Unraveling the Role of Monoolein in Fluidity and Dynamical Response

Subscriber access provided by WEBSTER UNIV

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

Unravelling the Role of Monoolein in Fluidity and Dynamical Response of a Mixed Cationic Lipid Bilayer Priya Singh, Veerendra Kumar Sharma, Subhankar Singha, Victoria GarcíaSakai, Ramaprosad Mukhopadhyay, RANJAN DAS, and Samir Kumar Pal Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.9b00043 • Publication Date (Web): 26 Feb 2019 Downloaded from http://pubs.acs.org on February 27, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 33 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

Langmuir

Unravelling the Role of Monoolein in Fluidity and Dynamical Response of a Mixed Cationic Lipid Bilayer

Priya Singh,1 Veerendra Kumar Sharma,2 Subhankar Singha,3 Victoria García Sakai,4 Ramaprosad Mukhopadhyay,2* Ranjan Das5* and Samir Kumar Pal1*

1Department

of Chemical, Biological & Macromolecular Sciences, S. N. Bose National Centre for Basic Sciences, Block JD, Sector III, Salt Lake, Kolkata 700106, India

2Solid

State Physics Division, Bhabha Atomic Research Centre, Mumbai 400085, India

3Department

of Chemistry, Pohang University of Science and Technology (POSTECH), 77 Cheongam-Ro, Nam-Gu, Pohang, Gyungbuk, Republic of Korea, 790784

4ISIS

Pulsed Neutron and MuonFacility, Science and Technology Facilities Council, Rutherford Appleton Laboratory, Didcot, UK

5Department

*To

of Chemistry, West Bengal State University, Barasat, Kolkata 700126, India

whom correspondence should be addressed:

E-mail: [email protected] E-mail: [email protected] E-mail: [email protected]

1 ACS Paragon Plus Environment

Langmuir 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

Abstract The maintenance of cell membrane fluidity is of critical importance for various cellular functions. At lower temperature when membrane fluidity decreases, plants and cyanobacteria react by introducing unsaturation to the lipids so that the membranes return to a more fluidic state.To probe how introduction of unsaturation leads to reduced membrane fluidity a model cationic lipid Dioctadecyldimethylammonium bromide (DODAB) has been chosen, and the effects of an unsaturated lipid monoolein (MO) on the structural dynamics and phase behaviour of DODAB has been monitored by quasi-elastic neutron scattering (QENS) and time-resolved fluorescence measurements. In the coagel phase fluidity of the lipid bilayer increases significantly in presence of MO relative to pure DODAB vesicles and becomes manifest in significantly enhanced dynamics of the constituent lipids along with faster hydration and orientational relaxation dynamics of a fluorophore. On the contrary, MO restricts both lateral and internal motions of the lipid molecules in the fluid phase (>330K), which is consistent with relatively slow hydration and orientational relaxation dynamics of the fluorophore embedded in mixed lipid bilayer. The present study illustrates how incorporation of an unsaturated lipid at lower temperature (below phase transition) assists the model lipid (DODAB) in regulating fluidity via enhancement of dynamics of the constituent lipids.


Page 2 of 33

Page 3 of 33 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

Langmuir

Introduction Biological membranes play an important role in maintaining cellular, structural and functional integrity. Usually the lipids in the membrane are often structurally heterogeneous having different acyl chain compositions.1 The persistence of fluidity in a cell membrane is important for optimum diffusion of the vital components including lipids and proteins in the membrane. The fluidity is also important in the maintenance of dynamics and function of membrane proteins leading to essential cellular functions including division, differentiation and general adaptation to the environment.2-4 One of the ways to maintain the “fluidity” in the cell membrane of an organism is reported to be through regulation of double bonds in the fatty acid of the membrane lipids.5 At a low temperature, when the membrane fluidity decreases, plants and cyanobacteria respond by insertion of unsaturation in the fatty acids of lipids so that the membranes attain a state of higher fluidity.6 In addition to membrane "fluidity" hydration of the lipid bilayer plays a crucial role in determining the function of biomembranes by affecting their integrity and dynamics.7 Therefore, it is of key interest to probe quantitatively how the presence of an unsaturated lipid modulates the membrane fluidity of a model bilayer forming lipid when a change in temperature is effected, and how the change in membrane fluidity is reflected on the dynamics of water molecules in the lipidwater interface. To this end, liposomes and surfactant vesicles are often regarded as adequate membrane models.8-9 Although, natural lipids e.g., phospholipids are important constituents of the cell membrane the typical phospholipid structure is by no means a prerequisite for vesicle formation to model cell membranes,8 because in many respects, vesicles prepared from simple synthetic lipids mimic the properties of vesicles prepared from natural phospholipids.10 In fact, the properties of synthetic lipids appear remarkably relevant for obtaining a better understanding of the fundamental aspects of the much more complex natural membranes.11-12 As for example, morphological transformations of cationic vesicles 3 ACS Paragon Plus Environment

Langmuir 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

based on Dioctadecyldimethylammonium bromide (DODAB, Figure 1) were found to be reminiscent of endocytosis and intracellular membrane traffic.11-12 So, in the present work we have chosen Dioctadecyldimethylammonium bromide (DODAB, Figure 1) as the model bilayer forming lipid which provides lamellar structure13-15 analogous to biological membranes, and monoolein (MO, figure 1) as the unsaturated lipid. Among several techniques used for studying the dynamical properties of lipid bilayers, Quasielastic Neutron scattering16-17 (QENS) is an appropriate technique to study molecular motions in the time window starting from few picoseconds to several nanoseconds.18-23 On the other hand time-dependent fluorescence Stokes shift (TDFSS) of an optically excited chromophore probe the dynamics with which the local environment (including solvent molecules) of the fluorophore relaxes following photoexcitation,24 and hence provides information concerning structural rigidity/flexibility of the probe environment.25-26 Therefore, a chromophore embedded in the lipid bilayer allows monitoring of the hydration and dynamics of the probed segment of the lipid molecules.27-28 In the present work neutron scattering techniques were employed to monitor the effect of an unsaturated lipid MO on the phase behaviour and dynamical characteristics of the bilayer of a model cationic lipid DODAB. Furthermore, we have carried out measurements of TDFSS and polarization-gated anisotropy of a novel fluorophore Acedan-18 (Figure 1) to probe the relaxation dynamics of the lipid bilayer of DODAB in the coagel phase and the fluid phase in presence and absence of an unsaturated lipid (MO). Quasielastic neutron scattering (QENS) and polarization-gated anisotropy measurements showed that MO significantly affects the dynamics of the DODAB bilayer and these effects are found to be strongly dependent on the phase of the lipid bilayer. In the coagel phase, MO acts as a plasticizer and enhances the dynamics of the cationic lipids (DODAB) in the bilayer, indicating significantly increased flexibility of the DODAB/MO lipid vesicles relative to pure DODAB. On the other hand, in 4 ACS Paragon Plus Environment

Page 4 of 33

Page 5 of 33 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

Langmuir

the fluid phase MO acts as a stiffening agent, restricting the dynamics of the lipid bilayer. From the TDFSS measurements, incorporation of unsaturated MO is found to correspond to faster hydration dynamics due to increased structural flexibility of the lipid membrane, whereas, it causes slower dynamics of water molecules for structurally rigid lipid vesicles in the fluid phase. EXPERIMENTAL DETAILS Chemicals DODAB, (C18H37)2N(CH3)2Br powder (>98%) is obtained from Sigma and 1-monooleoylrac-glycerol (MO) is procured from TCI Co. Ltd, respectively. The water used is from Millipore system. The experimental procedure for the synthesis of the Acedan-18 dye has been described earlier.29 Synthesis of the DODAB Vesicles DODAB vesicles of 70 mM concentration were prepared by mixing the suitable amounts of DODAB powder in water. The obtained mixture was kept under magnetic stirring at 70°C for 30–45 min, which produced an almost transparent fluid. The obtained DODAB dispersion was then equilibrated for a day. To prepare the aqueous dispersions of DODAB-MO (2:1), defined aliquots from the stock solutions of MO were mixed under vigorous stirring to an aqueous solution of DODAB at 70°C.30 We have used the (DODAB:MO = 2:1) composition, because, for DODAB-enriched formulations the lamellar phase is predominant14-15 and MO is distributed within the DODAB lamellar phase and because, for MO-enriched formulations (DODAB:MO (1:1 and 1:2)) multilamellar and non-lamellar inverted phases coexist which makes comparison of its dynamical properties with neat DODAB untenable.


Langmuir 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

Neutron Scattering Measurements Neutron scattering experiments were carried out on 70 mM DODAB vesicles in the absence and presence of 33 mol % of Monoolein (DODAB:MO = 2:1) using high energy resolution IRIS spectrometer31 at the ISIS pulsed Neutron and Muon source at the Rutherford Appleton Laboratory, UK. For neutron scattering measurements, we have used D2O (99.9% atom D purity) as a solvent to prepare the samples. We have employed IRIS spectrometer in the offset mode with a PG (002) analyzer which provides an energy resolution (ΔE) of 17 μeV. In the used configuration of the spectrometer, energy transfer and Q-range were from −0.3 to +1.0 meV and 0.5-1.8 Å-1, respectively. Two types of experiments (i) elastic fixed window scan (EFWS) and (ii) QENS were carried out. In EFWS, the elastic intensity within the ΔE of the spectrometer is measured with the temperature. EFWS experiments have been carried out in the temperature range of 285–345K in both heating and cooling cycles. Quasi-elastic Neutron Scattering (QENS) measurements were carried out at 310K during the heating cycle, and at 330K during the cooling cycle. The selection of temperatures for QENS measurements were based on the elastic intensity scan results, which show that at 310K (during the heating), and 330K (during the cooling), DODAB bilayer is in coagel and fluid phase respectively. For reference, QENS measurements were carried out on pure D2O at the same temperatures. For instrument resolution, QENS measurement was also carried out on a standard vanadium sample. MANTID software32-33 was used to perform standard data reduction. DODAB vesicles were placed in an annular aluminium sample holder for the neutron scattering experiments. In order to minimize the multiple scattering effects, sample thickness was kept at 0.5 mm. It may be noted that we used protonated MO and at 33 mol % concentration it would contribute about 20% of the incoherent scattering signal, though it is not negligible, but have not considered in the data analysis. We have used the DAVE software33 for QENS data analysis. 6 ACS Paragon Plus Environment

Page 6 of 33

Page 7 of 33 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

Langmuir

Differential scanning calorimetry (DSC) measurements DSC experiments were carried out on the 70 mM DODAB solution with and without 33 mol % of MO using a Mettler Toledo DSC instrument. The DSC measurements were carried out in the temperature range of 285–345 K with a rate of 5 K/min. Thermograms were measured in both heating and cooling cycles. Optical Spectroscopic Studies Steady-state emission was measured with a Jobin Yvon Fluorolog fluorimeter. All fluorescence transients were measured using a commercially available (Edinburgh instrument) time-correlated single-photon counting (TCSPC) setup having MCP-PMT as photo-detector.In order to excite the samples we have used 375 nm picosecond laser source. The instrument response function for our TCSPC setup is measured to be ~ 75 ps. Previous reports from our group depict the details of the time resolved fluorescence measurement techniques used in our present studies.34-35 The emission polarizer was adjusted parallel and perpendicular with respect to excitation polarization for the fluorescence anisotropy measurements.The fluorescence anisotropy decay was obtained by using equation, r(t) = (IVV ― G.IVH)

(IVV + 2GIVH),

where IVV and IVH are fluorescence transient when the emission polarizer was

adjusted parallel and perpendicular to that of the excitation source. The grating factor (G) was determined by using a long tail matching technique. In order to obtain time dependent fluorescence stokes shift, time resolved emission spectra were constructed following the methods described earlier.36-37 In brief, the solvent correlation function, C(t), is defined as, ν(t) ― ν(∞)

C(t) = ν(0) ― ν(∞), where ν(0), ν(t), and ν(∞) are the emission peak maxima (in cm-1) at time 0, t, and ∞ respectively.


Langmuir 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

RESULTS AND DISCUSSION Studies of phase behaviour by neutron scattering and differential scanning calorimetry Elastic intensity scan satisfactorily investigates phase transitions associated with changes in microscopic dynamics and mobility of a sample with variation in temperature.16 Any microscopic mobility in the sample shifts the scattering signal intensity away from zero energy transfer, hence, a discontinuous loss or gain of intensity in an elastic scan measurement is the signature of a phase transition, which is associated with a change in the microscopic dynamics of the system. Normalised Q-averaged elastic intensities for DODAB in presence and absence of MO are shown for heating as well as cooling cycles in Figure 2(a). In case of pristine DODAB, upon heating, a sudden fall in the elastic intensity is observed at 327K corresponding to the coagel to fluid phase transition.17 Upon cooling, pristine DODAB experiences transition from fluid to gel phase at 311K, and on further cooling it is converted to the coagel phase at ~299K. The formation of an intermediate ‘gel’ phase can be attributed to the loss of synchronicity between the change in the structure of the lipid headgroup and its alkyl chain. The headgroup of the DODAB lipid is positively charged, which interacts with the neighbouring headgroups via strong electrostatic interaction (mostly repulsive) whereas the alkyl chain of the lipid, being neutral, interact via weak van der Waals interaction with the neighbouring alkyl chains. The incongruity between the interactions among the lipid headgroups and those among the lipid alkyl chains lead to the formation of an intermediate gel phase. Since, the interaction among the alkyl chains is relatively weaker, the chain ordering happens independently and at distinct temperature leading to the formation of gel phase where the alkyl chains in the lipid bilayer are ordered while the headgroup is still disordered. Upon incorporation of MO the phase behaviour of the DODAB bilayer is significantly affected during the heating as well as the cooling cycle. In case of heating, the coagel to fluid phase transition gets broadened, and the phase transition temperature shifts to a lower temperature (~321K) which is indicative of increased disorder in the lipid vesicles 8 ACS Paragon Plus Environment

Page 8 of 33

Page 9 of 33 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

Langmuir

consistent with enhanced fluidity of the lipid bilayer. On the other hand, formation of the gel phase is inhibited during cooling and DODAB transforms directly from fluid phase to coagel phase indicating synchronous ordering of the lipid headgroups and the alkyl tails, which likely originates from screening of the electrostatic repulsion between the cationic headgroups of DODAB by MO promoting in turn the van der Waals attraction between the cationic lipid molecules. Differential scanning calorimetry (DSC) thermograms of DODAB vesicles, with and without MO, are shown in Figure 2(b). In the heating cycle, pure DODAB vesicles show a main phase transition (coagel to fluid) at 327K. On incorporation of MO the main phase transition (coagel to fluid) is shifted to a lower temperature by about ~6K. The transition enthalpy, Hm, was calculated from the area under the peak and were found to be 75 kJ and 77 kJ per mol of DODAB for pure DODAB and mixed DODAB/MO vesicles, respectively. The shape of the thermogram provides additional information concerning phase transition and the peak width at half-height (T1/2) is related to cooperativity of the transition. Upon incorporation of MO, the peak width increases from 2K to 4K suggesting that the phase transition becomes less cooperative in presence of the unsaturated lipid. The results from DSC thermograms are found to be consistent with the elastic intensity scan data (Figure 2(a)) and indicate to a significant effect of MO on the phase behaviour of the lipid bilayer.

Quasielastic Neutron Scattering (QENS) From elastic intensity scan measurements, phase transitions of the DODAB bilayer are found to be affected upon incorporation of MO, which is associated with a change in the microscopic dynamics of the lipid bilayer. To get insight into the effects of MO on the dynamical processes in the lipid bilayer, QENS measurements were carried out for DODAB vesicles with and without MO at 310K and 330K, respectively. Typical QENS spectra for DODAB bilayer with and without MO at Q = 1.4Å-1 are shown in Figure 3(a) at 310K. The 9 ACS Paragon Plus Environment

Langmuir 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 10 of 33

significant broadening of the QENS spectra in the coagel phase relative to instrumental resolution indicates to the presence of stochastic motion of the lipid molecules of DODAB. In presence of MO, this broadening is found to increase relative to pure DODAB, indicating an enhancement in the dynamics of the lipid molecules. On the length and time scales of QENS, lipid molecules can undergo (i) lateral motion in which the whole lipid molecule diffuses within the monolayer and (ii) a relatively faster internal motion.16-23 Thus, the scattering law for a lipid bilayer may be written in terms of Eq. 1

S Q, E   S lat Q, E   S int Q, E 

(1)

where, Slat(Q,E) and Sint(Q,E) are the scattering functions associated with lateral and internal motions of the lipid molecules, respectively. The lateral motion is characterized as a continuous diffusion21,38 and the corresponding scattering law could be described by a single Lorentzian function Slat(Q,E) = Llat(Γlat,E), where lat is the half-width at half-maximum (HWHM) of the Lorentzian function corresponding to lateral motion of the lipid molecules. As for the internal motion, which is localized in character, the scattering law is written by Eq. 2 as a linear combination of elastic and quasi-elastic components,

S int Q, E   AQ  E   1  AQ Lint int , E 

(2)

The quasi-elastic component is approximated as a single Lorentzian function with half-width at half-maximum (HWHM) Γint. The elastic fraction of the total scattering is called the elastic incoherent structure factor (EISF) and provides information about the geometry of the dynamical process. As evident from Eq. 2, A(Q) is nothing but the EISF. Hence, the scattering law (Eq. 1) for the lipid bilayer becomes S  Q, E    A  Q  Llat  lat , E   1  A  Q   Ltot (lat  int , E ) 


(3)

Page 11 of 33 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

Langmuir

Eq. 2 is found to describe the QENS data quite well for the DODAB bilayer in presence and absence of MO in the coagel phase, at 310K, indicating that only internal motion of the lipid molecules are active which is consistent with rigid or tight packing of the lipid molecules in the coagel phase giving rise to a very slow lateral motion to be observed within the time scale of the IRIS spectrometer. Typical fitted QENS spectra for DODAB bilayer with MO in the coagel phase at 1.4Å-1 are shown in Figure 3(b). The fluid phase is the disordered phase and its corresponding scattering function, Eq. 3, describes the DODAB bilayer spectra (Fig. 3(c)) reasonably well manifesting the presence of lateral as well as internal motions of the lipid molecules. Q-dependent QENS spectra for the DODAB with MO at 330K (fluid phase) are shown in Figure S1 (In supporting information). Components corresponding to lateral (dashed blue lines) and lateral + internal motions (dashed dotted magenta lines) of DODAB are also shown in the figure (3 (b), (c) and S1). The variations of the different parameters (e.g. EISF and HWHMs) correspond to the underlying dynamics involved in different phases are discussed in the later sections. Coagel Phase: The obtained fitting parameters, EISF (A(Q)) and HWHM (Γint) for DODAB in absence and presence of MO are shown in Figure 4(a) and (b) respectively. The lower EISF and the broader QENS signal relative to pure DODAB are indicative of increased flexibility of the lipid bilayer upon incorporation of MO. In the coagel phase, lipid molecules are more ordered and tightly packed but incorporation of MO induces disorder in the DODAB/MO (2:1) vesicles enhancing its flexibility. In order to investigate the dynamical behaviour in more details we have attempted to model the observed data. Based on the structure of the DODAB molecule, containing two methyl units in the head group and two octadecyl alkyl chains, the internal dynamics should consist of motion of the methyl units (3fold rotation) and that of the alkyl chains. The ordering and arrangement of the lipids in the bilayer manifest itself in the model of internal dynamics of the lipids. In the coagel phase, the 11 ACS Paragon Plus Environment

Langmuir 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 12 of 33

alkyl chain of the lipid has an almost all-trans conformation with almost no gauche defects. This feature translates into a uniaxial rotational model for internal dynamics wherein alkyl chains rotate uniaxially along their axes. It can be pictured as hydrogen atoms of CH2 units moving on the surface of a cylinder with a given radius, which corresponds to the radius of gyration in uniaxial rotational diffusion. Given the high ordering and close packing of lipids in the coagel phase, only a fraction of hydrogen atoms (px) was observed to be participating in the dynamical motion. The effective scattering law that reckons the above described model can be written as17 2 1  2 j0 (Qb)   74 px B0  Qa   74 1  px    E     1  N 1 S  Q, E    1    3   MG l 80   74 px  Bl  Qa  41  j0 (Qb)  2 2 2 2  9  E  MG 1  E  l  l 1  

(4)

where EISF is,

EISF 

1  2 1  2 j0 (Qb)   74 px B0  Qa   74 1  px   80 

(5)

Here b is the distance between the H-atoms (1.8 Å) in methyl group (MG) of the quaternary ammonium headgroup of DODAB. τMG is the mean residence time, a is the radius of gyration of the alkyl chain, and τl is related to rotational diffusion coefficient. Eq. 5 is found to describe the observed EISF (Figure 4(a)) very well for DODAB both in absence and presence of MO. The parameters, radius of gyration, a, and the mobile fraction of the dynamic lipid tail, px, have been obtained from least squares fitting of the EISF and are given in Table-1. It is found that for pure DODAB bilayer, only 13% of the lipid tails are undergoing uni-axial rotation in a circle of radius 1.7Å. However, in presence of MO, the fluidity of the membrane is significantly enhanced and the fraction of the mobile lipid tails increases to 41%. In addition, the radius of gyration (a) increases from 1.7Å to 2.3Å indicating enhancement in flexibility of the lipid tails. The rotational diffusion coefficient Dr and τMG are obtained from 12 ACS Paragon Plus Environment

Page 13 of 33 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

Langmuir

least-squares fitting of Eq. 4 to the HWHM (int). The values of Dr for the lipid tails in DODAB in absence and presence of MO are 14(±2) eV and 21(±2) eV, respectively, whereas, the mean residence time (τMG) for the hydrogen atom in the methyl group in absence and presence of MO, are 6.7 ± 0.3 ps and 4.7 ± 0.3 ps, respectively. These results in the coagel phase clearly show the role of MO as a plasticizer in enhancing the dynamics of cationic lipids in mixed vesicles. Fluid phase: In the fluid phase, lipids are more loosely packed with a large area per lipid molecules making them more mobile. There is a notable increase in the gauche defects in the alkyl chains of the lipids compared to the coagel phase. These changes in the structural feature of the lipids necessitate a more dynamically rich model involving both internal and lateral motion of the lipids. The variations in HWHM (Γlat) with Q2 associated with the lateral diffusion of the DODAB lipids in presence and absence of MO are shown in Figure 5(a). In the fluid phase, MO acts oppositely to that in the coagel phase. Γlat is found to decrease on the addition of MO. For pure DODAB as well as mixed cationic (DODAB/MO) vesicles, Γlat varies linearly with Q2 suggesting a continuous diffusion model Γlat = DlatQ2 following Fick’s law. The values of the diffusion coefficients, Dlat, of DODAB with and without MO are shown in Table 2. In the fluid phase, MO is found to act as a stiffening agent restricting lateral diffusion of the lipid molecules. As for example, Dlat decreases from 2.2(±0.1)  10-6 cm2/s in pure DODAB to 1.7(±0.1)  10-6 cm2/s in presence of 33 mol% of MO. The addition of MO reduces the available free area per lipid and hence hinders the lateral diffusion of the lipid molecules. The HWHM (int) is a Lorentzian function that describes internal motions of the lipid molecules with and without MO and are displayed in Figure 5(b) with corresponding EISFs (in the inset). EISF does not change much (Figure 5(b)) upon the addition of MO indicating a mild effect on the nature of the internal motion of the DODAB lipid molecules. However, int is reduced 13 ACS Paragon Plus Environment

Langmuir 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 14 of 33

on addition of MO indicating retardation of the internal dynamics of the lipid molecules. In the fluid phase, besides reorientational motion, lipid molecules can undergo various other internal motions (e.g. bending, stretching, etc.). The combination of these dynamical components can be approximated by a model in which the hydrogen atoms of a CH2 unit perform translational diffusion confined within a spherical domain. As the lipid tails have large gauche defects, that imposes restricted homogeneity in the dynamics along the alkyl chain. Hence, a linear distribution of radii and diffusivities along the alkyl chain is assumed. In this model, the CH2 units closest to the headgroup experience the highest restriction and therefore have the minimum radius (Rmin) and diffusivity (Dmin) within the linear distribution.17 Likewise, the radius and diffusivity would increase linearly along the alkyl chain as we move away from the head of the alkyl chain attaining maximum value of radius (Rmax) and diffusivity (Dmax) for the last (CH3) unit of the chain. A 3-fold jump motion of the headgroup methyl units are treated in the same way as in the case of coagel phase. The effective scattering law for internal dynamics in the fluid phase is17 2   74 NC  3 j1  QRi     2 1  2 j0 (Qb)       E   N C i 1  QRi      1  Sint  Q, E     80   3 MG 1 74 NC   2l  1Anl  QRi   4 1  j0 (Qb)    2 2    9 E N MG C i 1 l , n0,0    



      2  xnl  Di Ri2   2 l 2 2 2   x D R E  n i i 



(6)

where NC = 18 is the number of the CH2 units in the lipid tail. From Eq. 6 the EISF can be expressed as

1  74 EISF   2 1  2 j0 (Qb)   80  NC 

 3 j1  QRi      QRi  i 1  NC

2

  

(7)

where Ri and Di are the radius and the diffusion coefficient corresponding to the ith site of the lipid tail, and can be written as: Ri 

i 1 Rmax  Rmin   Rmin NC  1 14

ACS Paragon Plus Environment

Page 15 of 33 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

Langmuir

and Di 

i 1 Dmax  Dmin   Dmin NC  1

Least square fitting has been used to describe the EISF and int, assuming localized translational diffusion (LTD) model and are shown by the solid lines in Figure 5(b). This model is found to describe the experimental EISF and HWHM for both DODAB systems quite satisfactorily and the resulting fitting parameters are displayed in Table 2. The values of Rmin and Dmin are found to be unrealistically small for both the systems indicating negligible movement of the H-atoms in the first CH2 unit held by the headgroup. On addition of MO, the diffusivity corresponding to internal motion is reduced indicating that MO affects the internal motion of the lipid molecules. Steady-State Optical Studies: The steady state fluorescence spectra of Acedan-18 are sensitive to solvent polarity. For example, its emission peak maximum is significantly red shifted from 461 nm in dimethylformamide (DMF) to 522 nm in water (Figure S2, In supporting information) which is attributed to a highly polar excited state originating from an intramolecular charge transfer (ICT) process.29 Figure 6(a) shows temperature dependent fluorescence spectra of Acedan-18 in pure DODAB vesicles. The emission peak maximum is significantly red shifted (~30-35 nm) relative to DMF, but is moderately blue shifted (~20 nm) in comparison to aqueous buffer which is consistent with location of the dye in the interfacial region of the membrane. The fluorescence intensity is found to decrease with increase in temperature until up to 313K followed by a dramatic increase at 323K which corresponds closely to the melting temperature (327K) for transition from coagel to fluid phase. This may be attributed to increased disorder of the cationic lipid organizations due to increase of temperature which makes packing of the lipids in the membranes looser and enhances hydration of the bilayer interface region at 313K relative to 283K. As a result water induced non-radiative relaxation 15 ACS Paragon Plus Environment

Langmuir 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

process becomes enhanced leading to a decrease in fluorescence intensity. This is consistent with slightly red shifted (~ 4-5 nm) emission spectra of Acdean-18 at 313K relative to that at 283K. On further increase of temperature to 323K, near the phase transition, the dye likely relocates deeper in the lipid bilayer due to increased flexibility of the lipid chains, and becomes screened from perturbation of water molecules causing a significant increase of the fluorescence intensity relative to that at 313K along with a moderate blue shift (~10 nm). Incorporation of MO in DODAB gives rise to stronger fluorescence of Acedan-18 in the coagel phase (up to 313K) compared to that in pure DODAB (Figure 6a and 6b) due to more efficient screening of the dye from perturbation of water molecules which is consistent with moderately blue shifted (~7-8 nm) fluorescence spectra in DODAB/MO systems relative to pure DODAB. When MO molecules incorporate in the DODAB vesicles, Acedan-18 likely penetrates deeper in the lipid bilayer thus being less exposed to water. This is likely caused by increasing fluidity or flexibility of the lipid membrane in presence of the unsaturated lipid (MO) and this effect is found to be similar to the effect of phase transition in pure DODAB. Moreover, compared to pure DODAB, fluorescence intensity in the DODAB/MO mixed vesicles decreases continuously with a mild red shift in the peak maximum on an increase of temperature. This may be ascribed to increased non-radiative deactivation due to increasing temperature along with slightly increased exposure of the dye to water. It is also noteworthy that the effect of phase transition from coagel to fluid phase is not reflected in the fluorescence spectra of AC-18 in presence of MO unlike pure DODAB vesicles. This may be ascribed to weakening and broadening of the transition peak in DSC thermograms (Figure 2b) in accordance with Oliveira et al.14 They also failed to detect the effect of phase transition on the fluorescence spectra of pyrene in DODAB/MO system with 70 mol% of DODAB and attributed it to weakening and broadening of the transition peak in DSC thermograms. The steady state fluorescence spectra provides information on polarity of the lipid-water interface


Page 16 of 33

Page 17 of 33 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

Langmuir

as well as its change following phase transition in the absence and presence of MO. In order to probe the dynamics of the DODAB bilayer in presence of MO, we have carried out time resolved fluorescence measurements. Time-resolved Fluorescence Studies: Polarization-gated anisotropy studies Time-resolved anisotropy decays (Figure 7) of Acedan-18 were measured in the coagel and the fluid phase to probe the effect of MO on the fluidity or rigidity of the DODAB bilayer. The anisotropy decays, r(t), were analysed by the two-step wobbling in a cone model.39

( ) 𝑡

( ) 𝑡

(8)

𝑟(𝑡) = 𝑟0 [𝛽1exp - 𝜙1 + 𝛽2 exp - 𝜙2 ]

where r0 is initial anisotropy, 𝜙1 and 𝜙2are the orientational relaxation times of the probe40 in the lipid bilayer and β1, β2 are their associated amplitudes. The anisotropy decay of Acedan-18 is characterized by two time constants with a much longer average rotational relaxation time (𝜙) of 1330 ps for pure DODAB in the coagel phase (310K, Table 3) relative to buffer (~180 ps) (Figure S3, In supporting information) which is consistent with significantly hindered orientational motion of the probe in the highly rigid lipid vesicles compared to bulk water. Upon the incorporation of MO, the average rotational time constant decreases significantly to 510 ps in the coagel phase relative to pure DODAB, indicating much faster orientational relaxation of the dye in the former than the latter. This is consistent with remarkably increased flexibility of the dye environment in presence of the unsaturated lipid (MO). The average rotational time constant for pure DODAB in the fluid phase (~470 ps) is comparable to that of DODAB in presence of MO in the coagel phase, which clearly manifests the effect of the unsaturated lipid in enhancement of the membrane fluidity. In the fluid phase (330K), the average rotational relaxation time of Acedan-18 increases slightly (~6 %) in presence of MO relative to pure DODAB indicating a mild


Langmuir 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

increase in rigidity of the lipid bilayer. From the polarization-gated fluorescence anisotropy measurements, incorporation of MO is found to significantly increase fluidity of the DODAB bilayer in the coagel phase (310K) which becomes manifest in the faster orientational relaxation dynamics of the dye relative to that in pure DODAB. On the other hand, presence of MO in the fluid phase (330K) slightly increases rigidity of the lipid bilayer slowing down the orientational relaxation of Acedan-18 Time-dependent Fluorescence Stokes shift measurements Figures 8 and 9 display wavelength dependent fluorescence transients of Acedan-18 at three characteristic wavelengths. The decay and rise observed in the blue end (440 nm) and the red end (550 nm) of the emission spectra are characteristic of solvent relaxation25-26 around the excited state dipole of the fluorophore and is manifested by a time-dependent Stokes shift of the fluorescence spectrum. The kinetics of solvent relaxation in the lipid membrane is often interpreted in terms of the freedom of movement of water hydrating the membrane39 and is described by the solvation correlation function(C(t)). C(t), for the lipid vesicles decays (Figure 10) with two time constants (τ1, τ2) indicating mediation of two types of water dynamics in relaxation of the local environment of the probe (Table 4). The faster component (τ1) may be ascribed to loosely bound water (intermediate interlamellar water), whereas, the longer relaxation component (τ2) likely originates from water molecules strongly bound to the polar head groups of the lipid molecules.41,42 However, the dynamics of the overall solvation response is a better descriptor of environmental relaxation rather than the dynamics of the individual water molecules due to the coupled nature of surfactant/protein and water motions in micelles, proteins etc.24-26,43 In consequence, the overall solvation response described in terms of average or integrated relaxation time () serves as an efficient parameter to indicate flexibility/rigidity of the lipid bilayer.44 The decay of C(t) for DODAB (coagel phase, 310K) is characterized by two time constants of 100 ps (52%) and 1040 ps 18 ACS Paragon Plus Environment

Page 18 of 33

Page 19 of 33 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

Langmuir

(48%), respectively, with an average solvation time () of 550 ps (Table 4). Upon incorporation of MO the average relaxation time decreases significantly (330 ps) indicating faster relaxation dynamics of the probe environment in the coagel phase in presence of MO compared to pure DODAB. This may be ascribed to increased flexibility of lipid molecules in the bilayer of DODAB/MO vesicles compared to pure DODAB, which gives rise to much faster hydration (solvation) response in the former than the latter due to coupled nature of the dynamics of lipid/surfactant and associated water molecules.24-26,43 On the other hand, the average solvation time in the lipid bilayer of DODAB becomes moderately slower (~13%) in the fluid phase in presence of MO indicating the role of MO as a stiffening agent for lipid vesicles in the fluid phase. In the fluid phase, incorporation of MO slightly increases rigidity of the lipid bilayers resulting in slower relaxation dynamics of the associated water molecules, which becomes manifested in the longer average solvation time (680 ps) than that of pure DODAB (~600 ps, Table 4). The decrease of the average solvation time () in the coagel phase or its increase in the fluid phase on incorporation of MO is also consistent with the decrease or increase of the average orientational relaxation time (𝜙) (Table 3) confirming the effect of MO in increasing the fluidity or rigidity of the DODAB bilayer below and above the phase transition temperature, respectively. Finally, from measurements of quasi-elastic neutron scattering and polarization gated anisotropy in conjunction with time-dependent fluorescence Stokes shift measurements due to water dynamics, we conclude that MO strongly affects the dynamical and phase behaviour of the DODAB bilayers. In the ordered gel phase of DODAB, addition of MO enhances fluidity of the lipid bilayers, whereas in the fluid phase it causes stiffening. These results are in divergence with the effects of other additives such as cholesterol.45 As for example, for the DODAB membrane in the gel phase, steady state anisotropy as sensed by a probe DPH increases with increase in mol% of cholesterol consistent with increased rigidity or tighter


Langmuir 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

packing of lipids.45 So, the effect of the unsaturated lipid (MO) on the dynamics and phase behaviour of a membrane mimicking model cationic lipid DODAB are exclusive and could correlate with the situation observed in the nature when the organisms respond to low temperature by incorporating unsaturation via complex mechanisms to maintain the fluidity of cell membranes. In particular, the novelty of the present work lies in direct manifestation of the dynamical response of the model membrane (DODAB) on a timescale of picoseconds to nanoseconds in the presence of an unsaturated lipid monoolein (MO). Our work has clearly demonstrated that motional dynamics of the lipids is remarkably enhanced upon the incorporation of MO which is also reflected in faster hydration dynamics and orientational relaxation dynamics of a fluorophore embedded in the lipid bilayer. Conclusions The present work highlights clearly the role of an unsaturated lipid MO on the dynamical and the phase behaviour of a lipid (DODAB) bilayer. Elastic intensity scan measurements and DSC thermograms show that in the heating cycle, in presence of MO, coagel to fluid phase transition is found to take place at a lower temperature and the transition gets broadened. In the cooling cycle, in case of DODAB with MO, no intermediate gel phase is observed like pristine DODAB indicating that MO induces synchronous ordering between the polar headgroups and the non-polar tails of the cationic lipids. Quasi-elastic neutron scattering (QENS) studies and polarization-gated anisotropy measurements have revealed that in the coagel phase (310K), MO acts as a plasticizer enhancing significantly the dynamics of the lipid molecules and hence membrane fluidity of the DODAB bilayer. On the contrary, in the fluid phase, MO acts as a stiffening agent where it restricts lateral and internal motions of the lipid molecules and hinders orientational relaxation of the probe compared to the coagel phase. The dynamics of solvation response of the lipid bilayer is significantly faster for the structurally more flexible DODAB-MO vesicles relative to pure DODAB in the coagel phase


Page 20 of 33

Page 21 of 33 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

Langmuir

(310K), whereas, it becomes moderately slower in the fluid phase (330K) indicating increased rigidity of the DODAB bilayer in presence of MO. Our present work reflects how a specific (cationic) model lipid membrane (DODAB) restores fluidity in the coagel (ordered) phase at 310K upon incorporation of unsaturation (in the form of MO) and thus provides a coherent picture between the structure and the dynamics of biomimetic membranes. Acknowledgements Generous financial grant (ST/P/S&T/15G-41/2017) to RD from the dept. of science & technology and biotechnology, Govt. of West Bengal is gratefully acknowledged. Financial grants (SB/S1/PC-011/2013) from DST (India) and(BT/PR11534/NNT/28/766/2014) from DBT (India) to SKP are gratefully acknowledged.R. Mukhopadhyay is thankful to the Department of Atomic Energy, India,for the award of Raja Ramanna Fellowship, Supporting Information Fitted QENS spectra for DODAB with MO in the fluid phase at different Q values. Fluorescence emission spectra of Acedan-18 in water, ethanol and dimethylformamide and time resolved anisotropy decay of Acedan-18 in water.


Langmuir 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

References 1. Nyholm, T. K., Lipid-protein interplay and lateral organization in biomembranes. Chemistry and physics of lipids 2015,189, 48-55. 2. Hresko, R. C.; Kraft, T. E.; Quigley, A.; Carpenter, E. P.; Hruz, P. W., Mammalian glucose transporter activity is dependent upon anionic and conical phospholipids. Journal of Biological Chemistry 2016, jbc. M116. 730168. 3. Nicolson, G. L., The Fluid—Mosaic Model of Membrane Structure: Still relevant to understanding the structure, function and dynamics of biological membranes after more than 40years. Biochimica et Biophysica Acta (BBA)-Biomembranes 2014,1838 (6), 1451-1466. 4. Lipowsky, R., Remodeling of membrane compartments: some consequences of membrane fluidity. Biological chemistry 2014,395 (3), 253-274. 5. Russell, N. J., Mechanisms of thermal adaptation in bacteria: blueprints for survival. Trends in Biochemical Sciences 1984,9 (3), 108-112. 6. Vigh, L.; Los, D. A.; Horvath, I.; Murata, N., The primary signal in the biological perception of temperature: Pd-catalyzed hydrogenation of membrane lipids stimulated the expression of the desA gene in Synechocystis PCC6803. Proceedings of the National Academy of Sciences 1993,90 (19), 9090-9094. 7. Ball, P., Water is an active matrix of life for cell and molecular biology. Proceedings of the National Academy of Sciences 2017,114 (51), 13327-13335. 8. Engberts, J. B.; Hoekstra, D., Vesicle-forming synthetic amphiphiles. Biochimica et Biophysica Acta (BBA)-Reviews on Biomembranes 1995,1241 (3), 323-340. 9. Fonteijn, T. A.; Engberts, J. B.; Hoekstra, D., Asymmetric fusion between phospholipid vesicles and vesicles formed from synthetic di (N-alkyl) phosphates. Biochemistry 1991,30 (21), 53195324. 10. Fendler, J. H., Membrane mimetic chemistry: characterizations and applications of micelles, microemulsions, monolayers, bilayers, vesicles, host-guest systems, and polyions. John Wiley & Sons Inc: 1982. 11. Hubert, D. H.; Jung, M.; Frederik, P. M.; Bomans, P. H.; Meuldijk, J.; German, A. L., Morphology transformations of DODAB vesicles reminiscent of endocytosis and vesicular traffic. Langmuir 2000,16 (23), 8973-8979. 12. Duzgunes, N.; Goldstein, J. A.; Friend, D. S.; Felgner, P. L., Fusion of liposomes containing a novel cationic lipid, N-[2, 3-(dioleyloxy) propyl]-N, N, N-trimethylammonium: induction by multivalent anions and asymmetric fusion with acidic phospholipid vesicles. Biochemistry 1989,28 (23), 9179-9184. 13. Kepczynski, M.; Lewandowska, J.; Witkowska, K.; Kędracka-Krok, S.; Mistrikova, V.; Bednar, J.; Wydro, P.; Nowakowska, M., Bilayer structures in dioctadecyldimethylammonium bromide/oleic acid dispersions. Chemistry and physics of lipids 2011,164 (5), 359-367. 14. Oliveira, I. M.; Silva, J. P.; Feitosa, E.; Marques, E. F.; Castanheira, E. M.; Oliveira, M. E. C. R., Aggregation behavior of aqueous dioctadecyldimethylammonium bromide/monoolein mixtures: A multitechnique investigation on the influence of composition and temperature. Journal of colloid and interface science 2012,374 (1), 206-217. 15. Silva, J. N.; Oliveira, I.; Oliveira, A.; Lúcio, M.; Gomes, A.; Coutinho, P. J.; Oliveira, M. R., Structural dynamics and physicochemical properties of pDNA/DODAB: MO lipoplexes: effect of pH and anionic lipids in inverted non-lamellar phases versus lamellar phases. Biochimica et Biophysica Acta (BBA)-Biomembranes 2014,1838 (10), 2555-2567. 16. Sharma, V.; Mamontov, E.; Tyagi, M.; Urban, V., Effect of α-Tocopherol on the Microscopic Dynamics of Dimyristoylphosphatidylcholine Membrane. The Journal of Physical Chemistry B 2015,120 (1), 154-163. 17. Dubey, P.; Srinivasan, H.; Sharma, V.; Mitra, S.; Sakai, V. G.; Mukhopadhyay, R., Dynamical Transitions and Diffusion Mechanism in DODAB Bilayer. Scientific reports 2018,8 (1), 1862. 22 ACS Paragon Plus Environment

Page 22 of 33

Page 23 of 33 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

Langmuir

18. Swenson, J.; Kargl, F.; Berntsen, P.; Svanberg, C., Solvent and lipid dynamics of hydrated lipid bilayers by incoherent quasielastic neutron scattering. The Journal of chemical physics 2008,129 (4), 07B616. 19. Wanderlingh, U.; D’Angelo, G.; Branca, C.; Conti Nibali, V.; Trimarchi, A.; Rifici, S.; Finocchiaro, D.; Crupi, C.; Ollivier, J.; Middendorf, H., Multi-component modeling of quasielastic neutron scattering from phospholipid membranes. The Journal of chemical physics 2014,140 (17), 05B602_1. 20. Busch, S.; Smuda, C.; Pardo, L. C.; Unruh, T., Molecular mechanism of long-range diffusion in phospholipid membranes studied by quasielastic neutron scattering. Journal of the American Chemical Society 2010,132 (10), 3232-3233. 21. Sharma, V.; Mamontov, E.; Anunciado, D.; O’Neill, H.; Urban, V., Nanoscopic dynamics of phospholipid in unilamellar vesicles: effect of gel to fluid phase transition. The Journal of Physical Chemistry B 2015,119 (12), 4460-4470. 22. Sharma, V.; Mamontov, E.; Tyagi, M.; Qian, S.; Rai, D.; Urban, V., Dynamical and phase behavior of a phospholipid membrane altered by an antimicrobial peptide at low concentration. The journal of physical chemistry letters 2016,7 (13), 2394-2401. 23. Sharma, V. K.; Mamontov, E.; Anunciado, D. B.; O'Neill, H.; Urban, V. S., Effect of antimicrobial peptide on the dynamics of phosphocholine membrane: role of cholesterol and physical state of bilayer. Soft matter 2015,11 (34), 6755-6767. 24. Li, T.; Hassanali, A. A.; Singer, S. J., Origin of slow relaxation following photoexcitation of W7 in myoglobin and the dynamics of its hydration layer. The Journal of Physical Chemistry B 2008,112 (50), 16121-16134. 25. Choudhury, S.; Mondal, P. K.; Sharma, V.; Mitra, S.; Sakai, V. G.; Mukhopadhyay, R.; Pal, S. K., Direct observation of coupling between structural fluctuation and ultrafast hydration dynamics of fluorescent probes in anionic micelles. The Journal of Physical Chemistry B 2015,119 (34), 1084910857. 26. Maiti, J.; Kalyani, V.; Biswas, S.; Rodriguez-Prieto, F.; Mosquera, M.; Das, R., Slow solvation dynamics in supramolecular systems based on bile salts: Role of structural rigidity of bile salt aggregates. Journal of Photochemistry and Photobiology A: Chemistry 2017,346, 17-23. 27. Jurkiewicz, P.; Cwiklik, L.; Jungwirth, P.; Hof, M., Lipid hydration and mobility: an interplay between fluorescence solvent relaxation experiments and molecular dynamics simulations. Biochimie 2012,94 (1), 26-32. 28. Amaro, M.; Šachl, R.; Jurkiewicz, P.; Coutinho, A.; Prieto, M.; Hof, M., Time-resolved fluorescence in lipid bilayers: selected applications and advantages over steady state. Biophysical journal 2014,107 (12), 2751-2760. 29. Singha, S.; Kim, D.; Roy, B.; Sambasivan, S.; Moon, H.; Rao, A. S.; Kim, J. Y.; Joo, T.; Park, J. W.; Rhee, Y. M., A structural remedy toward bright dipolar fluorophores in aqueous media. Chemical science 2015,6 (7), 4335-4342. 30. Silva, J. N.; Coutinho, P. J.; Oliveira, M. R., Characterization of monoolein-based lipoplexes using fluorescence spectroscopy. Journal of fluorescence 2008,18 (2), 555-562. 31. Carlile, C.; Adams, M. A., The design of the IRIS inelastic neutron spectrometer and improvements to its analysers. Physica B Condensed Matter 1992,182, 431-440. 32. Arnold, O.; Bilheux, J.-C.; Borreguero, J.; Buts, A.; Campbell, S. I.; Chapon, L.; Doucet, M.; Draper, N.; Leal, R. F.; Gigg, M., Mantid—Data analysis and visualization package for neutron scattering and μ SR experiments. Nuclear Instruments and Methods in Physics Research Section A: Accelerators, Spectrometers, Detectors and Associated Equipment 2014,764, 156-166. 33. Azuah, R. T.; Kneller, L. R.; Qiu, Y.; Tregenna-Piggott, P. L.; Brown, C. M.; Copley, J. R.; Dimeo, R. M., DAVE: a comprehensive software suite for the reduction, visualization, and analysis of low energy neutron spectroscopic data. Journal of Research of the National Institute of Standards and Technology 2009,114 (6), 341.


Langmuir 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

34. Choudhury, S.; Naiya, G.; Singh, P.; Lemmens, P.; Roy, S.; Pal, S. K., Modulation of Ultrafast Conformational Dynamics in Allosteric Interaction of Gal Repressor Protein with Different Operator DNA Sequences. ChemBioChem 2016,17 (7), 605-613. 35. Singh, P.; Choudhury, S.; Chandra, G. K.; Lemmens, P.; Pal, S. K., Molecular recognition of genomic DNA in a condensate with a model surfactant for potential gene-delivery applications. Journal of Photochemistry and Photobiology B: Biology 2016,157, 105-112. 36. Singh, P.; Choudhury, S.; Singha, S.; Jun, Y.; Chakraborty, S.; Sengupta, J.; Das, R.; Ahn, K.-H.; Pal, S. K., A sensitive fluorescent probe for the polar solvation dynamics at protein–surfactant interfaces. Physical Chemistry Chemical Physics 2017,19 (19), 12237-12245. 37. Horng, M.; Gardecki, J.; Papazyan, A.; Maroncelli, M., Subpicosecond measurements of polar solvation dynamics: coumarin 153 revisited. The Journal of Physical Chemistry 1995,99 (48), 1731117337. 38. Armstrong, C.; Trapp, M.; Peters, J.; Seydel, T.; Rheinstädter, M., Short range ballistic motion in fluid lipid bilayers studied by quasi-elastic neutron scattering. Soft Matter 2011,7 (18), 8358-8362. 39. Dutt, G., Are the experimentally determined microviscosities of the micelles probe dependent? The Journal of Physical Chemistry B 2004,108 (11), 3651-3657. 40. Quitevis, E. L.; Marcus, A. H.; Fayer, M. D., Dynamics of ionic lipophilic probes in micelles: picosecond fluorescence depolarization measurements. The Journal of Physical Chemistry 1993,97 (21), 5762-5769. 41. Singh, P.; Choudhury, S.; Sharma, V.; Mitra, S.; Mukhopadhyay, R.; Das, R.; Pal, S. K., Modulation of Solvation and Molecular Recognition of a Lipid Bilayer under Dynamical Phase Transition. ChemPhysChem 2018,19 (20), 2709-2716. 42. Yamada, T.; Takahashi, N.; Tominaga, T.; Takata, S.-i.; Seto, H., Dynamical behavior of hydration water molecules between phospholipid membranes. The Journal of Physical Chemistry B 2017,121 (35), 8322-8329. 43. Nilsson, L.; Halle, B., Molecular origin of time-dependent fluorescence shifts in proteins. Proceedings of the National Academy of Sciences 2005,102 (39), 13867-13872. 44. Jurkiewicz, P.; Olżyńska, A.; Langner, M.; Hof, M., Headgroup hydration and mobility of DOTAP/DOPC bilayers: a fluorescence solvent relaxation study. Langmuir 2006,22 (21), 8741-8749. 45. Bhattacharya, S.; Haldar, S., Interactions between cholesterol and lipids in bilayer membranes. Role of lipid headgroup and hydrocarbon chain–backbone linkage. Biochimica et Biophysica Acta (BBA)-Biomembranes 2000,1467 (1), 39-53.


Page 24 of 33

Page 25 of 33 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

Langmuir

Table 1: The dynamical parameters of DODAB with and without MO in the coagel phase (310 K) Coagel phase

px(%)

a (Å)

τMG (ps)

Dr (μeV)

DODAB

13 ± 3

1.7 ± 0.2

6.7 ± 0.3

14 ± 2

DODAB-MO

41 ± 4

2.3 ± 0.2

4.7 ± 0.3

21 ± 2

Table 2: The dynamical parameters of DODAB with and without the MO in the fluid phase (330 K) Fluid

Lateral motion

Internal motion

Dlat (10-6 cm2/s)

Rmax(Å)

Dmax(10-6 cm2/s)

τMG (ps)

DODAB

2.2±0.1

4.2±0.2

17.3±0.4

4.5±0.2

DODAB-MO

1.7±0.1

4.5±0.2

14.3±0.4

4.9±0.2

Table 3. Rotational time constants of Acedan-18 in different systems System

𝛟𝟏/ns (β1)

𝛟𝟐/ns (β2)

𝜙/ns

DODAB (310 K)

0.05 (87)

10 (13)

1.33

DODAB-MO (310 K)

0.03 (95)

10 (5)

0.51

DODAB (330 K)

0.05 (37)

0.71 (63)

0.47

DODAB-MO (310 K)

0.14 (67)

1.21 (33)

0.50

𝜙1, 𝜙2 are rotational correlation times, respectively and β1 and β2 are associated amplitudes represented in % within parentheses. 𝜙 is average rotational relaxation time = β1𝜙1 + β2𝜙2

Table 4. Solvation time constants of Acedan-18 in different systems System

τ1/ns (a1)

τ2/ns (a2)

/ns

DODAB (310 K)

0.10 (52%)

1.04 (48%)

0.55

DODAB-MO (310 K)

0.06 (55%)

0.66 (45%)

0.33

DODAB (330 K)

0.12 (50%)

1.08 (50%)

0.60

DODAB-MO (330 K)

0.11 (28%)

0.90 (72%)

0.68

τ1,τ2 are solvation correlation time; a1,a2 are corresponding amplitudes shown in % within parentheses ; = a1τ1 + a2τ2 is average solvation time


Langmuir 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

A

B

Page 26 of 33

C

Figure 1. Molecular Structure of (A) DODAB, (B) MO and (C) Acedan-18

Figure 2. (a) Elastic intensity scans. (b) Differential scanning calorimetry (DSC) thermograms for 70 mM DODAB bilayer with and without 33 mol % MO in the heating and the cooling cycles.


Page 27 of 33 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

Langmuir

Figure 3.(a) Typical observed QENS spectra for DODAB bilayer with and without MO at 310K at Q = 1.4 Å-1. The contribution of the solvent (D2O) has been subtracted, and the resultant spectra are normalized to the peak intensities. The instrumental resolution measured from standard vanadium sample is shown by solid line, (b) Typical fitted QENS spectra for DODAB in presence of MO at Q =1.4 Å-1 in coagel phase (310 K) and in (c) fluid phase (330 K).

Figure 4.(a) Elastic incoherent structure factor (EISF) and (b) Γint for DODAB in presence and absence of MO in the coagel phase (310 K). The solid lines represent fits assuming the fractional uniaxial rotational model.


Langmuir 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

Figure 5.(a) Variation of HWHM (Γlat) with Q2 corresponding to lateral motion of DODAB with and without MO at 330 K. The solid lines correspond to the Fickian description. (b) The Q dependence of HWHM, Γint, corresponding to internal motion of DODAB bilayer in presence and absence of MO at 330 K. EISF’s for DODAB bilayer with and without MO are shown in inset. The solid line represents the fit obtained from localised translational diffusion model as described in text.

Figure 6. Fluorescence emission spectra of Acedan-18 in a) DODAB (70 mM) and b) DODAB (70 mM) in presence of 33 mol % MO at different temperatures 28 ACS Paragon Plus Environment

Page 28 of 33

Page 29 of 33 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

Langmuir

Figure7. Time-resolved anisotropy of Acedan-18 in DODAB in absence (a & c) and in presence (b & d) of 33 mol% MO in the coagel phase (310K) and liquid crystalline phase (330K).


Langmuir 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

Figure 8. Picosecond-resolved emission transients of Acedan-18 in DODAB in absence (a) and in presence (b) of MO in the coagel phase (310K). Time-resolved emission spectra (TRES) in DODAB in absence (c) and in presence of MO in the coagel phase.


Page 30 of 33

Page 31 of 33 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

Langmuir

Figure 9. Picosecond-resolved emission transients of Acedan-18 in DODAB in absence (a) and in presence of MO (b) in the fluid phase (330K). Time-resolved emission spectra (TRES) of Acedan-18 in DODAB in absence (c) and in presence of MO (d) in the fluid phase.


Langmuir 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

Figure 10. Decay of the Solvation correlation function, C(t), for Acedan-18 in DODAB in absence and presence of 33 mol% of MO in the coagel phase (a) and in the fluid phase (b).


Page 32 of 33

Page 33 of 33 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

Langmuir

TOC


Unraveling the Role of Monoolein in Fluidity and Dynamical Response

Recommend Documents