Bio-physicochemical analysis of ethylene oxide-linked diester-functionalized green cationic gemini surfactants

Abstract

A novel series of oxy-diester-functionalized gemini surfactants (C_m-E2O-C_m) was synthesized and the physicochemical parameters were evaluated by surface tension and steady-state fluorescence measurements. Negative values of Gibbs free energy change of adsorption and micellization indicate that adsorption and micellization of C_m-E2O-C_m gemini surfactants are spontaneous. These surfactants have lower Krafft points and thus better solubility. Besides, they exhibit excellent foam and micro-emulsion stability. Interestingly, these geminis show low cytotoxicity, as revealed by HC₅₀ analysis. FT-IR spectra after alkaline treatment confirm their cleavable nature. Moreover, a fascinating feature of these geminis is their considerably high biodegradability. Thus, a comprehensive study of the synthesized gemini surfactants has been carried out that may be significant for potential applications in various fields, more specifically in biomedicine and cosmetics, where efficiency and safety are strictly connected.

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Introduction

The discipline of surfactants has matured over the years, but there is still ample space for the synthesis of new surfactants with structural modifications in order to obtain desirable characteristics. In recent years, dimeric or gemini surfactants have drawn extensive attention of the scientific community, both at an academic as well as an industrial level, due to their fascinating properties over conventional (single tail/single head) surfactants. Gemini surfactants are a special class of surfactants that bear two hydrophobic chains and two polar head groups linked by a spacer. They are known to possess lower critical micelle concentration (CMC), better wetting, foaming, and dispersing properties. They offer promising applications in detergency, solubilization, soil remediation, gene transfection, enhanced oil recovery, environmental protection, antimicrobial activity, etc.^1–4 Hence, these surfactants are regarded as potentially the ‘next generation’ high quality surfactants.⁵

Cationic gemini surfactants are gaining keen interest of researchers as the strategies for their synthesis are relatively simple.⁶ Previous literature reports^7–9 have shown that majority of these surfactants are based on nitrogen carrying a positive charge and bis-quaternary ammonium halides which are designated as m–s–m. However, they are being limited by the environmental concern raised over their discharge after use into water bodies as industrial effluents and sewage.^10,11 Therefore, there is a dire requirement for synthesizing such novel surfactants that are not only efficient but are also extensively biodegradable. In addition, low cytotoxicity is very crucial for amphiphiles to be utilized as non-viral vehicle for drug delivery. One suitable approach to achieve this is to introduce polar or easily cleavable groups (such as ester, ethylene oxide, amide, carbonate, etc.) in the structure of surfactants.^11–16 Inspired by this, we wondered what new and diverse properties may appear if highly biocompatible diester groups along with polar ethylene oxide moiety are introduced together in the spacer. Although extensive literature is available^17–24 concerning synthesis and properties of gemini surfactants but to the best of our knowledge, there is no literature report addressing the comprehensive analysis involving synthesis, characterization, physicochemical and biological aspects of novel ethylene-oxide linked diester functionalized cationic gemini surfactants.

With the consideration of the above aspects, our research group has synthesized a novel series of quaternary ammonium based gemini surfactants containing flexible ester-linked (betaine type) ethylene oxide units as spacer. They are designated as C_m-E2O-C_m (where m (= 12, 14 and 16) is the number of carbon atoms in alkyl chains of cationic surfactants and E2O represents the ethylene oxide-linked diester spacer). As can be seen, the polar ester groups together with ethylene oxide moiety in the synthesized geminis have dramatically improved their self-assembling behavior along with other physicochemical properties.

The incorporation of betaine type ester groups into the spacer has been found to improve the biodegradability and cleavability of surfactants. In the new cationics, the combination of this feature with the polarity of ethylene oxide moiety leads to further enhancement of these properties. Besides, considerably low cytotoxicity of these geminis may be attributed to the significant polarity induced by the E2O moiety. Thus, the obtained results suggest that the new structural modifications have refined not only the physicochemical but also biological properties in the same molecule (gemini). This has widened the scope of application of these concerned geminis in various fields such as biomedical, industrial, environmental, etc. Therefore, our approach is interdisciplinary and thus, unique in its nature. Overall, this study will provide an improved and deeper understanding regarding the effect of tailored structure or functionalities on the interfacial activity, cleavability and biological properties of the gemini surfactants.

Experimental section

Chemicals and instruments

Chemicals. Chloroacetyl chloride (98%, CDH, India), diethylene glycol (>98%, TCI, Japan), ethyl acetate (99.8%, Rankem, India), ether (99%, Rankem, India), N,N-dimethyldodecylamine (>95%, TCI, Japan), N,N-dimethyltetradecylamine (>90%, TCI, Japan) and N,N-dimethylhexadecylamine (>98%, TCI, Japan) were used as received.

Instruments. Thermo Scientific (Flash 2000) Elemental Analyser, Perkin Elmer FT-IR Spectrometer (5700), Bruker Avance II-400 MHz Nuclear Magnetic Resonance Spectrometer, Waters Micromass Q-T (LC-MS) Spectrometer, Hardson Tensiometer (Kolkata, India) and Hitachi F-2700 Fluorimeter were used.

Synthesis and characterization

Synthesis of the concerned gemini surfactants has been carried out in two simple steps.²⁵ In the first step, the spacer, oxybis(ethane-1,2-diyl)bis(2-chloroacetate) was prepared. For this, chloroacetyl chloride (0.22 mol) was taken in a dried, three-necked, round-bottomed flask equipped with a magnetic stirrer, a thermometer, a condenser and a drying tube (calcium chloride). Then diethylene glycol (0.1 mol) was poured into it through additional funnel in the presence of N₂ atmosphere. Thereafter, the reaction mixture was heated at 50 °C for 8 h. The HCl gas (generated during the reaction) was removed under reduced pressure. The obtained organic phase was then washed with saturated brine. Lastly, to obtain the desired spacer, oxybis(ethane-1,2-diyl)bis(2-chloroacetate), the product was dissolved in ether and dried over MgSO₄.

In the second step, 0.21 mol of N,N-dimethyldodecylamine (or N,N-dimethyltetradecylamine/N,N-dimethylhexadecylamine) and 0.1 mol oxybis(ethane-1,2-diyl)bis(2-chloroacetate) were added in a three-necked round-bottomed flask with ethyl acetate (as solvent). The solution was heated to reflux and the reaction was allowed to continue for 10 h. The products were then recrystallized from ethyl acetate to finally obtain the desired geminis, 2,2′-[(oxybis(ethane-1,2-diyl))bis(oxy)]bis(N-dodecyl-N,N-dimethyl-2-oxoethanaminium) dichloride (C₁₂-E2O-C₁₂), 2,2′-[(oxybis(ethane-1,2-diyl))bis(oxy)]bis(N-tetradecyl-N,N-dimethyl-2-oxoethanaminium) dichloride (C₁₄-E2O-C₁₄) and 2,2′-[(oxybis(ethane-1,2-diyl))bis(oxy)]bis(N-hexadecyl-N,N-dimethyl-2-oxoethanaminium) dichloride (C₁₆-E2O-C₁₆) as white powder. The overall reaction protocol is illustrated in Scheme 1. The structure and purity of the products were confirmed by FT-IR, ¹H NMR, elemental analysis and LC-MS. All the results of the analysis were in accordance with the literature.^13,25

Scheme 1 Synthesis protocol of C_m-E2O-C_m (m = 12, 14 and 16) gemini surfactants.

The characterization data of all the three synthesized geminis are given below:

2,2′-[(oxybis(ethane-1,2-diyl))bis(oxy)]bis(N-dodecyl-N,N-dimethyl-2-oxoethanaminium) dichloride (C₁₂-E2O-C₁₂). M.p: 90–100 °C, IR (KBr) (cm⁻¹): 2921 (C–H), 2852 (C–H), 1749 (C=O), 1466 (C–O), 1127 (C–N), ¹H NMR (400 MHz, CDCl₃): δ (ppm) = 0.86–0.90 (t, 6H, 2 × CH₃, alkyl chain), 1.25–1.35 (m, 40H, −2 × (CH₂)₁₀, alkyl chain), 2.51 (s, 4H, −2 × CH₂–O–CH₂, spacer), 3.53–3.55 (s, 12H, −2 × N⁺(CH₃)₂), 3.74–3.81 (m, 4H, −2 × N⁺CH₂), 4.33–4.35 (m, 4H, −2 × CH₂OOC, spacer), 5.33 (s, 4H, N⁺CH₂COO), LC-MS(+) m/z(%): C₃₆H₇₄N₂O₅²⁺/2; 307 (calculated), 308.1 (observed), anal. calcd for C₃₆H₇₄N₂O₅Cl₂ (%): C, 63.07; H, 10.80; N, 4.09, found: C, 58.68; H, 10.77; N, 4.02.

2,2′-[(oxybis(ethane-1,2-diyl))bis(oxy)]bis(N-tetradecyl-N,N-dimethyl-2-oxoethanaminium) dichloride (C₁₄-E2O-C₁₄). M.p: 95–100 °C, IR (KBr) (cm⁻¹): 2920 (C–H), 2852 (C–H), 1747 (C=O), 1465 (C–O), 1126 (C–N), ¹H NMR (400 MHz, CDCl₃): δ (ppm) = 0.83–0.90 (t, 6H, 2 × CH₃, alkyl chain), 1.25–1.35 (m, 48, H, −2 × (CH₂)₁₂, alkyl chain), 2.10 (s, 4H, −2 × CH₂–O–CH₂, spacer), 3.52–3.62 (s, 12H, −2 × N⁺(CH₃)₂), 3.72–3.82 (m, 4H, −2 × N⁺CH₂), 4.32–4.34 (m, 4H, −2 × CH₂OOC, spacer), 5.40 (s, 4H, N⁺CH₂COO), LC-MS(+) m/z(%): C₄₀H₈₂N₂O₅²⁺/2: 335 (calculated), 335.4 (observed), anal. calcd for C₄₀H₈₂N₂O₅Cl₂ (%): C, 64.78; H, 11.07; N, 3.78, found: C, 60.66; H, 11.09; N, 3.87.

2,2′-[(oxybis(ethane-1,2-diyl))bis(oxy)]bis(N-hexadecyl-N,N-dimethyl-2-oxoethanaminium) dichloride (C₁₆-E2O-C₁₆). M.p: 100–106 °C, IR (KBr) (cm⁻¹): 2918 (C–H), 2851 (C–H), 1749 (C=O), 1468 (C–O), 1129 (C–N), ¹H NMR (400 MHz, CDCl₃): δ (ppm) = 0.86–0.90 (t, 6H, 2 × CH₃, alkyl chain), 1.25–1.34 (m, 48, H, −2 × (CH₂)₁₄, alkyl chain), 2.90 (s, 4H, −2 × CH₂–O–CH₂, spacer), 3.52–3.54 (s, 12H, −2 × N⁺(CH₃)₂), 3.74–3.81 (m, 4H, −2 × N⁺CH₂), 4.33–4.35 (m, 4H, −2 × CH₂OOC, spacer), 5.28 (s, 4H, N⁺CH₂COO), LC-MS(+) m/z(%): C₄₄H₉₀N₂O₅²⁺/2: 363 (calculated), 363.4 (observed), anal. calcd for C₄₄H₉₀N₂O₅Cl₂ (%): C, 66.25; H, 11.29; N, 3.51, found: C, 61.22; H, 11.15; N, 3.60.

Physicochemical properties (such as Krafft point, foaming ability, specific viscosity and emulsifying power) were obtained by employing the methods described in the literature.²⁵

Methods

Surface tension measurements. Surface tension measurements of aqueous solutions of all the three gemini surfactants were performed on SD Hardson Tensiometer, using the ring detachment method at 30 °C. The Pt–Ir ring was cleaned thoroughly, while the glassware was washed with double distilled water. The break points of the surface tension versus concentration plots were used to determine the CMC values.

Fluorescence measurements. All the fluorescence measurements were performed on Hitachi-2700 Spectrofluorimeter. The parameters were set as: excitation slit width = 2.5 nm, emission slit width = 2.5 nm, excitation wavelength = 337 nm and scan range = 350–450 nm. Here, pyrene and cetylpyridinium chloride (CPC) were used as a probe and quencher, respectively. For the CMC determination, surfactant solutions of 0.3 mM were used whereas for aggregation studies, 1 mM surfactant solutions containing pyrene (3 μM) were titrated with varying concentration of CPC (0.5 mM).

HC₅₀ analysis. The experiments were conducted on fresh heparinised blood (self donor), which was taken from a young, healthy and non-smoking individual. It was centrifuged at 1500 rpm for 10 min at 4 °C in a clinical centrifuge and the plasma and buffy coat were removed by aspiration. The erythrocyte pellets were washed thrice with phosphate buffered saline (PBS) (6.78 g NaCl, 1.42 g Na₂HPO₄ and 0.4 g KH₂PO₄ in 1 L distilled water) and re-suspended in PBS to give a 2% hematocrit. Erythrocytes (25 μL) were incubated with different concentrations of gemini surfactants (12.5 mg L⁻¹ to 400 mg L⁻¹) for 1 h at 37 °C. The treated RBCs were then centrifuged at 2500 rpm for 10 min at 4 °C. Thereafter, supernatants were collected and their absorbance was recorded at 540 nm by using a Beckman DU 40 spectrophotometer (USA).

Biodegradability test. The three C_m-E2O-C_m gemini surfactants were subjected to a three-day biodegradability test using the standard method.²⁶ 3 mL solution (1 mg mL⁻¹) of each surfactant was added to culture medium containing aerated bacterial cells in 300 mL B.O.D. bottles (dilution factor = 1000). To this was added manganous sulphate and alkali azide. The precipitate formed was then dissolved in sulphuric acid. Thereafter, this solution was titrated with 0.1 N sodium thiosulphate. The initial amount of dissolved oxygen was found by titrating immediately and the final amount was determined through titration after incubation (at 27 °C) for three days.

Cleavability test. The cleavability test was carried out by using sodium hydroxide/sodium hydrogen phosphate buffer (pH = 12)²⁷ and their FT-IR spectral data were recorded after at least 8 h. Further to support the FT-IR results, molecular docking was performed by using the literature method.²⁸

Results and discussion

CMC and other interfacial parameters through surface tension measurements

Generally, at lower concentrations, surfactants have the tendency to decrease the surface tension of water by their adsorption at air/solution interface, whereas at higher concentrations, they fully saturate it and any further addition leads to self-association of monomers into aggregated structures, called micelles. The particular concentration at which these micelles appear is called critical micelle concentration (CMC) and it is observed as a break in the surface tension isotherm. Beyond the CMC, with any further increase in surfactant concentration, there is no change in surface tension.

The surface tension profiles of C_m-E2O-C_m (C₁₂-E2O-C₁₂, C₁₄-E2O-C₁₄ and C₁₆-E2O-C₁₆) gemini surfactants are shown in Fig. 1, from which the CMC values were calculated (Table 1). It can be seen (Fig. 1, Table 1) that the C_m-E2O-C_m surfactants have dramatically lower CMCs, about 100 times lesser than the corresponding conventional surfactants^29,30 and 10 times lower than other functionalized gemini surfactants.^{5,14,15,23,31–34} This can be ascribed to the presence of oxy-diester moiety (E2O) which facilitates aggregation of surfactant monomers via hydrogen bonding. Moreover, this dramatic decrease in CMC brings them to the “green surfactant” category as a very small amount of gemini will be sufficient for the same level of performance in any application. Among the three C_m-E2O-C_m geminis, the CMC of C₁₆-E2O-C₁₆ (0.0062 mM) is lower than C₁₄-E2O-C₁₄ (0.0078 mM) and C₁₂-E2O-C₁₂ (0.0100 mM). This observed trend can be understood by considering the positive and negative contributing factors in the self-aggregation process. Out of these two factors, the primary driving force in self-aggregation is the hydrophobic interaction associated with the alkyl chains.³⁵ Due to this, there is an increase in the entropy of the system, which consequently reduces the CMC. In addition, this trend is supported by the reports given by Menger et al. and Negm et al.^36,37

Fig. 1 Surface tension profiles of C_m-E2O-C_m gemini surfactants at 30 °C.

Table 1 Physicochemical parameters of C_m-E2O-C_m gemini surfactants at 30 °C

Gemini surfactant	CMC (mM)	γCMC (mN m^−1)	ΠCMC (mN m^−1)	Γmax (×10^7 mol m^−2)	Amin (Å^2 per molecule)	ΔG^omic (kJ mol^−1)	ΔG^oads (kJ mol^−1)
a CMC determined by surface tension.b CMC determined by fluorescence measurement.
C12-E2O-C12	0.0100,^a 0.0102^b	41.0	31.0	9.44	175.88	−39.12	−39.30
C14-E2O-C14	0.0078,^a 0.0074^b	43.5	28.5	13.77	120.57	−39.74	−39.98
C16-E2O-C16	0.0062,^a 0.0059^b	39.0	33.0	17.16	96.75	−40.32	−40.66

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To get further insight into the interfacial adsorption of C_m-E2O-C_m geminis, interfacial parameters at air/solution interface were calculated and the results are given in Table 1. The surface pressure (Π_CMC) which is a measure of the effectiveness of surface tension reduction at CMC can be calculated by using eqn (1):

Π_CMC = γ₀ − γ_CMC (1)

where γ₀ and γ_CMC, respectively, represent the surface tension of pure water and the surface tension of surfactant solution at CMC.

Γ_max is the maximum concentration of surfactant molecules at air/solution interface in the saturated state and it is calculated by applying the Gibbs adsorption isotherm equation:³⁸

here, γ denotes the surface tension, R is the gas constant, T is the absolute temperature, C is the surfactant concentration and n is a constant which depends on the number of individual ions comprising the surfactant adsorbed at the interface. For bis(quaternary ammonium) surfactants, the value of n is taken as 3.^39–41 In earlier reports, to determine the surface excess, linear fitting of the pre-CMC data was done. This is a less accurate method, on account of well known Gibbs paradox. The mathematical solution to this problem is polynomial fitting.⁴² Thus, here we have applied second order polynomial fitting in the pre-CMC region.

The minimum area occupied per surfactant molecule (A_min) at the air/solution interface is obtained by using the following equation:⁴³

where N is the Avogadro's number. The values of Γ_max and A_min are given in Table 1. These surfactants have low A_min values indicating a tight packing of the surfactant monomers at the interface. Moreover, negative values of Gibbs free energy of adsorption and micellization, obtained by employing eqn (4) and (5), suggest spontaneity of the C_m-E2O-C_m adsorption and micellization:^27,44,45

ΔG^o_mic = RT ln X_CMC (4)

Evaluation of CMC and aggregation number by fluorescence measurements

To support the CMC results obtained in tensiometry, we have also estimated CMC by fluorescence spectroscopy using pyrene as a probe. Pyrene shows five predominant vibrational peaks in its fluorescence emission spectrum. The ratio of fluorescence emission intensities corresponding to the first and third vibrational peaks (I₁/I₃) is a sensitive parameter characterizing the polarity of the probe's environment.⁴⁶ Generally, higher I₁/I₃ ratio indicates polar environment whereas lower I₁/I₃ ratio depicts non-polar environment.

The plot of I₁/I₃ versus log[C_m-E2O-C_m] (Fig. 2(a)) gives a reverse sigmoid curve. Interestingly, it can be observed from the Fig. 2(a) that at lower surfactant concentrations, I₁/I₃ ratio is very high which indicates that pyrene resides in the hydrophilic environment (of water). Thereafter, increase in concentration of gemini surfactants causes decrease in I₁/I₃ ratio, indicating that the pyrene experiences a more hydrophobic environment (exerted by the surfactant monomers). Near the CMC, the curves show a sharp decrease in the micropolarity ratio (I₁/I₃) and the point of maximum inflection is taken as CMC. Beyond the CMC, I₁/I₃ ratio becomes roughly constant because of the solubilization of hydrophobic pyrene molecules in the hydrophobic core of the micelles.⁴⁷ The observed order of CMC values for the three geminis was: C₁₂-E2O-C₁₂ > C₁₄-E2O-C₁₄ > C₁₆-E2O-C₁₆ and their values are given in Table 1. All the CMC values obtained from fluorescence and surface tension measurements are in good agreement with each other and the same trend of decrease in CMC with the increase in alkyl chain length is observed. Moreover, the lower CMC values obtained in C₁₆-E2O-C₁₆ can be attributed to the decreased penetration of water molecules into the micelles of higher homologues of the cationic diester-bonded gemini series. As a result of this, the probability of experiencing the polar environment by pyrene is minimum in C₁₆-E2O-C₁₆ micelles and maximum in C₁₂-E2O-C₁₂.

Fig. 2 Plots of (a) I₁/I₃ versus log of concentration of C_m-E2O-C_m gemini surfactants and (b) ln(I₀/I) versus concentration of quencher.

The aggregation number (N_g) of C_m-E2O-C_m gemini surfactants was determined through steady-state fluorescence,⁴⁸ employing eqn (6):

where I₀ and I are the intensities of fluorescence emission (at 384 nm) in the absence and presence of the quencher, respectively. [Q] corresponds to the concentration of the quencher, C is the total surfactant concentration (1 mM) and CMC is the critical micelle concentration at the given concentration of quencher. The dependence of ln(I₀/I) on [Q] for the C_m-E2O-C_m geminis is shown in Fig. 2(b) through which N_g was evaluated and the trend in aggregation number was observed as: C₁₆-E2O-C₁₆ (49) > C₁₄-E2O-C₁₄ (46) > C₁₂-E2O-C₁₂ (39). As expected, N_g increases with the lengthening of alkyl chain. Here again, increased hydrophobicity of higher homologues may be attributive.⁴⁹

Krafft point

The Krafft temperature is an essential parameter of surfactants as it provides information relevant to their solubility. It is an accepted fact that the surfactants with lower Krafft points have higher solubility and vice versa. In our case, the Krafft points observed were same (i.e., 4 °C) for all the three C_m-E2O-C_m geminis. This indicates that solubilities of the three synthesized surfactants are similar and remain almost unaltered by an increase in the tail length. Moreover, substantial solubility of the three geminis can be inferred from their low Krafft points (slightly above 0 °C). This enhanced solubility may be ascribed to the polar ester groups and ethylene oxide moiety of the spacer, which form hydrogen bonds with water. The values of Krafft temperature are much lower than other synthesized geminis.^50–52 Thus, C_m-E2O-C_m geminis are potential candidates for various applications (e.g., spreading, wetting, detergency, etc.) at low temperatures.

Foaming ability

From the thermodynamic point of view, foams are highly unstable systems and their volume/height is related to foam stability. The foam height and corresponding foam stability of C_m-E2O-C_m geminis are given in Table 2. It can be observed that with an increase in the chain length of the gemini surfactants, foam stability increases; reason being the lower drainage/diffusion of gas from small bubbles to larger ones in the foams of C₁₆-E2O-C₁₆ than those of C₁₄-E2O-C₁₄ and C₁₂-E2O-C₁₂.

Table 2 Foam stability of C_m-E2O-C_m gemini surfactants

Gemini surfactant	Concentration (mg mL^−1)	Foam volume at 0 min (mL)	Foam volume after 10 min (mL)	Foam stability (%)
C12-E2O-C12	5	5.6	0	0
C14-E2O-C14	5	5.2	5.0	96
C16-E2O-C16	5	4.0	4.0	100

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Specific viscosity

To support the results of foaming ability test, specific viscosities of all the three geminis were determined. Surfactant solutions of higher viscosities are considered to generate foams with higher stability due to the lower chances of foam drainage. In the present case, the observed specific viscosity (0.0642) of C₁₆-E2O-C₁₆ was more than that of C₁₄-E2O-C₁₄ (0.0458) or C₁₂-E2O-C₁₂ (0.0366). Thus, the order of their viscosities was: C₁₆-E2O-C₁₆ > C₁₄-E2O-C₁₄ > C₁₂-E2O-C₁₂.

Emulsifying power

Further relevance to the above results was added by determining emulsifying power as indicated by the time (s) required for the separation of toluene/water emulsion (in the presence of the three geminis). The trend observed was: C₁₆-E2O-C₁₆ (1100 s) > C₁₄-E2O-C₁₄ (607 s) > C₁₂-E2O-C₁₂ (480 s). The reason for this order can be attributed to the higher chain length of C₁₆-E2O-C₁₆. Higher chain length geminis produce more viscous solutions, which, in turn, mitigates the chances of diffusion and other relevant phenomenon in micro-emulsions, leading to increase in micro-emulsion stability. The lower sedimentation rate of higher chain length geminis also accounts for higher emulsion stability.

Biological properties

Hemolytic assay. Low cytotoxicity of an amphiphile towards animal cell is very crucial for their potential application in drug delivery. Cytotoxicity of the synthesized geminis have been assessed through HC₅₀ analysis. HC₅₀ can be defined as the surfactant concentration that causes 50% hemolysis relative to its positive control⁵³ and it is a measure of cytotoxicity of a compound (surfactant). Higher the HC₅₀ value of a compound, lower will be its cytotoxicity. The HC₅₀ values obtained for the concerned geminis are given in Table 3. Clearly, it can be seen that C₁₂-E2O-C₁₂ is less hemolytic than C₁₄-E2O-C₁₄ and C₁₆-E2O-C₁₆ (as indicated by its higher HC₅₀). In agreement with the data available on hemolytic activity of other gemini surfactants, the capability of lysing red blood cells increases with increase in alkyl chain length.⁵⁴ Besides, when compared to conventional (0.05–0.1 mg L⁻¹) and other geminis (0.19 mg L⁻¹ or 8.7–110 mg L⁻¹), the synthesized gemini surfactants (235–305 mg L⁻¹) have lower cytotoxicity.^22,27 The presence of E2O moiety imparts polarity and is responsible for the increase in the hydrophilic character of the molecules, which in turn reduces the cytotoxicity of these gemini surfactants. Owing to their low toxicity to membranes, these concerned geminis can serve as good candidates for drug formulations.

Table 3 HC₅₀ values of C_m-E2O-C_m gemini surfactants

Gemini surfactant	HC50 (mg L^−1)
C12-E2O-C12	305.40
C14-E2O-C14	261.55
C16-E2O-C16	235.08

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Biodegradability assessment. Biodegradation is basically an oxidation process. Therefore, to evaluate the biodegradability of a substance, e.g., a surfactant we can determine the amount of oxygen required for its biodegradation i.e., its Biochemical Oxygen Demand (B.O.D.). B.O.D. is actually a measure of the amount of oxygen consumed by micro-organisms for the oxidation of organic part of the substance. In the laboratory, B.O.D. of a surfactant can be determined by the estimation of the amount of dissolved oxygen consumed from the closed bottle containing well-aerated water, fixed amount of surfactant sample and a small proportion of bacterial seeds along with the nutrients.

The B.O.D. values of C_m-E2O-C_m gemini surfactants were calculated by using the literature method,²⁶ which were then used for determining the percentage of biodegradability employing the equation given below:

Percentage (%) biodegradability of C_m-E2O-C_m gemini surfactants are given in Table 4, which indicate that their biodegradation rates are quite high (50–60% in 3 days). Furthermore, on comparing their % biodegradability with that of earlier synthesized geminis,^19,27,55 we can conclude that these concerned gemini surfactants have much higher biodegradability. This observation is at par with our expectations, owing to the presence of easily cleavable diester groups and polar ethylene oxide unit, which makes the concerned geminis highly susceptible to the attack of micro-organisms. Thus, the obtained results show that these new cationics can be potentially used for the formulation of high performance and eco-friendly detergents, paints, coatings, fabric softeners, etc.

Table 4 Biodegradability (%) of C_m-E2O-C_m gemini surfactants

Gemini surfactant	Biodegradability (%)
C12-E2O-C12	59.85
C14-E2O-C14	51.65
C16-E2O-C16	52.19

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Cleavable properties

The presence of diester groups and ethylene oxide moiety in the spacer part of the cationic gemini surfactants suggests that these might be cleavable through chemical means in alkaline condition. In the absence of alkaline medium, all the gemini surfactants were found to be stable (Fig. 3(a–c)). After alkaline treatment (pH 12), it is clear that typical ester peaks of all the three title geminis shifted to lower wave number regions (Fig. 3(d–f)). In addition, an increased intensity of the signals at 3200–3600 cm⁻¹ delineates the formation of easily degradable compounds (such as salts of carboxylic acids and respective diols or compounds with hydroxyl group). This confirms the cleavability of the synthesized gemini surfactants.

Fig. 3 FT-IR spectra of C_m-E2O-C_m gemini surfactants (a–c) in the absence and (d–f) in the presence of alkaline medium.

The lability in alkaline medium may be due to two reasons (1) inductive effect induced by the adjacent positively charged nitrogen (2) anchimeric assistance (neighboring group participation) provided by the second nitrogen. Both of these factors in combination make the carbonyl carbon of ester group sufficiently electron deficient for the nucleophilic attack by OH⁻. In addition, polar oxygen of ethylene oxide unit further reduces the electron density at carbonyl ester. To confirm this mechanism, we performed molecular docking of the C_m-E2O-C_m gemini surfactants.

The optimized conformations of the three gemini surfactants obtained through energy minimization are given in Fig. 4. The bond distances between the carbonyl carbon of ester group and each of the two nitrogens have also been calculated. In all the three geminis, the distances from the carbonyl carbon (ester) to the nearby nitrogen and to the other nitrogen are small (3 Å and 11 Å respectively), favoring both inductive effect and anchimeric assistance. The proximity of the other nitrogen to carbonyl is due to the betaine ester type arrangement, where carbonyl faces the nitrogen of head group (in contrast to esterquat gemini) which in turn promotes anchimeric assistance.¹⁹ These values are comparable to those of literature³¹ where similar reasons have been held accountable for the cleavability of ester-bonded geminis.

Fig. 4 Optimized conformations of (a) C₁₂-E2O-C₁₂, (b) C₁₄-E2O-C₁₄ and (c) C₁₆-E2O-C₁₆ gemini surfactants (nitrogen, oxygen and carbon atoms are represented by blue, red and white respectively).

Conclusions

In this work, we have proposed a simple strategy to synthesize high performance ethylene oxide-linked diester-functionalized gemini surfactants. The new structural hybrid of biocompatible diesters and polar ethylene oxide moiety in the spacer has significantly affected their bio-physicochemical properties. Interestingly, the synthesized geminis have extraordinarily lower CMCs. The order of CMC was found to be C₁₂-E2O-C₁₂ > C₁₄-E2O-C₁₄ > C₁₆-E2O-C₁₆, which is consistent with the increasing hydrophobicity. Other physicochemical properties (Krafft point, foam stability, specific viscosity, and emulsifying power) are quite appreciative and in relevance to each other. They have a quite high rate of degradation (about 50–60% in 3 days) and thus, it will not be an exaggeration to entitle them as ‘green surfactants’. Moreover, they have low cytotoxicity, since they possess easily cleavable E2O moiety. As compared to corresponding mono-chain surfactants and other geminis, these new cationics have 10–100 times lower CMC values.^{5,14,15,29–34} Their Krafft temperature is about 4 °C, which is much lower than other synthesized gemini surfactants.^50–52 They have far better biodegradability and lower cytotoxicity than earlier synthesized geminis.^19,22,27,55

The above mentioned outstanding bio-physicochemical properties make them suitable and promising candidates to be utilized in various fields (such as biomedical, industrial, environmental, etc.). Hence, their versatility gives them the potential to replace the conventional monomeric and dimeric surfactants. In future, these new geminis can serve as a model for engineering the properties of surfactants in a more efficient way.

Acknowledgements

F.A. and I.A.B. are highly thankful to UGC for BSR fellowship. S.A. acknowledges the financial support by UGC. The Environmental Laboratory, Civil Engineering Department, A.M.U. is acknowledged for B.O.D. test and Panjab University, Chandigarh is highly acknowledged for characterization of compounds. UGC (SAP, DRS-II) and DST (PURSE) are also acknowledged for providing assistance to our department.

References

1. A. A. Dar, G. M. Rather and A. R. Das, J. Phys. Chem. B, 2007, 111, 3122.
2. Kabir-ud-Din, M. Shafi, P. A. Bhat and A. A. Dar, J. Hazard. Mater., 2009, 167, 575.
3. W. H. Ansari, N. Fatma, M. Panda and Kabir-ud-Din, Soft Matter, 2013, 9, 1478.
4. A. J. Kirby, P. Camilleri, J. B. F. N. Engberts, M. C. Feiters, R. J. M. Nolte, O. Söderman, M. Bergsma, P. C. Bell, M. L. Fielden, C. L. G. Rodríguez, P. Guedat, A. Kremer, C. McGregor, C. Perrin, G. Ronsin and M. C. P. van Eijk, Angew. Chem., Int. Ed., 2003, 42, 1448–1457.
5. V. Kumar, A. Chatterjee, N. Kumar, A. Ganguly, I. Chakraborty and M. Banerjee, Carbohydr. Res., 2014, 397, 37–45.
6. S. M. Tawfik, J. Mol. Liq., 2015, 209, 320.
7. J. Zhang, Y. Zheng, P. Yu, L. He, H. Wang and R. Wang, J. Surfactants Deterg., 2010, 13, 155.
8. U. Siddiqui, J. Aslam, W. H. Ansari and Kabir-ud-Din, Colloids Surf., A, 2013, 421, 164.
9. M. Donkuru, S. D. Wettig, R. E. Verrall, I. Badeaa and M. Foldvari, J. Mater. Chem., 2012, 22, 6232.
10. M. Akram, S. Yousuf, T. Sarwar and Kabir-ud-Din, Colloids Surf., A, 2014, 441, 281.
11. M. Stjerndahl, C. G. van Ginkel and K. Holmberg, J. Surfactants Deterg., 2003, 6, 319.
12. C. F. J. Kuo, L. H. Lin, M. Y. Dong, W. S. Chang and K. M Chen, J. Surfactants Deterg., 2010, 14, 195.
13. A. Bhadani, T. Endo, K. Sakai, H. Sakai and M. Abe, Colloid Polym. Sci., 2014, 292, 1685.
14. S. D. Wettig, X. Li and R. E. Verrall, Langmuir, 2003, 19, 3666.
15. V. Chauhan, S. Singh, R. Mishra and G. Kaur, J. Colloid Interface Sci., 2014, 436, 122.
16. T. Banno, K. Kawada and S. Matsumura, J. Surfactants Deterg., 2010, 13, 387.
17. V. Klimavicius, Z. Gdaniec, J. Kausteklis, V. Aleksa, K. Aidas and V. Balevicius, J. Phys. Chem. B, 2010, 117, 10211.
18. S. A. Katsyuba, M. V. Vener, E. E. Zvereva, Z. Fei, R. Scopelliti, G. Laurenczy, N. Yan, E. Paunescu and P. J. Dyson, J. Phys. Chem. B, 2013, 117, 9094.
19. A. R. Tehrani-Bagha, H. Oskarsson, C. G. van Ginkel and K. Holmberg, J. Colloid Interface Sci., 2007, 312, 444.
20. M. Obrzud, M. Rospenk and A. Koll, Phys. Chem. Chem. Phys., 2014, 16, 3209.
21. C. Narayanan and C. L. Dias, J. Chem. Phys., 2013, 139, 115103.
22. L. Perez, M. T. Garcia, I. Ribosa, M. P. Vinardell, A. Manresa and M. R. Infante, Environ. Toxicol. Chem., 2002, 21, 1279.
23. S. Zhang, S. Ding, J. Yu, X. Chen, Q. Lei and W. Fang, Langmuir, 2015, 31, 12161.
24. A. Colomer, A. Pinazo, M. T. Garcia, M. Mitjans, M. P. Vinardell, M. R. Infante, V. Martinez and L. Perez, Langmuir, 2012, 28, 5900.
25. G. Zhinong, T. Shuxin, Z. Qi, Z. Yu, L. Bo, H. Li and T. Xiaoyan, Wuhan Univ. J. Nat. Sci., 2008, 13, 227.
26. APHA, AWWA and WEF, Standard Methods for the Examination of Water and Wastewater, Washington, D.C, 21st edn, 2005.
27. N. Fatma, M. Panda, W. H. H. Ansari and Kabir-ud-Din, J. Mol. Liq., 2015, 211, 247.
28. M. Akram, I. A. Bhat, S. Anwar and Kabir-ud-Din, J. Mol. Liq., 2015, 212, 641.
29. A. R. Tehrani-Bagha and K. Holmberg, Materials, 2013, 6, 580.
30. H. Naorem and S. D. Devi, J. Surf. Sci. Technol., 2006, 22, 89.
31. A. R. Tehrani-Bagha and K. Holmberg, Langmuir, 2010, 26, 9276.
32. R. Dominguez, A. Rodriguez, A. Maestre, I. Robina and M. L. Moya, J. Colloid Interface Sci., 2012, 386, 228.
33. A. Bhadani and S. Singh, Langmuir, 2011, 27, 14033.
34. M. Ao, G. Xu, Y. Zhu and Y. Bai, J. Colloid Interface Sci., 2008, 326, 490.
35. C. Tanford, The Hydrophobic Effect: Formation of Micelles and Biological Membranes, Wiley, New York, 2nd edn, 1980.
36. F. M. Menger and B. N. A. Mbadugha, J. Am. Chem. Soc., 2001, 123, 875.
37. N. A. Negm and A. S. Mohamed, J. Surfactants Deterg., 2008, 11, 215.
38. D. K. Chattoraj and K. S. Birdi, Adsorption and the Gibbs Surface Excess, Plenum Press, New York, 1984.
39. E. Alami, G. Beinert, P. Marie and R. Zana, Langmuir, 1993, 9, 1465.
40. L. Wang, Y. Zhang, L. Ding, J. Liu, B. Zhao, Q. Deng and T. Yan, RSC Adv., 2015, 5, 74764.
41. T. Patra, S. Ghosh and J. Dey, J. Colloid Interface Sci., 2014, 436, 138.
42. I. Mukherjee, S. P. Moulik and A. K. Rakshit, J. Colloid Interface Sci., 2013, 394, 329.
43. M. J. Rosen, Surfactants and Interfacial Phenomenon, Wiley-Interscience, John Wiley & Sons, New York, 3rd edn, 2004.
44. M. Akram, I. A. Bhat and Kabir-ud-Din, Int. J. Biol. Macromol., 2015, 78, 62.
45. M. Akram, I. A. Bhat, W. F. Bhat and Kabir-ud-Din, Spectrochim. Acta, Part A, 2015, 150, 440.
46. G. B. Ray, I. Chakraborty and S. P. Moulik, J. Colloid Interface Sci., 2006, 294, 248.
47. A. Domínguez, A. Fernández, N. González, E. Iglesias and L. Montenegro, J. Chem. Educ., 1997, 74, 1227.
48. N. J. Turro and A. Yekta, J. Am. Chem. Soc., 1978, 100, 5951.
49. S. Noori, A. Z. Naqvi, W. H. Ansari, M. Akram and Kabir-ud-Din, J. Surfactants Deterg., 2014, 17, 409.
50. R. Kamboj, S. Singh, A. Bhadani, H. Kataria and G. Kaur, Langmuir, 2012, 28, 11969.
51. D. A. Jaeger, Y. Wang and R. L. Pennington, Langmuir, 2002, 18, 9259.
52. J. V. Trillo, F. Meijide, J. V. Tato, A. Jover, V. H. Soto, S. de Frutos and L. Galantini, RSC Adv., 2014, 4, 6869.
53. M. Rasia, M. I. Spengler, S. Palma, R. Manzo, P. L. Nostro and D. Allemandi, Clin. Hemorheol. Microcirc., 2007, 36, 133.
54. M. Mitjans, V. Martinez, P. Clapes, L. Perez, M. R. Infante and M. P. Vinardell, Pharm. Res., 2003, 20, 1697.
55. K. A. Wilk, L. Syper, B. W. Domagalska, U. Komorek, I. Maliszewska and R. Gancarz, J. Surfactants Deterg., 2002, 5, 235.

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra28129f

‡ Present address: Department of Chemistry, Arba Minch University, Ethiopia.

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