Double-Tailed Cystine Derivatives as Novel Substitutes of

Article pubs.acs.org/JPCB

Double-Tailed Cystine Derivatives as Novel Substitutes of Phospholipids with Special Reference to Liposomes Ravi Bhattarai,† Tanushree Sutradhar,† Biplab Roy,† Pritam Guha,† Priyam Chettri,† Amit Kumar Mandal,‡ Alexey G. Bykov,∥ Alexander V. Akentiev,∥ Boris A. Noskov,∥ and Amiya Kumar Panda*,§ †

Department of Chemistry, University of North Bengal, Darjeeling 734013, West Bengal, India Department of Microbiology and §Department of Chemistry and Chemical Technology, Vidyasagar University, Midnapore 721102, West Bengal, India ∥ Department of Colloid Chemistry, St. Petersburg State University, Universitetskii pr. 26, 198504 St. Petersburg, Russia ‡

S Supporting Information *

ABSTRACT: Cystine-based gemini surfactants with dodecyl, tetradecyl, hexadecyl, and octadecyl hydrocarbon chains were synthesized, and their interactions with unsaturated (soy phosphatidylcholine, SPC)/saturated (hydrogenated SPC, HSPC) soy phosphatidylcholines in the forms of a monolayer and a model liposome were estimated for different combinations of the components in the mixed systems. Studies of Langmuir monolayers at the air−aqueous buffer interface revealed condensation of the monomolecular films with the addition of surfactants. The effect of surfactants decreased according to the following order: octadecyl > hexadecyl > tetradecyl > dodecyl homologs. The nonideal mixing between the components was estimated using the deviation of the experimental molecular area from the ideal area per molecule. The excess molecular area increased with the increase in the surfactant chain length and phospholipid saturation. The 50 mol % mixture of cystine derivatives and phospholipids formed thermodynamically stable monolayers. The surfactants increased the rigidity of SPC monolayers and decreased that of HSPC monolayers, as observed by the studies of surface dialational rheology. The film structure at the air− water interface could differentiate the SPC- and HSPC-comprising systems through the formation of organized regions, especially at a higher surface pressure. The constriction of surfactant/phospholipid hybrid vesicles was observed with an increase in the length of surfactant hydrocarbon chains. The negative zeta potential of vesicles took the highest values and did not change with time for 20 and 50 mol % surfactant. The spherical shape of the vesicles was confirmed by transmission electron microscopy. Differential scanning calorimetry revealed an increase in fluidity of HSPC bilayers and rigidity of SPS bilayers under the influence of surfactants. These effects were confirmed by fluorescence spectroscopy. All of the vesicle formulations were found to be nontoxic from the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide assay, suggesting their potential as a novel membranous system for the delivery of drugs, genetic materials, vaccines, and other therapeutic agents.

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INTRODUCTION Amphiphilic surfactants possess the ability to form selfassembled structures, namely, micelles, vesicles, liquid crystals, and microemulsions in aqueous medium depending on their structure and concentration.1−3 This is a reason why surfactants found various applications in the fields of food processing, pharmaceutical formulation, and other relevant areas such as paint, cosmetic, and textile industries.1,4,5 Among various classes of surfactants, gemini or dimeric surfactants have attracted special attention in recent years.6 They contain hydrophilic head and hydrophobic tail groups connected by a flexible or rigid spacer at or near the head-group region. Their attractive features include a lower critical micelle concentration and an enhanced surface activity as compared to those of the © 2016 American Chemical Society

conventional monomeric surfactants. The dimeric surfactants have also been explored as carriers for drug and genetic materials.5 Gemini surfactants derived from amino acids are proven to have superior properties, namely, biocompatibility, biodegradability, low toxicity, and efficient drug delivery, and are used as transfection agents.1,4,6−8 The structure of amino acid based gemini surfactants with relatively long hydrocarbon chains is similar to that of the vesicle-forming phospholipids. Therefore, these surfactants are capable of forming vesicular aggregates Received: June 24, 2016 Revised: September 21, 2016 Published: September 22, 2016 10744

DOI: 10.1021/acs.jpcb.6b06413 J. Phys. Chem. B 2016, 120, 10744−10756

Article

The Journal of Physical Chemistry B

usually biomembranes or mimetic systems are composed of lipid mixtures rather than individual pure lipids, as the mixed lipid systems are superior in terms of the biophysical functionality. Apart from the limited functionality, use of pure phospholipids will also be expensive. Hence, in the present study, we preferred to use a naturally occurring phospholipid mixture, soy lecithin. To understand the impact of hydrocarbon unsaturation on the membrane rigidity and fluidity, hydrogenated soy phosphatidylcholine (HSPC) was used for comparison. At the same time, to the best of our knowledge, there is no detailed information in the literature on the interactions of anionic cystine-based gemini surfactants with phospholipids in Langmuir monolayers and on the corresponding hybrid vesicles. The present study is directed to the evaluation of the physicochemical properties of hybrid vesicles prepared from cystine-derived gemini surfactants with different chain lengths (12, 14, 16, and 18) and two phosphocholines, soy phosphatidylcholine (SPC) and HSPC at different molar ratios. Furthermore, pure and mixed surfactant/phospholipid monolayers at the air−phosphate buffer interface were studied using a Langmuir balance in an attempt to gain insight into the interactions between the monolayer components. Cholesterol (30 mol %; with respect to the total surfactant) was used in combination with the phospholipids. Cholesterol, a principal sterol in mammalian cell membranes, modulates the fluidity/ rigidity of membranes.23,24 The type of mixing (ideal or nonideal), film compressibility, and the associated thermodynamic parameters of the interaction were evaluated by measurements of the surface properties. Studies of the surface dialational rheology of the monomolecular films were carried out to determine the surface elasticity for different component combinations and at different surface pressures. The morphology of monomolecular films was investigated by Brewster angle microscopy (BAM). The vesicles were formulated by careful mixing of the surfactants and phospholipids, and their hydrodynamic diameter (dh), zeta potential (Z.P.), and polydispersity index (PDI) values were determined by dynamic light scattering (DLS). The effects of the investigated surfactants on the thermotropic behavior of hybrid vesicles were examined by differential scanning calorimetry (DSC). The polarity and anisotropy of the palisade layer of the vesicles were determined by fluorescence spectroscopy using 7-hydroxycoumarin (7-HC) as a molecular probe. The results helped in the understanding of the head-group packing of the surfactants in vesicles and elucidated the effects of the hydrocarbon chain length of cystine-based surfactants on the properties of mixed vesicles and the interactions of synthesized surfactants with biomimetic membranes. Studies using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay were performed to evaluate the cytotoxicity of the formulations. The obtained results help in the selection of optimal compositions of mixed vesicles and can be used in the delivery of drugs, genetic materials, vaccines, and other therapeutic agents.

spontaneously upon exposure to excess aqueous environment.8−10 Yoshimura et al. reported the formation of vesicles in the case of the cystine-derived N-alkylamine gemini surfactant having a dodecyl hydrocarbon chain.8 At the same time, the formation of vesicles was not observed for octyl and decyl surfactant systems. Likewise, Fan et al. also observed the formation of vesicles of the cystine-based amide dimeric surfactant having an alkyl chain length of 12 carbon atoms.1 Liposomes are widely recognized as efficient delivery systems for drugs, genetic materials, vaccines, and other therapeutic agents due to their capacity to withhold both hydrophilic and hydrophobic agents to model biomembranes and also due to their biocompatibility, biodegradability, and low toxicity.11−13 Reports on the vesicles comprising naturally occurring phospholipids are not uncommon in literature.14,15 However, the medical application of such phospholipid-based vesicles, also known as liposomes, is seriously limited by their physicochemical instability, leading to a short shelf life of the systems due to oxidative degradation.7 According to the report of Ryan et al., the insertion and activity manipulation of membrane proteins are difficult due to the viscosity of the membrane bilayer prepared from naturally occurring phospholipids. A spongelike low-viscosity model membrane bilayer prepared from nonionic surfactants can significantly activate the transmembrane proteins like bacteriorhodopsin and sarcoplasmic reticulum Ca2+ATPase and its associated enzymes in the surfactant phase.16 The topological changes are possible using a judicious mixture of oppositely charged ionic surfactants in the presence of added salt.17 Formation of an ultrathin membrane bilayer by long-chain organic ion-pair amphiphiles, dodecyltrimethylammonium cation and perfluorooctanoate anion, has been reported by Chaban et al., whereby the aquaporin protein was incorporated with an aim to use such a system for water filtration.18 Faustino et al. synthesized and studied the properties of anionic urea-based gemini surfactants derived from cystine, belonging to the category of N-carbamoylamino acids, containing octyl hydrocarbon chains.4 The same group has also reported the mixed micelle formation in aqueous solution with similar surfactants and two phospholipids, 1,2-diheptanoyl-sn-glycero-3-phosphocholine and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) using conductivity measurements.2 However, to the best of our knowledge, the physicochemical characterization of vesicles on the basis of cystine-derived gemini surfactants (having longer hydrocarbon chains, greater than 12 carbon atoms) and studies on the effects of these surfactants on the physicochemistry of biomimetic membranes are not common in the literature. In the light of the aforementioned issues, the amino acid based surfactants are considered to act as a potential substitute of the classical phospholipid-derived vesicles. The lipid monolayers are considered to be effective models for studying the liposomal drug carriers because the bilayers are approximately a superimposition of two monolayers.19,20 One can characterize the physicochemical properties of lipid bilayers by the thermodynamic parameters obtained from the studies of Langmuir monolayers at the air−water interface.19,21 The lipid monolayer−bilayer models are believed to simulate the interaction of exogenous compounds with the biomembranes.20 The interaction studies of arginine-derived gemini surfactants with dipalmitoylphosphatidylcholine and DMPC monolayers have been reported by Lozano et al.,22 with special emphasis on the surfactant antimicrobial activity. It is worth mentioning that

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EXPERIMENTAL SECTION Materials. L-Cystine (C6H12N2O4S2) was purchased from Sisco Research Laboratories Pvt. Ltd., India. Dodecyl isocyanate (C 13 H 25 NO, 99%), tetradecyl isocyanate (C15H29NO, 97%), hexadecyl isocyanate (C17H33NO, 97%), octadecyl isocyanate (C19H37NO, 98%), sodium hydroxide (NaOH), hydrogenated soy lecithin (HSPC), (3β)-cholest-510745


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CO2−), 1466 (νs CO2−), 1405 (carboxylate), 720 (νs C−S), 613 (νs S−S). The observations were comparable with the previously published reports,2,4,5 thus confirming the formation of the derivatives. Preparation of Vesicles. Hybrid small unilamellar vesicles of phosphatidylcholines and gemini surfactants derived from cystine were prepared by the thin film hydration technique.25,26 Quantitative amounts of lipids and surfactants were dissolved in the mixture of chloroform/methanol (3:1, v/v). The solvents were then evaporated using a rotary evaporator to form a thin film in the round-bottom flask, which was kept overnight under vacuum for complete drying. Phosphate-buffered saline (PBS) (10 mM, pH 7.4) containing 150 mM sodium chloride was added to the resultant dried film followed by vigorous agitation at 70 °C for 1 h. The obtained dispersions were then subjected to six cycles of freeze−thaw sonication to form small unilamellar vesicles. The final concentration of the vesicle dispersion was maintained at 0.5 mM. Cholesterol equivalent to 30 mol % (with respect to the total amount of lipids and surfactants) was added in each composition. The concentrations of the vesicles were diluted to 10−4 M for DLS studies. Instrumentation. Langmuir Monolayer Studies. The surface pressure (π)−area (A) isotherms were recorded using a Langmuir Balance (Micro Trough X; Kibron, Finland) with a trough and barriers made up of Teflon and enclosed inside a Plexiglas box to avoid errors due to the air flow and dust particles. PBS solution (10 mM, pH 7.4) was used as a subphase. The trough was cleaned using a micropipette aspirator before starting the experiment. Solutions at different molar ratios were prepared in the chloroform/methanol (3:1, v/v) mixture to obtain a final concentration of 0.5 mM. The required amount of the solution was carefully spread on the surface of the aqueous subphase using a Hamilton microsyringe. The solvent was allowed to evaporate for 20 min prior to compression. The lateral compression speed of the barriers was set at 5 mm/min. The compositions of phospholipids (SPC/HSPC) and different cystine-based surfactants having dodecyl, tetradecyl, hexadecyl, and octadecyl chains were varied in the molar ratio of 10:0, 8:2, 6:4, 5:5, 4:6, 2:8, and 0:10 for the studies of the monolayers, in which 30 mol % cholesterol was incorporated. The experiments were conducted at a subphase temperature of 25 ± 0.5 °C. Each set of experiments was carried out at least twice to ensure the reproducibility of the results. Dilational Surface Rheology. The dynamic dilational surface elasticity was measured by the oscillating barrier method using the instrument supplied by KSV NIMA (Finland).27,28 The liquid in the Langmuir Teflon trough had a surface area of 75 × 15 cm2. The instrument was equipped with two Teflon barriers oscillating symmetrically at a given frequency and amplitude at the position chosen by the operator. The induced oscillations of the surface tension were measured by the Wilhelmy plate method. The plate was made from filter paper, had a width of 1 cm, and was positioned parallel to the barriers in the middle of the trough. All of the measurements were performed at a constant amplitude of area oscillations (±2%) and at a frequency of 0.1 Hz. If harmonic deformations of the surface area are small enough (the region of linear viscoelasticity), the induced surface tension oscillations are also sinusoidal. In this case, the complex dynamic dilational surface elasticity, E, can be defined in the following way

en-3-ol (cholesterol, CHOL), and 7-HC were procured from Sigma Aldrich Chemicals. SPC was a product of Calbiochem, Germany. The reagents used for the preparation of buffer solutions (disodium hydrogen phosphate (Na2HPO4·2H2O), sodium dihydrogen phosphate (NaH2PO4·2H2O), and sodium chloride (NaCl)) along with HPLC-grade solvents, that is, tetrahydrofuran (THF), chloroform, and methanol were purchased from Merck Specialities, India. All of the reagents of analytical grade having purity of ≥99.0%, unless otherwise stated, were used as received. Double-distilled water with a specific conductance of 2−4 μS (at 25 °C) was used for the preparation of aqueous solutions. Methods. Synthesis and Characterization of CystineBased Gemini Surfactants. The dimeric surfactants derived from cystine with alkyl chain lengths of 12, 14, 16, and 18 were synthesized by the methods proposed by Faustino et al.2,4,5 The compounds were synthesized by the condensation reaction between sodium salt of L-cystine and alkyl isocyanates of varying chain lengths (dodecyl, tetradecyl, hexadecyl, and octadecyl). The sodium salt of cystine was prepared by dissolving 0.01 mol of cystine in appropriate volume of 1 M sodium hydroxide solution to ensure complete neutralization of amino acid. After that, the alkyl isocyanate solutions, which had been prepared by dissolving stoichiometric amount of alkyl isocyanates (corresponding to 0.02 mol) in 10 mL of THF under stirring for 1 h, were separately added dropwise to the solution of sodium L-cystine. The reaction mixtures were then left overnight. The precipitated surfactants were collected by filtration and recrystallized using THF/water mixture. The synthesized surfactants were characterized using Fourier transform infrared (FT-IR) spectrometer (Spectrum RX I; PerkinElmer Inc.). The mixtures of different surfactants and potassium bromide (KBr) were prepared with a ratio of 1:5. These mixtures were subjected to KBr press to form pellets, which were then scanned in the range of 4000−400 cm−1. The 1 H NMR and FT-IR spectra of the cystine derivatives are presented in Figures S1 and S2, respectively (Supporting Information). Cystinedidodecyl derivative (Cys 12): Yield: 77.6%, mp: 192 °C. 1H NMR: δ (ppm) 0.88 (t, 6H, 2 × CH3), 1.25 (br s, 36H, 2 × CH3(CH2)9), 1.47 (m, 4H, 2 × CH2CH2NH), 3.09 (m, 4H, 2 × CH2NH), 3.13 (m, 4H, 2 × CH2S), 4.47 (q, 2H, 2 × CH). FT-IR (νmax, cm−1): 3342 (NH), 2919, 2842 (CH), 1612 (CO urea), 1577 (νass CO2−), 1462 (νs CO2−), 1366 (carboxylate), 720 (νs C−S), 612 (νs S−S). Cystineditetradecyl derivative (Cys 14): Yield: 72.8%, mp: 195 °C. 1H NMR: δ (ppm) 0.83 (t, 6H, 2 × CH3), 1.25 (br s, 44H, 2 × CH3(CH2)11), 1.49 (m, 4H, 2 × CH2CH2NH), 2.94 (m, 4H, 2 × CH2NH), 3.08 (m, 4H, 2 × CH2S), 4.28 (q, 2H, 2 × CH). FT-IR (νmax, cm−1): 3338 (NH), 2919, 2848 (CH), 1617 (CO urea), 1576 (νass CO2−), 1468 (νs CO2−), 1370 (carboxylate), 721 (νs C−S), 613 (νs S−S). Cystinedihexadecyl derivative (Cys 16): Yield: 67.5%, mp: 197 °C. 1H NMR: δ (ppm) 0.83 (t, 6H, 2 × CH3), 1.27 (br s, 52H, 2 × CH3(CH2)13), 1.45 (m, 4H, 2 × CH2CH2NH), 3.09 (m, 4H, 2 × CH2NH), 3.12 (m, 4H, 2 × CH2S), 4.46 (q, 2H, 2 × CH). FT-IR (νmax, cm−1): 3338 (NH), 2921, 2845 (CH), 1618 (CO urea), 1575 (νass CO2−), 1461 (νs CO2−), 1400 (carboxylate), 720 (νs C−S), 612 (νs S−S). Cystinedioctadecyl derivative (Cys 18): Yield: 75.8%, mp: 201 °C. 1H NMR: δ (ppm) 0.83 (t, 6H, 2 × CH3), 1.27 (br s, 60H, 2 × CH3(CH2)15), 1.45 (m, 4H, 2 × CH2CH2NH), 3.09 (m, 4H, 2 × CH2NH), 3.12 (m, 4H, 2 × CH2S), 4.23 (q, 2H, 2 × CH). FT-IR (νmax, cm−1): 3338 (NH), 2921, 2845 (CH), 1618 (CO urea), 1576 (νass 10746


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The Journal of Physical Chemistry B E(ω) = E′(ω) + iE″(ω) =

d γ (t ) d ln A(t )

mixture with a ratio of 1:100. The emission spectra were recorded in the 380−580 nm range by exciting the probe at 335 nm. The fluorescence anisotropy value (r) was determined by the following equation31

(1)

where γ is the surface tension, A is the surface area, ω is the angular frequency, and E′ and E″ are the real and imaginary parts of the dynamic surface elasticity, respectively. BAM. The morphology of monolayers was investigated in situ by Brewster angle microscope BAM1 (NFT, Göttingen, Germany) with a spatial resolution of 4 μM and equipped with a 10 mW He−Ne laser. DLS Studies. The hydrodynamic diameter (dh), PDI, and Z.P. of vesicles were measured by DLS using a Zetasizer Nano ZS90 (ZEN3690; Malvern Instruments Ltd., U.K.). A He−Ne laser of 632.8 nm wavelength was used, and the data were collected at a scattering angle of 90°. The instrument actually measures the translational diffusion coefficient (DT), from which the hydrodynamic radius of vesicles was determined using the Einstein−Stokes equation29 dh =

kBT 6πηDT

r=

IVV − G ·IVH IVV + 2GIVH

(3)

where IVV is the parallel polarized and IVH is the perpendicular polarized fluorescence intensities and G is the monochromator grating correction factor. Cytotoxicity Studies. Selection of Human Subjects for Collection of Lymphocytes. For separation of lymphocytes, healthy human subjects (n = 3) were selected to obtain a blood sample. In this study, the subjects, who were enrolled, were from the same geographical area. They were all asymptomatic, and none of them was abnormal or under medication, including antioxidant supplementation. The blood samples were collected satisfying the method of Hudson and Hay.32 The blood sample (5 mL) was diluted using PBS (1:1) and layered onto Histopaque 1077 (Sigma) using a Pasteur pipette and centrifuged at 400g for 40 min at ambient temperature, and the peripheral blood mononuclear cells (PBMCs) were collected as described previously.33 After the treatment schedule of PBMCs with various cystine derivatives (Cys 12 + HSPC, Cys 12 + SPC, Cys 14 + HSPC, Cys 14 + SPC, Cys 16 + HSPC, Cys 16 + SPC, Cys 18 + HSPC, and Cys 18 + SPC), the PBMCs (2 × 105 cells in each set) were washed with PBS (1×) at least three times. After that, the quantitative estimation for cytotoxicity was carried out using tetrazolium salt, MTT, as described elsewhere.29 The percentage of proliferation was calculated using the following equation

(2)

where kB is the Boltzmann constant, T is the absolute temperature of the dispersion medium, and η is the viscosity of the medium. Z.P. was measured using folded capillary cells. The size, PDI, and Z.P. measurements were carried out at the temperature of 25 ± 0.5 °C. The DLS studies were carried out for three different samples of liposome formulations. The compositions of phospholipids (SPC/HSPC) and four different cystine-based surfactants having dodecyl, tetradecyl, hexadecyl, and octadecyl chains were varied in the molar ratios of 10:0, 8:2, 6:4, and 5:5 for DLS measurements. The DLS result for each sample was an average of four consecutive measurements. Transmission Electron Microscopy (TEM). The morphology of the prepared vesicles was recorded by TEM. One drop of dilute (10−4 M) vesicular dispersion was placed over a Formver carbon-coated 300 mesh copper grid, whereby the excess liquid on the edge of the grid was removed using filter paper. The dried samples were then stained with 1% uranyl acetate, which was then further dried under vacuum at room temperature. The sample loaded on the grid was then dried in air for 20 min and finally analyzed by TEM (H 600, Hitachi, Japan).30 DSC. The thermal behavior of vesicles was assessed by differential scanning calorimeter DSC 1 (STARe; Mettler Toledo, Switzerland). The dried thin films were rehydrated using PBS solution in a 40 μL sealed Al pan. The prepared samples were then scanned two times at a scanning rate of 5 °C/min in the temperature range of −20 to 70 °C.30 PBS solution (pH 7.4) was used in the reference pan. The obtained results were evaluated using STARe software. DSC studies were undertaken for the vesicles of different surfactants at a constant phospholipid/surfactant molar ratio of 8:2 to observe the effects of surfactant chain length and the level of phospholipid saturation on the thermal behavior of vesicles in the same composition. UV−Visible Spectroscopy. The UV−visible spectrophotometer was used to record the absorption spectra of the 7-HCloaded vesicles (UVD-2950; Labomed Inc.). Measurements of the steady-state fluorescence spectra were carried out by a benchtop spectrofluorometer (Quantummaster-40; Photon Technology International Inc., NJ). 7-HC was used as the fluorescent probe because of its solvatochromic sensitivity and preferable residence over the palisade layer of the membrane.30 Spectral measurements were carried out using a lipid and probe

%proliferation = [absorbancesample − absorbancecontrol] × 100/absorbancecontrol

(4)

The average of three sets of experiments was reported. All of the experiments, unless otherwise stated, were carried out at a controlled ambient temperature (25 °C).

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RESULTS AND DISCUSSION Monolayers at the Air−Buffer Interface. Langmuir Monolayer Studies. In an attempt to understand the effects of alkyl chain length on the monolayer behavior of cystine-derived surfactants (along with 30 mol % cholesterol), π−A isotherms of pure cystine derivatives (Cys 12, Cys 14, Cys 16, and Cys 18) at the air−buffer interface were recorded. The results are summarized in Figures 1 and 2. The surface pressure isotherms of surfactant monolayers shifted progressively toward the lower molecular area with the increasing hydrocarbon chain length. Provided the head groups of all of the surfactants were similar, the variation of the isotherms was definitely due to the influence of alkyl chain length. The result could be explained on the basis of increasing extent of hydrophobic and van der Waals interactions between alkyl chains with the increase of their length.34 The isotherms exhibited lift-off area of 103, 100, 95, and 37 Å2 per molecule for Cys 12, Cys 14, Cys 16, and Cys 18 systems, respectively. An abrupt rise in the surface pressure at the molecular area of octadecyl > tetradecyl > dodecyl, due to the obvious rationale, that is, the increase in the hydrophobic/van der Waal interaction with the increase in chain length. Similarly, the higher negative ΔGex values were noted for HSPC-incorporated monolayers compared to those with SPC because of the higher rigidity of saturated HSPC.38 Thus, the results indicate increased intermolecular interactions between HSPC and cystine derivatives in mixed monolayers. Changes in the free energy of mixing (ΔGmix) were calculated to determine the thermodynamic stability of the mixed monolayers using the following equation35 ΔGmix = ΔGex + ΔGid

(8)

ΔGid = RT (x1 ln x1 + x 2 ln x 2)

(9) 10750


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The Journal of Physical Chemistry B almost purely elastic. Therefore, only the data on the elasticity modulus are discussed below. The modulus of the dynamic elasticity of HSPC monolayers increased much faster with surface pressure than in the case of SPC monolayers. At π = 35 mN/m, the elasticity modulus of the HSPC monolayer exceeded almost 2 times the value for the SPC monolayer, indicating a solidlike behavior in the former case. On the contrary, one can consider the SPC monolayer to be more fluidlike with loosely packed SPC molecules at the air−buffer interface. An obvious explanation behind the fact is the unsaturation of SPC, enhancing the fluidity of the monolayers with the addition of SPC. The dynamic elasticity of Cys derivative monolayers, for example, Cys 16 monolayer, is much higher than that of the monolayers of phosphatidylcholines. Therefore, even small additions of phosphatidylcholines to Cys derivatives decrease significantly the modulus of the dynamic surface elasticity, leading to a looser packing of surfactant molecules in the monolayer (Figure 5). This is especially noticeable in the case of SPC additions because the monolayers of this substance exhibit the lowest surface elasticity. The effects of HSPC are weaker especially at low surface pressures (≤15 mN/m). The increase in the length of surfactant hydrocarbon chain increases the dynamic elasticity of the mixed monolayers. This effect is more noticeable in the case of SPC/Cys derivative monolayers (Figure 5D). In the case of HSPC monolayers, the effect of Cys derivatives is weaker and the increase of the surface elasticity with the surfactant chain length becomes obvious only at high surface pressures (>30 mN/m, Figure 5C). In the middle range of surface pressures (15 < π < 30 mN/m), the elasticity of the mixed monolayers can be even lower than the values of pure HSPC monolayers. BAM Studies. BAM studies are considered to provide useful information on the morphology of monomolecular films at the air−water interface. Figure 6 shows some representative BAM images. The surface proves to be inhomogeneous for most systems. The increase in surface pressure results in the appearance of some surface aggregates, which can indicate some twodimensional phase transitions or insufficient miscibility of the components in the monolayer (panels A3, A4, C2−C3, and D2). The formation of surface aggregates occurs mainly in mixed monolayers with HSPC, and only in the mixed monolayers of SPC with Cys 14, one can clearly see the aggregates. The observed tendency is obviously caused by more condensed nature of the mixed monolayers with HSPC as compared to those with SPC. The addition of all Cys derivatives to HSPC monolayers leads to surface aggregation, but in the case of Cys 12, the aggregation proves to be less intensive (panel C1). Studies on Hybrid Vesicles. DLS Studies. The hydrodynamic diameter (dh), PDI, and Z.P. of the hybrid vesicles, prepared by separate mixing of Cys 12, Cys 14, Cys 16, and Cys 18 with SPC and HSPC, respectively, at different mole fractions (xCys: 0.0, 0.2, 0.4, and 0.5) along with 30 mol % cholesterol, were measured with respect to time (up to 100 days). Other combinations with a higher proportion of cystine derivative (xCys > 0.5) were also prepared; however, neither they were substantially stable nor the sizes of the vesicles were comparable with the aforementioned combinations. Variations in the size of different vesicles with time at different compositions are represented in Figures 7 and S7. The sizes of SPC and HSPC vesicles were around 200 and 250 nm,

Figure 6. BAM images for the monolayers of Cys derivative + HSPC and SPC (in the presence of 30 mol % cholesterol). Panels (A) and (B) represent HSPC and SPC, respectively, at mole fraction of Cys derivatives of 0.4 and surface pressures of 0, 10, 20, and 30 mN/m. Panels (C) and (D) represent HSPC and SPC, respectively, for Cys derivatives Cys 12, Cys 14, Cys 16, and Cys 18 at a surface pressure 30 mN/m.

Figure 7. Variation in the size (panel A), PDI (panel B), and Z.P. (panel C) of Cys 18 + SPC vesicles (in the presence of 30 mol % cholesterol) with time. Mole fractions of Cys 18 are indicated in the figure. Temperature is 25 °C.

respectively. A discrepancy in the size of SPC and HSPC vesicles was observed at the addition of different surfactants, and the size of hybrid vesicles depended on the relative content of cystine derivatives. The hydrodynamic diameters of SPC vesicles were smaller than those of HSPC vesicles due to the fluidizing property of SPC molecules. The size of SPC vesicles increased with the addition of surfactants except for the vesicles containing Cys 18 (20 mol %) when the size enhancement was noticed up to 10 days, after which the size constriction occurred throughout the storage period. In one of our previous studies on hybrid vesicles of SPC and ion-pair amphiphile (pseudo-double-tailed amphiphile, isolated from an equimolar mixture of oppositely charged single-tailed surfactants), it was observed that for certain mol %, 10751


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with 20 and 50 mol % surfactant can form stable hybrid vesicles, which is further confirmed by ΔGex and ΔGmix values obtained in the course of monolayer studies. Thereby, it is revealed that cystine-based surfactants have capability to enhance the stability of phospholipid vesicles. TEM Studies. The morphology of vesicles was examined by TEM studies to confirm the existence of bilayer structures. The representative electron micrographs of hybrid Cys 18 + SPC and Cys 18 + HSPC vesicles containing 50 mol % surfactant (along with 30 mol % cholesterol) are demonstrated in Figure 8.

the size was higher; also, for such systems, the size of vesicles decreased with storage time.30 In such cases, the arrangement of the lipidic components in the membrane bilayer initially was not very compact. During the storage, the amphiphile molecules can get reorganized, especially for the less-compact systems, leading to more ordered structures, for which there could occur size constriction. The size of hybrid SPC + Cys derivative vesicles followed the following sequence with respect to the surfactant chain length: octadecyl < hexadecyl < tetradecyl < dodecyl. With the increase in alkyl chain length, the surfactants effectively penetrated into the SPC monolayer as the similarity among the hydrocarbon chains increased.36 Furthermore, the attractive hydrophobic/van der Waals interactions also became stronger with the increase of the alkyl chain length.35 Thus, the packing of lipid bilayer became more compact, which was also supported by the surface pressure−area isotherms, as discussed earlier. In the case of HSPC vesicular systems, the size increment was observed with the incorporation of all of the surfactants (even with Cys 18). A similar trend in the size of vesicles with respect to alkyl chain length was observed for HSPC/surfactant hybrid vesicles. However, the sizes were larger compared to those for mixed SPC/surfactant vesicles, which could be rationalized on the basis that there occurred steric repulsions between the saturated HSPC and surfactant molecules such that the packing arrangement of the molecules is perturbed.35 All of the hybrid vesicular systems were found to be stable in terms of size for the time period up to 100 days. In all of the vesicular formulations, the size increased with the mole percentage of surfactants; the minimum size was recorded for systems with 20 mol % surfactants. The variation of PDI of vesicular dispersions with time is represented in panel B of Figures 7 (along with the size and Z.P. data) and S7. The PDI, an indicator of homogeneity/heterogeneity of the size distribution, was found to be less than 0.4 in all of the cases, thereby signifying the existence of fairly monodispersed vesicles. The PDI values did not vary significantly within the storage period of 100 days. However, the increase of PDI was observed with the addition of surfactants in SPC/HSPC vesicles. This finding suggests that the homogeneity of the size distribution was reduced upon surfactant addition to phospholipid vesicles, presumably due to the formation of micellar aggregates of the surfactants.5 Z.P. determines the stability of colloidal dispersions. The plot of Z.P. of vesicles versus time is graphically presented in panel C of Figures 7 and S7. Z.P. values for pure SPC and HSPC vesicles, although zwitterionic in nature, were found to be negative due to the presence of negatively charged phosphate groups in the head group of phospholipids.39 The magnitude of the negative charge increased with the progressive addition of surfactants in SPC/HSPC vesicles, owing to the presence of carboxylate anion in the head group of cystine derivatives. No significant variations in Z.P. were noticed with the variation of the chain length of surfactants in hybrid vesicles of SPC and HSPC. The magnitude of negative charge on the vesicle surface increased with the increase in the mole percentage of surfactants because of the obvious increase of the carboxylate anion in the dispersion medium. A significant decrease of negative Z.P. was observed with time in the case of pure SPC and HSPC vesicular formulations. However, no significant change in Z.P. for hybrid SPC/HSPC/surfactant vesicles with time was noted, especially with 20 and 50 mol % cystine derivatives, thereby excluding the possibility of vesicle fusion/ aggregation during the storage period. Thus, the formulations

Figure 8. Representative TEM images of (A) Cys 18 + SPC and (B) Cys 18 + HSPC with xCys = 0.5 (in the presence of 30 mol % cholesterol). Scale bar: 200 nm.

Formation of spherical aggregates within the size range of 150−200 nm was observed for hybrid Cys 18 + SPC systems and within the size range of 250−280 nm for hybrid Cys 18 + HSPC systems, thereby verifying the existence of vesicles. The size of the vesicles was found to be quite constricted in comparison to that measured by DLS studies. Such a discrepancy in the observations between the two methods is not uncommon in the literature.17 The observed distinctions can be attributed to the differences in the applied DLS and TEM methods.30 The aqueous dispersions of vesicles are used for the measurements of the hydrodynamic diameter by DLS; however, the dried samples are employed in TEM when water from the hydration skin of the vesicle surface is squeezed out, resulting in the size contraction of the vesicles. DSC Studies. The thermal behavior of the prepared vesicles was scrutinized using DSC studies by monitoring the phase transition temperature (Tm) and the associated thermodynamic parameters, namely, changes in enthalpy (ΔH), peak width at half transition height (ΔT1/2), and heat capacity (ΔCp) values. The effects of the alkyl chain length and phospholipid saturation on the thermotropic behavior of different hybrid vesicles containing 20 mol % cystine derivatives are graphically represented in Figure 9. Different thermodynamic parameters, namely, Tm, ΔH, ΔCp, and ΔT1/2, are presented in Table 1. The DSC thermograms of pure SPC vesicles exhibited two endothermic peaks in the temperature range of −20 to −19 °C (peak a) and 3−6 °C (peak c), respectively; the former one corresponds to the main phase transition temperature (Tm) of the lipid bilayer, and the latter one is the result of heat absorption due to the disruption of hydrated water crystals on the surface of vesicles. The exothermic peak (peak b) was also evidenced in the temperature range of 0−3 °C, which is the result of heat release due to the formation of a water overlayer at the head groups of phospholipids. 10752


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the increased peak “b” in Figure 9, the hydration of the head groups on the surface of the hybrid bilayer also increased with the incorporation of surfactants with the increased alkyl chain length, which can be correlated with the increasing hydrophobicity of head groups.30 The endothermic peak, “c”, which is the result of disorganization of water overlayer, also showed a similar trend as other peaks, indicating the increase in the headgroup packing order with the increase in the alkyl chain length of surfactants.30 A downshift in Tm was observed when the surfactants were incorporated into HSPC vesicles, which is indicative of the fluidizing effect of surfactants on the HSPC bilayer. A similar effect of surfactants was also evidenced in HSPC monolayer as indicated by the surface elasticity values. The steric repulsion between both molecules could increase the acyl chain mobility, thereby increasing the fluidity of the bilayer.41 This result is also corroborated by the increase in ΔT1/2 with the addition of surfactant (Table 1). However, the increase in both ΔH and ΔCp indicates the stability enhancement effect of the surfactants on the lipid bilayer. However, the fluidity exerted by the surfactant on the HSPC bilayer decreased with the increasing alkyl chain length, as indicated by the increase and decrease in ΔTm and ΔT1/2, respectively. Fluorescence Spectroscopic Studies. Fluorescence spectroscopic studies were carried out using 7-HC as a molecular probe with the aim to understand the packing of head groups. 7-HC is well known as a solvatochromic dye and has a great tendency to stay in the palisade layer of the membrane. The emission spectra of 7-HC in the vesicles of different chain lengths of cystine derivatives appeared at 451 nm and are presented in Figure 10. While considering the spectra of 7-HC in the hybrid Cys/ SPC vesicles with cystine derivatives of different chain lengths, it was observed that the fluorescence intensity was decreased. with the exception of some derivatives. In the case of SPC, the fluorescence intensity decreased with increasing chain length except the case of the Cys 18 derivative. For HSPC, the similar trend lines were observed with an exception for the Cys 12 derivative. The results indicated that there was a decrease in rigidity and an increase in the polarity of the membrane interfaces with the increase in chain length of cystine derivatives. This could be explained on the basis of chain mismatch. With increasing chain length, the incorporation of cystine derivatives probably softens the membrane through the breakage of head-group packing. Also, the impacts of SPC- and

Figure 9. DSC thermogram of Cys derivatives in combination with phosphocholines (in the presence of 30 mol % cholesterol). Panels A and B represent Cys with SPC and HSPC, respectively; the hydrocarbon chain length of the cystine derivatives is indicated in the figure. Scan rate: 2 °C min−1.

Similar thermograms of SPC vesicles were also reported by Guha et al.30 In the case of pure HSPC vesicles, a single main phase transition (Tm) of the lipid bilayer was observed at temperature ∼52 °C as reported by Liang et al.40 A noticeable difference in the phase transition temperatures was noted for SPC and HSPC vesicles with the addition of surfactants. Incorporation of cystine derivatives caused the upshift in Tm of SPC vesicles, suggesting the formation of a more rigid bilayer. As the surfactants make the monolayers more rigid, their intercalation within the phospholipid molecules could cause the restrictions of the acyl chain mobility of SPC bilayers. An upshift in the Tm was evidenced with the increase in acyl chain length of the surfactants. Such phenomena could be explained in terms of increased hydrophobicity of longer alkyl chain such that the strength of van der Waal/hydrophobic interaction increases with the chain length, thereby turning the bilayer from less rigid to more rigid states. The result is also demonstrated by the transition width at half peak width (ΔT1/2), which measures the stabilization/destabilization of the phospholipid assemblies. A sequential decrease in ΔT1/2 was noticed in the hybrid bilayer while moving from dodecyl to octadecyl homologs, thereby indicating the enhancement in the cooperativity of the transition. A similar increasing trend in ΔH and ΔCp indicated the increasing extent of the crystallinity of vesicle systems with the increase in the alkyl chain length of surfactants. These results agree with the surface pressure−area isotherms of the corresponding monolayers. As evident from

Table 1. Phase Transition Temperature (Tm), Transition Width at Half Peak Height (ΔT1/2), and Changes in the Enthalpy (ΔH) and Heat Capacity (ΔCp) of Different Phospholipid/Surfactant Hybrid Vesiclesa surfactant systems SPC SPC/Cys 12 SPC/Cys 14 SPC/Cys 16 SPC/Cys 18 HSPC HSPC/Cys 12 HSPC/Cys 14 HSPC/Cys 16 HSPC/Cys 18

Tm (°C) −19.46 −18.91 −18.01 −16.75 −16.16 52.69 49.57 50.87 51.37 51.92

± ± ± ± ± ± ± ± ± ±

0.12 0.36 0.23 0.15 0.18 0.14 0.11 0.12 0.16 0.14

ΔT1/2 (°C) 2.35 2.31 2.0 1.97 1.8 1.78 2.22 2.14 2.03 1.87

± ± ± ± ± ± ± ± ± ±

0.01 0.01 0.02 0.01 0.02 0.04 0.03 0.02 0.01 0.02

ΔH (kcal mol−1) 3.04 9.76 11.54 12.98 15.08 8.92 21.2 25.76 28.92 31.07

± ± ± ± ± ± ± ± ± ±

0.08 0.11 0.07 0.14 0.12 0.19 0.23 0.22 0.32 0.54

ΔCp (kcal mol−1 C−1) 0.65 2.10 2.89 3.29 4.19 2.5 4.77 6.01 7.12 8.31

± ± ± ± ± ± ± ± ± ±

0.04 0.07 0.05 0.08 0.11 0.10 0.14 0.12 0.09 0.17

a

The molar ratio of phospholipid/surfactants is 8/2 in all of the vesicular systems. Cholesterol (30 mol %; with respect to the total lipid concentration) was incorporated in all of the compositions. Scan rate: 2 °C min−1. 10753


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However, the progressive increase of the hydrocarbon chain length of cystine derivatives in combination with HSPC, except Cys 18, results in the rigidity of the packing of hydrocarbon chains, which brought some sort of crystallinity into the bilayer. Such an observation clearly implies that one can appreciably control the physical properties of vesicles through the incorporation of surfactant into the HSPC bilayer. Cytotoxicity of the Vesicles. The cell viability in terms of percentage in respect to the control (without any derivatives) is shown using a radar plot in Figure 12. Cytotoxity studies were Figure 10. Fluorescence spectra of 5 μM 7-HC in the hybrid vesicles of SPC (panel A) and HSPC (panel B) with 30 mol % cholesterol. xCys = 0.5. Excitation wavelength: 335 nm. Chain lengths of the cystine derivatives are indicated in the figure.

HSPC-comprising vesicles on the fluorescence intensities were different. In the case of HSPC-comprising vesicles, the fluorescence intensity followed the following order: tetradecyl > hexadecyl > dodecyl > octadecyl. With the present level of knowledge, the unusual behavior of the Cys 14 derivative for SPC and Cys 12 derivative for HSPC is beyond explanation. Further studies, like fluorescence lifetime measurements, are in progress to address this issue. With the aim to understand the fluidity/rigidity of the palisade layer of the vesicles, fluorescence anisotropy was evaluated as a function of the hydrocarbon chain length of cystine derivatives, as shown in Figure 11.

Figure 12. Dose−response radar plot of various cystine derivatives on human blood lymphocyte.

carried out on day 30 of the sample preparation. The cytotoxicity results obtained from MTT assay clearly demonstrate that the all seven combinations were completely nontoxic toward healthy PBMCs up to the concentration of 0.5 mM (as used for other physicochemical studies). Hence, the formulations could be considered safe in terms of drug administration. However, the stability of the vesicles was not checked in the PBMC media. Further studies on the influence of media, after the cytotoxicity studies, are in progress.

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CONCLUSIONS The effects of hydrocarbon chain length (dodecyl, tetradecyl, hexadecyl, and octadecyl) of cystine-derived gemini surfactants along with their concentration and degree of phospholipid saturation on the molecular interactions between the surfactants and phosphatidylcholines (SPC and HSPC) were investigated using models of biomembranes (lipid monolayers at the air−buffer interface and liposomes). The lipid monolayer studies suggested that the synthesized surfactants exhibited area-condensing effect on the phospholipid films and the extent of condensation increased with the alkyl chain length of surfactants because of stronger hydrophobic interactions exerted by longer hydrocarbon chains. Nonideal mixing behavior between the surfactants and phospholipids was revealed for all of the compositions. The stronger attractive interactions in the mixed films were exhibited by the HSPC system, which is connected with a more densely packed structure in this case. The mixed monolayers with 50 mol % surfactant were found to be the most thermodynamically stable, except Cys 16 + SPC and Cys 18 + SPC systems for which 60

Figure 11. Variation in the fluorescence anisotropy value of 7-HC (r) in hybrid vesicles with the chain length of Cys derivative (Cys 12, Cys 14, Cys 16, Cys 18). The excitation wavelength and emission wavelength were set at 335 and 451 nm, respectively. Temperature was 25 °C. Phosphatidylcholines are indicated in the figure.

The fluorescence anisotropy is a nonmonotonic function of the surfactant chain length with a maximum for the Cys 16 + HSPC system. For SPC-containing vesicles, all of the changes are in error bars. The initial increase in the anisotropy with the chain length for the HSPC-containing systems is probably due to the closer packing of HSPC groups with an increase in the chain length. The lower anisotropy for the Cys 18 + SPC/ HSPC system can be connected with the increase in repulsive forces, causing the breakage of head-group packing. The behavior for the Cys 12 + HSPC system also indicates the unfavorable packing of the components in the mixed system. 10754



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and 40 mol % surfactant yielded the most thermodynamically stable film. As evident from the surface dilational rheology studies, the surfactants showed differences in the impacts of the cystine derivatives among HSPC and SPC monolayers. BAM studies also revealed that in the case of HSPC the formation of domains was significant, whereas in the case of SPC, there was no significant formation of the condensed regions (domains). The DLS studies revealed that the size of hybrid vesicle formulations increased with the incorporation of surfactants; however, the size constriction with increasing hydrocarbon chains of surfactants was evidenced. The magnitude of the negative charge on the vesicle surface increased with the addition of surfactants due to its anionic nature. The high negative Z.P. of vesicular formulations with 20 and 50 mol % cystine derivatives almost did not vary with time, indicating the high stability of vesicles. The flexibility of SPC bilayer was reduced with the addition of surfactants and vice versa in the case of HSPC bilayer. The degree of crystallinity of hybrid vesicles was enhanced as the hydrocarbon chain length of the surfactant was increased. The above findings provide evidence that the cystine-based surfactants have propensity to stabilize the phospholipid monolayer as well as bilayer at appropriate compositions. The MTT assay supports the insignificant toxicity of the formulations. Thus, these surfactants can act as ideal substitutes for naturally occurring phospholipids in the preparation of vesicle formulations for the delivery of drugs, genetic materials, vaccines, and other therapeutic agents.

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REFERENCES

(1) Fan, H.; Han, F.; Liu, Z.; Qin, L.; Li, Z.; Liang, D.; Ke, F.; Huang, J.; Fu, H. Active Control of Surface Properties and Aggregation Behavior in Amino Acid-Based Gemini Surfactant Systems. J. Colloid Interface Sci. 2008, 321, 227−234. (2) Faustino, C. M.; Calado, A. R.; Garcia-Rio, L. Mixed Micelle Formation between Amino Acid-Based Surfactants and Phospholipids. J. Colloid Interface Sci. 2011, 359, 493−498. (3) Kunieda, H.; Masuda, N.; Tsubone, K. Comparison between Phase Behavior of Anionic Dimeric (Gemini-Type) and Monomeric Surfactants in Water and Water-Oil. Langmuir 2000, 16, 6438−6444. (4) Faustino, C. M.; Calado, A. R.; Garcia-Rio, L. Dimeric and Monomeric Surfactants Derived from Sulfur-Containing Amino Acids. J. Colloid Interface Sci. 2010, 351, 472−477. (5) Faustino, C. M.; Calado, A. R.; Garcia-Rio, L. s. Gemini Surfactant-Protein Interactions: Effect of pH, Temperature, and Surfactant Stereochemistry. Biomacromolecules 2009, 10, 2508−2514. (6) Infante, M. R.; Pérez, L.; Pinazo, A.; Clapés, P.; Morán, M. C.; Angelet, M.; García, M. T.; Vinardell, M. P. Amino Acid-Based Surfactants. C. R. Chim. 2004, 7, 583−592. (7) Marques, E. F.; Brito, R. O.; Silva, S. G.; Rodríguez-Borges, J. E.; Vale, M. L. s. d.; Gomes, P.; Araújo, M. J.; Söderman, O. Spontaneous Vesicle Formation in Catanionic Mixtures of Amino Acid-Based Surfactants: Chain Length Symmetry Effects. Langmuir 2008, 24, 11009−11017. (8) Yoshimura, T.; Sakato, A.; Tsuchiya, K.; Ohkubo, T.; Sakai, H.; Abe, M.; Esumi, K. Adsorption and Aggregation Properties of Amino Acid-Based N-Alkyl Cysteine Monomeric and N, N′-Dialkyl Cystine Gemini Surfactants. J. Colloid Interface Sci. 2007, 308, 466−473. (9) Bergsma, M.; Fielden, M. L.; Engberts, J. B. pH-Dependent Aggregation Behavior of a Sugar-Amine Gemini Surfactant in Water: Vesicles, Micelles, and Monolayers of Hexane-1, 6-bis (Hexadecyl-1′Deoxyglucitylamine). J. Colloid Interface Sci. 2001, 243, 491−495. (10) Danino, D.; Talmon, Y.; Zana, R. Alkanediyl- α, ω-bis (dimethylalkylammonium bromide) Surfactants (Dimeric Surfactants). 5. Aggregation and Microstructure in Aqueous Solutions. Langmuir 1995, 11, 1448−1456. (11) Samad, A.; Sultana, Y.; Aqil, M. Liposomal Drug Delivery Systems: An Update Review. Curr. Drug Delivery 2007, 4, 297−305. (12) Sardan, M.; Kilinc, M.; Genc, R.; Tekinay, A. B.; Guler, M. O. Cell Penetrating Peptide Amphiphile Integrated Liposomal Systems for Enhanced Delivery of Anticancer Drugs to Tumor Cells. Faraday Discuss. 2013, 166, 269−283. (13) Sawant, R. R.; Torchilin, V. P. Liposomes as ‘Smart’ Pharmaceutical Nanocarriers. Soft Matter 2010, 6, 4026−4044. (14) Chen, Y.; Wu, Q.; Zhang, Z.; Yuan, L.; Liu, X.; Zhou, L. Preparation of Curcumin-Loaded Liposomes and Evaluation of Their Skin Permeation and Pharmacodynamics. Molecules 2012, 17, 5972− 5987. (15) López-Pinto, J. M.; Gonzalez-Rodriguez, M. L.; Rabasco, A. M. Effect of Cholesterol and Ethanol on Dermal Delivery from DPPC Liposomes. Int. J. Pharm. 2005, 298, 1−12. (16) Rayan, G.; Adrien, V.; Reffay, M.; Picard, M.; Ducruix, A.; Schmutz, M.; Urbach, W.; Taulier, N. Surfactant Bilayers Maintain Transmembrane Protein Activity. Biophys. J. 2014, 107, 1129−1135. (17) Bergström, L. M.; Skoglund, S.; Edwards, K.; Eriksson, J.; Grillo, I. Spontaneous Transformations between Surfactant Bilayers of Different Topologies Observed in Mixtures of Sodium Octyl Sulfate and Hexadecyltrimethylammonium Bromide. Langmuir 2014, 30, 3928−3938. (18) Chaban, V. V.; Verspeek, B.; Khandelia, H. Novel Ultrathin Membranes Composed of Organic Ions. J. Phys. Chem. Lett. 2013, 4, 1216−1220. (19) Feng, S.-s. Interpretation of Mechanochemical Properties of Lipid Bilayer Vesicles from the Equation of State or Pressure-Area Measurement of the Monolayer at the Air-Water or Oil-Water Interface. Langmuir 1999, 15, 998−1010. (20) Karewicz, A.; Bielska, D.; Gzyl-Malcher, B.; Kepczynski, M.; Lach, R.; Nowakowska, M. Interaction of Curcumin with Lipid

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.6b06413. 1 H NMR spectra of cystine derivatives (Figure S1); FTIR spectra of cystine and its derivatives (Figure S2); variation of excess molecular area and excess free energy changes with the mole fraction of cystine derivatives (in combination with HSPC) (Figures S3 and S4); variation of changes in the free energy of mixing with cystine derivative mole fraction in combination with SPC and HSPC (Figures S5 and S6); variation in size, PDI, and ZP of different vesicles with time and at different compositions (Figure S7) (a, Cys 12 + SPC; b, Cys 12 + HSPC; c, Cys 14 + SPC; d, Cys 14 + HSPC; e, Cys 16 + SPC; f, Cys 16 + HSPC and g, Cys 18 + HSPC) (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +919433347210. Fax: +91322275329/297. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The research project was funded by an Indo-Russian Collaborative Research Program, supported by the Department of Science and Technology (DST), Govt. of India, and Russian Foundation of Basic Research RFBR (Project Numbers: INT/ RUS/RFBR/P-220 and 15-53-45043 IND_a). T.S. sincerely acknowledges the DST for a research fellowship. A.K.P. acknowledges the laboratory facility from the Department of Chemistry, University of North Bengal. 10755


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The Journal of Physical Chemistry B Monolayers and Liposomal Bilayers. Colloids Surf., B 2011, 88, 231− 239. (21) Blume, A. A Comparative Study of the Phase Transitions of Phospholipid Bilayers and Monolayers. Biochim. Biophys. Acta, Biomembr. 1979, 557, 32−44. (22) Lozano, N.; Pérez, L.; Pons, R.; Luque-Ortega, J.; FernándezReyes, M.; Rivas, L.; Pinazo, A. Interaction Studies of Diacyl Glycerol Arginine-Based Surfactants with DPPC and DMPC Monolayers, Relation with Antimicrobial Activity. Colloids Surf., A 2008, 319, 196− 203. (23) Dynarowicz-Łątka, P.; Hąc-Wydro, K. Interactions between Phosphatidylcholines and Cholesterol in Monolayers at the Air/Water Interface. Colloids Surf., B 2004, 37, 21−25. (24) Sułkowski, W. W.; Pentak, D.; Nowak, K.; Sułkowska, A. The Influence of Temperature, Cholesterol Content and pH on Liposome Stability. J. Mol. Struct. 2005, 744−747, 737−747. (25) Bangham, A.; Standish, M. M.; Watkins, J. Diffusion of Univalent Ions across the Lamellae of Swollen Phospholipids. J. Mol. Biol. 1965, 13, 238−IN27. (26) Zhu, M.; Li, J.; Fink, A. L. The Association of α-Synuclein with Membranes Affects Bilayer Structure, Stability, and Fibril Formation. J. Biol. Chem. 2003, 278, 40186−40197. (27) Bykov, A. G.; Loglio, G.; Miller, R.; Noskov, B. A. Dilational Surface Elasticity of Monolayers of Charged Polystyrene Nano- and Microparticles at Liquid/Fluid Interfaces. Colloids Surf., A 2015, 485, 42−48. (28) Bykov, A. G.; Noskov, B. A.; Loglio, G.; Lyadinskaya, V. V.; Miller, R. Dilational Surface Elasticity of Spread Monolayers of Polystyrene Microparticles. Soft Matter 2014, 10, 6499−505. (29) Pencer, J.; White, G. F.; Hallett, F. R. Osmotically Induced Shape Changes of Large Unilamellar Vesicles Measured by Dynamic Light Scattering. Biophys. J. 2001, 81, 2716−2728. (30) Guha, P.; Roy, B.; Karmakar, G.; Nahak, P.; Koirala, S.; Sapkota, M.; Misono, T.; Torigoe, K.; Panda, A. K. Ion-Pair Amphiphile: A Neoteric Substitute That Modulates the Physicochemical Properties of Biomimetic Membranes. J. Phys. Chem. B 2015, 119, 4251−4262. (31) Gidwani, A.; Holowka, D.; Baird, B. Fluorescence Anisotropy Measurements of Lipid Order in Plasma Membranes and Lipid Rafts from RBL-2H3 Mast Cells. Biochemistry 2001, 40, 12422−12429. (32) Koirala, S.; Roy, B.; Guha, P.; Bhattarai, R.; Sapkota, M.; Nahak, P.; Karmakar, G.; Mandal, A. K.; Kumar, A.; Panda, A. K. Effect of Double Tailed Cationic Surfactants on the Physicochemical Behavior of Hybrid Vesicles. RSC Adv. 2016, 6, 13786−13796. (33) Mandal, A. K.; Sen, I. K.; Maity, P.; Chattopadhyay, S.; Chakraborty, R.; Roy, S.; Islam, S. S. Structural Elucidation and Biological Studies of a Novel Exopolysaccaride from Klebsiella pneumoniae PB12. Int. J. Biol. Macromol. 2015, 79, 413−22. (34) Albalat, R.; Claret, J.; Ignés-Mullol, J.; Sagués, F.; Morán, C.; Pérez, L.; Clapés, P.; Pinazo, A. Langmuir Monolayers of Diacyl Glycerol Amino Acid-Based Surfactants. Effect of the Substitution Pattern of the Glycerol Backbone. Langmuir 2003, 19, 10878−10884. (35) Ngyugen, H.; McNamee, C. E. Determination and Comparison of How the Chain Number and Chain Length of a Lipid Affects its Interactions with a Phospholipid at an Air/Water Interface. J. Phys. Chem. B 2014, 118, 5901−5912. (36) Angelini, G.; Chiarini, M.; De Maria, P.; Fontana, A.; Gasbarri, C.; Siani, G.; Velluto, D. Characterization of Cationic Liposomes. Influence of the Bilayer Composition on the Kinetics of the Liposome Breakdown. Chem. Phys. Lipids 2011, 164, 680−687. (37) Nakahara, H.; Nakamura, S.; Kawasaki, H.; Shibata, O. Properties of Two-Component Langmuir Monolayer of Single Chain Perfluorinated Carboxylic Acids with Dipalmitoylphosphatidylcholine (DPPC). Colloids Surf., B 2005, 41, 285−298. (38) Broniatowski, M.; Flasiński, M.; Zięba, K.; Miśkowiec, P. Langmuir Monolayer Studies of the Interaction of Monoamphiphilic Pentacyclic Triterpenes with Anionic Mitochondrial and Bacterial Membrane Phospholipids-Searching for the Most Active Terpene. Biochim. Biophys. Acta, Biomembr. 2014, 1838, 2460−2472.

(39) Makino, K.; Yamada, T.; Kimura, M.; Oka, T.; Ohshima, H.; Kondo, T. Temperature-and Ionic Strength-Induced Conformational Changes in the Lipid Head Group Region of Liposomes as Suggested by Zeta Potential Data. Biophys. Chem. 1991, 41, 175−183. (40) Liang, C.-H.; Chou, T.-H. Effect of Chain Length on Physicochemical Properties and Cytotoxicity of Cationic Vesicles Composed of Phosphatidylcholines and Dialkyldimethylammonium Bromides. Chem. Phys. Lipids 2009, 158, 81−90. (41) Jurak, M.; Conde, J. M. Characterization of the Binary Mixed Monolayers of α-Tocopherol with Phospholipids at the Air-Water Interface. Biochim. Biophys. Acta, Biomembr. 2013, 1828, 2410−2418.

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Double-Tailed Cystine Derivatives as Novel Substitutes of

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