Article pubs.acs.org/Langmuir
Gemini Imidazolium Surfactants: Synthesis and Their Biophysiochemical Study Raman Kamboj,† Sukhprit Singh,*,† Avinash Bhadani,† Hardeep Kataria,‡ and Gurcharan Kaur‡ †
Department of Chemistry, UGC Sponsored-Centre of Advance Studies-1 and ‡Department of Biotechnology, Guru Nanak Dev University, Amritsar, India S Supporting Information *
ABSTRACT: New gemini imidazolium surfactants 9−13 have been synthesized by a regioselective epoxy ring-opening reaction under solvent-free conditions. The surface properties of these new gemini surfactants were evaluated by surface tension and conductivity measurements. These surfactants have been found to have low critical micelle concentration (cmc) values as compared to other categories of gemini cationic surfactants and also showed the tendency to form premicellar aggregates in solution at sufficiently low concentration below their cmc values. The thermal degradation of these surfactants was determined by thermograviometry analysis (TGA). These new cationic surfactants have a good DNA binding capability as determined by agarose gel electrophoresis and ethidium bromide exclusion experiments. They have also been found to have low cytotoxicity by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay on the C6 glioma cell line.
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INTRODUCTION
amphiphile with low toxicity has a greater probability of finding application as a synthetic vector for gene delivery.51,52 Pursuant to our continued research in this area, we have synthesized a new series of gemini imidazolium surfactants 9− 13 by a regioselective ring-opening reaction having different spacer lengths (i.e., (CH2)n−), where n = 3,4, 5, 6, and 8. The methodology described for the synthesis of new gemini surfactants in the present work involves a green, solvent-free approach.
In recent years, new classes of amphiphilic molecules have emerged and have attracted the attention of several industrial and academic research groups. One of these classes is the gemini or dimeric surfactants, which are generally made up of two hydrocarbon chains and two headgroups linked by a rigid or flexible spacer.1−15 These surfactants possess better physicochemical properties such as lower critical micelle concentration (cmc) values, higher solubilization power, and better wetting and foaming properties than the corresponding traditional single-chain surfactants.16−21 Cationic gemini surfactants have applications in skin care formulations,22,23 the construction of high-porosity materials,24,25 templates for the synthesis of nanoparticles,26−28 nanorods,29 biomedical application including gene delivery,30−32 drug entrapment/ release,33 and antimicrobial activity.34,35 Recent past syntheses and investigations of several new categories of gemini cationics such as pyridinium,36−40 imidazolium,40−44 piperidinum,45 pyrrolidinum,46 and amino acid-based cationics47−49 have been reported. Earlier, we reported the synthesis characterization and evaluation of the self-aggregation properties of gemini pyridinium36,40 and imidazolium surfactants.40,50 We have also observed that these gemini surfactants are much less toxic in nature and innocuous in human health. Recently, it has been demonstrated that an imidazolium moiety containing an © 2012 American Chemical Society
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EXPERIMENTAL SECTION
Materials. 1,2-Epoxydodecane, 1,3-dibromopropane, 1,4-dibromobutane, 1,5-dibromopentane, 1,6-dibromohexane, 1,8-dibromooctane, zinc perchlorate hexahydrate, and ethidium bromide were purchased from Sigma-Aldrich (U.S.) and used without any purification. Imidazole was purchased from Central Drug House (New Delhi, India). Agarose and Tris buffer were purchased from Sisco Research Laboratory Pvt Ltd. (Mumbai, India). Plasmid DNA pUC 18 was purchased from Bangalore GeNei (Bangalore, India). Millipore water was used in all experiments. Synthesis. In a typical procedure 1,2-epoxydodecane (1, 11.04 g, 60 mmol) was reacted with imidazole (2, 4.08 g, 60 mmol) in the presence of a catalytic amount of zinc perchlorate (Scheme 1). After addition, the reaction was stirred for 1 h at 80 °C under solvent-free Received: March 3, 2012 Revised: July 14, 2012 Published: July 30, 2012 11969
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Scheme 1
conditions. The progress of the reaction was monitored by thin layer chromatography (Silica gel G-coated (0.25-mm-thick) glass plates using 90:10 or 85:15 hexane/ethyl acetate as the mobile phase; the spots were visualized with iodine). The reaction was completed in 1 h. The reaction mixture was dissolved in 100 mL of chloroform and filtered to recover the catalyst. The catalyst was reused three to five times without any loss of activity.53,54 The chloroform layer was transferred to a separation funnel and washed twice with water, followed by a saturated solution of sodium chloride. Chloroform was removed from the crude reaction mixture under reduced pressure in a rotary flash evaporator at 40 °C. It was then allowed to cool. The purification of 1-(1H-imidazol-1-yl)dodecan-2-ol (3, white crystalline solid, 13.11 g, 87% yield) was carried out by recrystallization in hexane. The 1-(1H-imidazol-1-yl)dodecan-2-ol (3, 1.512 g, 6 mmol) was reacted with various dibromides {1,3-dibromopropane (4, 0.606 g, 3 mmol), 1,4-dibromobutane (5, 0.648 g, 3 mmol), 1,5-dibromopentane (6, 0.690 g, 3 mmol), 1,6-dibromohexane (7, 0.732 g, 3 mmol), and 1,8-dibromooctane (8, 0.816 g, 3 mmol)} at 80 °C for 30 min. The resulting crude mixtures were cooled to 25 °C. The product was washed three times with 50 mL of diethyl ether and cold precipitated in acetone to obtain the respective gemini imidazolium surfactants 9− 13. The structures of all of these products were confirmed by IR, NMR, and mass spectrometry. Infrared (IR) spectra were recorded as a thin neat film on a Fourier transform infrared (FT-IR) instrument (model 8400s, Shimadzu, Kyoto, Japan). Mass spectra were recorded on Waters Q-ToF micromass equipment using ESI as the ion source. 1 H and 13C NMR spectra were recorded on either an AL-300 (JEOL, Japan) FT-NMR (300 MHz) system or a Bruker Avance II (Switzerland) FT-NMR (400 MHz) system as a solution in CDCl3 using tetramethylsilane (TMS) as the internal standard. 1-(1H-Imidazol-1-yl)dodecan-2-ol (3). White crystalline solid. 300 MHz 1H NMR (CDCl3, TMS): δ 0.86−0.89 (t, 3 H), 1.26−1.37 (br. s, 16 H), 1.43−1.54 (m, 2H), 3.76−3.82 (dd, 2 H), 3.91−3.97 (dd, 1 H), 5.20 (br. s, 1 H), 6.84−6.88 (d, 2 H), 7.28−7.32 (s, 1 H). 75 MHz 13 C/DEPT-135 NMR (CDCl3): δ 137.46, 128.29, 119.71, 70.48, 53.65, 34.58, 31.19, 29.62, 29.34, 25.67, 22.69, 14.13. IR (cm−1) neat: 3408, 3230, 2919, 2850, 1739, 1647, 1563, 1459, 1350, 1237, 1108, 763. MS m/z (parent ions): 253 and 254 ([M+ + 1] and [M+ + 2]). 3,3′-(Propane-1,3-diyl)bis(1-(2-hydroxydodecyl)-1H-imidazol-3ium) Bromide (9). White paste. 300 MHz 1H NMR (CDCl3, TMS): δ 0.86−0.90 (t, 6 H), 1.26 (br. s, 32 H), 1.51 (m, 4 H), 2.57−2.69 (m, 4 H), 3.98 (m, 2 H), 4.07−4.14 (m, 2 H), 4.26−4.31 (m, 2 H), 4.57 (s, 4 H), 4.84 (br s, 2 H), 7.45 (s, 2 H), 7.95 (s, 2 H), 9.51 (s, 2 H). 75 MHz 13C/DEPT-135 NMR (CDCl3): δ 136.58, 123.07, 122.60, 69.12, 55.65, 46.59, 34.48, 31.81, 30.34, 29.66, 29.61, 29.28, 25.54, 22.56, 13.98. IR (cm−1) neat: 3462, 3299, 2922, 2851, 1739, 1643, 1568, 1452, 1348, 1242, 1100, 747. MS positive ions m/z (for C33H62BrN4O2+): 625.5 (base peak), 626.4, 627.5, 628.5. 3,3′-(Butane-1,4-diyl)bis(1-(2-hydroxydodecyl)-1H-imidazol-3ium) Bromide (10). White paste. 300 MHz 1H NMR (CDCl3, TMS): δ 0.86−0.89 (t, 6 H), 1.25−1.29 (br s, 32 H), 1.49 (br s, 4 H), 2.04 (br s, 4 H), 3.34 (s, 4 H), 3.95 (br s, 2 H), 4.09−4.15 (m, 2 H), 4.28−4.31
(m, 2 H), 4.44 (s, 4 H), 4.89 (br s, 2 H), 7.45 (s, 2 H), 7.84 (s, 2 H), 9.46−9.47 (s, 2 H). 75 MHz 13C/DEPT-135 NMR (CDCl3): δ 136.54, 122.91, 122.68, 69.34, 55.60, 48.99, 34.58, 31.95, 29.81, 29.78, 29.75, 29.72, 29.43, 26.65, 25.69, 22.71, 14.14. IR (cm−1) neat: 3400, 3262, 2912, 2886, 1746, 1662, 1567, 1461, 1356, 1240, 1112, 753. MS positive ions m/z (for C34H64BrN4O2+): 639.4 (base peak), 640.4, 641.5, 642.5. 3,3′-(Pentane-1,5-diyl)bis(1-(2-hydroxydodecyl)-1H-imidazol-3ium) Bromide (11). White paste. 300 MHz 1H NMR (CDCl3, TMS): δ 0.86−0.89 (t, 6 H), 1.20−1.35 (br. s, 32 H), 1.41−1.52 (m, 6 H), 1.99−2.02 (br s, 4 H), 2.95 (s, 2 H), 3.99 (br s, 2 H), 4.15−4.22 (m, 2 H), 4.33−4.41 (m, 6 H), 4.79−4.80 (br s, 2 H), 7.48 (s, 2 H), 7.77− 7.79 (s, 2 H), 9.69−9.72 (s, 2 H). 75 MHz 13C/DEPT-135 NMR (CDCl3): δ 136.77, 123.06, 122.28, 69.23, 55.44, 49.41, 34.67, 31.91, 29.72, 29.66, 29.37, 28.88, 25.65, 22.68, 22.47, 22.38, 14.13. IR (cm−1) neat: 3387, 3253, 2919, 2877, 1748, 1657, 1575, 1437, 1329, 1208, 1147, 733; MS positive ions m/z (for C35H66BrN4O2+): 653.4 (base peak), 654.4, 655.4, 656.4, 573.5, 574.5. 3,3′-(Hexane-1,6-diyl)bis(1-(2-hydroxydodecyl)-1H-imidazol-3ium) Bromide (12). White paste. 300 MHz 1H NMR (CDCl3, TMS): δ 0.85−0.88 (t, 6 H), 1.25 (br. s, 36 H), 1.45−1.53 (m, 4 H), 1.98 (m, 6 H), 4.02 (m, 2 H), 4.29−4.37 (m, 8 H), 4.73−4.78 (m, 2 H), 7.38 (s, 2 H), 7.57 (s, 2 H), 9.83 (s, 2 H). 75 MHz 13C/DEPT-135 NMR (CDCl3): δ 136.99, 123.33, 122.51, 69.64, 55.67, 49.77, 34.85, 32.19, 29.94, 29.64, 29.55, 25.94, 25.01, 22.96, 14.39. IR (cm−1) neat: 3430, 3287, 2867, 2823, 1742, 1650, 1557, 1432, 1326, 1248, 1134, 787. MS positive ions m/z (for C36H68BrN4O2+): 667.4, 668.4, 669.4, 670.4, 587.5, 588.5, 202.2 (base peak). 3,3′-(Octane-1,8-diyl)bis(1-(2-hydroxydodecyl)-1H-imidazol-3ium) Bromide (13). White paste. 300 MHz 1H NMR (CDCl3, TMS): δ 0.87−0.89 (t, 6 H), 1.26−1.52 (m, 44 H), 1.96−2.04 (m, 8 H), 4.00 (m, 2 H), 4.32−4.40 (m, 8 H), 4.73 (br. s, 2 H), 7.45−7.51 (dd, 4 H), 9.83 (s, 2 H). 75 MHz 13C/DEPT-135 NMR (CDCl3): δ 136.71, 123.14, 121.77, 69.29, 55.26, 49.72, 34.39, 31.81, 29.60, 29.55, 29.38, 29.26, 27.69, 25.58, 25.19, 22.58, 14.03. IR (cm−1) neat: 3396, 3215, 2907, 2857, 1749, 1657, 1573, 1448, 1334, 1246, 1143, 744. MS positive ions m/z (for C38H72BrN4O2+): 695.4, 696.5, 697.4, 698.5, 615.5, 616.5, 308.3 (base peak). The structures of gemini surfactants 9−13 have been established by 1 H, 13C, DEPT, 2D HETCOR, and 2D COSY experiments. The carbon protons (i.e., −N−CH2−) attached directly to heteroatom nitrogen are nonequivalent in nature, and each protons gives two independent signal at δ 3.95−4.40 and 4.26−4.41, respectively, for both protons (Ha and Hb). The signal for protons attached to the C atom bearing the hydroxyl group ((i.e., −CH−OH) appeared as a multiplet between δ 3.99 and 4.37. The protons attached to a spacer carbon (i.e., −N+−CH2−) appeared as multiplets between δ 4.29 and 4.57 for gemini imidazolium surfactants 9−13. The signals for imidazolium protons (i.e., −NCHCHN−) were observed between δ 7.38 and 7.48 and δ 7.51 and 7.84 as two independent singlets integrated for two protons each. The signal for the −NCHN− imidazolium proton appeared in the range of δ 9.46−9.83 as a distinct singlet. 11970
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C NMR spectra depicted sp3 carbon for terminal methyl at δ 13.98−14.39. The sp2-hybridized carbon (i.e., −N−CH2−) directly attached to heteroatom nitrogen was observed in the range of δ 55.26−55.67. The signal for the spacer carbon (i.e., −N+−CH2−) directly attached to the heterocyclic positively charged imidazolium nitrogen was observed at δ 46.59−49.77. Carbon attached to hydroxyl group (i.e., −CH−OH) was observed between δ 69.12 and 69.64. The carbons of the imidazolium rings (i.e., −NCHCHN−) were observed at δ 122.28−123.3, and those of −NCHN− were observed at δ 136.54−136.99. The formation of these gemini imidazolium surfactants has further been established by ESI-MS (positive ion) mass spectroscopy. The parent ion peak for gemini surfactants has been observed for a monopositive ion, where the direct loss of a bromide ion from the molecule led to the formation of positively charged parent ion [(M − Br−)]+. The [(M − Br−)]+ +1 and [(M-Br−)]+ +2 ions were also observed in each case. For surfactants 11 and 13, the peak corresponding to the loss of both bromide ions was also observed. Thermal Stability Measurements. The thermal stability of the gemini surfactants was measured with an SDT Q600 thermal gravimetric analyzer (TGA) using a nitrogen atmosphere. Thermograms were recorded using a heating rate of 5 °C/min from 25 to 400 °C. 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 thermal stability of the gemini imidazolium surfactants 9−13 were determined from a TGA graph. Krafft-Point Measurements. The Krafft temperatures of gemini surfactants 9−13 were determined using surfactant solutions of concentration 1 wt % (i.e., well above the cmc of the investigated gemini surfactants) using the electrical conductivity method.37 Each of the surfactant was dissolved in water and then left in a refrigerator at a temperature of 1.5 °C for 1 day until precipitation occurred. The precipitated surfactant solution thus obtained was introduced into the conductivity cell to measure the Krafft point. (A detailed procedure along with a plot (Figure S1) of conductivity (κ) versus temperature (T) for gemini surfactant 13 for the determination of the Krafft point has been included in the Supporting Information.) Conductivity Measurements. Conductivity was measured on a model EQ661 Equip-Tronics auto temperature conductivity meter equipped with a conductivity cell. The aqueous solutions were thermostatted in the cell at 25.0 ± 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 critical micelle concentration (cmc) to at least 1 to 2 times the cmc. The degree of counterion binding (β) has been calculated to be (1 − α), where α = Smicellar/Spremicellar (i.e., ratio of the slope after and before the cmc50,55). Surface Tension Measurements. The critical micelle concentration (cmc) and surface tension attained at the cmc were determined using a CSC (Central Scientific Co., Inc., USA) Du Nouy interfacial tensiometer with a platinum−iridium ring (circumference 5.992 cm) at 25.0 ± 0.1 °C. The tensiometer was calibrated using triply distilled water. Each of the surfactant solutions was aged for 24 h prior to the determination of surface activity.50,55 Agarose Gel Electrophoresis. pDNA (166 ng/well) and 10 μL of 12.5, 25, 50, and a 100 μM gemini imidazolium surfactant 9−13 solution were loaded with 5 μL of glycerol into 1% agarose gel containing 2 μL of ethidium bromide (0.5 mg/mL). 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.50 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 in the presence of pDNA (2 μg) from 530 to 700 nm at an excitation wavelength (λex) of 490 nm using a Perkin-Elmer LS 55 fluorescence spectrophotometer. Fifteen microliters of a 50 μM solution of gemini surfactants was added 12 times to a pDNA-EB intercalated system to obtain 12 observations. The percentage of quenching observed from 13
the replacement of EB by cationic gemini surfactants from the pDNA upon interaction with the cationic surfactants 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.56,57 Cytoxicity Assay. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide)-based cytotoxicity test was used to evaluate all of the Gemini surfactants, and the tests were carried out on C6 glioma (cancerous brain cell line, passage number 65). Cells were seeded in 96-well flat-bottomed microplates at a density of 5 × 104 per mL, 100 μL per well, and were allowed to grow for 24 h. The compounds dissolved in doubly distilled water were sterilized using a Millipore filter (pore size 0.22 μm) and were added to the culture media over a concentration range of 1−100 μM. The cytotoxicity of the compounds was assessed after 24 h of exposure. The absorbance was read at 550 nm using a Muliskan PLUS plate reader (Labsystem, Finland). The statistical analysis was performed using Sigma Stat 3.5.1 and Sigma Plot 11.0.36,58
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RESULTS AND DISCUSSION Self-Aggregation Studies in Aqueous Solution. The surface properties of the gemini imidazolium surfactants have been determined by surface tension measurements. Figure 1
Figure 1. Surface tension vs log C plot for gemini imidazolium surfactants.
shows the surface tension (γ) versus log (C) (i.e., C is the surfactant concentration) plots for five gemini imidazolium surfactants at 25 °C. The surface tension initially decreases with increasing concentration of surfactants and then reaches a plateau region, indicating that micelles are formed and the concentration of the break point corresponds to the critical micelle concentration. The cmc values of these surfactants increase with the elongation of the spacer length. The cmc values as determined by surface tension were found to be lower than that obtained by the conductivity method; however, the trend of increasing cmc values with the elongation of the spacer length remained the same. (A plot of surface tension vs log (C) of surfactants 11, 12, and 13 has also been included in the Supporting Information as Figure S3 for the visualization of the difference in their critical micelle concentration because there is only a small difference in the cmc's of these surfactants). Rosen et al.59 found different cmc values by two different techniques for a series of N-acyl-β-alaninate gemini surfactants. Similar results have also been obtained by Pinazo et al.60 for arginine-based gemini surfactants and Esumi et al.61 for trimeric surfactants. This behavior has already been discussed in detail 11971
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a cmc from surface tension. bcmc from conductivity; β, degree of counterion association; γcmc, surface tension at the cmc; Γmax, 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; ΔG°mic, Gibbs free energy of micellization; ΔG°ads, Gibbs free energy of adsorption; cmc/C20, cmc from surface tension/C20; TK, Krafft point. The values in parentheses are for n = 3.
0.57 0.82 0.56 0.20 0.36 ± ± ± ± ± −49.67 −50.63 −52.46 −44.78 −49.45 0.22 0.21 0.22 0.19 0.18 ± ± ± ± ± −32.85 −31.63 −35.21 −32.31 −31.14 5.8 9.8 5.3 2.9 4.3 1.23 0.77 1.90 3.63 2.63 ± ± ± ± ± 0.72 0.76 1.02 1.07 1.14
0.01 0.01 0.01 0.01 0.01
1.37 1.40 1.47 1.57 1.59 ± ± ± ± ±
0.01 0.01 0.01 0.01 0.01
0.75 0.70 0.84 0.74 0.70
30.0 28.1 32.9 35.2 37.6
± ± ± ± ±
0.3 0.3 0.4 0.2 0.3
2.53 2.33 2.29 2.98 1.90
± ± ± ± ±
0.06 0.09 0.05 0.02 0.02
(1.69 (1.55 (1.52 (1.98 (1.27
± ± ± ± ±
0.04) 0.06) 0.03) 0.02) 0.02)
0.65 0.71 0.72 0.55 0.87
± ± ± ± ±
0.02 (0.98 ± 0.02) 0.03 (1.07 ± 0.04) 0.02 (1.09 ± 0.02) 0.01(̀ 0.83 ± 0.01) 0.01 (1.30 ± 0.02)
cmc/C20a C20 × 10−4 Amin nm2
9 10 11 12 13
where N is Avogadro’s number and Amin is in nm2 (Table 1). Gemini imidazolium surfactants 9−13 reported in the present studies have been found to have lower Amin values as compared to those of other gemini cationic surfactants,39,59 including gemini imidazolium surfactants42,44 reported previously. Because of the low Amin values, these new gemini imidazolium surfactants have a greater tendency to form micelles instead of adsorbing at the air−water interface. Unlike the gemini pyridinium surfactants,39 Amin values of these new gemini imidazolium surfactants increase with increasing spacer length. Such a pattern of increase in Amin values with increasing spacer length was also observed by Ao et al.44 for gemini imidazolium surfactants and Zana et al.2 for gemini quaternary ammonium surfactants. Initially, lower Amin values were solely attributed to a tighter packing of the longer hydrophobic chains at the interface.62,64 However, recent studies by Fisicaro et al.39 revealed that surfactant molecules having lower Amin value may have a greater tendency to form premicellar aggregates instead of adsorbing at the air/water interface. A theortical explanation suggested that the dominant factor responsible for the variation in Amin values
ΔG°mic kJ/mol
ΔG°ads kJ/mol
(2)
106Γmax mol/m2
1 N Γmax
γ mN/m
A min =
β
Here, γ denotes the surface tension, R is the gas constant, T is the absolute temperature, and C is the surfactant concentration. Recent studies have been carried out by assuming that one counterion is associated with the ionic headgroup, and the value of n was taken to be 2.38,39 The value of n = 2 has been supported by the results obtained with neutron reflectivity studies.63 However, previous investigations on gemini imidazolium surfactants have been carried out by assuming a value of n = 3, considering a divalent surfactant ion and two univalent counterions.42,44 Therefore, it becomes essential to calculate the value of Γmax by assuming the value of n = 2 as well as n = 3. The area occupied per surfactant molecule (Amin) at the air− water interface2 has been obtained by using eq 2
cmc mMb
(1)
cmc mMa
T
surfactant
⎛ dγ ⎞ 1 ⎜ ⎟ 2.30nRT ⎝ d log C ⎠
Table 1. Surface Properties of Gemini Imidazolium Surfactants 9−13 as Determined from Surface Tension and Conductivity Measurements
Γmax = −
TK (°C)
by Fisicaro et al.39 for gemini pyridinium surfactants and has been attributed to the formation of non-surface-active premicellar aggregates by surfactants. Furthermore, the plot of Λ versus C0.5 also indicates the existence of such premicellar aggregates for gemini imidazolium surfactants 9−13, where there is a significant difference in the determination of the cmc values by surface tension and conductivity. We have found very peculiar behavior of gemini surfactants 9−13 being reported in a current study. It has been observed that the solution of the surfactant takes 30 min to stabilize after being transferred from a volumetric flask to a thermostated vessel before the set of five successive concordant readings can be recorded. Furthermore, the solution needs to be aged for at least 24 h prior to evaluation at a constant temperature of 25 °C to get uniform results. The initial sets of readings need to be completely ignored, and data obtained after the stabilization of the surfactant solution for 30 min in a thermostated vessel is considered to be accurate. The maximum surface excess concentration at the air/water interface,2,42,62 Γmax, has been calculated by applying the Gibbs adsorption isotherm (eq 1).
22.0 23.3 20.5 28.6 20.6
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Figure 2. Specific conductivity vs concentration and molar conductivity vs C0.5 of gemini imidazolium surfactants 9−13. The arrows indicate, from left to right, the onset of premicellar aggregate formation and the concentrations at which the maximum and the cmc are attained, respectively. The error estimate for the calculated value is ±0.5%. Individual points shown with error bars represent the mean value ± SEM.
of the surfactants is the size of the hydrophilic headgroup and the solvation of the imidazolium cation in water.65 The affinity to reduce the surface tension (γcmc) and the ability to reduce the surface tension by 20 mN m−1 (C20) for these gemini imidazolium surfactants have also been calculated from the plot of the decrease in surface tension versus the log of concentration (Table 1). The γcmc values of these gemini surfactants were found to increase with increasing length of the
spacer units with the exception of gemini imidazolium surfactant 10. The trend of increasing surface tension attained at the cmc for theis series of gemini imidazolium surfactants can be explained on the basis of the cmc/C20 ratio observed for these surfactants. The ability of a particular surfactant to reduce the surface tension depends upon the cmc/C20 ratio, with greater observed values having a greater surfactant tendency to reduce the surface tension of the system.66 Thus, gemini 11973
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Figure 3. (a) TGA graph of gemini surfactant 9 and (b) magnified TGA graph of surfactant 9 indicating the loss of water molecules, with the starting temperature of degradation and the onset temperature of degradation.
gemini imidazolium surfactants with respect to the increase in the number of spacer units (Table 1). Critical Micelle Concentration (cmc) and Degree of Counterion Binding. The cmc values of new gemini imidazolium surfactants 9−13 have also been evaluated by the conductivity method, and it has been observed that these values follow a similar trend of increasing cmc values with increasing spacer length as observed by surface tension measurements. However, the determined cmc values differ significantly by two different techniques. Compared to gemini pyridinium surfactants reported earlier by Quagliotto et al.37 and Zhao et al.,38 the cmc values of new gemini surfactants 9− 13 increase with increasing spacer length. Such a trend of increasing cmc values with increasing spacer length has also been observed previously for the gemini quaternary ammonium surfactant2 and gemini imidazolium surfactants.44 Another important parameter evaluated by a conductivity plot is the degree of counterion binding (β) that signifies the ability of counterions to bind micelles. Gemini imidazolium surfactants 9−13 show a degree of counterion binding of around 70−85% that is extremely high for gemini cationic surfactants having a bromide counterion. Our recent studies have shown that the degree of counterion binding (β) of gemini imidazolium surfactants50 is not influenced by increases in the alkyl chain length and spacer units, and similar results have also been observed in the present study. The molar conductivity (Λ) data has been plotted against the square root of concentration (C0.5). From these plots (Figure 2), it is evident that these new gemini surfactants show peculiar behavior at low concentration. Gemini imidazolium surfactants 9−13 show the occurrence of a maximum in these plots that would account for the formation of premicellar aggregates.60,69 The existence of premicellar aggregates at low concentration has been previously investigated by several research groups.59−61 Zana69 proposed dimer-type premicellar aggregates, with their hydrophobic chains oriented with respect to each other, leaving the two headgroups far apart from each other (at the edges of the dimer). Under these conditions, the dimer is fully ionized and the conductivity of the dimer should be higher than that of the surfactant monomers. Furthermore, Pinazo et al.60 also evaluated this kind of behavior and extended this discussion to the formation of oligomers, such as trimers, tetramers, and so on. In Λ versus C0.5 plots for gemini
imidazolium surfactant 10 has the maximum ability to reduce the surface tension of the aqueous system in the series of gemini surfactants being reported. The Gibbs free energy of micellization (ΔG°mic) has been calculated with the following equation42 ΔG°mic = RT (0.5 + β )ln Xcmc
(3)
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). Similarly, the Gibbs free energy of adsorption (ΔG°ads) has been calculated with the following equation:67 ΔG°ads = ΔG°mic −
πcmc Γ
(4)
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 results of the present study demonstrate a small energy gap between ΔG°mic and ΔG°ads in individual gemini imidazolium surfactants. A recent study has shown that the smaller the gap between these parameters, the greater the tendency of individual surfactants to aggregate in solution rather than to adsorb at the air/water interface.50,68 A direct relation between the calculated Amin value and the energy difference between ΔG°mic and ΔG°ads has been observed for gemini imidazolium surfactants. The smaller the Amin value, the smaller the energy difference between ΔG°mic and ΔG°ads. Similar results have also been observed by Bhadani and Singh.50 (The procedure to determine these surface parameters and a plot of surface tension versus log (C) (Figure S2) for surfactant 9 showing the error bars is included in the Supporting Information.) Krafft Points. The Krafft points of all of the gemini surfactants have been determined and found to be less than 25 °C. Even though the Krafft temperature for a 1 wt % solution of gemini surfactant 12 is around 28.6 °C, the stock solution of this surfactant at a concentration of C = 10cmc showed no visible surfactant precipitate when stored at room temperature for several weeks after being dissolved in water. No particular trend in the Krafft point has been observed for the series of 11974
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Table 2. Onset and Starting Temperatures for the Thermal Decomposition of Gemini Imidazolium Surfactants temperature (°C)
surfactant 9
surfactant 10
surfactant 11
surfactant 12
surfactant 13
Tonset Tstart
308.8 285.5
298.7 274.4
288.1 273.3
287.1 269.1
279.7 249.1
surfactants 9−13, the arrows from left to right indicate the onset of premicellar aggregate formation, the concentration at which the maximum is attained, and the cmc as determined in a conductivity versus concentration plot. At the onset point, the surfactant monomers start to form premicellar aggregates, and because the conductance of these premicellar aggregates is higher than that of surfactant monomers, they should stay in the solution bulk and are not absorbed at the air−water interface. The maximum in the molar conductivity plot probably came from the fact that as the concentration is further increased oligomers larger than dimers start to form and may bind counterions (the β value is found to be around 15− 20% for oligomers), after which surfactants 9−13 form regular micelles. Thermogravimetric Analysis. Gemini imidazolium surfactants 9−13 have been synthesized as their monohydrate or dihydrate salts. The water of hydration of these gemini surfactants has been determined by thermogravimetric analysis (TGA). 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. Thermal stability measurement shows that these gemini surfactants are stable to up to 310 °C. Figure 3a shows a characteristic curve for the decomposition of the gemini surfactants as measured by thermal gravimetric analysis. The onset temperature (Tonset) is the intersection of the baseline weight, either from the beginning of the experiment and from the tangent of the weight versus temperature curve as decomposition occurs. The starting temperature (Tstart) is the temperature at which the decomposition of the sample begins (Figure 3b).70 The onset and starting temperatures for the present gemini imidazolium surfactants are listed in Table 2. Thermal stability measurements designated that these new gemini surfactants have better thermal stability. Gemini imidazolium surfactant 9 was found to be the most thermally stable surfactant, having a Tstart of 285.5 °C and a Tonset of 308.8 °C whereas surfactant 13 was found to be the least thermally stable surfactant, having a Tstart of 249.1 °C and a Tonset of 279.7 °C among the imidazolium geminis. Furthermore, it has also been found that the thermal stability of these gemini surfactants decreases with increasing spacer length. Agarose Gel Electrophoresis. The DNA binding capability of gemini imidazolium surfactants 9−13 and reference conventional quaternary ammonium gemini surfactant 12-2-12 has been investigated by agarose gel electrophoresis. It has been observed that all gemini imidazolium surfactants were able to bind plasmid DNA at low concentration. All gemini surfactants 9−13 were able to retard the migration of DNA toward the positive electrode at a concentration of 50 μM (Figure 4). The interaction of pDNA with imidazolium surfactants takes place at an even lower concentration than 25 μM because these surfactants are able to replace ethidium bromide from DNA in ethidium bromide exclusion experiments (discussed later) at a concentration lower than 25 μM. However, effective binding occurs at a concentration of between 25 and 50 μM because there is complete neutralization of the partial negative charge of
Figure 4. Agarose gel electrophoresis of pDNA and gemini surfactants at different concentrations.
pDNA via the formation of a stable complex, which is evident from the retardation observed in gel electrophoresis and the complete displacement of EB in exclusion experiments. Because all of the imidazolium surfactants were able to bind pDNA to similar extents, it can be concluded that the increase in the spacer length plays little if any role in their binding with pDNA. The observed results were found to be in accordance with recent literature reports.40,50 This may be attributed to the fact that such molecules have a greater degree of flexibility as compared to other gemini surfactants and can bind the oppositely charged sites with ease. Ethidium Bromide Exclusion Experiment. The DNA binding capability of gemini imidazolium surfactants 9−13 has been further confirmed by EB exclusion experiments using fluorescence spectroscopy. The fluorescence emission of EB is enhanced as a result of intercalation between the DNA base pairs relative to that in water.71,72 The extent of binding of a particular surfactant can be determined by its ability to displace EB from the DNA−EB intercalated complex, hence causing a quenching of the fluorescence intensity.73 Figure 5 shows the tendency of gemini imidazolium surfactants 9−13 to displace 11975
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Figure 6. Absorbance vs concentration (μM) of gemini surfactants for the determination of the IC50 value. The values represent the mean IC50 of three different experiments done in triplicate.
Figure 5. Displacement of ethidium bromide from the pDNA−EB complex by gemini imidazolium surfactants at different charge ratios.
observed among gemini imidazolium surfactants 9−13 is surfactant 13 with an IC50 value of 10.06 μM, whereas surfactant 10 with an IC50 value of 25.43 μM has been found to be the least toxic.
EB from the DNA−EB intercalated complex with increasing N/ P charge ratio. The results of the experiment show that these gemini surfactants have an excellent binding capability. It has been found that gemini imidazolium surfactant 9 has the maximum ability to displace EB from DNA because it displaces about 82.31% of EB at an N/P charge ratio of 2.0, whereas 80.97% EB was displaced by surfactant 11 at the same N/P charge ratio. Gemini imidazolium surfactants 10 and 12 have the weakest capability to displace EB from pDNA compared to compounds in the same homologous series. Gemini surfactant 13 causes a significant decrease in fluorescence intensity at a low N/P charge ratio of 1.0−1.25, but at a higher charge ratio, the smallest displacement of EB from the DNA−EB complex is observed. Therefore, it can be stated that at a higher N/P charge ratio, DNA becomes saturated with surfactant and the exclusion of EB no longer occurs for surfactant 13. Cytoxicity Assay. Few reports are available regarding the cytotoxic effects of gemini imidazolium surfactants.40 The cytotoxicity of gemini imidazolium surfactants 9−13 has been assessed on C6 glioma cells and compared to that of reference conventional quaternary ammonium gemini surfactant 12−2− 12. A recent report74 demonstrated a lower toxicity of the hydroxyl group containing pyridinium surfactants compared to that of conventional cationic surfactants. Similar results have been observed in the case of a hydroxyl group containing gemini imidazolium surfactants because these molecules have been found to be less cytotoxic than quaternary ammonium gemini surfactant 12−2−12. Most of the gemini surfactants have been found to be less toxic than reference molecule 12− 2−12, with the exception of gemini surfactant 13. The presence of two hydroxyl groups in gemini imidazolium surfactants 9−13 imparts polarity and is responsible for the increase in the hydrophilic character of the molecules, which correspondingly reduces the toxicity of these surfactants. IC50 values of gemini surfactants 9−13 are given in Figure 6. The toxicity of these gemini surfactants increases with increasing spacer length with the exception of gemini imidazolium surfactant 10, which has been found to be the least cytotoxic among the series of gemini imidazolium surfactants being reported. These values indicate the micromolar concentration of gemini surfactants, which causes the death of 50% of the living cells. The most toxic geminis
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CONCLUSIONS In this study, five new gemini imidazolium surfactants with different spacer chain lengths have been synthesized by a regioselective ring-opening reaction under solvent-free conditions. The surface properties of these gemini surfactants were evaluated by surface tension and conductivity measurements. All gemini imidazolium surfactants have been found to have lower Amin values. Because of low Amin values, these surfactants have a tendency to form premicellar aggregates at sufficiently low concentration below their true critical micelle concentration. The formation of premicellar aggregates is supported by a small energy gap between ΔG°mic and ΔG°ads. The plot of molar conductivity versus C0.5 provided further evidence of the existence of premicellar aggregates. These surfactants were found to be thermally stable up to 279 to 308 °C. These surfactants have also been observed to have good binding capability toward pDNA as evaluated by agarose gel electrophoresis and ethidium bromide exclusion experiments. These gemini surfactants have also been found to have low cytotoxicity by an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide) assay on C6 glioma cells (cancerous brain cell line). The cytotoxicity of these new gemini surfactants increases with increasing spacer chain length. Overall, these new hydroxy groups containing gemini imidazolium surfactant have low cmc values, a better DNA binding capability, and low cytotoxicity, which can be utilized in several technical areas including the biomedical application of gene delivery.
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ASSOCIATED CONTENT
S Supporting Information *
1
H, 13C NMR, 13C DEPT, 2D COSY, and 2D HETCOR spectra for intermediate 3 and gemini imidazolium surfactant 10. Mass spectra (MS) of surfactants 9−13 and intermediate 3 studied in this research. Determination of surface parameters of gemini surfactant 13 from a surface tension plot and a Krafft temperature plot of gemini surfactant 13. Error bars for gemini surfactant 9 and figure showing the critical micelle concen11976
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tration of surfactants 11, 12, and 13. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS We are thankful to the Council of Scientific & Industrial Research (CSIR) India for providing a fellowship for R.K. and the Department of Science and Technology (DST), Government of India, for providing the research grant (SR/S1/OC35/2010) for this work. We are also thankful to the Sophisticated Analytical Instrumentation Facility (SAIF) at Panjab University (Chandigarh, India) for the 13C DEPT, 2D COSY, HETCORE, and mass spectral analyses of the compounds. 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.
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dx.doi.org/10.1021/la300920p | Langmuir 2012, 28, 11969−11978
