Nanoemulsion gel-based topical delivery of an antifungal drug: in vitro activity and in vivo evaluation.

http://informahealthcare.com/drd ISSN: 1071-7544 (print), 1521-0464 (electronic) Drug Deliv, Early Online: 1–16 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/10717544.2014.933284

Nanoemulsion gel-based topical delivery of an antifungal drug: in vitro activity and in vivo evaluation Afzal Hussain1, Abdus Samad2, S. K. Singh1, M. N. Ahsan1, M. W. Haque1, A. Faruk3, and F. J. Ahmed4

Drug Delivery Downloaded from informahealthcare.com by Memorial University of Newfoundland on 07/31/14 For personal use only.

1

Department of Pharmaceutical Sciences, Birla Institute of Technology, Mesra, Ranchi, Jharkhand, India, 2Department of Pharmacokinetic and Statistics, Fortis Clinical Research Ltd., Faridabad, Haryana, India, 3Depatment of Pharmaceutical Science, HNB Garhwal University (A Central University), Srinagar, Utterakhand, India, and 4Department of Pharmaceutics, Faculty of Pharmacy, Jamia Hamdard University, New Delhi, India

Abstract

Keywords

Objective: In this study, attempt has been focused to prepare a nanoemulsion (NE) gel for topical delivery of amphotericin B (AmB) for enhanced as well as sustained skin permeation, in vitro antifungal activity and in vivo toxicity assessment. Materials and methods: A series of NE were prepared using sefsol-218 oil, Tween 80 and Transcutol-P by slow spontaneous titration method. Carbopol gel (0.5% w/w) was prepared containing 0.1% w/w AmB. Furthermore, NE gel (AmB-NE gel) was characterized for size, charge, pH, rheological behavior, drug release profile, skin permeability, hemolytic studies and ex vivo rat skin interaction with rat skin using differential scanning calorimeter. The drug permeability and skin irritation ability were examined with confocal laser scanning microscopy and Draize test, respectively. The in vitro antifungal activity was investigated against three fungal strains using the well agar diffusion method. Histopathological assessment was performed in rats to investigate their toxicological potential. Results and discussion: The AmB-NE gel (18.09 ± 0.6 mg/cm2/h) and NE (15.74 ± 0.4 mg/cm2/h) demonstrated the highest skin percutaneous permeation flux rate as compared to drug solution (4.59 ± 0.01 mg/cm2/h) suggesting better alternative to painful and nephrotoxic intravenous administration. Hemolytic and histopathological results revealed safe delivery of the drug. Based on combined results, NE and AmB-NE gel could be considered as an efficient, stable and safe carrier for enhanced and sustained topical delivery for AmB in local skin fungal infection. Conclusion: Topical delivery of AmB is suitable delivery system in NE gel carrier for skin fungal infection.

Amphotericin B, gel, nanoemulsion, nephrotoxicity, topical delivery

Introduction Amphotericin B (AmB) is a broad-spectrum fungicidal antibiotic used primarily in the treatment of life-threatening systemic fungal infections (Kleinberg, 2006). However, the therapeutic efficacy is limited due to its poor aqueous solubility and its serious side effects (Nahar et al., 2008). The conventional AmB formulation used clinically is a micellar suspension of AmB with a bile salt-like sodium deoxycholate (FungizoneÕ ) (Wong-Beringer et al, 1998). Therefore, clinical utility of the micellar formulation of AmB is complicated by frequent and severe side effects including fever, chills, nausea, vomiting, anemia and nephrotoxicity (Espuelas et al., 1997). The novel lipid-containing formulations of AmB (i.e. liposomes and microemulsions) have been developed in an attempt to decrease its nephrotoxicity and increase its potential therapeutic effect. But, disadvantage of these liposomal colloidal carriers is the physical and chemical

Address for correspondence: Dr. Abdus Samad, Department of Pharmacokinetic and Statistics, Fortis Clinical Research Ltd, Sector 16-A, Faridabad, Haryana, India. Tel: +91 9988786698. Email: [email protected]

History Received 29 April 2014 Revised 6 June 2014 Accepted 6 June 2014

instability in aqueous dispersion (Glavas-Dodov et al., 2005) as a potential inherent challenge for formulation scientists. Interestingly, topical application of antifungal drug offers several advantages by targeted drug delivery to the infected site so as to maximize local side effects without concurrent systemic toxicity like nephrotoxicity. Even a single formulation of AmB in nanoemulsion (NE) form is not available in the market for the topical application. The most potential problem allied with topical delivery is the hydrophobic stratum corneum (SC) as barrier of intact skin that prevent percutaneous permeability of drugs. Therefore, it becomes a challenge to deliver hydrophilic or higher molecular weight therapeutic drugs through crystalline SC barrier to treat the cutaneous fungal infection. In this study, AmB has some suitable properties for topical application, yet, it exhibits low lipophilicity for insignificant permeability of the drug across the skin. Unfortunately, NEs as the topical carrier are rarely studied for AmB; it might offer several significant advantages including low skin irritation, powerful permeation ability and high drug-loading capacity for topical delivery when compared with the other carriers such as microemulsions, liposomes or solid lipid nanoparticles (LNPs; Sonneville-Aubrun et al., 2004;


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Tadros et al., 2004; Solans et al., 2005). Moreover, colloidal systems, such as microemulsions, have been investigated as targeted drug delivery. These systems can incorporate drug compounds modifying their bioavailability, stability and reducing their side effects (Formariz et al., 2005; Formariz et al., 2006). Microemulsion systems usually have reduced viscosity, while the more ordered hexagonal and cubic phases reveal the elastic properties of solids. Nonconventional NE has been developed to enhance the performance of hydrophobic-drug loaded NE delivery systems. But, in this study, we developed nanocarrier-based NE with the beauty of cost effectiveness and ease in scale up on industrial level. NE ensures adequate localization and penetration of the drug within or through the skin to enhance the local or systemic effect by adequate percutaneous absorption as compared to vesicular carrier system. To investigate, the effectiveness of diethylene glycol monoethyl ether (Transcutol-P) both as co-surfactant in the formulation and as a potential dermal enhancer of AmB permeation from NE already incorporated in carbopol gel was used. Transcutol-P is considered as a suitable co-surfactant for this study being non-toxic, biocompatible with the skin and significant solubilizing properties of both hydrophilic and lipophilic drugs. On the other hand, the development of lipid-based NE formulation for topical administration of AmB might therefore be desirable, presenting in monomeric forms leading to minimized nephrotoxicity and significant activity against fungal strains. Therefore, we already assessed aggregation behavior (published) of developed NE containing AmB, with strong evidence that its applicability could be more successful than conventional formulations (Hussain et al., 2014). Particularly, this study focused the development of a NE containing lipid components and its physicochemical characterization. All formulations were assessed in vitro with respect to fungicidal activity, hemolysis of the human erythrocytes and the most promising ones were assessment of ex vivo skin permeation study, irritation potential evaluation as well as in vivo toxicity.

Materials and methods Materials AmB was kindly gifted from Kwality Pvt. Ltd. (Amritsar, Punjab, India). Polyethylene glycol 400, propylene glycol (PG) and TweenÕ 80 were procured from Merck Chemicals (Mumbai, India). Diethylene glycol monoethyl ether (Transcutol-P) was obtained from Gattefosse Company, Cedex, France. The high-performance liquid chromatography (HPLC) Waters 515 HPLC system (US) equipped with 515 binary, 2998 Diode-Array Detector and Thermo BDS Hypersil-C18 column (150 mm 4.6 mm ID, 3 mm particle size of stationary packing in column). All other materials and solvents used in the experimental studies were of analytical grade. Double distilled water was used as aqueous solvent. Methods Screening of excipients and preparation of AmB NE The solubility of AmB in different oils (sefsol-218, cotton seed oil, soya oil and olive oil), surfactants (TweenÕ 80 and

Drug Deliv, Early Online: 1–16

TweenÕ 60) and co-surfactants (PG, polyethylene glycol-400 and Transcutol-P) were quantified by saturating the drug in 2 ml of each of the selected oils and mixed using vortex mixer for 10 min. The detail of the solubility studies has already been reported by our research group (Hussain et al., 2013). The mixture containing vials were kept in an isothermal shaker water bath set at 37 ± 1 C for 48 h in order to attain equilibrium. After 48 h, vials were centrifuged at 3000 rpm for 15 min, supernatant was taken out and filtered through 0.45 mm membrane filter. Finally, the drug content was determined in all excipients using UV-Vis spectrophotometer (Shimadzu, U-1800 spectrophotometer, Tokyo, Japan) at 382 nm. All the experiments were performed in triplicate. Thus, a number of exhaustive NE formulations were prepared by aqueous phase titration method (slow and spontaneous emulsification titration method) as reported by Azeem et al. (2009). In order to find out suitable oils, surfactants and co-surfactants for this study were based on the maximum solubility of the poorly soluble drug in these excipients. Sefsol-218 and dimethylsulfoxide (DMSO) mixture was found to have maximum solubility (55.82 ± 0.6 mg/ml). For the preparation of oil in water NE, surfactant was selected with HLB value greater than 11 to get clear and stable NE. TweenÕ 80 is hydrophilic, non-toxic and HLB value of 14.5, which was suitable for the study. From the solubility point of view, drug is least soluble (0.0074 mg/ml) in this surfactant. It has been taken in consideration of minimum concentration of surfactant as well as co-surfactant while selecting optimized formula. Only Transcutol-P (27 ± 0.8 mg/ml) was selected for use as co-surfactant due to significant solubility as compared to other co-surfactants. Surfactants and co-surfactants (Smix) were combined in such a ratio (1:1, 1:2, 1:3, 3:1, 2:1 and 1:4), which could give stable and maximum solubilized amount of drug in oil phase as shown in ternary phase Figure 1. Thus, by slow spontaneous aqueous phase titration, final combinations (Oil:Smix) were designed to delineate precisely the boundary of phase diagram with the Transcutol-P (1:2 and 1:3) (Figure 1A and B) as depicted in Figure 1, respectively. Briefly, a precisely weighed amount of AmB (10 mg) was dissolved easily in 1 ml of sefsol-218 oil and DMSO mixture in 1:1 ratio and considered as oil phase. All the Smix ratios were added to the oil phase and mixed with vortex mixer. Now obtained mixture was slowly titrated with aqueous phase to get clear and transparent NE. Thus, a series of NEs were prepared and optimized NE based on physic–chemical characterization and subjected to further study. NE gel preparation method Finally, NE F-V (5 ml) was selected as the optimized formulation to incorporate into the carbopol gel (1% w/w) formulation. Then, formulation F-V was incorporated into the previously prepared blank carbopol-980 gel (5 g) to get consistent and homogenous gel. For this, a weighed amount of carbopol-980 polymer was dispersed in distilled water and homogenized vigorously. Now, it was kept overnight after adding 2–3 drops of triethanolamine as cross linking agent using constant slow stirring and the final pH was adjusted to 7.4. By keeping the gel overnight, enable to remove the

Nanoemulsion gel for topical delivery of AmB


DOI: 10.3109/10717544.2014.933284

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Figure 1. Pseudoternary phase diagrams of the optimized nanoemulsions delineated in different combinations of Smix as Figure 1(A and B) reveals Smix ratio of 1:2 (TweenÕ 80:PEG-400) and 1:3 (TweenÕ 80:Transcutol P), respectively.

entrapped air and to complete cross linking of the polymeric gel with the base triethanolamine. AmB along with rhodamine 123 dye loaded NE gel (RA-AmB-NE gel) was formulated using AmB and rhodamine 123 dye, followed with the same procedure as described above. AmB and rhodamine 123 solutions were prepared separately by dissolving AmB and rhodamine 123 dye, respectively, in DMSO and distilled water (Table 2). Finally, the strength of drug in NE gel was 0.1% w/w of the gel (0.5% w/w).

Rheological study The flow characteristics of the AmB-NE gel formulation was performed in a Brookfield viscometer using rotary viscometer equipped with coaxial cone and plate (Bohlin Visco 88, Malvern, UK). In order to determine viscosity and thixotropic rheogram at shear rates between 12.28 and 120.5 s1, experimental values of viscosity (Z, Pa s) were a function _ s1) at 25 ± 1 C. The final rheograms of of the shear rate (y, the optimized AmB-NE gel consisting of the upward and downward curves were obtained and interpreted.

Physico–chemical characterization of NE and its gel The globular size of all selected NEs was measured using the dynamic light scattering technique (Malvern Zetamaster, ZEM 5002, Malvern, UK) (Attwood et al., 1992). The globular size evaluation of the prepared gel was analyzed with the same Zetasizer. Light scattering was monitored at 25 C temperature and a scattering angle of 90 . The oil globular surface charge density (Zeta potential) was determined using Zetasizer Nano ZS (Malvern Instruments, Worcestershire, UK). The gel architecture of the drug-loaded gel was visualized using Transmission Electron Microscopy (TEM) (morgagni 268D (FEI Company, Hillsboro, OR) operated at 20-60 KV at 1500X magnification. The pH measurements of the optimized AmBNE gel were obtained using a calibrated digital pH meter (Hanna Instrument HI 9321, Ann. Arbor, MI). The percentage drug entrapment efficiency (%EE) was obtained by calculating the amount of drug entrapped (E2) after removal of unentrapped drug using a dialysis bag (Himedia, Ltd., Mumbai, India, with molecular weight cutoff 12000-14000) by using the following Equation (1): EEð%Þ ¼ ðE2 =E1 Þ 100

ð1Þ

where E1 is the total amount of the drug added in the formulation. The concentration of the drug was determined by UV-Vis spectrophotometer (Shimadzu, U-1800 spectrophotometer) at 382 nm.

Spreadability To evaluate the rheological flowable and spreading properties of the developed NE formulations, a known weighed cellulose acetate filter paper (W1) was placed at the center of aluminum foil sheet (Khurana, 2013). The developed formulations were filled into the 5 ml syringe. Then, a fixed number of drops of the formulation to be tested (about 20 drops) were injected out of the syringe on the specific area at the centre of the cellulose acetate filter. After 10 min, the filter paper get saturated with the formulation was removed away from the unsaturated portion. The unsaturated portion was weighed out accurately (W2) and % spread by weight was calculated using the following Equation (2): % Speed by weight ¼ ½ðW1 W2 Þ=W1 100

ð2Þ

In vitro drug release assessment The In vitro drug release of AmB from AmB-NE gel formulation, drug solution (DS) and NE was carried out using dialysis membrane (Himedia, Ltd., Mumbai, India, with molecular weight cutoff 12000-14000). Limited solubility of AmB in water and phosphate buffer (pH 7.4), 2% v/v DMSO were added to maintain sink condition throughout the studies in the receptor chamber that facilitates the diffusion rate. The


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dialysis membrane was placed between the donor and receptor compartments of locally fabricated Franz diffusion cells (diffusion area 3.104 cm2; receptor volume of 22.5 ml). A weighed amount of drug (equivalent to 0.1 mg) from each formulation was loaded over the dialysis membrane and allowed to release in a receptor chamber containing phosphate buffer media (pH 7.4) at 32 ± 0.5 C with constant stirring (100 rpm) for 12 h (Skalko et al., 1998). Samples (1 ml) were withdrawn at 0.5, 1, 2, 4, 6, 8, 10 and 12 h time intervals, replaced with same volume of fresh buffer solution and analyzed the drug release by HPLC (kmax 382 nm) method. The HPLC analysis was performed using mobile phase composed of methanol, acetonitrile and 0.00125 M EDTA in ratio of 40:43:17, respectively. The mobile phase was run at flow rate of 0.8 ml/min with injection volume of 10 ll for 23 min, and retention time was 11 min. The detector response was linear in the range of 5.0–100 ng/mL. The drug release data was fitted to the several mathematical models (zeroorder, first-order and Higuchi) to extract the mechanism of AmB release from the formulations. In vitro skin permeation studies Skin permeation was studied with a Franz diffusion cells with a diffusion area of 3.104 cm2 and receptor volume of 22.5 ml using abdominal albino rat skin (Utreja et al., 2011). The rat was ethically sacrificed by cervical dislocation, and skin was made completely hairless. The abdominal skin was surgically excised and washed with isotonic saline solution. The excised skin was used after removing underlying fat and subcutaneous tissue. Then, this was mounted between the both chambers of the Franz diffusion cell with the dermis side in contact with the receptor medium and the SC side facing upward the donor chamber; 100 mg of the formulation containing 0.1 mg equivalent amount of AmB was uniformly placed on the skin. The lower receptor chamber containing phosphate buffer (pH 7.4) and 2% v/v DMSO was stirred constantly with magnetic bead and maintained at 32 ± 1 C to simulate the skin temperature throughout the experiment. Samples were withdrawn with subsequent replacement with the same buffer medium through the sampling port of the diffusion cell at predetermined time intervals of 0.5,1, 2, 4, 6, 8, 10, 12, 16, 20 and 24 h. The samples were filtered and analyzed at 382 nm using HPLC. Samples were centrifuged and analyzed for AmB content. The DS (0.1 mg/ml) served as control. For evaluation of the drug penetration into rat skin, the formulation remaining on the skin surface was removed by gentle washing with PBS (pH 7.4) at the end of the experiment (24 h). Ex vivo globule–skin interaction study Differential scanning calorimeter (DSC) study was performed to find out the interaction of the formulations with treated SC stratum corneum. Each desiccated as well as treated SC sample of rat skin (about 3 mg) was weighed in aluminum pan and packed tightly to ensure better thermal contact and hermetically sealed. The samples were subjected to heat from 0 to 200 C at the rate of 10 C/min using a DSC (TA-60W, DSC, Shimadzu). On completing the first cycle, samples in their pans were cooled to 0 C and then immediately reheated


again to 200 C at 10 C/min. This analysis study was performed in an absolute nitrogen atmosphere (100 ml min1) between 0 and 10 C with a constant rate of heating of 10 C min1 as described earlier. Transition temperatures were determined considering the minimum values of the endothermic peaks observed at the DSC curves. In vitro hemolysis assay Hemolysis was assessed as previously described method (Perkins et al., 1992). In vitro hemobiocompatibility assessment of the AmB-NE gel, NE, Tritone X-100 as positive control, blank NE and other excipients were studied using heparinized human blood freshly drawn (10 ml). The erythrocytes (separated from supernatant) were separated by centrifugation at 3000 rpm for 15 min. Then, collected RBCs were washed three times with buffer solution (pH 7.4) to remove debris and serum protein. Samples were serially diluted with phosphate buffer to get final concentration of 4% v/v in PBS. For control, 0.5 ml of RBCs suspension was mixed with 0.5 ml of phosphate buffer (as negative control) or Triton X100 5% v/v solution (as positive control). AmB-NE, AmB-NE gel and its components were subjected to interact with RBCs suspension. Then, samples were placed in a 37 C ± 1.0 C incubator and agitated for 24 h. After 24 h, tubes were centrifuged at low speed 4000 rpm for 15 min to pellet RBCs and other debris. The supernatants (2 ml) were analyzed for released hemoglobin content using UV-Vis spectrophotometer at a wavelength of 540 nm (Shimadzu U1800, spectrophotometer). One-hundred percent hemolysis was defined as the maximum amount of hemolysis obtained from Triton X-100 solution. Experiments were carried out in triplicate, and the mean values were reported.

Scanning electron microscopy of erythrocyte In order to visualize the samples in scanning electron microscopy (SEM), the treated erythrocyte pellets with NE, Triton X-100 treated and the control were fixed overnight at 4 C by adding 0.5 ml of each sample to the plastic tubes containing 2 ml glutaraldehyde (2.5% v/v), washed twice in distilled water, placed on siliconized alumina stubs and air dried at 37 ± 1 C for 12 h. The gold coating was done for 3 min, then coated gold grid specimens were examined under SEM (Carl Zeiss, EVO43, SEM, Stuttgart, Germany) at 1500 magnifications (Kumar et al., 2010). In vitro antifungal activity of NE containing AmB The in vitro activity of AmB-loaded NE was evaluated using the well diffusion method (Kadimi et al., 2007). The Aspergillus fumigatus (MTCC 6500) and Aspergillus niger (MTCC 282) strains were used for the in vitro test in the Czapek Dox media at pH 6.8. Candida albicans (MTCC 4748) was used as fungal strain to culture onto Sabouraud Dextrose agar media at pH 6.2. The media was dissolved in distilled water, mixed well and autoclaved at 121 C for 20 min. Then, it was transferred aseptically into the sterile Petridish under the laminar airflow chamber followed with mixing of strain at normal temperature just before solidification. In this study, appropriate dilutions (105 dilutions) of

DOI: 10.3109/10717544.2014.933284

0.1 ml of A. fumigatus strain were then mixed into the media. The plates were incubated at 35 C for 48 h (C. albicans, 4 105 CFU/ml), five days (A. fumigatus, 1.5 105 CFU/ml) and seven days (A. niger, 1 105 CFU/ml). The formulations were tested at a final concentration range of 0.125–25 mg/ml. Known concentrations of the samples were loaded into the well and incubated as per their earlier mentioned conditions of incubation in an inverted position.


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1 mm2 size and subjected evaluate for the probe penetration (depth) with CLSM (Fluorescence Correlation MicroscopeOlympus FluoView FV1000, Olympus, Melville, NY) with an argon laser beam with excitation at 488 nm and emission at 590 nm (Dubey et al., 2007). The sliced skin pieces were visualized under the normal light and fluorescence microscope. These two techniques were applied to obtain the images and to justify the distribution of the probe in the different strata of the skin.


In vivo studies All the animal studies were performed by using Wistar albino rats (27 in numbers) weighing 200–300 g of either sex. Animals were properly housed in polypropylene cages under standard laboratory conditions and had free access for food and water ad libitum. The animal protocol was approved by animal ethical committee of Birla Institute of Technology, Mesra, Ranchi-835215, and Jharkhand, India. Skin irritation studies Skin irritation potential of AmB-loaded NE and AmB-NE gel were evaluated by carrying out skin irritancy test on Wistar albino rats (200–300 g) (Draize et al., 1944; Pople & Singh, 2006). These rats were allowed to adopt the conditions for seven days before the commencement of the study. The dorsal surface of the rat was made hairless without damaging the skin surface, 4 h prior to the experiment. The animals were divided into four groups (n ¼ 3): Group I: plain gel (negative control); Group II: AmB DS; Group III: 0.8% v/v aqueous solution of formalin (positive control); and Group IV: AmBNE gel. The formulations (100 mg containing 0.1 mg equivalent amount of AmB) were topically applied to the hairless skin area (1 cm2). They were placed back to labeled respective cages and were inspected at 24, 48 and 72 h. Thus, the applied sites were observed for dermal reactions such as erythema and edema. The mean erythemal and edemal scores were recorded on the basis of their degree of severity caused by application of formulations: no erythema/edema ¼ 0, slight erythema/ edema ¼ 1, moderate erythema/edema ¼ 2 and severe erythema/edema ¼ 3. Visualization of the formulation into the skin penetrated in vivo Sefsol-218 oil containing AmB in NE and rhodamine 123 loaded NE gel (RA-AmB-NE gel) formulations has been designed to investigate the effective drug distribution and probe rhodamine 123 in the skin by confocal laser scanning microscopy (CLSM) where 0.1% w/v rhodamine 123-loaded dye nanoformulations were prepared. Rats were randomly grouped, and each group has three rats. The skin of the dorsal region was shaved. NE containing probe (100 ml), RA-AmB-NE gel (100 mg) and 0.1% w/v solution (100 ml) solely as marker rhodamine 123 were applied in defined area of 1 cm2 at the dorsal site of animals of group A, B and C, respectively, for 24 h. After applying probe-loaded AmB-NE gel and NE formulation, the dye was carefully dressed to remove the applied formulation with cotton bud at 24 h. Now, rats were subjected to sacrifice ethically. The rat’s excised skin was blotted and washed thrice using ethanol on inactive paper. The applied area was then removed into the pieces of

In vivo acute and repeated-dose dermal toxicity study As per guideline of OECD guideline 402 and 410 using Wistar rats weighing about 200–300 g were used for this study (OECD Guidelines, 1981, 1987). Before the commencement of the test, animals were distributed in different groups randomly and placed into the respective cages. Each group consisted of three rats. Fifteen rats were assigned into five equal groups (n ¼ 3). The group first received untreated control; second group received placebo gel; third group received Triton X-100 as positive control, fourth group received AmB-NE gel and final fifth group received NE (pH 7.4), respectively. Hundred times of the human higher dose (10 mg/kg) was used for the acute dermal toxicity study. Topically, dose of 0.1 mg/kg was used to apply for the repeated-dose dermal toxicity study. Hairs were properly shaved (dorsal area) without any abrasion at application site. The body surface (approx 10%) was used to test formulations. While application, the formulations were kept in contact with the skin using nonirritating dressing tape. The applied site was further covered to be adhered the test formulation. The formulations were applied over the period of 24 h for the acute dermal toxicity and observed up to 28 d. In this specific study, rats were treated with the formulation for 6 h per day on a six-day per week basis for a period of 28 d. The rats were observed frequently and individual records were made for changes in fur, eyes, mucous membranes, somatomotor activity respiratory, circulatory, autonomic and central nervous system and behavior changes. Rats were weighed before the study and at end of the study. Every surviving animal was sacrificed by cervical dislocation at the end of 28 d. By the necropsy study, the gross histopathological changes of the tested animal organs were observed and compared with normal anatomy of the major visceral organ. Major organs like liver, kidney and heart were collected and weighed carefully after dissection. The preserved tissue samples in formalin (10% v/v) were preserved and processed for the gross histopathological study by sectioning of 5 mm thickness. Then, these individual organ specimen was stained with hematoxylin and eosin. The excised skin and isolated organs were preserved in 10% formalin, and sections were fixed as well as blocks were made using the procedure as reported (Chen-Yu et al., 2012).

Results and discussion Physicochemical characteristics studies In the present investigation, various NE formulations were developed and screened out for further characterization and evaluation as listed in Table 1. The highest AmB solubility

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Table 1. Selected nanoemulsions with their globular size, zeta potential, polydispersity index and viscosity values. Formulation code F-I F-II F-III F-IV F-V F-VI

Oil (% w/w) 10 13 10 12 10 10

Smix ratioa (25) (22) (30) (30) (25) (25)

b

2:1 2:1c 1:2b 1:3c 1:3a 1:2c

Water (% w/w)

Mean droplet size (nm)

Polydispersity index

Zeta potential (mV)

Viscosity (cP)

pH

65 65 70 58 65 65

112.3 ± 1.8 167.7 ± 2.6 106.0 ± 1.5 86.6 ± 2.0 76.2 ± 1.4 172.6 ± 2.1

0.235 0.614 0.351 0.741 0.303 0.435

42.05 36.38 35.03 34.94 31.48 30.14

26.38 ± 1.2 32.52 ± 1.8 38.7 ± 1.3 41.51 ± 1.5 39.01 ± 1.4 33.11 ± 1.7

6.5 6.6 6.8 6.0 7.4 6.6

Value represented as mean ± SD (n ¼ 3). Transcutol-P as cosurfactant. Smix ¼ surfactant:co-surfactan ratio, and Tween 80 is common in each formulations as surfactant. c PEG-400 ¼ polyethylene glycol 400 as co-surfactant. a

b


Table 2. Composition of F-V gel and others formulations dye for CLSM study.

Formulations Placebo-NE gel AmB-NE gel RA AmB-NE gel AmB solution in DMSO Rhodamine 123 solution

Amphotericin B (mg)

Rhodamine 123 (RA) (mg)

Nanoemulsion (ml)a

Carbopol 980 gel (g)b

Final ratio (a:b)

– 10 mg 10 mg 0.1 mg/ml –

– – 5 – 0.1%w/v

5 5 5 – –

5 5 5 – –

1:1 1:1 1:1 – –

a: numerical value of nanoemulsion; b: numerical value of carbopol -980 gel.

was found in sefsol-218 (19.32 ± 0.5 mg/ml), and its combination with DMSO was 55.82 ± 0.6 mg/ml than other excipients. In the Table 1, it has been depicted that formulation F-V was the most suitable among all selected formulation for further characterization. It was evaluated for their globular size (76.2 ± 1.4 nm), zeta potential (–31.48 mV), minimum polydispersity index (0.303), optimum viscosity (39.01 ± 1.4 cP) and skin physiological pH (7.4). Pseudoternary phase diagram dictated the selection of TweenÕ 80 as surfactant and Transcutol-P as co-surfactant in development of NE. Placebo carbopol gel, NE loaded with AmB, AmB-NE gel incorporated with rhodamine 123 and AmB DS in DMSO were prepared using sefsol-218 oil as the organic phase. TweenÕ 80, Transcutol-P and carbopol-980 were as surfactant, cosurfactant and gel-forming gent, respectively (Table 2). The globular size (mean) of placebo-NE gel and AmB-NE gel were found to be 93.17 ± 8.5 nm and 97.04 ± 7.4 nm, respectively, with minimum polydispersity indices value as indicated in the Table 3. Moreover, the globular surface potential charge of placebo-NE gel and AmB-NE gel was found to be –34.84 ± 0.8 mV and –39.27 ± 0.25 mV, respectively (Table 3). Based on these results, it can be suggested that there is no significant variation (p40.05) between placeboNE gel and AmB-NE gel in terms of their globular size and zeta potential values. The percentage drug entrapment efficiency of AmB-NE gel was found to be 85.92 ± 3.25. The photograph of transmission electron microscopy in Figure 2(A and B) also revealed the morphological characteristics of spherical globular oil droplets in nanoscale in both Placebo gel as well as AmB-NE gel. These droplets are still non-aggregated in both formulations revealing its stability against Oswald ripening owing to globular collapse in heterogeneous globular sizes. The pH of both placebo gel and AmB-NE gel formulations was obtained 7.4 as depicted in the Table 3.

Table 3. Characterization of nanoemulsion gel (AmB-NE gel). Parameters

Placebo NE gel

AmB-NE gel

Particle size (nm) PI Zeta potential (mV) Drug entrapment efficiency (%) pH % Spread by weight Viscosity (cP) Steady state flux (Jss, mg/cm2/h)

93.17 ± 8.5 0.17 ± 0.02 34.84 ± 0.8 – 7.4 67.93 ± 1.7 2388 ± 12.83 Nil

97.04 ± 7.4 0.19 ± 0.01 39.27 ± 0.25 85.92 ± 3.96 7.4 68.73 ± 3.2 892 ± 9.64 18.09 ± 0.6

The reported are mean ± SD (n ¼ 3).

Rheological study The rheology is a significant parameter for evaluation of the gel when applied topically. Spreading and adherence behavior of topical formulations to the skin surface need to be evaluated. Finally, the gel was again held for seven days to reach pH equilibrium. After seven days, corresponding shear rate and shear stress values were made. The relationship between the stress (related to the force applied) and the shear rate on the samples were measured to study flow behavior and to determine the viscosity (Weyenberg et al., 2004a,b). A rotary viscometer equipped with coaxial cone and plat (Bohlin Visco 88) was used to determine viscosity at shear rates between 12.28 and 120.5 s1. Experimental values of _ s1), are viscosity (Z, Pa s) is a function of the shear rate (y, graphically represented in Figure 3, for a 0.5% (w/w) concentration gel at pH 7.4 neutralized with triethanolamine (0.1 ml). The rheogram of the placebo-NE gel and AmB-NE gel displayed thixotropic characteristics as indicated by the differences observed in ascending and descending curves of the rheogram (Figure 3). The viscosity of Placebo-NE gel (0.5% w/w) and AmB-NE gel (0.5% w/w) was found to be


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Figure 2. Transmission electron micrograph (TEM) of (A) nanoemulsion gel (placebo-NE gel) and (B) amphotericin B loaded nanoemulsio gel (AmB-NE gel).

Figure 3. Rheogram of nanoemulsion gel (placebo-NE gel) and amphotericin B-loaded nanoemulsion (AmB-NE gel) gel.

2388 ± 12.83 cP (coefficient of correlation ¼ 0.968) and 892 ± 9.64 cP (coefficient of correlation ¼ 0.9922), respectively. Thus, the viscosity of AmB-NE gel was reduced 2.677fold as compared to placebo-NE gel owing to incorporation of NE containing AmB. However, the curves were insignificant in both curves (p40.05) by the presence of AmB indicating constant consistency at same pH (7.4) and temperature (25.0 ± 1 C). It should also be noted that the correlation coefficient was excellent; the intercept and the slope was not statistically different from zero and one, respectively. Data for evaluation was fitted in Newtonian model using Bohlin software: Visco 88 to get the above Newtonian viscosity value and coefficient of correlation. Spreadability study Spreadability is a pivot for transdermal formulation (Chow et al., 2008). In this study, the optimized semisolid AmB-NE gel exhibited maximum percentage of spread by weight (68.73 ± 3.2) assuring and suggesting practically excellent spreadability behavior to the skin as shown in Table 3.

There were no significant differences (p40.05) between the spreadability of placebo-NE gel and AmB-NE gel (0.5% w/w). In vitro drug release assessment The release of the drug from the NE and AmB-NE gel formulations was absolutely significant (p50.001) in comparison to the DS (0.01% w/w). The AmB NE gel provided higher drug release rate over period of 24 h than DS (Figure 4). The in vitro drug release profile showed that 10.96 ± 1.5% w/w drug of the formulation from the AmB-NE gel, whereas 49.12 ± 1.8% w/w and 99.97 ± 2.3% w/w drug were released from the NE and AmB solution, respectively, within initial two hours. This revealed that rapid release of the drug from DS indicated absence any interaction of the drug with dialysis membrane in given set of experimental condition. Higher release rate of AmB from the NE could be attributed to the smaller globular size for larger surface area and permitting drug release rate. Although, the drug release rate from the AmB-NE gel and NE (pH 7.4) were slow as


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compared to DS, which was statistically significant (p50.05). Also among formulations, possible reason for this significantly different release rate of NE is its lower value of viscosity as well as its smaller globular size. Thus, NE (42.12%) and AmB NE gel (10.96%) formulation had shown 2.0- and 9.12-fold slower drug release, respectively, as compared to DS (99.97%) in first 2 h suggesting controlled and extended release profile. The release profile data as treated in zero-order, first-order, Higuchi and Korsmeyer– Peppas mathematical models to evaluate the release pattern from carrier systems and the drug release mechanism. The values of correlation coefficient of Korsmeyer–Peppas model for the obtained release data were greater than 0.96 in NE and AmB-NE gel except DS. The drug release pattern from AmBNE gel, NE and AmB NE-gel showed zero-order release kinetics with a best fit r2 value 0.9965, 0.9886 and 0.9815, respectively, whereas DS followed first order release kinetics (r2 ¼ 0.9997) during first initial 2 h. In vitro skin permeation study The in vitro skin permeation parameters of the test formulations and DS were determined as the results depicted in Figure 5. Permeation parameters are listed in the Table 4. The permeation profiles of AmB through rat skin from NE and


AmB-NE gel as shown in Figure 5 and compared with DS. It was observed that the cumulative amount of drug permeated at the end of 24 h was found to be 254.161 ± 1.45 mg, 870.42 ± 4.2 mg and 999.81 ± 7.3 mg for AmB DS, NE (pH 7.4) and AmB-NE gel, respectively (Figure 5). The NEs are known to provide increase permeation rate and decreased lag times by altering both the lipophilic and the polar pathway by synergistic interaction of vehicle component with the SC (Godwin et al., 1997; Shin & Choi 2005). Moreover, the permeation flux rate of AmB had been enhanced in AmB-NE gel (18.09 ± 0.6 mg/cm2/h), NE (15.74 ± 0.4 mg/cm2/h) as compared to DS (4.59 ± 0.01 mg/cm2/h) as listed in the Table 4. This might be due to greater extraction efficiency of skin lipid by Transcutol-P. The penetration enhancement of lipophilic drug by alcohol is due to higher solubilization power of the drug substance in the lipophilic area of the SC because of the presence of DMSO enhancer. Significant enhancement of the permeation was achieved by the use of 5% DMSO (Reddy & Ghosh 2001). The permeability potential of AmB-NE gel was determined and results clearly displayed that the flux rate of AmB-NE gel and NE were found to be 3.9- and 3.5-fold higher, respectively, with respect to the DS. The flux values obtained for NE and AmB-NE gel were significantly higher than the control DS (p50.01) indicating that the permeation parameters of the drug from NEs and gel were markedly influenced by the composition of the formulation. In this study, DS in 5% DMSO aqueous solution revealed slightly higher permeation flux as compared to aqueous DS reported in our earlier investigation.

Table 4. In vitro permeation parameters of different amphotericin B formulations across the albino rat skin after 24 h. Formulations AmB-NE gel Nanoemulsion Amphotericin B (drug) solution

Figure 4. In vitro cumulative amount of amphotericin B release through dialysis membrane. Figure 5. Cumulative amount of amphotericin B permeated through albino rat skin.

Jss1 (mg/cm2/h)

ER1

18.09 ± 0.6 15.74 ± 0.4 4.59 ± 0.01

10.42 8.97 0

Value represented as mean ± SD (n ¼ 3). Jss1 ¼ Transdermal flux, calculated from the slop of Cartestan plot of cumulative amount of drug present in receptor compartment versus time. ER1 ¼ Enhancement ratio; it is ratio of transdermal flux from formulation to drug solution.


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9

DSC mW 0.00

−1.00

(C)

114.38C

(B)

−2.00


−3.00

77.52C

−4.00 (A) −5.00

101.24C

50.00

100.00

150.00

Temp[C]

Figure 6. DSC thermogram of untreated (C), amphotericin B-loaded nanoemulsion gel (AmB-NE gel) treated (A) and Placebo (B) rat skin stratum corneum.

Ex vivo globule-skin interaction Ex vivo globule–skin interaction using the differential scanning calorimetry (DSC) was studied. To elucidate the mechanism of the drug permeation across the highly hydrophobic strata of the skin AmB gel and NE-treated formulation were subjected to DSC study. Furthermore, the SC intercellular lipids were also studied in term of thermal properties. The most common SC acts as the highly crystalline potential barrier to the diffusion of the drugs. It essentially consists of flattened keratinocytes embedded in a matrix of multi lamellar lipid bilayers (Elias, 1996). Furthermore, it was suggested that cholesterol and lipids with long saturated acyl chains (e.g. free fatty acids and ceramides) predominate in the barrier layer (Bowser & White, 1985). The DSC profile of the control, AmB-NE gel treated and Placebo (negative control) rat skin SC, respectively, are shown in Figure 6(A–C). In this investigation, it has been demonstrated that Figure 6(C) shows (control) the three typical endothermic transition temperatures at 59 C, 65 C and 114.38 C with the first peak being attributed to either lipid lamella phase transition from a crystalline to a gel-like phase or melting of sebaceous lipids, the second peak corresponding to the transformation from a lamellar to disordered state in the lipid structure and proteinassociated lipid transition from gel to liquid state, respectively, the third peak being allied with the conformation change of protein (Golden et al., 1986). In the rat skin treated with AmB-NE gel, it has been found that the SC lipid transition at 59 C (T1) and 65 C (T2) extremely reduced and broadened new characteristic transition at 101.24 C observed

in the DSC report (Figure 6A). The SC lipid transition at 59 C (T1) and 65 C (T2) were not appeared from the DSC thermogram of negative control SC samples (Figure 6B) with a broad new transition at 77.52 C. DSC results demonstrated that efficient conformational changes had been developed by the AmB gel formulation on SC lipids leading new responsible characteristics endothermic transition peaks at T1 (59 C) and T2 (65 C). The absence of peaks T1 and T2 in both the negative control skin samples and AmB-NE gel treated verified the intercellular lipid transition associated with the skin (Zellmer et al., 1995; Carelli et al, 1998; Changez, et al., 2006). Hence, our results suggested that AmB-NE gel caused significant modification in the lipid architectural structures of SC by lipids dissolution. The erythrocyte hemolytic study To find out the hemobiocompatibility, NE, AmB-NE gel, DS, sefsol-218 and others formulation components were subjected to hemolytic study. The extent of lysis induced by different formulations and its components after incubation with RBCs is shown in Figure 7. On incubating human erythrocytes with Triton X-100 (as positive control) led to 100% hemolysis over the period of 12 h. In case of negative control, normal saline caused no significant (p50.05) hemolysis. After incubation with 5 lg ml1 concentration of AmB-NE gel led to insignificant RBC lysis (19.3 ± 0.95%) compared to normal saline (negative control, 18.04 ± 0.1%) after 24 h. But, the erythrocytes lysis in the presence 5 lg/ml of NE (16.14 ± 0.07%) and sefsol-218 oil (12.6 ± 0.5%) was

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Figure 7. In vitro RBC lysis following incubation of RBCs with nanoemulsion and excipients.

100

% Hemolysis

80

60

40

n e lin

Tr

Sa

ito

n

so

X-

lu

10

tio

0

o eb ac Pl

N E

el -g N E B Am

D

ru

g

so

ol

lu

21

tio

8

n

0

Sa fs


20

Components and formulations

significantly (p50.01) low (Figure 6). Moreover, hemoglobin release in the placebo NE after 24 h incubation with the erythrocytes was found to be insignificant as compared to normal saline solution group. This suggested that NE components are unable to interact with human erythrocytes at physiological level. The controlled and extended release of AmB from NE and AmB-NE gel rationalized the minimum erythrocytes lysis as comparison to Triton X-100. This could be explained based on the fact that gel and NE formulation both are safe and biocompatible physiologically. Free drug induced hemolysis itself.

SEM of erythrocyte All of the lipids in erythrocytes are present in the stroma. These erythrocytes are analogous to large liposomes, and it seems that peroxidation was the main factor observed in lysis (Welles et al., 1977). To investigate, the capability of formulation and its component to do so that it leads to hemolysis was apparently determined by SEM. The morphological changes in RBCs cell dictates its stability in presence of formulations. Hence, to investigate erythrocytes changes were exposed to Triton X-100 and NE (pH 7.4) at a concentration of 5 mg/ml. SEM images for control RBCs, Triton X-100 and NE are Figure 8(A, B and C), respectively. As shown in Figure 8(A), RBCs exposed to the normal saline was found to be normal biconcave disc shaped. In contrast, positive control Triton X-100 caused significant changes at this concentration leading to development of spiny outgrowth. The presence of discocytes and many abnormal cells with spiny protuberances have been seen as shown in Figure 8(B). The NE formulation observed with absence of such outgrowth as lysis indicator. The role of sefsol-218 in protecting the system from hemolysis is not clear at this moment. However, sefsol-218 is thought to be non interacting component with

anionic RBCs membranes. Non-hemolytic characteristic of the NE system is to be considered as very attractive, especially when administered transdermally and topically.

In vitro antifungal activity studies of nanoformulation containing AmB The in vitro antifungal activities of AmB formulations were assayed against three different fungal strains such as C. albicans, A. niger and A. fumigatus. Among these fungal strains, C. albicans was found to be more sensitive fungal strain than others. All nanoformulations prepared in this study exerted significantly (p 0.001) higher antifungal activity in comparison with reference drug AmB solution in DMSO as shown in Table 5. The NE, DS, Transcutol-P (5%v/v) and AmB-NE gel formed uniform growth inhibition zones against A. niger strain (Figure 9C) as compared with that of other two fungal strain (Figures 9A and B), thus showing the higher sensitivity of the formulations. In the Figure 9(A), it has been seen that NE and AmB-NE gel caused extended growth inhibition zone due to nanoscale size globule leading to profound diffusion as compared to other two strains. The zone of inhibition was found to be enhanced with increasing the concentration of Amp-B. AmB-loaded NE and AmB-NE gel showed maximum antifungal activity than DS against C. albicans and A. fumigatus. Although least sensitivity was observed against A. niger as shown in the Figure 9(C) as compared to other strain. Moreover, in some literature, it has been reported that liposomal AmB and free AmB had comparable antifungal activities against various fungi such as Candida spp., Aspergillus spp. and Fusarium spp [Fukui et al, 2003a). The transfer mechanism of AmB from lipidic carriers to fungal cells was not fully understood. The similar or higher anti-fungal activities of AmB-loaded NE and AmBNE gel compared to DMSO and DS might be explained by the

Figure 8. SEM of red blood cells exposed to (a) untreated control (b) Triton X-100 (c) nanoemulsion concentration of 5 mg/mL for1 h.

difference in AmB release from the liposomal lipid carriers in the presence of fungal cells. Transcutol P showed very least ZOI against C. albicans and completely absent against A. fumigatus and A. niger. Drug incorporated in oily phase of NE are readily available to ergosterol composition of fungal hyphae, which might be the main potential cause of higher growth inhibition against fungal strain. Similar finding had been published in the previous literature. Hence, it has been reported by the author that AmB loaded into LNPs could be readily released in the presence of ergosterol-rich fungal cells due to its higher affinity to ergosterol in fungal cell membrane than to cholesterol in LNPs (Fukui et al., 2003a,b and c).

Skin irritation studies

The irritation potential of topical formulations was evaluated, and results have been listed in Table 6. Its utility and acceptability by the patients has been limited when any 1.3 ± 0.06 1.7 ± 0.05 2.9 ± 0.12 0.0 ± 0.0

3.2 ± 0.11 2.5 ± 0.09 1.3.0 ± 0.6

25.0 mg/ml

1.6 ± 0.2 2.8 ± 0.21 4.3 ± 0.28 0.0 ± 0.0

4.8 ± 0.24 2.9 ± 0.17 1.4 ± 0.5

50.0 mg/ml

0.0 ± 0.0 0.4 ± 0.03 0.4 ± 0.05 0.0 ± 0.0

0.3 ± 0.09 0.4 ± 0.07 1.0 ± 0.05

0.125 mg/ml

0.0 ± 0.0 1.4 ± 0.2 1.5 ± 0.8 0.0 ± 0.0

1.4 ± 0.13 0.6 ± 0.6 1.2 ± 0.06

12.5 mg/ml

0.0 ± 0.0 3.1 ± 0.1 2.7 ± 0.5 0.0 ± 0.0

2.6 ± 0.7 2.3 ± 0.32 1.1 ± 0.08

25.0 mg/ml

Aspergillus fumigatus (MTCC 6500)

0.0 ± 0.0 3.2 ± 0.2 3.5 ± 0.3 0.0 ± 0.0

3.5 ± 0.2 2.8 ± 0.8 1.2 ± 0.1

Values are mean ± SD (mm) from the experiments in triplicate. The diameter of the well (6 mm) is not included. bTranscutol P as co-surfactant.

0.3 ± 0.05 1.3 ± 0.03 1.7 ± 0.01 0.0 ± 0.0

0.2 ± 0.07 0.4 ± 0.1 0.5 ± 0.1 0.0 ± 0.0

a

2.2 ± 0.2 1.1 ± 0.23 1.2 ± 0.1

0.7 ± 0.01 0.3 ± 0.02 1.2 ± 0.08

Nanoemulsion DMSO (5% v/v) Placebo nanoemulsion Transcutol Pb AmB solution AmB-NE gel Placebo gel

12.5 mg/ml

0.125 mg/ml

Formulations

Candida albicans (MTCC 4748)

Growth inhibition zone (mm)a

Table 5. Comparison of inhibitory activities against different fungal species by the agar well diffusion method.

50.0 mg/ml

0.0 ± 0.0 0.6 ± 0.08 0.7 ± 0.05 0.0 ± 0.0

0.0 ± 0.0 0.7 ± 0.1 0.8 ± 0.02

0.125 mg/ml


0.0 ± 0.0 1.2 ± 0.2 1.1 ± 0.6 0.0 ± 0.0

1.2 ± 0.1 1.7 ± 0.8 1.0 ± 0.1

12.5 mg/ml

0.0 ± 0.0 1.8 ± 0.2 2.3 ± 0.6 0.0 ± 0.0

2.8 ± 0.4 2.3 ± 0.1 1.0 ± 0.2

25.0 mg/ml

Aspergillus niger (MTCC 282)

0.0 ± 0.0 2.2 ± 0.5 3.4 ± 0.12 0.0 ± 0.0

3.1 ± 0.7 2.8 ± 0.22 1.40 ± 0.1

50.0 mg/ml

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Nanoemulsion gel for topical delivery of AmB 11


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Figure 9. Photographs of the zones of inhibition, the Petriplates with amphotericin B drug solution (DS), nanoemulsion (NE), Transcutol-P (TP) and amphotericin B nanoemulsion gel (AmB-NE gel) formulation. The data represents the mean ± SD (n ¼ 3). Table 6. Mean erythemal scores observed at the end of 24, 48 and 72 h.

around the application site. Thus, the developed formulation can be classified as a non-irritant and safe to the rat skin.

Erythema scores (n ¼ 3) Formulations Plain gel (negative control group I) Drug solution (group II) Aqueous formalin solution 0.8% v/v (group III) AmB-NE gel (group IV)

24 h

48 h

72 h

0 0 2 0

0 0 3 0

0 1 3 0

irritation or erythema is observed on topical application. Hence, any topical delivery system of AmB should be free of these erythematic reactions. In this study, the results showed that no severe irritation symptoms such as erythema (redness) and edema (swelling) during 72 h except reference positive control group III. After application, all the scores were zero for placebo/plain gel (group I), DS (group II) and AmB-NE gel (group IV) (Table 6). The reference formalin aqueous solution triggered itching and redness at the applied area, resulting in visible skin irritation and inflammation with group III, calculated to be 1 score. Hence, skin irritation study revealed that neither polymeric plain gel alone nor NE containing drug incorporated in gel formulation (AmB-NE gel) exhibited any noticeable irritation or inflammation on or

Visualization of formulations into the skin penetrated in vivo Presently, CLSM has become a well established technique to observe the drug distribution within the skin (Borgia et al., 2005; Chen et al., 2006). The skin penetration potential of RA-AmB-NE gel and to relate the in vitro skin permeation parameters, CLSM was performed. Figure 10 reveals the fluorescence images of sections (vertical) of albino rat skin after being treated with rhodamine-loaded NE, RA-AmB-NE gel and rhodamine 123 solution at 24 h (Figures 10A–C). Apparently, it was observed that dye content in sefsol-218 oil of the NE depended on dye distribution in the skin. After 24 h, the fluorescence images of dye distribution from NE and RA-AmB-NE gel was significantly high in the SC, viable epidermis and deeper area of dermis. On the other hand, the dye solution was merely confined to the SC and upper viable epidermis as shown in Figures 10(A–C). The amount of drug released from NE and its gel formulation mainly depends on the organic phase content in the NE. The higher the oil loading, the higher the drug released is obtained

DOI: 10.3109/10717544.2014.933284


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Figure 10. CLSM images revealing the penetration and distribution of Rhodamine 123 within albino rat skin when treated with nanoemulsion (NE), AmB-NE gel and Rhodamine 123 dye solution after being applied for 24 h: (A) NE; (B) AmB-NE gel; and (C) rhodamine 123 solution.

(Hu et al., 2006). Other possible mechanism for enhanced drug release into the deeper area of rat skin could be explained that the dye solution application on the skin is insufficient to form any film or creamy texture. Feasibility of fabricating NE and RA-AmB-NE gel of AmB was improved drug penetration into the skin. This was achieved by formation of a monolayer film on the skin and simultaneously the loss of water induced the lipid modification leading to the drug expulsion from the formulation. In this study, drug penetration was significant. The results are also consistent with the in vitro drug permeation study through the albino rat skin for the same formulations. In vivo acute and repeated-dose dermal toxicity study The most important aim in designing of NE based topical delivery of AmB formulation was to resolve its potential

nephrotoxicity. Plasma drug concentration should be minimized and topical formulation fabricated to enhance epidermal drug permeation. However, alternative novel carrier systems like particulate carrier are rapidly removed from the systemic circulation by the cells of reticulate endothelial phagocyte system (Lemke et al., 2005). On acute and repeated application of the NE, AmB-NE gel and Placebo gel did not reveal any abnormalities in the test animal groups at the end of 28 d of study. The test groups were free from any reaction at the application site and mortality. Moreover, the histopathological examination results of major organs (Figures 11 and 12) did not reveal any adverse effect on repeated dermal exposure to any groups. The results were found to be insignificant changes (p40.05). This could be owing to very small quantity of drug being absorbed into the blood circulation after topical application of AmB from NE and NE gel, and major proportion of drug being remained into


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Figure 11. Representative histopathological sections of various organs showing effect of repeated topical application of amphotericin B in 28-d dermal toxicity study. (A) Heart, (B) kidney and (C) liver. (1) Untreated control, (2) treated NE-7.4 and (3) AMB-NE gel.

Figure 12. Photomicrographs of rat skin sample: (a) control group showing normal epidermis, dermis and subcutaneous tissues at high power view (magnification 400 ); (b) NE-treated group; (c) skin sample from AmB-NE gel-treated group; and (d) Placebo gel-treated group.

DOI: 10.3109/10717544.2014.933284

the skin as evident of confocal laser microscopy results of the rat skin (Figure 10).


Conclusion AmB is gold standard antifungal drug and used in the treatment of wide variety of dermatological fungal infection. Higher molecular weight, least aqueous solubility and potential nephrotoxicity like problems have limited its clinical application in systemic treatment. The objective of the investigation was achieved by adopting alternative topical route of administration of AmB and loading in the nanoscale NE carrier system for enhanced skin permeation. The in vitro drug release profile results showed sustained release of AmB effectively as compared to free DS. Ex vivo skin permeation improved enhanced skin permeation of NE and AmB NE-gel to facilitate drug permeation after fabricating AmB NE gel of drug using Transcutol-P and TweenÕ 80 combination. This combination had synergistic effects on improved drug permeation and penetration. Ex vivo skin interaction results revealed maximum perturbation caused by the skin penetration enhancer (DMSO), which further supported the result. In vivo histopathological examination suggested that formulations for cutaneous infection are safe and efficacious than oral delivery. Hence based on the above results, it can be concluded that NE gel mediated delivery is an economic approach for effective as well as safe localized delivery of AmB against fungal infection.

Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.

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Nanomiemgel--a novel drug delivery system for topical application--in vitro and in vivo evaluation.

Correction: Nanomiemgel--a novel drug delivery system for topical application--in vitro and in vivo evaluation.

Development of a novel in vitro onychomycosis model for the evaluation of topical antifungal activity.

Antifungal activity of Cleome gynandra L. aerial parts for topical treatment of Tinea capitis: an in vitro evaluation.

Nanoemulsion: an advanced mode of drug delivery system.

Polymeric nanocarriers for magnetic targeted drug delivery: preparation, characterization, and in vitro and in vivo evaluation.

Evaluation of the in vitro antifungal activity of allicin.

Anti HIV nanoemulsion formulation: optimization and in vitro-in vivo evaluation.

In vitro and in vivo evaluation of the delivery of topical formulations containing glycoalkaloids of Solanum lycocarpum fruits.

In vitro and in vivo release characteristics of Tacrolimus (FK506) from an episcleral drug-delivery implant.

Antifungal and anthelmintic activity of novel benzofuran derivatives containing thiazolo benzimidazole nucleus: an in vitro evaluation.

Liposomes in topical ophthalmic drug delivery: an update.

In vitro evaluation of antifungal drug combinations against Sarocladium (Acremonium) kiliense, an opportunistic emergent fungus resistant to antifungal therapies.

in vivo evaluation.

In vitro and in vivo topical delivery studies of tretinoin-loaded ultradeformable vesicles.

In vitro activity of protoanemonin, an antifungal agent.

Evaluation of vaginal antifungal formulations in vivo.

in vitro model.

In Vitro and In Vivo Antifungal Activity of Lichochalcone-A against Candida albicans Biofilms.

In vitro and in vivo evaluation of novel implantation technology in hydrogel contact lenses for controlled drug delivery.

In vitro and in vivo activity of a novel antifungal small molecule against Candida infections.

Formulation, characterization, in vitro, in vivo, and histopathological evaluation of transdermal drug delivery containing norfloxacin and Curcuma longa.

In vitro and in vivo antifungal activities of BMS-181184.

An in-vivo evaluation of a MEMS drug delivery device using Kunming mice model.