RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology
Coencapsulation of Hydrophobic and Hydrophilic Antituberculosis Drugs in Synergistic Brij 96 Microemulsions: A Biophysical Characterization GURPREET KAUR,1 S.K. MEHTA,1 SANDEEP KUMAR,2 GAURAV BHANJANA,2 NEERAJ DILBAGHI2 1 2
Department of Chemistry and Centre of Advanced Studies in Chemistry, Panjab University, Chandigarh 160014, Punjab, India Department of Bio and Nano Technology, Guru Jambheshwar University of Science and Technology, Hisar 125001, Haryana, India
Received 21 January 2015; revised 1 April 2015; accepted 2 April 2015 Published online 7 May 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.24469 ABSTRACT: A microemulsion has been formulated to coencapsulate antituberculosis drugs to solve the issue of stability of rifampicin (RIF) in the presence of isoniazid (INH) and pyrazinamide (PZA). The structural transition, solubilization locus, and quantitative release of drugs without interference have been estimated. Derivative absorbance spectroscopy, especially ratio derivative and double divisor ratio derivative methods, has been employed for estimating the release. The coencapsulation of the anti-tuberculosis drugs were carried out in single, binary, or ternary mixtures and occupy the same solubilization sites in multiple drugs microemulsion systems as in the case of single drug-loaded systems. INH and PZA obey the diffusional (Fickian) release mechanism, whereas RIF shows anomalous release. Resazurin assay and agar well diffusion method were adopted for cytotoxicity analysis and antimicrobial activity, respectively. Cytotoxicity was C 2015 Wiley Periodicals, Inc. and the American found to be dependent on concentration and on colloidal structure of microemulsion. Pharmacists Association J Pharm Sci 104:2203–2212, 2015 Keywords: nonionic microemulsions; anti-TB drugs; stability; in vitro release; Brij 96; derivative absorbance spectroscopy; UV-visible spectroscopy; solubility; toxicity; preformulation
INTRODUCTION Colloidal dispersions of oil and water represent an interesting prospect for the development of formulations for use as vehicles to control the release of drugs to the human body. Many forms of colloidal assemblies, namely, SMEEDS (self-microemulsifying drug delivery systems), microemulsions, emulsions, liquid crystals, micelles, liposomes, niosomes, and so on, have been evaluated for delivery of drugs.1,2 Taking about microemulsions, they may behave differently when loaded with drugs, diluted in in vivo conditions and also show dependence on their colloidal structure.3–6 Many microemulsion formulations, particularly SMEDDS, are presently used in several commercial formulations (cyclosporine A, ritonavir, saquinariz).7 The research in microemulsions is focused on control of particle size and surface properties for specific applications, development of core shell particles, drug delivery systems, and their uses in biomedical applications.8–11 However, rare literature is available for coencapsulation of multiple drugs and their simultaneous delivery.12 Multiple drug therapy is widely used for the treatment of tuberculosis (TB). The first line drugs used are rifampicin (RIF), isoniazid (INH), and pyrazinamide (PZA).13 RIF is a borderline class II drug of Biopharmaceutical Classification System (BCS); however, INH and PZA are hydrophilic drugs. In fixed dose combinations, INH onsets the degradation of RIF (in acidic medium) and hydrolyzes it to insoluble 3-formyl rifamycin Correspondence to: Gurpreet Kaur (Telephone: +91-9872800434; Fax: +91172-2545074; E-mail: [email protected]) This article contains supplementary material available from the authors upon request or via the Internet at http://wileylibrary.com. Journal of Pharmaceutical Sciences, Vol. 104, 2203–2212 (2015) C 2015 Wiley Periodicals, Inc. and the American Pharmacists Association
SV.14 Other factors that are believed to affect the bioavailability of drug are particle size, crystallinity, excipients, and so on.15 The idea is to explore the advantage of internal structure of the microemulsions for cosolubilization of hydrophobic and hydrophilic drugs. The work has been carried out to make the factors explicit that will affect microemulsions efficiency as carrier for multiple drugs. The formulations composed of polyoxyethylene 10 oleoyl ether (Brij 96) using ethyl oleate (pharmaceutical accepted oil) have been prepared and assessed as a carrier for anti-TB drugs [i.e., RIF, INH, and PZA; (Fig. S1) in single and multiple drugs combinations]. Similar studies have been reported earlier using Tween 80/oleic acid/ethanol/phosphate buffer (pH 7.4).16 The idea here is to use another nonionic surfactant-based microemulsion system, that is, Brij 96/ethyl oleate/butanol/water. Main difference between the chosen microemulsions is the solubility of RIF. In Tween-based system, RIF is more soluble in oleic acid than Tween 8017 ; however, in present case, RIF is more soluble in Brij 96 than ethyl oleate.18 Moreover, higher solubility of RIF is observed in Brij 96 in comparison to Tween 80. The characterization and stability have been verified by using various physicochemical, spectroscopic methods, and cytotoxicity analysis. The release of drugs both as single and multiple formulations has been carried out in in vitro conditions. The order of the release kinetics (zero order, first order, or Higuchi) has also been determined by ratio derivative and double divisor ratio derivative method16 for estimating concentrations in binary or ternary mixtures, respectively. Further, to access the idea of efficacy of these formulations carrying drug in variable combinations, antimicrobial studies have been carried out using gram-positive bacteria, gram-negative bacteria, and fungal strains.
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MATERIALS AND METHODS Materials Brij 96 was purchased from Fluka. Butanol (purity >99.8%) was obtained from Spectrochem, Maharashtra, INDIA. Ethyl oleate (EO), RIF (purity >98.0%), PZA (purity >98.0%), and INH (purity >98.0%) were supplied by Sigma (MO, USA). Resazurin sodium, nutrient agar, and potato dextrose agar were purchased from HiMedia, Mumbai India. The chemicals were used as received. Doubly distilled water (specific conductance, 2–4 :Scm−1 at 30 ± 0.01°C) was used for all the preparations. Microemulsion Preparation and Phase Behavior The microemulsion consisting of oil (ethyl oleate), surfactant (Brij 96), cosurfactant (butanol), and double distilled water with constant surfactant–cosurfactant mass ratio (Km 1.5) was formulated. For sample preparation, the oil was mixed to the surfactant–cosurfactant mixture followed by the addition of required amount of water to obtain the desired microemulsion compositions. The elucidation of the microstructure was carried out along a dilution line of constant oil–surfactant mixing ratio of 1.2:2 by weight, using phase behavior mapping of microemulsions as reported previously.19 Drug Incorporation in Microemulsion For the preparation of drug-loaded microemulsion in single, binary (RIF+INH and RIF+PZA), or ternary (RIF+INH+PZA) formulations, RIF was dissolved in lipophilic phase, and other two drugs were dissolved in hydrophilic component, followed by the addition of remaining components of the microemulsion. Each drug was 1% (wt) of the total weight of microemulsion. Conductivity Measurements The electric conductivity (F) was measured by means of a PICO digital conductivity meter (Labindia Instruments) operating at 50 Hz, maintained by a RE320 Ecoline thermostat at 30 ± 0.01°C. The error limit of conductance measurements was ±3%. The conductivity of selected and drug-loaded microemulsions was measured as a function of T. Viscosity Measurements Viscosity measurements for pure and drug-loaded microemulsions were carried out using a Brookfield DV viscometer (model DV-II+; Brookfield Engineering Laboratories, MA, U.S.A.) with a SC4-DIN-82 spindle (shear rate = 1.29N) and SC4-13RPY chamber (w/RTD temperature probe and cable) while maintaining the speed at 200 rpm. The experiment was repeated (in duplicate) at 30°C, with accuracy up to ±1%. Fourier Transform Infrared Fourier transform infrared (FT-IR) spectra of pure microemulsions and of the drug-loaded microemulsion (1% wt drug) were recorded on Perkin-Elmer (RX1) FT-IR spectrometer using AgCl plates, in the frequency range of 4000–350 cm−1 with 100 number scans and 4 cm−1 spectral resolution. Dilutability and Particle Size Measurements The dilutability of the microemulsions was assessed to know whether these systems would be diluted with the aqueous phase of the system without separation or not. For this purpose, selected and drug-loaded microemulsions were diluted with waKaur et al., JOURNAL OF PHARMACEUTICAL SCIENCES 104:2203–2212, 2015
ter and their transparency was assessed visually. The particle size measurements (at infinite dilution) were made using laser diffraction analyzer [CILAS 1180 liquid range (0.04–2500 :m)]. Absorbance Spectroscopy UV-visible spectra were recorded over the wavelength range of 200–600 nm using JASCO-530 UV-visible spectrophotometer. RIF was varied in the absence and presence of INH and PZA, for this three sets of experiments were performed. Spectra of RIF loaded in microemulsion were recorded against empty microemulsion to rule out the interference of components. Sample Preparation 1. Stability test of RIF in the presence of other drugs: three sets of six standard solutions each containing 3.0 g microemulsion loaded with RIF (varying concentration, 0.01–0.04 mM in microemulsion) were prepared. INH and PZA were coencapsulated with RIF at a constant concentration of 0.23 and 0.25 mM, respectively. 2. Stability test of RIF with time: 6.0 g microemulsion was prepared containing 1% (wt %) of each drug (both in single and fixed combinations). 0.1 g of each drug-loaded microemulsion were transferred into 10 mL volumetric flask and diluted to volume with ethanol. The absorbance of RIF (in single, binary, or ternary mixture formulations) was analyzed using spectrophotometer initially for 2 h and thereafter for 20 days. Samples were kept at room temperature (25°C). In Vitro Release Release Behavior and Kinetics The dialysis bag method20 using cellulose tubing [average flat width 33 mm (1.3 in)] was followed to determine the in vitro release. For the purpose, 1.0 g microemulsion containing 1% (wt) of drug-loaded microemulsion formulation (single, binary or ternary mixtures) was introduced in 50 mL of phosphate buffer (PB) (pH 7.4) maintained at 37 ± 0.1°C in a release cell. The system was stirred at constant speed of 100 rpm. The aliquot of 2 mL was withdrawn at regular intervals for 5 h (i.e., aliquot was taken at 2, 5, 10, 15, 20, 30, 45, 60, 90, 120, 150, 180, 210, 240, 270, and 300 min) and was filtered through 0.45:m membrane. Each time the same amount of fresh phosphate buffer (kept at the same temperature) was added to maintain the volume of the release medium. The drug concentrations were determined spectrophotometrically at their characteristic wavelengths (i.e., RIF 475 nm, INH 262 nm, and PZA 268 nm) for single loaded formulations. The ratio derivative and double divisor ratio derivative method were used for determination of concentrations in binary or ternary mixtures, respectively.16 Drug release was determined by two parameters, first cumulative percent drug released after 5 h and second the release rate of drug expressed as slope of the linear regression line. The order of the release kinetics was determined by comparing linear regression line for different rate equations. All the experiments were performed in triplicate and SD is ±2%. Cytotoxicity Analysis Microemulsion loaded with anti-TB drugs (1%, wt/wt) were screened for cytotoxicity analysis at two different concentrations and were compared with solid (API) in (1%, wt/wt) using DOI 10.1002/jps.24469
RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology
Resazurin assay.21 Vero cells at a concentration of 1 × 104 cells per well were cultured in a 100-:L volume of EMEM media containing 5% fetal bovine serum in a 96-well cell culture plate. The experiment was performed in triplicate. Cells were allowed to incubate for proliferation at 37°C in CO2 (5%) incubator for 24 h. Water was used as solvent except for RIF, which is soluble in dimethyl sulfoxide. Cells were further treated followed by incubation for 24 h with 20 and 40 :L of solutions of pure drugs, single drug-loaded Brij 96 microemulsions, and multiple drug-loaded Brij 96 microemulsions. Other constituents such as butanol, ethyl oleate, and Brij 96 were also evaluated for toxicity analysis using same volumes. Untreated cells were taken as reference. Background coloration was corrected with the help of media without cells. After incubation, plate was removed from incubator and 10 :L of resazurin solution prepared in EMEM media was added in all wells and incubated for 4 h. After 4 h, the pink-colored resorufin is formed and absorbance was taken by spectrophotometer (ELISA plate reader; BMG Labtech, (Ortenberg) Germany ) at wavelength of 573 nm. Cytotoxicity percentage was calculated with reference to untreated cells after normalizing the background coloration of media. Cytotoxicity (%) =
Absu − Abst ×100 Absu
(1)
where Absu is the absorbance of cells not treated with drug formulations, and Abst is the absorbance of cells treated with formulated drug-loaded microemulsions.
surements have been used to elucidate the solubilization loci of these studied drugs in Brij 96 microemulsions, which were correlated with partition coefficient studies.24 Conductivity In this work, the stability and structural changes in microstructure of microemulsions are assessed in the presence of both hydrophobic and hydrophilic anti-TB drugs in single as well as multiple combinations. In principle, microemulsion undergoes an intriguing phenomenon of electric percolation and this increase in conductivity is related to clustering of the microemulsion droplets and migration of charges within the aggregates.25 The variation of electric conductivity of pure and drug-loaded microemulsion (1.0, wt %) as a function of T (water to surfactant) is represented in Figure 1a. The drug incorporation does not affect the microstructure of the microemulsion, it only initiates the early onset of the process of percolation (at lower T) than in pure system. However, a dip in the conductivity has been seen for RIF-loaded microemulsion at higher T = 16 (Fig. 1a), which helps in locating the position of drug.18 On the contrary, the two hydrophilic drugs INH and PZA show increment in the conductivity values from the region where the transition of swollen micelles into short cylinders have just begun (T 4). A significant change is observed in the bicontinuous region. Taking into account the energetics of clustering of the droplets, in different microemulsion systems, Gocl (standard free energy of clustering) is obtained from the relation: Gocl = RT ln X d
Antimicrobial Studies Antimicrobial studies were carried out using growth inhibitory zone well method against pathogenic microorganisms,22 that is, a gram-positive (Escherichia coli) and a gram-negative (Staphylococcus aureus) bacteria and fungus strains, namely, Aspergillus niger, Aspergillus fumigatus, Cladosporium herbarium, Curvularia lunata, and Helminthosporium oryzae. Bacteria were cultured on nutrient agar and fungi were cultured on potato dextrose agar media. One-hundred microliter of test samples (concentration 0.05 g/mL) was added to 6 mm well bored on agar plates after homogeneous spreading of freshly prepared broth of various microbial stains. After incubation of 24 h for bacteria and 48 h for fungus at 37°C and 25°C, respectively, diameters of inhibition zones were measured and average value of triplicate measurement is reported. Diameter of the well was excluded from inhibition zone diameter.
RESULTS AND DISCUSSION The detailed investigation of microstructural changes of Brij 96 microemulsions on dilution with dispersion media has been carried out in our previous results.18,19,23,24 It has been found that this nonionic microemulsion undergo a microstructural change from w/o to o/w via bicontinuous phase upon dilution at T = 7.5 and 13.3, respectively. The effect of change of oil, cosurfactant (alcohols), phase behavior, water solubilization, and states of water of prepared Brij 96 microemulsion has been thoroughly investigated.23 Keeping in view the significance of microemulsions as drug delivery vehicles, these prepared compositions have been analyzed as drug carriers for anti-TB drugs.18,24 Differential scanning calorimetry (DSC) and fluorescence meaDOI 10.1002/jps.24469
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(2)
where Xd is mole fraction of droplets at percolation threshold and defined as Xd = nd /(nd +noil ), where nd and noil are the number of moles of droplet and oil, respectively. The calculated standard free energy of clustering Gocl using Eq. (2) shows a value of −48.80 kJ/mol for pure system and −48.8, −46.04, and −42.97 kJ/mol for INH, RIF, and PZA drug-loaded microemulsions, respectively. The data support the spontaneity of the droplet clustering. In case of multiple drugs cosolubilized in microemulsions, the conductivity profile reveals that in the w/o region, the presence of RIF+PZA, RIF+INH, and the combination of three drugs do not have any effect on the microstructure. However, binary mixtures show diminished conductance in o/w region, whereas ternary mixture displays high conductance. Viscosity The viscosity measurements of drug-loaded and drug-unloaded microemulsions are depicted in Figure 1b. The general trend in the viscosity remains similar for both empty and drug-loaded samples. In w/o type region, the magnitude of viscosity is nearly same in all the systems. However, as the dilution progress and system reverts to bicontinuous phase, a significant enhancement in the viscosity takes place. The measurements give a clear indication of interactions between the drug molecules and microemulsion components. The RIF-loaded Brij microemulsions display higher viscosity values in bicontinuous and o/w region.18 Similar trend is observed for INH. However, PZAincorporated microemulsion do not show any change in the viscosity values. This gives an inference that PZA mainly resides in the bulk water phase throughout the dilution. The viscosity results of binary and ternary mixtures of drug-loaded Kaur et al., JOURNAL OF PHARMACEUTICAL SCIENCES 104:2203–2212, 2015
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Figure 1. Comparison of (a) conductivity and (b) viscosity of Brij 96 and drug-loaded microemulsions as a function of T single and multiple drug formulations.
microemulsions reveal that in w/o or swollen reverse micelle region, the drug incorporation in the microemulsion does not create any significant effect on the structure. However, increment has been observed in the bicontinuous region for binary (RIF+INH) and ternary mixture while viscosity decreases for RIF+PZA. Fourier Transform Infrared We previously reported23,24 an in-depth analysis of states of water and effect of dilution on Brij 96 microemulsions. Here, the main emphasis has been laid on the OH band, as it is indicative of water and surfactant head group’s interactions and influence of drugs on it. Table 1 depicts the wavenumber of OH of empty and drug-loaded Brij 96 microemulsions. The higher frequency than pure water (3400 cm−1 ) indicates weak interaction of surfactant molecules with that of water.26 As long as water molecules are present in w/o or bicontinuous type microemulsion, bands are observed at higher wavenumber. However, as soon as water becomes the dispersion phase, the band appears at the same position as that of bulk water. In w/o type region, a shift of up to 4, 12, and 20 cm−1 in the presence of RIF, PZA, and INH, respectively, has been obTable 1. Infrared Frequencies of Various Weight Fractions of Aqueous Phase in Anti-TB Drug-Loaded Brij 96 Microemulsions wt Fraction/T
Structure
8.5/1.3 w/o 27.0/5.40 Bicontinuous 39.6/9.50 Bicontinuous 48.4/15.00 o/w 58.0/20.00 o/w 63.2/25.00 o/w
Wavenumber RIF PZA INH (OH) (1 wt %) (1 wt %) (1 wt %) 3432 3431 3424 3400 3400 3400
3428 3412 3416 3429 3430 3467
3420 3420 3412 3406 3421 –
3412 3416 3392 3409 3412 3411
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served. As RIF is mainly believed to be present in the oil region, therefore, it shows such a weak influence on the water surfactant interaction. On the contrary, the presence of INH and PZA strengthens the interaction between water and surfactant head group by being present nearby. In bicontinuous region, a great deal of difference is found between empty and all three drug-loaded microemulsions. However, trend is similar for hydrophilic as well as hydrophobic drugs. This is probably because in such type of microemulsions, zero curvature is found27 and almost the same amount of water and oil coexist. Therefore, most likely, the comparable amounts of drugs are available. A contrast has been observed once the microemulsion system reverts into o/w type. The presence of drugs enhances the wavenumber in comparison to that of pure system. It has been observed that as the dilution of o/w region occurs, the presence of drug weakens the interaction of water and the surfactant head groups. As RIF is hydrophobic in nature and prefers to stay near hydrophobic environment (i.e. oil phase and hydrophobic tails of the surfactant), it might interact with the surfactant hydrophobic tails and also interfere with the surfactant and water interactions. On the contrary, the enhancement of the wavenumber in INH- and PZA-loaded microemulsions in the o/w region leads to the fact that these drugs are probably interacting with the bound water, as both drugs are hydrophilic in nature. The partition coefficient results, DSC and fluorescence measurement,24 support this observation. Thus, we can firmly comment on the location of these drugs in Brij 96 microemulsion: RIF is present at interphase toward oil, INH in bound water, and PZA in free water.18,24 Conductivity and viscosity measurements also support the observation. In order to observe the concentration effect, the FT-IR spectra for drug-loaded microemulsions have been carried out with 0.5 and 1 wt % of drugs. However, change in the concentration of all the drugs does not show any effect. DOI 10.1002/jps.24469
RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology
Dilutability and Particle Size Analyses The droplet size of the microemulsions has been measured after addition of 250 mL water to 1 g of mixture. No further treatment of the samples was necessary. From physicochemical and other solubility studies, it may be concluded that the Brij 96 microemulsions are stable up to N(wt %) = 68. It is also believed that on dilution, the microemulsion turns into a emulsion, that is, o/w (heterogeneous coarse dispersion).28 In comparison to the particle size of 0.17 :m for pure microemulsion, the addition of drugs increases the particle size in the order of 12, 2.81, and 0.27 :m in the presence of RIF, INH, and PZA, respectively (Fig. S2). Interaction of INH with the bound water (water molecules with surfactant molecules) is the probable reason behind the increase in the size of o/w emulsion droplet. PZA prefers to be in the free water phase and does not influence the particle size. However, the RIF-loaded system shows the highest increase by being present at the core of the droplet. Stability Analysis In this study of single and multiple drugs loaded in Brij microemulsions, the stability of RIF in the presence of drugs (INH and PZA) and with span of time is analyzed using UV-visible spectroscopy.29 The UV-visible spectra of RIF in combination with other drugs in the formulated drug-loaded microemulsion system are shown in Figure 2. The slopes of the working curves of RIF in single or in binary mixture (RIF + constant INH and RIF + constant PZA)-loaded microemulsion system do not illustrate any significant difference (Table S1). Figure S3 shows that single, binary, or ternary mixture-loaded microemulsion formulation show maxima at 8max = 480 nm for RIF. The presence of other two hydrophilic drugs, in all the three mixed drug formulations, does not produce any change in 8max of RIF, neither any other peak has emerged in the UV-visible spectra. This demonstrates the stability of RIF in all the drug-loaded microemulsions. The stability study has been observed for 20 days, to assess the stability of RIF. For single (RIF) and binary (RIF + PZA) formulations, the maxima has been obtained at 8max = 480 nm as shown in Figure 3 (near the characteristic peak of RIF 475 nm). After day 1, 8max for binary (RIF + INH) and ternary (RIF + PZA + INH) formulations produce a red shift (487 nm) that is probably indicative of the formation of 3-formyl RIF SV that gives a characteristic peak at 492 nm because of the hydrolysis of RIF in the presence of INH.30 Following days show the emergence of a new peak near 425 nm for both the formulations. However, quantitative analysis in terms of rate and extent of degradation is still in progress. Release Behavior In vitro release studies with artificial cellulose membrane provide information about the diffusion of the drug, which depends on vehicle internal structure, diffusion of the drug inside the carrier system, and pH of the water phase.31,32 Anti-Tb drug release through an artificial membrane has been characterized by two parameters: the amount of drug released after 5 h and the rate of release of the drug. In order to investigate the mode of release from the microemulsion system, the release data have been analyzed with different mathematical models: zero-order kinetic, first-order kinetic, Higuchi kinetics, Korsmeyer and Peppas equation, and first-order exponential DOI 10.1002/jps.24469
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(Eqs. (3)–(7). Q = k0 t
(3)
ln Q = ln Q 0 − k1 t
(4)
Q = kH t1/2
(5)
Mt = kp tn M
(6)
Q = Q 0 − be−kt
(7)
where Q is the cumulative percent of drug released at time t, Mt /M is the fraction of drug released at time t, and k0, k1, kH , kp , and k are the rate constants for respective models. Q0 and b in Eqs. (4) and (7) are constants. Comparing the release profile of the anti-TB drugs from the Brij 96 microemulsion at pH 7.4 depicts that the fastest release has been for the hydrophilic drugs, that is, INH and PZA, with almost 80% release has been observed in first half an hour (Fig. 4). However, RIF shows a slow diffusion of drug because of its high affinity with the hydrophobic components. Therefore, only 8% release is seen in first 3 h.18 Table 2 represents the rate constants and correlation coefficient values estimated using Eqs. (3)–(7). The best agreement of the fitting has been attained with the equation of first-order exponential decay. The cumulative percent of drug released versus time t (Fig. 4) depicts the controlled release of drugs.33,34 Using Korsmeyer and Peppas equation i.e. Eq. (6), the values of n for INH and PZA in the drug-loaded microemulsion formulations indicate a diffusional (Fickian) release mechanism. On the contrary, RIF shows anomalous release from the single drug formulation. The comparative release behavior of anti-TB drugs loaded in the Brij 96 microemulsions in different binary and ternary shows that the extent of release of RIF is maximum when all the three drugs are present as ternary mixture. The release is retarded in the case of binary mixtures, both in the presence of PZA and INH (Fig. 4b). The release profile of INH in single and ternary mixture-loaded formulation is nearly same, but decrease in the extent of release has been observed for INH in the presence of RIF (as compared with single drug formulation) (Fig. 4b). The PZA release profile does not show any considerable effect on its release behavior in the presence of binary or ternary mixtures in the formulation (Fig. 4c). From the correlation constants, it has been evaluated that RIF in binary mixtures obeys zero and Higuchi kinetics in the presence of PZA and INH, respectively. In ternary mixture, however, the release of RIF follows first-order kinetics. In case of INH and PZA, exponential first order gives best fit for both the binary and ternary mixtures. As far as the release mechanism of INH and PZA from mixture-loaded microemulsions is concerned, they follow Fickian mechanism. To conclude, it is appropriate to articulate that solubilization loci of drugs especially in binary and ternary combinations play some role as far as release rates are concerned. However, mechanism of drug release is not greatly affected. Kaur et al., JOURNAL OF PHARMACEUTICAL SCIENCES 104:2203–2212, 2015
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Figure 2. UV-visible spectra of selected microemulsion (ME) containing (a) RIF, (b) RIF coencapsulated with INH (0.28 mM), and (c) RIF coencapsulated with PZA (0.26 mM). The inset shows corresponding regression line of each plot.
In contrast to Tween 80 microemulsions, Brij 96 microemulsions show high dilutability because of the most appropriate choice of cosurfactant. Moreover, in the Brij 96 microemulsions, PZA prefers to be in bulk water phase and INH near the palisade layer or trapped water region, which is opposite to Tween 80 microemulsions. Furthermore, both the microemulsion systems give different results as far as release mechanism of these drugs is concerned. Hydrophilic drugs show non-Fickian release and hydrophobic drugs shows Fickian release from Tween 80 embedded microemulsions, whereas reverse is true for Brij 96 embedded microemulsions. The probable reasons behind the different release behavior is because of difference in solubilization loci of these drugs due to disparity in the interface backbone, HLB value of the surfactants, and partition coefficient of drugs in oil/surfactant. Cytotoxicity Analysis In vitro cytotoxicity assays are useful to define basal cytotoxicity and are also necessary to define the concentration range for further and more detailed in vitro testing of any formulation to provide meaningful information on parameters such as genotoxicity or programmed cell death.35 The possible cytotoxic effect of microemulsions (both drug loaded and empty) and its components are evaluated on Vero cells treated for 24 h at different concentrations. The toxic effect of microemulsions and its components are dose-dependent, and percentage cytotoxicity increases with increase in concentration. Maximum cytotoxicity is found for ethyl oleate and minimum for Brij 96. In comparison to pure components, microemulsions show less percentage cytotoxicity. Three compositions, one from each Kaur et al., JOURNAL OF PHARMACEUTICAL SCIENCES 104:2203–2212, 2015
region, that is, w/o (ME 1), bicontinuous (ME 2), and o/w (ME 2) are selected. Interestingly, minimum cytotoxicity is observed for bicontinuous microemulsion (ME 2). Warisnoicharoen et al.36 have investigated the toxicity of nonionic surfactant including Brij 96 and its combination with various oils to human bronchial (16-HBE14o-) epithelium cells. They have correlated various factors such as aggregation concentration, hydrophobicity, and so on, which leads to damage of the cell membrane. Among other results, they revealed that Brij 96 in combination with a high-molecular-volume oil (e.g., ethyl oleate, Miglyol 812, or soybean oil) are toxic only at concentrations significantly greater than their critical aggregation concentration. Authors also suggested that surfactant toxicity is mediated by the aggregated form of the surfactant that solubilizes components of the cell membrane. In the present study, if the structure of the microemulsion is related to the toxicity, it follows the order of o/w> w/o> bicontinuous (Fig. 5a), which can be very well corelated with the high solubilizing capacity of membrane components by o/w droplets. As far as bicontinuous structure is concerned, it has zero curvature with oil and water trapped in channels and Brij 96 at the boundary, thus protecting the cell from damage caused by oil and other components. The cytotoxicity analysis of drug-loaded microemulsion both single and multiple formulations are also made. The comparison is made among pure drug (solid 1 wt %), microemulsion formulation, and drug-loaded microemulsion formulation (1 wt/wt %). For INH, IC50 is 20 :L and effect is found to be dose dependent (Fig. 5b). The drug-loaded formulations show almost comparable effects as that of vehicle. Similar trend is observed for PZA and its formulations (Fig. 5c). For the hydrophobic drug, RIF, the pure drug shows cytotoxicity of 70%; however, all DOI 10.1002/jps.24469
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Figure 3. UV-visible spectra of time study of selected microemulsion (ME) containing (a) RIF, (b) RIF + INH, (c) RIF + PZA, and (d) ternary mixture-loaded Brij 96 ME formulations.
RIF-loaded (single and multiple drugs) formulations show decrease in toxicity effect in comparison to the pure drug. Antimicrobial Activity As far as antimicrobial activity of the studied drugs is concerned, RIF has potent activity against gram-positive bacteria; on the contrary, gram-negative bacteria are generally less sensitive.37 Although the used drugs are popular as first line drugs for tuberculosis; however, RIF is a broad spectrum antibiotic. It is therefore important to evaluate these drugs in single and mixed drug microemulsion formulations for their action against microbes. INH and PZA have high bactericidal activity against Mycobacterium tuberculosis; however, other mycobacteria are resistant to both the drugs except for some strains of M. xenopi and M. bovis, respectively. The phylogenetic position of M. tuberculosis relative to other bacteria is controversial. Its cell wall has characteristics of both gram-positive and gram-negative bacteria.38 Therefore, both gram-positive and gram-negative strains, that is, E. coli and S. aureus are selected, respectively. For the purpose, different fungus strains, namely, A. niger, A. fumigatus, C. herbarium, C. lunata, and H. oryzae are also selected to evaluate antifungal activity of these microemulsion loaded with drugs (Table 3). A dose of 0.05 g/mL is selected; pure components, INH, and PZA DOI 10.1002/jps.24469
did not show activity against any bacterial and fungal strains. However, RIF encapsulated in microemulsions is active against microbes except for C. lunata. Activity of RIF loaded in microemulsion is evaluated in the presence of other drugs, too. As expected, RIF microemulsion showed high activity against E. coli, and its activity remains same even in the presence of INH and PZA. However, ternary mixture of drug-loaded microemulsion show reduced inhibition zone and S. aureusis is totally resistant to RIP microemulsion. Single RIF-loaded microemulsion show highest activity for fungal strains A. niger, A. fumigatus, and C. herbarium. Reduced activity is observed for INH (binary) and ternary drugs loaded in microemulsion. For H. oryzae, maximum zone of inhibition is found for RP mixture and single RIF.
CONCLUSIONS Microemulsions are very successful as carrier for different types of drugs (hydrophobhic, hydrophicic, and amphiphilic).1–3 However, this work reports coencapsulation of hydrophobic and hydrophilic first line anti-Tb drugs in single, binary, and ternary mixtures using Brij 96-based microemulsion. The conductivity and viscosity measurements have been helpful in elucidating the structural transition of anti-Tb drug-loaded Brij Kaur et al., JOURNAL OF PHARMACEUTICAL SCIENCES 104:2203–2212, 2015
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Figure 4. Release profiles of various single and multiple combinations of drugs from prepared microemulsions (n = 3, SD = ±2%) where: (a) release of single drug from microemulsion, (b) RIF, (c) INH, and (d) PZA in various combinations, respectively. Table 2. Rate Constants (k) and Regression Correlations (R2 ) Using Rate Eqs. (3)–(7) for the Release of Drugs from Selected Microemulsions Zero-Order Kinetics Drugs RIF PZA INH RIF + PZA RIF+ INH RIF+ INH + PZA PZA + RIF PZA + RIF+ INH INH + RIF INH + PZA+ RIF
First-Order Kinetics
Higuchi Kinetics
KP Model
Exponential First Order
R2
k0
R2
k1
R2
kH
R2
n
kp
R2
k
0.9572 0.4872 0.4975 0.9929 0.9841 0.8592 0.5985 0.4139 0.6566 0.6223
0.0893 0.4199 0.4572 0.0182 0.0257 0.0380 0.3999 0.4113 0.4576 0.4645
0.9859 0.8517 0.9542 0.9772 0.9745 0.9983 0.8935 0.8265 0.9838 0.9104
0.0217 0.0192 0.6712 0.0204 0.0210 0.0158 0.0052 0.0043 0.0275 0.0296
0.9899 0.6437 0.6440 0.9855 0.9988 0.9431 0.7443 0.7932 0.8953 0.7647
0.5060 6.8678 7.4718 0.2537 0.3689 0.5862 6.3910 9.3208 9.5365 7.5022
0.9751 0.9334 0.8909 0.9928 0.9971 0.9785 0.9731 0.8975 0.9620 0.9688
0.5776 0.2318 0.0976 0.7664 0.6673 0.2606 0.4892 0.3240 0.4726 0.2707
0.0036 0.3404 0.5940 0.0006 0.0016 0.0199 0.0990 0.2423 0.1407 0.3155
0.9753 0.9914 0.9692 0.9677 0.9867 0.9493 0.9738 0.9956 0.9873 0.9886
0.0106 0.0912 0.1361 0.0071 0.0079 0.0183 0.0509 0.1122 0.0571 0.0523
Table 3. Average Inhibition Zones for Different Combinations of Drugs in Microemulsions (ME) and Their Components (Diameter in cm)
R-ME RI-ME RP-ME RIP-ME P-ME I-ME Ethyl oleate Butanol B96
Concentration Used (g/mL)
E. coli MTCC 40
S. aureus MTCC 2901
A. niger 302
A. Fumigates 1628
C. herbarium 3137
C. lunata 6257
H. oryzae 5559
0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05
16 15 15 14 0.0 0.0 0.0 0.0 0.0
15 15 16 0.0 0.0 0.0 0.0 0.0 0.0
21 18 20 15 0.0 0.0 0.0 0.0 0.0
21 18 20 18 0.0 0.0 0.0 0.0 0.0
18 15 16 17 0.0 0.0 0.0 0.0 0.0
NA NA NA NA NA NA NA NA NA
20 20 23 17 0.0 0.0 0.0 0.0 0.0
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DOI 10.1002/jps.24469
RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology
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Figure 5. Percentage cytotoxicity of pure drug (solid 1 wt %), microemulsion formulation, and drug-loaded microemulsion formulation (1%, wt/wt).
96 microemulsion that undergoes a continuous transition on dilution. Results indicate the presence of RIF’s molecules at the interface toward oil side and INH toward hydrophilic side. On the contrary, PZA mainly remains in the bulk water. FTIR helped in evaluating the head group and OH (water) interactions. Absorption and FT-IR spectroscopic data indicate that entrapment of the studied drugs can also be carried out in different binary or ternary mixtures without any physical or chemical instability. Anti-Tb drugs occupy the same solubilization sites in multiple drug-loaded systems as in the case of single drug-loaded microemulsion systems. Derivative absorbance spectroscopy has been utilized to quantitatively estimate the three drugs in microemulsion system in the presence of each other in combination of two or three. The release behavior shows that INH and PZA in the Brij 96-loaded microemulsion formulations obey the diffusional (Fickian) release mechanism, whereas RIF shows anomalous release. The results obtained in the present report were compared with previous studies carried on with Tween 80 embedded microemulsions and significant difference is obtained in terms of solubilization, release mechanism, and partition coefficients.16 The cytotoxicity has been analyzed using Vero cells and it is found the percentage cytotoxicity increases with increase in the dose concentration. Interestingly, the percentage cytotoxicity is also found to depend on microstructure of the microemulsion. Microbial activity of these drug-loaded formulations has also been assessed using various pathogenic microbes including various bacteria and fungi. The combination drugs in microemulsion formulation gave promising results as far as antimicrobial activities are concerned. The present report advances the utility of microemulsions as vectors for drugs in multiple drug therapy.
DOI 10.1002/jps.24469
ACKNOWLEDGMENTS G. Kaur is thankful to DST, India for INSPIRE faculty award (IFA-12-CH41) and UGC for start-up grant [2031(12)/2012(BSR)]. S.K.M. is thankful to DST India for financial assistance under project scheme (SERB/F/3596/2013-14 dated 09.09.2013).
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8. Mouri A, Diat O, Alain Lerner D, El Ghzaoui A, Ajovalasit A, Dorandeu C, Maurel J, Devoisselle JM, Legrand P. 2014. Water solubilization capacity of pharmaceutical microemulsions based on Peceol, lecithin and ethanol. Int J Pharm 475:324–334. 9. Spernath A, Aserin A. 2006. Microemulsions as carriers for drugs and nutraceuticals. Adv Colloid Interface Sci 47:128–130. 10. Cooney GF, Jeevanandam V, Choudhury S, Feutren G, Mueller EA, Eisen HJ. 1998. Comparative bioavailability of neoral and sandimmune in cardiac transplant recipients over 1 year. Transplant Proc 30:1892– 1894. 11. Porter CJH, Charman WN. 2001. In vitro assessment of oral lipid based formulations. Adv Drug Deliv Rev 50:S127. 12. Lee PJ, Langer R, Shastri VP. 2003. Novel microemulsion enhancer formulation for simultaneous transdermal delivery of hydrophilic and hydrophobic drugs. Pharm Res 20:264–269. 13. Singh S, Mariappan TT, Sankar R, Sarda N, Singh B. 2001. A critical review of the probable reasons for the poor/variable bioavailability of rifampicin from anti-tubercular fixed-dose combination (FDC) products, and the likely solutions to the problem. Int J Pharm 228:5–17. 14. Shishoo CJ, Shah SA, Rathod IS, Savale SS, Vora MJ. 2001. Impaired bioavailability of rifampicin in presence of isoniazid from fixed dose combination (FDC) formulation. Int J Pharm 228:53–67. 15. Agrawal S, Panchagnula R. 2004. Dissolution test as a surrogate for quality evaluation of rifampicin containing fixed dose combination formulations. Int J Pharm 287:97–112. 16. Mehta SK, Kaur G, Bhasin KK. 2010. Entrapment of multiple anti TB drugs in microemulsion system: Quantitative analysis, stability and In vitro release studies. J Pharm Sci 99:1896–1911. 17. Mehta SK, Kaur G, Bhasin KK. 2007. Analysis of Tween based microemulsion in the presence of TB drug rifampicin. Colloids Surf B Biointerfaces 60:95–104. 18. Kaur G. 2014. Assessment of Brij 96 embedded microemulsions as carrier for anti-tuberculosis drug rifampicin. Mat Focus 3:18–22. 19. Kaur G, Chiappisi L, Pr´evost S, Schweins R, Gradzielski M, Mehta SK. 2012. Probing the microstructure of nonionic microemulsions with ethyl oleate by viscosity, ROESY, DLS, SANS, and cyclic voltammetry. Langmuir 28:10640–10652. 20. Omotoshjo A, Whatelety L, Florenac ET. 1989. Methotrexate transport from internal phase of multiple w/o/w emulsion. J Microencapsul 6:183–192. 21. Kumar S, Bhanjana G, Sharma A, Sidhu MC, Dilbaghi N. 2014. Synthesis, characterization and on field evaluation of pesticide loaded sodium alginate nanoparticles. Carbohydr Polym 101:1061–1067. 22. Kumar S, Bhanjana G, Kumar R, Dilbaghi N. 2013. Synthesis, characterization and antimicrobial activity of nano titanium (IV) mixed ligand complex. Mat Focus 2:475–481.
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23. Mehta SK, Kaur G, Mutneja R, Bhasin KK. 2009. Solubilization, microstructure, and thermodynamics of fully dilutable U-type Brij microemulsion. J Colloid Interface Sci 338:542–549. 24. Kaur G, Mehta SK. 2014. Probing location of anti-TB drugs loaded in Brij 96 microemulsions using thermoanalytical and photophysical approach. J Pharm Sci 103:937–944. 25. Lagourette B, Peyrelasse J, Boned C, Clausse M. 1979. Percolative conduction in microemulsion type systems. Nature 281:60–62. 26. Aliotta F, Migliardo P, Donato DI, Liveri VT, Bardez E, Larry B. 1992. Local hydration effects in reversed micellar aggregation. Progr Colloid Polym Sci 89:258–262. 27. Kaler EW, Bennett KE, Davis HT, Scriven LE. 1983. Toward understanding microemulsion microstructure II. J Chem Phys 79:5685. 28. El-Laithy HM. 2003. Preparation and physicochemical characterization of dioctyl sodium sulfosuccinate (aerosol OT) microemulsion for oral drug delivery. AAPS PharmSciTech 4(1):E11. 29. Gursoy A, Senyucel B. 1997. Characterization of ciprofloxacin liposomes: Derivative ultraviolet spectrophotometric determinations. J Microencapsul 14:769–776. 30. Agrawal S, Singh I, Kaur KJ, Bhade SR, Kaul CL, Panchagnula R. 2004. Comparative bioavailability of rifampicin, isoniazid and pyrazinamide from a four drug fixed dose combination with separate formulations at the same dose levels. Int J Pharm 276:41–49. 31. Jurkovˇıc P, Sentjurc M, Gˇasperlin M, Kristl J, Pˇecar S. 2003. Skin protection against ultraviolet induced free radicals with ascorbyl palmitate in microemulsions. Eur J Pharm Biopharm 56:59–66. 32. Kreilgaard M. 2002. Influence of microemulsions on cutaneous drug delivery. Adv Drug Deliv Rev 54:S77–S98. 33. Zhang L, Sun X, Zhang ZR. 2005. An investigation on livertargeting microemulsions of norcantharidin. Drug Deliv 12:289– 295. 34. Dai H, Chen Q, Qin H, Guan Y, Shen D, Hua Y, Tang Y, Xu J. 2006. A temperature-responsive copolymer hydrogel in controlled drug delivery. Macromolecules 39:6584–6589. 35. Eisenbrand G, Pool-Zobel B, Balls M, Blaauboer BJ, Boobis A, Carere A, Kevekordes S, Lhuguenot JC, Pieters R, Kleiner J. 2002. Methods of in vitro toxicology. Food Chem Toxicol 40:193–236. 36. Warisnoicharoen W, Lansley AB, Lawrence MJ. 2003. Toxicological evaluation of mixtures of nonionic surfactants, alone and in combination with oil. J Pharm Sci 92:859–868. 37. Finch RG, Greenwood D, Norrby SR, Whitley RJ. 2000. Antibiotic and chemotherapy—Anti-infective agents and their use in therapy. 9th ed. Saunders Elsevier, Edinburgh, New York. 38. Fu LM, Liu CSF. 2002. Is Mycobacterium tuberculosis a closer relative to Gram-positive or Gram-negative bacterial pathogens? Tuberculosis 82:85–90.
DOI 10.1002/jps.24469
Coencapsulation of Hydrophobic and Hydrophilic Antituberculosis Drugs in Synergistic Brij 96 Microemulsions: A Biophysical Characterization GURPREET KAUR,1 S.K. MEHTA,1 SANDEEP KUMAR,2 GAURAV BHANJANA,2 NEERAJ DILBAGHI2 1 2
Department of Chemistry and Centre of Advanced Studies in Chemistry, Panjab University, Chandigarh 160014, Punjab, India Department of Bio and Nano Technology, Guru Jambheshwar University of Science and Technology, Hisar 125001, Haryana, India
Received 21 January 2015; revised 1 April 2015; accepted 2 April 2015 Published online 7 May 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.24469 ABSTRACT: A microemulsion has been formulated to coencapsulate antituberculosis drugs to solve the issue of stability of rifampicin (RIF) in the presence of isoniazid (INH) and pyrazinamide (PZA). The structural transition, solubilization locus, and quantitative release of drugs without interference have been estimated. Derivative absorbance spectroscopy, especially ratio derivative and double divisor ratio derivative methods, has been employed for estimating the release. The coencapsulation of the anti-tuberculosis drugs were carried out in single, binary, or ternary mixtures and occupy the same solubilization sites in multiple drugs microemulsion systems as in the case of single drug-loaded systems. INH and PZA obey the diffusional (Fickian) release mechanism, whereas RIF shows anomalous release. Resazurin assay and agar well diffusion method were adopted for cytotoxicity analysis and antimicrobial activity, respectively. Cytotoxicity was C 2015 Wiley Periodicals, Inc. and the American found to be dependent on concentration and on colloidal structure of microemulsion. Pharmacists Association J Pharm Sci 104:2203–2212, 2015 Keywords: nonionic microemulsions; anti-TB drugs; stability; in vitro release; Brij 96; derivative absorbance spectroscopy; UV-visible spectroscopy; solubility; toxicity; preformulation
INTRODUCTION Colloidal dispersions of oil and water represent an interesting prospect for the development of formulations for use as vehicles to control the release of drugs to the human body. Many forms of colloidal assemblies, namely, SMEEDS (self-microemulsifying drug delivery systems), microemulsions, emulsions, liquid crystals, micelles, liposomes, niosomes, and so on, have been evaluated for delivery of drugs.1,2 Taking about microemulsions, they may behave differently when loaded with drugs, diluted in in vivo conditions and also show dependence on their colloidal structure.3–6 Many microemulsion formulations, particularly SMEDDS, are presently used in several commercial formulations (cyclosporine A, ritonavir, saquinariz).7 The research in microemulsions is focused on control of particle size and surface properties for specific applications, development of core shell particles, drug delivery systems, and their uses in biomedical applications.8–11 However, rare literature is available for coencapsulation of multiple drugs and their simultaneous delivery.12 Multiple drug therapy is widely used for the treatment of tuberculosis (TB). The first line drugs used are rifampicin (RIF), isoniazid (INH), and pyrazinamide (PZA).13 RIF is a borderline class II drug of Biopharmaceutical Classification System (BCS); however, INH and PZA are hydrophilic drugs. In fixed dose combinations, INH onsets the degradation of RIF (in acidic medium) and hydrolyzes it to insoluble 3-formyl rifamycin Correspondence to: Gurpreet Kaur (Telephone: +91-9872800434; Fax: +91172-2545074; E-mail: [email protected]) This article contains supplementary material available from the authors upon request or via the Internet at http://wileylibrary.com. Journal of Pharmaceutical Sciences, Vol. 104, 2203–2212 (2015) C 2015 Wiley Periodicals, Inc. and the American Pharmacists Association
SV.14 Other factors that are believed to affect the bioavailability of drug are particle size, crystallinity, excipients, and so on.15 The idea is to explore the advantage of internal structure of the microemulsions for cosolubilization of hydrophobic and hydrophilic drugs. The work has been carried out to make the factors explicit that will affect microemulsions efficiency as carrier for multiple drugs. The formulations composed of polyoxyethylene 10 oleoyl ether (Brij 96) using ethyl oleate (pharmaceutical accepted oil) have been prepared and assessed as a carrier for anti-TB drugs [i.e., RIF, INH, and PZA; (Fig. S1) in single and multiple drugs combinations]. Similar studies have been reported earlier using Tween 80/oleic acid/ethanol/phosphate buffer (pH 7.4).16 The idea here is to use another nonionic surfactant-based microemulsion system, that is, Brij 96/ethyl oleate/butanol/water. Main difference between the chosen microemulsions is the solubility of RIF. In Tween-based system, RIF is more soluble in oleic acid than Tween 8017 ; however, in present case, RIF is more soluble in Brij 96 than ethyl oleate.18 Moreover, higher solubility of RIF is observed in Brij 96 in comparison to Tween 80. The characterization and stability have been verified by using various physicochemical, spectroscopic methods, and cytotoxicity analysis. The release of drugs both as single and multiple formulations has been carried out in in vitro conditions. The order of the release kinetics (zero order, first order, or Higuchi) has also been determined by ratio derivative and double divisor ratio derivative method16 for estimating concentrations in binary or ternary mixtures, respectively. Further, to access the idea of efficacy of these formulations carrying drug in variable combinations, antimicrobial studies have been carried out using gram-positive bacteria, gram-negative bacteria, and fungal strains.
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MATERIALS AND METHODS Materials Brij 96 was purchased from Fluka. Butanol (purity >99.8%) was obtained from Spectrochem, Maharashtra, INDIA. Ethyl oleate (EO), RIF (purity >98.0%), PZA (purity >98.0%), and INH (purity >98.0%) were supplied by Sigma (MO, USA). Resazurin sodium, nutrient agar, and potato dextrose agar were purchased from HiMedia, Mumbai India. The chemicals were used as received. Doubly distilled water (specific conductance, 2–4 :Scm−1 at 30 ± 0.01°C) was used for all the preparations. Microemulsion Preparation and Phase Behavior The microemulsion consisting of oil (ethyl oleate), surfactant (Brij 96), cosurfactant (butanol), and double distilled water with constant surfactant–cosurfactant mass ratio (Km 1.5) was formulated. For sample preparation, the oil was mixed to the surfactant–cosurfactant mixture followed by the addition of required amount of water to obtain the desired microemulsion compositions. The elucidation of the microstructure was carried out along a dilution line of constant oil–surfactant mixing ratio of 1.2:2 by weight, using phase behavior mapping of microemulsions as reported previously.19 Drug Incorporation in Microemulsion For the preparation of drug-loaded microemulsion in single, binary (RIF+INH and RIF+PZA), or ternary (RIF+INH+PZA) formulations, RIF was dissolved in lipophilic phase, and other two drugs were dissolved in hydrophilic component, followed by the addition of remaining components of the microemulsion. Each drug was 1% (wt) of the total weight of microemulsion. Conductivity Measurements The electric conductivity (F) was measured by means of a PICO digital conductivity meter (Labindia Instruments) operating at 50 Hz, maintained by a RE320 Ecoline thermostat at 30 ± 0.01°C. The error limit of conductance measurements was ±3%. The conductivity of selected and drug-loaded microemulsions was measured as a function of T. Viscosity Measurements Viscosity measurements for pure and drug-loaded microemulsions were carried out using a Brookfield DV viscometer (model DV-II+; Brookfield Engineering Laboratories, MA, U.S.A.) with a SC4-DIN-82 spindle (shear rate = 1.29N) and SC4-13RPY chamber (w/RTD temperature probe and cable) while maintaining the speed at 200 rpm. The experiment was repeated (in duplicate) at 30°C, with accuracy up to ±1%. Fourier Transform Infrared Fourier transform infrared (FT-IR) spectra of pure microemulsions and of the drug-loaded microemulsion (1% wt drug) were recorded on Perkin-Elmer (RX1) FT-IR spectrometer using AgCl plates, in the frequency range of 4000–350 cm−1 with 100 number scans and 4 cm−1 spectral resolution. Dilutability and Particle Size Measurements The dilutability of the microemulsions was assessed to know whether these systems would be diluted with the aqueous phase of the system without separation or not. For this purpose, selected and drug-loaded microemulsions were diluted with waKaur et al., JOURNAL OF PHARMACEUTICAL SCIENCES 104:2203–2212, 2015
ter and their transparency was assessed visually. The particle size measurements (at infinite dilution) were made using laser diffraction analyzer [CILAS 1180 liquid range (0.04–2500 :m)]. Absorbance Spectroscopy UV-visible spectra were recorded over the wavelength range of 200–600 nm using JASCO-530 UV-visible spectrophotometer. RIF was varied in the absence and presence of INH and PZA, for this three sets of experiments were performed. Spectra of RIF loaded in microemulsion were recorded against empty microemulsion to rule out the interference of components. Sample Preparation 1. Stability test of RIF in the presence of other drugs: three sets of six standard solutions each containing 3.0 g microemulsion loaded with RIF (varying concentration, 0.01–0.04 mM in microemulsion) were prepared. INH and PZA were coencapsulated with RIF at a constant concentration of 0.23 and 0.25 mM, respectively. 2. Stability test of RIF with time: 6.0 g microemulsion was prepared containing 1% (wt %) of each drug (both in single and fixed combinations). 0.1 g of each drug-loaded microemulsion were transferred into 10 mL volumetric flask and diluted to volume with ethanol. The absorbance of RIF (in single, binary, or ternary mixture formulations) was analyzed using spectrophotometer initially for 2 h and thereafter for 20 days. Samples were kept at room temperature (25°C). In Vitro Release Release Behavior and Kinetics The dialysis bag method20 using cellulose tubing [average flat width 33 mm (1.3 in)] was followed to determine the in vitro release. For the purpose, 1.0 g microemulsion containing 1% (wt) of drug-loaded microemulsion formulation (single, binary or ternary mixtures) was introduced in 50 mL of phosphate buffer (PB) (pH 7.4) maintained at 37 ± 0.1°C in a release cell. The system was stirred at constant speed of 100 rpm. The aliquot of 2 mL was withdrawn at regular intervals for 5 h (i.e., aliquot was taken at 2, 5, 10, 15, 20, 30, 45, 60, 90, 120, 150, 180, 210, 240, 270, and 300 min) and was filtered through 0.45:m membrane. Each time the same amount of fresh phosphate buffer (kept at the same temperature) was added to maintain the volume of the release medium. The drug concentrations were determined spectrophotometrically at their characteristic wavelengths (i.e., RIF 475 nm, INH 262 nm, and PZA 268 nm) for single loaded formulations. The ratio derivative and double divisor ratio derivative method were used for determination of concentrations in binary or ternary mixtures, respectively.16 Drug release was determined by two parameters, first cumulative percent drug released after 5 h and second the release rate of drug expressed as slope of the linear regression line. The order of the release kinetics was determined by comparing linear regression line for different rate equations. All the experiments were performed in triplicate and SD is ±2%. Cytotoxicity Analysis Microemulsion loaded with anti-TB drugs (1%, wt/wt) were screened for cytotoxicity analysis at two different concentrations and were compared with solid (API) in (1%, wt/wt) using DOI 10.1002/jps.24469
RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology
Resazurin assay.21 Vero cells at a concentration of 1 × 104 cells per well were cultured in a 100-:L volume of EMEM media containing 5% fetal bovine serum in a 96-well cell culture plate. The experiment was performed in triplicate. Cells were allowed to incubate for proliferation at 37°C in CO2 (5%) incubator for 24 h. Water was used as solvent except for RIF, which is soluble in dimethyl sulfoxide. Cells were further treated followed by incubation for 24 h with 20 and 40 :L of solutions of pure drugs, single drug-loaded Brij 96 microemulsions, and multiple drug-loaded Brij 96 microemulsions. Other constituents such as butanol, ethyl oleate, and Brij 96 were also evaluated for toxicity analysis using same volumes. Untreated cells were taken as reference. Background coloration was corrected with the help of media without cells. After incubation, plate was removed from incubator and 10 :L of resazurin solution prepared in EMEM media was added in all wells and incubated for 4 h. After 4 h, the pink-colored resorufin is formed and absorbance was taken by spectrophotometer (ELISA plate reader; BMG Labtech, (Ortenberg) Germany ) at wavelength of 573 nm. Cytotoxicity percentage was calculated with reference to untreated cells after normalizing the background coloration of media. Cytotoxicity (%) =
Absu − Abst ×100 Absu
(1)
where Absu is the absorbance of cells not treated with drug formulations, and Abst is the absorbance of cells treated with formulated drug-loaded microemulsions.
surements have been used to elucidate the solubilization loci of these studied drugs in Brij 96 microemulsions, which were correlated with partition coefficient studies.24 Conductivity In this work, the stability and structural changes in microstructure of microemulsions are assessed in the presence of both hydrophobic and hydrophilic anti-TB drugs in single as well as multiple combinations. In principle, microemulsion undergoes an intriguing phenomenon of electric percolation and this increase in conductivity is related to clustering of the microemulsion droplets and migration of charges within the aggregates.25 The variation of electric conductivity of pure and drug-loaded microemulsion (1.0, wt %) as a function of T (water to surfactant) is represented in Figure 1a. The drug incorporation does not affect the microstructure of the microemulsion, it only initiates the early onset of the process of percolation (at lower T) than in pure system. However, a dip in the conductivity has been seen for RIF-loaded microemulsion at higher T = 16 (Fig. 1a), which helps in locating the position of drug.18 On the contrary, the two hydrophilic drugs INH and PZA show increment in the conductivity values from the region where the transition of swollen micelles into short cylinders have just begun (T 4). A significant change is observed in the bicontinuous region. Taking into account the energetics of clustering of the droplets, in different microemulsion systems, Gocl (standard free energy of clustering) is obtained from the relation: Gocl = RT ln X d
Antimicrobial Studies Antimicrobial studies were carried out using growth inhibitory zone well method against pathogenic microorganisms,22 that is, a gram-positive (Escherichia coli) and a gram-negative (Staphylococcus aureus) bacteria and fungus strains, namely, Aspergillus niger, Aspergillus fumigatus, Cladosporium herbarium, Curvularia lunata, and Helminthosporium oryzae. Bacteria were cultured on nutrient agar and fungi were cultured on potato dextrose agar media. One-hundred microliter of test samples (concentration 0.05 g/mL) was added to 6 mm well bored on agar plates after homogeneous spreading of freshly prepared broth of various microbial stains. After incubation of 24 h for bacteria and 48 h for fungus at 37°C and 25°C, respectively, diameters of inhibition zones were measured and average value of triplicate measurement is reported. Diameter of the well was excluded from inhibition zone diameter.
RESULTS AND DISCUSSION The detailed investigation of microstructural changes of Brij 96 microemulsions on dilution with dispersion media has been carried out in our previous results.18,19,23,24 It has been found that this nonionic microemulsion undergo a microstructural change from w/o to o/w via bicontinuous phase upon dilution at T = 7.5 and 13.3, respectively. The effect of change of oil, cosurfactant (alcohols), phase behavior, water solubilization, and states of water of prepared Brij 96 microemulsion has been thoroughly investigated.23 Keeping in view the significance of microemulsions as drug delivery vehicles, these prepared compositions have been analyzed as drug carriers for anti-TB drugs.18,24 Differential scanning calorimetry (DSC) and fluorescence meaDOI 10.1002/jps.24469
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(2)
where Xd is mole fraction of droplets at percolation threshold and defined as Xd = nd /(nd +noil ), where nd and noil are the number of moles of droplet and oil, respectively. The calculated standard free energy of clustering Gocl using Eq. (2) shows a value of −48.80 kJ/mol for pure system and −48.8, −46.04, and −42.97 kJ/mol for INH, RIF, and PZA drug-loaded microemulsions, respectively. The data support the spontaneity of the droplet clustering. In case of multiple drugs cosolubilized in microemulsions, the conductivity profile reveals that in the w/o region, the presence of RIF+PZA, RIF+INH, and the combination of three drugs do not have any effect on the microstructure. However, binary mixtures show diminished conductance in o/w region, whereas ternary mixture displays high conductance. Viscosity The viscosity measurements of drug-loaded and drug-unloaded microemulsions are depicted in Figure 1b. The general trend in the viscosity remains similar for both empty and drug-loaded samples. In w/o type region, the magnitude of viscosity is nearly same in all the systems. However, as the dilution progress and system reverts to bicontinuous phase, a significant enhancement in the viscosity takes place. The measurements give a clear indication of interactions between the drug molecules and microemulsion components. The RIF-loaded Brij microemulsions display higher viscosity values in bicontinuous and o/w region.18 Similar trend is observed for INH. However, PZAincorporated microemulsion do not show any change in the viscosity values. This gives an inference that PZA mainly resides in the bulk water phase throughout the dilution. The viscosity results of binary and ternary mixtures of drug-loaded Kaur et al., JOURNAL OF PHARMACEUTICAL SCIENCES 104:2203–2212, 2015
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Figure 1. Comparison of (a) conductivity and (b) viscosity of Brij 96 and drug-loaded microemulsions as a function of T single and multiple drug formulations.
microemulsions reveal that in w/o or swollen reverse micelle region, the drug incorporation in the microemulsion does not create any significant effect on the structure. However, increment has been observed in the bicontinuous region for binary (RIF+INH) and ternary mixture while viscosity decreases for RIF+PZA. Fourier Transform Infrared We previously reported23,24 an in-depth analysis of states of water and effect of dilution on Brij 96 microemulsions. Here, the main emphasis has been laid on the OH band, as it is indicative of water and surfactant head group’s interactions and influence of drugs on it. Table 1 depicts the wavenumber of OH of empty and drug-loaded Brij 96 microemulsions. The higher frequency than pure water (3400 cm−1 ) indicates weak interaction of surfactant molecules with that of water.26 As long as water molecules are present in w/o or bicontinuous type microemulsion, bands are observed at higher wavenumber. However, as soon as water becomes the dispersion phase, the band appears at the same position as that of bulk water. In w/o type region, a shift of up to 4, 12, and 20 cm−1 in the presence of RIF, PZA, and INH, respectively, has been obTable 1. Infrared Frequencies of Various Weight Fractions of Aqueous Phase in Anti-TB Drug-Loaded Brij 96 Microemulsions wt Fraction/T
Structure
8.5/1.3 w/o 27.0/5.40 Bicontinuous 39.6/9.50 Bicontinuous 48.4/15.00 o/w 58.0/20.00 o/w 63.2/25.00 o/w
Wavenumber RIF PZA INH (OH) (1 wt %) (1 wt %) (1 wt %) 3432 3431 3424 3400 3400 3400
3428 3412 3416 3429 3430 3467
3420 3420 3412 3406 3421 –
3412 3416 3392 3409 3412 3411
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served. As RIF is mainly believed to be present in the oil region, therefore, it shows such a weak influence on the water surfactant interaction. On the contrary, the presence of INH and PZA strengthens the interaction between water and surfactant head group by being present nearby. In bicontinuous region, a great deal of difference is found between empty and all three drug-loaded microemulsions. However, trend is similar for hydrophilic as well as hydrophobic drugs. This is probably because in such type of microemulsions, zero curvature is found27 and almost the same amount of water and oil coexist. Therefore, most likely, the comparable amounts of drugs are available. A contrast has been observed once the microemulsion system reverts into o/w type. The presence of drugs enhances the wavenumber in comparison to that of pure system. It has been observed that as the dilution of o/w region occurs, the presence of drug weakens the interaction of water and the surfactant head groups. As RIF is hydrophobic in nature and prefers to stay near hydrophobic environment (i.e. oil phase and hydrophobic tails of the surfactant), it might interact with the surfactant hydrophobic tails and also interfere with the surfactant and water interactions. On the contrary, the enhancement of the wavenumber in INH- and PZA-loaded microemulsions in the o/w region leads to the fact that these drugs are probably interacting with the bound water, as both drugs are hydrophilic in nature. The partition coefficient results, DSC and fluorescence measurement,24 support this observation. Thus, we can firmly comment on the location of these drugs in Brij 96 microemulsion: RIF is present at interphase toward oil, INH in bound water, and PZA in free water.18,24 Conductivity and viscosity measurements also support the observation. In order to observe the concentration effect, the FT-IR spectra for drug-loaded microemulsions have been carried out with 0.5 and 1 wt % of drugs. However, change in the concentration of all the drugs does not show any effect. DOI 10.1002/jps.24469
RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology
Dilutability and Particle Size Analyses The droplet size of the microemulsions has been measured after addition of 250 mL water to 1 g of mixture. No further treatment of the samples was necessary. From physicochemical and other solubility studies, it may be concluded that the Brij 96 microemulsions are stable up to N(wt %) = 68. It is also believed that on dilution, the microemulsion turns into a emulsion, that is, o/w (heterogeneous coarse dispersion).28 In comparison to the particle size of 0.17 :m for pure microemulsion, the addition of drugs increases the particle size in the order of 12, 2.81, and 0.27 :m in the presence of RIF, INH, and PZA, respectively (Fig. S2). Interaction of INH with the bound water (water molecules with surfactant molecules) is the probable reason behind the increase in the size of o/w emulsion droplet. PZA prefers to be in the free water phase and does not influence the particle size. However, the RIF-loaded system shows the highest increase by being present at the core of the droplet. Stability Analysis In this study of single and multiple drugs loaded in Brij microemulsions, the stability of RIF in the presence of drugs (INH and PZA) and with span of time is analyzed using UV-visible spectroscopy.29 The UV-visible spectra of RIF in combination with other drugs in the formulated drug-loaded microemulsion system are shown in Figure 2. The slopes of the working curves of RIF in single or in binary mixture (RIF + constant INH and RIF + constant PZA)-loaded microemulsion system do not illustrate any significant difference (Table S1). Figure S3 shows that single, binary, or ternary mixture-loaded microemulsion formulation show maxima at 8max = 480 nm for RIF. The presence of other two hydrophilic drugs, in all the three mixed drug formulations, does not produce any change in 8max of RIF, neither any other peak has emerged in the UV-visible spectra. This demonstrates the stability of RIF in all the drug-loaded microemulsions. The stability study has been observed for 20 days, to assess the stability of RIF. For single (RIF) and binary (RIF + PZA) formulations, the maxima has been obtained at 8max = 480 nm as shown in Figure 3 (near the characteristic peak of RIF 475 nm). After day 1, 8max for binary (RIF + INH) and ternary (RIF + PZA + INH) formulations produce a red shift (487 nm) that is probably indicative of the formation of 3-formyl RIF SV that gives a characteristic peak at 492 nm because of the hydrolysis of RIF in the presence of INH.30 Following days show the emergence of a new peak near 425 nm for both the formulations. However, quantitative analysis in terms of rate and extent of degradation is still in progress. Release Behavior In vitro release studies with artificial cellulose membrane provide information about the diffusion of the drug, which depends on vehicle internal structure, diffusion of the drug inside the carrier system, and pH of the water phase.31,32 Anti-Tb drug release through an artificial membrane has been characterized by two parameters: the amount of drug released after 5 h and the rate of release of the drug. In order to investigate the mode of release from the microemulsion system, the release data have been analyzed with different mathematical models: zero-order kinetic, first-order kinetic, Higuchi kinetics, Korsmeyer and Peppas equation, and first-order exponential DOI 10.1002/jps.24469
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(Eqs. (3)–(7). Q = k0 t
(3)
ln Q = ln Q 0 − k1 t
(4)
Q = kH t1/2
(5)
Mt = kp tn M
(6)
Q = Q 0 − be−kt
(7)
where Q is the cumulative percent of drug released at time t, Mt /M is the fraction of drug released at time t, and k0, k1, kH , kp , and k are the rate constants for respective models. Q0 and b in Eqs. (4) and (7) are constants. Comparing the release profile of the anti-TB drugs from the Brij 96 microemulsion at pH 7.4 depicts that the fastest release has been for the hydrophilic drugs, that is, INH and PZA, with almost 80% release has been observed in first half an hour (Fig. 4). However, RIF shows a slow diffusion of drug because of its high affinity with the hydrophobic components. Therefore, only 8% release is seen in first 3 h.18 Table 2 represents the rate constants and correlation coefficient values estimated using Eqs. (3)–(7). The best agreement of the fitting has been attained with the equation of first-order exponential decay. The cumulative percent of drug released versus time t (Fig. 4) depicts the controlled release of drugs.33,34 Using Korsmeyer and Peppas equation i.e. Eq. (6), the values of n for INH and PZA in the drug-loaded microemulsion formulations indicate a diffusional (Fickian) release mechanism. On the contrary, RIF shows anomalous release from the single drug formulation. The comparative release behavior of anti-TB drugs loaded in the Brij 96 microemulsions in different binary and ternary shows that the extent of release of RIF is maximum when all the three drugs are present as ternary mixture. The release is retarded in the case of binary mixtures, both in the presence of PZA and INH (Fig. 4b). The release profile of INH in single and ternary mixture-loaded formulation is nearly same, but decrease in the extent of release has been observed for INH in the presence of RIF (as compared with single drug formulation) (Fig. 4b). The PZA release profile does not show any considerable effect on its release behavior in the presence of binary or ternary mixtures in the formulation (Fig. 4c). From the correlation constants, it has been evaluated that RIF in binary mixtures obeys zero and Higuchi kinetics in the presence of PZA and INH, respectively. In ternary mixture, however, the release of RIF follows first-order kinetics. In case of INH and PZA, exponential first order gives best fit for both the binary and ternary mixtures. As far as the release mechanism of INH and PZA from mixture-loaded microemulsions is concerned, they follow Fickian mechanism. To conclude, it is appropriate to articulate that solubilization loci of drugs especially in binary and ternary combinations play some role as far as release rates are concerned. However, mechanism of drug release is not greatly affected. Kaur et al., JOURNAL OF PHARMACEUTICAL SCIENCES 104:2203–2212, 2015
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Figure 2. UV-visible spectra of selected microemulsion (ME) containing (a) RIF, (b) RIF coencapsulated with INH (0.28 mM), and (c) RIF coencapsulated with PZA (0.26 mM). The inset shows corresponding regression line of each plot.
In contrast to Tween 80 microemulsions, Brij 96 microemulsions show high dilutability because of the most appropriate choice of cosurfactant. Moreover, in the Brij 96 microemulsions, PZA prefers to be in bulk water phase and INH near the palisade layer or trapped water region, which is opposite to Tween 80 microemulsions. Furthermore, both the microemulsion systems give different results as far as release mechanism of these drugs is concerned. Hydrophilic drugs show non-Fickian release and hydrophobic drugs shows Fickian release from Tween 80 embedded microemulsions, whereas reverse is true for Brij 96 embedded microemulsions. The probable reasons behind the different release behavior is because of difference in solubilization loci of these drugs due to disparity in the interface backbone, HLB value of the surfactants, and partition coefficient of drugs in oil/surfactant. Cytotoxicity Analysis In vitro cytotoxicity assays are useful to define basal cytotoxicity and are also necessary to define the concentration range for further and more detailed in vitro testing of any formulation to provide meaningful information on parameters such as genotoxicity or programmed cell death.35 The possible cytotoxic effect of microemulsions (both drug loaded and empty) and its components are evaluated on Vero cells treated for 24 h at different concentrations. The toxic effect of microemulsions and its components are dose-dependent, and percentage cytotoxicity increases with increase in concentration. Maximum cytotoxicity is found for ethyl oleate and minimum for Brij 96. In comparison to pure components, microemulsions show less percentage cytotoxicity. Three compositions, one from each Kaur et al., JOURNAL OF PHARMACEUTICAL SCIENCES 104:2203–2212, 2015
region, that is, w/o (ME 1), bicontinuous (ME 2), and o/w (ME 2) are selected. Interestingly, minimum cytotoxicity is observed for bicontinuous microemulsion (ME 2). Warisnoicharoen et al.36 have investigated the toxicity of nonionic surfactant including Brij 96 and its combination with various oils to human bronchial (16-HBE14o-) epithelium cells. They have correlated various factors such as aggregation concentration, hydrophobicity, and so on, which leads to damage of the cell membrane. Among other results, they revealed that Brij 96 in combination with a high-molecular-volume oil (e.g., ethyl oleate, Miglyol 812, or soybean oil) are toxic only at concentrations significantly greater than their critical aggregation concentration. Authors also suggested that surfactant toxicity is mediated by the aggregated form of the surfactant that solubilizes components of the cell membrane. In the present study, if the structure of the microemulsion is related to the toxicity, it follows the order of o/w> w/o> bicontinuous (Fig. 5a), which can be very well corelated with the high solubilizing capacity of membrane components by o/w droplets. As far as bicontinuous structure is concerned, it has zero curvature with oil and water trapped in channels and Brij 96 at the boundary, thus protecting the cell from damage caused by oil and other components. The cytotoxicity analysis of drug-loaded microemulsion both single and multiple formulations are also made. The comparison is made among pure drug (solid 1 wt %), microemulsion formulation, and drug-loaded microemulsion formulation (1 wt/wt %). For INH, IC50 is 20 :L and effect is found to be dose dependent (Fig. 5b). The drug-loaded formulations show almost comparable effects as that of vehicle. Similar trend is observed for PZA and its formulations (Fig. 5c). For the hydrophobic drug, RIF, the pure drug shows cytotoxicity of 70%; however, all DOI 10.1002/jps.24469
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Figure 3. UV-visible spectra of time study of selected microemulsion (ME) containing (a) RIF, (b) RIF + INH, (c) RIF + PZA, and (d) ternary mixture-loaded Brij 96 ME formulations.
RIF-loaded (single and multiple drugs) formulations show decrease in toxicity effect in comparison to the pure drug. Antimicrobial Activity As far as antimicrobial activity of the studied drugs is concerned, RIF has potent activity against gram-positive bacteria; on the contrary, gram-negative bacteria are generally less sensitive.37 Although the used drugs are popular as first line drugs for tuberculosis; however, RIF is a broad spectrum antibiotic. It is therefore important to evaluate these drugs in single and mixed drug microemulsion formulations for their action against microbes. INH and PZA have high bactericidal activity against Mycobacterium tuberculosis; however, other mycobacteria are resistant to both the drugs except for some strains of M. xenopi and M. bovis, respectively. The phylogenetic position of M. tuberculosis relative to other bacteria is controversial. Its cell wall has characteristics of both gram-positive and gram-negative bacteria.38 Therefore, both gram-positive and gram-negative strains, that is, E. coli and S. aureus are selected, respectively. For the purpose, different fungus strains, namely, A. niger, A. fumigatus, C. herbarium, C. lunata, and H. oryzae are also selected to evaluate antifungal activity of these microemulsion loaded with drugs (Table 3). A dose of 0.05 g/mL is selected; pure components, INH, and PZA DOI 10.1002/jps.24469
did not show activity against any bacterial and fungal strains. However, RIF encapsulated in microemulsions is active against microbes except for C. lunata. Activity of RIF loaded in microemulsion is evaluated in the presence of other drugs, too. As expected, RIF microemulsion showed high activity against E. coli, and its activity remains same even in the presence of INH and PZA. However, ternary mixture of drug-loaded microemulsion show reduced inhibition zone and S. aureusis is totally resistant to RIP microemulsion. Single RIF-loaded microemulsion show highest activity for fungal strains A. niger, A. fumigatus, and C. herbarium. Reduced activity is observed for INH (binary) and ternary drugs loaded in microemulsion. For H. oryzae, maximum zone of inhibition is found for RP mixture and single RIF.
CONCLUSIONS Microemulsions are very successful as carrier for different types of drugs (hydrophobhic, hydrophicic, and amphiphilic).1–3 However, this work reports coencapsulation of hydrophobic and hydrophilic first line anti-Tb drugs in single, binary, and ternary mixtures using Brij 96-based microemulsion. The conductivity and viscosity measurements have been helpful in elucidating the structural transition of anti-Tb drug-loaded Brij Kaur et al., JOURNAL OF PHARMACEUTICAL SCIENCES 104:2203–2212, 2015
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Figure 4. Release profiles of various single and multiple combinations of drugs from prepared microemulsions (n = 3, SD = ±2%) where: (a) release of single drug from microemulsion, (b) RIF, (c) INH, and (d) PZA in various combinations, respectively. Table 2. Rate Constants (k) and Regression Correlations (R2 ) Using Rate Eqs. (3)–(7) for the Release of Drugs from Selected Microemulsions Zero-Order Kinetics Drugs RIF PZA INH RIF + PZA RIF+ INH RIF+ INH + PZA PZA + RIF PZA + RIF+ INH INH + RIF INH + PZA+ RIF
First-Order Kinetics
Higuchi Kinetics
KP Model
Exponential First Order
R2
k0
R2
k1
R2
kH
R2
n
kp
R2
k
0.9572 0.4872 0.4975 0.9929 0.9841 0.8592 0.5985 0.4139 0.6566 0.6223
0.0893 0.4199 0.4572 0.0182 0.0257 0.0380 0.3999 0.4113 0.4576 0.4645
0.9859 0.8517 0.9542 0.9772 0.9745 0.9983 0.8935 0.8265 0.9838 0.9104
0.0217 0.0192 0.6712 0.0204 0.0210 0.0158 0.0052 0.0043 0.0275 0.0296
0.9899 0.6437 0.6440 0.9855 0.9988 0.9431 0.7443 0.7932 0.8953 0.7647
0.5060 6.8678 7.4718 0.2537 0.3689 0.5862 6.3910 9.3208 9.5365 7.5022
0.9751 0.9334 0.8909 0.9928 0.9971 0.9785 0.9731 0.8975 0.9620 0.9688
0.5776 0.2318 0.0976 0.7664 0.6673 0.2606 0.4892 0.3240 0.4726 0.2707
0.0036 0.3404 0.5940 0.0006 0.0016 0.0199 0.0990 0.2423 0.1407 0.3155
0.9753 0.9914 0.9692 0.9677 0.9867 0.9493 0.9738 0.9956 0.9873 0.9886
0.0106 0.0912 0.1361 0.0071 0.0079 0.0183 0.0509 0.1122 0.0571 0.0523
Table 3. Average Inhibition Zones for Different Combinations of Drugs in Microemulsions (ME) and Their Components (Diameter in cm)
R-ME RI-ME RP-ME RIP-ME P-ME I-ME Ethyl oleate Butanol B96
Concentration Used (g/mL)
E. coli MTCC 40
S. aureus MTCC 2901
A. niger 302
A. Fumigates 1628
C. herbarium 3137
C. lunata 6257
H. oryzae 5559
0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05
16 15 15 14 0.0 0.0 0.0 0.0 0.0
15 15 16 0.0 0.0 0.0 0.0 0.0 0.0
21 18 20 15 0.0 0.0 0.0 0.0 0.0
21 18 20 18 0.0 0.0 0.0 0.0 0.0
18 15 16 17 0.0 0.0 0.0 0.0 0.0
NA NA NA NA NA NA NA NA NA
20 20 23 17 0.0 0.0 0.0 0.0 0.0
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Figure 5. Percentage cytotoxicity of pure drug (solid 1 wt %), microemulsion formulation, and drug-loaded microemulsion formulation (1%, wt/wt).
96 microemulsion that undergoes a continuous transition on dilution. Results indicate the presence of RIF’s molecules at the interface toward oil side and INH toward hydrophilic side. On the contrary, PZA mainly remains in the bulk water. FTIR helped in evaluating the head group and OH (water) interactions. Absorption and FT-IR spectroscopic data indicate that entrapment of the studied drugs can also be carried out in different binary or ternary mixtures without any physical or chemical instability. Anti-Tb drugs occupy the same solubilization sites in multiple drug-loaded systems as in the case of single drug-loaded microemulsion systems. Derivative absorbance spectroscopy has been utilized to quantitatively estimate the three drugs in microemulsion system in the presence of each other in combination of two or three. The release behavior shows that INH and PZA in the Brij 96-loaded microemulsion formulations obey the diffusional (Fickian) release mechanism, whereas RIF shows anomalous release. The results obtained in the present report were compared with previous studies carried on with Tween 80 embedded microemulsions and significant difference is obtained in terms of solubilization, release mechanism, and partition coefficients.16 The cytotoxicity has been analyzed using Vero cells and it is found the percentage cytotoxicity increases with increase in the dose concentration. Interestingly, the percentage cytotoxicity is also found to depend on microstructure of the microemulsion. Microbial activity of these drug-loaded formulations has also been assessed using various pathogenic microbes including various bacteria and fungi. The combination drugs in microemulsion formulation gave promising results as far as antimicrobial activities are concerned. The present report advances the utility of microemulsions as vectors for drugs in multiple drug therapy.
DOI 10.1002/jps.24469
ACKNOWLEDGMENTS G. Kaur is thankful to DST, India for INSPIRE faculty award (IFA-12-CH41) and UGC for start-up grant [2031(12)/2012(BSR)]. S.K.M. is thankful to DST India for financial assistance under project scheme (SERB/F/3596/2013-14 dated 09.09.2013).
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DOI 10.1002/jps.24469
