Introduction

Tazarotene (TZRT), 6-[2-(4,4-dimethylthiochroman-6-yl)ethynyl] nicotinic acid ethyl ester, a member of a new generation of receptor selective synthetic retinoids is used for the topical treatment of mild to moderate plaque psoriasis, acne vulgaris and photoaging. Dermal safety studies have indicated that TZRT did not show phototoxic or photoallergic potential (Russell 2000; Zaenglein 2008; Patel et al. 2010). However, the course of treatment which is usually prolonged (weeks or months) may lead to adverse reactions like pruritus, burning/stinging and erythema in a significant subset of users. These may often result in the interruption or discontinuation of the treatment regimen. Further, extremely low solubility limits TZRT incorporation into an acceptable vehicle and its tolerability results in either discontinuation of treatment or poor compliance in patients (Zaenglein 2008; Patel et al. 2010). Therefore, it is prudent to improve the topical delivery and reduce adverse effects of TZRT using an appropriate carrier having desired skin targeting properties that may reduce the adverse reactions substantially and improve users’ compliance to the novel regimen.

Acne, a cutaneous disorder of multifactorial origin, is a disease whose cause and severity depends on relationship between hormones, keratinization, sebum and bacteria. The objectives of acne therapy usually include controlling acne lesions, preventing scaring and minimizing morbidity (Russell 2000). The conventional anti-acne formulations could be less efficacious due to insignificant skin penetration properties. In comparison, the novel drug delivery systems of recently available anti-acne formulations have potential to reduce the adverse effects without reducing the efficacy. Microemulsion (ME) offers a powerful way for drug delivery as colloidal drug carrier due to its versatility and favorable advantages (Patel et al. 2009, 2011, 2013a, b; Nasr and Abdel-Hamid 2015).

The application of ME systems to skin, distributes the anti-acne agent(s) steadily and could reduce the irritancy. These systems have also shown to promote follicular targeting and thus creating high local concentrations of the active compound in the pilosebaceous unit and efficient penetration in hair follicles. Thus, microemulsions (MEs) could play a pivotal role in improving topical delivery of anti-acne agents by enhancing their dermal localization with concomitant reduction in their adverse effects (Patel et al. 2009, 2011; Nasr and Abdel-Hamid 2015).

In order to find out innovative ways for administering TZRT and assuaging their disadvantages, the present study investigated the development of ME containing TZRT. It was hypothesized that the oily core present around TZRT may result in reduction of irritation when presented in the form of ME. The combination of effects viz. equal and/or enhanced effectiveness, reduction in adverse reactions and regimen adherence may provide better platform of novel drug delivery with improved therapeutic efficacy. Thus, the present study focuses on novel formulation consideration, characterization and evaluation of ME containing TZRT.

Materials and methods

Materials

TZRT was procured on gratis from Dr. Reddy’s Laboratory Ltd., (Andhra Pradesh, India). Labrafac CC, Labrasol, Plurol oleique (Gattefosse, Saint-Priest, France); Cremophor RH 40 (BASF India Ltd., Mumbai, India) and Capmul MCM (Abitec Corporation, Janesville, USA) were procured on gratis. Isopropyl myristate was purchased from National Chemicals (Vadodara, India). Ethanol was purchased from Baroda Chemical Ind. Ltd (Dabhoi, India). Double distilled water was used throughout the study. All other chemicals and solvents were of analytical reagent grade and were used as received without further purification.

Methods

High performance thin layer chromatography method and chromatographic conditions

TZRT standard and test samples solutions were spotted on pre-coated high performance thin layer chromatography (HPTLC) plates in the form of narrow bands of 6 mm length, with 10 mm from the bottom and left margin and with 9 mm distance between two bands. Samples were applied using Linomat V autosprayer at constant application rate of 150 nL/s. Linear ascending development was carried out in 10 cm × 10 cm twin trough glass chamber equilibrated (20 min; 25 ± 2 °C) with mobile phase (toluene–methanol (9:1, v/v)) for migration distance of 70 mm. After development, the HPTLC plates were dried completely. Densitometric scanning was performed on Camag TLC Scanner III (Camag, Muttenz, Switzerland) in absorbance mode and operated by winCATS Planar Chromatography version 1.3.4. The source of radiation utilized was deuterium lamp. The spots were analyzed at a wavelength of 327 nm. The slit dimensions used in the analysis were length and width of 5 mm and 0.45 mm respectively, with a scanning rate of 20 mm/s. These were selected as recommended by the CAMAG TLC Scanner III Manual. Concentrations of compound chromatographed were determined from the intensity of diffusely reflected light and evaluated as peak areas against concentrations using linear regression equation. The developed method was validated in accordance with International Conference on Harmonization Guideline (Patel et al. 2010).

Tazarotene solubility determination

The solubility of TZRT was determined in various oils, emulsifiers, and co-emulsifiers. In this experiment, excess amount of TZRT was added in 1 mL of each excipients and mixed thoroughly using vortexer at ambient temperature. The mixed samples were kept for 72 h to allow equilibration. Then the samples were centrifuged at 5000×g for 30 min to separate the un-dissolved drug. Supernatants were diluted and analysed by HPTLC method (Patel et al. 2010) in order to estimate concentration of soluble TZRT. Excipients in which TZRT showed good or excellent solubility were chosen for preparing MEs (Patel et al. 2009).

Construction of phase diagrams

The pseudo-ternary phase diagrams were constructed using spontaneous microemulsification method at ambient temperature. The mixture of Labrasol (LBS) and Cremophor RH-40 (CMP40) (1:1) was used as mixed emulsifier. Three phase diagrams were prepared with the 1:1, 2:1 and 3:1 weight ratios (Km) of mixture of LBS/CMP40 as emulsifier and Capmul MCM (CAPM) as co-emulsifier (Emix), respectively. For each phase diagram at a specific emulsifier/co-emulsifier mixing ratio (Km), the ratio of Isopropyl myristate (IPMS) to the Emix were varied as 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1. Spontaneous formation of ME was evaluated by the addition of known amount of water at once to the known amount of oil, emulsifier and co-emulsifier with controlled stirring. The ease of formation of clear ME was taken as the criteria of spontaneity of microemulsification (Patel et al. 2009, 2011).

Preparation of tazarotene microemulsions

Eight potential ME formulations, which were different from each other by water volume fraction, were selected and formulated at Km 1:1 by spontaneous microemulsification method. The calculated amount of TZRT (0.05 %w/w) was added to the oily phase of ME and magnetically stirred until dissolved and followed by adding Emix in a fixed proportion to produce clear mixture. Then a defined proportion of water was added and stirred to produce clear ME containing TZRT (Patel et al. 2009, 2011).

Characterization of microemulsions

The average droplet size and polydispersity index (PDI) of ME was measured by photon correlation spectroscopy (PCS) with in-built Zetasizer (Nano ZS, Malvern Instruments, UK) at 633 nm. Helium–neon gas laser having intensity of 4mW was the light source. The droplet size was calculated using Stokes–Einstein relationship by Zetasizer Software. Electrophoretic mobility (µm/s) was measured using small volume disposable zeta cell and converted to zeta potential by in-built software using Helmholtz–Smoluchowski equation. All determinations were made in triplicate. Transmission electron microscopy (TEM) was used to characterize the microstructure of TZRT loaded ME. The TEM images were obtained using a Tecnai G2 20 TEM (Philips, Holland). TZRT content in the formulation was assayed using a HPTLC (Patel et al. 2010). The percent transmittance of the formulation was measured using a colorimeter (Digital Colorimeter, D-801, Photocon, India) at 570–590 nm. Isotropic nature of ME formulations were verified using cross-polarized light microscopy (Polarizing Microscope RPL-55 Series, Radical Instuments, India). The pH value of the formulation was determined using a digital pH meter (HI 98107, Hanna Instruments, India.) which was standardized using pH 4 and 7 buffers before use. The electric conductivity of ME was measured using a conductivity meter (Equip-Tronics, EQ—664, Mumbai, India) equipped with inbuilt magnetic stirrer. The viscosity of ME was measured using a Brookfield Viscometer LVDV—IIIU (Brookfield Engineering LBS, Stoughton, USA) with spindle SC 18 at 100 rpm using interval of 30 s (Bachhav and Patravale 2009; Patel et al. 2009, 2011, 2013a, b).

In vitro skin permeation study

The in vitro skin permeation study was carried out as per the guidelines proposed by Committee for the Purpose of Control and Supervision of Experiments on Animal (CPCSEA) and study protocols were approved by the Local Institutional Animal Ethics Committee (IAEC). The abdominal skins obtained from male Wistar rats weighing 230 ± 20 g (age, 6–8 weeks) were used for in vitro permeation experiments of prepared formulations. After hair was shaved carefully with an electric clipper, the skin was excised from the abdominal region of each sacrificed rat and the subcutaneous fat and other extraneous tissues were removed without damaging the epidermal surface. The excised rat skins were rinsed with physiological saline and the thickness of each skin tissue was kept almost similar (Bachhav and Patravale 2009; Patel et al. 2009)

The excised skins were then placed in refrigerator at 4 °C and then used for permeation experiments. The skin membranes were first hydrated for 30 min in buffer solution (pH 7.4) at room temperature to remove extraneous debris and leachable enzymes. The permeation experiments were performed using Franz diffusion cells fitted with excised rat skins such that the dermal side of the skin was exposed to the receptor fluid and the stratum corneum remained in contact with the donor compartment. The effective permeation area was 3.14 cm2 (20 mm diameter orifice) and the receptor compartment was filled with 12 mL of physiological saline: ethanol (7:3 v/v) for ensuring pseudo-sink conditions by increasing the solubility of TZRT in the receiving phase. The temperature of the receiver chamber containing 12 mL of receptor media was controlled at 32 ± 1 °C using circulating equibath (Model 8506, Medica Instrument Mfg. CO, Mumbai, India). Diffusion media was continuously stirred with a Teflon-coated magnetic bar at a constant rate, in a way that the rat skin surface just flushes the diffusion fluid. The formulation (1 g) was gently placed in a donor chamber and then the diffusion cells were covered with an aluminium foil to prevent light exposure. At 1, 2, 4, 6, 8, 10 and 12 h aliquot of 1 mL sample were withdrawn from the receptor compartment and replaced immediately with an equivalent volume of receptor fluid. The concentration of TZRT in receptor fluid was analyzed using HPTLC method (Patel et al. 2010). Each experiment was performed in triplicate. Cumulative corrections were made to obtain the total amount of TZRT permeated at interval each time.

The permeation rate was determined using the equation—Jss = 1/A(dQ/dt)ss = C0Kp; where J ss is the permeation rate at steady-state (μg/cm2/h), A is the effective diffusion area (cm2), (dQ/dt)ss is the amount of drug permeated through the skin per unit time at steady-state (μg/h), C 0 is the applied drug concentration in the donor compartment (μg/mL) and K p is the permeability coefficient (cm/h). The average cumulative amount of drug permeated per unit surface area of the skin was plotted versus time. The permeation rate of TZRT at steady-state (J ss , μg/cm2/h) through rat skin was calculated from the slope of linear portion of the cumulative amount permeated through the rat skins per unit area versus time plot. In order to obtain the permeability coefficient K p (cm/h), the following equation was used K p  = J ss /C 0 (Bachhav and Patravale 2009; Patel et al. 2009, 2011).

Skin retention study

Skin retention study was performed in order to analyse the content of the drug in the skin. At the end of the in vitro skin permeation study, the skin samples were washed with water and methanol on both sides and carefully dried. Then a defined amount of methanol was added to each piece of skin (Bachhav and Patravale 2009). The samples were vortexed for 10 min in order to extract its drug content and stirred overnight. The samples were analysed by HPTLC (Patel et al. 2010) after centrifugation.

Histopathological investigation of skin

The rat abdominal skin was mounted on Franz diffusion cell. The optimized ME was applied similar to method for permeation study and the effects were compared against control. A piece of fresh excised untreated skin sample was used as control. The skin was fixed in 10 % neutral formalin for 24 h and then cut vertically against the surface at the central region (4 mm width). Each section was dehydrated using graded solutions of ethanol and then embedded in paraffin wax. Tissues were divided into small pieces and stained with haematoxylin and eosin. The sections were observed under 100× magnifications and photographed (Bachhav and Patravale 2009; Patel et al. 2009, 2011, 2013b).

Infrared study

The infrared (IR) spectra of TZRT pure powder, unloaded ME and optimized TZRT-ME were taken using an IR spectrophotometer (Spectrum GX FT-IR, Perkin Elmer, Norwalk, CT). The formulations were spread as a thin layer on potassium bromide cell and then scanned between 4000–400 cm−1. The resulting IR spectra of TZRT and plain ME were then compared with TZRT-ME to detect any possible interaction between the drug and different components used (Patel et al. 2013b).

Stability studies

The stability of ME formulations was studied through various parameters such as clarity, phase separation observation, droplet size determinations, viscosity measurements, conductivity measurements, pH and zeta potential determinations for 6 months at room temperature. In order to estimate metastable systems, the selected ME vehicles were centrifuged (Remi Lab, Mumbai) at 3000×g for 30 min (Bachhav and Patravale 2009; Patel et al. 2009, 2011, 2013a, b). Beside the physicochemical properties, the chemical stability of the investigated drug in the vehicle plays a major role. Therefore, the drug content was analysed at defined time intervals during the observation period of 6 months. During the observation period the formulations were stored at room temperature in tubes to simulate patient usage conditions (Zaenglein 2008). During stability studies the samples were taken at fixed time intervals.

Statistical analysis

All the skin permeation studies were carried out in triplicate and the data were expressed as the mean value ± SD. Statistical data were analyzed by one-way analysis of variance (ANOVA). A multiple comparison test was used to compare different formulations and p < 0.05 was considered to be significant.

Results and discussion

HPTLC method development

The HPTLC separation was achieved on an aluminium-backed layer of silica gel 60F254 using toluene-methanol (9.0 + 1.0 v/v) as mobile phase. Quantitation was achieved by densitometric analysis at 327 nm over the concentration range of 100–500 ng/mL (Patel et al. 2010). Figure 1 represents densitogram of TZRT indicating single compact band at Rf value 0.75 ± 0.01. The linear regression analysis data for the calibration plots showed a good linear relationship with r2—0.9995. The method was validated successfully as per the International Conference on Harmonization guidelines. The minimum detectable amount was found to be 18.59 ng, whereas the limit of quantitation was found to be 56.34 ng (Patel et al. 2010). Statistical analysis of the data showed that the method is precise, accurate, reproducible, robust and specific for the analysis of TZRT (Table 1).

Fig. 1
figure 1

Densitogram of standard tazarotene (400 ng/spot) using mobile phase toluene–methanol (9.0 + 1.0, v/v)

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Table 1 Validation results of the determination of tazarotene using proposed HPTLC method (Patel et al. 2010)
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Screening of microemulsion components

The solubility of TZRT was found highest in IPMS (59.14 ± 7.44 mg/gm) followed by Labrafac-CC (38.66 ± 7.21 mg/gm) and Captex 200 (20.45 ± 5.32 mg/gm). IPMS is a low molecular volume fatty acid ester used as a permeation enhancer in topical formulations, and hence it was chosen as oil phase for the preparation of MEs (Warisnoicharoen et al. 2000; Shah et al. 2007; Djekic and Primorac 2008). Interest in using non-ionic emulsifiers is increasing due to high stability, low toxicity, low irritancy and biodegradability of many non-ionic emulsifiers. So the solubility of TZRT was determined in various non-ionic emulsifiers in order to develop ME systems based on non-ionic emulsifiers as potential drug delivery vehicle. It is evident from the results that TZRT has maximum solubility in LBS (80.17 ± 8.17 mg/gm) followed by tween 80 (75.47 ± 6.77 mg/gm) and CMP40 (18.49 ± 3.19 mg/gm). The use of LBS for the stabilization of ME vehicles for cutaneous delivery of drugs is an area of growing interest due to its low potential to induce skin irritation and penetration enhancing effects. Therefore, it was selected as emulsifier for formulation of ME. The use of mixed emulsifiers increases the water solubilising capacity and as reported in literature the addition of CMP40 increases the HLB of LBS (Shah et al. 2007). Thus, CMP40 was further selected as another emulsifier inspite of its low solubilising capacity for drug as compared to tween 80. Both LBS and CMP40 are non-ionic, GRAS listed excipients and widely used in pharmaceutical preparations. It can be inferred from the results that the drug has significant solubility in coemulsifiers such as plurol oleique (14.38 ± 4.95 mg/gm) and CAPM (11.61 ± 4.57 mg/gm). But during preliminary studies it was observed that only CAPM could result in formation of stable MEs with above selected oils and mixture of emulsifiers. So CAPM was selected as co-emulsifier for preparation of MEs.

From these results, IPMS, LBS, CMP40 and CAPM were subsequently used as the oil phase, emulsifiers and co-emulsifier for the formulation of MEs containing TZRT in this study.

Phase studies

The pseudoternary phase behaviour of the system of water, LBS and/or CMP40, CAPM and IPMS was studied by constructing pseudoternary phase diagrams adopting a simple titration technique (Patel et al. 2009). The phase boundary was determined by observing the changes of the sample appearance from turbid to transparent or from transparent to turbid based on visual identification. The mixture (blend) of emulsifiers changes the polarity of polar and apolar phases and also enhances the emulsifier partitioning at the interface by increasing the stability of emulsifier film. This will affect the packing parameter of the mixed emulsifiers in a manner that more water can be solubilized in the ME droplets. Therefore the increased emulsifier partitioning at the interface in the mixture of emulsifiers is synergistic. Hiuberts and Shah (1997) have also demonstrated a strong synergism in non-ionic mixture of emulsifiers similar to the findings in this study. The combined use of emulsifiers showed apparent advantages over the single use of emulsifier; the ME region was greatly increased in the phase diagram. As shown in Figs. 2a, b and 3a the isotropic region of ME was increased when combination of emulsifiers (LBS + CMP40 in Fig. 3a) were used along with coemulsifier (CAPM) as compared to single emulsifier (CMP40 in Fig. 2a and LBS in Fig. 2b). It is reported by Rhee et al. (2001) that for CMP40 in combination with LBS the ME existence area becomes enlarged as their weight ratio decreases. Same observations were made by us with the ME area reaching maximum at weight ratio of 1:1. Hence the weight ratio of emulsifiers (LBS: CMP40) was fixed at 1:1. Addition of co-emulsifier decreases the interfacial tension between water and oil phase and reduces the spontaneous curvature leading to increased water solubilization. The co-emulsifier penetrates into the interface and increases the flexibility of emulsifier film, because the liquid crystalline phase is destabilized in favor of ME phase that even connects the aqueous with the oil phase (Mohammed and Manoj 2000). The area of ME isotropic region changed in size with ratio of emulsifiers to co-emulsifier (Km) in the order of: 1:1 > 2:1 > 3:1 (Fig. 3). The maximum area of single phase ME region was obtained when ratio of Emix (LBS/CMP40: CAPM) was in the order of 1:1 (Km = 1) and so it was fixed at 1:1 for further studies. Further, eight potential ME vehicles, ME 1–ME 8 (Table 2) were selected to study the effects of formulation components on skin permeation of TZRT. The entire drug loaded ME formulations containing TZRT were prepared in the same ratios and the proportion of TZRT was kept constant at 0.05 % w/w for all the batches.

Fig. 2
figure 2

The pseudoternary phase diagram of the isopropyl myristate, cremophor RH 40/capmul MCM (Emix, 1:1), water (a) and isopropyl myristrate, labrasol/capmul MCM (Emix, 1:1), water (b) at ambient temperature. The one phase region is designated by 1Φ and the multiphase region is designated by MΦ

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Fig. 3
figure 3

The pseudoternary phase diagrams of the isopropyl myristate, labrasol/cremophor RH 40 (1:1)/capmul MCM (Emix) water at the 1:1 (a), 2:1 (b) and 3:1 (c) weight ratios of Emix (Km) at ambient temperature. The one phase region is designated by 1Φ and the multiphase region is designated by MΦ

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Table 2 Compositions of the selected microemulsion formulations (% w/w)
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In vitro skin permeation studies

Percutaneous penetration of a drug into and through the skin depends on its characteristics in an appropriate vehicle. IPMS is a known penetration enhancer. LBS, CMP40 and CAPM are assumed to act as permeation enhancers by reducing the diffusional barrier of the stratum corneum (Hiuberts and Shah 1997). So in the present study suitable vehicle in form of ME comprising oil (IPMS), mixed emulsifiers (LBS and CMP40), co-emulsifier (CAPM) and aqueous phase was selected to alter the characteristics of TZRT. The effect of composition of different ME vehicles on TZRT permeation through the skin was evaluated using the excised Wistar rat skin as the model membrane.

The permeation parameters of the tested ME formulations with various compositions are presented in Table 3. A steady increase of TZRT in the receptor chambers with time (Fig. 4) was observed due to reservoir effect of ME for TZRT. The cumulative amount of TZRT that had permeated through excised rat skin (μg/cm2) was plotted as a function of time (hours). The steady state flux (Jss) was obtained from the linear portion (2–8 h) of the graph. The flux in different compositions of MEs ranged from 0.0227 to 0.0490 μg/cm2/h, indicating that drug permeation characters through the rat skin were significantly affected by ME formulation. As shown in Table 3 and Fig. 4 the permeation of TZRT was highest from ME 4 which is composed of 6 % IPMS, 15 % LBS/CMP40 (1/1) mixture, 15 % CAPM and 64 % aqueous phase. The skin permeation rate of TZRT from this ME was 0.0490 ± 0.00075μg/cm2/h. The permeation rate from ME 5 composed of 12 % IPMS, 30 % LBS/CMP40 (1/1) mixture, 30 % CAPM and 28 % aqueous phase was lowest with a value of 0.0227 ± 0.00058 μg/cm2/h. Statistical comparison of flux throughout 12 h showed that ME 4 provided significantly higher fluxes as compared to other ME formulations (p < 0.001). Same observations were also made while considering permeability co-efficient (Kp) of different MEs with ME 4 having highest Kp (0.979 × 10−4 ± 0.138 × 10−5) and ME 5 having lowest Kp (0.453 × 10−4 ± 0.116 × 10−5). Thus from Table 3, it was concluded that the permeation parameters of TZRT from MEs were affected by the content of Emix, water and oil.

Table 3 The permeation parameters and particle size of tazarotene loaded microemulsion formulations (mean ± SD, n = 3)
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Fig. 4
figure 4

In vitro permeation profiles of tazarotene through rat skin from microemulsion formulations (mean ± SD; n = 3)

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The flux value was significantly increased (p < 0.01), from 0.0237 ± 0.00064 μg/cm2/h to 0.0490 ± 0.00075 μg/cm2/h in MEs containing 6 % w/w IPMS and from 0.0227 ± 0.00058 μg/cm2/h to 0.0422 ± 0.00076 μg/cm2/h in MEs containing 12 % w/w IPMS, as the content of Smix was decreased from 60 to 30 % (w/w). Similarly the skin permeation rate was significantly increased by 2 fold as the content of water was increased from 34 to 64 % (w/w) in MEs containing 6 % w/w IPMS and 28 to 58 % (w/w) in MEs containing 12 % w/w IPMS. However the increase of IPMS from 6 % w/w to 12 % w/w resulted in decrease of skin permeation rate. A previous study reported that the increase in the concentration of the internal phase of ME causes a gradual rise in the viscosity of the system. The viscosity of formulations plays an important role in controlling the diffusion of the drug through the double layer ME into the receptor phase. It is also considered that high content of Emix in the ME make the effect of oil on the skin permeation less pronounced (Kantarci et al. 2007; Amabade et al. 2008).

Moreover, the particle size of the ME may also affect its efficiency, where its small particle size makes it an excellent carrier for promoting TZRT percutaneous uptake as the number of vesicles that can interact on a fixed area of stratum corneum will increase when the particle size decreases (Patel et al. 2010). The particle size of all the formulations was in nanorange between 20 and 70 nm (Table 3), thus providing a large surface area for transfer of TZRT to skin due to very small droplet size.

Skin retention study

The accumulative amount of TZRT in skin was determined with an aim to formulate a vehicle with suitable penetration as well as uptake in the skin. MEs are believed to be in close contact of the skin lipid due to low interfacial tension values leading to faster permeation (Eicke et al. 1994; Kantarci et al. 2007; Bumajdad and Eastoe 2004; Amabade et al. 2008). As shown in Table 3 the amount of TZRT retained in skin from MEs was between 24.52 ± 1.45 μg (4.7 % of applied dose) and 37.33 ± 0.67 μg (7.5 % of the applied dose). The highest amount of TZRT was retained in formulation ME8 which did not show highest skin permeation rate. Hence it was observed that there was no significant correlation between skin content and flux. The ME 8 could significantly increase the skin content as compared to other formulations (p < 0.01 for ME 1, ME 2, ME 3, ME 6 and p < 0.05 for ME 5, ME 7) except ME 4 (p > 0.05). There are some results indicating that ME can increase the uptake of drug in skin (Desai 2004; Chen et al. 2007). ME can act as nanocarrier for drug due to small particle diameter and the controlled release of drug from ME may induce the increase of drug accumulation. However it was observed that along with difference in particle size the concentration of different components might have also influenced the uptake of drug in skin. As such the exact mechanism by which the uptake of drug is increased is still unclear and needs further investigation in future.

Thus, the ME formulation (ME 8) containing 12 % IPMS, 15 % LBS/CMP40 (1/1) mixture, 15 % CAPM and 58 % aqueous phase having appropriate skin permeation rate and skin uptake was considered as optimized formulation and was further evaluated.

Histopathological investigation of skin

The rat skin is a multilayered organ with many histological layers. The histology of excised rat skin in control and treated with optimized ME indicate that the optimized ME has no significant effect on the microscopic structure of the skin (Fig. 5). The surface epithelium lining and the granular cellular structure of the skin were totally intact. No major changes in the ultra structure of skin morphology could be seen and the epithelial cells appeared mostly unchanged (Patel et al. 2009).

Fig. 5
figure 5

Light microscopic photographs of in vitro histopathological study. a Control and b the rat skin treated with optimized tazarotene microemulsion

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Infrared study

The infrared spectra of TZRT pure powder, plain ME and TZRT loaded ME are as shown in the Fig. 6. TZRT spectrum shows absorption bands at 3055 cm−1 due to aromatic C–H stretching and at 2908 cm−1 and 2961 cm−1 due to aliphatic C–H stretching. A absorption band at 2201 cm−1 corresponds to C≡C stretching of the di-substituted alkyne and prominent absorption band at 1719 cm−1 related to C=O stretching of ester. Band at 1585 cm−1 is due to aromatic C–C stretching. Absorption bands at 1365 and 1448 cm−1 are due to aliphatic C–H bending. Other bands at 823 and 777 cm−1 corresponds to aromatic C-H bending. The IR spectrum of TZRT loaded ME is entirely different from TZRT powder, while it closely resembles the spectrum of plain ME. The characteristic bands of TZRT either have disappeared or few bands of TZRT reduced in intensity probably due to the restriction inside the formulation matrix. In IR spectrum of TZRT loaded ME no additional peak was observed which emphasized the absence of any possible interaction between the drug and formulation components used (Patel et al. 2013b).

Fig. 6
figure 6

Infra red spectra of a tazarotene (TZRT) pure powder, b unloaded microemulsion and c tazoretene loaded microemulsion (TZRT-ME)

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Physicochemical characterization of optimized formulation

The physicochemical parameters of unloaded and drug loaded optimized ME are as shown in Table 4. The zeta potential of unloaded and drug loaded ME formulations are less than −30 mv (Table 4). It can be seen that the ME system is negatively charged and on the addition of drug the zeta potential values in the ME did not change significantly. TZRT content of ME formulations was found to be 99.56 ± 1.85 % w/w of the theoretical value (0.05 % w/w). The % T values of unloaded and drug loaded ME formulation were between 98 and 100 % (Table 4) confirming its optical clarity. pH values of unloaded and TZRT loaded ME formulation was 6.77 ± 0.04 and 6.81 ± 0.03 respectively. It was observed that pH values were within the physiological range and the incorporated drug did not affect the optical texture or the pH values of the ME formulation. The average particle size of the plain and drug loaded ME was 32.61 ± 1.34 nm and 28.08 ± 1.35 nm, respectively. The particle size of drug loaded ME was small as compared to unloaded ME, this may be due to the emulsified effect of the undissolved drug (Desai 2004; Chen et al. 2007). All the formulations had droplets in the nano range which is very well evident from the low PDI values for both unloaded and drug loaded vehicles. The PDI values of both formulations were very low in the range of 0.109 ± 0.017 to 0.343 ± 0.048 suggesting the higher uniformity of the droplet size within the formulation (Patel et al. 2011, 2013a, b). The TEM image of drug loaded ME (Fig. 7) revealed that the particle size was in the nanometric range and that particle had nearly spherical morphology without aggregation which is in accordance with particle size analysis. The samples were examined by ocular inspection in a cross polarizer for sample homogeneity and birefringence. All ME formulations appeared completely dark when observed under cross polarizer which confirmed its optically isotropic nature (Patel et al. 2013a). Electrical conductivity is a structure sensitive property and there are some studies reported about the systems of non-ionic emulsifiers. In present study, the conductivity measurements were done without incorporation of an electrolyte as it can lead to phase separation as reported in the literature (Mohammed and Manoj 2000). The conductivity results obtained for unloaded and drug loaded ME formulations (Table 4), showed that loading TZRT and the addition of water phase into the formulation had no negative effects on system stability. When an unstable emulsion system and phase separation occurs, the conductivity values are greatly reduced (Chen et al. 2007). However the conductivity values for unloaded vehicle was about 2–3 factor lower than those of drug loaded formulation. In previous literature, it has been reported that the lowest conductivity plots extend to the largest droplet radii. The size of droplet for unloaded ME with low conductivity values was greater than those of drug loaded formulation with higher conductivity values (Desai 2004; Yamaguchi et al. 2005; Liu et al. 2007; Heuschkel et al. 2008; Huang et al. 2008; Moreira et al. 2010). The results of viscosity measurements indicate that the viscosity value of the drug loaded ME was higher than the unloaded vehicles. Thus, the conductivity and viscosity studies suggest that the ME formulations are of o/w type.

Table 4 Physicochemical characterization of optimized unloaded and tazarotene loaded microemulsion formulations (mean ± SD, n = 3)
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Fig. 7
figure 7

The TEM image of tazarotene loaded microemulsion

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Stability studies

Stability studies of the ME samples were carried out under stressed (centrifugation) and unstressed conditions by visual inspection as well as determination of physicochemical parameters such as particle size, polydispersity index, zeta potential, drug content, percentage transmittance, pH, conductivity and viscosity. The ME showed good shelf stability showing no phase separation, either cloudiness or any flocculation or precipitation for 6 months. These samples also showed no sign of phase separation under stress when subjected to centrifugation at 5000×g for 30 min. Thus centrifuge tests showed that all MEs had good physical stability. All physicochemical parameters were determined at weekly intervals for the first month and monthly interval for the subsequent months. However, no significant change in the physicochemical parameters was observed. The physicochemical parameters of ME formulation is as shown in Table 5 at the end of 6 months.

Table 5 Physicochemical characterization of optimized microemulsion formulation containing tazarotene at the end of 6 months at room temperature (mean ± SD, n = 3)
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Conclusion

Collectively, the results demonstrate that ME may be a promising approach for the topical delivery of TZRT. It is assumed that ME due to its appropriate physicochemical properties, high skin permeation ability, higher accumulative uptake in skin and improved skin tolerability may be more suitable for targeted therapy and could be developed as a novel regime for topical administration of TZRT. Nevertheless, significant work is yet to be conducted to elucidate the possible mechanism(s) of drug delivery into the skin. The clinical investigations with emphasis on desired therapeutic efficacy and favorable safety profile followed by pilot scale studies on manufacturing the product for commercial use also needs to be considered.