Journal of Scientific & Industrial Research Vol. 74, February 2015, pp. 88-92
Nanoemulsion based Hydrogels of Itraconazole for Transdermal Drug Delivery S Sampathi1*, S K Mankala2, J Wankar1 and S Dodoala1 *1
Department of Pharmaceutics, National Institute of Pharmaceutical Education and Research (NIPER-HYD), Balanagar, Hyderabad, A.P. - 500037, India 2 Department of Pharmaceutics, Sri Krupa Institute of Pharmaceutical Sciences, Vill: Velkatta, Siddipet. District: Medak, A.P. - 502103, India Received 29 January 2013; revised 20 January 2014; accepted 31 October 2014 The present study aimed to formulate a nanoemulsion and nanoemulsion based hydrogel of itraconazole for transdermal delivery in the treatment of on chomycosis. The nanoemulsions were prepared using lecithin and sodium cholate as surfactant and co-surfactant. The prepared nanoemulsions were characterized for particle size and zeta potential. The optimized nanoemulsion was incorporated into 3% carbopol-934 solution to get a gel for improving convenience in superficial application. In vitro and ex vivo drug penetration studies of nanoemulsions and gels were determined using dialysis membrane and rat skin. The particle size was found around 223.9 nm to 154.3 nm. The viscosity of the nanoemulsions and nanoemulsion gel were found around 1964.89 mPa.S to 1644.82 mPa.S and 28.3 mPa.S to 8.58 mPa.S respectively. The polydispersibility value was found very low indicating uniformity of droplet size of the formulations. The drug content in gels was found in between 86.2% to 98.26%. The drug release was found to be 44.33 % to 73.6% after 24 h with permeation flux around 296.3 to 203.1 (μg/cm2/hr1). The results indicated that nanoemulsion based hydrogels as promising vehicle for transdermal delivery of itraconazole. Further in vivo studies are to be performed to access its suitability for topical application. Keywords: Itraconazole, nanoemulsion, nanoemulsion based hydrogel, lecithin, sodium cholate, carbopol.
Introduction Recently research has been focused on the colloidal drug delivery systems such as micro emulsions, solid lipid nano-particles and liposomes for topical delivery of drugs because of low side effects high bioavailability, good patient compliances1,2. Nanoemulsions are non-equilibrium heterogeneous systems consisting of two immiscible liquids in which one liquid is dispersed as droplets in another liquid with droplet diameter in the range of 10-100 nm. Due to their unique physicochemical properties NE offer many advantages over traditional topical and transdermal drug delivery formulations3-7. Previously attempts have been made in the development of ITZ loaded nano-particles for parenteral application8. This article is intended to demonstrate the feasibility of new nanoemulsion system for transdermal delivery of anti-fungal drug itraconazole (ITZ). Methods Materials
Itraconazole was obtained as gift sample from Dr. Reddys Labs., Pvt. Ltd. (Hyderabad, India). Lecithin —————— *Author for correspondence E-mail:
[email protected]
and sodium cholate were purchased from Sigma Aldrich, eugenol was purchased from Loba chemicals. Pvt. Ltd. (Mumbai, India) and all other reagents and solvents used were of analytical grades. Analytical method development for itraconazole
A spectrophotometric method was developed for analysis of itraconazole. Briefly, the stock solution was prepared by dissolving 100 mg of itraconazole in 100 mL of saline phosphate buffer with 2% SLS and subsequent dilutions were made to get concentrations of 5 - 25 µg/mL. The absorbance was measured using double beam Jasco UV spectrophotometer at 263 nm. Preparation of Nanoemulsion (Ultrasonication method)
The aqueous phase was prepared by dissolving weighed amount of lecithin and sodium cholate in 4.5 mL of deionized distilled water (Table 1). Itraconazole (50 mg) was accurately weighed and dissolved in 0.5 mL of eugenol, heated to 75-80˚C for 2-3 min. The aqueous phase was added slowly to the oil phase and mixed using electronic stirrer at 15000 rpm for a period of 5 min. The droplet size in the course emulsion was further reduced by ultrasonication at 30% and 50% amplitude duty cycle using ultrasound instrument (Vibra-Cell VC 505,
89
SAMPATHI et al.: NANOEMULSION BASED HYDROGELS FOR DRUG DELIVERY
Sonic Instruments, Newtown, CT) for 15 min and stored at 4˚C for further studies. Preparation of nanoemulsion based hydrogel (NBH)
The best nanoemulsion formulation was incorporated into 3% of carbopol 934 to get a gel of the nanoemulsion. Weighed quantity of the carbopol 934 was dissolved in 15 mL of distilled water and stirred thoroughly to get homogenous slurry. The best and stable nanoemulsion was incorporated and mixed thoroughly and the pH was adjusted to neutral with triethanolamine. Limonene 0.1% was added as the permeation enhancer and as odor masking agent in the formulations.
for the measurement of droplet size. The average diameter and polydispersity index of samples were measured by photon correlation spectroscopy (Nano ZS90, Malvern Instruments, U.K) at 633 nm. The measurement was performed at 25˚ C using a He-Ne laser. Drug content
The drug content of the nanoemulsion based gel was determined by dissolving an amount of gel containing 5mg of drug. The drug content was determined after appropriate dilutions at 263 nm by UV Spectrophotometer. Viscosity determination
The viscosity of the formulations was determined by using a Brookfield viscometer using spindle #S16 at 25 ± 0.3˚C.
Evaluation Characterization of nanoemulsions Photon correlation spectroscopy
The mean particle size and the size distribution were determined by photon correlation spectroscopy. The prepared nanoemulsions were 40 times diluted
Zeta potential
The surface charge was analyzed using a Zeta-sizer at 25˚C by measuring the zeta potential of the prepared formulation. The formulations were diluted with distilled water and then the zeta potential was measured. Scanning electron microscopy (SEM)
Morphological characterization of the nanoemulsion was carried using scanning electron microscopy under the reduced pressure (0.001torr). The nanoemulsion were viewed at an accelerating voltage of 15-20kv. Thermodynamic stability study
Fig. 1—Globule size distribution curve of ITZ nanoemulsion F4
The optimized NE was centrifuged at 4000 rpm for 30 min and observed for phase separation, creaming, and cracking. Then it was subjected to heating and cooling cycle. Six cycles between the refrigerator temperature (4°C) and 45°C temperature were performed with storage at each temperature
Table 1—Composition, Particle size, Polydispersity index and Zeta potential of the ITZ nanoemulsion formulations Formulation
Lecithin (mg)
Sodium cholate (mg)
Particle size (nm)
Polydispersity index (PDI)
Zeta potential
Viscosity
F1 F2 F3 F4 F5 F6 F7 F8 F9
21.03 40.98 60.11 80.56 101.05 121.59 140.53 161.04 180.92
20.55 21.08 21.53 20.44 21.23 21.83 21.03 20.40 20.64
199.2 193.3 181.6 178.7 164.3 223.9 159.7 154.3 163.6
0.0091 0.082 0.079 0.134 0.136 0.252 0.145 0.203 0.192
-20.6 -19.9 -19.4 -18.4 -17.5 -14.9 -18.0 -18.0 -17.7
73.5 34 46.8 35.1 102 113.2 109.5 122.7 117.6
90
J SCI IND RES VOL 74 FEBRUARY 2015
for not less than 48 h. Finally it was subjected to freeze thaw cycle. Formulations were exposed for three freeze thaw cycles between -21˚C and +25˚C with storage at each temperature for not less than 48 h to check the thermodynamic stability of the formulations. In vitro diffusion studies using dialysis membrane
The in vitro study was performed by using Franz diffusion cells with an effective diffusion area of 2cm2. The dialysis membrane (12,000 MW) was soaked for 12 h and clamped between the donor and receptor compartment of the cell. The nanoemulsion and the NBH containing itraconazole were placed in the donor compartment. The receptor compartment was filled with saline phosphate buffer with 2% SLS as the medium and maintained at 37 ± 0.5 ºC and stirred at 600 rpm and samples were withdrawn at regular intervals for 24 h and analyzed by UVSpectrophotometer. The experiment was conducted in triplicates. Ex vivo studies
The best nanoemulsion was taken and studied for the ex vivo release studies by using albino rat skin as the membrane. Ex vivo skin permeation study was performed by using Franz diffusion cells as mentioned earlier. Adult Wistar rats of 180 -200 g were depilated with trimmer and skin samples were excised and were clamped between the donor and the receptor chamber of Franz diffusion cells with the stratum corneum facing the donor chamber. The nanoemulsion and the NBH containing itraconazole were placed in the donor compartment. The study was performed in the similar way as invitro diffusion study for 24h and analyzed the samples by UV spectrophotometer. Steady state flux (Jss/Cm/h) and lag time (T-lag/h) were calculated from the slope and intercept of the straight line obtained by plotting the cumulative amount of itraconazole permeated versus time in steady condition. Permeability Coefficient (Kp.Cm/h) was calculated by dividing the flux obtained by the initial concentration of drug in the donor compartment9,10. Kinetic analysis of drug release
To analyze the mechanism of drug release from itraconazole nanoemulsion and nanoemulsion based gel the in vitro dissolution data were fitted to zero order, first order, Higuchi release model, and
Korsemeyer and Peppa’s model and the model with higher correlation coefficient was considered to be the best model. Skin irritation test
The test was performed on male albino rats weighing around 180 - 200 g and all the procedures were approved by institutional animal ethical committee [Ethical Committee Reg. No: 1548/PO/a/11/CPCSEA]. The animals were divided into 4 groups each group consists of 4 rats: Group A served as control, Group B received 0.5ml of 0.8% V/V aqueous formalin solution as a standard irritant. Group C and D received ITZ-NE and ITZ-NBH prepared in plain carbopol gel. Standard irritant was applied on the left dorsal surface of each rat and formulations were applied on the right dorsal surface of the albino rat. The formulation was removed after period of 24 h with the help of an alcohol swab. The skin was examined for erythema or edema and primary irritancy index (PII) was scored as per Draize et al.,11.The results obtained were statistically compared using student’s T- test and statistical significance was set at p < 0.05. Results and discussion Itraconazole nanoemulsions with different concentrations of lecithin and sodium cholate as surfactant and co-surfactant were formulated. The mean particle size of the nanoemulsion formulation of ITZ was found to be 154.3 - 223.9 nm. The polydispersity index was found to range from 0.0091 to 0.203 which indicates uniformity of droplet size within the formulations with zeta potential values between -14.9 to -20.6 respectively (Table 1). The small mean particle size and PdI values below 0.2 (Table 1), indicated a narrow droplet size distribution and thus assured better stability of the formulation. The viscosity of all nanoemulsion was ranging from 34 to 122.7 mPa.S. The optimized nanoemulsion was incorporated 3 % carbapol 934 as the gelling agent to obtain the desired NBH and evaluated for drug content, which showed uniformity of 5 mg in all the prepared NBH formulations, hence the data has not been included.In vitro drug release studies from all the 9 formulations of nanoemulsions were determined and higher release was observed with the formulation F5 73.6% and lowest with formulation F4 i.e. around 44.33%.
91
SAMPATHI et al.: NANOEMULSION BASED HYDROGELS FOR DRUG DELIVERY
The significant difference in the release between formulations was probably due to the mean size of internal phase droplets. Hence, the maximum drug release found with F5 and F8 formulations having least size of internal phase droplets (Figure 2). Fitting of the release data into first order, Higuchi´s, and Peppa´s equations were done for formulations to known the mechanism of drug release and almost all formulations were following first order release kinetics by fickian transport (Table 2). In order to evaluate the skin targeting potential of the
NE and NBH, the skin permeation and penetration ability of ITZ cross the rat skin were examined ex vivo. Flux and permeability coefficients for optimized formulations are given in Table 3 and cumulative drug permeated in Figure 3. Compared to ITZ solution, the skin permeation and penetration ability were more pronounced with ITZ NE and ITZ NBH. The nanoemulsions can interact with the lipid bilayers of the stratum corneum and thereby contribute significantly to the penetration enhancing effect which was the reason for enhanced flux and permeation coefficient with nanoemulsions as compared with the gels as the consistency of the gels inhibit the drug permeation. The skin irritation studies shows that the ITZ-NE and ITZ-NBH to be non-irritant with the PII of ITZ-NE 1.25 ± 0.4 and1.8 ± 0.38 for ITZ-NBH (p > 0.05) when compared to the control group treated with formalin solution with a PII of 6.86 ± 0.89 (p < 0.05). The innovated formulations were safe to be applied to the skin for the intended period of time. Table 3—Flux values and permeability coefficient values of the formulations Sl. No Formulation code
Fig. 2—In vitro drug release profiles of itraconazole nanoemulsion formulations F1-F9
1 2 3 4
F8 NE F8 NE Gel F5 NE Flux of F5 Gel
Flux (μg/cm2/hr1).
KP (Cm.hr-1)
296.3 203.1 296.5 211.1
29.63 20.31 29.65 21.11
Table 2—Curve fitting data for all formulations of itraconazole nanoemulsions using lecithin as surfactant Formulation
ZERO ORDER 2
0.959 0.923 0.913 0.965 0.959 0.943 0.855 0.960 0.974 0.982 0.974 0.919 0.835
r F1 F2 F3 F4 F5 F6 F7 F8 F9 F5 (ex vivo) F8 (ex vivo) F5 Gel (ex vivo) F8 Gel(ex vivo)
FIRST ORDER
K
r
2
9.050 6.770 8.586 4.617 9.050 7.550 7.505 6.018 3.734 4.776 10.51 13.95 16.25
0.976 0.981 0.986 0.991 0.995 0.995 0.917 0.996 0.998 0.987 0.996 0.880 0.955
HIGUCHI
K
r
2
2.002 1.973 1.970 1.988 1.975 1.984 1.973 1.993 1.990 2.040 1.995 1.881 1.911
0.985 0.994 0.994 0.981 0.972 0.992 0.944 0.981 0.984 0.928 0.902 0.93 0.954
PEPPA’S K
r
2
K
-10.63 -6.780 -7.655 -7.548 -10.08 -7.923 -9.689 -12.76 -9.510 -7.532 3.604 10.24 8.869
0.611 0.840 0.823 0.776 0.682 0.787 0.834 0.735 0.772 0.688 0.732 0.888 0.727
2.029 2.017 2.026 2.018 2.054 2.025 2.034 2.053 2.027 2.240 2.222 1.971 1.991
92
J SCI IND RES VOL 74 FEBRUARY 2015
frequency of administration and improves the patient compliance. References 1
Fig. 3—Ex vivo drug release of ITZ nanoemulsions and hydrogel based nanoemulsions of F8 and F5 formulations
Conclusion The results obtained in the present work show that NE based hydrogel containing eugenol, lecithin and sodium chlolate as a suitable carrier system for incorporation of itraconazole and satisfies the best attributes for transdermal application with good particle size in the nano range with negative zeta potential, viscosity, good spread ability, and with suitable release profile. Prepared nanoemulsion based gel formulations are highly stable and safe for the transdermal delivery. The developed system could able to release the drug in sustained pattern and thereby it might reduce the
Rosen H & Abribat T, The rise and rise of drug delivery, Nat Rev Drug Discov, 4 (2005) 381-385. 2 Garg AK, Negi LM & Chauhan M ,Gel containing ethosomal vesicles for transdermal delivery of aceclofenac, Int J Pharm Pharm Sci, 2 (2010) 102-108. 3 Dixit N, Kohili K & Baboota S, Nanoemulsion system for the transdermal delivery of a poorly soluble cardiovascular drug, PDA J Pharm Sci Technol, 62 (2008) 46-55. 4 Khandavilli S & Panchagnula R, Nanoemulsions as versatile formulations for paclitaxel delivery: peroral and dermal studies in rats, J Invest Dermatol, 127 (2007) 154-162. 5 Shevachman M, Garti N, Shani A & Sintov AC, Enhanced percutaneous permeability of diclofenac using a new U-type dilutable micro emulsion, Drug Dev Ind Pharm, 34 (2008) 403-412. 6 Babooota S, Al-Azaki A, Kohli K, Ali J, Dixit N & Shakeel F, Development and evaluation of a micro emulsion formulation for transdermal delivery of terbinafine, PDA J Pharm Sci Technol, 61 (2007) 276-285. 7 Kantarci G, Ozguney I, Karasulu HY, Arzik S & Guneri T, Comparison of different water/oil microemulsions containing diclofenac sodium: Preparation, characterization, release rate, and skin irritation studies, AAPS PharmSciTech, 8 (2007) E91. 8 Karande P & Mitragotri S, Enhancement of transdermal drug delivery via synergistic action of chemicals, Biochim Biophys Acta, 1788 (2009) 2362–2373. 9 Pinner RW, Teutsch SM, Simonsen L, Klug LA, Graber JM, Clarke MJ & Berkelman RL, Trends in infectious diseases mortality in the United States, JAMA, 275 (1996) 189-193. 10 Armstrong GL, Conn LA & Pinner RW,Trends in infectious disease mortality in the United States during the 20th century, JAMA, 281 (1999) 61-66. 11 Draize JH, Woodard G & Calvery HO, Methods for the study of irritation and toxicity of substances applied topically to the skin and mucous membranes, J Pharmacolo Exp Ther, 82 (1944) 377-390.
