J Mater Sci: Mater Med (2014) 25:703–721 DOI 10.1007/s10856-013-5112-1

Olive oil based novel thermo-reversible emulsion hydrogels for controlled delivery applications Vinay K. Singh • Sowmya Ramesh • Kunal Pal • Arfat Anis • Dillip K. Pradhan Krishna Pramanik

•

Received: 25 May 2013 / Accepted: 29 November 2013 / Published online: 11 December 2013 Ó Springer Science+Business Media New York 2013

Abstract Gels have been considered as a popular mode of delivering medicament for the treatment of sexually transmitted diseases (STDs) (e.g. human immunodeficiency virus, bacterial vaginosis, epididymitis, human papillomavirus infection and condylomata acuminata etc.). The present study discusses the development of novel olive oil based emulsion hydrogels (EHs) using sorbitan monopalmitate as the structuring agent. The developed EHs may be tried as drug delivery vehicle for the treatment of STDs. The formation of EHs was confirmed by fluorescence and confocal microscopy. FTIR studies suggested intermolecular hydrogen bonding amongst the components of the EHs. X-ray diffraction study suggested the amorphous nature of the EHs. The developed EHs have shown nonNewtonian flow behavior. The EHs were found to be

Electronic supplementary material The online version of this article (doi:10.1007/s10856-013-5112-1) contains supplementary material, which is available to authorized users. V. K. Singh  K. Pal (&)  K. Pramanik Department of Biotechnology & Medical Engineering, National Institute of Technology, Rourkela 769008, Odisha, India e-mail: [email protected] V. K. Singh e-mail: [email protected] S. Ramesh Sastra University, Tirumalaisamudram, Thanjavur 613401, Tamil Nadu, India A. Anis SABIC Polymer Research Center, Department of Chemical Engineering, King Saud University, Riyadh 11421, Saudi Arabia D. K. Pradhan Department of Physics, National Institute of Technology, Rourkela 769008, Odisha, India

biocompatible. The formulations were able to effectively deliver two model antimicrobial drugs (e.g. ciprofloxacin and metronidazole), commonly used in the treatment of the STDs.

1 Introduction Hydrogels may be defined as polymeric constructs which immobilizes aqueous phase. If the polymeric constructs are formed due to physical interactions, the developed gels are regarded as physical hydrogels [1]. The mechanism of physical interactions may include H-bonding, Van der Waals forces and/or electron transfer amongst the components of the hydrogels. Since there are no chemical reactions involved, the physical hydrogels often show gel-tosol transition with the increase in the temperature of the formulation and subsequently show sol-to-gel transition as the temperature of the formulation is decreased. Due to this thermo-reversible nature, the physical hydrogels have been described to posses self-healing capacity. The stability of such formulations has been reported to be sensitive under varied environmental conditions, viz. pH, salt concentration and temperature. The physical hydrogels quite often show pseudoplastic flow behavior, i.e. they are present as solid-like structure at lower shear rates while they behave as fluid at higher shear rates [2]. The hydrogels, either made from polymers or by interactions of different surfactants, have been a centre of attraction of research since centuries for their potential applications in product development for everyday use [3]. If the hydrogels contain internal oil phase, then the hydrogels are regarded as EHs and such hydrogels have not yet been studied extensively. In this study, olive oil (OO) was selected as the internal oil phase. Olive oil is obtained from the fruit of olive trees

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(Olea europaea). It is mainly a mixture of triglyceride esters of oleic acid and palmitic acid and other fatty acid. It contains squalene and sterols in trace amounts [4]. Due to the presence of the natural phenolic compounds (e.g. glycoside oleuropein, hydroxytyrosol and tyrosol), OO has been reported to possesses potent antioxidant properties [5, 6]. Topical application of virgin OO has been reported to delay the onset of UV-light induced skin tumors in mice. This has been attributed to the presence of the phenolic compounds in OO. The study concluded that OO has a UVprotective nature [7]. Moreover, there are no reports suggesting any side effects of topical applications of OO based formulations and are widely used in cosmetics, soaps and pharmaceutical industries [8, 9]. Sorbitan monopalmitate (SMP) was selected as the surfactant based gelator for this study. SMP is a solid gelator and is generally known to promote water-in-oil type of emulsions. It has been studied extensively for the development of organogels [10–12]. SMP forms a 3D network structure during the formation of organogels. SMP based formulations have been used to develop various cosmetic and pharmaceutical products [13, 14]. Our group has previously reported the development of OO based organogels as carriers for controlled drug delivery [15]. The main disadvantage of the organogels based products is the greasy feeling when the gels are applied topically. In this context, hydrogels provide a soothing effect over the skin, when applied topically. Due to this reason, hydrogels has been extensively used to develop products of pharmaceutical, cosmetics and food industries [16]. The EHs are special category hydrogels which comprise of a continuous aqueous phase and a dispersed oil phase [17]. Physical hydrogels possess a wide range of applications, including tissue engineering, cell encapsulation [18] and skin regeneration [19, 20]. Chen et al. [21] reported formation of high internal phase EH prepared from water borne poly(N-isopropylacrylamide) nanogel dispersions for food and nutraceutical applications. Gelatin-based hydrogels crosslinked with genipin has been developed and characterized for controlled drug delivery [22–24]. The use of gels for the treatment of STDs has been extensively studied [25–27]. Swinehart et al. [28] developed sustained release injectable gels, containing fluorouracil and adrenaline, for treating condylomata acuminata. Tasoglu et al. [29] reported topical microbicidal gels for the treatment of HIV. Sobel et al. [30] reported reduction in recurrent symptomatic episodes of bacterial vaginosis using metronidazole (0.75 %). Ferris et al. [31] achieved nearly equivalent cure rates while comparing oral metronidazole, metronidazole vaginal gel and clindamycin vaginal cream for the treatment of bacterial vaginosis. Anukam et al. [32] compared probiotic Lactobacillus GR-1 and RC-

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14 with metronidazole vaginal gel for the symptomatic treatment of bacterial vaginosis. Center for Disease Control and Prevention (CDC) proposed the use of ciprofloxacin for the treatment of gonococcal infections [33–35]. Knapp et al. [36] proposed criteria for interpretation of susceptibilities of strains of Neisseria gonorrhoeae to ciprofloxacin, ofloxacin, enoxacin, lomefloxacin, and norfloxacin. Mostly EHs are developed using chemical crosslinker. In the present study, we report the development of physical EHs, which can allow eliminating the need for crosslinking agents, thereby; avoiding the cytotoxic properties associated with the crosslinking agents. Taking inspiration from the above, it seems justified to develop OO based formulations for the treatment of STDs. The present study reports the development and a through characterization of novel OO based EHs for controlled delivery of model antimicrobials, viz. metronidazole and ciprofloxacin, commonly used antimicrobials for the treatment of STDs.

2 Materials and method 2.1 Materials SMP and nutrient agar were purchased from Loba Chemie Pvt. Ltd., Mumbai, India. Food garde OO was purchased from the local market. Metronidazole (MZ) was provided as a gift by Aarti drugs, Mumbai, India. Ciprofloxacin (CP) was purchased from Fluka, China. Microbial culture of Bacillus subtilis (NCIM 2699) was obtained from National Collection of Industrial Microorganisms (NCIM), Pune, India. Nutrient agar was purchased from Himedia laboratories Pvt. Ltd., Mumbai, India. All experimental studies were carried out using double distilled water (DW). 2.2 Methods 2.2.1 Preparation of gels The optimization for the gels was carried out by trial and error method, in which the proportions of DW, SMP and OO were varied. In short, DW (maintained at 60 °C) was added drop-wise to the hot solution of SMP and OO, kept on stirring at 500 RPM at 60 °C. The stirring was further continued for 30 min after the addition of the specified amount of DW under same experimental conditions. This resulted in the formation of a hot homogenous emulsion. The hot emulsions were subsequently cooled down to room-temperature (RT, 25 °C). Depending on the composition of the SMP-GNO-DW mixture, the biphasic system either formed a semi-solid formulation or remained as liquid emulsions, as the biphasic system was cooled down to RT [37]. The formation of the gel was confirmed by

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inverted tube method [38, 39]. 1 % w/w MZ and CP (model antimicrobial drugs) were incorporated within the optimized gel formulations for drug release and antimicrobial efficiency studies. The methods of preparation of the drug-loaded gels were similar to the blank gels except that the drug dispersion in OO was used for making the gels instead of OO. 2.2.2 Stability studies Thermocycling (freeze–thaw cycles; FT) method is one of the accepted and widely reported methods to determine the accelerated stability for emulsion based formulations [40, 41]. One cycle of freeze–thaw consisted of 15 min of freezing at -5 ± 1 °C and 15 min of thawing at 70 ± 1 °C. A typical test consists of 5 cycles. After each cycle, the gels were analyzed for any signs of destabilization. Bright field microscopy was carried out after each cycle to study the effect of thermocycling on the structural integrity of the gels. A sample is regarded as stable if there are no indications of instability after the completion of the study [42]. The optimized gels were incubated at 30 ± 2 °C/65 % RH ± 5 % RH for 6 months (intermediate stability, International Conference on Harmonisation ICH guidelines) and checked at regular intervals of time for any signs of instability (e.g. phase separation or syneresis) [43]. 2.2.3 Organoleptic evaluation The organoleptic properties such as color, odor, texture, appearance and taste were studied [44]. Measurement of pH is very essential as these formulations are meant to come in contact with the body tissues. Even a smallest variation in the pH can lead to various immunological reactions [12]. ATC pH meter (EI instruments, model no132E) was used to determine the pH of the sample. pH of the sample should remain in the range of 6.0–7.5 to prevent skin irritation [45].

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(Leica make; model: M205 FA) and confocal (Olympus make; model: Fluoview FV-1000) to confirm the organization of the immiscible phases in the gels. 0.1 % fluoral yellow solution in OO was used for the development of gels for analysis using fluorescence and confocal microscope. Green filter was used for visualization under fluorescence microscope. Multi argon laser was used for exciting fluoral yellow (emission peak = 515 nm) and the microstructure was visualized using a screening filter of 527 nm. The topology of the gels was studied using scanning electron microscopy (SEM; Jeol JSM-6480LV, Japan). The gels were converted into xerogels by removing the external solvent phase. The xerogels were prepared by carefully washing with acetone and subsequent drying under vacuum for 6 h. The dried gels were coated with platinum before analysis [12]. The xerogels were subsequently analyzed using atomic force microscopy (AFM; Veeco di innova) in the ‘‘contact mode’’ using etched silicon probes operated at their fundamental resonance frequencies (xo) of 18.0–23.0 kHz. The images were recorded at a scan rate of 20 lm/s and a scan angle of 0° [49]. 2.2.5 Molecular properties The identification of the functional groups in the raw materials and their interaction in gels, if any, was studied using FTIR (AlphaE ATR-FTIR, Bruker, USA) in the range of 4,000–400 cm-1 [50, 51]. X-ray diffractometer (XRD-PW 1700, Philips, Rockville, USA) was used to study the effect of the gel components on the crystalline nature of the gels. Monochromatized Cu Ka radiation (k = 0.154 nm) was used as the x-ray source. The analysis was done in the diffraction angle range of 5°–50° 2h. The scan rate for the analysis was 2° 2h/min [52] . 2.2.6 Mechanical properties

2.2.4 Microscopic studies An upright bright field microscope was used to study the microstructure of the developed gels (Leica DM 750 equipped with ICC50HD Camera). The effect of the variation of the concentration of DW, SMP and OO on the formation of gels was studied in-depth [46, 47]. The micrographs of the gels were processed and the size distribution pattern of the dispersed phase was analyzed using NI Vision Assistant-2010, National Instruments, USA [42, 48]. The microstructure of the optimized gels was further analyzed by automated Fluorescence Stereo Microscope

Cone-and-plate viscometer (Bohlin visco 88, Malvern, UK) was used to study the viscosity profile of the freshly prepared gels [53]. The experiments were conducted at RT. The rheological measurements were performed using a controlled-strain rheometer (TrustrainTM, Rheometer, Anton Paar, Austria) using a parallel plate geometry (25 mm diameter, 1 mm gap) by small-deformation oscillatory measurement [54]. A sequence of the following sweeps was used: (1) a strain sweep from 0.01 to 500 % at a frequency of 1 rad/s and (2) a frequency sweep between 0.1 and 100 rad/s at fixed strain of 0.5 %. Both the tests were conducted at RT. The storage modulus (G’) and the

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loss modulus (G’’) were recorded. Complex viscosity (g) was calculated from the results [55]. The mechanical behavior of the gels were studied by TAHD Plus Texture Analyser (Stable Microsystems, TAHDplus, U.K) [56]. 2.2.7 Thermal properties Melting point (MP) apparatus (MP apparatus 931, Environmental & Scientific Instruments Co., Panchkula, Haryana, India) was used for the determination of the MP (Tm) of the gels. Drop-ball method was used for the analysis as per the reported literature [57]. The thermal properties of the developed gels was analyzed using differential scanning calorimeter (DSC 200F3 Maia, Netzsch, Germany) in the temperature range of 25 and 150 °C. Aluminium pans, with pierced lids were used under nitrogen environment. The flow-rate of the nitrogen gas was maintained at 40 ml/min. The heating-rate of the gels was 2 °C/min [48, 58, 59]. 2.2.8 Electrical properties The electrical properties of the developed gels were carried using a computer-controlled impedance analyzer (Phase sensitive multimeter, Model: PSM1735, Numetriq, UK). The tan delta, impedance, phase angle and parallel capacitance were obtained as a function of frequency (0.1 Hz– 1 MHz) at RT [60]. 2.2.9 Hemocompatibility study The hemocompatibility study was done as per the literature reported earlier [15, 61]. In short, the optimized gels were placed in dialysis bags and were incubated in 50 ml saline, kept on stirring at 100 rpm and maintained at 37 ± 2 °C for 1 h. 0.5 ml of the leachant was mixed with 0.5 ml of citrated goat blood. The final volume was made up to 10 ml using saline. For positive control, 0.5 ml of citrated blood was mixed with 0.5 ml of 0.1 N HCl and the final volume was made up to 10 ml by adding normal saline. In the similar manner, negative control was prepared by mixing 0.5 ml saline to 0.5 ml of diluted blood followed by the dilution of the mixture to 10 ml using normal saline. The test and the control samples were incubated at 37 ± 2 °C for 1 h. The samples were subsequently centrifuged at 3000 rpm for 10 min and the supernatant was collected. The supernatant was analyzed using UV–vis spectrophotometer (Shimadzu UV 1601 R) at 545 nm. Percentage hemolysis of the blood was calculated using the formula given below [62]:

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% Hemolysis =

ODTest  ODNegative  100 ODPositive  ODNegative

ð1Þ

where, ODTest = optical density of test sample, OD Positive = optical density of ?ve control and ODNegative = optical density of -ve control. % hemolysis \5 is considered as extremely hemocompatible while a value less than 10 indicates hemocompatible. If the % hemolysis exceeds more than 20, the sample is considered as non-hemocompatible [63]. 2.2.10 In vitro drug release studies The drug release from the MZ and CP loaded gels was carried out by using a two-compartment modified Franz’s diffusion cell as per the reported literature [64]. In short, accurately weighed (*1.0 g) drug-containing gels were taken in the donor attached with previously activated dialysis membrane (MW cut-off—60 kDa, Himedia, Mumbai), which served as semi-permeable membrane. 50 ml of DW served as the dissolution media. The dissolution media was kept on stirring at 100 rpm and was maintained at 37 ± 1 °C. The samples were collected at regular intervals, 15 min for the first 1 h, 30 min for the next 3 h and 60 min for the next 8 h. At each sampling time, the receptor fluid was completely replaced with fresh DW to ensure sink conditions. The sampled aliquots were analyzed using UV–vis spectrometer (Shimadzu UV 1601 R) at 321 nm and 271 nm for MZ and CP, respectively [65]. The cumulative percentage of drug release (CPDR) was calculated from the results. The release kinetic models (e.g. zero order, first order, Higuchi kinetics and KP model) were studied in-depth. The drug release studies were carried out in duplicate [66, 67]. Iontophoresis employs enhanced drug diffusion under the influence of electric current. An in-house developed ionotophoretic drug delivery setup was used to perform the in vitro drug release studies. The donor compartment was filled with accurately weighed (*15 g) drug loaded organogels. The dummy compartment contained DW. Stainless steel electrodes (diameter 2 cm) were connected to the donor and the dummy chambers. The donor compartment was separated from the receptor compartment using pre-activated dialysis membrane (MW cut-off— 60 kDa, Himedia, Mumbai). The receptor compartment contained 130 ml of DW as the dissolution media which was kept on stirring at 100 rpm. The temperature of the receptor fluid was maintained at 37 °C to simulate the physiological conditions. A sinusoidal current of 8.04 lA (Irms), which provided a current density of 0.644 lA/cm2, was used for the study. The voltage controlled constant current source was developed using a computer-controlled microprocessor (Model: speedy-33, National Instruments,

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Fig. 1 Mechanism of gel formation. a Homogenous mixture of OO and SMP, b Milky white mixture after addition of water, and c Gel formed when cooled to RT

USA). The microprocessor generated a sinusoidal voltage of 1.414 V (Vrms). 3 ml of the releasate was collected at regular intervals of 15 min for 2 h which was subsequently replaced with 3 ml of fresh DW. The collected samples were analyzed spectrophotometically (UV 3200 double beam, Labindia) at 321 nm [68, 69]. 2.2.11 Antimicrobial studies Antimicrobial efficacy of the MZ and CP loaded gels were studied against B. subtilis, a model gram positive bacteria [70, 71]. 100 ll of Bacillus strain (2 9 106 CFU/ml) was spread over the petri-plates containing solid nutrient agar medium using a L-shaped spreader (spread plate method) [72]. 9 mm diameter wells were made with the help of a borer. The blank gels, MZ loaded gels, CP loaded gels, marketed gel (Metrogyl) and the pure drug in powdered form were poured in the wells [12, 73]. Petri-plates were then incubated at 37 ± 1 °C for 24 h. The zone of inhibition was measured using a scale [74, 75].

3 Results and discussion 3.1 Preparation of gels Biphasic systems, containing SMP, OO and DW, were developed with the aim to formulate a more acceptable and stable gel based formulations for controlled delivery applications [62]. SMP was dissolved in OO in a culture tube, kept

Table 1 Optimization of gel formulations SMP (% w/w)

DW (% w/w) 20

40

60

80

2

X

X

X

X

4

X

X

X

X

5

X

H

H

X

6

X

H

H

X

8

X

H

H

H

10

X

H

H

H

on stirring at 500 rpm and maintained at 60 °C. The clear solution turned into white turbid mixture when warm DW was added drop-wise. As the temperature of the system was lowered to RT, the mixture either formed a gel or remained as liquid mixture (Fig. 1). The critical gelation concentration (CGC) of SMP for the gelation of OO is 19 % (w/w), as reported in our previous paper [15]. The proportion of SMP: OO: DW was varied in such a way so as to reduce the SMP concentration. The concentration of SMP was varied from 1 to 10 % (w/w) whereas the proportion of DW was varied from 20 to 80 % (w/w) to modulate the properties of the biphasic formulations (Table 1). It was observed that the gelation happened when the concentration of SMP was C5 % w/w and the concentration of DW was C40 % w/w. The CGC of the SMP-OO-DW formulations was obtained when the proportion of SMP: OO: DW was 5:55:40.

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Table 2 Accelerated stability testing of the stable gel formulations SMP (% w/w)

DW (% w/w) 20

40

60

80

2

X

X

X

X

4

X

X

X

X

5

X

X

X

X

6

X

H

H

X

8

X

H

H

H

10

X

H

H

H

When the concentration of DW was B40 % (w/w), the prepared formulations were non-homogenous, inconsistent and were gritty in appearance. They failed to maintain their integrity when the culture tubes were inverted, which is a primary requirement to be considered as a gel [76]. Increase in the concentration of DW resulted in the improvement of the gel properties (e.g. formation and consistency). The consistency and smoothness of the gels was found to increase with the increase in the DW concentration. At lower concentrations of SMP (5 and 6 %), as the concentration of DW was increased, there was formation of gels when the concentration of DW was 40 and 60 %. But, when the concentration of DW was increased beyond 60 %, the formation of gels did not happen. As the concentration of SMP was increased [6 %, there was an improvement in the qualitative aspects of the gels. It was also observed that at higher proportions of SMP, the gelation was quicker [77]. Also at higher proportions of SMP, the formation of gels happened even at higher proportions of DW under the experimental conditions. 3.2 Stability studies The developed gels were analyzed for their stability by accelerated stability test. The test employs 5 cycles of thermo-cycling at – 5 and 70 °C. The test aims at the analysis of the physical stability of the gels by altering the physical interactions at – 5 °C and promoting the chemical degradation reactions which occurs at higher temperatures (70 °C) [78]. The results showed that the gels containing 5 % of SMP failed to pass the test. These gels lost their structural integrity and started flowing at the end of the test (Fig. S1). All the other gel formulations were found to be stable, i.e., when the concentration of the SMP was [6 %, the gels were able to maintain their structural integrity (Fig. S1; Table 2). Since our main objective was to reduce the concentration of SMP to the lowest extent possible without compromising the stability of the gels, the gels having 6 % SMP were chosen for further studies. The gels having SMP: OO: DW of 6:56:40 and 6:36:60 were termed as

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GW1 and GW2, respectively. MZ loaded GW1 and GW2 were named as GW1M and GW2M, respectively, whereas CP loaded GW1 and GW2 were named as GW1C and GW2C, respectively. The GW1 and GW2 gels were incubated at 30 ± 2 °C and 75 ± 5 % RH for 12 months. Both types of the gels did not show any signs of destabilization during the experimental period and hence can be regarded as stable gels. 3.3 Organoleptic evaluation The gels (GW1 and GW2) were white in color, slightly greasy and had a smooth texture. The pH of all the samples were in the range of 6.5–7.5, i.e., within the pH range of the human skin (Table S1). 3.4 Microscopic studies Bright field micrographs have been shown in Fig. 2. The micrographs suggested the presence of spherical globules, which were uniformly dispersed throughout the matrix of the gels. Though the average size of the dispersed phase of both GW1 and GW2 was *17 lm, the span of the droplets present in the GW2 was higher as compared to GW1 (Fig. 3). GW2 showed droplet sizes up to 100 lm while GW1 showed droplets up to 200 lm size. The dispersed phase of GW1 was closely packed while inter-particulate distances amongst the droplets were higher in GW2. This may be explained by the fraction of DW present in the formulations. Since the amount of DW was higher in GW2, the fraction of the internal phase was lower in GW2. This resulted in the increased inter-particulate distance of the dispersed phase in GW2. On the other hand, the proportion of DW was lower in GW1. Hence the fraction of the dispersed phase was higher in GW1 which resulted in the formation of a more compact microstructure. At higher magnifications, fibrous structures were visible. Since the SMP molecules are usually present at the interface of the oil and water, it is expected that the fibrous structures were due to the presence of SMP molecules at the OO and DW interface [40]. The dispersed phase size analysis of the gels was carried out as per the reported method (Fig. S2) [76]. The bright field microscopy failed to explain the nature of the emulsion. Fluorescent and confocal microscopic analysis was performed to have a clear understanding about the distribution of the phases [79]. Fluoral yellow was incorporated within the oil phase for the study. The fluorescent and confocal micrographs have been shown in Fig. 4a–d. The micrographs showed the presence of fluorol yellow within the dispersed phase thereby indicating that OO was present as the internal phase in the developed formulations. This confirmed that the developed gels had a

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Fig. 2 Bright field micrographs showing spherical shaped droplets. a GW1 (109 magnification), b GW1 (409 magnification), c GW2 (109 magnification), and d GW2 (409 magnification)

Fig. 3 Droplet size distribution of the gels

water continuum phase and hence may be regarded as oilin-water emulsion gels (EHs) [80]. The surface topology of the gels was studied by scanning electron microscopy by converting them into xerogels. The xerogels were sputter coated with platinum, so as to make the xerogels electrically conducting, before imaging. The micrographs showed the presence of uniformly distributed globular particles (Fig. 4e, f). The sizes of the

globular particles were bigger in GW1 as compared to the GW2 and were in accordance with the micrographs obtained by other microscopic techniques. Similar results were also obtained by AFM microscopy (Figs. 4g, 5h). The AFM micrographs of the xerogels suggested the presence of globular structures which were arranged in a more dense fashion in GW1 as compared to GW2. FTIR spectra of the blank (GW1 and GW2) and the drug loaded (GW1M, GW1C, GW2M and GW2C) gels have been shown in Fig. 5. The FTIR spectra of the raw materials were in exact match with the spectrograms reported earlier [12, 81, 82]. OO showed absorption peaks at *2,930 and *2,867 cm-1 which may be associated with the C–H stretching vibrations. The absorption peak at *1,460 cm-1 was associated with the CH bending vibrations in the fatty acids [83–85]. The peak at *714 cm-1 may be explained due to the CH rocking vibration. The peak at *1,763 cm-1 was associated with the C=O stretching of the triglycerides [86]. An absorption peak at *1,140 cm-1 was associated to the C=O stretching vibration of the ester group in OO. A broad peak was observed in SMP molecule at *3,398 cm-1 and was attributed to the presence of intramolecular hydrogen bonding amongst the SMP molecules [12]. Two major absorption peaks were observed at *2,930 and *2,850 cm-1 and was associated with the C–H stretching vibrations in CH2 and CH3 present in the alkanes. The

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Fig. 4 Microscopic studies. Fluorescent micrographs: (a) GW1 and (b) GW2; Confocal micrographs: (c) GW1 and (d) GW2; Scanning electron micrographs: (e) GW1 and (f) GW2; Atomic force micrographs: (g) GW1 and (h) GW2

absorption peak at *3,220 and at *3,535 cm-1 was observed in MZ and CP, respectively. These peaks were associated to the –OH stretching vibrations [87]. The absorption peaks at *3,100 and *1,732 cm-1 was due to

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the C–H stretching vibration and the presence of C=C group, respectively in MZ. The peak in the range of 1,400–1,535 cm-1 was due to the imidazole ring [88]. The absorption peak at 1,348 cm-1 was associated with the N=O

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components. A broad peak was observed at *3,300 cm-1 in both the blank and drug loaded gels suggesting the presence of strong intra/intermolecular hydrogen bonding amongst the components of the gels. The quantification of the hydrogen bonding (3,714–3,029 cm-1) was done by calculating the area under the peak (AUC; Table S2). The AUC gives an indication about the extent of inter/intra-molecular hydrogen bonding. The results suggested that the addition of drugs resulted in the reduction of the AUC. This indicated that there was a decrease in the intermolecular hydrogen bonding as the drugs were incorporated within the gels. The XRD profiles of the blank and the drug loaded gels (GW1M, GW1C, GW2M and GW2C) have been shown in Fig. 6. The XRD profile of CP has shown characteristic peaks at 14.29°, 16.42°, 20.34° and 25.40° 2h [91, 92] whereas MZ showed peaks at 13.60°, 29.00° and 33.00° 2h [43]. SMP showed a characteristic peak at 21° 2h [93]. The blank and CP loaded gels showed a broad peak at *20.00° 2h, which suggested the amorphous nature of the gels. The MZ loaded gels has shown characteristic prominent peak of MZ at 13° 2h indicating the presence of the drug within the gels in its native form. Apart from the characteristic peak of MZ, the gels also showed a broad peak at *20.00° 2h suggesting the predominant amorphous nature of the gels. 3.5 Mechanical properties

Fig. 5 FTIR spectra of (a) GW1 and (b) GW2 gels

symmetrical stretching in the imidazole ring [89]. The absorption peak at 3,375 cm-1 was due to the NH stretching vibrations associated with the presence of secondary amine. The characteristic absorption peaks of CP at 3,375 cm-1 and 1,178 cm-1 was due to –OH and C=O stretching vibration of carboxylic acid groups, respectively. The absorption peaks at 3,535 and 1,624 cm-1 may be explained due to the stretching and bending vibration of amines, respectively [90]. The developed gels have shown almost all the absorption peaks present in the components of the gels. The absorption peak at 2,928 cm-1, present in both OO and SMP, was shifted towards the lower wavenumber in the gels. Similarly, the peaks at 2,867 and 1,762 cm-1 in OO also shifted towards the lower wavenumber. This might be due to the formation of physical bonds within the gel

The viscosity profiles of GW1 and GW2 have been shown in Fig. 7. The viscosity profile of the gels showed shear thinning phenomena thereby suggesting the pseudoplastic (non-Newtonian) flow behaviour of the gels. GW1 has shown higher viscosity as compared to GW2. This may be associated to the presence of lower amount of DW in GW1. The deformation of the gel structure was recovered (to a great extent) as the shear rate was decreased. An overlapping recovery profile in GW2 suggested almost complete recovery of the gel architecture as compared to GW1 where complete recovery was not possible. This phenomena of complete recovery of the gel structure have been reported to assist the retention of the applied formulation at the site of topical application [94]. The variations of the G’ (storage modulus) and G’’ (loss modulus) of GW1 and GW2 as a function of strain have been shown in Fig. 8a. The strain was applied at a constant frequency of 1 rad/s. The rheograms of both the gels showed crossover of G’–G’’. The crossover point is the minimum strain required to make the semi-solid formulation flow and is regarded as the gel-to-sol transition point (GST). The gels retain their structural integrity up to the GST and start flowing beyond this point [95]. Beyond GST, there was a pronounced decrease in the G’ and G’’ values.

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Fig. 6 XRD profile. a GW1 and b GW2

Fig. 7 Viscosity profile of GW1 and GW2

The crossover point of GW1 and GW2 was achieved at 9 and 8 % strain, respectively. From the results it may be inferred that the mechanical properties of GW1 were better as compared to GW2. The results were in accordance with the viscosity profile where it was also found that the viscosity of GW1 was higher as compared to GW2. The rheological behavior of the gels was also studied by varying the angular frequency at a constant strain of 0.5 %. The test was conducted in the linear viscoelastic range. The plot of G’–G’’ with respect to angular frequency has been shown in Fig. 8b. The results suggested an increase in the G’ and G’’ with the increase in the angular frequency. This indicated a predominant elastic

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behavior of the gels rather than viscous behavior thereby suggesting the mechanical rigidity of the gels. The G’ of GW1 was higher as compared to GW2 indicating better mechanical property of GW1. The complex viscosity (g) as a function of frequency showed a linear relationship. The complex viscosity of GW1 was more as compared to GW2 and was in accordance with the viscosity measurement [96]. The mechanical behavior of the gels was thoroughly studied by texture analysis studies. The viscoelastic properties of the gels were studied from the stress relaxation (SR) study. To understand the SR properties, two forces were defined. The force recorded after the probe has travelled a distance of 5 mm (after a trigger force of 5 g) was regarded as F0. The probe was allowed to stay at the same position for a period of 30 s and the change in the force was recorded. The force recorded at the end of 30 s was regarded as F30. The % reduction in the force between F0 and F30 is defined as %SR and is given by the formula [97]: % SR ¼

F0  F30  100 F0

ð2Þ

The SR profiles of the gels has been shown in Fig. 9a and the parameters have been tabulated in Table 3 [98]. GW2 has shown higher %SR as compared to GW1. The observed result may be attributed to the lower mechanical properties of GW2 due to the presence of higher concentration of DW in GW2. This, in turn, might have lead to a greater molecular rearrangement when a stress was applied [99, 100].

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Fig. 8 Rheological behavior. a Strain dependence of the G’ and G’’ at a constant angular frequency (1 rad/s), and b Angular frequency dependence of the G’ at a constant strain (0.5 %)

The spreadability profiles of the gels have been shown in Fig. 9b. Spreadability study provides information about the firmness and the stickiness of the gels. The positive and negative peak forces in the spreadability profiles are defined as the firmness and the stickiness of the formulations, respectively. The positive and the negative area gives information about the cohesiveness and the adhesiveness of the formulations, respectively (Table 4) [101]. GW1 has shown higher firmness, cohesiveness, stickiness and adhesiveness as compared to GW2. The results were in accordance to the viscosity and SR studies which showed that the mechanical properties of GW1 were better as compared to GW2. The backward extrusion (BE) test is a type of compression-extrusion test in which a force is applied to the sample until it starts flowing through the space available between the outer-diameter of the compression platen and the inner diameter of the sample holder. The gels were poured into a 50 ml beaker (inner diameter: 42 mm) so as to fill up the beaker *75 % of its volume. A flat probe (perpex made) of 40 mm diameter was made to move to a target distance of 20 mm after a trigger force of 3 g was achieved. The test was performed at a speed of 0.5 mm/s. Index of viscosity (given by the area under the negative

peak) was calculated from the graph (Table 5) [102]. Index of viscosity of GW1 was higher as compared to GW2 and was in accordance with the observed viscosity and rheology results (Fig. 9c). Creep is a phenomena related to the time-dependant deformation of a material when a constant force is applied over the material. The test provides an insight about the structural changes occurring at the molecular level of the material, when a constant force is applied [58]. The test was performed by applying a constant force of 20 g for a period of 65 min. The creep profile of the gels has been shown in Fig. 9d. The results indicated that the distance travelled by the probe to maintain a constant force of 20 g during the period of 65 min was more for GW2 as compared to GW1. This may be accounted to the presence of a dense network arrangement in GW1 which resulted in the lower creep indentation in GW1. 3.6 Thermal properties The MP of GW1 and GW2 was found to be 42.5 ± 0.5 °C and 41.0 ± 0.5 °C, respectively. The lower MP of GW2 was due to the lower thermal stability of the GW2 and may be accounted to the presence of higher proportions of DW.

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Fig. 9 Textural properties. a Stress relaxation profile, b Spreadability profile, c Backward extrusion profile, and d Creep profile

In general, if a gel is having lower mechanical properties then it is expected that the thermal properties will also be on the lower side. The thermal properties of the gels were further analyzed using DSC (Fig. 10; Table 6). Both the gels have shown a broad endothermic peak at *100 °C. These endothermic peaks are associated with the loss of moisture from the gels. The change in enthalpy (DHm) and the change in Table 3 Parameters studied from stress relaxation studies Formulations

F0 (g)

F30 (g)

% SR

GW1

28.794

9.156

68.201

GW2

24.416

7.165

70.654

entropy (DSm) was calculated from the thermograms. The DHm and DSm were higher for GW2 and may be associated to the higher water-retention capacity of GW2 [103]. 3.7 Electrical properties The Nyquist plots of GW1, GW2 and drug loaded gels have been shown in Fig. 11a. All the gels have shown the formation of two semicircular arcs indicating the presence of both bulk and grain boundary effect [104]. The formation of high frequency semicircle is due to the bulk properties of the material arising due to a parallel combination of bulk resistance (Rb) and bulk capacitance (Cb). The low frequency semicircle is due to the grain boundary effect arising due to a parallel combination of grain boundary

Table 4 Parameters studied from spreadability studies Formulations

Firmness (kg)

Cohesiveness (kg s)

Stickiness (kg)

Adhesiveness (kg s)

GW1 GW2

0.619 0.309

0.985 0.499

-0.475 -0.187

-0.044 -0.177

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Table 5 Parameter studied from backward extrusion study Formulations

Index of viscosity* (kg s)

GW1

-9.437

GW2

-7.917

formulations. A typical frequency dependence of ,ac spectrum has been shown the Fig. 11c. The conductivity profiles exhibited three distinct regions: low frequency dispersion, an intermediate frequency plateau and an extended dispersion at high frequency. The low frequency dispersion was due to the space charge polarization at the material-electrode interface [107]. In the intermediate frequency plateau region, the conductivity of the gels was tending to be frequency-independent which has been reported to provide information about the dc conductivity (,0). In the high frequency region, the conductivity increases with the frequency. The frequency dependence of the conductivity may be best expressed by Jonschers power law given by the following equation [104]: Rac ¼ r0 þ Axs

Fig. 10 Thermal profiles obtained from differential scanning calorimetry

Table 6 Thermal analysis Formulations

Peak

DHm (J/g)*

DSm (J/g K)*

GW1

99.9

937.6

4597.72

GW2

101.6

1,124

5512.48

resistance and grain boundary capacitance [105]. The Rb of the sample was obtained from the intersection of the high frequency impedance semicircle with the real axis [106, 107]. It was observed that GW1 was having higher resistance as compared to GW2. Since the pockets of OO (as visualized under the microscope) were the non-conducting elements and were mainly responsible for the electrical resistance, the higher resistance of GW1 as compared to GW2 was quite expected. This can also be attributed to the presence of lower amount of DW in GW1. Incorporation of drugs within the gels resulted in the increase in the bulk resistance of the gels. The tangent loss (tan d) profiles of the gels have been shown in Fig. 11b. Tan d peaks of both GW1 and GW2 were found to be in the range of *102–103 Hz. It was observed that the tangential loss was more in GW1 compared to GW2. The higher tangent loss suggests increased viscoelastic properties [108]. A nearly overlapping ac conductivity (,ac) profile was observed in all the

where, rac: ac conductivity; r0: dc conductivity; A is a preexponential constant; x = 2pf: angular frequency; and s: power law exponent, where 0 \ s \ 1. The dc conductivity of the gels has been tabulated in Table 7. GW2 showed higher ,dc as compared to GW1. This is due to the presence of higher proportions of DW in GW2. Incorporation of MZ and CP in the gels resulted in the decrease in the ,dc of the gels. This may be associated with the increase in crystallinity of the gels (as was observed from the XRD studies) [109]. The dielectric constant (e0 ) profiles of the gels have been plotted in Fig. 11d [110]. The e0 of all the gels showed a higher e0 at lower frequencies. As the frequency was increased, there was a sharp exponential decrease in the e0 of the gels. This was due to the capacitive dominant nature of the gels. At lower frequencies, there is an accumulation of charge which causes electrode polarization thereby resulting in an increased e0 . With the increase in the frequency, there is a quick reversal of the ac field which results in the constant e0 value [111]. There was no significant variations in the e0 profiles of the drug loaded gels. 3.8 Hemocompatibility study The preliminary biocompatibility of the optimized gels was studied by calculating the % hemolysis of goat blood. The % hemolysis was found to be \3 % in all the formulations. The results suggested that the gels were biocompatible (Fig. 12) [63, 112]. 3.9 In-vitro drug release The cumulative percentage drug release (CPDR) of MZ loaded gels have been shown in Fig. 13a. GW1M and GW2M have shown releases of *71 %/w and *81 %, respectively, in a period of 12 h. The release pattern can be explained based on the partition coefficient effect of the drug

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Fig. 11 Electrical properties. a Nyquist plot, b Frequency dependent tangential loss, c Frequency dependent ac conductivity, and d Frequency dependent dielectric constant

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Table 7 DC conductivity (,0) of various gels -5

-1

Formulations

dc conductivity (,0) (910 ) (Scm )

GW1

1.82

GW1FT

1.79

GW1M

1.87

GW1C

1.02

GW2

1.97

GW2FT

2.22

GW2M

2.71

GW2C

1.64

Fig. 12 Hemocompatibility study

[113, 114]. A higher amount of MZ was released from GW2M and might be accounted to the quick partitioning of the drug into the aqueous phase of GW2M. Since the concentration of DW was less in GW1M, the partitioning of the drug into the aqueous phase was lower and hence the release of the drug. Also, the higher degree of amorphousity of GW2M might have played an important role in higher MZ release from GW2M [115]. CP loaded gels have shown a release of *12 and *10 % for GW1C and GW2C, respectively, in a span of 12 h (Fig. 13b). This can be attributed to the lipophilic nature of CP, which resulted in the poor partitioning of CP into the water continuum phase of the gels thereby resulting in the lower amount of release. The release kinetics of the drugs from the gels suggested that the release of the drugs from the gels followed Higuchian kinetics. The mathematical model confirmed that the gels behaved as homogeneous-planar matrices and did not lose their structural integrity during the study [116]. The mechanism of drug release from the formulations was understood by calculating the ‘n’ value from Korsmeyer– Peppas (KP) model [117, 118]. The n value was found to be 0.42 for GW1M suggesting Fickian diffusion of MZ from the

gel matrix. The n-values for GW2M, GW1C and GW2C were in the range of 0.45- 0.85 suggesting non-Fickian diffusion of the drugs (Fig. S3) [117, 119]. The results suggested that the drug release was diffusion-predominant release [116]. The results from the impedance spectroscopy suggested that the developed organogels were electroconductive in nature. The electrical properties of the formulations could be altered by tailoring the composition of the formulations. Since the gels were electroconductive in nature, it was expected that the gels may be used as matrices for iontophoretic drug delivery. The dispersed hydrophobic phase will act as reservoir for the drugs. Hence the drugs may be delivered to the systemic circulation without compromising the skin integrity. Also, the elcectroconductive nature of the gels may help improving the release rate of the drugs from the formulations. To justify the same and determine the suitability of the developed gels to be used as matrices for iontophoretic drug delivery, a preliminary release study from MZ loaded gels was done using an in-house built iontophoretic drug delivery setup. Iontophoretic drug delivery employs the diffusion of the drug through the skin into the systemic circulation under the influence of an electric field [120, 121]. The release profiles of MZ from the gels have been shown in Fig. 13c. The release of MZ from GW2M was higher under both active and passive conditions over a period of 2 h as compared to GW1M. This may be associated with the higher proportion of DW in GW2M. The release of the drug under the influence of the electrical current was higher in both the gels as compared to the passive diffusion. The increase in the release of the drug from the hydrogel was less prominent in GW2M as compared to GW1M. The % increase in the released drug from GW1M and GW2M was [90 and *20 %, respectively (Fig. 13d). This may be explained by the increased partitioning of the drug molecules from the gels into the dissolution medium when the concentration of DW was higher in GW2M. Though there was an increase in the drug release in the presence of externally applied electrical field, the effect was not prominent. This study suggested that the developed gels may be used as carriers for iontophoretic drug delivery [121]. The release profile of the drugs may be tailored by altering the composition of the formulations. 3.10 Antimicrobial assay The efficiency of the drug loaded gels was studied against B. subtilis, a model gram positive bacterium. The zone of inhibition was measured after 24 h of incubation at 37 ± 1 °C. Pure drugs (MZ and CP) were taken as the positive control whereas the blank gels served as the negative controls. The results indicated that the developed

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Fig. 13 Cumulative % drug release (CPDR). a MZ loaded gels, b CP loaded gels, and c Iontophoretic delivery of MZ. d % increase in CPDR during iontophoresis

Table 8 Antimicrobial test against B. subtilis Formulations

Zone of inhibition (cm ± SD)

Negative control

0.00 ± 0.00

Positive control–MZ

3.45 ± 0.39

Positive control–CP

4.50 ± 0.28

Metrogyl

Ò

GW1M

3.40 ± 0.21 3.40 ± 0.14

GW2M

3.30 ± 0.18

GW1C

3.80 ± 0.20

GW2C

4.00 ± 0.15

formulations showed sufficient antimicrobial activity to be used as topical antimicrobial formulation (Table 8).

found to be smooth in texture, stable and hemocompatible in nature. The stability tests suggested that the optimized gels should be stable for prolonged periods. The gels were found to be viscoelastic in nature. The gels acted as planar matrices for the drugs and the drug release followed Higuchian kinetics. Iontophoretic drug delivery study suggested that the developed gels may be used as matrices for iontophoretic drug delivery. The drug loaded gels showed sufficient antimicrobial efficacy to be used as a topical antimicrobial gels. From the present studies, we can conclude that the developed gels can be used as controlled drug delivery matrices for the delivery of antimicrobial drugs. Acknowledgments The authors acknowledge the financial support received from Department of Biotechnology, New Delhi, India vide sanction order (BT/PR14282/PID/06/598/2010) for this study.

4 Conclusion The current study explains the development of OO and SMP based EHs. These hydrogels can be prepared by an easy and economical procedure. The developed gels were

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