Self-Assembly, Supramolecular Organization, and Phase Behavior of l

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Self-Assembly, Supramolecular Organization, and Phase Behavior of L‑Alanine Alkyl Esters (n = 9−18) and Characterization of Equimolar L‑Alanine Lauryl Ester/Lauryl Sulfate Catanionic Complex D. Sivaramakrishna and Musti J. Swamy* School of Chemistry, University of Hyderabad, Hyderabad-500 046, India S Supporting Information *

ABSTRACT: A homologous series of L-alanine alkyl ester hydrochlorides (AEs) bearing 9−18 C atoms in the alkyl chain have been synthesized and characterized with respect to self-assembly, supramolecular structure, and phase transitions. The CMCs of AEs bearing 11−18 C atoms were found to range between 0.1 and 10 mM. Differential scanning calorimetric (DSC) studies showed that the transition temperatures (Tt), enthalpies (ΔHt) and entropies (ΔSt) of AEs in the dry state exhibit odd−even alternation, with the odd-chain-length compounds having higher Tt values, but the even-chain-length homologues showing higher values of ΔHt and ΔSt. In DSC measurements on hydrated samples, carried out at pH 5.0 and pH 10.0 (where they exist in cationic and neutral forms, respectively), compounds with 13−18 C atoms in the alkyl chain showed sharp gel-to-liquid crystalline phase transitions, and odd−even alternation was not seen in the thermodynamic parameters. The molecular structure, packing properties, and intermolecular interactions of AEs with 9 and 10 C atoms in the alkyl chain were determined by single crystal X-ray diffraction, which showed that the alkyl chains are packed in a tilted interdigitated bilayer format. d-Spacings obtained from powder X-ray diffraction studies exhibited a linear dependence on the alkyl chain length, suggesting that the other AEs also adopt an interdigitated bilayer structure. Turbidimetric, fluorescence spectroscopic, and isothermal titration calorimetric (ITC) studies established that in aqueous dispersions L-alanine lauryl ester hydrochloride (ALE·HCl) and sodium dodecyl sulfate (SDS) form an equimolar complex. Transmission electron microscopic and DSC studies indicate that the complex exists as unilamellar liposomes, which exhibit a sharp phase transition at ∼39 °C. The aggregates were disrupted at high pH, suggesting that the catanionic complex would be useful to develop a base-labile drug delivery system. ITC studies indicated that ALE·HCl forms a strong complex with DNA, suggesting that the AEs may find use in DNA therapeutics as well.

1. INTRODUCTION Phospholipid vesicles (or liposomes), first reported in the 1960s by Bangham and co-workers,1 have shown considerable potential as carriers for the delivery of drugs and therapeutics to specific target tissues/organs.2−5 However, in view of the high cost of phospholipids, there has been considerable interest in synthesizing newer, inexpensive amphiphiles that give liposomes with improved stability and encapsulation efficiency. In this context, the observation that mixing cationic and anionic surfactants leads to the spontaneous formation of liposomes is of considerable interest.6,7 Mixing cationic and anionic surfactants even at concentrations considerably lower than the critical micellar concentrations of the individual surfactants leads to the formation of different kinds of aggregates.8 The resulting mixtures, termed © 2015 American Chemical Society

catanionic mixtures, show a remarkable synergistic behavior compared to the individual surfactants, due to the strong attractive forces between oppositely charged species.9,10 Vesicle formations from mixtures of cationic and anionic surfactants were first reported about 25 years ago.11,12 The size, shape and stability of the catanionic vesicles were found to be affected by a variety of parameters including headgroup size, ionic strength, temperature, chain length of the two surfactants, as well as their molar ratio.13−16 The formation of catanionic vesicles from micellar dispersions of the surfactants appears to proceed via mixed micelles and disk-like lamellar structures.17,18 Received: April 18, 2015 Revised: August 13, 2015 Published: August 20, 2015 9546

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Langmuir Scheme 1. Synthesis of L-Alanine Alkyl Esters

infrared (FTIR), 1H NMR, and 13C NMR spectroscopy and highresolution mass spectrometry. Capillary melting points of AEs were recorded on a Superfit (Mumbai, India) melting point apparatus as described earlier.39 IR spectra (on KBr pellet) were recorded on a Jasco FTIR 5300 Spectrometer. 1H- and 13C NMR spectra were recorded on a Bruker Avance NMR spectrometer operating at 400 and 100 MHz, respectively, using samples dissolved in CDCl3. High-resolution mass spectra were obtained in the positive ion mode on a Bruker Maxis mass spectrometer. Critical Micellar Concentration (CMC). The CMCs of AEs with 11−18 C atoms in the chains were determined by fluorescence spectroscopy using polarity ratio changes of pyrene monomer. Fluorescence spectra were recorded at a scan rate of 5 nm/s on a Horiba Jobin-Yvon Fluoromax-4 spectrofluorometer with 3 and 5 nm slits on excitation and emission monochromators, respectively. Experiments were carried out by adding small aliquots of the AEs from 10 to 200 mM stock solutions to a 2.0 mL solution of 2 μM pyrene solution. After each addition, the fluorescence spectrum was recorded between 350 and 500 nm, keeping the excitation wavelength at 335 nm. Pyrene monomer exhibits five bands in the emission spectrum.40 The intensity ratio of first and third bands (I1/I3, referred to as polarity ratio) was considered for further analysis. The polarity ratio was then plotted as a function of amphiphile concentration. The CMC was estimated as the concentration where a distinct break in the slope was observed. Differential Scanning Calorimetry (DSC). DSC experiments with dry AEs were performed on a PerkinElmer Pyris Diamond differential scanning calorimeter.39 Samples prepared as described earlier,39,41 were subjected to three heating and two cooling scans at a scan rate of 2°/min. First heating scans were considered for data analysis of all samples. Transition enthalpy values were determined by integrating the peak area under the transition curve. Transition entropies were calculated from transition enthalpies assuming a firstorder transition according to eq 1.42

Catanionic vesicles have considerable potential for application in the delivery of drugs and DNA to cells.19 They exhibit reduced toxicity compared to those consisting only of cationic surfactants, and are hence more suitable for use in formulating liposomes for drug delivery.20 Electrostatics plays a key role in their interaction with biomacromolecules.21−23 Catanionic mixtures have been also used as stationary phases for electrokinetic separations.24 A number of catanionic systems have been demonstrated to form vesicles, and new systems are being discovered.25−31 Catanionic systems containing amphiphiles derived from amino acids have attracted considerable interest in recent years in view of their low toxicity and environmental impact. A number of single-chain and gemini surfactants derived from lysine and arginine have been synthesized and evaluated with respect to membrane interaction, antimicrobial activity, cytotoxicity and biodegradability.32−37 In the present study, we synthesized a homologous series of L-alanine alkyl esters (AEs) and characterized their structures and phase transitions by X-ray diffraction and DSC, respectively. Crystal structures of AEs containing 9 and 10 C atoms in the alkyl chain showed that molecules of these two alanine esters are packed in an interdigitated manner in the crystal lattice, whereas DSC studies revealed different types of odd−even alternation in the transition temperatures and transition enthalpies (as well as entropies). Studies on the interaction of L-alanine lauryl ester hydrochloride (ALE·HCl) with sodium dodecyl sulfate (SDS) revealed the formation of an equimolar complex between the two amphiphiles, which is disrupted at higher pH. This catanionic complex can potentially be used to develop a basetriggerable, liposomal drug delivery system. Finally, the strong association of ALE·HCl with DNA, inferred from titration calorimetric studies, suggests that AEs may have considerable application potential in the intracellular delivery of nucleic acids.

ΔSt = ΔHt /Tt

(1)

where Tt refers to the transition temperature. DSC studies with hydrated samples were performed using a VPDSC calorimeter (MicroCal LLC, Northampton, MA, USA). Accurately weighed samples (7−8 mg) were hydrated with 1.0 mL of acetate buffer (pH 5.0) or glycine/NaOH buffer (pH 10.0), containing 1.0 M NaCl. The final concentration of AEs with different alkyl chains ranged between 19 and 25 mM. For measurements with ALE·HCl/SDS complex, an equimolar mixture containing 7.5 mM of each amphiphile was prepared by weighing the required amounts of the two components into a sample tube, followed by hydrating the mixture with double-distilled water. The sample was them vortexed vigorously, and loaded into the DSC cell. After incubating for 30 min in the DSC cell, thermograms were recorded between 1 and 80 °C at a scan rate of 60°/h. Three successive heating scans performed on each sample were found to be reproducible. Transition temperatures, enthalpies, and widths at half height were determined using Origin 7.0 software provided by MicroCal. Transition entropies were calculated from the transition enthalpies assuming a first-order transition according to eq 1.

2. MATERIALS AND METHODS Synthesis of L-Alanine Alkyl Esters. L-Alanine alkyl esters were prepared by the reaction of 1-alcohols of different chain lengths with Lalanine as reported earlier.38 A representative procedure is given here for reaction with 1-decanol; reactions with other alcohols were carried out in an essentially similar manner. Briefly, a mixture containing Lalanine (1.0 mmol), 1-alcohol (1.0 mmol), and p-toluenesulfonic acid monohydrate (1.2 mmol) was refluxed at 110 °C in toluene for 5−6 h (Scheme 1). After evaporation of the solvent, the residue was dissolved in chloroform and washed with 10% sodium carbonate solution and dried with anhydrous Na2SO4. The solvent was removed by rotary evaporation, and the residue was dissolved in acetone containing HCl and kept for recrystallization at −20 °C. The product (L-alanine decyl ester hydrochloride) was recrystallized again from acetone at −20 °C. The final product thus obtained was filtered, dried by vacuum desiccation, and characterized by melting point, Fourier transform 9547

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Figure 1. Determination of the CMC of ATDE (A) and APE (B) in water. A plot of chain length (n) versus log CMC (C). The log CMC values in (C) were calculated from CMC values taken in mM units. From these values, the thermodynamic parameters, ΔGb and ΔSb, are calculated according to the following basic thermodynamic equations:

Crystallization and X-ray Diffraction. Thin, plate-type colorless crystals of alanine nonyl ester hydrochloride (ANE·HCl) and alanine decyl ester hydrochloride (ADE·HCl) were grown from acetone at −20 °C. X-ray diffraction measurements on ANE·HCl were carried out at room temperature with an Oxford Gemini X-ray diffractometer, whereas measurements with ADE·HCl were carried out at 100 K on a Bruker Smart Apex X-ray diffractometer. Both diffractometers were equipped with Mo−Kα radiation source (λ = 0.71073 Å). The crystal parameters and unit cell dimensions for ANE·HCl and ADE·HCl as well as the details of structure solution and refinement are given in the Supporting Information. Turbidimetric and Fluorescence Spectroscopic Studies on ALE−DS Complexation. Complexation of alanine lauryl ester hydrochloride (ALE·HCl) with SDS was investigated by Job’s method using turbidimetry and fluorescence spectroscopy. Turbidimetric studies were performed at room temperature on a Shimadzu UV3600 UV−vis−NIR spectrophotometer using 1 cm path length cells. Aqueous solutions of ALE·HCl and SDS (concentration = 0.2 mM for each surfactant, which is well below their CMCs) were mixed at different ratios, vortexed, and incubated for 30 min. In order to investigate the pH dependence of the stability of these catanionic aggregates, samples were prepared in a similar manner in the following buffers: 20 mM KCl/HCl (pH 2.0), 20 mM citric acid/sodium phosphate dibasic (pH 3.0−6.0), 20 mM sodium phosphate (pH 7.0), 20 mM Tris/HCl (pH 8.0−9.0), 20 mM glycine/NaOH (pH 10.0). After incubation at room temperature for 30 min, optical density was recorded from 220 to 400 nm, and absorbance (turbidity) at 330 nm was considered for further analysis. Fluorescence studies were carried out on a Horiba Jobin Yvon Fluoromax-4 spectrofluorometer using the instrumental settings described above for CMC determination. Sample solutions were prepared as described above for UV measurements, but containing 2 μm pyrene. The height ratio of the first and third peaks (I1/I3) was used for further analysis. Isothermal Titration Calorimetry (ITC). ITC studies were performed using the VP-ITC equipment from Microcal, LLC (Northampton, MA). Titrations for investigating the interaction between ALE·HCl and SDS were performed by injecting 10 μL aliquots from a 1 mM stock solution of SDS via a rotating stirrersyringe to 1.455 mL of 0.1 mM ALE·HCl solution in the sample cell. The duration of each injection was 20 s, and an interval of 300 s was given between consecutive injections. Throughout the experiment, the solution in the reaction cell was stirred at a speed of 300 rpm. Generally, the first injection was found to be imprecise; therefore, a 5 μL injection was added, and the resultant point was deleted before the analysis of the rest of the data to a “one set of sites” model using the MicroCal Origin software provided by the ITC manufacturer as described earlier.30 The analysis yields values of stoichiometry of binding (n), binding constant (Kb), and enthalpy of binding (ΔHb).

ΔG b = − RT ln Kb

(2)

ΔG b = ΔHb − T ΔS b

(3)

Calorimetric titrations on the interaction of ALE·HCl with salmon testes DNA (ST-DNA) were carried out at 25 °C by injecting a 5 μL first injection of the ALE·HCl from a 2.5 mM stock solution in water followed by a series of 10 μL aliquots via a rotating stirrer-syringe to 1.455 mL of 0.2 mM ST-DNA solution (in monomeric units) in water in the sample cell. All other instrumental settings were identical to those described above. Powder X-ray Diffraction (PXRD) Studies. PXRD measurements were carried out on a Bruker SMART D8 Advance powder Xray diffractometer (Bruker-AXS, Karlsruhe, Germany) with Cu−Kα radiation at 40 kV and 30 mA. Samples of the AEs (n = 9−18) were ground to a fine powder with mortar and pestle before measurement. To obtain PXRD data for the equimolar ALE−DS complex, samples were prepared by mixing solid samples of the two compounds, dissolving in a mixture of dichloromethane-methanol (1:1, v/v), followed by evaporation of the solvent. Then the sample was subjected to vacuum desiccation for 8 h, and the resultant mixture was ground thoroughly before measurement. The resulting fine powder was placed on a circular rotating disk of the sample holder of the instrument. Diffraction patterns were collected at room temperature using a LynxEye PSD data collector over a 2θ range of 1−50° with a step size of 0.0198° and a measuring time of 1.5 s for each step. Transmission Electron Microscopy. TEM studies on ALE−DS complex were carried out with samples containing 0.1 mM each of ALE·HCl and SDS in water, prepared as described above for turbidimetric studies. A 10 μL aliquot of the sample was placed on a carbon-coated copper grid, and after ∼5 min, excess liquid was removed with the aid of a filter paper at the periphery of the grid. The sample was then allowed to air-dry for 10 min. Grids were stained with 2% uranyl acetate for 30 s, followed by removal of the staining solution by the aid of an absorbent paper at the periphery of the grid. After drying for 10 min, samples were examined with FEI model TECNAI G2 S-Twin TEM at an accelerating voltage of 120 kV.

3. RESULTS AND DISCUSSION The L-alanine alkyl esters synthesized in this study were extensively characterized by various spectroscopic (FTIR, 1Hand 13C NMR) methods as well as by high-resolution mass spectrometry, and the details are given in the Supporting Information (Figures S1−S4, Tables S1−S4). The results obtained from these spectral analyses are fully consistent with 9548

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Langmuir the structures of L-alanine alkyl esters and indicate that they are all highly pure. Critical Micellar Concentrations of AEs. The CMCs of AEs with alkyl chains bearing 11−18 C atoms were determined by monitoring changes in the fluorescence emission characteristics of pyrene. Addition of successive small aliquots of the AE from a concentrated stock solution to pyrene solution in water results in a linear decrease in the polarity ratio of the probe up to a certain concentration of the surfactant, where the slope changes and the linear trend continues. This is shown for Lalanine tridecyl ester hydrochloride (ATDE·HCl) and L-alanine palmityl ester hydrochloride (APE·HCl) in Figure 1A,B, respectively. From the intercepts of the linear least-squares fits of the two parts of such plots, the CMCs of AEs with 11− 18 C atoms in the alkyl chains were determined. These values are listed in Table S5. The plot shown in Figure 1 and the data presented in Table S5 indicate that the CMC values of AEs decrease with increase in alkyl chain length. A plot depicting the chain length dependence of log (CMC) is shown in Figure 1C. For a number of homologous series of surfactants, it has been reported that log (CMC) exhibits a linear dependence on the alkyl chain length.43 The plot in Figure 1C shows linear dependence for n = 11−14, but the values deviate from linearity at higher n values, which possibly arise due to differences in the characteristics of the micelles (for example, size and shape) formed by the AEs with longer alkyl chains vis-à-vis those with shorter chains. Thus, it is likely that the shape of micelles formed by the shorter chain length AEs (n = 11−14) differ from those formed by AEs with n = 15−18. DSC of Dry AEs. Heating thermograms of dry alanine esters with even and odd number of C atoms in the alkyl chain are shown in Figure 2A,B, respectively. All even-chain AEs show two transitions in the first heating scan. Since the higher temperature transition corresponds to the capillary melting

point of the compound, the lower temperature transition must correspond to a solid−solid transition, indicating polymorphism. All odd-chain AEs showed a single sharp transition, which coincided with the melting point of the compound. When samples were subjected to a second heating scan it was observed that the transition enthalpy decreased slightly. Therefore, in all cases the first heating scans were considered for further analysis. Transition temperatures (Tt), transition enthalpies (ΔHt) and transition entropies (ΔSt) for all AEs are presented in Table S6. DSC of Hydrated AEs. In the fully hydrated state, AEs bearing shorter alkyl chains (n = 9−12) did not show any phase transition above 0 °C, whereas those with longer alkyl chains yielded broad thermograms, suggesting low cooperativity of the transition, which could be due to electrostatic repulsion between the head groups of the amphiphile. In order to screen the electrostatics and also to investigate the phase transitions of the AEs in the protonated and deprotonated conditions, we performed DSC studies using samples hydrated with acetate buffer (pH 5.0) and Glycine−NaOH buffer (pH 10.0), both containing 1 M NaCl. The pKa of L-alanine palmityl ester was determined as 7.94 from DSC studies carried out at various pH values between 3.0 and 11.0 (see Figure S5 and related discussion in the Supporting Information). Heating thermograms corresponding to the AEs containing alkyl chains of 13− 18 C atoms hydrated with buffers of pH 5.0 and 10.0 are shown in Figure 2C,D, respectively. While at pH 5.0, all AEs gave single sharp transitions, at pH 10.0 some of the AEs gave two well-defined and sharp transitions. When the same samples were reheated upon cooling, we found that in some cases the intensity of the lower-temperature transition decreased while in others it disappeared altogether. Therefore, the higher temperature transition was assigned as the chain-melting phase transition. In the first heating scan, some of the thermograms gave slightly higher values of Tt and ΔHt, whereas the second and third heating scans gave reproducible values, which could be due to incomplete hydration of the lipid before the first heating scan. Therefore, the data from the second heating scan were used in all cases to determine Tt, ΔHt and ΔSt, and the values obtained from measurements at pH 5.0 and 10.0 are given in Tables S7 and S8, respectively. Chain Length Dependence of Transition Enthalpy and Entropy. The chain length dependence of transition enthalpy (ΔHt) and transition entropy (ΔSt) of dry AEs are shown in Figure 3A,B. When the total enthalpy corresponding to both the major and minor transitions is considered, an odd−even alternation is seen in the ΔHt as well as ΔSt, with the values corresponding to the even chain length series and odd chain length series independently exhibiting a linear dependence on the chain length (Figure 3A,B). When the ΔHt and ΔSt values for the entire chain length series were viewed together, a zigzag pattern is seen in each case, with the values corresponding to the even chain length AEs being higher than those of odd chain length compounds. Such odd−even alternation was seen earlier in a variety of homologous series of long chain compounds including hydrocarbons, alcohols, fatty acids, N-acylethanolamines, N,O-diacylethanolamines with matched as well as mixed chains, and N-acyl conjugates of dopamines and serotonins.39,41,44−48 Interestingly, in N-acyl conjugates of Lalanines (NAAs), the ΔHt and ΔSt values of odd chain length molecules are higher than those of the even chain length ones.49 The transition enthalpies and entropies of AEs with odd

Figure 2. DSC thermograms of dry AEs of even (A) and odd (B) alkyl chains. DSC thermograms of hydrated AEs (n = 13−18) at pH 5.0 (C) and pH 10.0 (D). 9549

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structures of the compounds within each series would be very similar in the solid state. Therefore, the molecular packing and intermolecular interactions that govern the crystal packing of all the compounds in each series of AEs are likely to be very similar, and determination of the crystal structure of any one AE in each series is likely to give an idea of the molecular packing and intermolecular interactions present in the crystal lattice in that series. In the present case, we obtained the 3dimensional structure of two different AEs with even and odd alkyl chain lengths by single-crystal X-ray diffraction and found that their structures are very similar. This will be discussed in detail below. Chain length dependences of transition enthalpy and entropy for AEs hydrated with buffers of pH 5.0 and pH 10.0 are shown in Figure 3C,D, respectively. These pH conditions were chosen since at pH 5.0 the amino group of the AEs would be protonated, whereas at pH 10.0 they would be in the deprotonated form. The data presented in Figure 3 show that the odd−even alternation seen in the dry state is absent in the hydrated state. In both cases, values of the transition enthalpy and entropy increase linearly with the alkyl chain length for n = 13−18, except for the tridecyl ester at pH 5.0. Values of ΔHt and ΔSt corresponding to the protonated AEs are consistently higher than those of the deprotonated compounds. Linear leastsquares analysis of the transition enthalpies and entropies (Tables S7 and S8) yielded the incremental values and end contributions to the total enthalpy and entropy. These values are listed in Table 1. Linear dependence of transition enthalpies and entropies upon hydration with pH 5.0 buffer suggests that all the protonated AEs would have similar type of molecular packing and exhibit similar intermolecular interactions. The lack of odd−even alternation indicates that in the hydrated state the alkyl chains are most likely aligned perpendicular to the bilayer plane. Similarly, in the case of deprotonated AEs, the molecular packing and intermolecular interactions would also be expected to be similar for all the members of the chain length series, with the alkyl chains oriented perpendicular to the bilayer plane. It is observed that the incremental values of transition enthalpy and entropy of the deprotonated AEs (pH 10.0) are somewhat higher than those of the protonated samples (pH 5.0), suggesting stronger interaction between the alkyl chains in the supramolecular assemblies formed by the deprotonated AEs. Chain Length Dependence of Transition Temperature. Plots of transition temperature versus alkyl chain length of dry and hydrated AEs are shown in Figure 4A,B, respectively. In the dry state, a distinct odd−even alteration is seen in the transition temperatures, with the odd chain length AEs displaying higher Tt values as compared to the even chain length homologues. However, within each series (odd or even) the Tt values increase in a smooth progression but the

Figure 3. Chain-length dependence of ΔHt (A, C) and ΔSt (B, D) of AEs. Data for dry samples are shown in panels A and B. Data for hydrated samples are given in C and D, where filled symbols (●) correspond to pH 5.0 and open symbols (○) correspond to pH 10.0. See text for more details.

and even chain lengths could be independently fit well to expressions 4 and 5, respectively.44 ΔHt = ΔHo + (n − 2)ΔHinc

(4)

ΔSt = ΔSo + (n − 2)ΔSinc

(5)

where n is the number of C atoms in the alkyl chains, and ΔH0 and ΔS0 are the end contributions to ΔHt and ΔSt, respectively, arising from the terminal methyl group and the polar region of the molecule. ΔHinc and ΔSinc are the average incremental values of ΔHt and ΔSt contributed by each CH2 group. It was previously observed that the values of ΔHt and ΔSt for diacyl phosphatidylcholines and phosphatidylethanolamines with matched, saturated acyl chains as well as a number of homologous series of amphiphiles with saturated acyl/alkyl chains fit well to eqs 4 and 5.39,41,45−48,50−53 Interestingly, when the enthalpies of only the major transitions are considered, it is seen that the transition enthalpies and entropies of the entire chain length series of AEs exhibit a linear dependence on the chain length with no odd−even alternation (Figure S6A, B). This data could also be fit well to eqs 4 and 5. Linear least-squares analysis of the ΔHt and ΔSt values of AEs with even- and odd alkyl chain lengths yielded the incremental values (ΔHinc and ΔSinc) and end contributions (ΔH0 and ΔS0) for the two series, which are listed in Table 1. A linear chain length dependence of ΔHt and ΔSt values of even chain and odd chain AEs suggests that the three-dimensional

Table 1. Incremental Values (ΔHinc, ΔSinc) of Chain Length Dependence and End Contributions (ΔHo, ΔSo) to Phase Transition Enthalpy and Entropy of AEs in the Dry and Hydrated Statesa DSC of dry AEs thermodynamic parameter ΔHinc (kcal/mol) ΔH0 (kcal/mol) ΔSinc (cal/mol/K) ΔS0 (cal/mol/K) a

even chain length 0.60 2.02 1.52 6.24

(0.03) (0.37) (0.08) (0.97)

DSC of hydrated AEs (n = 13−18)

odd chain length 0.52 2.06 1.32 6.18

(0.02) (0.27) (0.06) (0.64)

only major peak 0.53 1.94 1.34 5.84

(0.02) (0.22) (0.05) (0.60)

pH = 5.0

pH = 10.0 (only major peak)

0.45 (0.03) −1.64 (0.44) 1.06 (0.10) −0.25 (1.43)

0.67 (0.02) −6.56 (0.35) 2.02 (0.08) −19.35 (1.08)

Values in parentheses correspond to fitting errors obtained from linear least squares analysis. 9550

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tilted chain packing observed in the crystal structures of ANE· HCl and ADE·HCl, discussed below. As the alkyl chain length increases, the total contribution towards transition enthalpy and entropy arising from the polymethylene chain would become very large as compared to the end contributions. Therefore, at infinite alkyl chain length, eqs 4 and 5 can be reduced to44 ΔHt = (n − 2)ΔHinc

(6)

ΔSt = (n − 2)ΔSinc

(7)

Then the Tt for infinite chain length, Tt∞, can be obtained from Tt∞ = ΔHinc/ΔSinc

(8)

Tt∞

From the data presented in Table 1 the values for the AEs with odd and even alkyl chain lengths in the dry state have been estimated as 393.94 and 394.7 K, respectively. Similarly, from the ΔHinc and ΔSinc values of the AEs hydrated with pH 5.0 and 10.0, the corresponding Tt∞ values were estimated as 424.5 and 331.7 K, respectively. For a variety of amphiphiles with one and two hydrophobic alkyl/acyl chains that exhibit linear dependence of ΔHt and ΔSt with respect to the chain length, it has been shown that these values can be fit to the following equation:54

Figure 4. Chain length dependence of chain-melting phase transition temperatures of AEs. (A) Dry state. (B) In the fully hydrated state at pH 5.0 (●) and pH 10.0 (○).

Tt = ΔHt /ΔSt = Tt∞[1 − (no − n′o )/(n − n′o )]

increments between successive values in each series decrease with increase in the alkyl chain length. Odd−even alternation in Tt, ΔHt and ΔSt has been observed in many homologous series of long chain compounds.39,41,44−47 Usually in such cases values of the above parameters are higher for the even chain length compounds than those with odd chain lengths. In contrast to this, in N-acyl L-alanines the odd chain length molecules exhibit higher values of Tt, ΔHt, and ΔSt than those with even chain lengths.49 Interestingly, the present results show that for AEs only the Tt values are higher for the odd chain length series, whereas the ΔHt and ΔSt values are higher for the even chain length series. In long-chain fatty acids in which the acyl chains are tilted with respect to the planes of the terminal methyl groups, it was shown that differences in the methyl end group structure between the odd and even chain-length compounds can lead to differences in the physical properties.44 Such packing differences are not seen if the chains are perpendicular to the plane of the terminal methyl groups. The odd−even alternation in the values of Tt, ΔHt and ΔSt of dry AEs is consistent with the

(9)

where no (= −ΔH0/ΔHinc) and n′o (= −ΔS0/ΔSinc) are the values of n at which the ΔHt and ΔSt extrapolate to zero. It can be seen from Figure 4A that the Tt values of dry AEs containing odd as well as even number of C atoms in the alkyl chains independently fit well to eq 9. The Tt∞ values for dry AEs with odd as well as even alkyl chain lengths were obtained from the fitting parameters as 399.5 and 394.7 K. These values are in good agreement with the Tt∞ values estimated using eq 9. In the fully hydrated state, transition temperatures of AEs with 13−18 C atoms in the alkyl chains increase with the chain length at pH 5.0 as well as pH 10.0. In contrast to the trends observed in the dry samples, the hydrated AEs do not show any odd−even alternation in the transition temperatures. Protonated AEs show higher Tt values than the deprotonated samples. Fits of the Tt values to eq 9 in both cases are shown in Figure 4B. From these fits the Tt∞ values for AEs hydrated with buffers of pH 5.0 and 10.0 were obtained as 397.3 and 377.8 K, respectively. Although these values differ considerably when compared with those obtained from the incremental values of

Figure 5. ORTEP of ANE·HCl (A) and packing along the a-axis and b-axis ANE·HCl (B,C). 9551

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Figure 6. Characterization of the ALE/DS catanionic complex. (A) Turbidity and (B) polarity ratio (I1/I3) of pyrene in various mixtures of ALE·HCl and SDS. (C) Raw ITC profile of the interaction between ALE·HCl and SDS. (D) Integrated data obtained from the raw data shown in C. Solid line in D represents the best curve fit of the experimental data to the “one set of sites” model in MicroCal Origin. (E) PXRD patterns of ALE·HCl, SDS and their equimolar mixture. (F) TEM images of the equimolar mixture. See text for details.

exposed to the aqueous medium, the α-methyl group is oriented inward and faces the terminal methyl group of the alkyl chain of another molecule from the opposite leaflet of the interdigitated bilayer, with the methyl−methyl distance in ANE and ADE being 3.935 and 3.81 Å, respectively, which indicates considerable dispersion interaction. The bilayer thickness (N1− N1 distance) in the structures of ANE and ADE is 17.015 and 17.42 Å, respectively. The corresponding repeat distances are 18.744 and 18.919 Å, respectively, which are in good agreement with the d-spacings of 18.88 Å (ANE) and 20.07 Å (ADE), estimated from PXRD (see below). The tilt angles of the hydrocarbon chains with respect to the bilayer normal (where the bilayer plane is defined by the N1 atoms), observed in the crystal structures of ANE and ADE, are 17.35° and 32.34°, respectively. Interdigitation is usually seen in single chain lipids such as lysophosphatidylcholine (3-dodecanoyl-propandiol-1phosphorylcholines·H2O) with bulky headgroup.55 In the crystal lattice of both ANE and ADE, each chloride ion is hydrogen bonded to three N−H hydrogen atoms (two from the same leaflet and one from the opposite leaflet), and the chloride ion acts as a bridge between the head groups coming from the adjacent and opposite layers. The hydrogen bond distances (H···Cl) and angles for the three types of N−H···Cl interactions in ANE are 2.32, 2.35, and 2.29 Å, and 157°, 163° and 160°. The corresponding values for ADE are 2.30, 2.30, and 2.24 Å, and 160°, 153° and 159°.

enthalpy and entropy, the trends in both estimates are similar as the Tt∞ at pH 5.0 is higher in both cases. Description of Structures. The molecular structures of ANE·HCl and ADE·HCl are shown in the ORTEPs given in Figure 5A and Figure S7A, respectively, along with the atomic numbering for the non-hydrogen atoms. The atomic coordinates and equivalent isotropic displacements for all nonhydogen atoms together with the bond distances, bond angles, and torsion angles of ANE·HCl and ADE·HCl are given in Tables S9−S11 and S12−S14, respectively. It can be seen from Figure 5 and Figure S7 that the hydrocarbon portion of the alkyl chains of ANE (C2−C9) and ADE (C2−C10) are in all-trans conformation, and the observed torsion angles are close to 180°. The torsion angles observed for ANE and ADE at O2−C1−C2−C3 linkage are −7° and 61°, which correspond to eclipsed and gauche conformations. As a result of these, a bend is seen in both ANE and ADE, giving the molecules an ‘L’ shape. Molecular Packing. Packing diagrams of ANE·HCl along the a-axis and b-axis are given in Figure 5B,C, respectively. Corresponding packing diagrams of ADE·HCl are shown in Figure S7B,C, respectively. Both the molecules are packed in a head-to-tail manner with the alkyl chains arranged in an interdigitated fashion with two hydrocarbon chains being accommodated per headgroup. While the amino group of the alanine moiety is oriented away from the bilayer and is fully 9552

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Figure 7. DSC thermogram of ALE·HCl/SDS equimolar mixture in water (A). Temperature dependence of polarity ratio (I1/I3) of pyrene in equimolar mixture (B). Effect of pH on equimolar aggregates formed by ALE·HCl and SDS monitored by (C) turbidity and (D) polarity ratio of pyrene.

Powder X-ray Diffraction. Since our efforts to obtain single crystals for higher chain AEs did not yield any success, in order to derive information on the structures adopted by these alkyl esters we carried out PXRD studies. The PXRD data obtained for AEs of different chain lengths are shown in Figure S8A,B. All AEs (n = 9−18) gave several sharp diffraction peaks in the 2θ range of 1−50°. From the diffraction peak position the d-spacings were calculated, with 3−4 peaks being used to estimate the average d-spacing of each AE. The average values obtained together with standard deviations are given in Table S15 and the plot depicting chain length dependence of the dspacings is shown in Figure S8C. The d-spacing data exhibit a linear dependence on the alkyl chain length with a slope of 0.985 Å/CH2. Since the crystal structures of ANE and ADE have clearly shown that the molecules are packed in an interdigitated bilayer structure, the linear dependence of dspacings on the alkyl chain length indicates that chain interdigitation would be present in all the AEs investigated here. Since the projected distance for each C−C bond in an untilted chain is 1.27 Å, the incremental value of 0.985 Å/CH2 suggests that the interdigitated chains would also be tilted with respect to the bilayer normal. From the value of 0.985 Å for increase in d-spacing per CH2, the tilt angle of the alkyl chains with respect to the bilayer normal was calculated as 39.1°. Although this value is somewhat higher than the angle of tilt observed in the crystal structure of ADE·HCl, it is broadly in agreement with the notion that all the AEs form interdigitated bilayer structures in the solid state. ALE and DS Form an Equimolar Complex. In view of the high potential of catanionic mixed surfactant systems in developing drug delivery systems, we investigated the interaction of ALE with DS by turbidimetric, fluorescence spectroscopic, and isothermal titration calorimetric studies. As discussed below, the results obtained from all the three methods show that ALE and DS form an equimolar complex. ALE·HCl forms an optically clear solution in water. Upon addition of small aliquots of SDS solution, the sample becomes turbid, most likely due to formation of larger aggregates. This suggests that cationic ALE interacts with negatively charged DS,

resulting in the formation of larger aggregates compared to those formed by ALE·HCl or SDS alone. This was investigated by Job’s method employing turbidimetry and fluorescence spectroscopy. In these studies, the total surfactant concentration was kept constant while concentrations of ALE·HCl and SDS were varied. The turbidity (measured as optical density at 330 nm) of ALE·HCl/SDS mixtures of different molar ratios is shown in Figure 6A. It is seen from the plot that the turbidity of the mixed surfactant solution increases with increase in SDS content up to an XSDS value of 0.5, but decreases with further increase in SDS content. The higher turbidity at XSDS = 0.5 indicates the formation of large aggregates containing the two components in equimolar ratio. The formation of catanionic complex was also investigated by fluorescence spectroscopy, by monitoring the polarity ratio (I1/ I3) of pyrene (probe). The pyrene polarity ratio decreases initially when SDS concentration is increased up to an XSDS value of 0.5 (Figure 6B). At higher XSDS, the polarity ratio (I1/ I3) increases with further increase in SDS concentration. The minimum in the I1/I3 ratio, seen at XSDS = 0.5, supports the above interpretation that ALE·HCl and SDS form an equimolar complex. Further evidence for the formation of ALE−DS equimolar complex and its thermodynamic characterization was obtained from ITC. A representative calorimetric titration shown in Figure 6C indicates that the exothermic heat of binding is initially nearly constant, but decreases rather steeply with successive injections until saturation is achieved. The incremental heat released as a function of the ratio of SDS to ALE·HCl together with a nonlinear least-squares fit of the data to a “one set of sites” model is shown in Figure 6D. The fit yielded the following values for various parameters characterizing the binding interaction: n = 0.86 (±0.07), Kb,= 6.14 (±0.86) × 105 M−1, ΔHb = −8.86 (±0.62) kcal/mol, ΔSb = −3.25 (±2.37) cal/mol/K. These values are consistent with the formation of a strong equimolar complex between ALE·HCl and SDS. Powder X-ray Diffraction Studies. Although we tried to crystallize the equimolar mixture of ALE·HCl and SDS, 9553

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decreases with increase in salt concentration (Figure S9). This is consistent with decreased electrostatic attraction between the two surfactants in the presence of high salt, which is known to screen the electrostatic forces. Interaction of ALE·HCl with DNA. In recent years, cationic lipids and amphiphiles are under active investigation as nonviral carriers of nucleic acids for gene therapy.56−58 Therefore, with the objective of exploring the possibility of AEs in such applications, we carried out preliminary investigations on the interaction of ALE·HCl with ST-DNA using ITC. A typical calorimetric titration of ALE·HCl from a 2.5 mM stock solution into 0.2 mM ST-DNA in water is shown in Figure 8.

unfortunately we did not get single crystals of the complex. Therefore, we collected a PXRD pattern of the mixture and compared it with the PXRD patterns of the individual compounds (see Figure 6E). It is clear from this figure that the diffraction pattern of the complex is different from the patterns of ALE·HCl and SDS, suggesting that the supramolecular structure of the complex is distinctly different from that of SDS as well as ALE·HCl. The d-spacing of the mixture calculated from the diffraction pattern is 32.08 Å (±0.41), whereas the d-spacing values of ALE·HCl and SDS are 22.19 and 37.75 Å, respectively. Transmission Electron Microscopy. The above observations indicate that catanionic aggregates are formed by mixing ALE and SDS in equimolar ratio. The morphology and size of these aggregates have been characterized by transmission electron microscopy. A TEM image of the ALE·HCl/SDS equimolar mixture is given in Figure 6F, which clearly shows that unilamellar vesicles are formed by the hydrated catanionic lipid mixture. The circular patterns seen in the image have a diameter ranging between 0.3 and 0.6 μm. Phase Transition of ALE−DS Complex. Figure 7A shows a DSC thermogram of a hydrated equimolar mixture of ALE· HCl and SDS. The thermogram shows a sharp transition centered at 38.7 °C, which was reproducible in second and third heating cycles. Individually, hydrated samples of ALE·HCl and SDS did not show any thermotropic phase transitions. The samples of the mixture were turbid at room temperature, but became optically clear above phase transition, suggesting that the mixture exists most likely in a micellar form at high temperatures (i.e., above Tt). This shows that the equimolar mixture forms stable liposomes, which undergo gel-micellar phase transition in a reversible manner. The phase transition of the equimolar ALE−DS complex was also investigated by florescence spectroscopy by monitoring changes in the polarity ratio of pyrene (see Figure 7B). The polarity ratio value does not change noticeably up to 35 °C, but exhibits a steep increase with temperature above 40 °C. This suggests that at high temperatures, the microenvironment of the probe gradually becomes more polar, which can be ascribed to the increased penetration of water molecules into the micelle interior as the temperature is increased. Effect of pH. The stability of the equimolar catanionic aggregates at different pH was studied by turbidimetry and fluorescence spectroscopy. The turbidity of the solution decreases slowly with increase in pH up to 7.0, but shows a sharp decrease above pH 7.0. This suggests that the catanionic aggregates are disrupted at high pH due to transformation of cationic lipid to neutral lipid (due to deprotonation of the ammonium group). Figure 7C shows turbidity of the equimolar ALE·HCl/SDS mixtures of fixed concentration as a function of pH. The changes in the polarity ratio (I1/I3) of pyrene probe in equimolar aggregates at different pH was also studied using fluorescence spectroscopy. The polarity ratio (I1/I3) of pyrene in the 1:1 complex increases with increase in pH of the sample (Figure 7D), suggesting that below pH 7.0 the pyrene probe is located in hydrophobic region of the aggregates, whereas at higher pH due to disruption of the aggregates and formation of micelles, due to which there is an increased penetration of solvent water molecules into the micelle as compared to the vesicles that exist at pH ≤ 7.0, the microenvironment of pyrene becomes more polar. When NaCl was included in the medium, it was observed that turbidity of the equimolar mixtures

Figure 8. Isothermal titration calorimetry of ALE·HCl binding to STDNA. (A) Raw data for the titration of 0.2 mM ST-DNA (nucleotide concentration) with 2.5 mM ALE.HCl at 25 °C. (B) Integrated heats of binding obtained from the raw data shown in panel A.

Three independent titrations gave essentially similar profiles. The ITC profile consists of two components; the initial injections resulted in exothermic heat release with slightly increasing amounts of heat released for successive injections, indicating cooperative and very strong binding. Such cooperativity was also observed for the binding of the cationic surfactant cetyltrimethylammonium bromide to DNA.59 When the charge ratio (Z±) was ∼0.7, the process became endothermic, and the incremental heat changes decreased with successive injections, reaching a near zero value at a molar ratio of 1.5. Since the binding process is rather complex, estimation of binding constants for the interaction is not straightforward, as observed earlier for the titration of plasmid DNA with other cationic lipids and amphiphiles.60 Notwithstanding this, the present results, which indicate rather strong interaction between ALE·HCl and DNA, suggest that AEs are promising candidates for further investigations in gene delivery applications.

4. CONCLUSIONS The present work reports investigations on the self-assembly, supramolecular organization, and phase behavior of a homologous series of L-alanine alkyl esters and characterization of ALE·HCl/SDS catanionic complex as well as the interaction of ALE·HCl with DNA. DSC studies on dry AEs revealed an 9554

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(6) Bramer, T.; Dew, N.; Edsman, K. Pharmaceutical Applications for Catanionic Mixtures. J. Pharm. Pharmacol. 2007, 59, 1319−1334. (7) Tondre, C.; Caillet, C. Properties of the Amphiphilic Films in Mixed Cationic_Anionic Vesicles: A Comprehensive View from a Literature Analysis. Adv. Colloid Interface Sci. 2001, 93, 115−134. (8) Tsuchiya, T.; Ishikake, J.; Kim, T.-S.; Ohkubo, T.; Sakai, H.; Abe, M. Phase Behavior of Mixed Solution of a Glycerin-Modified Cationic Surfactant and an Anionic Surfactant. J. Colloid Interface Sci. 2007, 312, 139−145. (9) Khan, A.; Marques, E. F. Synergism and Polymorphism in Mixed Surfactant Systems. Curr. Opin. Colloid Interface Sci. 1999, 4, 402−410. (10) Bergstrom, L. M.; Bramer, T. Synergistic Effects in Mixtures of Oppositely Charged Surfactants as Calculated from the Poisson− Boltzmann Theory: A Comparison Between Theoretical Predictions and Experiments. J. Colloid Interface Sci. 2008, 322, 589−595. (11) Kaler, E. W.; Murthy, A. K.; Rodriguez, B. E.; Zasadzinski, J. A. Spontaneous Vesicle Formation in Aqueous Mixtures of Single-Tailed Surfactants. Science 1989, 245, 1371−1374. (12) Kaler, E. W.; Herrington, K. L.; Murthy, A. K.; Zasadzinski, J. A. N. Phase Behavior and Structures of Mlxtures of Anionic and Cationic Surfactants. J. Phys. Chem. 1992, 96, 6698−6707. (13) Dubois, M.; Demé, B.; Gulik-Krzywicki, T.; Dedieu, J.-C.; Vautrin, C.; Désert, S.; Perez, E.; Zemb, T. Self-Assembly of Regular Hollow Icosahedra in Salt-Free Catanionic Solutions. Nature 2001, 411, 672−675. (14) Marques, E. F. Size and Stability of Catanionic Vesicles: Effects of Formation Path, Sonication, and Aging. Langmuir 2000, 16, 4798− 4807. (15) Xia, Y.; Goldmints, I.; Johnson, P. W.; Hatton, T. A.; Bose, A. Temporal Evolution of Microstructures in Aqueous CTAB/SOS and CTAB/HDBS Solutions. Langmuir 2002, 18, 3822−3828. (16) Andreozzi, P.; Funari, S. S.; La Mesa, C.; Mariani, P.; Ortore, M. G.; Sinibaldi, R.; Spinozzi, F. Multi- to Unilamellar Transitions in Catanionic Vesicles. J. Phys. Chem. B 2010, 114, 8056−8060. (17) Shioi, A.; Hatton, T. A. Model for Formation and Growth of Vesicles in Mixed Anionic/Cationic (SOS/CTAB) Surfactant Systems. Langmuir 2002, 18, 7341−7348. (18) Coldren, B.; van Zanten, R.; Mackel, M. J.; Zasadzinski, J. A.; Jung, H.-T. From Vesicle Size Distributions to Bilayer Elasticity via Cryo-Transmission and Freeze-Fracture Electron Microscopy. Langmuir 2003, 19, 5632−5639. (19) Rosa, M.; Miguel, M. D.; Lindman, B. DNA Encapsulation by Biocompatible Catanionic Vesicles. J. Colloid Interface Sci. 2007, 312, 87−97. (20) Lozano, N.; Pérez, L.; Pons, R.; Pinazo, A. Diacyl Glycerol Arginine-Based Surfactants: Biological and Physicochemical Properties of Catanionic Formulations. Amino Acids 2011, 40, 721−729. (21) Bonincontro, A.; La Mesa, C.; Proietti, C.; Risuleo, G. A. Biophysical Investigation on the Binding and Controlled DNA Release in a Cetyltrimethylammonium Bromide-Sodium Octyl Sulfate CatAnionic Vesicle System. Biomacromolecules 2007, 8, 1824−1829. (22) De Persiis, F.; La Mesa, C.; Pons, R. Protein-Covered Silica Nano-Particles Adsorbing onto Synthetic Vesicles. Soft Matter 2012, 8, 1361−1368. (23) Letizia, C.; Andreozzi, P.; Scipioni, A.; La Mesa, C.; Bonincontro, A.; Spigone, E. Protein Binding onto Surfactant-Based Synthetic Vesicles. J. Phys. Chem. B 2007, 111, 898−908. (24) Hong, M.; Weekley, B. S.; Grieb, S. J.; Foley, J. P. Electrokinetic Chromatography Using Thermodynamically Stable Vesicles and Mixed Micelles Formed from Oppositely Charged Surfactants. Anal. Chem. 1998, 70, 1394−1403. (25) Wang, X.; Danoff, E. J.; Sinkov, N. A.; Lee, J.-H.; Raghavan, S. R.; English, D. S. Highly Efficient Capture and Long-Term Encapsulation of Dye by Catanionic Surfactant Vesicles. Langmuir 2006, 22, 6461−6464. (26) Danoff, E. J.; Wang, X.; Tung, S.-H.; Sinkov, N. A.; Kemme, A. M.; Raghavan, S. R.; English, D. S. Surfactant Vesicles for HighEfficiency Capture and Separation of Charged Organic Solutes. Langmuir 2007, 23, 8965−8971.

unusual odd−even alternation wherein the odd chain length series showed higher Tt values, whereas the even chain length series displayed higher values of ΔHt and ΔSt. Crystal structures of AEs with 9 and 10 C atoms in the alkyl chain revealed tilted, interdigitated bilayer packing of the alkyl chains similar to that observed with the chain packing in the crystal structure of lysophosphatidylcholine. Turbidimetric, isothermal titration calorimetric and fluorescence spectroscopic studies on the interaction of L-alanine lauryl ester with sodium dodecyl sulfate provided strong evidence for the formation of an equimolar catanionic complex by these two surfactants. The catanionic complex was found to be base-labile, which should make it useful in designing liposomal formulations for drug delivery to tissues/organs at high pH, e.g., colon.30 The strong binding of ALE·HCl with DNA suggests that the alanine alkyl esters may also find use as carriers for genetic material in therapeutic applications.

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ASSOCIATED CONTENT

S Supporting Information *

Parts of Materials and Methods, Results and Discussion, 15 Tables (S1−S15) and 9 Figures (S1−S9) are given as Supporting Information. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.5b02475. (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Address: School of Chemistry, University of Hyderabad, Hyderabad-500 046, India. Tel: +91-40-2313-4807; Fax: +9140-2301-2460/0145; E-mail: [email protected]; Website: http://www.uohyd.ernet.in. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by a research grant from the DST (India) to M.J.S. D.S. was supported by a Senior Research Fellowship from the CSIR (India). We thank the Centre for Nanotechnology, supported by the DST (India), for the use of transmission electron microscope. The UGC (India) is acknowledged for their support through the UPE and CAS programs, to the University of Hyderabad, and School of Chemistry, respectively.

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REFERENCES

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