RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology

Ultrasonication-Assisted Preparation and Characterization of Emulsions and Emulsion Gels for Topical Drug Delivery VINAY K. SINGH, BAIKUNTHA BEHERA, KRISHNA PRAMANIK, KUNAL PAL Department of Biotechnology and Medical Engineering, National Institute of Technology, Rourkela 769008, Odisha, India Received 31 July 2014; revised 14 October 2014; accepted 21 October 2014 Published online in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.24260 ABSTRACT: The current study describes the use of ultrasonication for the preparation of biphasic emulsions and emulsion gels for topical drug delivery. Sorbitan monostearate (SMS) was used as the surfactant for stabilizing the interface of sesame oil (apolar phase) and water (polar phase). Emulsions were formed at lower concentrations of SMS, whereas emulsion gels were formed at higher concentrations of SMS. The formulations were characterized by fluorescent microscopy, X-ray diffraction, viscosity, stress relaxation, spreadability, and differential scanning calorimetry studies. Fluorescence microscopy suggested formation of oil-in-water type of formulations. There was an increase in the viscosity, bulk resistance, and firmness of the formulations as the proportions of SMS was increased. The emulsion gels were viscoelastic in nature. Thermal studies suggested higher thermodynamic stability at higher proportions of either SMS or water. Metronidazole, a model antimicrobial drug, was incorporated within the formulations. The release of the drug from the formulations was found to be diffusion mediated. The drug-loaded formulations showed sufficient antimicrobial efficiency to be used as carriers for topical antimicrobial drug C 2014 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci delivery.  Keywords: Emulsions; Calorimetry; FTIR; Biocompatibility; Ultrasound

INTRODUCTION Emulsions are thermodynamically unstable biphasic formulations. This may be associated with the destabilization of the interfacial membranes, formed by emulsifiers, which ultimately leads to the rupture of the membrane layer, thereby resulting in the loss of the internal phase.1 The stability of the emulsions may be improved by increasing the stability of this interfacial membrane layer. It has been noted that the method of emulsification plays an important role in improving the stability of the emulsions.2 In general, the formation of metastable emulsions has been reported when external energy is supplied to the pre-emulsion mixtures. Commonly, energy may be provided either by mechanical agitation (stirrer, colloid mill, mixer, valve homogenizer) or by ultrasonic probes. In recent years, ultrasonication has evolved as one of the most easy-to-handle and powerful homogenization technique for formulating biphasic formulations.3 Ultrasonic transducer-based homogenizers stimulate the piezoelectric crystals, attached to the ultrasonic probes, to oscillate at a very high speed (>20,000 cycles/s).4 The pre-emulsion flows toward the ultrasonic probe during the contraction phase, whereas it is pushed away from the probe during the expansion phase. As the speed of the probe is much higher than the speed of the flow of the pre-emulsion, this results in the generation of microscopic shockwaves within the liquids. The cavities, so formed, collapse within a fraction of a second. This releases a large amount of energy within the preemulsion. This phenomenon helps in the formation of smaller droplets within a continuous phase.5 The use of ultrasonication has been reported by many researchers for the preparation of emulsions (microemulsions/nanoemulsions).6,7

Emulsion gels may be defined as the biphasic formulations, similar to the emulsions, but the formulations are semisolid in consistency.8 The stability of the emulsion gels is much better than the emulsions and may be explained by the semisolid nature of the formulations. This restricts the movement of the internal phase droplets, thereby reducing the chances of coalescence of the droplets.9 In recent years, emulsion gels have gained much importance because of their ability to control the release profile of the incorporated drugs. The preparation and the properties of the emulsion gels using ultrasonic-assisted method have not been explored much yet. In the current study, an attempt was made to prepare biphasic formulations with improved properties using ultrasonic homogenization. The formulations were tested for the in vitro delivery of the antimicrobial drugs. Sesame oil was used as the apolar phase for the preparation of the formulations. Sesame oil is obtained from the seeds of the plant Sesamum indicum. The oil has very good antioxidant properties because of the presence of sesamin, sesamolin, and sesamol.10 Even though the oil is easily available in the Indian subcontinent, the use of the sesame oil in formulating pharmaceutical products is rare. Sorbitan monostearate (SMS) is a nonionic surfactant and is being commonly used in pharmaceutical and food industries.11 Keeping a note of the above, it seemed justified to develop sesame oil and SMS-based formulations. Emulsions and emulsion gels were prepared and characterized thoroughly. Metronidazole was incorporated within the prepared formulations and their drug release properties were studied in-depth under in vitro conditions.

EXPERIMENTAL Correspondence to: Kunal Pal (Telephone: +91-876-336-6085; Fax: +91-6612472926; E-mail: [email protected], [email protected]) This article contains supplementary material available from the authors upon request or via the Internet at http://onlinelibrary.wiley.com/. Journal of Pharmaceutical Sciences  C 2014 Wiley Periodicals, Inc. and the American Pharmacists Association

Materials Sorbitan monostearate (Loba Chemie, Mumbai, India), a nonionic surfactant, was used as an emulsifier. Edible grade sesame oil (Tilsona ; Recon Oil Industries Ltd., Mumbai, India) was R

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used as apolar phase to prepare the biphasic formulations (emulsions and emulsion gels). Nutrient agar and dialysis tubing (MW cutoff: 60 kDa) were obtained from Himedia (Mumbai, India). Microbial culture of Bacillus subtilis (NCIM 2699) was obtained from National Collection of Industrial Microorganisms (NCIM) (Pune, India) for conducting the antimicrobial studies. Metronidazole was obtained from Aarti drugs (Mumbai, India) as a gift sample and was used as received. Milli-Q Ultrapure water was used throughout the study. Methods

Mechanical Behavior A cone-and-plate viscometer (Bohlin visco 88; Malvern, Worcestershire, UK) was used for the viscosity measurements. During the measurements, the viscosity of the emulsions and emulsion gels were recorded at room temperature (RT, 25.0◦ C) in the shear rate range of 10–100 s−1 .16 Large-scale deformation properties (stress relaxation and spreadability) of the formulations were studied using Texture Analyser (TA-HDplus; Stable Microsystems, Godalming, UK).17 The test parameters are enlisted in Table S1.

Preparation of Emulsions and Emulsion Gels The formulations were developed by varying the proportions of SMS, sesame oil, and water. Initially, a pre-emulsion was prepared by homogenous mixing of the three components in a magnetic stirrer (500 rpm, 70◦ C). The pre-emulsion was subsequently homogenized using a probe sonicator (6 mm probe, Syclon ultrasonic cell crusher, SKL-250IIDN; Ningbo Haishu Sklon Electronics Instruments Company Ltd., Zhejiang, China), operating at a ultrasonic frequency of 20–25 KHz and 150 W. The sonication was carried out for 20 cycles (6 s on time and 2 s off). The homogenization was carried out in an ice-bath to compensate the heat generated. Metronidazole (1%, w/w)-loaded emulsions and emulsion gels were prepared similarly. Accurately weighed metronidazole was uniformly homogenized (by ultrasonication) in sesame oil and used as the apolar phase. The rest of the procedure remained same. The prepared formulations were checked for their organoleptic behavior such as odor, color, texture, and pH. The nature of the formulations (oil-in-water or water-in-oil) was checked by dilution test.12 Stability Studies The stability of the prepared emulsions and emulsion gels were checked by accelerated stability study and intermediate stability study. The accelerated stability testing was carried out by freeze–thaw thermocycling method reported elsewhere.13 The intermediate stability of the formulations was studied by incubating the formulations at 30 ± 2◦ C/65 ± 5% RH for 6 months (ICH guidelines). The formulations were checked for any changes in the physical properties at regular intervals.14 Microscopic Evaluation The microstructure of the emulsions and emulsion gels was studied using Fluorescence Stereo Microscope (M205 FA; Leica, Germany). Samples were prepared by incorporating an oilsoluble dye (0.1% fluoral yellow) in sesame oil. The thin smears were prepared on glass slides and were visualized under green filter. The fluorescence micrographs were further used to predict the droplet size distribution of the formulations.13 The diameter of the droplets was measured by ImageJ 1.43U software (National Institute of Health, Maryland, USA).

Thermal Analysis Drop-ball method is the most commonly used method to determine the melting point (Tm ) of the semisolid formulations. The melting point of the emulsion gels was determined using melting point apparatus-931 (EI Instruments, Haryana, India).18 The thermal profile of the formulations was studied using differential scanning calorimetry (DSC 200F3 Maia; Netzsch, Selb, Germany). Samples were accurately weighed (∼15 mg) in the aluminum pans and were sealed with pierced lids. The test was carried out under inert atmosphere by purging nitrogen gas at a flow rate of 40 mL/min. The heating and the cooling thermal profiles were recorded in the temperature range from 25◦ C to 150◦ C at a scan rate of 5◦ C/min.15

Biocompatibility Study Tetrazolium-based MTT [3-(4,5-dimethyltiazol-2-yl)-2,5diphenyltetrazolium bromide] assay was performed to estimate the cytocompatibility of the developed formulations using HaCaT cells.19 The leachants of the formulations were collected in phosphate buffer saline (pH 7.2). 1.0 × 104 cells per well of the cell suspension were seeded in 96-well plates and incubated for 24 h to allow cell adhesion. Thereafter, 20 :L of the leachant of the formulations was added and further incubated for 24 h. The cell viability was then measured using MTT assay as per the reported literature.20,21

In Vitro Drug Release Studies The release of metronidazole from the metronidazole-loaded formulations was studied in vitro using two-compartment modified Franz’s diffusion cell. The receiver compartment contained 50 mL of phosphate buffer saline (pH 7.2). The release media was completely replaced with an equivalent amount of the fresh media at regular intervals of time. The replaced release media was analyzed at 321 nm using UV–Vis spectrophotometer (UV 3200 Double Beam; Labindia, Thane (West), Maharashtra, India). The study was conducted for 12 h.22,23

X-ray Diffraction Study

Antimicrobial Assay

The relative changes in the crystallinity of the emulsions and emulsion gels were predicted by X-ray diffraction studies (PW3040; Philips, Almelo, Holland). Monochromatized CuK" radiation (8 = 0.154 nm) was used as the X-ray source and the spectrum was recorded in the range of 5◦ –50◦ 22 at a scan rate of 2◦ 22/min.15

The prepared emulsions and emulsion gels were evaluated for their antimicrobial efficiency against a gram-positive Bacillus subtilis according to the method reported elsewhere. The zone of inhibition for the metronidazole-loaded formulations was compared against commercially available metronidazole gel (Metrogyl ).15

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RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology

RESULT AND DISCUSSION Preparation of Emulsions and Emulsion Gels The composition of the formulations was optimized by varying the proportions of SMS, sesame oil, and water. The concentration of SMS was varied from 2.5%–10% (w/w), whereas the proportion of water was varied from 20%–80% (w/w) (Table S2). At very low concentrations (2.5%, w/w) of SMS, phase separation was observed. Stable formulations (emulsions/emulsion gels) were obtained when the concentration of SMS was ≥5% (w/w). The pre-emulsions formed were white and was a homogenous mixture. After ultrasonic homogenization, the hot mixture was cooled down to room temperature. The mixture either remained as physically separated system or formed a stable formulation (emulsion/emulsion gel) based on the concentration of SMS (emulsifier). The emulsions were easily flowing under the gravity, whereas the emulsion gels did not flow upon inversion of the culture bottles (Fig. S1, Supporting Data). Among the stable formulations, emulsions were formed at lower concentrations of SMS, whereas at higher concentrations of SMS, emulsion gels were formed. The concentration of SMS played an important role in the formation of the formulations. In general, formulations with improved stability were obtained at higher concentration of the SMS. This may be associated with the increased crystallinity of the formulations at higher concentrations of SMS. Two representative formulations of each emulsions and emulsion gels were selected for further characterization (Table 1). Both emulsions and emulsion gels were milky white in color and had a smooth texture. The drug-loaded formulations were prepared in a similar manner and showed similar color and texture as compared with the blank formulations. The nature of the formulations is an important criterion to assess the properties of the biphasic formulations. Dilution test was performed to determine the nature of the formulations (Fig. S2, Supporting Data). All the representative emulsions and emulsion gels were dispersed homogeneously in water, but settled down at the bottom when mixed with oil. This suggested that the oil phase might be the dispersed phase while water was the continuum phase.24 The pH of the formulations was in the range 6.2–6.6 (Table S3). Results suggested that the developed formulations might be nonirritant as the pH of the formulations are within the physiological pH limit.25 Accelerated Stability Testing Biphasic formulations suffer from a major drawback of their lower stability. Emulsion is a thermodynamically unstable system and with time shows destabilization with eventual phase separation. Gels are comparatively more stable because of their three-dimensional network structure. All the emulsions and emulsion gels passed the accelerated and intermediate stability

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tests. Results suggested that the physical appearance and the texture of all the samples did not change when passed through harsh temperature treatments (accelerated stability test) and on a time scale. This may be explained by the improved stability of the formulations because of ultrasonication. Li and Chiang7 prepared stable d-limonene-based nanoemulsion using ultrasonic emulsification method. Kaltsa et al.6 reported improved stability of the olive oil emulsions prepared by ultrasonication. Microscopic Evaluation Fluorescent microscopy helps understanding the arrangement of the oil and the aqueous phase in a biphasic formulation. Fluorescent dyes soluble in either of the phase are used for the purpose. In the present study, fluoral yellow dye was dissolved in the oil phase. The fluorescent micrographs showed that the dispersed droplets acquired the fluorescent dye (green) and the background remained as black, that is, unstained (Figs. 1a–1d). Results confirmed that the oil phase was dispersed within the aqueous phase in both the emulsions and the emulsion gels. Similar results were also obtained using the dilution test. The higher proportions of water within the formulations might have resulted in the formation of oil-in-water systems. In both the emulsions and emulsion gels, the droplets were densely packed and were spherical in shape. The droplet sizes were bigger in emulsions in comparison to the emulsion gels. As emulsions are thermodynamically unstable system, the bigger droplet sizes might have resulted because of the coalescence of the adjacent droplets. The droplet size distributions were evaluated by calculating the SPAN factor of the particle size for the formulations. The SPAN factor is a dimensionless number that represents the width of the droplet size distribution based on 10%, 50%, and 90% population of the droplets.26 It is calculated by the formula given below: SPAN = (D90 − D10 )/D50

(1)

where, D10 , D50 , and D90 represent 10%, 50%, and 90% of the cumulative particle size distribution at the given size.27 In general, a wide droplet size distribution yields a comparatively large SPAN factor, whereas a narrow size distribution yields a small SPAN factor.28 The size distribution of the droplets is shown in Figures 1e and 1f and the corresponding SPAN factor of the formulations is tabulated in Table 2. The emulsions have shown bimodal size distribution that may be associated with the coalescence, a most frequently found phenomenon in emulsions. The emulsion gels showed unimodal size distributions. The mean droplet sizes of the emulsions and the emulsion gels were in the range of approximately 8–16 and 4–6 :m, respectively. X-ray Diffraction Study

Table 1. 100 g)

Formulations Selected for Further Characterization (for

Formulations E1 E2 G1 G2

DOI 10.1002/jps.24260

SMS (g)

Sesame Oil (g)

Water (g)

5 5 10 10

35 15 50 10

60 80 40 80

Emulsions and emulsion gels are amorphous formulations. X-ray diffraction studies have often been used to study the molecular architecture of these formulations. The formulations showed a sharp crystalline peak at approximately 21.4◦ 22 that may be associated with the gelator (SMS) molecule (Fig. S3, Supporting Data).29 The crystalline phase is periodically arranged as three-dimensional arrangement of atoms. When Xrays hit the lattice planes formed by the atoms, they scatter only in certain directions. Because of this ordering, they possess Singh et al., JOURNAL OF PHARMACEUTICAL SCIENCES

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RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology

Figure 1. Fluorescent micrographs of the emulsions and emulsion gels. (a) E1, (b) E2, (c) G1, and (d) G2. Droplet size distribution analysis of the emulsions and emulsion gels (e) percent frequency distribution, and (f) cumulative frequency distribution. Table 2.

Droplet Size Distribution Analysis of the Emulsions and Emulsion Gels (Mean ± SD, n = 3)

Formulations

Mean Particle Size (:m)

Size Distribution (:m) D10

E1 E2 G1 G2

15.5 8.25 5.5 4.75

± ± ± ±

0.75 0.34 0.15 0.11

3.5 3 3.25 2

± ± ± ±

D50 0.06 0.12 0.11 0.09

rigid structural periodicity.30 On the contrary, the amorphous phase atoms are randomly distributed in the three-dimensional space.31 Hence, they do not possess periodicity as compared with the crystalline phase. When the X-rays hit the amorphous phase, they scatter in many directions leading to the formation of broadened and distorted hump in a wide 22 range instead of formation of high-intensity narrow peaks.32,33 The distance between the periodic arrangements of the crystalline phase is commonly regarded as d-spacing. Crystalline Singh et al., JOURNAL OF PHARMACEUTICAL SCIENCES

15.5 8.25 5.5 4.75

± ± ± ±

SPAN Factor D90

0.75 0.34 0.15 0.11

19.75 15 8.25 7.5

± ± ± ±

1.1 0.81 0.67 0.54

1.05 1.45 0.90 1.16

± ± ± ±

0.04 0.02 0.05 0.06

material possesses unique periodicity, which is dependent on the size and shape of the unit cells. Each phase present in the formulation can be analyzed by quantifying the periodicity (separation or d-spacing of the crystal planes). The periodicity of any crystalline material extends to more than three set directions along which the arrangements of atoms repeat itself regularly.34 The corresponding d-spacing values to those directions can be used to characterize the periodicity in each of those directions.35 To get correct peak positions for the observed DOI 10.1002/jps.24260

DOI 10.1002/jps.24260

0 0 − 490.17 ± 19.42 − 318.99 ± 21.62 0.61 0.53 21.2 18.4 ± ± ± ± 9.82 7.96 670.23 537.41 – – 15.64 ± 1.12 17.11 ± 0.95 – – 0.35 ± 0.01 0.38 ± 0.02 – – 66.8 ± 2.63 61.8 ± 1.77 – – 10.75 ± 0.84 9.02 ± 0.72 – – 32.37 ± 1.17 23.62 ± 1.26 0.45 0.27 3.2 0.68 ± ± ± ± 7.73 6.16 113.59 8.7 0.98 0.98 0.97 0.98 0.009 0.017 0.18 0.011

Firmness (g) S (g/s) Fr * (g) Relaxation (%) Fr (g) R2

Yield Stress (Pa)

F0 (g)

Un-normalized Curve Herschel–Bulkley Model

5

E1 E2 G1 G2

where J0 is the yield stress, K is the consistency coefficient, and “n” is the flow behavior index.40 The yield stress gives indication about the strength of the fluids. The yield stress value was in the order of G1 > G2 > E1 > E2 (Table 3).

n Value

(3)

Formulations

J = J0 + K(n

Power Law

where J is the shear stress at ( shear rate, K is the flow consistency coefficient, and “n” is the power law (or rate) index. The n value is a measure of the shear-thinning property. The n < 1 value confirmed the shear-thinning behavior of the emulsions and the emulsion gels. The power law model is fitted for fluids showing zero shear stress at zero strain rate. Fluids that possess nonzero shear stress at zero strain rate are analyzed by Herschel–Bulkley model.38 The Herschel–Bulkley model combines the effects of Bingham and power-law behaviour in a fluid.39 Herschel–Bulkley model is a slight modification from the power law that incorporates yield stress value in the expression. It is given by the equation:

Viscosity Analysis

(2)

Mechanical Properties of the Emulsions and Emulsion Gels (Mean ± SD, n = 3)

J = K(n

Table 3.

The mechanical properties of the developed formulations were studied by viscosity, stress relaxation, and spreadability studies. The viscosity profile of the emulsions and the emulsion gels has been shown in Figures 2a and 2b. The shear rate was linearly ramped from 10 to 100 s−1 and the effect of the shear rate on the viscosity and the resultant shear stress were investigated. All the formulations showed the desired shearthinning behavior where the viscosity decreased with the increased shear rate. There was an increase in the viscosity with the corresponding increase in the SMS concentration.37 The increase in water concentration resulted in decreased viscosity. The viscosity of the formulations was in the order of G1 > G2 > E1 > E2. The shear-thinning behavior may provide beneficial effects in topical applications, as it imparts easy and uniform spreadability of the formulations. The viscometric data was further characterized by fitting Ostwald-de Wale Power law16 :

Texture Analysis

Mechanical Behavior

Normalized Curve

Stress Relaxation Study

Spreadability Study

sharp crystalline peak at approximately 21.4◦ 22, the X-ray diffractograms were fitted with multipeak algorithm. In the present investigation, the X-ray diffraction profiles are very broad, indicating long-range ordering (i.e., amorphous nature of the formulations). The d-spacing values suggested that E2 and G2 were more periodic as compared with E1 and G1, respectively (Table S4, Supporting Data). Similar observation was also reported by Kikuma et al.36 , where the periodicity of polyimide thin film was increased with the increase in the peak intensities. The formulations containing higher proportions of water showed higher full width at half maximum (FWHM) values (Table S4, Supporting Data). A reverse trend was obtained when the proportions of SMS was increased from 5% (E1) to 10% (w/w, G1). As a matter of fact, higher FWHM value suggests higher amorphous nature of the formulations. From the observations, it may be concluded that the increase in SMS proportion increased the crystallinity of the formulations whereas an increase in the water proportion increased the amorphosity of the formulations.

Stickiness (g)

RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology

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Figure 2. Mechanical properties of the emulsions and emulsion gels. (a and b) Viscosity studies. (c and d) Stress relaxation study. (e and f) Spreadabilty study. The error bars in the graph 2a represents mean ± SD, n = 3 (independent experiments).

Large-scale deformation properties of the formulations were further studied by stress relaxation and spreadability studies. Stress relaxation study was performed for the emulsion gels to investigate their viscoelastic properties (Figs. 2c and 2d). The force-time graph of the emulsions was below the detectable range; hence, stress relaxation study was performed only for the emulsion gels. The study was performed according to the protocol tabulated in Table S1. F0 (maximum force attained after loading trigger force of 5 g) and Fr (residual force) values were noted down and the percent relaxation was calculated by

Singh et al., JOURNAL OF PHARMACEUTICAL SCIENCES

the formula41 :  Relaxation(%) =

F0 − Fr F0

 ∗ 100

(4)

Both the emulsion gels showed nearly similar stress relaxation (61%–67%) behavior, which confirmed their viscoelastic nature.42 The percent stress relaxation of G1 was higher than G2.

DOI 10.1002/jps.24260

RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology

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Figure 3. Differential scanning calorimetry study of the emulsions and emulsion gels. (a) Complete thermogram showing the heating and cooling cycle. (b) Thermograms showing the endothermic peaks in the temperature range 40–70◦ C. (c) Thermograms showing the exothermic peaks in the temperature range 35–60◦ C.

The stress relaxation curves were normalized. The parameters such as residual force (Fr *) and area under the curve (S) were calculated from the normalized stress relaxation data (Table 3). G2 showed higher residual force compared with G1. The area under the curve (S) during relaxation provides information about the viscoelastic property of the formulations. Higher S value in G2 indicated higher elastic nature of G2 as compared with G1. The spreadability study was performed for both emulsions and emulsion gels (Figs. 2e and 2f). The firmness (positive peak) and stickiness (negative peak) of the formulations were calculated (Table 3).43 The firmness of the formulations was in the order of G1 > G2 > E1 > E2. In general, formulations containing higher concentrations of SMS showed higher firmness and stickiness. The emulsions showed zero stickiness under the experimental conditions.

Table 4.

Thermal Analysis The melting point (Tm ) of the emulsion gels was measured using drop-ball method. The melting points of the prepared emulsion gels were in the temperature range of 52◦ C–57◦ C (Table S5). The melting point of G2 was higher in comparison to G1, which may be associated with the higher-water concentration in G2. Increase in the water content might have resulted in the increase in the thermal stability of the emulsion gels. The thermal events (melting and crystallization) of the emulsions and the emulsion gels were thoroughly studied using differential scanning calorimetry (Fig. 3). The analysis of the events was carried out using Netzsch Proteus software (Table 4). The thermograms of the formulations showed two endothermic peaks during the heating cycle; one in the temperature range of 50◦ C–58◦ C and another in the temperature range of 108◦ C–119◦ C. The thermograms of the formulations

Thermal Properties of Formulations Heating DSC Curve, Endotherm

Formulations E1 E2 G1 G2

DOI 10.1002/jps.24260

Tm

(◦ C)

50.5 57.7 53.1 55.5

Hm (J/g)

Sm (J.K/g)

0.05157 0.63442 0.09902 0.66696

0.00102 0.011 0.00186 0.01202

Cooling DSC Curve, Exotherm Tevap

(◦ C)

118.4 111.5 108.7 111.6

Tc

(◦ C)

46.3 47.2 45.9 48.1

Hc (J/g)

Sc (J.K/g)

0.02492 0.03591 0.11608 0.26481

0.00054 0.00076 0.00253 0.00572

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Figure 4. In vitro drug release study of emulsions and the emulsion gels. (a) Cumulative percent drug release. (b) Higuchian fitting. (c) Korsmeyer–Peppas model fitting. (d) Antimicrobial assay. The error bars in the graph 4a and 4d represent mean ± SD, n = 3 (independent experiments).

showed one exothermic peak during the cooling cycle in the temperature range of 45◦ C–49◦ C. The endothermic peaks at approximately 53◦ C and the exothermic peaks at approximately 46◦ C indicated the melting (Tm ) and the crystallization (Tc ) temperatures of SMS, respectively.44 The position of the peaks varied during the melting and the crystallization events and may be explained by the thermal hysteresis.45 The enthalpy (Hm and Hc ) and entropy (Sm and Sc ) of the melting and the crystallization events were calculated from the amount of heat absorbed and released during the events, respectively. Both enthalpy and entropy during the events were higher in the formulations containing higher concentrations of SMS and water. Hence, it may be stated that the thermodynamic stability of the system increased with the increase in the concentrations of either SMS or water. The second endothermic peak obtained during the heating DSC in the temperature range of 108◦ C–119◦ C may be associated with the evaporation of the water present in the formulations (Tevap ).46 There were numerous small peaks connected to the second endothermic peak in that temperature range and in the nearby temperature regions. This may be associated with the loss of aqueous components according to their ease of dissociation from their molecular bonding at different temperatures.47 Singh et al., JOURNAL OF PHARMACEUTICAL SCIENCES

Biocompatibility Study The biocompatibility of the emulsions and the gels was tested using HaCaT cell. The viability of the cells was checked using extracts of the formulations. The viability of the cells was measured by MTT assay after 24 h of incubation (Fig. S4, Supporting Data). The extracts of the formulations did not show any cytotoxic effect and the variation in the cell viability was statistically insignificant (p > 0.05) in comparison to the control, indicating biocompatible nature of the prepared formulations.48 In Vitro Drug Release Studies The release studies of metronidazole from the emulsions and the emulsion gels were conducted for 12 h in triplicates. The cumulative percent drug release from the formulations has been shown in Figure 4a. Results indicated that the release of the drug was dependent on the gelator and the water concentrations. The release rate decreased with the increase in the gelator concentration and a corresponding decrease in the water concentration. E1 and E2 showed approximately 100% release of metronidazole in 12 and 9 h, respectively. Emulsion gels have shown lesser release in comparison to the emulsions. In 12-h time period, G1 and G2 showed approximately 93% and DOI 10.1002/jps.24260

RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology

99% release of the drug, respectively. Irrespective of the gelator concentration, both E2 and G2 showed approximately 100% of drug release in 12 h, suggesting that the effect of the gelator was nullified at higher proportions of water. Metronidazole is a poorly water-soluble drug. The solubility of metronidazole is 10 mg/mL in water, whereas it is approximately 0.0045 mg/mL in vegetable oil (palm kernel oil, palm oil, sesame oil, etc.).49 Hence, based on the inner or outer phase, the release of the drug was affected. The release of the metronidazole was higher from the formulations containing higher proportions of water. The partial hydrophilic nature of metronidazole might have caused higher partitioning of metronidazole into the aqueous phase of the formulations that resulted in quicker diffusion into the dissolution media. Results suggested that the formulations with higher water content showed higher diffusion of the drugs.50 This can be explained by the higher partitioning of the drug into the aqueous phase as compared with the oil phase. Results suggested that the release rate may be modulated by altering the components of the formulations. The kinetics of the drug release from the emulsions and the emulsion gels was evaluated using different drug release kinetic models (Figs. 4b and 4c). The formulations followed Higuchian kinetics that suggested that the release of metronidazole was diffusion-mediated. The release kinetic data have been tabulated in Table S6. The 60% of the total drug released data was fitted in Korsmeyer–Peppas kinetic model. The “n” value (Fickian value) obtained from the Korsmeyer–Peppas model fitting gives information about the type of drug diffusion from the formulations. The Fickian value was less than 0.45 in E1 suggesting Fickian diffusion, whereas it was in between 0.45 and 0.85 for E2, G1, and G2 suggesting non-Fickian diffusion of metronidazole.51 In general, non-Fickian diffusion is followed when the release mechanism is not well known or when more than one mechanism are involved.51 The possible mechanisms involved in the release behavior of metronidazole could be a combination of the diffusion and erosion. Antimicrobial Assay All the formulations showed higher zone of inhibition in comparison to the commercially available metronidazole gel, Metrogyl (positive control). Blank emulsions and emulsion gels did not show any inhibitory action against the microbe (Fig. 4d). Results suggested the release of the drug in its active form. Hence, it may be stated that the developed formulations can be used as carriers for delivery of antimicrobial drugs. R

CONCLUSIONS The study explained the use of ultrasonication for the development of emulsions and emulsion gels with good thermal stability. Oil-in-water type of emulsions and emulsion gels were formed when the gelator (SMS) concentration was >5% (w/w). The formulations showed good mechanical properties and thermodynamic stability at higher concentrations of gelator and water. The formulations showed shear-thinning behavior that is an essential property of the topical formulations. The formulations were biocompatible. The release of metronidazole from the formulations followed Higuchian kinetics. The drugloaded emulsions and gels have shown good inhibitory activity against B. subtilis. In conclusion, the developed emulsions and DOI 10.1002/jps.24260

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emulsion gels may be used as matrices for controlled delivery of bioactive agents.

ACKNOWLEDGMENT The authors would like to acknowledge the financial support received from Department of Biotechnology, New Delhi, India, vide sanction order (BT/PR14282/PID/06/598/2010).

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DOI 10.1002/jps.24260