 3639–3653. (17) Szostak, J. W.; Bartel, D. P.; Luisi, P. L. Synthesizing life, Nature 2001, 409, 387–390. (18) Hanczyc, M. M.; Fujikawa, S. M.; Szostak, J. W. Experimental Models of Primitive Cellular Compartments: Encapsulation, Growth, and Division. Science 2003, 302, 618–622. (19) Walde, P.; Ichikawa, S. Enzymes inside Lipid Vesicles: Preparation, Reactivity and Applications, Biomol. Eng. 2001, 18, 143–177. (20) Stano, P.; Wehrli, E.; Luisi, P. L. Insights into the Self-Reproduction of Oleate Vesicles. J. Phys.: Condens. Matter 2006, 18, S2231–S2238. (21) Chen, I. A.; Szostak, J. W. A Kinetic Study of the Growth of Fatty Acid Vesicles.Biophys.J .2004, 87, 988−998. (22) Markvoort, A.; Pfleger, N.; Staffhorst, R.; Hilbers, P.; van Santen, R.; Killian, J.; de Kruijff, B. Self-Reproduction of Fatty Acid Vesicles: A Combined Experimental and Simulation Study. Biophys.J. 2010, 99, 1520−1528. (23) Cistola, D. P.; Hamilton, J. A.; Jackson, D.; Small, D. M. Ionization and Phase Behavior of Fatty Acids in Water: Application of the Gibbs Phase Rule. Biochemistry 1988, 27, 1881– 1888. (24) Dejanović, B.; Mirosavljević, K.; Noethig-Laslo, V.; Pečar, S.; Šentjurc, M.; Walde, P. An ESR Characterization of Micelles and Vesicles Formed in Aqueous Decanoic acid/Sodium Decanoate Systems using Different Spin Labels Chem. Phys. Lipids 2008, 156, 17−25. (25) Fukuda, H.; Goto, A.; Yoshioka, H.; Goto, R; Morigaki, K.; Walde, P. Electron Spin Resonance Study of the pH-Induced Transformation of Micelles to Vesicles in an Aqueous Oleic Acid/Oleate System. Langmuir 2001, 17, 4223−4231. (26) Ngo, V. A.; Kalia, R. K.; Nakano, A.; Vashishta, P. Molecular Mechanism of Flip-Flop in Triple-Layer Oleic-Acid Membrane: Correlation between Oleic Acid and Water. J. Phys. Chem. B 2012, 116, 13416−13423. (27) Kamp, F.; Zakim, D.; Zhang, F.; Noy, N.; Hamilton, J. A. Fatty Acid Flip-Flop in Phospholipid Bilayers is Extremely Fast. Biochemistry 1995, 34, 11928–11937. (28) Caschera, F.; de la Serna, J. B.; Löffler, P. M. G.; Rasmussen, T. E.; Hanczyc, M. M.; Bagatolli, L. A.; Monnard, P.-A. Stable Vesicles Composed of Monocarboxylic or Dicarboxylic Fatty Acids and Trimethylammonium Amphiphiles. Langmuir 2011, 27, 14078−14090.


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(29) Xu, W.; Song, A.; Dong, S.; Chen, J.; Hao, J. A Systematic Investigation and Insight into the Formation Mechanism of Bilayers of Fatty Acid/Soap Mixtures in Aqueous Solutions. Langmuir 2013, 29, 12380−12388. (30) Suga, K.; Yokoi, T.; Kondo, D.; Hayashi, K.; Morita, S.; Okamoto, Y.; Shimanouchi, T.; Umakoshi, H. Systematical Characterization of Phase Behaviors and Membrane Properties of Fatty Acid/Didecyldimethylammonium Bromide Vesicles. Langmuir 2014, 30, 12721−12728. (31) Day, E.P.; Kwok, A.Y.; Hark, S.K.; Ho, J.T.; Vail, W.J.; Bentz, J.; Nir, S. Reversibility of Sodium-Induced Aggregation of Sonicated Phosphatidylserine Vesicles. Proc. Natl. Acad. Sci. USA 1980, 77, 4026 – 4029. (32) Henderson, I.A.; Paxton, W.F. Salt, Shake, Fuse—Giant Hybrid Polymer/Lipid Vesicles through Mechanically Activated Fusion. Angew. Chem. Int. Ed. 2014, 53, 3372 –3376. (33) Ohki, S.; Arnold, K. A Mechanism for Ion-Induced Lipid Vesicle Fusion. Colloids Surf. B 2000, 18, 83–97. (34) Kantor, H. L.; Prestegard, J. H. Fusion of Phosphatidylcholine Bilayer Vesicles: Role of Free Fatty Acid. Biochemistry 1978, 17, 3592–3597. (35) Banerjee, C.; Roy, A.; Kundu, N.; Banik, D.; Sarkar, N. A New Strategy to Prepare Giant Vesicles from Surface Active Ionic Liquids (SAILs): a Study of Protein Dynamics in a Crowded Environment using a Fluorescence Correlation Spectroscopic Technique. Phys. Chem. Chem. Phys. 2016, 18, 14520—14530. (36) Sasmal, D.K.; Mandal, A.K.; Mondal, T.; Bhattacharyya, K. Diffusion of Organic Dyes in Ionic Liquid and Giant Micron Sized Ionic Liquid Mixed Micelle: Fluorescence Correlation Spectroscopy. J. Phys. Chem. B 2011, 115, 7781–7787. (37) Dey, S.; Mandal, U.; Mojumdar, S.S.; Mandal, A.K.; Bhattacharyya, K. Diffusion of Organic Dyes in Immobilized and Free Catanionic Vesicles. J. Phys. Chem. B 2010, 114, 15506–15511. (38) Ghosh, S.; Adhikari, A.; Mojumdar, S.S.; Bhattacharyya, K. A Fluorescence Correlation Spectroscopy Study of the Diffusion of an Organic Dye in the Gel Phase and Fluid Phase of a Single Lipid Vesicle. J. Phys. Chem. B 2010, 114, 5736–5741. (39)Khan, M.F.; Singh, M.K.; Sen, S. Measuring Size, Size Distribution, and Polydispersity of Water-in-Oil Microemulsion Droplets using Fluorescence Correlation Spectroscopy: Comparison to Dynamic Light Scattering. J. Phys. Chem. B 2016, 120, 1008−1020. (40) Deptuła, T.; Buitenhuis, J.; Jarzębski, M.; Patkowski, A.; Gapinski, J. Size of Submicrometer Particles Measured by FCS: Correction of the Confocal Volume. Langmuir 2015, 31, 6681−6687. (41) Becker, W.; Bergmann, A.; Biskup, C.; Zimmer, T.; Klöcker, N.; Benndorf, K. MultiWavelength TCSPC Lifetime Imaging. In: AmmasiPeriasammy, Peter T So; (eds) Proc. SPIE- Multiphoton Microscopy in Biomedical Sciences II2002, 4620, 79-84. (42) Mojumdar, S.S.; Ghosh, S.; Mondal, T.; Bhattacharyya, K. Solvation Dynamics under a Microscope: Single Giant Lipid Vesicle. Langmuir 2012, 28, 10230−10237. 15 ACS Paragon Plus Environment


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(43) Sykora, J.; Kapusta, P.; Fidler, V.; Hof, M. On What Time Scale Does Solvent Relaxation in Phospholipid Bilayers Happen? Langmuir 2002, 18, 571−574. (44) Pal, S. K.; Sukul, D.; Mandal, D.; Bhattacharyya, K. Solvation Dynamics of DCM in Lipid. J. Phys. Chem. B 2000, 104, 4529−4531. (45) Ghosh, S.; Ghatak, C.; Banerjee, C.; Mandal, S.; Kuchlyan, J.; Sarkar, N. Spontaneous Transition of Micelle−Vesicle−Micelle in a Mixture of Cationic Surfactant and Anionic Surfactant-like Ionic Liquid: A Pure Nonlipid Small Unilamellar Vesicular Template Used for Solvent and Rotational Relaxation Study. Langmuir 2013, 29, 10066−10076. (46) Ribeiro, A.M.C.; Chaimovich, H.Salt-induced Aggregation and Fusion of Dioctadecyldimethylammonium Chloride and Sodium Dihexadecylphosphate Vesicles. Biophys. J. 1986, 50, 621-628. (47) Karatekin , E.; Rothman, J. E. Fusion of Single Proteoliposomes with Planar, Cushioned Bilayers in Microfluidic Flow Cells. Nature Protocols 2012, 7, 903–920. (48) Terasawa, H.; Nishimura, K.; Suzuki, H.; Matsuura, T.; Yomo, T. Coupling of the Fusion and Budding of Giant Phospholipid Vesicles Containing Macromolecules Proc. Natl. Acad. Sci. USA. 2012, 109, 5942–5947. (49) Hennig, R.; Heidrich, J.; Saur, M.; Schmu¨ser, L.; Roeters, S.J.; Hellmann, N.; Woutersen, S.; Bonn, M.; Weidner, T.; Mark, J. et al. IM30 Triggers Membrane Fusion in Cyanobacteria and Chloroplasts. Nat Commun. 2015, 6, 7018. (50) Versluis, F.; Voskuhl, J.; Kolck, B.V.; Zope, H.; Bremmer, M.; Albregtse, T.; Kros, A. In Situ Modification of Plain Liposomes with Lipidated Coiled Coil Forming Peptides Induces Membrane Fusion. J. Am. Chem. Soc. 2013, 135, 8057−8062. (51) Chernomordik, L.V.; Kozlov, M.M. Membrane Hemifusion: Crossing a Chasm in Two L1eaps, Cell 2005, 123, 375-382. (52) Chernomordik, L.V.; Kozlov, M.M. Protein-Lipid Interplay in Fusion and Fission of Biological Membranes. Annu. Rev. Biochem. 2003, 72, 175–207. (53) Ribeiro, A.M.C.; Yoshida, L.S.; Chaimovich, H. Salt Effects on the Stability of Dloctadecyldlmethylammonium Chloride and Sodium Dlhexadecyi Phosphate Vesicles, J. Phys. Chem. 1985,89, 2928-2933. (54) Wilschut, J.; Diizgiines, N.; Papahadjopoulos, D. Calcium/Magnesium Specificity in Membrane Fusion: Kinetics of Aggregation and Fusion of Phosphatidylserine Vesicles and the Role of Bilayer Curvature. Biochemistry 1981,20, 3127-3133. (55) Smeijers, A.F.; Markvoort, A.J.; Pieterse, K.; Hilbers, P.A.J. A Detailed Look at Vesicle Fusion. J. Phys. Chem. B 2006, 110, 13212-13219. (56) Arai, N.; Yoshimoto, Y.; Yasuoka, K.; Ebisuzaki, T. Self-assembly Behaviours of Primitive and Modern Lipid Membrane Solutions: a Coarse-Grained Molecular Simulation Study, Phys. Chem. Chem. Phys. 2016, 18, 19426-19432. (57) Blumenthal, R.; Clague, M.J.; Durell, S.R.; Epand, R.M. Membrane Fusion, Chem. Rev. 2003, 103, 53-69.


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(58) Diao, J.; Grob, P.; Cipriano, D.J.; Kyoung, M.;Zhang, Y.; Shah, S.; Nguyen, A.; Padolina, M.; Srivastava, A.; Vrljic, M. et al. Synaptic Proteins Promote Calcium-Triggered Fast Transition from Point Contact to Full Fusion. eLife 2012, 1, 1-21.

Scheme 1. Chemical structures of oleic acid, oleate ethyl amine (OEA) and different fluorophores which have been used in different experiments. Table 1. Translational Diffusion Parameter of DCM in different Vesicles. System

!"#$ % &

$ !"#$ % &

water oleic acid/oleate vesicle OEA vesicle

302.00 302.00 (0.16) 302.00 (0.05)

1.67±0.06 (0.84) 0.55±0.05(0.95)

Table 2. Fitted Fluorescence Lifetimes of DCM in different vesicles collected in different wavelengths obtained from the FLIM imagesa System

Wavelength (nm)

' !(&

' !(&

')*+ !(&

,

606-620 731-744

0.7 (0.75) 1.13 (-0.43)

1.63 (0.25) 2.28 (1.43)

0.93 2.77

1.06 1.04

606-620 731-744

0.86 (0.62) 1.18 (-0.46)

4.31 (0.38) 3.46 (1.46)

2.17 4.51

1.08 1.30

Oleic acid/oleate vesicle

OEA vesicle a

experimental error ±8%



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Figure 1. (a,b) FLIM images of OEA vesicles; the left panel shows the lifetime image and the right panel shows the intensity image. (c) 3d surface view of figure (b) shows the formation of different sized vesicles (d) DLS intensity profile of 0.1(M) OEA vesicle in water. (a)

(b)

Figure 2. FLIM Z stack recording of (a) OEA vesicles and (b) Oleic acid/oleate vesicles stained with DCM excitation at 488 nm. Z step width ±5"# (scale bar in the image a and b represent 1.5 "# and 1 "# respectively). 18 ACS Paragon Plus Environment

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Figure 3. (a) FCS traces of DCM in water, bulk OEA vesicle and oleic acid/oleate vesicle. The red line indicates the actual values of - !.& and the black line indicates the best fit. (b) Distribution of values obtained by performing the FCS experiments more than 10 times and the mean value of was determined by fitting the distribution to Gaussian function.


5


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(i)

(ii)

Figure 4. Multi-wavelength FLIM images of single (i) OEA vesicle and (ii) oleic acid/oleate vesicle stained with DCM fluorophore. The upper panel in each image represents the lifetime image and the lower panel represents the intensity image. The scale bar in image (i) and (ii) represent 2 "# and 1.8 "# respectively. Images are collected in 6 different wavelengths regions: (a) 606-620 nm, (b) 630-645 nm, (c) 655-669 nm, (d) 680-694 nm, (e) 706-720 nm, and (f) 730-744 nm.


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Figure 5.Lifetime decays of DCM in OEA vesicle collected in two different channels. Decays are collected at single point obtained from the MW-FLIM images which is indicated by the red dot in the image. The black line indicate the prompt and the red and blue line indicates the lifetime decays of DCM collected in 606-620 nm and 730-744 nm respectively.

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Figure 6. Lifetime distribution of DCM in (i) OEA vesicle and (ii) oleic acid/oleate vesicle collected in 6 different wavelengths regions: (a) 606-620 nm, (b) 630-645 nm, (c) 655-669 nm, (d) 680-694 nm, (e) 706-720 nm, and (f) 730-744 nm.


3200


OEA Vesicle OEA Vesicle + 0.1 M NaCl

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Figure 7.Size distribution of 0.1 (M) OEA vesicle and 0.1 (M) oleic acid/oleate vesicular assemblies in absence and in presence of 0.1 (M) NaCl. (the neat vesicular solutions were probe sonicated for 15 minutes).

Figure 8. FLIM images of OEA vesicle in presence of 60 mM NaCl collected in different time scale. (time zero indicates the time when the image of the sample is started to record; it does not imply the starting time of the reaction after addition of NaCl into the medium.)


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