Thermally Stable Ionic Liquid-Based Microemulsions for High

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Interface Components: Nanoparticles, Colloids, Emulsions, Surfactants, Proteins, Polymers

Thermally Stable Ionic Liquid Based Microemulsions for High Temperature Stabilization of Lysozyme at Nano-interfaces Manvir Kaur, Gurbir Singh, Anupreet Kaur, Pushpender Kumar Sharma, and Tejwant Singh Kang Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.9b00106 • Publication Date (Web): 27 Feb 2019 Downloaded from http://pubs.acs.org on February 27, 2019

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Thermally Stable Ionic Liquid Based Microemulsions for High Temperature Stabilization of Lysozyme at Nano-interfaces Manvir Kaur,a Gurbir Singh,a Anupreet Kaur,a Pushpender Kumar Sharma,b Tejwant Singh Kanga,* a

Department of Chemistry, UGC Sponsored Centre for Advanced Studies-II, Guru Nanak Dev University, Amritsar-143005 b Department of Biotechnology, Sri Guru Granth Sahib World University, Fatehgarh Sahib-140406, India.

Abstract: The development of new strategies for thermal stability and storage of enzymes is very important considering the non-retention of catalytic activity by enzymes under harsh conditions of temperature. Following this, herein, a new approach based on interfacial adsorption of Lysozyme (LYZ) at nano-interfaces of ionic liquid (IL) based microemulsions, for enhanced thermal stability of LYZ, is reported. Microemulsions (MEs) composed of dialkyl imidazolium based surface active ionic liquids (SAILs) as surfactants, ionic liquid (IL) as non-polar phase and ethylene glycol (EG) as polar phase, without any co-surfactant, have been prepared and characterized in detail. Various regions corresponding to Polar-in-IL, bi-continuous and IL-inPolar phase have been characterized using conductivity measurements. Dynamic light scattering (DLS) measurements have provided insights into size distribution of micro-droplets, whereas temperature dependent DLS measurements established the thermal stability of MEs. Nanointerfaces formed by SAILs with EG in thermally stable reverse MEs acts as fluid scaffolds to adsorb and provide thermal stability, up to 120 oC, to LYZ. Thermally treated LYZ upon extraction into buffer shows enzyme activity owing to negligible change in active site of LYZ as marked by retention of microenvironment of Trp residues present in active site of LYZ. The present work is expected to establish a new platform for development of novel nano-interfaces utilizing bio-based components for other biomedical applications.

*To whom correspondence should be addressed: e-mail: [email protected]; [email protected]; Tel: +91-183-2258802-Ext3207

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Introduction Generally, enzymes remain stable for a short duration under in vitro conditions and freeze drying is used to enhance their shelf life.1 However, the effective use of enzymes in biomedical processes requires structurally stable and catalytically active enzymes under different storage conditions.2-4 This, along with unique physico-chemical properties of ionic liquids (ILs)5, has prompted the researchers to explore solubilization, stabilization and maintenance of enzyme activity in neat or hydrated ILs.6-13 Different strategies, to stablize inherent strcure of enzymes and hence maintaining enzyme activity in hydrophilic ILs,9-11 have been implemented.9 One such strategy, that could be implemented for stabilization of enzymes, is utilization of nano-interfaces created by ILs as exemplified for different applications14-17 that include preparation of protein microcapsules.14,15 Such IL based nano-interfaces are easy to develop in the form of microemulsions,18-24 and could be utilized for enzyme storage and stabilization. The solubilization, and catalysis by enzymes, inside aqueous core of reverse MEs comprising hydrophobic ILs and ionic or non-ionic surfactants have been reported.25,26 However, the hydrophobic IL (non-polar) residing away from the polar-surfactant interface, in these MEs, is not expected to effect the solubilization and activity of enzyme trapped in the polar core. The efficacy of reverse MEs may be increased by using surface active ILs (SAILs)27-34 in place of conventional ionic surfactants as nano-interfaces of self-assembled SAILs could stabilize enzymes.33,34 Following that the studies on stabilization of bovine serum albumin (BSA) in aqueous pool of MEs comprising bio-based SAIL and cyclohexane has been derived.35 Nevertheless, the use of cyclohexane renders the MEs toxic and thermally unstable. Therefore, the nano-interfaces of relatively less toxic and thermally stable MEs comprising ILs (non-polar) or SAILs (surfactant) in conjunction with organic solvents with very low vapour pressure and


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cytotoxicity36-39 e.g. ethylene glycol (EG) (polar phase) are expected to offer new platform for high temperature enzyme stabilization. Herein, we have conceptualized and developed thermally stable IL based microemulsions (MEs) for enhanced thermal stability of Lysozyme (LYZ). LYZ is chosen as a model antimicrobial enzyme that can survive in wide pH range and exhibit fluorescence owing to the presence of Trp residues, which is easy to monitor for investigating secondary (2o) structural changes

in

LYZ.40-42

MEs

comprising

an

IL,

1-ethyl-3-methylimidazolium

bis(trifluromethylsulfonyl)imide, [C2mim][Tf2N] (non-polar), dialkylimidazolium based SAILs, 1-butyl-3-alkylimidazolium bromide, [C4Cnim][Br] (n = 4, 8 and 12) (surfactant) and ethylene glycol (EG) (polar) without using any co-surfactant, are prepared and characterized. Thus prepared MEs are employed for enhancing the thermal stability of LYZ, which upon extraction into buffer showed biological activity. The stability and activity of LYZ was confirmed by fluorescence, circular dichroism (CD) and enzyme activity on Micrococcus lysodeikticus and Bacillus cereus, respectively. The present work, along with previous studies on macroscopic interfaces formed by ILs with other solvents,14,17 is expected to provide a new direction for successful utilization of scarcely explored IL based nano-interfaces.

Materials and Methods Materials 1-butylimidazole (99%), 1-bromobutane (99%), 1-bromooctane (98%), 1-bromododecane (99%), EG (>99%) were purchased from Sigma-Aldrich and used without further purification. [C2mim][Tf2N] (99%) was purchased from IOLITEC. Different SAILs, [C4Cnim][Br] (n = 4, 8 and 12) have been prepared by the method reported by Chabba et al.43 The prepared SAILs were characterized using 1H NMR and HR-Mass Spectroscopy and corresponding characterization


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data is provided in Annexure S1 (Supporting Information, SI). Lysozyme (LYZ) from chicken egg white (>90%), Micrococcus luteus (ATCC No. 4698) and Bacillus cereus bacteria were purchased from Sigma-Aldrich and used without any purification.

Methods Preparation and Characterization of Microemulsions: Ternary phase diagrams of microemulsions (MEs) comprising EG/SAIL/[C2mim][Tf2N] as polar/surfactant/non-polar phase, respectively, were constructed at 25˚C by titrating the mixtures of EG and SAIL at various component ratios with [C2mim][Tf2N]. The mass ratio of EG to SAIL (R value) was fixed for each titration and phase transitions from transparent to turbid phase were observed with the help of naked eye. Thus formed MEs were characterized using conductivity measurements, which helped in marking different regions in ternary systems at varying compositions. Specific conductance (κ) was measured using a digital conductivity meter (systronics 308) using cell of unit cell constant after calibrating the conductivity meter using aqueous KCl solutions of different concentrations at 25˚C. Conductivity measurements were made in triplicate and found to be accurate within an uncertainty of ± 1%. For specific conductivity measurements, mass ratio of different SAILs to [C2mim][Tf2N] was kept constant as 0.85:0.15. Size distribution of investigated MEs was explored by dynamic light scattering (DLS) measurements using a light scattering apparatus (Zeta sizer, nanoseries, nano-ZS), Malvern Instruments, equipped with a built-in temperature controller having an accuracy of ± 0.1 K at a scattering angle of 173˚. An average of 5 measurements was considered as the data. For all measurements, the mass ratio of SAIL/[C2mim][Tf2N] was kept constant. For EG/[C4C4im][Br]/[C2mim][Tf2N] system, the amount of [C4C4im][Br] was kept at 67 w/w% and the amount of non-polar phase, [C2mim] [Tf2N] was kept at 33 w/w%. For EG/[C4C8im][Br]/[C2mim] [Tf2N] system, the amount of


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surfactant [C4C8im][Br] was kept at 68 w/w% and amount of non-polar was kept at 32 w/w%. For EG/[C4C12im][Br]/[C2mim] [Tf2N] system, the amount of surfactant [C4C12im][Br] was kept at 78 w/w% and amount of non-polar was kept at 22 w/w%. The mass ratio was fixed depending on the single phase ME region in ternary phase diagram. DLS measurements were made by varying R values i.e. the molar ratio of EG and SAIL, along line a, b and c as shown in Figure 1(B-D), respectively. Steady-state fluorescence measurements were performed using a Perkin Elmer LS-55 spectrophotometer using quartz cuvette of path length 1 cm. Intrinsic fluorescence of MEs was measured using an excitation wavelength of 315 nm and the spectra were measured in the wavelength range of 340-500 nm using excitation and emission slit width of 5 nm, each. Solubility of Lysozyme in MEs: To load LYZ into MEs, stock solution of LYZ (1.4 mM) in EG was used to prepare MEs in place of neat EG, keeping the composition of other components same. The composition of MEs, for evaluating thermal stability of LYZ, was kept same as employed in DLS measurements. A critical amount of LYZ that can be solubilized without any precipitation, immediate aggregation or without phase separation, in MEs comes out to be 5 mmol L-1, 35 μmol L-1 and 25 μmol L-1 in case of EG/[C4C4im][Br]/[C2mim][Tf2N], EG/[C4C8im][Br]/[C2mim][Tf2N] and EG/[C4C12im][Br]/[C2mim][Tf2N], respectively. It is important to mention that beyond respective critical concentration, different ME systems under investigation become turbid suggesting the presence of a phase separated system. Thermal Stability and Activity of Lysozyme: Stability of LYZ, as a function of temperature in MEs, was monitored by fluorescence spectroscopy. For this, the fluorescence spectra of LYZ in MEs was recorded by using excitation wavelength of 280 nm and the spectra were measured in the wavelength range of 290-500 nm as a function of temperature, using excitation and emission slit width of 5 nm, each. A quartz cuvette of path length 1 cm was used for fluorescence measurements. LYZ in MEs was heat treated at a given temperature for 15 minutes and cooled


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down to 25 oC before fluorescence measurements. After the heat treatment of LYZ in MEs at different temperatures of interest, LYZ was extracted from the ternary systems using liquidliquid extraction method employing phosphate buffer (10 mM). To remove any remained IL from the extracted solution of LYZ thus extracted LYZ in buffer was passed through amicon membrane (3 kDa). Removal of any SAIL from LYZ solution was confirmed by conductivity measurements, where specific conductance came out to be very close to that of used buffer, within the limits of accuracy of instrument employed. Secondary (2o) structure of recovered LYZ was monitored using circular dichroism (CD) spectroscopy (Jasco J-810 spectrometer) using a cuvette of path length 1 mm at a scan speed of 50 nm/min. Enzyme activity of extracted LYZ was monitored using Micrococcus lysodeikticus and Bacillus cereus. Bacterial culture were grown overnight in 5 ml LB at 37˚C with a shaking speed of 220 rpm, next day the culture were re inoculated in fresh medium. The activity of extracted LYZ from MEs prepared by using different SAILs and control sample was determined by spreading overnight grown culture on agar plate. Well diffusion assay was carried out by punching well in solid medium and dispensing of various samples into these wells. Agar plates were kept at 37˚C for 24 hrs, and removed thereafter. The zone of inhibition around the well was measured for evaluating enzyme activity.

Results and Discussion Phase Behavior, Size and Thermal Stability of Microemulsions: The study of phase behaviour is the foremost step to investigate microemulsion (ME) systems. For the sake of simplicity, different SAILs employed for ME formation are abbreviated as SAIL-1, SAIL-2 and SAIL-3, respectively as shown in Figure 1A along with other components of MEs. EG is taken as polar solvent in place of water due to its high boiling point, which would provide thermal


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stability to MEs. EG is relatively much less toxic36,37 as compared to most of the organic solvents and can solubilize LYZ,38,39 required for incorporation of LYZ into MEs. A relatively smaller ME region was obtained when water was used as a polar solvent (Figure S1A, Supporting Information, SI) instead of EG, which further supported the use of EG as a polar solvent. Dialkylimidazolium based SAILs are employed in expectation of better stability of EG-SAIL interface via balanced solvophilic/solvophobic interactions, even in the absence of a cosurfactant.

Figure 1. (A) Different components of MEs; and (B-D) Obtained phase diagrams for different microemulsions comprising EG, IL and (B) SAIL-1; (C) SAIL-2; and (D) SAIL-3.

This is justified by observation of relatively lower area under ME region (Figure S1B, SI) when a co-surfactant (1-decanol) was used in conjunction with SAILs. [C2mim][Tf2N] (IL) is chosen as non-polar medium due to its highly hydrophobic nature, relatively low viscosity, high thermal


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and electrochemical stability along with hydrolysis stability towards moisture.44 Henceforth, MEs formed by SAIL-1, SAIL-2 and SAIL-3 would be abbreviated as ME-1, ME-2 and ME-3, respectively, if not stated, otherwise. In phase diagrams (Figure 1B-D), two regions, one transparent ME- (single-phase region) and a turbid multi-phase region, are observed. It is important to mention that the term “singlephase region” refers to a transparent appearance, however, different ME regions are observed in this phase depending upon the composition (discussed later). An increase in length of alkyl chain of SAILs led to a decrease in area under ME region, which is in line with earlier report24 for similar ME systems comprising single tailed SAILs. The observation, of decrease in the area of ME region with increase in alkyl chain length, is in contrast with that reported for ME systems comprising TX-100 (surfactant), cyclohexane (non-polar) and alkyl sulfate based ILs (polar).45 This is assignable to the fact that with increase in alkyl chain length, while going from 1-ethyl-3methylimidazolium ethylsufate, [C2mim][C2SO4], to 1-ethyl-3-methylimidazolium octylsufate, [C2mim][C8SO4], the role of IL changes from a polar solvent to a co-surfactant.45 Therefore, the varying set of interactions between different constituents of MEs led to opposite behavior. It is natural to assume that H-bonding interactions and ion-dipole interactions along with hydrophobic interactions plays an important role in stabilization of MEs. Different ME regions i.e. Polar-in-IL, bi-continuous and IL-in-Polar in MEs are characterized by conductivity measurements (Figure 2A & S2, SI) using percolation theory.46


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Figure 2: (A) Dependence of specific conductivity (κ) of MEs on EG wt%; and (B-D) Calculation of percolation threshold, EGp, from d(κ)/ d(EG wt%)Vs. EG wt% graph of (B) ME-1; (C) ME-2 and (D) ME-3. Inset of Figure (B-D) shows scaling dependence of κ on (EG-EGp)/EGp.

With increase in the content of EG, an increase in specific conductivity (κ) in Polar-in-IL (P/IL) region is observed (Figure 2A).24 It is natural to assume that an increase in EG content decreases intermolecular interactions between SAILs and IL at the cost of enhanced but weaker ion-dipole as well as H-bonding interactions with EG that lead to an increase in κ in P/IL region. The presence of bi-continuous (BC) region in MEs is generally observed in continuation with P/IL region and can be confirmed using percolation theory. Percolation threshold (EGp), the concentration of EG where transition from one-region to another region occur, is obtained from inflection point of graph between d(κ)/d(EG wt%) and EG wt% as shown in Figure 2(B-D). κ, below and after EGp follows the following scaling laws:46


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𝜅 = 𝐴(𝐶& − 𝐶))*

Eq. 1

𝜅 = 𝐵(𝐶& − 𝐶),

Eq. 2

where κ is specific conductivity, Cp is percolation threshold (Cp = EGp), C is content of polar component, A, and B are constants, s and t are scaling parameters. Thus obtained values of scaling parameter, s, for MEs comprising SAIL-1, SAIL-2 and SAIL-3 comes out be -0.68, 0.67, -0.76, respectively. The obtained values of s are very close to the reported value 0.7 for MEs having bi-continuous phase in continuation with P/IL phase.2,46,47 κ continuous to increase in BC phase but with a relatively lower slope due to partially restricted movement of constituent ions as a consequence of formation of continuous solvent channels. Further addition of EG beyond BC phase results in transition to IL-in-polar (IL/P) phase, where normal micelle forms with hydrophobic component in core of ME droplets. Decreased interactions between different components and a changeover in size of self-assembled system from bi-continuous to normal micellar phase add to κ. Following that, κ falls due to transition to a dilute solution as also reported earlier.24,46 Hydrodynamic diameter (Dh) of micro-droplets is monitored (Figure 3A & S3, SI) along line ‘a’ (Figure 1B), line b (Figure 1C) & line c (Figure 1D) for ME-1, ME-2 and ME-3, respectively. As can be seen from Figure 3A, an increase in the content of EG results in initial contraction of droplet, due to reduced repulsions between SAIL head groups, as a consequence of penetration of EG molecules towards SAILs.48 This would result in enhanced hydrophobic interactions between the alkyl chains of SAILs leading to increased interfacial curvature49 and hence a contraction of ME droplet is observed, which is in line with earlier reports.48,49 This contraction of ME droplets is followed by swelling of ME droplet after a certain value of R, in case of SAIL-1 and SAIL-2. It is natural to assume that the presence of relatively smaller alkyl chains in SAIL-1 and SAIL-2 offers increased hydrophobic interactions limited up to critical


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value of R, beyond which the addition of EG don’t influence these interactions, and enlargement of polar pool of EG results in swelling of ME droplet. In case of SAIL-3, greater extent of solvophobic interactions between EG and long alkyl chain along with the possibility of structural rearrangement (enhanced packing or coiling) of relatively flexible alkyl chain could result in contraction of ME droplet with increasing content of EG within the limits of composition of observed single-phase ME region. Another reason for observed decrease in size of ME droplets could be an increase in the number of micro-droplets.48

Figure 3: Variation in hydrodynamic diameter (Dh) of microdroplets of MEs as a function of (A) ethylene glycol concentration (R value = [EG]/[SAIL]) and (B) temperature. The R values for different MEs corrosponds to line a, b and c in Figure 1B, C and D, respectively.

Thermal stability of MEs was investigated in the temperature range of 25-75 oC using DLS measurements. In general, MEs are found to be thermally stable up to 75 oC (Figure 3B) and a marginal decrease in Dh is observed, in case of ME-1 and ME-2. On the other hand, ME-3 although remains stable in the investigated temperature range, however displays a relatively larger decrease in Dh with rise in temperature. Thermally induced percolation of EG towards the alkyl chains of SAILs, facing IL from EG/SAIL interface, which is accompanied by movement of EG molecules from bulk EG (core of reverse MEs) towards EG/SAIL interface, is expected to govern the decrease in Dh, when MEs are heated. It is inferred that the SAIL-3 with relatively


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longer alkyl chain offers more surface for percolation of EG accompanied by greater solvophobic interactions as compared to that offered by SAILs having smaller alkyl chains. Further the butyl chain attached to one of the N of imidazolium head group of SAIL, facing the EG core, remains solvated by EG in reverse MEs due to appreciable solvophobicity of EG. Such a solvation is also assignable to thermal stability of MEs. Interestingly, MEs regain Dh upon cooling to 25 oC (Figure S4, SI) even after heating up to 180o C. This along with the absence of phase separation in this temperature range establishes the thermodynamic stability of investigated MEs. Location and Thermal Stability of Lysozyme in MEs: Thus prepared MEs were explored for testing the thermal stability of LYZ in a temperature range of 25-150 oC. LYZ was loaded into MEs using stock solution of LYZ. The solubilization of LYZ in different SAILs, non-polar IL, [C2mim][Tf2N], mixtures of [C2mim][Tf2N] with SAILs and EG (Figure 4A) was first investigated by naked eye. The formation of clear solution only in case of EG establishes the solubilization of LYZ in EG, whereas LYZ remains suspended in all other solvents. To confirm the observation about solubilization of LYZ, fluorescence measurements were made exploiting the inherent fluorescence of LYZ. As can be seen from Figure 4B, LYZ solubilized in EG shows appriciable fluorescence whereas negligible emission of LYZ was observed when dispersed in other solvent systems. It is important to mention that SAILs are fluorescent in nature due to the symmetrical/partial symmetrical conjugating structure.50 Figure S4, SI shows the absorption and fluorescence spectra of SAILs which confirms the fluorescent nature of SAILs. However the non-interefence of fluorescence of SAILs with that of LYZ (Figure 5A, Figure S5, SI) rules out the possibility of any misinterpretation of results.


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A

B

C

Figure 4. (A) Solubilization of lysozyme in Ethylene glycol (EG), Non-solubilization of Lysozyme in investigated SAILs, non-polar IL, [C2mim][Tf2N], and its mixtures with SAILs; (B) Emission spectra of lysozyme (λex = 280 nm) in ethylene glycol (EG); [C2mim][Tf2N] (IL); SAIL-1; SAIL-2; SAIL-3. Figure 4C shows a schematic representation of adsorption of LYZ at nano-interface of EG and SAIL.

The solubilization of LYZ only in EG suggests that in reverse MEs, LYZ could be localized either in polar phase (EG) or at EG-SAIL interface in MEs. SAIL ions are not expected to be highly dissociated in EG having low dielectric constant than water, however rather weaker electrostatic forces of interaction between head group of SAILs and oppositely charged amino acid residues of LYZ could help in adsorption of LYZ at EG-SAIL interface in reverse MEs. The presence of LYZ at EG-SAIL interface is suggested by the reduced solubility of LYZ in MEs as compared to that in neat EG. A small increase in the size of ME droplet, as shown in Figure S6 (SI) upon the addition of LYZ, confirms the encapsulation of LYZ in ME droplet. A red shift in


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inherent fluorescence of LYZ41,42, from 340 nm in EG to 341-353 nm in different MEs, (Figure 5A, Figure S7, SI) supports the presence of LYZ near EG-SAIL interface. Further increase in concentration of LYZ in MEs results in quenching of inherent fluorescence of SAILs (Figure 5A, Figure S7, SI). This is only possible if the presence of LYZ either affects the molecualr arrangement of SAILs or is drectly in contact with SAIL head groups in MEs. There exists an isobestic point in the fluorescence spectra of LYZ and SAIL in MEs.

Figure 5: (A) Fluorescence spectra of LYZ (λex = 280 nm) in ME-3 as a function of LYZ concentration; (B) Variation in fluorescence intensity of LYZ as a function of temperature. Fluorescence spectra of LYZ (λex = 280 nm) in (C) ME-3 & (D) EG at 25 oC after heating at different temperatures.

The presence of an iosbestic point between LYZ and fluorescent SAILs (Figure 5A, Figure S7, SI) confirms the complexation of LYZ with SAILs at EG-SAIL interface as shown in Figure 4C. Thermal stability of LYZ, dissolved in MEs (along line a, b and c at R value = 0.8, Figure 1 ), was monitored by fluorescence spectroscopy as a function of temperature up to 75 oC


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(Figure 5B, Figure S8, SI) in different MEs. A decrease in emission intensity (Iem) of inherent fluorescence of LYZ with rise in temperature is observed, which regains on cooling (Figure 5B & S8, SI). LYZ is reported to remain stable up to 70 oC in buffer and in EG,40,51 and therefore such a decrease in Iem is assigned to the polarity changes around hydrophobically buried Trp residues, which could be accompanied by 2o structural changes in LYZ.12,41,42 Another reason for such decrease in Iem may be an obvious decrease in viscosity of the system at higher temperatures. To further investigate the thermal stability of LYZ beyond its unfolding-refolding temperature of ≈ 70 oC,40 LYZ in MEs was subjected to different temperatures and cooled down to 25 oC followed by fluorescence measurements. Relatively smaller loss of Iem for LYZ present in ME-2 (Figure S9B, SI) and ME-3 (Figure 5C), when heated up to 120 and 150 oC, respectively is observed as compared to that observed in ME-1 (Figure S9A, SI). On the other hand, LYZ loses about 50% of Iem after heating to 120 oC when present in EG (Figure 5D) and is assigned to substantial change in microenvironment around Trp residues of LYZ. This suggests that Trp residues of LYZ adsorbed at EG-SAIL interface experience lesser change in polarity of surrounding environment as compared to that offered by EG in MEs. Such contrasting behaviour could be due to varying extent of solvophillic/solvophobic interactions between LYZ, EG and SAIL at EG-SAIL interface. It seems that EG is not fully able to penetrate into the LYZ adsorbed at EG-SAIL interface contrary to that in neat EG. Modulation in 2o structure of LYS: To have insight into structural modifications of LYZ after heat treatment, CD spectroscopy measurements were performed (Figure 6). LYZ was extracted in phosphate buffer for structural analysis as CD spectra in MEs offered saturation of detection voltage owing to high concentration of UV absorbing IL and SAILs. CD spectra of LYZ exhibit spectra peculiar of a+b character and shows two negative CD bands at ≈ 209 and a shoulder ≈


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220 nm, resulting from proportion of a-helical and b-sheet content, respectively.38,52 There is almost no change in CD spectra of LYZ treated up to 80 oC in all MEs (Figure 6A). Further CD spectra were analysed for its shape corresponding to relative content of a-helices and b-sheets.53

Figure 6: Far-UV CD spectra of extracted lysozyme (A) from different microemulsions after 80 oC; from (B) ME-1; (C) ME-2; and (D) ME-3 after heating at different teperatures.

After heat-treatment up to 120 oC, LYZ present in ME-2 (Figure 6C) and ME-3 (Figure 6D) maintains a+b character, however the content of a-helical structure decreases marginally with subsequent rise in the content of b-sheet structure. On the other hand, 2o structure of LYZ transforms to a/b structure having a higher content of b-sheets in case of ME-1 (Figure 6B). LYZ adopts all b-sheet structure with further rise in temperature to 150 oC in case of ME-2 and ME-3, whereas LYZ maintains a/b character with increased content of b-sheet structure in ME1. This shows that LYZ maintains its inherent structure up to 120 oC in ME-2 and ME-3,


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however a change in relative content of a-helix and b-sheet is observed. This along with results obtained from fluorescence measurements establishes that although there is a marginal change in 2o structure of LYZ after heating to 120 oC, however the local environment around fluorescent Trp residues (Trp-62, Trp-63 and trp-108) does not change. This is manifested by retention of enzyme activity as discussed later, in which, Trp residues present near active site, plays an important role.54 On the other hand, LYZ present in EG seems to retain its native structure when heated up to 120 oC, with a decrease in content of b-sheet (Figure S10, SI), whereas a drastic perturbation of local environment around Trp residues was observed from fluorescence measurements (Figure 6D). This is assigned to percolation of EG into LYZ near Trp residues present in hydrophobic cavity and is visible from enzyme activity measurements. It is inferred that, thermal stability of EG-SAIL interface along with varying extent of percolation of EG towards SAIL in different MEs affects the stability of LYZ. The presence of butyl chain at one end of cationic head group of SAIL hinders solvophilic interactions between LYZ and imidazolium cation of SAIL. This provides interfacial stability to LYZ, whereas solvophobicity of EG helps in retaining inherent structure of LYZ.38,39 SAIL at EG-SAIL interface are expected not to dissociate fully into ions considering the relatively low dielectric constant of EG. Therefore, Br- ions present at EG-SAIL interface, having affinity for LYZ, would intermediate the adsorption of positively charged LYZ at EG-SAIL interface along with screening of electrostatic interactions between head group of SAIL and oppositely charged amino acid residues of LYZ. The solvophobic effect of EG bearing –OH groups at interface may offer less competition for SAILs to adsorb and hence helps in retaining the structure of LYZ. Antimicrobial Activity of Extracted Lysozyme: The extracted LYZ in buffer after heating up to 120 oC in ME-2 and ME-3 is found to be active and displayed inhibitory activity against Micrococcus luteus and Bacillus cereus (Figure 7). Interestingly, it was observed that LYZ when


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stored in MEs shows high activity compared to the control sample employed in equal concentration. This observation may be attributed to the change in 2o structure as evidenced by CD measurements where content of b-sheet increases after heat treatment along with negligible change in microenvironment of Trp residues present in the active site.51 Another reason for such behaviour could be the aggregation of LYZ in EG as also suggested by DLS measurements (Figure S11), when employed at same concentration.

Figure 7: (A) & (B) Images showing the activity of extracted lysozyme against bacterial cell culture of (A) micrococcus luteus; and (B) bacillus cereus (C1, C2, C3, C4 are the control samples of lysozyme in buffer, lysozyme in EG, buffer & EG respectively; T1, T2, T3 are the test samples of lysozyme extracted from ME-1, ME-2 & ME-3, respectively).

As discussed earlier, when LYZ is heat treated in EG, a change in the microenvironment of these Trp residues along with aggregation of LYZ results in loss of enzyme activity (Figure 7). Hence, it is of paramount interests to observe that the LYZ extracted from MEs is active and don’t aggregate while maintaining its active conformation, whereas it loses its activity when heat treated in EG. Therefore, it is natural to assume that the adsorption of LYZ at EG-SAIL interface suppresses the aggregation of LYZ. This suppression of aggregation along with enhanced refolding ability of LYZ involving a partial change in 2o structure leads to improved enhanced


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enzyme activity of LYZ. Therefore, the role of suppression of aggregation of LYZ when adsorbed at EG-SAIL interface, in maintaining the enzyme activity, cannot be ruled out. In nut-shell, as shown Figure 4C, LYZ gets adsorbed at EG-SAIL interface in MEs, which provides stability to LYZ against high temperature. LYZ is found to retain its enzyme activity with partial change in 2o structure when extracted into buffer after heat treatment. The results obtained here along with previous reports on reverse micelles,55,56 bilayer structures,34 microemulsions35 and aqueous ILs57,58 showing enzyme and protein stability, respectively, at room temperature provides a new platform to design and construct novel thermally stable MEs comprising bio-based ILs as polar, non-polar and SAIL for diverse biomedical applications.

Conclusion Thermally stable MEs23,24 based on ILs, SAILs and EG, having relatively low vapour pressure, are prepared and investigated for their phase behaviour. Their utility, as providers of thermal stability, to LYZ is explored. Very low solubility of LYZ in used IL and SAILs along with loss of enzyme activity in EG limits their use as thermally stable media. However, thermally stable nano-interfaces formed by investigated MEs act as a suitable platform to provide thermal stability to LYZ up to 120 oC. The heat treated LYZ is extracted into buffer, which upon extraction show enhanced enzyme activity and is ascribed to marginal change in secondary structure of LYZ. The present work along with earlier reported hydrophilic or hydrated hydrophilic ILs25,26 for storage of enzymes or proteins is expected to provide a new platform for commercial use of ILs for enzyme/protein stability and storage. It is expected that the work would open up a new research avenue for devising all IL based non-toxic MEs for other therapeutic applications in future.


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Supporting Information The Supporting Information is available free of charge on the ACS Publications website at www.pubs.acs.org. The synthetic procedure for SAILs and Figures S1-S11.

Acknowledgement: This work was supported by the DST, Govt. of India wide project number EMR/2017/002656. M. K. and G. S. are thankful to CSIR, Govt. of India for fellowship. The infrastructure facility provided for this work under the UPE grant is highly acknowledged.

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Surface active ionic liquid based nano-interfaces in microemulsion are used to provide enhanced thermal stability to Lysozyme. 165x77mm (300 x 300 DPI)


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Thermally Stable Ionic Liquid-Based Microemulsions for High

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