Synthesis and Properties of Thioether Spacer Containing Gemini

ARTICLE pubs.acs.org/Langmuir

Synthesis and Properties of Thioether Spacer Containing Gemini Imidazolium Surfactants Avinash Bhadani and Sukhprit Singh* Department of Chemistry, UGC Sponsored-Centre for Advance Studies
I, Guru Nanak Dev University, Amritsar, India

bS Supporting Information ABSTRACT: Twelve new gemini imidazolium surfactants have been synthesized, having dodecyl, tetradecyl, hexadecyl, and octadecyl chain lengths and three different spacers (i.e.,
S
(CH2)n
S
), where n = 2, 3, and 4 and their surface properties have been evaluated by surface tension and conductivity methods. The thermal degradation of these new gemini surfactants was determined by thermogravimetric analysis (TGA). These surfactants have low cmc values as compared to other categories of gemini cationic surfactants and exhibit peculiarities at sufficiently low concentration because they were able to form premicellar aggregates over a wide range of concentration below their cmc values. The DNA binding affinity of these gemini surfactants determined by agarose gel electrophoresis and ethidium bromide exclusion experiments established their strong interaction with DNA, thereby protecting it against enzymatic degradation.

’ INTRODUCTION Gemini surfactants compared with conventional monochain surfactants are more effective at reducing surface tension and forming micelles at relatively low concentration.1,2 Cationic gemini surfactants are an important category of gemini surfactants that contain two positively charged headgroups (i.e., quaternary ammonium, pyridinium, imidazolium, etc.) and two aliphatic chains linked by a rigid or flexible spacer. The conventional quaternary ammonium group bearing gemini surfactants has been extensively studied.3
13 In recent years, several new categories of gemini cationics have been developed and investigated; these include pyridinium-,14
18 imidazolium-,18
21 pyrrolidinium-,22 piperidinium-,23 and amino acid-24
26 based cationic gemini surfactants. An investigation of these novel molecular derivatives has shown some interesting behavioral trends. The cationic gemini surfactants are witnessing ever increasing attention because of their varied applications, such as soft templates for the synthesis of mesoporous materials,27,28 capping agents for the synthesis of nanoparticles29
31 and nanorods,32 and biomedical applications including gene delivery,33,34 drug delivery,35 and antimicrobial activity.36 More than 10 000 international patents on gemini amphiphiles have been filed in the recent past, and investigations for newer costeffective products are in progress.37 Currently, imidazolium ring bearing ionic liquids have been widely investigated because of their inherent nature and possibility of use in several applications.38
41 This fact has attracted surfactant chemists to design and synthesize relatively new categories of cationic surfactants bearing the imidazolium moiety and exploit their self-aggregation behavior for potential applications in several areas.42 The presence of the imidazolium moiety in the surfactant molecule has several advantages as compared to r 2011 American Chemical Society

other gemini surfactants because they demonstrate a stronger tendency toward self-aggregation, owing to the distinct polarizability of imidazolium headgroups. A thorough survey of the recent literature concerning the synthesis and investigation of imidazolium-based surfactants suggests that considerable work has been done in recent years on both monomeric43
50 and gemini imidazolium surfactants18
21,51,52 to establish their properties. More in-depth physicochemical studies on relatively more complex molecular structures are required to establish the set of self-aggregation behavior displayed by this group of gemini surfactants and compare their properties with the other type of gemini cationic surfactants. In the past decade, gemini cationic amphiphiles have found new applications as carriers for therapeutic DNA.53 A biophysical investigation of gemini imidazolium surfactants and their interaction with DNA needs to be conducted because several imidazolium-based cationic amphiphiles have been used as gene-delivery agents.54
56 We have reported the synthesis of gemini pyridinium14,18 and imidazolium amphiphiles18 in the past. In the present work, 12 new gemini imidazolium surfactants having different alkyl chain lengths and spacers (i.e.,
S
(CH2)n
S
, where n = 2, 3, and 4) have been synthesized. These gemini bisimidazolium surfactants have been characterized by NMR, mass spectrometry, IR, and elemental analysis. The surface properties, such as the surface excess concentration (Γcmc), surface area occupied by a molecule at the air/water interface (Amin), efficiency in surface tension reduction by 20 mN 3 m
1 (C20), effectiveness of surface tension reduction (γmin), critical micelle concentration (cmc), degree of Received: June 11, 2011 Revised: October 10, 2011 Published: October 17, 2011 14033

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Scheme 1

counterion binding (β), standard free energy of micellization (ΔG0mic), and adsorption (ΔG0Ads) have been evaluated by surface tension measurements and a conductivity method. Furthermore, the DNA binding capabilities of these surfactants were evaluated by agarose gel electrophoresis and EB exclusion experiments using fluorescence spectroscopy. These gemini surfactants have excellent surface properties and form micelles at very low concentration. The cmc values of these surfactants decrease with increasing spacer length as well as alkyl chain length. The DNA binding capability of gemini surfactants as determined by agarose gel electrophoresis and EB exclusion experiments demonstrate the strong interaction of imidazolium gemini surfactants toward DNA, protecting it against enzymatic degradation by DNase I.

’ EXPERIMENTAL SECTION Materials. 1-Dodecene, 1-tetradecene, 1-hexadecene, 1-octadecene, ethane-1,2-dithiol, propane-1,3-dithiol, butane-1,4-dithiol, and ethidium bromide were purchased from Sigma-Aldrich (U.S.) and used without any purification. N-Bromosuccinimide (NBS) and magnesium chloride were purchased from Central Drug House (New Delhi, India). NMethylimidazole was obtained from Acros. Agarose and Tris buffer were purchased from Sisco Research Laboratory Pvt. Ltd. (Mumbai, India). Plasmid DNA pUC 18 and DNase I were purchased from Bangalore GeNei (Bangalore, India). Millipore water was used in all experiments. Synthesis. Ethane-1,2-dithiol (1; 0.94 g, 10 mmol), propane-1,3dithiol (2; 1.08 g, 10 mmol), or butane-1,4-dithiol (3; 1.22 g, 10 mmol) was slowly added to the stirred suspension of 1-dodecene (4; 3.36 g, 20 mmol)/1-tetradecene (5; 3.93 g, 20 mmol)/1-hexadecene (6; 4.45 g, 20 mmol)/1-octadecene (7; 5.05 g, 20 mmol) and N-bromosuccinimide (8; 3.56 g, 20 mmol) in chloroform (200 mL) at 5 °C under inert conditions (a nitrogen atmosphere). After addition, the reaction was

stirred for 30 min. The progress of the reaction was monitored by thin layer chromatography (silica gel G-coated (0.25-mm-thick) glass plates using hexane/ethyl acetate (98:2) as the mobile phase; the spots were visualized by iodine). The reaction was complete in 30 min in all the cases. Chloroform was removed from the crude reaction mixture under reduced pressure in a rotary flash evaporator (Heidolph Labrota 4000 efficient, Germany) at 40 °C. It was then allowed to cool. The crude reaction mixture was stirred with 60 mL of hexane and filtered to remove the precipitated succinimide. Hexane was removed at 45 °C using a vacuum rotary flash evaporator. The crude reaction mixture was washed with 20 mL of acetonitrile and dried under vacuum at 45 °C. The resulting intermediate compounds containing unreacted α-olefins and 1,2-bis(2-bromoalkylthio)alkane (9
20) were reacted with N-methylimidazole (21; 1.64 g 20 mmol) at 80 °C for 3 h (Scheme 1). The resulting crude mixture was cooled to 20 °C. The product was washed three times with 50 mL of diethyl ether and then cold precipitated in acetone to get gemini imidazolium surfactants (22
33). The structures of all of the products were confirmed by IR, NMR, mass spectra, and elemental analysis. IR spectra were recorded as a thin neat film on a Shimadzu 8400s FT-IR (Kyoto, Japan) instrument. Mass spectra of the surfactants were recorded on a Waters Q-Tof micromass using ESI as an ion source. 1H and 13C NMR spectra were recorded on a 300 MHz JEOL AL-300 FT-NMR (JEOL, Japan) as a solution in CDCl3 using tetramethylsilane (TMS) as an internal standard. Elemental analysis was recorded on a Thermo Electron (U.K.)-made Flash EA 1112 series CHNSO analyzer. TGA/DSC Measurements. Simultaneous measurements of weight change (TGA) and true differential heat flow (DSC) were determined by an SDT Q600 simultaneous TGA/DSC analyzer. Thermograms were recorded using a heating rate of 5 οC/min from 25 to 400 οC. The experiments were carried out on an alumina sample pan by using a nitrogen flow rate of 100 mL/min. The water of hydration and the thermal stability of gemini imidazolium surfactants were determined from a TGA graph whereas the phase-transition temperatures 14034

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Langmuir of gemini surfactants were determined from a simultaneous differential heat flow (DSC) graph.19,57 Conductivity Measurements. Conductivity was measured on an Equip-Tronics auto temperature conductivity meter model EQ661 equipped with a conductivity cell. The solutions were thermostated in the cell at 25 ( 0.1 °C. For the determination of the cmc, an adequate quantity of a concentrated surfactant solution was added in order to change the surfactant concentration from concentrations well below the cmc to up to at least 1 to 2 times the cmc. The degree of counterion binding (β) has been calculated as (1
α), where α = Smicellar/Spremicellar (i.e., ratio of the slope before and after the cmc14,58). Surface Tension Measurements. The cmc and surface tension attained at the cmc were determined using a CSC Du Nouy interfacial tensiometer (Central Scientific Co., Inc., U.S.) with a platinum
iridium ring (circumference 5.992 cm) at 25 ( 0.1 °C. The tensiometer was calibrated using triply distilled water. The surfactant solution was aged for 12 h prior to the determination of surface activity.14,58 Agarose Gel Electrophoresis. pDNA and gemini imidazolium surfactants (22
33) were loaded with 5 μL of glycerol in a 1% Agarose gel containing 2 μL of ethidium bromide (0.5 mg/mL) at N/P charge ratios of 0.5:1, 1:1, 2:1, and 4:1. Electrophoresis was carried out at 100 V in Tris buffer for 30 min. The DNA band was visualized under UV transillumination with an Alpha Imager HP (Alpha Innotech Corporation, U.S.) Photographs were taken using the Alpha Imager.14 Ethidium Bromide Exclusion. A 2 μL solution of 0.25 mM EB was mixed with 3 mL of Millipore water, and the fluorescence spectra of water
EB were recorded in the absence of pDNA and the presence of pDNA from 530 to 700 nm at an excitation wavelength (λex) of 490 nm using a Varian Cary Eclipse fluorescence spectrophotometer. We added 2.5 μL of a 100 μM solution of gemini amphiphiles 10 times to the pDNA
EB intercalated system to get 10 observations. The percentage of quenching observed due to the replacement of EB by cationic gemini amphiphiles from pDNA upon interaction with the cationic amphiphiles was calculated according to (I0
I)/(I0
IEB)  100, where I0 and IEB are the fluorescence intensities of free and pDNA-bound EB and I is the fluorescence intensity in the presence of different amounts of surfactants.59,60 DNase I Sensitivity Assay. pDNA was complexed with varying amounts of cationic gemini surfactants (using the indicated surfactant/ DNA charge ratios in Figure 6). It was treated with 10 μL of DNase I (5 μg/mL) in the presence of 20 mM of MgCl2 and incubated for 10 min at 37 °C. The reaction was then halted by adding EDTA (to a final concentration of 100 mM) and incubating at 60 °C for 20 min in a water bath. Lipids were then extracted from the aqueous DNA sample with 30 μL of phenol/chloroform/isoamyl alcohol (25:24:1 v/v) and centrifuged at 10 000g for 5 min. The aqueous DNA-containing supernatants were separated, and 15 μL was loaded onto a 1% agarose gel (prestained with ethidium bromide); 50% w/v glycerol in H2O was added to each complex, and gel electrophoresis was carried out at 100 V for 25 min in TAE buffer at pH 8 (20 mM tris-acetate, 1 mM EDTA). As a control, the same preparations, treated identically but without DNase I, were submitted to agarose gel electrophoresis under the same conditions as for the treated samples.61
63

’ RESULT AND DISCUSSION Synthesis and Characterization. Gemini imidazolium surfactants have been prepared by the regioselective cobromination of α-olefins (4
7) using N-bromosuccinimide (8) to get reactive intermediates 1,2-bis((2-bromoalkyl)thio)alkane (9
20). N-Bromosuccinimide forms a cyclic bromonium ion at low temperature. Dithiols (1
3), which are strong nucleophiles, attack the less-hindered terminal carbon in a regioselective manner via anti-Markovnikov addition to form reactive

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intermediate β,β0 -dibromoditioethers (9
20). These reactive intermediates are easily quarternized by N-methylimidazole under neat conditions to give the corresponding gemini imidazolium surfactants. The structures of these new gemini imidazolium surfactants have been established by 1H and 13C-DEPT NMR, mass spectrometry, IR spectroscopy, and elemental analysis. The resonance for the protons directly attached to the heterocyclic positively charged imidazolium nitrogen was observed as a broad singlet between δ 3.13 and 3.43. The chemical shifts for methylene protons in between the carbon directly attached to the cationic heterocyclic moiety and sulfur (i.e., imid+CH
CH2
S
) were observed between δ 4.48 and 4.54 for gemini imidazolium surfactants. The protons attached to spacer carbon (i.e.,
S
CH2
) appeared as a multiplet between δ 2.70 and 2.89 for gemini imidazolium surfactants. The signal for the methyl group attached to the imidazolium ring was observed as a singlet between δ 4.12 and 4.14 representing six protons. The signals for imidazolium protons (i.e.,
NCHCHN
) were observed between δ 7.40 and 7.75 as two multiplets for [12-(S-2-S)-12]im, [14-(S-2-S)-14]im, [16-(S-2-S)-16]im, and [18-(S-2-S)-18]im denoting two protons each. This typical behavior has recently been reported for a gemini imidazolium system having short spacers64 and is essentially due to coupling with adjacent protons. This signal for other gemini imidazolium protons appeared as a singlet. The signal for the
NCHN
imidazolium proton appeared in the range of δ 10.17
10.43. These values are typical for
NCHN
protons.65 In the 13C NMR spectra of gemini imidazolium surfactants, the signal for the sp3-hybridized terminal methyl carbon was observed between δ 13.78 and 14.03. The sp3 carbons for
CH2
were observed in the range of δ 22.31 to 32.27, including the carbons of the spacer. The carbon directly attached to the heterocyclic positively charged imidazolium nitrogen was observed at δ 45.67
46.33. The sp2-hybridized carbon directly attached to sulfur and carbon, which in turn is attached to a cationic heterocyclic moiety (i.e., imid+CH
CH2
S
), was observed at δ 53.17
53.96. The carbon of the methyl group attached to the imidazolium ring is observed at δ 36.47
36.83. The carbons of imidazolium rings (i.e.,
NCHCHN
) were observed between δ 122.77 and 123.27, and those of
NCHN
are observed between δ 137.28 to 138.26. The structure of these gemini imidazolium surfactants has been further established by ESI-MS (positive ion) mass spectroscopy. The parent ion peak for gemini surfactants has been observed for the monopositive ion, where the loss of one bromide ion from each molecule led to the formation of positively charged parent ion [M+
Br]. The [M+
Br]+ and [M+
Br]+2 peaks were also observed in each case. Similarly, the base ion peak for gemini surfactants was observed for dipositive molecular ions, lacking both bromides, as observed by the ratio mass/charge (where charge = 2). The spectral details of the mass spectrometry analysis have been summarized in the spectral section of the Supporting Information. TGA/DSC Analysis. All of these gemini imidazolium surfactants have been synthesized as their monohydrate or dihydrate stable salts. The hydrated salts are solid in nature and nonhygroscopic as compared to their nonhydrated derivative, which are hygroscopic in nature. These hydrated salts enable us to determine the surface properties to high accuracy as compared to those for the nonhydrated form because of the accuracy in weighing and in the preparation of their stock solutions. 14035

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Figure 1. (a) TGA/DSC graph of [12-(S-3-S)-12]im and (b) magnified TGA graph of [12-(S-3-S)-12]im indicating the loss in weight due to the loss of water molecules, with the start temperature of degradation and the onset temperature of degradation.

The water of hydration of these gemini surfactants has been determined by thermogravimetric analysis. The observed loss in weight due to the presence of water molecules in the gemini surfactant corresponds to the signal in the 1H NMR having the exact integration for water molecules. The onset and start decomposition temperatures of these gemini imidazolium surfactants have been determined by TGA analysis.66 The onset temperature (Tonset) is the intersection of the baseline weight after the loss of the water of hydration and the tangent of the weight versus temperature curve as decomposition occurs. The start temperature (Tstart) is the temperature at which the decomposition of the surfactant begins. These gemini imidazolium surfactants do not have a sharp melting point, as do most of the typical organic molecules; instead, they undergo a phase transition at a certain temperature. The temperature maximum of the main phase change in DSC run is represented as the phase-transition temperature. Figure 1a shows the simultaneous DSC curve of [12-(S-3-S)-12]im, which undergoes a phase transition at 83.4 °C. No particular trend is observed regarding the phase transition of gemini surfactants; however, all surfactants undergo a phase transition between 73.4 and 88.6 °C. Figure 1b shows the characteristic curve for the decomposition of [12-(S-3-S)-12]im, as measured by TGA. This surfactant starts to degrade at 202.9 °C, shown as Tstart in the figure. However, it undergoes rapid degradation at 241.5 °C, denoted as Tonset in the figure. Similarly, all gemini imidazolium surfactants were evaluated by TGA analysis to determine their thermal stability. [12-(S-2-S)-12]im was found to be the most thermally stable surfactant, having a Tstart of 221.6 °C and a Tonset of 253.3 °C, whereas [14-(S-4-S)-14]im was found to be the least thermally stable surfactant, having a Tstart of 192.3 °C and a Tonset of 231.2 °C among the imidazolium geminis. The Tstart and Tonset temperatures of other gemini imidazolium surfactants lie between the two extreme values. In general, the thermal stability of these gemini imidazolium surfactants decreases with increases in the hydrophobic alkyl chain length from dodecyl to hexadecyl chain units (for a particular spacer length with the exception of [14-(S-4-S)-14]im). However, the last members of the series having octadecyl hydrophobic alkyl chain lengths do not obey

this trend and have higher Tstart and Tonset values. Furthermore, it has been established that the thermal stability of these gemini imidazolium surfactants decreases with increases in the spacer units. This trend is applicable for all series of gemini imidazolium surfactants having similar hydrophobic alkyl chain lengths. The Tstart temperature of gemini imidazolium surfactants having a hydrophobic dodecyl chain length decreases from 221.6 °C for [12-(S-2-S)-12]im to 202.9 °C for [12-(S-3-S)-12]im, which further decreases to 197.3 °C for [12-(S-4-S)-12]im. Similarly, the Tonset temperature for this series decreases in the order [12-(S-2-S)-12]im (Tonset = 253.3 °C) > [12-(S-3-S)-12]im (Tonset = 241.5 °C) > [12-(S-4-S)-12]im (Tonset = 233.7 °C). All three parameters (i.e., the onset temperature of degradation (Tonset), the start temperature of degradation (Tstart), and the phase transition temperature) have been described in the spectral section of the Supporting Information. Surface Tension Measurements. The plot of surface tension versus the log of concentration is shown in Figure 2. The cmc values of these surfactants decreases with the increase in spacer length as well as the hydrophobic alkyl chain length. The surface activity parameters of these surfactants are given in Table 1. The cmc values determined by the surface tension measurement significantly differ from the values observed by the conductivity method; however, the decreasing trend in cmc values with increasing spacer length and hydrophobic alkyl chain length remains the same. Recent report have shown that cmc values may differ significantly when evaluated by two different techniques, depending upon the self-aggregation properties of the surfactant under consideration.17 Considerable peculiarities have been observed at sufficiently low concentration when these geminis were evaluated by surface tension measurements. Variations in readings of the decrease in surface tension for a particular concentration of surfactant solution are often observed even after prolonged aging of the surfactant solution. To avoid such experimental errors, initial readings of every experiment were completely ignored and a set of five successive readings were taken into account for a particular concentration for which the standard deviation was less than 0.1 mN/m
1. The surface activity parameters of these surfactants are given in Table 1. 14036

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Figure 2. Surface tension vs log C plot for (a) the gemini imidazolium surfactants and (b) the gemini imidazolium surfactants.

The maximum surface excess concentration at the air/water interface,5,20,67 Γmax, is calculated by applying the Gibbs adsorption isotherm equation: ! 1 dγ Γmax ¼
ð1Þ 2:30nRT d log C T

Here, γ denotes the surface tension, R is the gas constant, T is the absolute temperature, and C is the surfactant concentration. The value of n is taken to be 2 because recent studies have been carried out by assuming that one counterion is associated with the ionic headgroup.16,17 Further gemini surfactants with flexible spacers with n = 2 have been found to be better with neutron reflectivity.68 However, previous investigations on gemini imidazolium surfactants have been carried by assuming a value of n = 3, considering a divalent surfactant ion and two univalent counterions;20,52 therefore, it becomes essential to calculate the value of Γmax by assuming n to equal 2 as well as 3. The area occupied per surfactant molecule (Amin) at the air
water interface5 is obtained by using following equation Amin ¼

1 Γmax N

ð2Þ

where N is Avogadro’s number and Amin is in nm2. The values of Γmax and Amin are shown in Table 1. The Amin values of these new gemini imidazolium surfactants have been found to be exceptionally low compared to other gemini cationic surfactants,5,17,69,70 including gemini imidazolium surfactants previously reported.20,52 Significantly lower Amin values of the gemini imidazolium surfactant evaluated in the current studies show that these surfactants have a greater tendency to form micelles instead of adsorbing at the air
water interface. Furthermore, both series of gemini imidazolium surfactants bearing dodecyl and tetradecyl chain exhibit different sets of behavior when studied for the change in Amin with respect to increasing spacer length. For series of gemini imidazoliums bearing dodecyl chains, the values of Amin increases with increases in the thioether spacer methylene units; however, the reverse trend is observed in the case of gemini with the tetradecyl chain length. Recent studies have shown that Amin values of gemini imidazolium surfactants of type [Cn-s-Cnim]Br2

increase with increasing alkyl chain length and spacer units;20,52 the same set of results is also evident for the gemini quaternary ammonium surfactant.5 In contrast to these results, there is a continuous decrease in the value of Amin for the series of gemini pyridinium surfactants.17 Initially it was believed that the lower Amin value is due to tighter packing of the longer chains at the interface.67,71 However, recent report by Fisicaro et al. have shown that the lower Amin value may also be due to the greater tendency of surfactant molecules to form premicellar aggregates instead of adsorbing at the air/water interface.17 Thus, lower Amin values can be attributed to either tighter packing of the longer chains at the interface or the tendency of surfactants to form premicellar aggregates in bulk solution. Experimental results of our findings have established lower Amin values (obtained from the surface tension vs log c curves) along with a greater tendency to form premicellar aggregates (obtained from the plot of molar conductivity vs C0.5) by these new gemini imidazolium surfactants. The results are supported by the fact that the size of the hydrophilic headgroup is a dominant factor in the determination of Amin values of the surfactants and the imidazolium cation is solvated to a greater extent in water and therefore is responsible for forming premicellar aggregates.72 The continual formation of premicellar aggregates at low concentration prevents the adsorption of surfactant at the surface. Other important parameters such as γcmc (affinity to reduce the surface tension at the cmc) and C20 (affinity of the surfactant to reduce the surface tension of water by 20 mN m
1) of these gemini surfactants have been calculated from the plot of the decrease in surface tension versus the log of concentration and are shown in Table 1. The γcmc values of these gemini surfactants increase with increases in the length of the spacer units. The Gibbs free energy of micellization (ΔG°mic) is calculated with the following equation20 ΔG°mic ¼ RTð0:5 þ βÞln Xcmc

ð3Þ

where Xcmc is the molar fraction of the cmc and Xcmc = cmc/55.4, where cmc is in mols/L and 55.4 comes from 1 L of water corresponding to 55.4 mols of water at 25 °C. β is the degree of counterion binding to micelles (discussed later). 14037

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42.26 ( 0.22

0.67

0.66

0.65

0.022 ( 0.001

0.021 ( 0.001

0.020 ( 0.001

[16-(S-2-S)-16]im

[16-(S-3-S)-16]im

[16-(S-4-S)-16]im

cmc from conductivity. b cmc from surface tension; β, degree of counterion association; Γ, maximum surface excess concentration; Amin, area per molecule at the interface; C20, surfactant concentration required to reduce the surface tension of the solvent by 20 mN/m; γcmc, surface tension at the cmc; ΔG°mic, Gibbs free energy of micellization; and ΔG°ads, Gibbs free energy of adsorption. The values in parentheses are for n = 3. Surface properties of [18-(S-2-S)-18]im, [18-(S-3-S)-18]im, and [18-(S-4-S)-18]im cannot be evaluated because of their solubility limit in water. a


42.72 ( 0.20


42.49 ( 0.21


42.31 ( 0.23
42.82 ( 0.24


47.80 ( 0.22


33.83 ( 0.21
34.62 ( 0.22 0.52 0.53 0.063 ( 0.002 0.058 ( 0.002 0.085 ( 0.002 0.071 ( 0.002 [14-(S-3-S)-14]im [14-(S-4-S)-14]im

45.8 ( 0.1 46.6 ( 0.1

3.09 ( 0.11 (2.06 ( 0.074) 3.10 ( 0.075 (2.07 ( 0.050)

0.53 ( 0.019 (0.80 ( 0.028) 0.53 ( 0.014 (0.80 ( 0.021)

3.83  10
5 4.10  10
5


47.51 ( 0.22


34.08 ( 0.20 0.54 0.072 ( 0.002 0.10 ( 0.002 [14-(S-2-S)-14]im

42.9 ( 0.1

2.12 ( 0.12 (1.41 ( 0.08)

0.78 ( 0.043 (1.17 ( 0.064)

2.93  10
5


48.21 ( 0.21


32.36 ( 0.20 7.58  10
5 0.58 0.22 ( 0.01 0.31 ( 0.01 [12-(S-4-S)-12]im

40.8 ( 0.1

2.06 ( 0.069 (1.37 ( 0.046)

0.80 ( 0.026 (1.21 ( 0.039)


33.52 ( 0.19


45.57 ( 0.21
33.14 ( 0.19 1.17  10
4 0.63 ( 0.009 (0.95 ( 0.012)

8.74  10
5 2.13 ( 0.096 (1.42 ( 0.064)

2.60 ( 0.036 (1.73 ( 0.024) 39.7 ( 0.1

40.7 ( 0.1 0.63

0.32 ( 0.01

0.26 ( 0.01

0.40 ( 0.01

0.35 ( 0.01

[12-(S-2-S)-12]im

[12-(S-3-S)-12]im

0.63

0.77 ( 0.034 (1.16 ( 0.051)

ΔG°mic kJmol
1 C20 (M) Amin (nm2) 106Γmax (mol 3 m
2) γcmc mN 3 m
1 β cmc (mM)b cmc (mM)a surfactant

Table 1. Surface Properties of Gemini Imidazolium Surfactants as Determined by Surface Tension and Conductivity Measurements

ΔG°ads kJmol
1

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Similarly, the Gibbs free energy of adsorption (ΔG°ads) is calculated with the following equation:73 πcmc ð4Þ ΔG°ads ¼ ΔG°mic
Γ Here, πcmc denotes the surface pressure at the cmc (πcmc = γo
γcmc, where γo and γcmc are the surface tensions of water and the surfactant solution at the cmc, respectively). The free energy of adsorption (ΔG°ads) represents the free energy of transfer of 1 mol of surfactant in solution to the surface, and the free energy of micellization (ΔG°mic) represents the work done to transfer the surfactant molecules from the monomeric form at the surface to the micellar phase.74 The standard free energy of micellization (ΔG°mic) and adsorption (ΔG°ads) is always negative, indicating tendencies to form micelles in solution and to adsorb at the air/ water interface.75 If the value of ΔG°ads is more negative and greater than the difference between ΔG°ads and ΔG°mic, then the adsorption of surfactant molecules at the interface becomes more favorable because of the greater freedom of motion of hydrocarbon chains at the planar air/aqueous solution interface than in the interior of the micelle. However, if the energy difference is small, then less work has to be done to transfer surfactant molecules from the monomeric form at the surface to the micellar phase. When the difference in the free energies is small, the surfactant undergoes aggregation more readily than when the difference in the free energies is large. This is evident from the results obtained by Yoshimura et al.70 The ΔG°mic and ΔG°ads values of gemini imidazolium surfactants are summarized in Table 1. The difference in the freeenergy gap is small for gemini imidazolium surfactants; therefore, these surfactants have a greater tendency to aggregate in solution as compared to other surfactants. As such, these gemini surfactants form premicellar aggregates more readily in water and have low Amin values. An important observation that justifies the trend of increasing Amin values with increasing spacer length in the case of a gemini bearing a hydrophobic dodecyl chain (i.e., [12-(S-2-S)12]im, [12-(S-3-S)-12]im, and [12-(S-4-S)-12]im) can be attributed to the difference in their values of ΔG°mic and ΔG°ads. The difference between the free energies of micellization and adsorption increases with the increase in spacer length in the case of geminis bearing a hydrophobic dodecyl chain length enabling [12-(S-2-S)-12]im to aggregate quickly in water as compared to other analogues of this series. Thus, this surfactant readily transfers to bulk solution from the air/water interface to form premicellar aggregates and has its lowest Amin value in the series. As the difference between the gaps in free energy becomes larger, the affinity to remain at the air/water interface becomes more prominent and the value of Amin increases in this series of surfactants. However, the reverse trend is observed in the case of a gemini imidazolium surfactant bearing hydrophobic tetradecyl chain (i.e., [14-(S-2-S)-14]im, [14-(S-3-S)-14]im, and [14-(S-4-S)-14]im); consequently, the ability to remain at the air/water interface decreases with increasing spacer length. As a result, [14-(S-4-S)-14]im has the lowest Amin value among the series because it is quickly able to undergo a transition from the air/water interface to form micelles. Conductivity Method. The cmc values of gemini imidazolium surfactants have also been evaluated by the conductivity method. The cmc value decreases with increasing spacer length and hydrophobic alkyl chain length. The cmc values of gemini imidazolium surfactants are shown in Table 1. The cmc values of these surfactants are lower than for conventional gemini 14038

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Figure 3. (a
c) Specific conductivity vs concentration plot. (d
f) Molar conductivity vs C0.5 plot of gemini imidazolium surfactants. Left and right arrows denote individual surfactants in d. (e, f) Onset of premicellar aggregate formation, and the concentrations at which the maximum and the cmc are attained, respectively. The error estimate for the calculated onset and maximum value is (1%. (d
f) Individual points shown with error bars represent the mean value ( SEM.

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Figure 4. Agarose gel electrophoresis of pDNA and gemini surfactants at different charge ratios.

imidazolium surfactants20,52 of type [Cn-s-Cnim]Br2 and other categories of gemini cationic surfactants (i.e., gemini pyridinium surfactants15,16 and gemini quaternary ammonium surfactants5 having similar hydrophobic alkyl chain lengths). Unlike the conventional gemini quaternary ammonium surfactant5 of type 12-s-12.2Br, where s is 3, 4, 6, and gemini imidazolium surfactant52 of type C12-s-C12im.2Br, where s = 2, 4, or 6, cmc values of these new gemini surfactants decreases with increasing spacer unit length. Such a pattern of decreasing cmc values with increasing spacer length has been previously observed by Quagliotto et al.15 and Zhao et al.16 for a series of gemini pyridinium surfactants. Another important parameter evaluated by a conductivity plot is the degree of counterion binding (β) that signifies the ability of counterions to bind micelles. The calculated β values of the gemini imidazolium surfactant are shown in Table 1. In our recent studies on gemini pyridinium surfactants,14 we have observed that β values decreases with increasing hydrophobic alkyl chain length. However, in contrast to our previous findings we have found that the β parameter is independent for a series of gemini imidazolium surfactants, and increases in the hydrophobic alkyl chain length and spacer units do not influence the degree of counterion binding (β). The plot of molar conductivity versus C0.5 (Figure 3d
f) gives significant information about the formation of premicellar aggregates. Gemini imidazolium surfactants show peculiar behavior at low concentration. Apart from [14-(S-2-S)-14]im, all gemini imidazolium surfactants were able to form premicellar aggregates at low concentration. The existence of premicellar aggregates at low concentration has been previously reported by several research groups.22,76
78 Left and right arrows denoting each individual surfactant in Figure 3d
f indicate the onset of premicellar aggregate formation, the concentrations at which the maximum and the cmc point are attained, respectively. At the onset point, the concentration of surfactant at the interface remain practically constant; thereafter, there is an increase in the conductance of the solution. At this point, surfactant monomers start to form

premicellar aggregates, and because the conductance of the premicellar aggregate is higher than that of the simple surfactant, they stay in the bulk solution and are not adsorbed at the air
water interface.79 [12-(S-2-S)-12]im, [12-(S-3-S)-12]im, [12-(S-4-S)-12]im, [14-(S-4-S)-14]im, [16-(S-2-S)-16]im, and [16-(S-3-S)-16]im attain their maximum values in the Λ versus C0.5 plot and grow further to form regular micelles. However, [14-(S-4-S)-14]im and [16-(S-4-S)-16]im behave differently because no maximum is observed for these surfactants. Instead, they show fluctuating behavior in the Λ versus C0.5 plot. The premicellar aggregates never attain their maximum in these surfactants because they tend to collapse. The ability to form premicellar aggregates by these surfactants can be attributed to the presence of the thioether functional moiety near the positively charged imidazolium headgroup. The thioether spacer group is relatively polar compared to methylene spacers present in conventional gemini cationic surfactants and significantly influences the aggregation properties of these gemini imidazolium surfactants in aqueous solution. [14-(S-2-S)-14]im is exceptional among the series of gemini being reported because no premicellar aggregates are observed in this case. The low Amin value of this surfactant can be explained on the basis of the tighter packing of the longer chains at the interface.67,71 Agarose Gel Electrophoresis. The DNA binding capability of these gemini imidazolium surfactants has been evaluated by agarose gel electrophoresis (Figure 4). All these gemini imidazolium surfactants were able to completely bind the DNA above an N/P charge ratio of 1:1. (The N/P charge ratio is the ratio between the positively charged nitrogen of the surfactant molecule and the negatively charged phosphates of the DNA molecule.) The DNA binding capability of these gemini imidazoliums is not influenced by the increase in the hydrophobic alkyl chain length or the spacer chain length. This DNA interaction pattern is opposite to the DNA binding behavior of similar groups of surfactants containing pyridinium headgroups.14 Unlike the gemini pyridinium surfactant, where the DNA binding 14040

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Langmuir capability increases with increasing hydrophobic alkyl chain length and the hydrophobic interactions played a significant role in DNA binding, gemini imidazolium surfactants exhibited little dependence on the hydrophobic interactions. It has also been observed that gemini imidazolium surfactants have a greater tendency to bind with DNA as compared to other categories of gemini cationic surfactants. Recent investigations of dodecyl alkyl chain-containing bisquaternary ammonium gemini surfactants and gemini pyridinium surfactant by agarose gel electrophoresis14 have shown that neither surfactant was able to retard the migration of DNA toward the positive electrode at an approximate charge ratio (N/P) of 1:1. Nevertheless, all gemini imidazolium surfactants having a certain dodecyl chain length were able to retard the migration of DNA at similar charge ratios. However, for the series of gemini pyridinium surfactants the hydrophobic interactions come into play with increasing alkyl chain length, and all other geminis are able to effectively bind with DNA at an N/P charge ratio of 1:1. In contrast, all imidazolium geminis exhibit the same capability of DNA binding affinity irrespective of the different alkyl chain lengths. This pattern of behavior has been investigated in further detail by EB exclusion experiments. This unexpected behavior of gemini imidazolium surfactants can be attributed to the greater electrostatic interaction of the positively charged imidazolium headgroup with the negatively charged phosphate of DNA. Figure 4 shows the DNA binding capability of different gemini imidazolium surfactants with DNA at different charge ratios. Ethidium Bromide Exclusion Assay. The DNA binding capability of gemini imidazolium surfactants is evaluated by ethidium bromide (EB) exclusion experiments via fluorescence spectroscopy. The fluorescence intensity of the DNA
EB intercalated complex is enhanced with respect to that of only ethidium ions in solution.80,81 The addition of surfactant solution to the DNA
EB intercalated complex causes a decrease in fluorescence intensity because of the displacement of EB from the DNA
EB intercalated complex and the formation of a new DNA
surfactant complex.82 Figure 5 shows the tendency of different gemini imidazolium surfactants to displace EB from the DNA
EB intercalated complex with increasing N/P charge ratio. The 3D comparative plot of various surfactants provides a significant amount of information about their binding capability. All of the gemini imidazolium surfactants have been found to have an excellent DNA binding capability. The area of the plot has been divided into several colored zones, where the orange region depicts negligible EB displacement when no surfactant has been added. Yellow and green regions of the plot demonstrate low EB displacement whereas sky blue and indigo blue areas show moderate EB displacement. The purple region demonstrates a high EB exclusion region. [12-(S-2-S)-12]im, [12-(S-3-S)12]im, and [12-(S-4-S)-12]im have dodecyl alkyl chains and cause significant decreases in fluorescence intensity at lower N/P charge ratios but were unable to displace EB completely from the DNA
EB intercalated complex at higher charge ratios. The binding of [12-(S-2-S)-12]im, [12-(S-3-S)-12]im, and [12-(S-4-S)12]im to DNA phosphate sites was observed until an N/P charge ratio of 1 to 1.25 was reached, where these surfactants displace about 79
81% of EB from the DNA
EB intercalated complex. Further addition of surfactant solution does not bring about a significant change in the quenching pattern as DNA becomes saturated with surfactants and the exclusion of EB from DNA no longer occurs. [18-(S-2-S)-18]im has greater ability to displace EB than do other gemini surfactants from

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Figure 5. Displacement of ethidium bromide from the pDNA
EB complex by the gemini imidazolium surfactant at different charge ratios.

the DNA
EB intercalated complex because it is able to displace EB completely at an N/P charge ratio of 1.75. All other gemini imidazolium surfactants (i.e., [14-(S-2-S)-14]im, [14-(S-3-S)14]im, [14-(S-4-S)-14]im, [16-(S-2-S)-16]im, [16-(S-3-S)16]im, [16-(S-4-S)-16]im, and [18-(S-4-S)-18]im) completely displace EB from the DNA
EB intercalated complex at an N/P charge ratio of 2.0. DNase I Sensitivity Assay. The association of gemini imidazolium surfactants with pDNA protects plasmids from DNase I digestion, which has been confirmed by the DNase I sensitivity assay. pDNA undergoes enzymatic degradation in the absence of gemini imidazolium surfactants. When plasmid DNA was incubated with DNase I, it was completely digested within 10 min. (The band intensity of naked DNA was no longer visible in Figure 6.) However, the surfactant/DNA complex at N/P 1:1 and 3:1 charge ratios remains unaffected by the DNase I activity. In a typical experiment, the complexed surfactant/DNA system was treated with DNase I, and then the DNA was separated from gemini surfactant and DNase I and subsequently loaded onto a 1% agarose gel. Band intensities of DNA after treatment at 1:1 and 3:1 N/P charge ratios demonstrate the protection of DNA versus DNase I digestion (Figure 6) in the presence of gemini imidazolium surfactants. At a low N/P charge ratio (1:1), a weaker band was observed in all cases, suggesting the weak protection of DNA, whereas at a higher charge ratio (3:1), a relatively stronger band was observed, suggesting greater protection of the complexed system toward DNase I digestion. It can be clearly seen from the results of agarose gel electrophoresis that most of the surfactants are able to retard the migration of DNA at an N/P charge ratio of 1:1. Although the electrostatic interaction of negatively charged DNA with positively charged gemini surfactants is enough to retard the migration of DNA toward the positive electrode, this is not enough to protect the DNA completely from the enzymatic degradation of DNase I. The results of EB exclusion experiments also support 14041

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Figure 6. Electrophoretic gel patterns for DNase I sensitivity assays. N/P charge ratios are indicated at the top of each lane.

these results because none of the gemini surfactants are able to displace ethidium bromide completely at an N/P charge ratio of 1:1 and relatively more surfactant is required to displace the ethidium bromide completely from the DNA
EB intercalated complex. Thus, at a higher N/P charge ratio, the gemini imidazolium surfactants form stable complexes with DNA.

’ CONCLUSIONS In this study, 12 new gemini imidazolium surfactants have been synthesized and characterized by 1H and 13C-DEPT NMR, mass spectrometry, IR spectroscopy, and elemental analysis. The surface properties of these new surfactants have been evaluated by surface tension and conductivity methods. All of these surfactants have been found to have excellent surface properties with low cmc values compared to other gemini cationic surfactants,14
21 including conventional quaternary ammonium gemini surfactants having similar hydrophobic alkyl chain lengths.83 The calculated physical parameters demonstrate the peculiar behavior of the surfactants at low concentration, where the values for Amin have been found to be exceptionally low compared to those for other gemini cationic surfactants. This behavior is due to the tendency of imidazolium gemini cationics

to form premicellar aggregates at sufficiently low concentration below their true cmc values, which is supported by the small energy gap between the Gibbs free energy of micellization (ΔG°mic) and the Gibbs free energy of adsorption (ΔG°ads). The plot of molar conductivity versus the square root of concentration further provided the evidence for the existence of premicellar aggregates at low concentration. The DNA binding capabilities of these surfactants determined by agarose gel electrophoresis and ethidium bromide exclusion experiments by fluorescence spectroscopy established the strong interaction of imidazolium gemini surfactants toward DNA, thereby protecting the DNAsurfactant complex against enzymatic degradation by DNase I. The physicochemical and biophysical evaluation of novel series of gemini imidazolium surfactants have established superior properties of these new surfactants, which can be useful for a wide range of applications, including gene delivery.

’ ASSOCIATED CONTENT

bS

H and 13C-DEPT NMR, mass spectrometry, IR spectral data, elemental analysis data, yields, onset temperature of degradation (Tonset), start temperature of degradation (Tstart), phase-transition temperature, and 1H and

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Supporting Information.

1

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C-DEPT NMR spectra of gemini imidazolium surfactants. Determination of surface parameters of [12-(S-2-S)-12]im from the surface tension graph and Tonset temperature from the TGA graph. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]. Mobile telephone: +919855557324. Fax: +911832258820.

’ ACKNOWLEDGMENT We are thankful to the DST (Department of Science and Technology, Government of India) for providing the research grant (SR/S1/OC-35/2010) for this work and the Sophisticated Analytical Instrumentation Facility (SAIF), Panjab University, Chandigarh, for the mass spectra of the compounds. A.B. is thankful to the DST for the DST-INSPIRE fellowship. We also thank UGC, India, for their UGC CAS program and DST, India, for the FIST program awarded to the department of chemistry, Guru Nanak Dev University, Amritsar. ’ REFERENCES (1) Menger, F. M.; Keiper, J. S. Angew. Chem., Int. Ed. 2000, 39, 1906–1920. (2) Tehrani-Bagha, A. R.; Holmberg, K. Langmuir 2010, 26, 9276. (3) Zana, R.; Benrraou, M.; Rueff, M. Langmuir 1991, 7, 1072. (4) Alami, E.; Levy, H.; Zana, R.; Skoulios, A. Langmuir 1993, 9, 940. (5) Alami, E.; Beinert, G.; Marie, P.; Zana, R. Langmuir 1993, 9, 1465. (6) Frindi, M.; Michels, B.; Levy, H.; Zana, R. Langmuir 1994, 10, 1140. (7) Kern, F.; Lequeux, F.; Zana, R.; Candau, S. J. Langmuir 1994, 10, 1714. (8) Danino, D.; Talmon, Y.; Zana, R. Langmuir 1995, 11, 1448. (9) Zana, R., Xia, J., Eds. Gemini Surfactants: Synthesis, Interfacial and Solution-Phase Behavior, and Applications; Dekker: New York, 2003. (10) Zana, R. Adv. Colloid Interface Sci. 2002, 97, 205. (11) Wattebled, L.; Laschewsky, A. Langmuir 2007, 23, 10044– 10052. (12) Laschewsky, A.; Wattebled, L.; Arotcarena, M.; Habib-Jiwan, J.-L.; Rakotoaly, R. H. Langmuir 2005, 21, 7170–7179. (13) Wattebled, L.; Laschewsky, A.; Moussa, A.; Habib-Jiwan, J.-L. Langmuir 2006, 22, 2551–2557. (14) Bhadani, A.; Singh, S. Langmuir 2009, 25, 11703–11712. (15) Quagliotto, P.; Viscardi, G.; Barolo, C.; Barni, E.; Bellinvia, S.; Fisicaro, E.; Compari, C. J. Org. Chem. 2003, 68, 7651–7660. (16) Zhou, L.; Jiang, X.; Li, Y.; Chen, Z.; Hu, X. Langmuir 2007, 23, 11404–11408. (17) Fisicaro, E.; Compari, C.; Biemmi, M.; Duce, E.; Peroni, M.; Barbero, N.; Viscardi, G.; Quagliotto., P. J. Phys. Chem. B 2008, 112, 12312–12317. (18) Bhadani, A.; Kataria, H.; Singh, S. J. Colloid Interface Sci. 2011, 361, 33–41. (19) Ding, Y.-S.; Zha, M.; Zhang, J.; Wang, S.-S. Colloids Surf., A 2007, 298, 201–205. (20) Baltazar, Q. Q.; Chandawalla, J.; Sawyer, K.; Anderson, J. L. Colloids Surf., A 2007, 302, 150–156. (21) Ao, M.; Xu, G.; Zhu, Y.; Bai, Y. J. Colloid Interface Sci. 2008, 326, 490–495. (22) Akbay, C.; Hoyos, Y.; Hooper, E.; Arslan, H.; Rizvi, S. A. A. J. Chromatogr., A 2010, 1217, 5279–5287.

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