Micro-structural investigations on oppositely charged mixed surfactant gels with potential dermal applications

Dicarboxylic amino acid-based surfactants (N-dodecyl derivatives of -aminomalonate, -aspartate, and -glutamate) in combination with hexadecyltrimethylammonium bromide (HTAB) form a variety of aggregates. Composition and concentration-dependent mixtures exhibit liquid crystal, gel, precipitate, and clear isotropic phases. Liquid crystalline patterns, formed by surfactant mixtures, were identified by polarizing optical microscopy. FE-SEM studies reveal the existence of surface morphologies of different mixed aggregates. Phase transition and associated weight loss were found to depend on the composition where thermotropic behaviours were revealed through combined differential scanning calorimetry and thermogravimetric studies. Systems comprising more than 60 mol% HTAB demonstrate shear-thinning behaviour. Gels cause insignificant toxicity to human peripheral lymphocytes and irritation to bare mouse skin; they do not display the symptoms of cutaneous irritation, neutrophilic invasion, and inflammation (erythema, edema, and skin thinning) as evidenced by cumulative irritancy index score. Gels also exhibit substantial antibacterial effects on Staphylococcus aureus, a potent causative agent of skin and soft tissue infections, suggesting its possible application as a vehicle for topical dermatological drug delivery.


Results and discussion
Structures of C 12 MalNa 2 and HTAB are shown in (Fig. 1a) along with other information. Manifestation on the Gibbs ternary phase diagram (Fig. 1b) demonstrates the occurrence of gel, viscous, precipitate and clear fluid states. With increasing proportion of HTAB, surfactant mixtures form gels where the relative proportion of viscous and gel states increase following the order: C 12 MalNa 2 + HTAB > C 12 AspNa 2 + HTAB > C 12 GluNa 2 + HTAB (Fig. S1, supplementary section). Hydrophobic interaction between AASs and HTAB is the predominant factor for the formation of different types of aggregates besides the electrostatic attraction 8 . AASs interact with HTAB at a 1:2 mol ratio and form gels at equimolar region due to the dominance of the HTAB molecules 44 . Microstructural investigations on surfactant mixtures at different concentrations (AAS + HTAB) and different compositions (AAS/HTAB) were further investigated through polarizing optical microscopy (POM) and field emission scanning electron microscopic (FE-SEM) studies. POM studies reveal the occurrence of liquid crystal and associated textures as shown in Fig. 1c. Gels exhibit different textures in the surfactant concentration range of 3-5 wt%. With increasing proportions of HTAB, C 12 MalNa 2 + HTAB gels display nematic, smectic, spherulite, cholesteric, calamitic, and flower-like textures (Fig. S2, Table S1A) [44][45][46][47] . Nematic liquid textures originate from thread-like shapes, that correspond to surfactant gels aligning themselves in threadlike shapes as reported earlier 48 . The patterns become more complex with enhanced sizes due to the aggregation and associative interactions between AASs and HTAB. Texture size increases with increasing proportions of HTAB, which has a higher cross-sectional area than the AASs 49 .
Features become more prominent with increasing mixed surfactant concentrations, common in the case of lyotropic liquid crystals 16,47 (Fig. S2, Supplementary Section). In the case of C 12 AspNa 2 + HTAB gels for 50, 60, and 80 wt% of HTAB, smectic (Fig. S3a 1 ), spherulite (Fig. S3b 1 ), and flower-like (Fig. S3c 1 ) patterns are observed 50,51 , while in the case of C 12 GluNa 2 + HTAB mixtures, discotic ( Fig. S3a 2 ), calamitic ( Fig. S3b 2 ), and flower-like ( Fig. S3c 2 , Table S1B) textures are observed due to the formation of sterically favourable seven and eight-member rings. Two carboxylate groups get progressively separated by one methylene group while moving from C 12 MalNa 2 to C 12 AspNa 2 to C 12 GluNa 2 . Accordingly, two carboxylate groups of AASs can electrostatically interact with one HTAB to form six, seven, and eight-member rings 49 . Smectic textures designate ordered and rigid layer structure whereby C 12 MalNa 2 + HTAB can closely interact with HTAB to exhibit smectic textures (Fig. S2b 1 ,c 2 ,a 4 ,c 4 ). The nematic texture (Fig. S2a 1 ,b 2 ,a 3 ), is characteristic of stacked layer and positional order whereby the discotic texture (Fig. S2b 4 ) is due to rigid disk-like core, according to Fan Shao et al. 52,53 . Spherulite textures ( Fig. S2a 5 and b 3 ) are larger bundles and the hexagonal shapes caused by close packing are even more defined, as also reported by Haas et al. 54 C 12 MalNa 2 + HTAB gels display a more prominent spherulite texture ( Fig. S2a 5 ,b 3 ) than C 12 AspNa 2 + HTAB due to the formation of strongly aggregated structure and associative interaction with HTAB. Calamitic textures, exhibited by C 12 GluNa 2 + HTAB gels (Fig. S3b 2 , Table S1B) have a relatively flexible core, due to weak hydrophobic interaction between the oppositely charged C 12 GluNa 2 and HTAB system.
Microstructures of the aggregates were further investigated with FE-SEM studies, which display interconnected morphologies (Fig. 1d) 27,55,56 . To achieve optimal solvation and swelling, the pore of the gels can provide  S5c 1 ,a 2 ) due to the emergence of micropores at the surface of gels 60 . C 12 AspNa 2 + HTAB gels have fibre network-like morphologies (Fig. S5a 1 ,b 1 ) that can hold water molecules due to assisted surface tension enhancement. Irregular (Fig. S5b 2 ) and sheet-like structures (Fig. S5c 2 ) for C 12 GluNa 2 + HTAB indicate the entrapment of water molecules into the gels. Maximum number of HTAB accumulated in gels indicate that HTAB plays a fundamental role in demonstrating higher aggregation and formation of porous-like morphology, which are in consonance with the phase manifestation, and POM studies. Phase transition and associated weight loss of gels were investigated by thermogravimetry analysis (TGA) [61][62][63] . Results on the TGA of the pure components, as well as AAS + HTAB aggregates, have been summarized in Fig. S6 and Table S2. HTAB decomposes to produce some solid carbon along with the production of long-chain hydrocarbon, nitrogen, and hydrogen 61,64 , whereby decomposition of C 12 MalNa 2 , C 12 AspNa 2 and C 12 GluNa 2 to produce dodecane (or smaller alkyl fragments) and free aminomalonic, aspartic, and glutamic acid 63 . AAS + HTAB gels show endothermic peaks in the temperature range of 40 to 100 °C due to dehydration (Fig. 2a) 65 . Two carboxylate groups of C 12 MalNa 2 electrostatically interact with HTAB that result in higher ionicity and subsequent moisture absorption capability than C 12 AspNa 2 and C 12 GluNa 2 . In the case of C 12 GluNa 2 , two ionic carboxylate groups are separated by three methylene groups; so its interaction capability with HTAB and magnitude of hydration is lower. Formation of rigid aggregates result in the higher chain melting temperature (T m ) as determined from the DSC studies.
Thermotropic behaviour and associated parameters were evaluated by DSC studies 44 . Variation of phase transition temperature (T m ), width at half peak height (ΔT 1/2 ), enthalpy change (ΔH) and corresponding heat capacity changes (ΔCp) were determined as functions of α AAS as summarized in (Fig. 2). HTAB exhibits two endothermic peaks at 64 °C and 84 °C indicating transition from solid to liquid crystalline phase and the phenomenon www.nature.com/scientificreports/ of dehydration 44 , common in the amphiphiles (Fig. 2b) 63 . With increasing α AAS , T m values increase due to the incorporation of HTAB in the aggregates and assimilation of HTAB with AASs; relatively sharp peaks indicate favourable hydrocarbon chain packing (Fig. 2c) 49 . Lowering of T m is due to size reduction, decreased specific surface area, and interaction between oppositely charged surfactants also known as Kelvin effect 66 . Widening of the melting peaks designates multi-crystallinity and heterogeneity 66 . The extent of hydrophobic interaction between AAS and HTAB is lower in C 12 AspNa 2 and C 12 GluNa 2 , where the T m values follow the sequence: C 12 MalNa 2 + HTAB > C 12 AspNa 2 + HTAB > C 12 GluNa 2 + HTAB. With increasing α AAS , ΔT 1/2 values decrease indicating better packing of the hydrophilic overlayer as well as oppositely charged head groups (Fig. 2d). With increasing associative interaction between AAS and HTAB increased crystal imperfection results in higher ΔT 1/2 values. Increasing magnitude of interaction with increasing proportion of HTAB induces the formation of ionpair amphiphiles, resulting in higher ΔH values for the surfactant mixtures (Fig. 2e). With increasing α AAS , ΔCp values gradually increase and exhibit endothermicity due to the formation of water overlayer around surfactant aggregates (Fig. 2f). Lower values of ΔCp are due to the increase in multicrystallinity. Viscosity studies on AAS + HTAB mixtures at different combinations exhibit shear-thinning (results not shown) 49 . Zero shear viscosity (η 0 ) vs. concentration profile for the gels comprising 60 mol% HTAB are shown in Fig. 3a.
With increasing surfactant concentrations, vis cosity increas es monotonously in the range of 10 to 100 mM. The sequence in the viscosity variation follows the order: C 12 MalNa 2 + HTAB > C 12 AspNa 2 + HTAB > C 12 GluNa 2 + HTAB, which are in consonance with the previous studies. Due to the stronger packing and subsequent formation of rigid-structured aggregates, the viscosity of C 12 MalNa 2 + HTAB system is higher than the other two.
AAS + HTAB aggregates exhibit less toxicity against human blood lymphocyte up to 20 mM; although, cytotoxicity increases with increasing surfactant concentration (Fig. 3b). Results suggest that cytotoxicities are in consonance with the corresponding viscosity of the system.
Dermal responses towards the gels were assessed through topical application of the experimental gels on the bare mice skin surface where sterile distilled water and 5% (w/v) phenol-water were used as a negative (NC) and positive control (PC), respectively. Dermal irritability, as evidenced by the development of edema, and erythema were tested carefully and were scored according to the Organization for Economic Co-operation and Development (OECD) guidelines (Fig. 4A,C).
Simultaneously, developments of skin inflammation upon the application of gels were studied histologically by examining the hematoxylin-eosinY stained gel-treated skins for neutrophil invasion (Fig. 4B) 68,69 . It was found that gels do not induce symptoms related to skin irritation and inflammation (erythema, edema, and neutrophilic invasion) as shown in Fig. 4A(ii),B(ii). Effects of gels on mice skin appeared similar to the effect of sterile distilled water, used as negative control (NC). On the other hand, 5% (w/v) phenol-water (PC) caused considerable erythema, edema, and neutrophilic invasion Fig. 4A(iii),B(iii), resulting in high CII score (> 5.00) that corresponds to moderate skin irritation 67 . The CII score, for each of the gel treatment, was calculated to be as low as < 0.49 (Fig. 4C). These values, along with the CII values observed in NC group, correspond to the scores representing no potential skin irritation according to the OECD guidelines 67 . Besides, mice treated with 5% (w/v) phenol-water (PC group) visibly suffered from post-treatment erythema and edema resulting in higher CII score (> 5.00) that corresponds to moderate skin irritation 67 . Similarly, Fig. 4C reveals that there were significant differences (P < 0.05) in CII scores between 5% (w/v) phenol-water-treated group and each of the gel-treated groups. Based on these findings, it could be concluded that gels used in this study do not have significant skin irritation potential and thus may safely be used for topical dermal applications.
Gels also exhibited substantial antibacterial activities against Staphylococcus aureus, a gram-positive pathogenic bacteria. In spite of ~ 90% lymphocyte viability, 20 mM surfactant mixtures of AAS + HTAB (40:60, M/M) could substantially inhibit bacterial growth 67 . The minimum inhibitory concentration (MIC) is the lowest concentration of a substance that renders no turbidity in bacterial culture, corresponding to 99% bacterial growth inhibition 70 . The MIC value of individual surfactant mixture was determined for different clinical isolates of S. aureus, i.e., American Type Culture Collection (ATCC) 25923 and four other clinical isolates: AK1, AK2, AK8, and AK10 with the broth dilution method. Vancomycin was also used as a reference drug for growth  Table 1.
Results indicate that gels can potentially be used in the treatment of bacteria-borne dermatological infections. In addition, the gels are expected to exhibit higher entrapment efficiency and sustained release of dermatological drugs in terms of topical applications. However, further in vitro and in vivo studies are warranted to substantiate the aptitude of the gels as potential drug delivery systems. Besides, studies involving composition structure and functional correlationships are also considered to be worthy.

Summary and conclusions
Microstructures of AAS + HTAB aggregates were investigated by combined phase manifestation, polarizing optical microscopy and field-emission scanning electron microscopic studies. Texture of liquid crystals depended on the concentration and proportion of constituent surfactants. Energetics of phase transition processes were evaluated by TGA and DSC studies. Cytotoxicity could be correlated with the viscosity of the gels. Gels impart insignificant skin irritation although they possess substantial antibacterial activities that project their potential as dermal drug delivery systems. However further in vitro and in vivo studies by incorporating appropriate drugs into the gels are necessary and is considered as the future perspectives.
In order to draw final conclusions about solvation, water channels, and pores, drawn from the morphological studies, further characterization techniques, viz., small-angle X-ray scattering and small-angle neutron scattering studies, combined with the molecular dynamics simulation studies are worthy to be investigated. These are considered as the future perspectives.  67 . Overall comparison of CII scores among the groups were carried out by the Kruskal-Wallis test followed by post-hoc Dunn's test for multiple comparisons between each pair of groups. Significant differences in CII scores at P < 0.05 were obtained between groups PC vs. NC, PC vs. C 12 MalNa 2 + HTAB, PC vs. C 12 AspNa 2 + HTAB, and PC vs. C 12 GluNa 2 + HTAB gels.
Methods. Phase manifestation. Composition, close to the boundary of the two-phase regions, was detected by homogeneous mixing of aqueous stock solution of oppositely charged surfactants at 25 °C. The exact boundary of the two-phase region was detected by further step wise addition (using a calibrated micropipette under constant stirring) of a higher concentration of AASs into the HTAB solution. On the basis of visual observation, more than one hundred samples were collected at different AASs and HTAB weight ratios and phase boundaries were identified 75 . The different phases were recorded consecutively for a longer time-period (upto fifteen days, after which the samples started microbial degradation). All the experiments were repeated thrice to ensure reproducibility.
Microscopic studies. The texture of different combinations of the mixed surfactant systems were recorded with a polarizing optical microscope (POM, Nikon ECLIPSELV100POL, Japan) set with a CCD camera. The sample was placed onto a glass slide and thereafter the POM images were recorded. Morphology of the surfactant aggregates were investigated with field emission-scanning electron microscopy (FE-SEM, ZEISS EVO 18, Germany). Samples were prepared by the drop-casting of the gel on a freshly cleaved mica foil and kept in air for two hours for solvent evaporation. Those were further dried at reduced pressure for two hours. The gold-sputtered samples were then analysed in FE-SEM at the operating voltage of 20-30 kV.
Thermogravimetric analysis (TGA). Weight loss and thermal stability of the gels were investigated by TGA, performed using Pyris 6 TGA-DTA-8000 (Perkin Elmer, USA). Samples were scanned in the temperature range of 50-500 °C with a scan rate of 2 °C min −1 under nitrogen gas flowing conditions.
Differential scanning calorimetric (DSC) studies. DSC studies were performed to evaluate the chain melting temperature (T m ) and associated thermodynamic parameters of mixed surfactant systems that control its physical states. DSC measurements were recorded using a Pyris 6 DSC-8000 (Perkin Elmer, USA) differential scanning calorimeter with indium as a calibrator before performing the experiment. After equilibrating for 10 min, the sample was scanned in the temperature range 0-100 °C with a scan rate of 2 °C min −1 during the heating cycle. From the thermogram the peak temperature and enthalpy of phase transition were evaluated. Endothermic peak vs. temperature in evaluating different physicochemical parameters of mixed surfactant system were considered 76 .
Rheology studies. Viscosities of different surfactant mixtures were determined with a DV II-Pro rotoviscometer (Brookfield, USA) with a stated accuracy of ± 0.01 cP. 1.0 mL surfactant solution of different concentrations (40,60,80, and 100 mm) were taken in a cone and plate type rotoviscometer separately 77 . Viscosities were measured at different shear rates (ranging from 76 to 380 s −1 ). Zero shear viscosity (η 0 ) was determined from the intercept of the plot of viscosity vs. shear rate by fitting polynomial regression. Temperature during the viscosity measurement was controlled by a circulatory water bath MIC-255 (Hanntech Corporation, South Korea).
Biological activities. All the biological experiments were performed in accordance with relevant guidelines and regulations, duly approved by the Institutional Ethics Committee, Vidyasagar University. All the methods were carried out in accordance with relevant guidelines and regulations.
Cytotoxicity studies. Cytotoxicity studies were carried out following the method of Sun et al. 67  www.nature.com/scientificreports/ Skin irritation test. The biocompatibility of the experimental gels (AAS + HTAB) were investigated through skin irritation tests on swiss albino mice, in compliance with the Animal Research: Reporting of in vivo Experiments (ARRIVE) guidelines 80 . The tests were performed following the method mentioned in the Good Laboratory Practice Standards (GLPS) manual and the guidelines of OECD for acute dermal irritation 81 . Thirty healthy swiss albino mice were divided into five groups, each group consisting of six mice. Group A was negative control (NC, treated with sterile distilled water) and group B was positive control (PC, treated with 5% (w/v) phenol-water). Mice of group C, D, and E were treated individually with 100 mM C 12 MalNa 2 + HTAB, C 12 AspNa 2 + HTAB and C 12 GluNa 2 + HTAB gels, respectively. Prior to the application of gels, hairs of the dorsal area of the trunk region of all mice were removed followed by the topical application of 500 mL of the gels and the controls, respectively. After 1 h, signs of erythema or edema in individual animal was recorded. The entire procedure of dermal application of gels and subsequent recording for any irritation was continued for seven consecutive days. The CII score for each of the treated groups were calculated according to OECD guidelines 81 . CII score of each treated group is the average score, i.e., score of a total of erythema and edema divided by the number of animals and testing days.
Histological studies of mice skin. Animals were sacrificed post euthanasia by carbon dioxide asphyxiation after the completion of skin treatment with gels and controls for seven consecutive days. The treated skins were processed in wax blocks and transverse sections were prepared, followed by staining with hematoxylin-eosinY (HE) 69 . The prepared skin sections were further examined under a light microscope (Axioscope A1; Carl Zeiss, Germany). Histology of the gel-treated skins were compared with the controls.
Studies on antibacterial activity. Antibacterial efficacies of the gels against the gram-positive bacterial pathogen, Staphylococcus aureus, grown in Luria Bertani (LB) broth, were evaluated. MIC of each gel against the five clinical isolates of S. aureus (AK1, AK2, AK8 and AK10 along with ATCC 25923) was determined by broth dilution method. 10 µL gelatinous suspension of surfactant mixture at particular composition was added to 1 mL bacterial culture in LB having approximately 1 × 10 7 CFU. Each surfactant mixture (AAS + HTAB) was added to the bacterial culture in the concentration range of 5 to 100 μM (with an increment of 5 μM in each step) in a serial manner for individual bacteria and were incubated at 37 °C for 18 h [70][71][72]82 . Vancomycin was used as reference drug for growth inhibition. All the experiments were repeated thrice.
Statistical analysis. The mean value and standard deviation of the quantitative variables were calculated after repeating each quantification three times. Overall comparisons of data among the groups were carried out by the Kruskal-Wallis test. This was followed by post hoc Dunn's test for multiple comparisons of data between each pair of groups. Differences were considered significant at P < 0.05 83 .