Being a poorly water-soluble drug, solubility of NDFX in the oily phase of microemulsion is very critical because it may affect the stability as well as the percutaneous delivery performance of the formulation. In order to screen appropriate solvents for the preparation of microemulsions, the solubility of NDFX in various oils, nonionic surfactants and cosurfactant was measured and the results are shown in figs. –, respectively. The solubility of NDFX in Capryol 90 was 2065.26±0.04 μg/ml, which was the highest amongst the oils investigated. Amongst the various surfactants and cosurfactants screened, NDFX was found to exhibit good solubility in surfactants such as Tween 80 (12.73 μg/ml) and Labrafil M 2125 CS (36.18 μg/ml), whereas Transcutol P (7123.58±0.54 μg/ml) exhibited superior solubility amongst the cosurfactants screened in the preliminary studies. The selection of surfactant or cosurfactant in the further study was governed by their emulsification efficiency of oil in which NDFX had maximum solubility rather than their ability to solubilise NDFX.
Solubility of Nadifloxacin in various oily phases.
Solubility of Nadifloxacin in cosurfactants.
Solubility of Nadifloxacin in 5% w/w surfactant solution.
Emulsification studies were performed to evaluate the ability of various surfactants to emulsify the selected oily phase. For the oil–surfactant mixture which forms microemulsion, it is essential to determine the condition under which they could disperse efficiently to form microemulsions with the selected oil. The percentage transmittance values of various dispersions are given in Tables and . Emulsification studies with various surfactants showed that SolutolHS-15 had very good ability to emulsify Capryol 90 followed by Tween 80 and Tween 20, whereas, Labrasol appeared to be a poor emulsifier for Capryol 90. These observations are in line with the investigations reported by Malcolmson et al
] and Warisnoicharoen et al
] who concluded that microemulsification is also influenced by the structure and chain length of the surfactant. In case of Polaxomer 407, good transmittance (99.7%) was observed, but was not selected as it showed less solubility for NDFX as compared to Solutol HS-15 and Tween 80. whereas, Labrafil M 2125 CS which exhibited good solubility for NDFX, was not able to show good % transmittance indicating poor microemulsifying ability for the selected oily phase and hence rejected.
EMULSIFICATION EFFICIENCY OF VARIOUS NONIONIC SURFACTANTS
EMULSIFICATION STUDIES ON SURFACTANT/COSURFACTANT COMBINATIONS
The short-chain alcohols and Transcutol P were widely used as cosurfactants[23
]. In this experiment, Lauroglycol, PEG-400, n-butanol, Akomed E, Labrafil M 1944 CS, propane-1,2-diol, Akolin MCM, ethanol, isopropyl alcohol and Transcutol P as cosurfactants were investigated with Capryol 90 as the oil phase, Tween 80/Solutol HS 15 as surfactant at the fixed km
of 1:1. All the hydrophilic cosurfactants were equivalent in improving the microemulsification ability of Solutol HS 15 and Tween 80. In case of lipophilic cosurfactants based on emulsification ability, Akomed E and Labrafil M 1944 CS were found to be the best followed by Akolin MCM whereas Lauroglycol 90 was less effective as a cosurfactant. Thus, there was good correlation between the structure and chain length of cosurfactant and transmittance value. This correlation was applicable to all lipophilic cosurfactants except Akomed E and Labrafil M 1944 CS. Labrafil M 1944 CS (oleic and linoleic acid backbone) was superior than Lauroglycol 90 (lauric acid backbone) due to its hydrophilic and surfactant-like properties. These investigations are in line with the investigation reported by Date et al
]. Though all cosurfactants showed good spontaneity of microemulsions, due to high solubility of NDFX, Transcutol P was selected as cosurfactant.
The pseudoternary phase diagrams were constructed in order to obtain the concentration range of components for the existence range of microemulsions. The phase diagrams of Capryol 90–Solutol SH 15–Transcutol P–water and Capryol 90–Tween 80–Transcutol P–water are shown in figs. and . The transparent or translucent o/w microemulsion area was presented as a shaded region in the phase diagrams. It is evident that Capryol 90–Tween 80–Transcutol P–water system had larger microemulsification region as compared to Capryol 90–Solutol SH 15–Transcutol P–water system (figs. and ). The optimum surfactant/cosurfactant ratio of microemulsion system was found at km
1:1 for Capryol 90–Tween 80–Transcutol P–water system. The emulsified area was the lowest at km
3:1 as the concentration of cosurfactant was less. System at km
1:1 formed a larger single-phase region than the systems at other km
. It was reported that at the optimum km
value, the cosurfactant gets entrapped into the cavities between the surfactant molecules, and the formed microemulsion will have the maximum solubilisation capacity[25
]. In the current investigation, due to larger microemulsion region Capryol 90–Tween 80–Transcutol P–water system at km
value 1:1 was selected for further studies.
Pseudoternary phase diagrams of microemulsions.
Pseudoternary phase diagrams of microemulsions.
After the microemulsion regions in the phase diagrams were identified, the microemulsion formulations were selected at different component ratios as described in . The microemulsions containing excess amount of drug were prepared and optimised for drug-loading efficiency and effect of amount of surfactant:cosurfactant and oil phase on globule size and PI of drug-loaded microemulsions were investigated. Formulations in which the oil content was 10% and surfactant–cosurfactant km (1:1) were 30, 35, 40 and 60%, respectively, were investigated for drug loading.
COMPOSTION OF NDFX MICROEMULSION FORMULATION
The drug-loading efficiency was found to be increased, as the content of surfactant:cosurfactant mixture was varied from 30 to 60% for microemulsion system. The results demonstrated that the increased drug load ing of microemulsion was obtained due to the higher concentration of surfactant and cosurfactant in the microemulsion system. Hence, this ensured that concentration of surfactant–cosurfactant had a major effect on the drug loading. This could be due to the highest solubilisation capacity of surfactant and cosurfactant for NDFX as investigated in the preliminary studies.
In order to determine the effect of the amount of oily phase on drug-loading efficiency, the content of oily phase varied from 10 to 20%, whereas the content of surfactant and cosurfactant mixture was fixed at 35% as in formula 2, 5, 6 in microemulsion system. The increase in drug loading with increase in oil content could be attributed to enhanced solubility of NDFX in oil. The amount of oily phase affected the drug-loading efficiency.
The amount of oily phase also affected the globule size. The average globule sizes were found to increase significantly with more oil, which can be attributed to the expansion of oil drop of microemulsion by increased amount of oil. This finding was consistent with a previous report that the average droplet sizes of tripolide microemulsion containing 1.5 and 60% oil were 12.5 and 9.8 nm, respectively[10
]. Small droplet size was preferred in terms of skin permeation, so the oil content was selected as 10%.
The globule size was varied from 120 to 57 nm as the content of surfactant–cosurfactant mixture was varied from 30 to 60% microemulsion system. The result showed that the smaller globule size of microemulsion was obtained due to the higher concentration of surfactant and cosurfactant in microemulsion system. The decrease in the globule size can be attributed to the solubilisation of internal phase within a larger number of surfactant micelles, which are consequently swollen to a lesser extent[26
]. When the content of oil was at 10% and surfactant cosurfactant mixture 60%; the formulations exhibited the smallest globule sizes as in case of formula 4. Based on the high drug-loading efficiency formula 3, 4 and 6 were further characterised and investigated for thermal stability.
Microemulsions were found to be robust to all dilutions and did not show any separation even after 24 h of storage. Optical isotropicity of the microemulsion was confirmed by visualisation through crossed polarisers. Birefringence is a light-scattering phenomenon, which is a characteristic of liquid crystalline systems. Birefringence results from unequal refractive indices of light in which the light passing through the matter is divided into two components having different velocities. Therefore, when liquid crystalline phase is observed between crossed polarisers, an intense bands of colours, i.e., birefringence is observed whereas, microemulsions appear completely black. Formulations appeared completely dark when observed between polarising plates in crossed position because of inability of light to pass. These observations, therefore, confirmed the optical isotropy of the resulting microemulsions[8
]. The globule size for all NDFX-ME formulations revealed from light-scattering experiments were in the range 65.83-121.21 nm, which is generally considered to be the globule size of microemulsion (≈10-150 nm). From , NDFX-ME had slightly increased globule size of the internal droplet as compared to those without drug indicating that drug would immerse in the surfactant film around oily droplet of o/w microemulsion. PI is the measure of globule homogeneity and it varies from 0 to 1. PI value to zero is the indication of higher the homology between the globules. PI for all formulations was around one. The selected NDFX-ME formulations () were subjected to various thermodynamic stability tests, which included centrifugation and freeze–thaw cycle tests. No phase separation, creaming or drug precipitation was observed while performing these tests and all the formulations survived these stress tests. The results showed that all the formulations had a good physical stability. Very low interfacial tension between oil and water and small droplet size made these systems thermodynamically stable. The increased concentration of oil and surfactant in microemulsions may retain the drug in droplets of microemulsion formulation that will decrease its permeation in the skin[29
]. Hence, based on these studies, the microemulsion formulation containing low oil and surfactant: cosurfactant content, i.e., F3 was formulated into gel.
CHARACTERIZATION OF NDFX MICROEMULSION
From the results shown in , it was noted that there was no significant change in globule size when microemulsion was formulated in gel form using xanthan gum and Carbopol ETD 2020. When the microemulsion was gelled by incorporating a gelling agent, globule aggregation was not observed which was indicated by no change in PI. The pH values of all formulations were found to be compatible for topical formulations. The pH range for skin is 6-8. The spreadability diameter of all microemulsion-based gels was in the range 7-8.6 cm, whereas the diameter for marketed formulation was 6.32 cm, indicating that the spreadability of microemulsion-based gels was better than that of marketed formulations. This could be because of the loose gel matrix nature of microemulsion-based gel due to presence of oil globules rather than the conventional cream formulation. Although good spreadability was observed for all the formulations, the formulation gelled with xanthan gum showed better spreadability and had a diameter that ranged 8.2-8.7 cm as compared to those gelled with Carbopol ETD 2020 with diameter ranged 7.1-7.6 cm.
CHARACTERIZATION OF NDFX MICROEMULSION GEL
The drug content in the NDFX-MEG is as shown in the , and was well within limits. There was no degradation of drug in the microemulsion when formulated as microemulsion-based gels. Rheological data obtained from microemulsion gel showed non-Newtonian, thixotropic behaviour. The thixotropic system is very advantageous and is more convenient than an ideally viscous system from the technological as well as the topical application viewpoints of the formulation[32
]. Viscosity of microemulsion formulation gelled with Carbopol ETD 2020 was higher, i.e., in the range 199,000-220,000 cps as compared to those formulation which were gelled with xanthan gum had viscosity in the range 97,000-99,900 cps. Viscosity results at 20 rpm are shown in . The lower viscosity of xanthan gum NDFX-MEG could be attributed to ease of spreading. This was confirmed in a spreadability test where in xanthan gum NDFX-MEG showed larger diameter compared to Carbopol ETD 2020 NDFX-MEG. Ex vivo
permeation studies were carried out using abdominal skin of male Wistar rat to assess the release of selected NDFX-MEG and compared with commercial formulation. Phosphate buffer (pH 6.8) containing 20% ethanol was selected as a diffusion medium on the receptor side. Ethanol was added in the release media to maintain the sink condition. The result of percutaneous penetration of marketed cream formulation (Nadoxin®
) containing 1% NDFX and o/w microemulsion-based gel containing 0.2% w/w NDFX were shown in . In the ex vivo
skin permeation studies, it was demonstrated that microemulsion-based gels improved the permeation as compared to the marketed formulation at the end of 10 h.
Cumulative amount of drug permeated in μg/cm2 versus time in hours.
Conventional creams have a mean droplet size ranging 10-100 μm. Such formulations have demonstrated poor penetration of drug-loaded oil droplets into deep skin layers. Gupta and Garg[4
] have reported that microparticles with diameters ranging from 3 to 10 μm selectively penetrate follicular ducts, whereas particles >10 μm remain on the skin surface, and those <3 μm are distributed randomly into hair follicles and stratum corneum. As shown in , microemulsion gel improved the skin permeation of NDFX over the commercial creams. This could be due to the presence of surfactant and cosurfactant in microemulsion which may affect the stratum corneum structure and reduce the diffusional barrier by acting as a permeation enhancer[30
]. The microemulsion is expected to penetrate the stratum corneum and exist intact in the whole horny layer. Once it enters into the stratum corneum, the microemulsion may simultaneously alter both the lipid and the polar pathways[6
]. The lipophilic domain of the microemulsion can interact with the stratum corneum in many ways. NDFX dissolved in the lipid domain of a microemulsion can directly partition into the lipids of the stratum corneum or the lipid vesicles themselves can intercalate between the lipid chains of the stratum corneum, thereby destabilising its bilayer structure. In effect, these interactions would lead to an increase in the permeability of the lipid pathway to NDFX. On the other hand, the hydrophilic domain of the microemulsion can hydrate the stratum corneum to a great extent[29
]. There is a general experience that hydration of the skin plays an important role in the percutaneous uptake of poorly soluble drug. When the aqueous fluid of the microemulsion enters the polar pathways, it increases the interlamellar volume of stratum corneum lipid bilayers, resulting in disruption of the interfacial structure. Since some lipid chains are covalently attached to corneocytes, hydration of these proteins will also lead to disorder of lipid bilayers. Similarly, swelling of the intercellular proteins may also disturb the lipid bilayers; a lipophilic drug like NDFX can then permeate more easily through the lipid pathway of the stratum corneum. The greater drug penetration enhancing activity of microemulsions may be attributed to the combined effects of both the lipophilic and hydrophilic domains of microemulsions[33
The permeability parameters of different formulations are given in . The results showed that the optimised microemulsion formulation when gelled with xanthan gum, the flux (Jss) and permeability coefficient (Kp) was greatly increased as compared to the marketed formulation, i.e., xanthan gum NDFX-MEG showed highest flux of 2.761±0.051 μg/cm2/h. At the end of 10 h, the cumulative drug release by xanthan gum NDFX-MEG was found to be 65±0.45 μg/cm2and 2.7-fold increase in flux as compared to marketed formulation with highest enhancement ratio (Er) 2.832. Also steady-state flux and permeability coefficient and enhancement ratio were significantly increased in Carbopol ETD 2020 NDFX-MEG as compared with marketed formulation. Results of statistical analysis of permeation studies also showed that NDFX-MEG were statistically significant as compared to marketed cream.
PERMEABILITY PARAMETERS OF DIFFERENT FORMULATIONS
The xanthan gum formulations showed good permeability as compared to Carbopol ETD 2020 formulations, so optimised microemulsion formulation gelled with xanthan gum was selected for antibacterial studies. Mueller-Hinton agar was selected as the media from the available literature, supporting the growth of the aerobic microorganisms. The inoculum was grown on nutrient agar broth and turbidity (indicating growth of the aerobic microorganisms) was observed. When the microorganisms were inoculated in petri plates containing Mueller-Hinton agar, followed by incubation, a matted growth was obtained. This confirmed the growth of the organisms, suitability of the media and maintenance of successful conditions for the growth. After addition of microemulsion-based gels, the zone of inhibition was visible and prominent at the end of 48 h and reported in . A distinct zone of inhibition was observed in the petri plate containing standard solution of the drug and the drug-loaded microemulsion-based gels as well as one containing marketed cream. Petri plates with placebo microemulsion gel formulation as well as DMSO blank did not show any zone of inhibition. Results of disc plate method are as shown in figs. and indicates an antibacterial effect of NDFX-MEG compared with marketed cream and NDFX in DMSO. The One-way ANOVA followed by Bonferroni's multiple comparison test showed that the NDFX-MEG had statistically significant antimicrobial activity as compared to marketed cream and NDFX in DMSO (P
<0.05). The enhanced in vitro
antibacterial activity of NDFX-MEG may be attributed to enhanced penetration of oil globules containing NDFX through bacterial cell wall to inhibit the enzyme DNA gyrase, thus inhibiting bacterial multiplication[15
DIAMETER OF ZONE OF INHIBITIONS RECORDED IN THE ANTIMICROBIAL STUDY
Zone of inhibition antibacterial activity.
Effect of antibacterial activity of different formulations of Nadifloxacin.
Thus, foregoing results indicate that under optimised conditions, NDFX can be successfully incorporated in microemulsion system using GRAS-listed and topically acceptable surfactants, cosurfactants and oily phase. The results provide a basis for the successful design of NDFX microemulsion which resulted in improved penetration of drug and antimicrobial activity in comparison with commercial formulation of NDFX.