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Detection of Curvature Radius Dependent Interfacial pH/polarity for Amphiphilic Self-assemblies: Positive vs Negative Curvature Yeasmin Sarkar, Rini Majumder, Sanju Das, Ambarish Ray, and Partha Pratim Parui Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b03888 • Publication Date (Web): 21 Dec 2017 Downloaded from http://pubs.acs.org on December 31, 2017

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Detection of Curvature Radius Dependent Interfacial pH/polarity for Amphiphilic Selfassemblies: Positive vs Negative Curvature

Yeasmin Sarkar,† Rini Majumder,† Sanju Das, †,‡ Ambarish Ray,‡ and Partha Pratim Parui*,†

†

Department of Chemistry, Jadavpur University, Kolkata 700032, India.

‡

Department of Chemistry, Maulana Azad College, Kolkata 700013, India.

Keywords: Interfacial pH/polarity; anionic amphiphilic self-assemblies; positive/negative curvature radius; Spiro-rhodamine pH-probe.

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Abstract It is possible that a defined curvature at the membrane interface controls its pH/polarity to exhibit specific bioactivity. By utilizing interface interacting spiro-rhodamine pH-probe and the Schiffbase polarity-probe, we have shown that the pH-deviation from the bulk phase to the interface (pH)/interfacial dielectric constant ((i)) for amphiphilic self-assemblies can be regulated by the curvature geometry (positive/negative) and its radius. According to 1H-NMR and fluorescence anisotropy investigations, the probes selectively interact with anionic interfacial Stern layer. The pH/(i) for the Stern layer are estimated by UV-Vis absorption and fluorescence studies. For anionic sodium bis-2-ethylhexyl-sulfosuccinate (AOT) inverted micellar (IM) negative interface, the highly restricted water and proton penetration into the Stern layer owing to tight surfactant packing or reduced water-exposed headgroup area may responsible for the much lower pH ~ – 0.45 and (i) ~ 28 in comparison to ~ –2.35 and ~ 44, respectively, for anionic sodium dodecyl sulphate (SDS) micellar positive interface with close similar Stern layer. With increasing AOT IM water-pool radius (1.7–9.5 nm) or [water]/[AOT] ratio (w0) (8.0–43.0), the pH and (i) increase maximally up to ~ –1.22 and ~ 45, respectively, due to greater water-exposed headgroup area. However, the unchanged pH ~ 0.65 and (i) ~ 53.0 within radii ~ 3.5–8.0 nm for the positive interface of mixed triton X-100 (TX-100)/SDS (4:1) micelle justify its packing flexibility. Interestingly, the continuous increasing pH trend for IM up to its largest possible water-pool radius ~ 9.5 nm may rationalize the increase of pH (~ –1.4 to –1.6) with change in curvature radii (~ 15 to 50 nm) for sodium 1,2-dimyristoyl-sn-glycero-3-phosphorylglycerol (DMPG)/1,2dimyristoyl-sn-glycero-3-phosphocholine (DMPC) (2:1) large unilamellar vesicles (LUV) owing to its negative interface. Whereas, similar to that micellar positive interface, the unchanged pH at the positive LUV interface was confirmed by fluorescence microscopic studies with giant 2 ACS Paragon Plus Environment

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unilamellar vesicle of identical lipid composition. The present study offers a unique and simple method to monitor the curvature radius dependent interfacial pH/polarity for biologically related membranes.


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INTRODUCTION The curvature of phospholipid membrane plays most decisive role in biology for defining the morphology of cell, organelles and the structure of local membrane subdomains.1,4 Lipids adopt inherent 2D-shapes depending on the size of their polar headgroups and nonpolar acyl chain compositions. The side-by-side lipids packing generates a specific shape in the monolayer selfassemblies to produce a spontaneous curvature of the local lipids in the membrane structure. The membrane curvature is generally ascribed by positive and negative bending depending on the area of lipid polar headgroup relative to that of acyl chain.5 Lipids with a large area ratio of polar headgroups to nonpolar acyl chains spontaneously produces positive (convex) curvature, while lipids with the opposite ratio generates negative (concave) curvature.6.7 In addition, inter- and/or intra-cellular various biological processes, such as movement, division, extension and vesicle trafficking also manipulate the local membrane bending by the change of lipid conformations.5,811

The curvature dependent evolution of each shape at the membrane interface arises because of specific physiological causes. The intracellular membrane compartment with a defined local interfacial curvature allows the cell to compartmentalize specific protein(s), supporting numerous biochemical reactions required for eukaryotic life.5,12 On the other hand, the plasma membranes of cells that have internal membranes undergo major curvature transformations when they develop intercellular contacts or spread/move on a substrate.13,14 Again, different transmembrane proteins have an intrinsic ability to bend their associated membranes interface and modify the local membrane curvature for specific in vivo reactivity.3,15-17 Interestingly, the studies for the role of membrane curvature on the mechanistic action of antimicrobial and amyloid peptides have revealed that the peptide binding at the membrane interface generates adequate amount of


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curvature strain, which activates lipids phase separation process and finally disrupts the membrane structures.18-22 It has also been reported that the fusogenic mechanism of viral fusion is also mediated by negative curvature strain on the lipid bilayer.23,24 Therefore, the protection or generation of specific membrane curvature strain is extremely essential in biology. Nevertheless, except few elegant studies, it is still a matter of debate how membrane curvature in its subdomain stimulates a particular biological event.3,25-28 Meanwhile, a vast amount of studies have shown that the local cellular pH/polarity controls membrane biochemical reactivities, such as membrane transport, ion transport across the membrane, insertion of protein/molecules into membranes and their translocation across the membrane.29-35 In this context, we believe that a certain pH/polarity at the membrane interface is regulated by preserving a certain membrane curvature. It has been reported that the curvature geometry and its radius affect the surfactant packing in a self-assembly to control the Stern layer solvation or the extent of headgroup surface area exposed to aqueous medium.36-39 As the more water-exposed headgroup is associated with the increased interfacial water and solvated H+/OH– penetration, the self-assembly curvature nature or radius value may govern the magnitude of pH/polarity deviation between the bulk and the interface. However, no investigation has ever been attempted to identify the interfacial curvature radius with its pH/polarity for the amphiphilic self-assemblies to address weather pH/polarity at the membrane interface is affected by a modulation of its shape. Most probably, the monitoring of interfacial properties themselves for self-assemblies are extremely difficult due to highly complex micro-heterogeneous compartmentalization including the uncertainty about its bulk phase contribution. In the recent past, several investigations have identified a large shift in acid/base pKa for small organic molecules due to their interaction with various self-assembled systems and therefrom speculated a difference in pH between the bulk


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aqueous phase and the probe localized interface.40-42 However, quantitative pH estimation at a specific interfacial depth and its deviation from bulk medium for amphiphilic self-assemblies has rarely been addressed. Tahara and co-workers have established a heterodyne-detected electronic sum frequency generation (HD-ESFG) spectroscopic method to estimate the self-assembled system induced acid/base pKa shift, and monitored the interfacial pH values.43,44 It has been identified that the interfacial pH is substantially altered from the bulk pH. Recently, we have introduced a new interfacial pH/polarity monitoring method for biologically important micelles and vesicles by utilizing interface interacting pH/polarity detecting probes.45-47 The estimated values of the pH/polarity differences between the bulk phase and the interface are observed to be similar to that obtained from HD-ESFG method. Most importantly, the inherent simplicity of our detection technique can be highly effective to monitor the shape and structure dependent manipulation of the pH/polarity at the complex biological membrane interfaces. Herein, a water soluble glucose derivative of spiro-rhodamine molecule and Schiff-base molecule acting as a highly sensitive interfacial pH and polarity probe, respectively, were synthesized. The specific interfacial location of the probes provide the shape and structure dependent a minute change of pH-deviation from the bulk to the interface and interfacial dielectric constant for different self-assembled systems. As the shape of biological membrane subdomain transforms from one to another by changing the positive and/or negative curvature radii,5 we determine the variation of interfacial pH/polarity and its deviation from bulk phase value with a change of positive curvature radius for micelle and negative curvature radius for inverted micelle. Furthermore, the interfacial pH/polarity detection by simultaneous variation of positive and negative curvature radii in the phospholipid vesicle have also shown that curvature radius induced change in interfacial pH occurs preliminary at its negative interface and remain invariant for the


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positive interface. The present study may be important to estimate the difference in interfacial pH/polarity due to change of its curvature radius for various complicated biological membranes.

MATERIALS AND METHOD General procedure. Unless otherwise stated, all solvent and chemicals of analytical grade were purchased from Sigma Aldrich Chemicals (USA) and used without further purification. However, the phospholipid sodium 1,2-dimyristoyl-sn-glycero-3-phosphorylglycerol (DMPG) and 1,2dimyristoyl-sn-glycero-3-phosphocholine (DMPC) were purchased from Avanti Polar Lipids (USA). Sodium bis-2-ethylhexyl-sulfosuccinate (AOT), triton X-100 (TX-100) and sodium dodecyl sulphate (SDS) were dried under vacuum overnight before use. For preparation of buffers and other analytical measurements, milli-Q Millipore® 18.2 MΩ.cm water was used. Different buffer compositions were used to attain a particular medium pH: sodium citrate/sodium phosphate for pH 2.06.0; sodium cacodylate-HCl for pH 5.06.0; HEPES-NaOH for pH 6.08.0. 1H-NMR spectra was performed in D2O both in presence and absence of various self-assembled systems (SDS, DMPG/DMPC (2:1) and TX-100/SDS (4:1)) with a Bruker 300-MHz NMR Spectrophotometer using tetramethylsilane ( = 0) as a standard. The spectrum in DMSO-d6 was also performed as the reference. To maintain various acidic pH, appropriate amount of CF3COOH was added in D2O. The average particle size for AOT inverted micelles (IMs) and TX-100/SDS (4:1) micelles were determined by dynamic light scattering (DLS) measurement with Malvern Instruments, DLS-nano ZS, Zetasizer, Nanoseries. Synthesis of spiro-rhodamine-glucose molecule (s-RHG) and Schiff-base molecule (PMP). The s-RHG was prepared according to the earlier procedure with some modifications.48,49 In brief, first, spiro-rhodamine 6G hydrazide was prepared by the reaction of rhodamine 6G hydrochloride


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with a large excess of hydrazine in ethanol. Rhodamine 6G hydrazide (0.857 g, 2.00 mmol) and glucose (0.540 g, 3.00 mmol) were mixed in the mixture of 20 mL toluene and 60 mL methanol in the presence of p-toluene sulfonic acid (0.20 mmol, 0.038 g) with a constant starring over a period of 60 min. After being reflux for 36 h the reaction mixture was cooled. The crude product was purified by column chromatography (CH3COOC2H5:CH3OH = 15:1, v/v) followed by rotary evaporation to get solid product (s-RHG) in 25% yield. 1H NMR (DMSO-d6, 300MHz): 7.79 (d, 1H, J=6.0 Hz, imine-H), 7.49 (d, 2H, J=3.0Hz, Ar-H), 6.91(d, 2H, J=6.0 Hz, Ar-H), 6.24 (m, 2H, Ar-H), 6.10 (m, 2H, Ar-H), 3.36 (m, 7H, glucose-H), 3.12 (s, 6H, CH2–CH3(4H), Glucose-H(2H)), 2.88 (m, 2H, CH2–OH), 1.89 (s, 6H, Ar-CH3), 1.19 (s, 6H, CH2–CH3) ppm (For details, see Supporting Information). The Schiff base molecule 2-((2-(pyridine-2-yl)ethylimino)methyl)-6-(hydroxymethyl)-4methylphenol (PMP) was synthesized by following our method reported previously.46 In brief, to an methanolic solution of 2-hydroxy-3-(hydroxymethyl)-5-methylbenzaldehyde (HHMB) (0.166 g, 1.00 mmol), 2-(2-aminoethyl)-pyridine (AEP) (0.122 g, 1.00 mmol) was added drop-wise at ambient temparature with a constant stirring and 2 drops of AcOH were further added to it. The mixture was refluxed for 2 h at 40°C and then filtered. The filtrate was then evaporated under reduced pressure to get the crude product as a gel. The product was purified by column chromatography followed by rotary evaporation and dried over CaCl2 under vacuum: 1H-NMR (CDCl3, 300MHz): 2.26 (s, 3H, ArCH3), 3.17 (t, J = 6.9 Hz, 2H, CH2CH2), 4.02 (t, J = 6.9 Hz, 2H, CH2CH2), 4.7 (s, 2H, CH2), 6.92 (s, 1H, H-3), 7.10-7.18 (4H, ArH), 7.26 (due to trace amount of CHCl3 in the solvent CDCl3), 7.57-7.62 (dd, J = 7.6 and 1.7 Hz, H-1), 8.23 (s, 1H, imine-H), 8.5 (s, 1H,-OH) ppm (For details, see Supporting Information). Preparation of AOT inverted micellar (IM) solution. A 0.3 M AOT stock was prepared by


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dissolving AOT in n-heptane solution. About 44.0 µL of s-RHG (23.0 µM) or PMP (46.0 µM) in 2.0 mM cacodylate-HCl buffer, pH 5.2 was mixed with 956 µL stock to obtain AOT IM with the water to AOT ratio (w0) = 8.0 (final concentration: s-RHG, 1.0 and PMP, 2.0 µM). Furthermore, a 1.0 µM s-RHG or 2.0 µM PMP in 2.0 mM cacodylate-HCl buffer, pH 5.2 solution maximally up to 231.0 µL were added stepwise into the same solution to generate AOT IM of different waterpool radii (1.7–9.5 nm) or w0 (8.042.0) so that the concentration of s-RHG/PMP was remain constant (1.0/2.0 µM) among all w0. Preparation of DMPG/DMPC (2:1) large unilamellar vesicles (LUVs). Required amount of DMPG/DMPC (2:1) mixture was dissolved in 1.0 mL chloroform/methanol (5:1) mixed solvent. Thin layer of lipid film was prepared on the wall of the round bottom flask by removing organic solvent with rotary evaporator at 35°C. Any residual amount of organic solvent in thin lipid film was completely removed in vauco for 3 h. For hydration of prepared thin film, 5.0 mM buffer solution with or without the presence of s-RHG (1.0 µM)/PMP (2.0 µM) was added at 45°C. The solution was votex for 2.0 min for complete dissolution of the lipids to form vesicles. Seven cycles of freeze-and-thaw were performed between 196°C and 50°C to produce giant multilamellar vesicles (MLVs). To obtain unilamellar vesicles (LUVs) of defined pore size, the liposome dispersion was extruded 15-31 times depending on the final LUV size (radii: ~ 15, 25, 50 and 100 nm) through two stacked polycarbonate membrane filters (Whatman) with different pore sizes (30, 50, 100 and 200 nm) equipped in a Mini-Extruder system (Avanti Polar Lipid, USA). The temperature throughout the LUVs preparation process before and after lipid film hydration was maintained above 30C. The final particle size distribution was confirmed by DLS measurement. UV-Vis absorption and fluorescence studies. The UV-Vis optical absorption and fluorescence were performed with a UV-2450 spectrophotometer (Shimadzu, Japan) and Perkin Elmer LS-55


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spectro-fluorimeter (Perkin Elmer, USA), respectively. Quartz cells with 1 cm path-lengths were used for absorption and fluorescence measurements. All solutions were filtered through Millipore membrane filter (0.22 µm) before spectroscopic measurements. During temperature variation experiment, each measuring solution was equilibrated at the desired temperature for 5 min. All measurements were repeated at least three times to check the reproducibility. Giant unilamellar vesicles (GUVs) preparation and microscopic observation. A gentle hydration of a lipid film was performed to obtain cell-sized GUVs. Appropriate amount of lipid stock in chloroform/methanol (5:1) was dried in vauco for 3 h to produce the lipid thin film. The lipid film was gently hydrated in absence and presence of s-RHG (0.5 M) with 1.0 mM HEPES, pH 6.0 containing 200 mM sucrose solution at 30°C for overnight. The final concentration of DMPG/DMPC (2:1) mixture in solution was set to 500 µM. Furthermore, the s-RHG (0.5 M) was added into the GUV suspension formed in absence of s-RHG before microscopic measurement. The supernatant GUV solution was used for microscopic observation. A microscopic image was captured using an Olympus IX71 inverted microscope coupled with Hamamatsu Photonics, ORCS-Flash 2.8 CMOS Camera. The temperature of the sample was maintained at 30°C. Binding studies. The DMPG/DMPC (2:1) lipid vesicle (3.0 mM) in 5.0 mM buffer solution (pH 5.2) was prepared in presence of s-RHG (1.0 µM) or PMP (2.0 µm). A 100K MW cut-off centrifugal mini-filter (Amicon Ultra-0.5 mL Centrifugal Filters, Millipore) was used to collect the unbound s-RHG/PMP in the bulk phase. Approximately 200 µL of the filtrate was collected from the initial 400 µL lipid solution after centrifugation for about 2 min at 5000 g. The amount of residual probe in the filtrate was estimated by the UV-Vis absorption spectra, and the amount of unbound s-RHG/PMP was calculated (See Supporting Information for details).


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Scheme 1. Schematic views of solvent (A) pH and (B) polarity dependent interconversion equilibrium between two molecular forms for spiro-rhodamine-glucose derivative (s-RHG) and Schiff base molecule (PMP), respectively. (C) Different amphiphilic self-assembled molecules. (C) Amphiphilic molecules

(A) Interfacial pH sensing probe (s-RHG) OH H

CH2OH OH H

N

N H

O

OH OH

OH OH

O H

N

H

OH H

OH pKa~ 4.35

N H

O

(SDS)

H

N

O O O Na+

N H

O

S

O O

(AOT)

O H

O

(so-RHG) Abs ~ 535 nm; FL ~ 555 nm

(sc-RHG) No visible Abs and FL

O Na+

O

N H

N H

S

CH2OH

O

+

O

O

OH H

O 9

(TX-100)

(B) Interfacial polarity sensing probe (PMP) O

O

Polar solvent N

O

OH

H

Non-polar solvent

N

H

O

OH

(PMP) Abs ~ 420 nm

OH

(Lipid: DMPG)

O O

O

(PMP0)

Abs ~ 330 nm

O 10

N N

O

10

HO

P O O Na+ O

10

O

O

P O O

N

O

(Lipid: DMPC)

10 O

RESULTS Interface interacting pH and polarity sensing probe. It has been reported that the acid induced protonation of colorless/non-fluorescent spiro-rhodamine amide derivatives exhibit a large amount of visible absorption/fluorescence intensity due to cleavage of the spiro-ring.50 The N-center in the spiro-ring accepts proton reversibly in acidic pH with pKa ~ 2.53.0 to revive the loss of conjugation in the rhodamine moiety and the visible absorption/fluorescence intensity (Scheme 1A). Such acid/base equilibria can be exploited for a fluorometric and colorimetric detection of pH. However, to satisfy the interfacial pH (pH(i)) detection mainly for anionic amphiphilic selfassembled systems close to a neutral bulk pH, an interface interacting spiro-rhodamine-glucose derivative (s-RHG) exhibiting a large aqueous solubility (~ 0.1 mM in buffer, pH 7.0) with 11 ACS Paragon Plus Environment

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relatively higher acid/base pKa ~ 4.35 was synthesized (Scheme 1A). Notably, the synergic polarity effect between polar glucose residue and nonpolar spiro-rhodamine moiety probably plays the critical role to interact s-RHG with the water/oil separating self-assembly interfaces. With decreasing pH of the medium, the large amount of visible absorption (extinction coefficient () ~ 6.59.5104 M-1cm-1) and fluorescence (quantum yield (F) ~ 0.60.9) intensity generation due to conversion from its spiro-close (sc-RHG) to spiro-open (so-RHG) species was utilized to monitor pH(i) and its negative pH deviation from the bulk (pH) for amphiphilic self-assembled systems (Scheme 1A, Figure S1). On the other hand, the interfacial polarity ((i)) was estimated utilizing a interface interacting Schiff-base polarity-probe (PMP) as reported previously by us (Scheme 1B).46 The nonionic form (PMP0) of PMP stabilized at low medium polarity () participates an intramolecular ground state proton transfer reaction (GSIPT) from phenolic-OH to imine-N to generate zwitterionic species (PMP) at an increased value of . The ratiometric absorption changeover from 330 to 420 nm with increasing  of the medium for interface interacting PMP was exploited to estimate the (i).46 To identify precise probe localized depth within the anionic water/oil interface, the local environment of the imine proton in s-RHG during its interaction with the self-assembled systems were monitored by

1

H-NMR studies.51,52 The anionic self-assembled system induced a

considerable amount of up-field chemical shift from ~ 7.86 to 7.74 (SDS micelle) or 7.76 (DMPG/DMPC (2:1) LUV) or 7.80 ppm (mixed TX-100/SDS (4:1) micelle) indicates that the sRHG locates in its opposite charge environment (Figure S2S5). As the s-RHG exists mostly as cationic so-RHG species in acidic pH, it involves electrostatic interaction with anionic selfassembly headgroups to locate in the interfacial Stern layer. The interfacial probe location depth may also be assessed from the fluorescence anisotropic decay analysis.53,54 The large increase of 12 ACS Paragon Plus Environment

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rotational correlation time (c) from ~ 1.0 to 3.74.2 ns for s-RHG or ~ 0.8 to 2.83.0 ns for PMP in presence of various anionic self-assembled system (SDS micelle or AOT IM of different AOT IM water-pool radii (1.7–9.5 nm) or DMPG/DMPC (2:1) LUVs) indicates that the s-RHG/PMP participates a strong electrostatic interaction with oppositely changed anionic polar headgroup of the self-assembled system to justify the interfacial Stern layer probe location (Figure S6 and S7, Table S1). However, a relatively small increase of c value from ~ 1.0 to 2.5 ns (s-RHG) or ~ 0.8 to 2.0 ns (PMP) in presence of TX-100/SDS (4:1) micelle shows that the probe is weakly interacted with this micellar headgroup for its interfacial location (Figure S6 and S7, Table S1). To ensure large probe binding affinity to the interface of the self-assemblies, the binding between sRHG/PMP and DMPG/DMPC (2:1) LUVs (radius: 100 nm) were investigated by determining the residual fraction of the unbound probe in the bulk phase. More than 9095% of the s-RHG/PMP molecules were attached to LUVs for the solution containing s-RHG (1.0 µM) or PMP (2.0 M) and DMPG/DMPC (2:1) (4.0 mM, total lipid) LUVs (Figure S8). The result shows that almost all probe molecules were bound to the LUV interface. Detection of interfacial pH (pH(i)) and its deviation from bulk pH (pH) with s-RHG. With increasing concentration of amphiphilic molecule, the gradual optical (absorption/fluorescence) intensity changes at an identical bulk pH for the pH-probe (s-RHG) indicates that more amount of s-RHG change in its location from bulk to interface with adapting a different sc-RHG to so-RHG mole-ratio (Figure S9). The intensity saturation after a certain concentration of amphiphilic molecule justifies that nearly all s-RHG are interacted with the interface. The change in sc-RHG to so-RHG mole-ratio between the bulk and the interface was estimated to obtain pH(i)/pH. Since, only so-RHG species exhibits visible absorption and fluorescence intensities, the moleratio of so-RHG and sc-RHG in their mixture can be obtained directly by judging visible optical 13 ACS Paragon Plus Environment

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Figure 1. Normalized (A) UV-Vis absorption and (B) fluorescence spectra of s-RHG (1.0 M) in presence of different amphiphilic molecules at their intensity-saturated concentrations at pH 4.0 (broken) and 5.0 (solid) in 20 mM citrate/phosphate buffer (except 5.0 mM for LUV): green, SDS (4.0 mM) micelle; pink, DMPG/DMPC (2:1) (3.0 mM, total lipids) LUVs of radius ~ 100 nm; purple, TX-100/SDS (4:1) (5.0 mM, total surfactants) micelle. The spectra in absence of amphiphilic molecules at pH 5.0 (black, solid) and pH 4.0 (black, dotted) are also shown for comparison. Each spectrum is normalized by dividing with the corresponding highest value of extinction coefficient () or fluorescence intensity at pH 3.0 for buffer and TX-100/SDS (4:1) micelle or pH 4.0 for SDS micelle and DMPG/DMPC (2:1) LUV. (C) The mole-ratio of so-RHG (Xso-RHG) are plotted against various bulk pH values in presence and absence of different amphiphilic systems (color code same as A and B) or wt% ethanol containing buffer system (gray, 45 and light gray, 85%). The combined data (absorption method: hollow symbol; fluorescence method: solid symbol) for each system are fitted with a single sigmoidal curve.

intensities (Scheme 1A, Figure 1). The unit mole-ratio of so-RHG is ensured from acid induced no further increase i.e. the saturated 535-nm extinction coefficient (0) or 555-nm fluorescence intensity (F0). Therefore, the pH dependent mole-ratio of so-RHG is represented by the observed optical intensity at a particular pH (FpH or pH) divided with F0 or 0 for identical composition of 14 ACS Paragon Plus Environment

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Table 1. The pKa of so-RHG to sc-RHG interconversion equilibrium and polarity correction factor () for the pH-probe (s-RHG), difference in the interfacial pH from the bulk value (pH), and the interfacial dielectric constant ((i)) in presence of ethanol/buffer or different amphiphilic systems.

System

pKa



pH(i)a

(i)b

Buffer

4.35

-

0.00

78.5c

45% EtOH/buffer

3.85

-

0.00

52.5c

85% EtOH/buffer

3.53

-

0.00

30.0c

SDS

6.11

0.60

2.35

43.5

DMPG/DMPC (2:1)d

5.45

0.52

1.60

50.0

TX-100/SDS (4:1)

5.02

0.50

0.65

53.8

a

Deduced by using eqn. 3.

b

From references (46).

c

Bulk 

d

LUV curvature radius ~ 100 nm.

the medium. The so-RHG mole-ratios at various bulk pH in absence and presence of intensity saturated concentration of amphiphilic molecules were plotted and therefrom self-assembled systems induced the change in acid/base pKa was correlated with pH or pH(i) (Figure 1, Table 1). The anionic self-assembled SDS (4.0 mM) or TX-100/SDS (4:1) (5.0 mM, total surfactant) micelles and DMPG/DMPC (2:1) (4.0 mM, total lipid) LUVs (radius ~ 100 nm) induced the large increase of visible intensity or the increase in pKa for the s-RHG (1.0 µM) suggests that anionic micelles/LUVs interface are more acidic than the bulk phase acidity (Figure 1, Table 1).55,56 Presumably, the interfacial negative charge for an anionic self-assemblies attracts H3O+/H+ ions and at the same time repels OH ions electrostatically causing a higher interfacial H3O+/H+ ion 15 ACS Paragon Plus Environment

Langmuir

Scheme 2. Pictorial representation for the difference in H+/OH– distribution between the anionic interface and the neutral bulk phase. The conversion of one to another molecular species for the pH/polarity-probe from the bulk to the interface location are depicted (pH-probe, s-RHG: colorless/non-fluorescent spiroclose (sc-RHG) to purple/fluorescent spiro-open (so-RHG) form; polarity-probe, PMP: (zwitterionic)

H+ OH– OH– H+ H+ – + OH OH– H OH– OH– + OH– H+ H H+

H+ H+ H+ OH– + H+ H+ – – – – – – H– – – –

Bulk More Acidic Lesspolar

pH sensor

PMP (yellow/fluorescent) to (nonionic) PMP0 (colorless/non-fluorescent) form. Polarity sensor

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

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Interface (anionic)

Hydrocarbon phase

concentration to result more acidic pH(i) than the bulk value pH(b) (Scheme 2). The pH(i) may be different by an amount same with the varying extent of the bulk pH (/pH) induced by the selfassembled systems to obtain identical amount of so-RHG between interface and bulk.45,47 The /pH may be expressed by self-assembled system induced difference in pKa (pKa) owing to unchanged /pH under different bulk pH values (Figure 1C, Table S2). However, for precise measurement of pH(i)/pH, a polarity correction factor () equal to the pKa difference between aqueous medium and mixed solvents (ethanol/buffer) of identical  to that of respective interface needs to be subtracted from the /pH:45 pH = pH(i)  pH(b) = /pH  

(1)

or, pH (i) = pH(b) + /pH  

(2)

pH(i) = pH(b) + pKa(b)  pKa(i)  

(3)

About 0.500.60 unit decrease in pKa was detected due to a decrease in the solvent  from 78.5 (pure aqueous medium) to a value similar with the (i) of SDS micelle (~ 44.0) or DMPG/DMPC (2:1) LUV (~ 50.0) or TX-100/SDS (4:1) micelle (~ 53.0) (Table 1, Figure 1C).46 By using eq. 16 ACS Paragon Plus Environment

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Langmuir

Table 2. The self-assemble curvature radius (r) dependent 420-nm UV-Vis extinction coefficient (w420) of PMP evaluated to estimate (i), polarity correction factor () for the pH-probe (s-RHG), and mole-fraction of so-RGH species (Xso-RGH) obtained from absorption (Abs) and fluorescence (Fl) studies to estimate the pH-deviation from the bulk to the interface (pH).

Self-assembled system

b

AOT

TX-100 /SDS (4:1)c

DMPG /DMPC (2:1)c

r/nm

w420

(i)a

Xso-RHG Abs

Fl



pH

1.7

0.294

28.0

0.041

0.040

0.85

 0.45

2.4

0.344

33.4

0.077

0.075

0.80

 0.67

3.1

0.384

36.5

0.133

0.131

0.74

 0.84

4.0

0.425

40.1

0.186

0.189

0.67

 0.94

5.5

0.455

44.3

0.253

0.251

0.61

 1.02

7.5

0.462

44.8

0.287

0.288

0.60

 1.11

8.8

0.461

45.0

0.324

0.322

0.60

 1.16

9.5

0.460

45.0

0.351

0.350

0.60

 1.22

3.5

0.575

53.6

0.503

0.499

0.50

 0.65

4.5

0.563

53.3

0.501

0.500

0.50

 0.64

7.1

0.560

53.1

0.498

0.497

0.50

 0.63

8.0

0.558

53.2

0.501

0.500

0.50

 0.65

15.0

0.499

47.7

0.431

0.429

0.52

1.40

25.0

0.501

48.2

0.502

0.518

0.55

1.52

50.0

0.518

49.7

0.577

0.575

0.56

1.59

100.0

0.532

50.0

0.579

0.578

0.57

1.60

(i) deduced by using eqn. 4. pH deduced with respect to bulk pH 5.2 by using eqn. 1 and 2. c pH deduced with respect to bulk pH 5.0 (for TX-100/SDS (4:1) micelle) and 5.2 (for DMPG/DMPC (2:1) LUV) by using eqn. 1 and 2. a

b

13, the pH(i) of anionic SDS micelle, TX-100/SDS (4:1) and DMPG/DMPC (2:1) LUVs (radius 17 ACS Paragon Plus Environment

Langmuir

~ 100 nm) are more acidic than pH(b) by an amount ~ −2.35, −0.65 and −1.60, respectively (Table 1). Negative curvature radius dependent pH/(i) for inverted micelles. An inverted micelle (IM) contains negatively curved (concave/inward) interface surrounded by a spherical nanometer size water-pool in a bulk hydrocarbon medium.57-61 The water-pool size dependent curvature radius can be selectively tuned by varying water to surfactant mole-ratio (w0). The AOT IM with the close similar headgroup as that of SDS micelle has been considered to distinguish interfacial pH/polarity between positive and negative curvatures (Scheme 1). Moreover, the AOT molecules can form a stable IM up to a very high w0 (~ 45.0) with maintaining a linear correlation between w0 and water-pool radius.59 To obtain sufficient amount of water molecules located around the interface (interfacial water) in addition to the bulk water away from the interface, the water-pool radius was varied from ~ 1.7 (w0 = 8.0) to 9.5 nm (w0 = 42.0) with 0.3 M AOT in n-heptane

1.0 0.8

45

A

0.4

35 30

0.2 0.0 300

B

40

0.6

i)

10-4 (M-1cm-1)

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

Page 18 of 44

25

350

400 450 500 Wavelength (nm)

550

2.0

4.0

6.0 8.0 radius (nm)

10.0

Figure 2. (A) UV-Vis absorption spectra of PMP (2.0 M) in presence of AOT (0.3 M) inverted micelle (IM) in n-heptane at different water to AOT mole-ratio (w0) dependent water-pool radius using 2 mM buffer, pH 5.2: purple, 1.7; cyan, 2.4; violet, 3.1; brown, 4.0; pink, 5.5; dark yellow, 7.5; blue, 8.8; red, 9.5 nm. The black (broken) spectrum is for 2 mM buffer, pH 5.2 without AOT IM. (B) The interfacial dielectric constant ((i)) are plotted against water-pool radius.


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Langmuir

medium (Table 2).59 The AOT IM radius for each w0 was estimated from DLS measurement. To obtain water-pool radius, the alkyl chain length37 ~ 1.1 nm was further subtracted from the DLS estimated radius. Notably, the high AOT concentration ensures that all s-RHG/PMP (1.0/2.0 M) molecules are interacted with the interface. The difference in ratio’s between the two molecular forms of s-RHG/PMP from AOT/buffer/heptane to bulk buffer medium were estimated to evaluate pH(i)/pH and (i). With increasing radius from 1.7 nm (w0 = 8.0), the 420-nm intensity for the zwitterionic PMP species of PMP was enhanced gradually in expense of 330-nm intensity for the non-ionic form (PMP0) until a saturated intensity at either wavelength was detected after radius from 5.5 nm (w0 = 30) (Figure 2A). The (i) at each w0 or radius value was deduced according to the following linear relation (Table 2).46 420/0420 = 0.44  2.0

(4)

Where,  at 420-nm of measuring solution (420) is normalized by  at the same wavelength for solvent THF (0420). The slope and intercept for the 420/0420 vs  linear plot are represented by 0.44 and –2.0, respectively. The (i) for AOT IM increases sharply from 28.0 maximally up to a similar value of SDS micelle ~ 44.0 with the increase of radius from 1.7 to 5.5 nm or more (Table 2, Figure 2B). On the other hand, the pH(i)/pH at various water-pool radius was evaluated from the equilibrium between sc-RHG and so-RHG molecular forms of s-RHG. In compare with aqueous medium, the value of 0 or F0 for the s-RHG molecule (defined previously), which corresponds to the unit so-RHG mole-ratio, was larger by a factor ~ 1.50 in a solvent of  less than 55.0. Therefore, the 0 or F0 is to be larger by a factor ~ 1.50 when s-RHG change its location from bulk to the AOT IM interface of various (i) ~ 28.045.0 (water-pool radii: 1.79.5 nm) (Figure S10 and S11). The ratio of so-RHG at various water-pool size can be obtained by its size dependent 19 ACS Paragon Plus Environment

Langmuir

Normalized fluorescence

A

0.3

Normalized

0.2

0.1

550 500 Wavelength(nm) -1.2

600

B

0.3

0.2

0.1

0.0 540

0.0

600 570 Wavelength(nm)

630

C

-1.0 pH(i)

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 20 of 44

-0.8 -0.6 -0.4 2.0

4.0

8.0 6.0 radius (nm)

10.0

Figure 3. Normalized (A) UV-Vis absorption and (B) fluorescence spectra of s-RHG (1.0 M) in presence of AOT (0.3 M) IM in n-heptane at different water to AOT mole-ratio (w0) dependent water-pool radius using 2 mM buffer, pH 5.2: purple, 1.7; cyan, 2.4; violet, 3.1; brown, 4.0; pink, 5.5; dark yellow, 7.5; blue, 8.8; red, 9.5 nm. The black (broken) spectrum is for 2 mM buffer, pH 5.2 without AOT IM. (A,B) Each spectrum is normalized by the corresponding highest value of extinction coefficient () or fluorescence intensity in 55% (w/w) ethanol/buffer medium at pH 2.0. (C) Plot for the deviation of interfacial pH from the buffer pH 5.2 against water-pool radius are depicted: (hollow symbols) absorption method and (solid symbols) fluorescence method.

extinction coefficient band intensity (w) or fluorescence band intensity (Fw) divided by 0 or F0 for the buffer/ethanol medium with  ~ 2845.0. On increasing radius from 1.7 to 9.5 nm with a buffer of pH 5.2, the normalized intensity maxima or the mole-ratio of so-RHG species was increased gradually from a value ~ 0.3-fold with respect to that of the buffer medium to ~ 3.5-fold at the highest possible radius ~ 9.5 nm without showing any intensity saturation behavior (Figure


Page 21 of 44

3A and B, Table 2). The increasing so-RHG ratio for larger radius indicates that the interfacial acidity increases with increasing water-pool size or negative curvature radius. To evaluate pH(i)/pH accurately, the polarity correction factor (), i.e., the pKa due to change in  from the buffer to the mixed ethanol/buffer medium of  same as the (i) was determined for each radius (Table 2). About 0.25 unit lowering in the pKa from

variation of (i) from 28 to 45, and subsequently

0.6 0.4

)

0.2

the  value was deduced at each radius (Figure 1C,

0.0 300

Table 2). Since the pKa shift for the s-RHG at

1.0

different radii are difficult to obtain, the eq.1 or 2 is more useful to estimate water-pool radius

A

0.8

-1 -1

-4

 similar as the water-pool radius dependent

10 (M cm

0.85 to 0.60 was detected due to change of solvent

1.0

10-4 (M-1cm-1)

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

Langmuir

350

400 450 Wavelength (nm)

500

550

B

0.8 0.6 0.4

dependent pH(i)/pH. It has been observed that the

0.2

pH changes from ~ 0.45 to 1.22 with

0.0 300

350

400 450 500 Wavelength (nm)

550

increasing water-pool radius from 1.7 to 9.5 nm (Table 2, Figure 3C). In the plot of pH vs waterpool radius, the pH increases more rapidly with

Figure 4. UV-Vis absorption spectra of (A) PMP (2.0 M) in presence of intensitysaturated concentration of TX-100/SDS (4:1) (5.0 mM, total surfactants) micelles at

increasing radius for smaller water-pool radii (~ 1.74.0 nm) compared to that of larger radii (~

different temperatures in 20 mM buffer, pH 5.0: red, 15C; blue, 28C; purple, 40C; violet, 50C and (B) DMPG/DMPC (2:1)

5.59.5 nm) (Figure 3C).

LUV of different radii (3.0 mM, total lipid) in

Effect for the variation of micellar positive curvature radius on pH/(i). To interrelate the pH/(i) with the positive curvature radius, we

5.0 mM buffer, pH 5.2: red, 100 nm; blue, 50 nm; purple, 25 nm; violet, 15 nm. The spectra in absence of amphiphilic molecules (black, broken) are also shown for comparison.


Langmuir

0.6

0.6 fluorescence Normalized fluo. int. Normalized

Normalized

A 0.4

0.2

0.0 500 550 Wavelength(nm)

B 0.4

0.2

0.0 540

600

0.6

Normalized fluoresecnce

Normalized 


630

0.6

C 0.4

0.2

0.0 500

550 Wavelength(nm)

D 0.4

0.2

0.0 540

600

0.6


630

Normalized fluoresecnce

0.6

E Normalized 

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 22 of 44

0.4

0.2

0.0 500 550 Wavelength(nm)

F 0.4

0.2

0.0 540

600


630

Figure 5. Normalized (A,C,E) UV-Vis absorption and (B,D,F) fluorescence spectra of s-RHG (1.0 M) in presence of intensity-saturated concentration of (A,B) TX-100/SDS (4:1) (5.0 mM, total surfactant) micelles at different temperatures in 20 mM buffer, pH 5.0: red, 15C; blue, 28C; purple, 40C; violet, 50C and (C–F) DMPG/DMPC (2:1) (3.0 mM, total lipid) LUV of different radii in 5.0 mM buffer, pH 5.2 at 30C: red, 100 nm; blue, 50 nm; purple, 25 nm; violet, 15 nm. s-RHG were added


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Langmuir

(C,D) before and (E,F) after formation of LUV. (AF) The spectra in absence and presence of TX-100/SDS (4:1) micelle or DMPG/DMPC (2:1) LUV are normalized by dividing with the respective highest value of extinction coefficient or fluorescence intensity in buffer at pH 3.0 for TX-100/SDS (4:1) micelle and 4.0 for DMPG/DMPC (2:1) LUV, respectively, at 28°C. The spectra in absence of amphiphilic molecules (black, broken) are also shown for comparison.

exploited temperature induced size variation of TX-100 micelle as reported previously by Phillies and co-worker.62 However, to avoid the pH detection in a highly acidic bulk pH and improve detection sensitivity, ~ 20% SDS was mixed with TX-100 to obtain SDS-doped TX-100 micelle. In compare with TX-100 micelle without SDS, the TX-100/SDS (4:1) shows an increase of s-RHG acid/base pKa from 3.4 to 5.0 (Figure 1 and S12). Therefore, the effective pH for TX-100/SDS (4:1) micelle would be ~ 0.65 instead of 0.45 for TX-100 on presuming unchanged  parameter between them (Table 2). As the detection sensitivity for a pH-probe is maximum in the bulk pH near to its pKa, we maintained the bulk pH at ~ 5.0 to observe the change in pH due to temperature induced variation of micellar size. The DLS measurement with TX-100/SDS (4:1) micelle have shown that the micelle radius increases from ~ 3.5 to 8.0 nm by increasing temperature from 15 to 50 °C with fairly high polydispersity index (Figure S13). As both pH and (i) depend on specific interfacial depth, temperature dependent

1

H-NMR and transient fluorescence anisotropy

measurement in presence of TX-100/SDS (4:1) micelle were performed to identify temperature induced change of probe location within the micellar interface (Figure S5).51-54 The unaffected upfield  value for the imine-proton in s-RHG (2.0 mM) and c value for s-RHG/PMP for the change of TX-100/SDS (4:1) (5.0 mM) micelle containing buffer temperature (1550 C) indicates that the interfacial probe location depths are similar among different micelle radii (Figure S4S7, Table S1). Nearly unchanged optical intensities for s-RHG/PMP (1.0/2.0 M) between the presence and absence of TX-100/SDS (4:1) (5.0 mM) at bulk pH 5.0 for the increase of temperature from 15 to


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

50°C suggests that the pH/(i) is independent on size or micellar curvature radius (Table 2, Figure 4 and Figure 5A,B). Large unilamellar vesicle (LUV) curvature radius dependent pH/(i). The spherical LUV contains both micelle like positive and IM like negative interfaces, where its curvature radius can be tuned from nm to m range. The size variation of LUVs produces a simultaneous change in both positive and negative radii. To obtain similar negatively charged LUV interface as that of cellular membrane, the DMPG, an extensively studied model for negative membranes, was mixed with DMPC in a 2:1 mole-ratio in order to obtain highly stable LUVs of different sizes. The lipid extrusion technique was utilized to obtain variable LUV size from radius ~ 15 to 100 nm.63,64 The pH/(i) for different LUVs size were estimated to obtain its correlation with interfacial curvature radius of LUVs. A minute ~ 5% increase of the 420-nm UV-Vis intensity for PMP with an increase of DMPG/DMPC (2:1) (4.0 mM, total lipid) radius from ~ 15 to 100 nm at pH 5.2 results only a little increase of (i) from ~ 48.0 to 50.0, respectively (Figure 4B). The LUV size induced such a small change in (i) also suggests a similar interfacial probe location depth for various interfacial curvature radii of LUVs, since the (i) largely depends on various interfacial depths along the water- to oil-exposed surface. Indeed, the LUV size dependent an unaffected 1H-NMR peak position of imine-H for s-RHG and fluorescence anisotropic decay constant (c) for s-RHG/PMP strongly justify our proposition of the unchanged probe (s-RHG/PMP) location depth for different interfacial curvature radii (Figure S4, S6 and S7). To estimate LUV curvature radius dependent change in pH, the DMPG/DMPC (2:1) LUVs of different radii were generated in s-RHG (1.0 M) containing buffer medium. The 0 or F0 normalized optical intensity maxima (defined previously) representing the so-RHG mole-ratio increases gradually up to ~ 25% in pH 5.2 buffer with an increase of DMPG/DMPC (2:1) (4.0 24 ACS Paragon Plus Environment

Page 24 of 44

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Langmuir

mM, total lipid) LUV curvature radius from 15 to 50 nm and the intensity saturation was identified on further increase of LUV radius up to ~ 100 nm (Figure 5C and D). The results propose that the interfacial acidity can increase with increasing LUV radius up to a certain value and after that the pH(i) or pH is unaffected for any further increase of LUV radius. To estimate the pH according to eqn. 1 or 2, about 0.04 unit decrease in the polarity correction factor () due to increase of (i) from ~ 48.0 to 50.0 for the change in LUV radius from 15 to 50 was considered (Table 2). Such increase of LUV radius exhibits a change in pH from 1.40 to 1.60 (Table 2). However, to discriminate the probe location and subsequently the difference in pH from the positive to the negative LUV interface, the different LUVs of identical size (radius: 15 to 100 nm) were generated in absence of s-RHG which was added after formation of LUVs with an expectation that s-RHG will interact mostly with the positive interface. Interestingly, the addition of s-RHG after formation of LUVs shows only 6% intensity enlargement for s-RHG for the increase of LUV radius (Figure 5E and F), indicating curvature radius induced a minute difference in pH. Therefore, the s-RHG mostly locates only at the positive LUV interface with producing no curvature radius dependent change in pH. To distinguish specific s-RHG location between positive and negative LUV interfaces, fluorescence microscopic observations were performed with the DMPG/DMPC (2:1) GUVs generated by hydration with 1.0 mM HEPES, 200 mM sucrose buffer in pH 6.0 both in presence and absence of s-RHG (0.5 M).65 The appreciable amount of fluorescence intensity generation from the GUVs interface indicates that the non-fluorescent sc-RHG molecular form of the probe (s-RHG) existing mostly in the bulk pH 6.0 converts into fluorophoric so-RHG species by its interaction with the anionic interface (Figure 6A(a,b)). Moreover, the result not only supports that the s-RHG interacted selectively with the interface, but also the anionic DMPG/DMPC (2:1) GUV 25 ACS Paragon Plus Environment

Langmuir

A a

b

B c

d

Normalized fluorescence

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 26 of 44

Cu(ClO4)2/Na2S

a,b

c,d

Length

Figure 6. (A) Fluorescence microscopic observation for DMPG/DMPC (2:1) GUV generated in (a,c) presence and (b,d) absence of s-RHG (0.5 M) in 1 mM HEPES-NaOH, 200 mM sucrose buffer, pH 6.0; (b,d) s-RHG (0.5 M) was added in the buffer medium after formation of GUV. The fluorescence were monitored in (a,b) absence and (c,d) presence of fluorescence quencher Cu(ClO4)2/Na2S (1:2 mM). The excitation and emission slit-width are identical throughout all observation. White bar represents 5 M. (B) Relative fluorescence intensities for the selected lengths (yellow bar in A) along the LUV diameter are plotted. The intensities at the center of GUVs are normalized to an identical intensity value.

interface as similar to the LUV interface is more acidic than that of bulk medium. In case of lipid hydration without s-RHG, an equal concentration of s-RHG (0.5 M) was added after formation of GUVs to maintain identical s-RHG concentration during microscopic observation for two different lipid hydrations processes. Irrespective of different probe addition methods, a homogeneous distribution of fluorescence intensity at the surface of GUVs was detected (Figure 6A(a,b)). However, the fluorescence quenching studies have shown that the intensity was quenched completely by the addition of Cu(ClO4)2 (1.0 mM) followed by Na2S (2.0 mM) when the probe (s-RHG) was added after formation of GUVs (Figure 6A(d)). The result indicates that sRHG locates mostly at the positive interface of GUVs, since the ionic quencher is only effective to quench the fluorescence intensity of the positive (outer) interface localized s-RHG. Evidently, 26 ACS Paragon Plus Environment

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the penetration of s-RHG from the outer buffer medium into the GUV water-pool to contribute its interaction with negative (inner) interface is negligibly small. On the other hand, a partial quenching of fluorescence intensity in presence of same amount of Cu(ClO4)2 (1.0 mM) + Na2S (2.0 mM) for the GUVs formed with s-RHG suggests that the fluoresce intensity originated from both the positive and negative interfaces, where the intensity at the negative interface is probably unaffected in presence of quencher ions (Figure 6A(c)). Indeed, nearly 40% residual intensity justify that the s-RHG is distributed between positive and negative interfaces during lipid hydration in presence of s-RHG (Figure 6B). All these results strongly suggests that the addition of s-RHG after formation of LUVs in buffer provides the pH value for the positive interface, whereas a combined pH for the positive and the negative interfaces can be obtained by generating the LUVs in presence of s-RHG. The LUV radius (1550 nm) dependent the minute optical intensity changes (~ 6%) for the LUVs prepared in absence of s-RHG followed by its addition also justify that pH for positive interface does not change with the variation of its curvature radius. Therefore, when the LUVs prepared by hydrating the lipid film with s-RHG containing buffer followed by extrusion, the curvature radius dependent change in pH at the LUV negative interface preliminary attributes to the overall change in pH from 1.40 to 1.60 (Table 2).

DISCUSSION As the interface of an amphiphilic self-assembled system separates the nonpolar hydrophobic phase from the bulk aqueous phase, a considerable decrease in water concentration at the interface contributes for the large decreases of (i) from that of the bulk value irrespective of difference in curvature geometries (Scheme 3). Moreover, the extensive H-bonding network in the bulk water structure responsible for its large  is greatly distressed when the water molecules localize at the 27 ACS Paragon Plus Environment

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Scheme 3. Schematic representation of curvature radius dependent change in pH-deviation from the bulk to the interface (pH) and interfacial polarity ((i)) for anionic (I) positively and (II) negatively curved interfaces under neutral bulk pH condition. The pH and (i) increases with increasing penetration of water molecules and solvated H+/OH– ions across water/oil separating interfacial Stern layer (yellow) containing anionic headgroups (orange), respectively. The penetration ability depends on the Stern layer headgroup packing (P) or average headgroup surface area (a0) for each surfactant. The water-penetration dependent solvation of anionic polar headgroups are depicted by diffuse blue color.

interface, which may further enlarge the polarity difference from the bulk to the interface.59,67 Unlike the positive interface of TX-100/SDS (4:1) micelle, the pH and (i) for the negative interface of AOT IM were shown to depend on its curvature radius (Figure 26, Table 2). It has also been shown that the pH differs only at the negative interface for a simultaneous change in the positive and the negative curvature radii of DMPG/DMPC (2:1) LUVs (Figure 5, Table 2). Therefore, the nature of interfacial curvature geometry and its magnitude may be important to determine pH and (i). 28 ACS Paragon Plus Environment

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The nature of self-assembly curvature geometry is widely been rationalized and predicted by invoking the surfactant molecule packing parameter (P) defined as V0/a0.l0, where V0 and l0 are the volume and length of surfactant tail, respectively, and a0 is the effective headgroup surface area for each surfactant molecule at the aggregate-solution interface.36,37 As the V0/l0 is constant and independent on tail length, the area a0 will be inversely related with P.37 The increased value of P is associated with a lowering in the solvated headgroup surface area a0 (Table 3 and Scheme 3). It has been shown that highly flexible headgroup packing (P < 0.3) for a positively curved interface of oil-in-water microemulsion with a similar curvature property of micelles or exterior of vesicles can increase with increasing curvature radius until unity for infinitely large radius (Table 3).36 However, very tight headgroup packing (P > 3.0) for a low radius (~ 1.3 nm) of water-in-oil microemulsion with a similar curvature of IM or interior of vesicles was shown to decrease maximally up to unity with increasing radius (Table 3). From the correlation between P and area a0, it has been identified that the area a0 increases from ~ 19 to 51 Å2 with increasing negative curvature radius from 1.3 to 10.5 nm for a typical double tail surfactant in water-in-oil microemulsion compatible with the AOT IM system (Table 3). Although the change in majority area a0 was recognized up to radius ~ 5.0 nm.36,68 On the other hand, for a single tail surfactant forming positive interface, such as TX-100/SDS (4:1) micelle, the minimum possible area a0 is not much lower compare to the largest area a0 for negative interface with double tail surfactant.36 Therefore, the headgroup packing in TX-100/SDS (4:1) micellar interface for different radii are considered to be highly flexible in nature (Scheme 3). Conversely, the tight headgroup packing for the negative interface of AOT IM with low area a0 restricts the water penetration into the interfacial Stern layer from the bulk water-pool (Scheme 3). Consequently, an inadequate water solvation of headgroup with decreasing (i) should observe. With increasing negative curvature radius of AOT


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Table 3. Comparison of theoretically calculated curvature radius (r), water-exposed headgroup surface area (a0) and surfactant packing parameter (P) for microemulsion drops for single and double trail surfactants.a The observation for increasing () or unchanging () nature of pH/(i) due to increase of IM/LUV negative or LUV positive curvature radius from that of the theoretically calculated value are shown.

Surfactant nature

r/nmb

a0/Å2

P

Variation of (i)c

Variation of pHc

Doubletail

 1.3

19.3

3.10





 2.7

33.4

1.80





 5.0

43.0

1.40





 10.5

51.0

1.18





 47.9

58.0

1.04





+ 27.6

64.0

0.94





+ 21.1

69.5

0.86





+ 6.2

80.0

0.74

-

-

+ 4.8

88.0

0.68

-

-

+ 1.2

126.0

0.24

-

-

+ 1.5

105.0

0.28

-

-

+ 1.8

88.0

0.34

-

-

+ 2.9

56.0

0.52

-

-

+ 16.5

35.0

0.90

-

-

Singletail

a

Comparison of microemulsion drop parameters are taken from reference (36).

b

Negative radii correspond to inverted drops. Radii are labeled by signs for the conveniences.

c

Experimentally achievable radii region are mentioned.

IM, the expected increase in area a0 associated with lower inter-headgroup overlap may enhance 30 ACS Paragon Plus Environment

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the water penetrating ability to justify the increase of (i) from ~ 28 to 45 with increasing negative interfacial curvature radius (Figure 2, Table 2 and 3, Scheme 3B). Presumably, the small change in area a0 within 5.5 to 9.5 nm does not influence significantly on the interfacial water penetrating ability, which justifies the saturation in (i) ~ 45 (Figure 2B). On the other hand, the area a0 for the TX-100/SDS (4:1) micelle within radii 3.5–8.5 nm may be large enough to produce curvature radius dependent no changes in interfacial water penetration or (i) value (Figure 4A). Similarly, the unchanged water penetration among various DMPG/DMPC (2:1) LUV radii is interpreted from curvature radius dependent almost constant (i) value (Figure 4B). Therefore, in contrary to the positive interface, negative (convex) curvature bending is essential to influence the interfacial water concentration dependent (i) up to certain increased value of its curvature radius (Table 2 and 3). Notably, the close similarity between the largest value of (i) (~ 45) for the negative interface of AOT IM (radius > 5.5 nm) and the (i) (~ 44) for the positive interface of SDS micelle (radius ~ 2.0 nm) may incidentally similar possibly owing to close similar Stern layer composition for two self-assembles (Table 1 and 2). The change in area a0 for surfactant headgroup with self-assembly curvature radius may also play crucial role to dictates the overall interfacial H+/OH– ion concentration or the pH (Scheme 3). As mentioned before, the strong electrostatic interaction between charged headgroups in the Stern layer and solvated H+/OH– near to them generates a difference in H+/OH– distribution at the interface than the bulk phase (Scheme 2). In compare to positive micellar interface, the smaller area a0 due to close headgroup packing for negatively curved AOT IM may restrict to allow the solvated H+/OH– to interact with anionic sulphonate headgroups electrostatically. Moreover, it can also possible that the electrostatic interaction for the solvated H+/OH– species due to its large water solvation periphery will be hindered more compared to the penetration of free water molecule in 31 ACS Paragon Plus Environment

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the Stern layer (Scheme 3). As a result, the negative curvature radius induced a reduced electrostatic interaction between H+/OH– and IM headgroup may be responsible for small difference in H+/OH– from the bulk to the interface or low pH value (–0.45 to –1.22) compared to that of positive interface of SDS micelle (pH ~ –2.35) with similar headgroup composition of Stern layer (Figure 3C, Table 2). With increasing AOT IM water-pool radius, the larger solvent exposed headgroup area a0 is more effective for interfacial penetration of solvated H+ or move away of solvated OH– by enhancing its electrostatic attractive/repulsive interaction with the anionic Stern layer to develop the increasing interfacial H+ to OH– ratio compared to that of the bulk medium (Scheme 3, Table 3). Therefore, a gradual increasing magnitude of pH was detected with increasing AOT IM curvature radius (Figure 3C). However, in contrary to the curvature radius dependent (i) variation, no saturation behavior in pH was found for entire AOT IM curvature radii (Figure 2B and 3C). A comparatively large increase in pH ~ –0.47 from –0.45 to –1.02 with increasing AOT IM radius within 1.7–5.5 nm followed by only a small increase ~ –0.2 unit for further increase up to the maximum possible radius ~ 9.5 nm were identified (Table 2). Notably, the curvature radius range 1.7–5.5 nm for the larger pH increasing phase is closely similar to that for the entire change in (i) (Table 2). As the penetration of solvated H+ species increases both of the interfacial water and H+ concentration, the curvature radius induced a simultaneous large change in pH and (i) were identified for AOT IM within radii 1.7–5.5 nm (Table 3). Although the water penetration dependent (i) remains unaffected for radius > 5.5 (Figure 2B), the increasing pH trend for further increasing AOT IM radius > 5.5 nm suggests that the penetration of solvated H+ due to its large solvent periphery is affected considerably even by the small change in the area a0 within radii 5.5–9.5 nm36,55 (Table 3 and Scheme 3). Perhaps, the solvated water molecules and the AOT counter cations surrounded by anionic sulphonate headgroup are 32 ACS Paragon Plus Environment

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exchanged by the electrostatic penetration of water solvated H+ species in such a fashion so that the effective Stern layer water concentration and (i) remain unaffected for radius > 5.5. No saturation trend in pH up to the highest possible radius ~ 9.5 for AOT IM also predicts that the solvated H+ penetration dependent more increase in pH magnitude is to be expected if further increase of curvature radius is possible. However, the unchanged pH value with variation of positive curvature radius of TX-100/SDS (4:1) micelle suggests that the headgroup area a0 for each radius is large enough to influence the curvature radius induced similar solvated H+ penetrating ability (Figure 5A and B, Table 3). Indeed, in spite of both positive and negative curvature radii are affected by the DMPG/DMPC (2:1) LUV variation from 15 to 50 nm, only negative interface is responsible for the change in pH from 1.40 to 1.60 (Figure 5C–F, Table 2). However, the constant value of pH for more increase of LUV radius from 50 to 100 nm suggests that the negative curvature radius induced solvated H+ penetration ability can effectively change up to certain increased radius value, and beyond that it becomes saturated to exhibit a similar value as that of the positive interfaces (Table 3). All these results justify the fact that the pH or (i) can susceptible to change with a variation of self-assembly curvature radius at the negative interface up to its certain increased value, then the pH/(i) value between the positive and the negative interfaces will be similar to each other (Scheme 3). It is relevant to mention here that the curvature radius induced change in pH/(i) is monitored for various self-assembled systems with different interfacial curvature geometry to identify relative change in pH/(i) with a variation of the curvature radius irrespective of specific self-assembly dependent its absolute magnitude.

CONCLUSIONS We demonstrate a simple method to detect positive and/or negative curvature radii dependent 33 ACS Paragon Plus Environment

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interfacial pH/polarity for different amphiphilic self-assemblies utilizing interface interacting pH/polarity sensing molecules. The change in probe location from the bulk buffer to the Stern layer of interface and corresponding difference in UV-Vis absorption and fluorometric response for the probe molecule provide the pH deviation from the bulk to the interface (pH) or interfacial dielectric constant ((i)) values. The negative pH and (i) increase with increasing of negative curvature radius of AOT inverted micellar (IM) water-pool, while those parameters remain unchanged with the variation of positive TX-100/SDS (4:1) micellar interface. The nature of curvature geometry and its radius value dependent water and solvated H+ penetration abilities into the Stern layer are considered to explain the curvature radius dependent pH and (i) differences between micellar positive and IM negative interfaces. On the other hand, the change in negative membrane curvature in the DMPG/DMPC (2:1) unilamellar phospholipid vesicles is mostly responsible for the observed difference in pH during simultaneous variation of positive as well as the negative curvature radii for the vesicle. The simplicity of the detection methodology for different model systems may also be highly effective for real-time pH measurement of various complex biological interfaces. However, a suitable probe designing to estimate pH under physiological bulk pH as well as to eliminate the effect of inhomogeneous distribution of the probe molecules within cellular membrane interface is essential. For this, the synthesis of interfacial probe operable under neutral bulk pH with comprising of pH-induced multiple interconvertible optical intensities in the visible region is in progress. With this probe, pH along the different locations of cellular membrane interface with various curvature strain can be mapped from the ratio between optical band intensities, without knowing actual distribution of probe molecules at the heterogeneous membrane interface.


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ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI:

AUTHOR INFORMATION *Corresponding author Partha Pratim Parui, PhD E-mail: [email protected]. Fax: +91-33-24146223, Phone:+91-9433490492 Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENT This work was supported by Research Projects (SB/FT/CS-089/2013) (PPP), UGC (ERO) MRP (PSW-196/13-14) (AR), UGC (ERO) MRP (PSW-197/13-14) (SD). RM and YS acknowledges UGC for JRF fellowship. Authors acknowledges Dr Kazuma Yasuhara (NAIST, Japan) and Nayim Sepai (JU, India) for the microscopic experiment and NMR data interpretation, respectively.

ABBREVIATIONS AOT, Sodium bis-2-ethylhexyl-sulfosuccinate; SDS, Sodium dodecyl sulphate; Polyethylene glycol tert-octylphenyl ether or titron X-100, TX-100; Sodium 1,2-dimyristoyl-sn-glycero-3phosphorylglycerol, DMPG; 1,2-dimyristoyl-sn-glycero-3-phosphocholine, DMPC; spirorhodamine-glucose

derivative,

(s-RHG);

2-((2-(pyridine-2-yl)ethylimino)methyl)-6-

(hydroxymethyl)-4-methylphenol, PMP; 2-hydroxy-3-(hydroxymethyl)-5-methylbenzaldehyde,


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HHMB; 2-(2-aminoethyl)-pyridine, AEP; Large unilamellar vesicle, LUV; Gaint unilamellar vesicle, GUV; Inverted micelle, IM; Molar extinction coefficient, ; spiro-close form of s-RHG, sc-RHG; spiro-open form of s-RHG, so-RHG; Nonionic form of PMP, PMP0; Zwitterionic form of PMP, PMP; pH-induced largest extinction coefficient or fluorescence intensity at max, 0 or F0; pH dependent extinction coefficient or fluorescence intensity at, pH or FpH; Interfacial pH, pH(i); Bulk pH, pH(b); The deviation of pH from the bulk to interface, pH; Dielectric constant, ; Interfacial dielectric constant, (i); Polarity correction factor, ; [water]/[surfactant] mole-ratio, w0; Water-pool size dependent  or F, w or Fw. References (1) Fischer, T.; Lu, L.; Haigler, H. T.; Langen, R. Annexin B12 is a sensor of membrane curvature and undergoes major curvature-dependent structural changes J. biol. Chem. 2007, 282, 9996. (2) Bigay, J.; Antonny, B. Curvature Lipid packing, and electrostatics of membrane organelles: defining cellular territories in determining specificity Dev. Cell 2012, 23, 886. (3) Petrov, A. G. Electricity and mechanics of biomembrane systems: Flexoelectricity in living membranes Anal. Chim. Acta. 2006, 568, 70. (4) Tsukamoto, M.; Kuroda, K.; Ramamoorthy, A.; Yasuhara, K. Modulation of raft domains in a lipid bilayer by boundary-active curcumin Chem. Commun. 2014, 50, 3427. (5) McMahon, H. T.; Gallop, J. L. Membrane curvature and mechanisms of dynamic cell membrane remodeling Nature 2005, 438, 590. (6) Brown, W. J.; Chambers, K.; Doody, A. Phospholipase A2 (PLA2) enzymes in membrane trafficking: mediators of membrane shape and function Traffic 2003, 4, 214.


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(17) Fertuck, H. C.; Salpeter, M. M. Localization of acetylcholine receptor by 125I-labeled aBungarotoxin binding at mouse motor endplates Proc. Natl. Acad. Sci. USA 1974, 71, 1376. (18) Hallock, K. J.; Lee, D. K.; Ramamoorthy, A. MSI-78, An analogue of the magainin antimicrobial peptides, disrupts lipid bilayer structure via positive curvature strain Biophys. J 2003, 84, 3052. (19) Hallock, K. J.; Lee, D. K.; Omnaas, J.; Mosberg, H. I.; Ramamoorthy, A. Membrane composition determines pardaxin’s mechanism of lipid bilayer disruption Biophys. J 2002, 83, 1004. (20) Matsuzaki, K.; Sugishita, K. I.; Harada, M.; Fujii, N.; Miyajima, K. Interactions of an antimicrobial peptide, magainin 2, with outer and inner membranes of Gram-negative bacteria Biochim. Biophys. Acta 1997, 1327, 119. (21) Sciacca, M. F. M.; Lolicato, F.; Mauro, G. D.; Milardi, D.; D’Urso, L.; Satriano, C.; Ramamoorthy, A.; Rosa, C. L. The role of cholesterol in driving IAPP-membrane interactions Biophys. J 2016, 111, 140. (22) Sciacca, M. F. M.; Milardi, D.; Messina, G. M. L.; Marletta, G.; Brender, J. R.; Ramamoorthy, A.; Rosa, C. L. Cations as switches of amyloid-mediated membrane disruption mechanisms: Calcium and IAPP Biophys. J 2013, 104, 173. (23) Epand, R. M. Fusion peptides and the mechanism of viral fusion, Biochim. Biophys. Acta 2003, 1614, 116. (24) Kasson, P. M.; Pande, V. S. Control of membrane fusion mechanism by lipid Composition: predictions from ensemble molecular dynamics, PLOS Comput. Biol 2007, 3, 2228.


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(51) Gaidamauskas, E.; Cleaver, D. P., Chatterjee, P. B.; Crans, D. C. Effect of micellar and reverse micellar interface on solute location: 2,6-pyridinedicarboxylate in CTAB micelles and CTAB and AOT reverse micelles Langmuir 2010, 26, 13153. (52) Lorenz, B. B.; Crans, D. C.; Johnson, M. D. Electron-transfer rate enhancements in nanosized waterpools Eur. J. Inorg. Chem. 2014, 27, 4537. (53) Lakowicz, J. R. in Principles of Fluorescence Spectroscopy Springer, 3rd edn, 2006, ch. 11. (54) Sonu; Kumari, S.; Saha, S. K. Solvation dynamics and rotational relaxation of coumarin 153 in mixed micelles of Triton X-100 and cationic gemini surfactants: effect of composition and spacer chain length of gemini surfactants Phys. Chem. Chem. Phys. 2016, 18, 1551. (55) Crans, D. C.; Schoeberl, S.; Gaidamauskas, E.; Baruah, B.; Roess, D. A. Antidiabetic vanadium compound and membrane interfaces: interface-facilitated metal complex hydrolysis J. Biol. Inorg. Chem. 2011, 16, 961. (56) Sripradite, J.; Miller, S. A.; Johnson, M. D.; Tongraar, A.; Crans, D. C. How interfaces affect the acidity of the anilinium ion Chem. Eur. J. 2016, 22, 3873. (57) Levinger, E. N.; Rubenstrunk, C. L.; Baruah, B.; Crans, C. D. Acidification of reverse micellar nanodroplets by atmospheric pressure CO2 J. Am. Chem. Soc. 2011, 133, 7205. (58) Kitchens, L. C.; Bossev, P. D.; Roberts, B. C. Solvent effects on AOT reverse micelles in liquid and compressed alkanes investigated by neutron spin-echo spectroscopy J. Phys. Chem. B 2006, 110, 20392. (59) Moilanen, E. D.; Fenn, E. E.; Wong, D.; Fayer, D. M. Water dynamics in large and small reverse micelles: From two ensembles to collective behavior J. Chem. Phys. 2009, 131, 014704.


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TOC Detection of Curvature Radius Dependent Interfacial pH/polarity for Amphiphilic Selfassemblies: Positive vs Negative Curvature

Yeasmin Sarakar, Rini Majumder, Sanju Das, Ambarish Ray, and Partha Pratim Parui*

H+-penetration: pHb+ = pHa+ > pHb– > pHa– H2O-penetration: (i)b+ = (i)a+ = (i)b– > (i)a– − OH− H+ OH

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b

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H+

ACS Paragon Plus Environment

H+ OH− H+ + OH+ − H H

H+ H+ H+

polarity for

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