Bile salt assisted morphological changes of cationic gemini surfactant (12-4-12) micelles

Abstract

Microstructural evolution of a cationic gemini surfactant butanediyl-1,4-bis(dodecyldimethylammonium bromide) (hereafter referred to as 12-4-12) micelles in the presence of two bile salts viz. sodium deoxycholate (NaDC) and sodium cholate (NaC) was investigated using surface tension, viscosity, nuclear magnetic resonance (NMR), and small angle neutron scattering (SANS) measurements. A negative value of interaction parameter (β) evaluated from surface tension measurements is a signature of strong synergistic interaction between oppositely charged surfactants. Other micellar parameters have also been calculated at different mole fractions. Both the bile salts induced a shape transitions in 12-4-12 micelles on account of their hydrophobicity. Viscosity measurements disclose that loading of bile salts induces morphological changes in gemini micelles; NaDC is more efficient in altering the aggregation behaviour compared to NaC and presents a pronounced increase in viscosity and micellar growth which is suppressed at elevated temperatures. A remarkable growth observed in micelles in the presence of NaDC at low pH has been ascribed to the solubilization of bile acids formed in acidic medium. The size and shape of gemini mixed micelles obtained from SANS measurements are explicated on the basis of the hydrophobicity of bile salts. The location of bile salts in the micelle was determined from NOESY. The present study characterizes gemini–bile salt mixed systems which significantly enriches our knowledge and such a structural transition provides an opportunity to use these bioamphiphiles (bile salt containing mixed micelles) as delivery vehicles and in some pharmaceutical formulations.

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1. Introduction

Bile salts are imperative biological amphiphiles that contain a nonpolar planar steroid ring on a convex β-face as a hydrophobic part and carboxylic and polar hydroxyl (–OH) groups as a hydrophilic part on a concave α-face. Such a distinct structure offers surface activity and aggregation behaviour to bile salts in aqueous solutions. However, the formation of bile salt aggregates is somewhat diverse from those of ordinary surfactants; at low bile salt concentration primary micelles having low aggregation number do form as a result of hydrophobic interaction. At higher concentration, hydrogen bonding between the –OH groups and side chain groups of bile salt primary micelles are held together and form secondary micelles.^1–3 The acceptable CMCs of NaC and NaDC in water are ∼16 and ∼6 mM, respectively.⁴ The presence of one extra –OH group in NaC is responsible for higher aqueous solubility and consequently higher CMC compared to NaDC.⁵ Bile salts play physiological role in fat digestion and solubilization of cholesterol and other lipolytic products.

Quaternary ammonium type cationic surfactants in addition to their interfacial activity show antibacterial, antimicrobial, antistatic activity and corrosion inhibiting action which find their numerous applications in the diverse fields.^6–8 Gemini type cationic surfactants (dimeric) contain two polar heads and two nonpolar tails (identical or different) joined through a flexible hydrocarbon chain of variable length or rigid moiety known as spacer.^9,10 These surfactants have gained much interest due to their low CMC, improved solubilization capacity, interfacial activity, efficient adsorption and unusual viscosity as compared to conventional single chain surfactants and are therefore considered as surfactants with improved performance.^11–13 Gemini surfactants of type 12-s-12 (s is oligomethylene spacer) have been extensively examined for their surface activity and micelle formation.^9,10,14,15 Gemini surfactants find prospective applications in cleaning agents, cosmetics and personal care products, enhanced oil recovery, gene transfection, DNA extraction, drug delivery agents and protein refolding.^11,16

Mixed surfactants are often preferred in finished formulations of technological interest due to their superior surface activity and micellization tendency over their single constituents. Surfactants with similar charge viz. cationic–cationic, anionic–anionic and nonionic–nonionic generate weak interaction while strong interaction has been observed for oppositely charged surfactants. However, this strong interaction limits their use due to the formation of precipitate/coacervates over some concentration range particularly close to equimolar. The interaction of bile salts with different surfactants viz. dodecydimethyl-amine oxide,¹⁷ Triton X-100,^18,19 Tweens,^20,21 Pluronics®,^22,23 sodium oleate²⁴ and Brij²⁵ has been reported. Mixed systems containing bile salts as the anionic component and cationic surfactants have also been studied. The innovative work on mixed systems from bile salts and cationic surfactants was done by Barry and Gray.^26,27 Jiang et al.²⁸ reported that incorporation of bile salts favors vesicle to micelle transition in catanionic surfactant–bile salt mixed systems in which steric interaction is found to be decisive factor in inducing such morphological changes. Interaction of bile salts with conventional cationic surfactants has been studied by Bahadur and coworkers.^29–31

Microstructures of alkyltrimethylammonium bromide with varying chain length in the presence of NaC have been investigated earlier. Addition of NaC to micellar solution of cetyltrimethylammonium bromide (C₁₆TAB) in the presence of sodium chloride shows concentration aided morphological change which is otherwise not shown by its lower homologues.³² A study on NaDC induced micelle to rod/vesicle transition in aqueous solution of CTAB has been reported.³³ George et al.³⁴ and Jana et al.³⁵ have proposed that bile salts interact with CTAB strongly depending on composition of surfactants. Manna et al.³⁶ have reported the microstructural changes for mixed system of C_nmimBr (n = 12, 14, 16) and bile salts; NaDC being more hydrophobic, pierces inside micelles and forms rod-like micelles while NaC with an extra –OH group prefers to stay on micelle interface giving rise to formation of spherical micelles.

Studies showing the interaction of bile salts with gemini surfactants reported by Wang et al.³⁷ clearly reveal strong synergistic interaction. The formation of bile salt-rich mixed micelles, precipitates and their dissolution and formation of gemini surfactant-rich mixed micelles was observed as a function of gemini surfactant concentration. Akram et al.³⁸ from mixed system of bile salt and biodegradable diester bearing cleavable gemini surfactants found that NaC with one extra –OH group produces preferentially stronger synergistic interactions compared to NaDC by forming H-bond with the diester group of gemini surfactant. Such H-bonding results in more flexible gemini surfactant–NaC mixed micelles. The hydrodynamic diameter of aggregates manifests that attractive interaction between gemini surfactant and NaC gives rise to the formation of vesicles leading to conclusion that NaC displays pronounced micelle growth with gemini surfactant compared to NaDC. Mixed micelle formation between amino acid based-gemini surfactant and NaC and NaDC has also been studied.³⁹ A negative value of interaction parameter clearly demonstrates synergistic interaction between gemini and bile salts which changes to antagonistic one at higher gemini composition. Such interactions are found to be due to hydrophobic effects and hydrogen bonding. Recently, Li et al.⁴⁰ reported the phase behaviour of a gemini surfactant 12-2-12 and NaDC and also examined the effect of temperature and pH. Worm-like micelles formed at low NaDC concentration transformed to ellipsoidal micelles at higher temperature while lamellar aggregates are formed at higher NaDC concentration which can be transformed to micelles by increasing temperature. It was proposed that tuning the pH of solution drives worm-like micelle to lamellar shaped aggregates. Such a transition is associated with the formation of deoxycholic acid molecules by protonation of NaDC followed by their subsequent solubilization in worm-like micelles.

Despite available literature on gemini–bile salt system, there is dearth of systematic study showing bile salt-induced nanostructural changes in cationic gemini surfactant micelles which can be further tuned by varying pH. In this context, herein we report a study describing the effect of NaDC and NaC in inducing micellar transition of 12-4-12 micelles as a function of concentration and pH induced micellar growth of 12-4-12–bile salt mixed micelles. Both these bile salts are very much similar in structure except one more –OH group in NaC still they exhibit peculiar behavior with 12-4-12 micelles which encouraged us for a detailed investigation. The intention of the present research was to perform a systematic study on the interaction of bile salts with 12-4-12 and to examine influence of external factors like temperature and pH. A special attention is paid on pH induced morphological changes in mixed micelles. To the best of our knowledge, such a comparative study portraying bile salt induced dissimilar morphological changes in gemini surfactant micelles is of its first kind in gemini surfactant–bile salt mixed system.

2. Experimental

2.1 Materials

12-4-12 was synthesized using the reported procedure.^11,12,41 The purity of 12-4-12 was checked by ¹H NMR and surface tension measurement. An absence of minimum in surface tension plot and extra peak in ¹H NMR spectrum clearly designates the absence of any impurity. The CMC obtained from surface tension measurement is ∼1.19 mM which meets to reported value.¹² Sodium deoxycholate (NaDC) and sodium cholate (NaC) were purchased from Sigma Aldrich and Fluka, respectively and used without further purification. Solutions for SANS and NMR were prepared in D₂O (99%). Deionized water from a Millipore Milli-Q system was used to prepare solutions for viscosity measurements and then kept on shaker for few hours for homogeneity. Prior to the measurements, samples were kept for 48 hours to attain equilibrium. The details of surfactants used for the study are presented in Table 1.

Table 1 Molecular structures of gemini surfactant and bile salts

Surfactant	CMC mM	Chemical structure
		NaDC	NaC	12-4-12
a Data taken from ref. 12.b Data taken from ref. 4.
12-4-12	∼1.2^a
NaDC	∼6^b
NaC	∼16^b

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2.2 Methods

2.2.1 Surface tension. The surface tension measurements were carried out on Kruss Model K9 Tensiometer, Germany. All the experiments were performed at constant temperature 30 °C (±0.1 °C). The surface tension of Millipore water was 71.4 mN m⁻¹.

2.2.2 Viscosity. Calibrated Cannon Ubbelohde viscometers were used to get the viscosity of solutions. Viscometers are suspended vertically in a thermostat with a temperature stability of ±0.1 °C. Relative viscosities of the solutions were obtained as per detailed procedure is given in literature.⁴² Flow times are reproducible. Viscosity measurements as a function of concentration and pH were performed at 30 °C. For temperature dependant study, viscosities of solutions were measured by varying temperature.

2.2.3 Small angle neutron scattering (SANS). SANS experiments were performed at the SANS-I diffractometer at the Guide Tube Laboratory, Dhruva reactor, BARC, Mumbai, India. The solutions were prepared in D₂O (99%). The mean incident wavelength was 5.2 Å with Δλ/λ = 10%. The scattering data were measured in the wave vector transfer (Q = 4π sin(θ/2)/λ, where θ is scattering angle) range of 0.017 to 0.35 Å⁻¹. The measured SANS data were corrected for the background, empty cell contributions and transmission and normalized to absolute cross-sectional unit using standard protocols. All the SANS measurements were performed at 30 °C.

2.2.4 Nuclear magnetic resonance (NMR). ¹H NMR and nuclear overhauser enhancement spectroscopy (NOESY) experiments were performed on a Bruker Avance-II 400 MHz NMR spectrometer at St. Francis Xavier University, Antigonish, Canada. The spectrum was calibrated by setting the HDO peak at a chemical shift of 4.65 ppm at 298 K. Solvent suppression eliminated the HDO peak due to residual water. For the NOESY experiments the mixing times and the delay times were estimated from the spin-lattice relaxation times. In all cases, an acquisition delay of ≫3 × T₁ and a mixing time of ≫1 × T₁ were used to obtain the NOESY spectra. All samples were prepared in D₂O and measurements were performed at 30 °C.

3. Results and discussion

3.1 Critical micelle concentration

12-4-12 is a cationic gemini surfactant having lower CMC, higher solubilization efficiency and surface activity than conventional surfactants. In order to retrieve information on interaction between 12-4-12 and bile salts, surface tension measurements were performed for single surfactants viz. 12-4-12, NaDC, NaC, and mixed 12-4-12 + bile salt system at different composition and results are presented in Fig. 1. The CMCs of 12-4-12, NaDC and NaC at 30 °C were found to be 1.19 mM, 5.2 mM and 12.3 mM, respectively.^12,43 Fig. 1 represent the surface tension–log concentration plot for individual surfactants and their mixtures. The CMC ∼1.19 mM, degree of counter dissociation ∼0.29 and area per molecule (A_min) ∼110 m² for 12-4-12 are consistent with the reported value.^44,45

Fig. 1 Surface tension plots for 12-4-12 solution in the presence of (a) NaDC and (b) NaC at various mole fractions () 0.0 () 0.25 () 0.5 () 0.75 () 1.0 at 30 °C.

3.2 Interaction and interaction parameter (β)

Fig. 1 clearly reveals that both the bile salts (NaDC and NaC) are capable of lowering the surface tension of 12-4-12 solution. The CMCs of mixed surfactant systems were measured from surface tension measurements at 30 °C. The equations and procedure used to calculate different parameters are reported in literature.⁴⁶ All the parameters are presented in Table 2. Generally, a strong interaction is observed for oppositely charged mixed surfactants. The synergistic interaction between 12-4-12 and bile salts was confirmed from lower experimental CMC (CMC_exp.) than ideal CMC (CMC_ideal) (negative deviation). Extent of interaction between 12-4-12 and bile salts can also be linked with the value of interaction parameter (β) shown in Table 2. Accordingly, a large negative value of (β) clearly demonstrates the strong synergistic interaction between cationic 12-4-12 and negatively charged bile salts. Such a synergism is mainly due to electrostatic interaction. Due to more hydrophilic nature, NaC compared to NaDC, it generates strong synergistic interactions with 12-4-12 as evident from more negative value of (β).¹⁸ A value of γ_CMC denotes the surface tension at CMC and is an indicator of effectiveness of surfactants in lowering the surface tension of solution. Accordingly, more hydrophobic NaDC having lower CMC lowers the surface tension of solution to significant extent compared to NaC. NaC generates strong electrostatic interaction with 12-4-12, consequently it is more difficult for them to aggregate. It is also obvious from packing parameter (P) that NaDC display more compact packing due to it's hydrophobic nature. Size of micelles can also be evaluated from value of packing parameter (P). The value of P between 0 and 0.33 clearly suggests that micelles are spherical but P becomes 0.41 which manifests the existence of ellipsoidal micelles in the absence of bile salt.⁴⁶ An idea of close packing at air–water interface can be obtained from minimum area per molecule (A_min). A_min increases with increase in mole fraction of 12-4-12 and becomes maximum at 0.75 mole fractions clearly suggesting the molecules are loosely packed which may be due to increased charge repulsion at higher 12-4-12 concentration. Hydrophilicity of NaC also contributs in higher value of A_min (Table 3).

Table 2 Critical micellar concentrations (CMC), (CMC_ideal) and (CMC_experimental), interaction parameter (β), activity coefficients (f^m) for 12-4-12-bile salts mixed system at 30 °C^a

α12-4-12	CMCideal mM	CMCexp mM	β	X12-4-12	f^m12-4-12	f^mBS
a CMC = critical micelle concentration in mM, α = mole fraction.
12-4-12 − NaDC
0.00
0.25	2.58	0.060	−14.8	0.51	0.028	0.021
0.5	1.77	0.051	−14.8	0.55	0.050	0.011
0.75	1.36	0.045	−15.6	0.57	0.056	0.006
1.00
12-4-12 − NaC
0.00
0.25	3.73	0.070	−16.0	0.55	0.030	0.009
0.5	2.06	0.062	−15.7	0.57	0.055	0.006
0.75	1.43	0.053	−16.5	0.61	0.062	0.003
1.00

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Table 3 Interfacial composition (X⁰₁), interaction parameter (β⁰), surface pressure at CMC (Π_CMC), packing parameter (P), minimum area per molecule (A_min), surface tension at CMC (γ_CMC) of 12-4-12-bile salts mixtures at 30 °C

α12-4-12	X^01	β^0	f^01	f^02	γCMC	Amin (Å^2)	P	ΠCMC
12-4-12 + NaDC
0.00					48.5	324.7	0.13	22.8
0.25	0.51	−16.9	0.017	0.012	38.6	143.5	0.30	32.8
0.50	0.54	−17.1	0.026	0.007	38.1	145.2	0.29	33.2
0.75	0.57	−17.7	0.028	0.003	38.5	169.5	0.25	32.9
1.00					38.0	102.9	0.41	33.4
12-4-12 + NaC
0.00					47.7	140.3	0.30	23.7
0.25	0.54	−16.9	0.027	0.007	47.1	139.9	0.31	24.2
0.50	0.58	−17.1	0.048	0.003	47.2	174.6	0.24	24.1
0.75	0.59	−17.7	0.051	0.002	49.7	192.3	0.22	21.7
1.00					39.1	102.9	0.41	32.3

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3.3 Effect of concentration

Being cationic surfactant, 12-4-12 forms positively charged micelles (N_agg = 78). An electrostatic interaction is observed between 12-4-12 micelles and negatively charged bile salts. Addition of bile salt to 12-4-12 solution facilitates growth by neutralizing positive charge of micelles. Viscosity is a simple but important technique to visualize morphological changes in the solution. In order to emphasize micellar growth, viscosity measurements were performed on 50 mM 12-4-12 in the presence of varying concentration of bile salts and the results are displayed in Fig. 2. It is obvious from these results that initially at lower bile salt concentrations upto 5 mM, none of the bile salts has any modulating effect on viscosity. Fig. 2 clearly manifests that NaC interacts with 12-4-12 micelles at lower concentration and induces micellar growth compared to NaDC. It may be due to the fact that NaC possesses higher polarity compared to NaDC. But with further increase in bile salt concentration around 25–30 mM phase separation occurs. In analogy to NaC, NaDC displays interaction at relatively higher concentration and quite interestingly there is no increase in viscosity upto 20 mM NaDC but increase in solution viscosity of 2 orders of magnitude could be achieved by further addition of 13 mM NaDC. Such an increase in viscosity can be correlated with the increase in hydrodynamic diameter of micelles but as the system contains multiple morphologies, polydispersity will be very higher. Consequently, DLS measurements did not give the exact idea about alteration in micelle size as a function of bile salt concentration. Anticipation made from viscosity data can further be confirmed from corresponding SANS measurements. These measurements performed on 50 mM 12-4-12 as a function of NaDC are shown in Fig. 3. The SANS result present the influence of NaDC concentration ranging from 0 to 30 mM with fixed concentration (50 mM) of 12-4-12. With progressive addition of NaDC, there is a strong build up of scattering intensity in the low-Q region; however, they overlap at higher-Q region irrespective of the NaDC concentration. It is a symptom of the morphological changes in the system. The SANS distribution of 50 mM 12-4-12 solution generally shows a characteristic correlation peak which designates the presence of repulsive interaction due to charge in the system.^47,48 The analysis of data suggests that 50 mM 12-4-12 micelles have semi-major axis, semi-minor axis and aggregation number (N_agg) 53.7 Å, 16.4 Å and 65, respectively.⁴⁴ It is apparent from axial ratio ∼3.3 that at 50 mM, 12-4-12 micelles are prolate ellipsoidal in shape. In the presence of 10 mM NaDC, increase in semi-major axis with nominal change in semi-minor axis presents extended ellipsoidal micelles with axial ratio ∼4.3. With added 20 mM NaDC, axial ratio becomes ∼4.8 still presenting extended ellipsoidal micelles. A shift in correlation peak towards low-Q region clearly manifests micellar growth. But at 30 mM NaDC, one dimensional micellar growth was observed; axial ratio jumps to ∼9.8 suggesting the existence of long rod-like aggregates. It is also reflected in significant increase in N_agg of both 12-4-12 and NaDC. The change in N_agg of surfactants as a function of NaDC concentration is shown in Table 4. Here, the disappearance of the correlation peak may be due to neutralization of charge from the system by negatively charged NaDC. A close look of Fig. 3a also revealed that with added NaDC, scattering also increased which tends to attain the scattering profile of rod-like micelles (at 30 mM NaDC). As shown in inset of Fig. 3a, a typical slope of (−1) in log–log scale in low-Q region is observed which indicates to the formation of rod-like micelles.^49,50 Micellar parameters obtained from SANS data are summarized in Table 4. It is obvious from the data that the fractional charge decreases with increase in NaDC concentration. In analogy to NaDC, addition of NaC also leads to increase in the scattering intensity which is an indicative of micellar growth, but it is also observed that NaC is less efficient in inducing the growth of 12-4-12 micelles compared to NaDC (Fig. 3b). Table 4 clearly depicts that extended ellipsoidal micelles with larger dimensions are formed in the presence of 20 mM NaC. The values of N_agg is summarized in Table 4 and are lower compared to NaDC which accounts to more hydrophilic nature of NaC. It can be safely concluded that added NaC also favours the growth of 50 mM 12-4-12 micelles but to a lesser extent. At higher bile salt concentrations, precipitates are formed which is a characteristic of oppositely charged system. It is concluded that added bile salts exhibit the concentration dependant micelle growth; NaDC is more efficient and leads to one dimensional micelle growth thereby producing rod-like micelles while NaC results in the formation of extended ellipsoidal micelles.

Fig. 2 Viscosity of 50 mM 12-4-12 in the presence of varying amount of () NaDC and () NaC at 30 °C.

Fig. 3 SANS pattern for 50 mM 12-4-12 in the presence of (a) () 0 mM () 10 mM () 20 mM () 30 mM NaDC (b) () 0 mM () 10 mM () 20 mM NaC at 30 °C. The solid lines represent the fits to the experimental data.

Table 4 Micellar parameter for 50 mM 12-4-12 in the presence of bile salts obtained from SANS measurements^a,b

[BS], mM	a (Å)	b (Å)	a/b	α	Nagg		Shape
					12-4-12	Bile salt
a a = semi-major axis, b = semi-minor axis, a/b = axial ratio, α = fractional charge.b Nagg = aggregation number.
0 NaDC	53.7 ± 1.5	16.4 ± 0.6	3.3	0.46 ± 0.05	65 ± 7	—	Prolate ellipsoidal
10 NaDC	66.3 ± 1.7	15.5 ± 0.5	4.3	0.38 ± 0.04	63 ± 6	13 ± 2	Extended ellipsoidal
20 NaDC	71.8 ± 1.8	15.1 ± 0.5	4.8	0.33 ± 0.04	58 ± 5	23 ± 3	Extended ellipsoidal
30 NaDC	130.2 ± 2.2	13.3 ± 0.5	9.8	—	74 ± 8	44 ± 5	Rod-like
10 NaC	54.2 ± 1.4	13.2 ± 0.4	4.1	0.41 ± 0.05	37 ± 4	7 ± 1	Extended ellipsoidal
20 NaC	58.3 ± 1.6	15.5 ± 0.5	3.8	—	48 ± 5	19 ± 2	Extended ellipsoidal
20 NaDC pH ∼ 2	108.5 ± 2.0	17.7 ± 0.8	6.1	—	117 ± 10	47 ± 5	Rod-like
20 NaC pH ∼ 2	82.1 ± 1.8	19.3 ± 0.6	4.3	—	109 ± 9	44 ± 5	Extended ellipsoidal

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3.4 Effect of temperature

For ionic surfactants, with rise in temperature increased repulsion between surfactant monomers favors demicellization which is reflected in decrease in solution viscosity and it is a common trend.^51,52 With this aim, we performed viscosity measurements for aqueous solution of 50 mM 12-4-12 in the presence of 20 mM bile salts in the temperature range 30–60 °C and results are displayed in Fig. 4. It shows that temperature has no perceptible effect on viscosity of 12-4-12 micelles in the presence of 20 mM NaC while for 20 mM NaDC viscosity exponentially decreases. It is reported that with increase in temperature, length of rod-like micelles is decreased that results in lowering of solution viscosity.⁴⁰ Initially, slight increase of temperature has pronounced effect on solution viscosity for solution containing NaDC than NaC. With slight increase in temperature, 12-4-12–NaC mixed micelles have smaller size compared to NaDC and therefore decrease in viscosity is less pronounced. While at elevated temperature change in viscosity as a function of temperature is almost same. SANS study shows the existence of oblate ellipsoidal micelles in the presence of 20 mM NaDC which may transform to smaller aggregates at higher temperature which is reflected from lower solution viscosity.⁵³

Fig. 4 Viscosity of 50 mM 12-4-12 in the presence of 20 mM () NaDC and () NaC at varying temperature.

3.5 Effect of pH

Bile acids possess planar nonpolar steroid ring with two or three polar –OH groups along with carboxylic acid at the end which offer them pH sensitivity. They can be hydrolysed to controlled degree by varying the pH. Fig. 5 shows the influence of pH on micelles at 50 mM 12-4-12 in the presence of 20 mM bile salts at 30 °C. Originally, at pH ∼ 7.2, 12-4-12 micelles are extended ellipsoidal in shape in the presence of both salts and is evident from higher viscosity (earlier proved by SANS). Moving from pH ∼ 7.2 to acidic medium leads to the formation of corresponding bile acids generating hydrophobicity in the system. These bile acids are solubilized in 12-4-12 micelles and display the micellar growth on account of their hydrophobicity. Such a micellar growth is apparent from increase in viscosity of solution. It is evident from Fig. 5 that change of pH has greater modulating effect on 12-4-12 micelles containing NaDC compared to NaC. A polarity of bile salts is crucial factor in inducing micellar growth. It was also noticed that change of pH has almost negligible influence on 12-4-12 micelles containing NaC till pH ∼ 3. Below pH ∼ 2, NaC shows micellar growth which is due to the fact that NaC becomes completely hydrophobic in this pH range.⁴² It is obvious from Fig. 5 that extent of increase in viscosity is higher for NaDC compared to NaC. The orientation of NaC is another important aspect for micellar growth; NaC molecule with an extra –OH group displays higher polarity and is not able to penetrate inside micelles and mostly remains flat on micelle interface (location of bile salts further proved by NOESY study in later part of manuscript). NaDC with one less –OH group displays preferential modulating effect on 12-4-12 micelles. Here, NaDC molecules orient in such a way that deoxycholate anion inserts in cationic micelles such that only the negatively charged –COO⁻ group remains near to the head group region. Such intercalation of NaDC between charged head groups of 12-4-12 surfactant micelles diminishes the charge repulsion between them and overall charge on micelle surface and facilitates micellar growth as reflected in high solution viscosity which is found to be 2 orders of magnitude compared to NaC at pH ∼ 3. From these observations it was manifested that slight tuning of pH towards acidic medium has quite dissimilar effect on 12-4-12 micelles in the presence of bile salt with minimal structural difference. In order to justify our anticipation from viscosity data and to examine pH-induced micellar growth of 12-4-12 micelles in the presence of bile salts, SANS measurements were performed. Fig. 6 depicts the effect of pH on SANS profile for 50 mM 12-4-12 in presence of 20 mM bile salts at 30 °C. The figure clearly signify that in case of NaDC there is a strong build up of scattering in low-Q region clearly portraying the micellar growth and (−1) slope in log–log scale in low-Q region in SANS spectrum suggests the presence of rod-like micelles.^49,50 Analysis of SANS data also suggests that in the presence of NaDC at pH ∼ 2, micelles have semi-major axis, semi-minor-axis and axial ratio ∼108.5 Å, 17.7 Å and 6.1, respectively clearly presenting rod-like micelles. A large increase in N_agg is a consequence of formation of rod-like micelles (Table 4). These results are equally quantified by the high solution viscosity seen earlier (Fig. 5). NaC being more hydrophilic in nature, displays less pronounced effect on micellar dimensions of 12-4-12 micelles. Consequently, in the presence of NaC at pH ∼ 2, micelles having semi-major axis ∼82.1 Å and semi-minor-axis ∼19.3 Å, respectively are formed. The axial ratio ∼4.3 indicates the presence of extended ellipsoidal micelles at pH ∼ 2 presenting the higher solution viscosity. A significant increase in N_agg is a reflects the formation of extended ellipsoidal micelles (Table 4). So from SANS measurements, it was concluded that change of pH towards acidic medium has prominent effect on morphology of 12-4-12 micelles in the presence of NaDC compared to NaC which can be linked with higher hydrophobicity of deoxycholic acid than cholic acid. From SANS measurements, it was manifested that at pH ∼ 2, rod-like micelles are formed in the presence of NaDC while change of pH towards acidic medium gives rise to the formation of extended ellipsoidal micelles for NaC. This presents a quite diverse effect of the structurally similar bile salts which is ultimately an aim of the present study.

Fig. 5 Viscosity of 50 mM 12-4-12 in the presence of 20 mM () NaDC and () NaC as a function of pH at 30 °C.

Fig. 6 Effect of pH on SANS pattern for 50 mM 12-4-12 in the presence of 20 mM bile salts at 30 °C. The solid lines represent the fits to the experimental data.

NMR is a very useful technique that provides an idea about small change in micro-environment of surfactant protons. In ¹H NMR of 50 mM 12-4-12 in D₂O, chemical shift obtained in much hydrophobic region of spectrum at ∼0.85 ppm corresponds to terminal methyl (–CH₃) protons (a) which forms core of micelles (ESI-SM1).⁵⁴ Whereas internal chain protons (b and c) show chemical shift ∼1.3 ppm. Chemical shifts appeared at ∼1.7 ppm and ∼1.9 ppm are of spacer protons (d) and chain protons (e), respectively. Protons which are in close proximity of positively charged quaternized N atom experience hydrophilic environment and shows chemical shift in downfield region of spectrum. The head group protons show chemical shift ∼3.1 ppm while that for other protons nearer to N atom (g and h) appear at 3.4 ppm. Similarly, protons of bile salts are labelled and shown in Table 1. As reported in literature, ¹H NMR spectrum for NaC shows chemical shifts at ∼4.1, ∼3.9 and ∼3.5 ppm for three (–OH) groups positioned at C₁₂, C₇ and C₃, respectively while intense NMR signals concerned with (–CH₃) groups oriented at C₂₁, C₁₉ and C₁₈ appeared at ∼1.0, ∼0.9 and ∼0.7 ppm, respectively. Other steroid protons of NaC appeared between ∼1–2.2 ppm.⁵⁵ Similarly, hydrophobic analogue of NaC shows NMR signals for (–OH) and (–CH₃) groups in slight upfield region with can be correlated with it's more hydrophobic nature.⁵⁶ A dynamic location of solubilizates added to surfactant can be at different interiors of micelle viz. micelle water interface, core of micelle, palisade layer, between head groups depending solution conditions like temperature, pH, solvents and can be tuned easily.^57,58 Interaction of solubilizates with any part of surfactant micelles is reflected in cross peaks in NOESY spectrum which is more reliable tool to study the intra- and inter micellar interaction in surfactant systems. An accurate measurement of amplitude of such cross peaks gives better idea about probable location of solubilizates in the micelle. In this context, we have performed NOESY experiments on gemini/bile salt mixed micelle system and from it the most probable location of NaDC/NaC was anticipated. The NOESY contour plots for 50 mM 12-4-12 in the presence of NaDC and NaC are displayed in Fig. 7. From the first look of both the spectra, it is clearly seen that more number of peaks are observed for NaDC compared to NaC. Consequently, it has been postulated NaDC generates stronger interaction with 12-4-12 micelles compared to NaC. Many intra- and intermolecular cross peaks in NOESY spectra suggest the interaction of NaDC and NaC with different interior of 12-4-12 micelles.

Fig. 7 NOESY spectra of 50 mM 12-4-12 in the presence of (a) NaDC and (b) NaC at 30 °C.

In case of 12-4-12 + NaDC system, most importantly, there are no any cross peak showing the interaction of head group protons (f) with NaDC protons clearly implying that NaDC molecules penetrate inside 12-4-12 micelle (Fig. 7a). Cross peaks are observed for (g) and (h) protons with C₂₂ protons of NaDC at ∼1.2 ppm which implies that NaDC penetrates in 12-4-12 micelles and resides in such a way that C₂₂ protons remains inside 12-4-12 micelles near head group (f) and near spacer protons (g). Many intra-molecular cross peaks are observed for (e) and (d) protons showing interaction between them. Very intense cross peaks are observed for (e) protons with C₂₃ protons ∼1.9 ppm stating the location of C₂₃ protons of NaDC inside 12-4-12 head group. Similarly, weak cross peaks are seen ∼1.7 ppm for C₂₂ protons with (d) protons depicting that these protons are in close proximity. The –OH group of NaDC at C₃ and (g) and (h) protons of 12-4-12 show chemical shifts at ∼3.6 ppm which appear as merged peaks. Strong interaction between –OH group of C₃ and protons of hydrophobic chain (b) of 12-4-12 gives an intense cross peak at ∼1.3 ppm in NOESY spectrum. A hydrophobic chain protons (b) of 12-4-12 shows cross peaks with C₅ protons at ∼1.3 ppm. The terminal methyl protons (a) of 12-4-12 micelles show cross peaks with C₂ and C₄ protons ∼1.3 and 1.4 ppm, respectively. These clearly manifest that NaDC molecules orient in 12-4-12 micelle in such a way that –OH group of C₃ along with C₄ and C₅ protons reside close to hydrophobic chain protons (a and b) which form core of 12-4-12 micelles. From all above observations, it was conveyed that NaDC molecules penetrate inside 12-4-12 micelles in such a way that –OH group and hydrophobic steroid domain remains in the core while alkyl protons (C₂₃) remain near 12-4-12 head group inside micelles.

In case of 12-4-12 + NaC system, many intense intramolecular cross peaks are observed for NaC protons and internal chain and spacer protons (Fig. 7b). There are no cross peaks for (g) and (h) protons with any NaC protons intimating that NaC do not penetrate inside 12-4-12 micelle. There are no cross peaks for internal chain protons of 12-4-12 with NaC protons but some cross peaks observed for chain protons (a and b) of 12-4-12 with NaC protons ∼0.8 and ∼1.3 ppm and internal chain protons (e) with C₂₃ protons ∼2.2 ppm which may be due to bending of hydrocarbon chain of 12-4-12.^59,60 There are three perfect reasons to support our postulation that NaC remains flat on 12-4-12 micelles; (i) cross peaks observed for spacer protons (d) with C₁₆, C₁₇ protons ∼1.8 ppm (ii) spacer protons (g) generates cross peaks with C₇ protons ∼3.5 ppm (iii) cross peaks are observed for head group protons (f) with C₂₂ protons ∼1.2 ppm, with C₂₁ and C₁₉ protons ∼0.8 ppm and with C₃ protons at ∼3.1 ppm. A combination of all these observations prompted us to conclude that NaC molecules do not penetrate inside micelles due to their hydrophilic nature and orient itself on micelle interface. Our anticipation made for location of NaDC and NaC in 12-4-12 micelles is in excellent accordance with literature.^29,34

4. Conclusions

In this report, we systematically studied the interaction of biologically important bile salt analogues with cationic gemini surfactant 12-4-12 micelles using surface tension, viscosity, SANS and NMR studies. A large negative value of interaction parameter (β) for 12-4-12–NaC system clearly represents strong preferential synergistic interaction between them compared to NaDC. Doping of bile salts drives to morphological changes of 12-4-12 micelles in which one dimensional micellar growth is observed for NaDC which was further subsided with increase in temperature. A micellar growth proposed by viscosity measurements was further quantified by SANS measurements which manifests that addition of NaDC leads to ellipsoidal to rod-like micellar transition of 12-4-12 gemini surfactant micelles while for NaC formation of extended ellipsoidal micelles are facilitated. Furthermore, desired morphology of mixed micelles can be conveniently achieved by tuning of solution pH; rod-like micelles are formed in the presence of NaDC in acidic pH while ellipsoidal micelles with higher axial ratio are observed in case of NaC. Location of these bile salts was obtained from NOESY spectra; NaDC molecules orient in micelle in such a way that alkyl chain of NaDC remains near the head group inside surfactant micelle whereas hydrophobic part remains in the core of 12-4-12 micelles while NaC owing more hydrophilic nature due to the presence of an extra –OH group remains flat on micelle–water interface. The present study highlights that the micellar growth observed in acidic medium and as function of concentration gradient of bile salts is more prominent for NaDC compared to NaC on account of its higher hydrophobic nature. It also validates the pH and concentration dependant micellar growth of cationic gemini surfactant (12-4-12) in the presence of biologically essential bile salts.

Acknowledgements

PB and SP thank UGC New Delhi Fellowships and CSIR, New Delhi, India for financial assistance [Project No. F. No. 01(2763)/13/EMR-II].

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra17212a

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