Drug Delivery

ISSN: 1071-7544 (Print) 1521-0464 (Online) Journal homepage: www.tandfonline.com/journals/idrd20

Nanoemulsion gel-based topical delivery of an antifungal

drug: in vitro activity and in vivo evaluation

Afzal Hussain, Abdus Samad, S. K. Singh, M. N. Ahsan, M. W. Haque, A. Faruk

& F. J. Ahmed

To cite this article: Afzal Hussain, Abdus Samad, S. K. Singh, M. N. Ahsan, M. W.

Haque, A. Faruk & F. J. Ahmed (2016) Nanoemulsion gel-based topical delivery of an
antifungal drug: in vitro activity and in vivo evaluation, Drug Delivery, 23:2, 642-657, DOI:
10.3109/10717544.2014.933284

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ISSN: 1071-7544 (print), 1521-0464 (electronic)

Drug Deliv, 2016; 23(2): 642–657

! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/10717544.2014.933284

RESEARCH ARTICLE

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
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 Amphotericin B, gel, nanoemulsion,
topical delivery of amphotericin B (AmB) for enhanced as well as sustained skin permeation, nephrotoxicity, topical delivery
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 History
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, Received 29 April 2014
pH, rheological behavior, drug release profile, skin permeability, hemolytic studies and ex vivo Revised 6 June 2014
rat skin interaction with rat skin using differential scanning calorimeter. The drug permeability Accepted 6 June 2014
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.

Introduction as a potential inherent challenge for formulation scientists.

Interestingly, topical application of antifungal drug offers
Amphotericin B (AmB) is a broad-spectrum fungicidal
several advantages by targeted drug delivery to the infected
antibiotic used primarily in the treatment of life-threatening
site so as to maximize local side effects without concurrent
systemic fungal infections (Kleinberg, 2006). However, the
systemic toxicity like nephrotoxicity. Even a single formula-
therapeutic efficacy is limited due to its poor aqueous
tion of AmB in nanoemulsion (NE) form is not available in
solubility and its serious side effects (Nahar et al., 2008).
the market for the topical application.
The conventional AmB formulation used clinically is a
The most potential problem allied with topical delivery is
micellar suspension of AmB with a bile salt-like sodium
the hydrophobic stratum corneum (SC) as barrier of intact
deoxycholate (FungizoneÕ ) (Wong-Beringer et al, 1998).
skin that prevent percutaneous permeability of drugs.
Therefore, clinical utility of the micellar formulation of AmB
Therefore, it becomes a challenge to deliver hydrophilic or
is complicated by frequent and severe side effects including
higher molecular weight therapeutic drugs through crystalline
fever, chills, nausea, vomiting, anemia and nephrotoxicity
SC barrier to treat the cutaneous fungal infection. In this
(Espuelas et al., 1997). The novel lipid-containing formula-
study, AmB has some suitable properties for topical applica-
tions of AmB (i.e. liposomes and microemulsions) have been
tion, yet, it exhibits low lipophilicity for insignificant
developed in an attempt to decrease its nephrotoxicity and
permeability of the drug across the skin. Unfortunately,
increase its potential therapeutic effect. But, disadvantage of
NEs as the topical carrier are rarely studied for AmB; it might
these liposomal colloidal carriers is the physical and chemical
offer several significant advantages including low skin
instability in aqueous dispersion (Glavas-Dodov et al., 2005)
irritation, powerful permeation ability and high drug-loading
Address for correspondence: Dr. Abdus Samad, Department of capacity for topical delivery when compared with the other
Pharmacokinetic and Statistics, Fortis Clinical Research Ltd, Sector carriers such as microemulsions, liposomes or solid lipid
16-A, Faridabad, Haryana, India. Tel: +91 9988786698. Email:
[email protected] nanoparticles (LNPs; Sonneville-Aubrun et al., 2004;
DOI: 10.3109/10717544.2014.933284 Nanoemulsion gel for topical delivery of AmB 643

Tadros et al., 2004; Solans et al., 2005). Moreover, colloidal TweenÕ 60) and co-surfactants (PG, polyethylene glycol-400
systems, such as microemulsions, have been investigated as and Transcutol-P) were quantified by saturating the drug in
targeted drug delivery. These systems can incorporate drug 2 ml of each of the selected oils and mixed using vortex mixer
compounds modifying their bioavailability, stability and for 10 min. The detail of the solubility studies has already
reducing their side effects (Formariz et al., 2005; Formariz been reported by our research group (Hussain et al., 2013).
et al., 2006). Microemulsion systems usually have reduced The mixture containing vials were kept in an isothermal
viscosity, while the more ordered hexagonal and cubic phases shaker water bath set at 37 ± 1  C for 48 h in order to attain
reveal the elastic properties of solids. equilibrium. After 48 h, vials were centrifuged at 3000 rpm
Nonconventional NE has been developed to enhance the for 15 min, supernatant was taken out and filtered through
performance of hydrophobic-drug loaded NE delivery sys- 0.45 mm membrane filter. Finally, the drug content was
tems. But, in this study, we developed nanocarrier-based NE determined in all excipients using UV-Vis spectrophotometer
with the beauty of cost effectiveness and ease in scale up on (Shimadzu, U-1800 spectrophotometer, Tokyo, Japan) at
industrial level. NE ensures adequate localization and pene- 382 nm. All the experiments were performed in triplicate.
tration of the drug within or through the skin to enhance the Thus, a number of exhaustive NE formulations were
local or systemic effect by adequate percutaneous absorption prepared by aqueous phase titration method (slow and
as compared to vesicular carrier system. To investigate, the spontaneous emulsification titration method) as reported by
effectiveness of diethylene glycol monoethyl ether Azeem et al. (2009). In order to find out suitable oils,
(Transcutol-P) both as co-surfactant in the formulation and surfactants and co-surfactants for this study were based on
as a potential dermal enhancer of AmB permeation from NE the maximum solubility of the poorly soluble drug in
already incorporated in carbopol gel was used. Transcutol-P is these excipients. Sefsol-218 and dimethylsulfoxide
considered as a suitable co-surfactant for this study being (DMSO) mixture was found to have maximum solubility
non-toxic, biocompatible with the skin and significant (55.82 ± 0.6 mg/ml). For the preparation of oil in water NE,
solubilizing properties of both hydrophilic and lipophilic surfactant was selected with HLB value greater than 11 to get
drugs. On the other hand, the development of lipid-based NE clear and stable NE. TweenÕ 80 is hydrophilic, non-toxic and
formulation for topical administration of AmB might there- HLB value of 14.5, which was suitable for the study. From the
fore be desirable, presenting in monomeric forms leading to solubility point of view, drug is least soluble (0.0074 mg/ml)
minimized nephrotoxicity and significant activity against in this surfactant. It has been taken in consideration of
fungal strains. Therefore, we already assessed aggregation minimum concentration of surfactant as well as co-surfactant
behavior (published) of developed NE containing AmB, with while selecting optimized formula. Only Transcutol-P
strong evidence that its applicability could be more successful (27 ± 0.8 mg/ml) was selected for use as co-surfactant due
than conventional formulations (Hussain et al., 2014). to significant solubility as compared to other co-surfactants.
Particularly, this study focused the development of a NE Surfactants and co-surfactants (Smix) were combined in such a
containing lipid components and its physicochemical charac- ratio (1:1, 1:2, 1:3, 3:1, 2:1 and 1:4), which could give stable
terization. All formulations were assessed in vitro with respect and maximum solubilized amount of drug in oil phase as
to fungicidal activity, hemolysis of the human erythrocytes shown in ternary phase Figure 1. Thus, by slow spontaneous
and the most promising ones were assessment of ex vivo skin aqueous phase titration, final combinations (Oil:Smix) were
permeation study, irritation potential evaluation as well as in designed to delineate precisely the boundary of phase diagram
vivo toxicity. with the Transcutol-P (1:2 and 1:3) (Figure 1A and B) as
depicted in Figure 1, respectively. Briefly, a precisely
Materials and methods weighed amount of AmB (10 mg) was dissolved easily in
1 ml of sefsol-218 oil and DMSO mixture in 1:1 ratio and
Materials
considered as oil phase. All the Smix ratios were added to the
AmB was kindly gifted from Kwality Pvt. Ltd. (Amritsar, oil phase and mixed with vortex mixer. Now obtained mixture
Punjab, India). Polyethylene glycol 400, propylene glycol was slowly titrated with aqueous phase to get clear and
(PG) and TweenÕ 80 were procured from Merck Chemicals transparent NE. Thus, a series of NEs were prepared and
(Mumbai, India). Diethylene glycol monoethyl ether optimized NE based on physic–chemical characterization and
(Transcutol-P) was obtained from Gattefosse Company, subjected to further study.
Cedex, France. The high-performance liquid chromatography
(HPLC) Waters 515 HPLC system (US) equipped with 515
NE gel preparation method
binary, 2998 Diode-Array Detector and Thermo BDS
Hypersil-C18 column (150 mm  4.6 mm ID, 3 mm particle Finally, NE F-V (5 ml) was selected as the optimized
size of stationary packing in column). All other materials and formulation to incorporate into the carbopol gel (1% w/w)
solvents used in the experimental studies were of analytical formulation. Then, formulation F-V was incorporated into the
grade. Double distilled water was used as aqueous solvent. previously prepared blank carbopol-980 gel (5 g) to get
consistent and homogenous gel. For this, a weighed amount of
Methods carbopol-980 polymer was dispersed in distilled water and
homogenized vigorously. Now, it was kept overnight after
Screening of excipients and preparation of AmB NE
adding 2–3 drops of triethanolamine as cross linking agent
The solubility of AmB in different oils (sefsol-218, cotton using constant slow stirring and the final pH was adjusted to
seed oil, soya oil and olive oil), surfactants (TweenÕ 80 and 7.4. By keeping the gel overnight, enable to remove the
644 A. Hussain et al. Drug Deliv, 2016; 23(2): 642–657

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 Rheological study
gel with the base triethanolamine. AmB along with rhoda-
The flow characteristics of the AmB-NE gel formulation was
mine 123 dye loaded NE gel (RA-AmB-NE gel) was
performed in a Brookfield viscometer using rotary viscometer
formulated using AmB and rhodamine 123 dye, followed
equipped with coaxial cone and plate (Bohlin Visco 88,
with the same procedure as described above. AmB and
Malvern, UK). In order to determine viscosity and thixotropic
rhodamine 123 solutions were prepared separately by
rheogram at shear rates between 12.28 and 120.5 s1,
dissolving AmB and rhodamine 123 dye, respectively, in
experimental values of viscosity (Z, Pa s) were a function
DMSO and distilled water (Table 2). Finally, the strength of
_ s1) at 25 ± 1  C. The final rheograms of
of the shear rate (y,
drug in NE gel was 0.1% w/w of the gel (0.5% w/w).
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 Spreadability
dynamic light scattering technique (Malvern Zetamaster, ZEM
5002, Malvern, UK) (Attwood et al., 1992). The globular size To evaluate the rheological flowable and spreading properties
evaluation of the prepared gel was analyzed with the same of the developed NE formulations, a known weighed cellulose
Zetasizer. Light scattering was monitored at 25  C temperature acetate filter paper (W1) was placed at the center of aluminum
and a scattering angle of 90  . The oil globular surface charge foil sheet (Khurana, 2013). The developed formulations were
density (Zeta potential) was determined using Zetasizer Nano filled into the 5 ml syringe. Then, a fixed number of drops of
ZS (Malvern Instruments, Worcestershire, UK). The gel the formulation to be tested (about 20 drops) were injected out
architecture of the drug-loaded gel was visualized using of the syringe on the specific area at the centre of the
Transmission Electron Microscopy (TEM) (morgagni 268D cellulose acetate filter. After 10 min, the filter paper get
(FEI Company, Hillsboro, OR) operated at 20-60 KV at 1500X saturated with the formulation was removed away from the
magnification. The pH measurements of the optimized AmB- unsaturated portion. The unsaturated portion was weighed out
NE gel were obtained using a calibrated digital pH meter accurately (W2) and % spread by weight was calculated using
(Hanna Instrument HI 9321, Ann. Arbor, MI). The percentage the following Equation (2):
drug entrapment efficiency (%EE) was obtained by calculating % Speed by weight ¼ ½ðW1  W2 Þ=W1   100 ð2Þ
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) In vitro drug release assessment
by using the following Equation (1): The In vitro drug release of AmB from AmB-NE gel
formulation, drug solution (DS) and NE was carried out using
EEð%Þ ¼ ðE2 =E1 Þ  100 ð1Þ
dialysis membrane (Himedia, Ltd., Mumbai, India, with
where E1 is the total amount of the drug added in the molecular weight cutoff 12000-14000). Limited solubility of
formulation. The concentration of the drug was determined by AmB in water and phosphate buffer (pH 7.4), 2% v/v DMSO
UV-Vis spectrophotometer (Shimadzu, U-1800 spectropho- were added to maintain sink condition throughout the studies
tometer) at 382 nm. in the receptor chamber that facilitates the diffusion rate. The
DOI: 10.3109/10717544.2014.933284 Nanoemulsion gel for topical delivery of AmB 645

dialysis membrane was placed between the donor and again to 200  C at 10  C/min. This analysis study was
receptor compartments of locally fabricated Franz diffusion performed in an absolute nitrogen atmosphere (100 ml
cells (diffusion area 3.104 cm2; receptor volume of 22.5 ml). min1) between 0 and 10  C with a constant rate of heating
A weighed amount of drug (equivalent to 0.1 mg) from each of 10  C min1 as described earlier. Transition temperatures
formulation was loaded over the dialysis membrane and were determined considering the minimum values of the
allowed to release in a receptor chamber containing phosphate endothermic peaks observed at the DSC curves.
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 In vitro hemolysis assay
withdrawn at 0.5, 1, 2, 4, 6, 8, 10 and 12 h time intervals,
replaced with same volume of fresh buffer solution and Hemolysis was assessed as previously described method
analyzed the drug release by HPLC (kmax 382 nm) method. (Perkins et al., 1992). In vitro hemobiocompatibility assess-
The HPLC analysis was performed using mobile phase ment of the AmB-NE gel, NE, Tritone X-100 as positive
composed of methanol, acetonitrile and 0.00125 M EDTA in control, blank NE and other excipients were studied using
ratio of 40:43:17, respectively. The mobile phase was run at heparinized human blood freshly drawn (10 ml). The erythro-
flow rate of 0.8 ml/min with injection volume of 10 ll for cytes (separated from supernatant) were separated by centri-
23 min, and retention time was 11 min. The detector response fugation at 3000 rpm for 15 min. Then, collected RBCs were
was linear in the range of 5.0–100 ng/mL. The drug release washed three times with buffer solution (pH 7.4) to remove
data was fitted to the several mathematical models (zero- debris and serum protein. Samples were serially diluted with
order, first-order and Higuchi) to extract the mechanism of phosphate buffer to get final concentration of 4% v/v in PBS.
AmB release from the formulations. For control, 0.5 ml of RBCs suspension was mixed with
0.5 ml of phosphate buffer (as negative control) or Triton X-
100 5% v/v solution (as positive control). AmB-NE, AmB-NE
In vitro skin permeation studies
gel and its components were subjected to interact with RBCs
Skin permeation was studied with a Franz diffusion cells with suspension. Then, samples were placed in a 37  C ± 1.0  C
a diffusion area of 3.104 cm2 and receptor volume of 22.5 ml incubator and agitated for 24 h. After 24 h, tubes were
using abdominal albino rat skin (Utreja et al., 2011). The rat centrifuged at low speed 4000 rpm for 15 min to pellet
was ethically sacrificed by cervical dislocation, and skin was RBCs and other debris. The supernatants (2 ml) were
made completely hairless. The abdominal skin was surgically analyzed for released hemoglobin content using UV-Vis
excised and washed with isotonic saline solution. The excised spectrophotometer at a wavelength of 540 nm (Shimadzu U-
skin was used after removing underlying fat and subcutaneous 1800, spectrophotometer). One-hundred percent hemolysis
tissue. Then, this was mounted between the both chambers of was defined as the maximum amount of hemolysis obtained
the Franz diffusion cell with the dermis side in contact with from Triton X-100 solution. Experiments were carried out in
the receptor medium and the SC side facing upward the donor triplicate, and the mean values were reported.
chamber; 100 mg of the formulation containing 0.1 mg
equivalent amount of AmB was uniformly placed on the Scanning electron microscopy of erythrocyte
skin. The lower receptor chamber containing phosphate buffer
(pH 7.4) and 2% v/v DMSO was stirred constantly with In order to visualize the samples in scanning electron
magnetic bead and maintained at 32 ± 1  C to simulate the microscopy (SEM), the treated erythrocyte pellets with NE,
skin temperature throughout the experiment. Samples were Triton X-100 treated and the control were fixed overnight at
withdrawn with subsequent replacement with the same buffer 4  C by adding 0.5 ml of each sample to the plastic tubes
medium through the sampling port of the diffusion cell at containing 2 ml glutaraldehyde (2.5% v/v), washed twice in
predetermined time intervals of 0.5,1, 2, 4, 6, 8, 10, 12, 16, distilled water, placed on siliconized alumina stubs and air
20 and 24 h. The samples were filtered and analyzed at dried at 37 ± 1  C for 12 h. The gold coating was done for
382 nm using HPLC. Samples were centrifuged and analyzed 3 min, then coated gold grid specimens were examined under
for AmB content. The DS (0.1 mg/ml) served as control. SEM (Carl Zeiss, EVO43, SEM, Stuttgart, Germany) at
For evaluation of the drug penetration into rat skin, the 1500  magnifications (Kumar et al., 2010).
formulation remaining on the skin surface was removed by
gentle washing with PBS (pH 7.4) at the end of the In vitro antifungal activity of NE containing AmB
experiment (24 h).
The in vitro activity of AmB-loaded NE was evaluated using
the well diffusion method (Kadimi et al., 2007). The
Ex vivo globule–skin interaction study
Aspergillus fumigatus (MTCC 6500) and Aspergillus niger
Differential scanning calorimeter (DSC) study was performed (MTCC 282) strains were used for the in vitro test in the
to find out the interaction of the formulations with treated SC Czapek Dox media at pH 6.8. Candida albicans (MTCC
stratum corneum. Each desiccated as well as treated SC 4748) was used as fungal strain to culture onto Sabouraud
sample of rat skin (about 3 mg) was weighed in aluminum pan Dextrose agar media at pH 6.2. The media was dissolved in
and packed tightly to ensure better thermal contact and distilled water, mixed well and autoclaved at 121  C for
hermetically sealed. The samples were subjected to heat from 20 min. Then, it was transferred aseptically into the sterile
0 to 200  C at the rate of 10  C/min using a DSC (TA-60W, Petridish under the laminar airflow chamber followed with
DSC, Shimadzu). On completing the first cycle, samples in mixing of strain at normal temperature just before solidifi-
their pans were cooled to 0  C and then immediately reheated cation. In this study, appropriate dilutions (105 dilutions) of
646 A. Hussain et al. Drug Deliv, 2016; 23(2): 642–657

0.1 ml of A. fumigatus strain were then mixed into the media. 1 mm2 size and subjected evaluate for the probe penetration
The plates were incubated at 35  C for 48 h (C. albicans, (depth) with CLSM (Fluorescence Correlation Microscope-
4  105 CFU/ml), five days (A. fumigatus, 1.5  105 CFU/ml) Olympus FluoView FV1000, Olympus, Melville, NY) with an
and seven days (A. niger, 1  105 CFU/ml). The formulations argon laser beam with excitation at 488 nm and emission at
were tested at a final concentration range of 0.125–25 mg/ml. 590 nm (Dubey et al., 2007). The sliced skin pieces were
Known concentrations of the samples were loaded into the visualized under the normal light and fluorescence micro-
well and incubated as per their earlier mentioned conditions scope. These two techniques were applied to obtain the
of incubation in an inverted position. 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 In vivo acute and repeated-dose dermal toxicity study
rats (27 in numbers) weighing 200–300 g of either sex.
As per guideline of OECD guideline 402 and 410 using
Animals were properly housed in polypropylene cages under
Wistar rats weighing about 200–300 g were used for this study
standard laboratory conditions and had free access for food
(OECD Guidelines, 1981, 1987). Before the commencement
and water ad libitum. The animal protocol was approved by
of the test, animals were distributed in different groups
animal ethical committee of Birla Institute of Technology,
randomly and placed into the respective cages. Each group
Mesra, Ranchi-835215, and Jharkhand, India.
consisted of three rats. Fifteen rats were assigned into five
equal groups (n ¼ 3). The group first received untreated
Skin irritation studies
control; second group received placebo gel; third group
Skin irritation potential of AmB-loaded NE and AmB-NE gel received Triton X-100 as positive control, fourth group
were evaluated by carrying out skin irritancy test on Wistar received AmB-NE gel and final fifth group received NE (pH
albino rats (200–300 g) (Draize et al., 1944; Pople & Singh, 7.4), respectively. Hundred times of the human higher dose
2006). These rats were allowed to adopt the conditions for (10 mg/kg) was used for the acute dermal toxicity study.
seven days before the commencement of the study. The dorsal Topically, dose of 0.1 mg/kg was used to apply for the
surface of the rat was made hairless without damaging the repeated-dose dermal toxicity study. Hairs were properly
skin surface, 4 h prior to the experiment. The animals were shaved (dorsal area) without any abrasion at application site.
divided into four groups (n ¼ 3): Group I: plain gel (negative The body surface (approx 10%) was used to test formulations.
control); Group II: AmB DS; Group III: 0.8% v/v aqueous While application, the formulations were kept in contact with
solution of formalin (positive control); and Group IV: AmB- the skin using nonirritating dressing tape. The applied site
NE gel. The formulations (100 mg containing 0.1 mg equiva- was further covered to be adhered the test formulation. The
lent amount of AmB) were topically applied to the hairless formulations were applied over the period of 24 h for the
skin area (1 cm2). They were placed back to labeled respective acute dermal toxicity and observed up to 28 d. In this specific
cages and were inspected at 24, 48 and 72 h. Thus, the applied study, rats were treated with the formulation for 6 h per day on
sites were observed for dermal reactions such as erythema and a six-day per week basis for a period of 28 d.
edema. The mean erythemal and edemal scores were recorded The rats were observed frequently and individual records
on the basis of their degree of severity caused by application were made for changes in fur, eyes, mucous membranes,
of formulations: no erythema/edema ¼ 0, slight erythema/ somatomotor activity respiratory, circulatory, autonomic and
edema ¼ 1, moderate erythema/edema ¼ 2 and severe ery- central nervous system and behavior changes. Rats were
thema/edema ¼ 3. weighed before the study and at end of the study. Every
surviving animal was sacrificed by cervical dislocation at the
Visualization of the formulation into the skin end of 28 d. By the necropsy study, the gross histopatho-
penetrated in vivo logical changes of the tested animal organs were observed and
Sefsol-218 oil containing AmB in NE and rhodamine 123 compared with normal anatomy of the major visceral organ.
loaded NE gel (RA-AmB-NE gel) formulations has been Major organs like liver, kidney and heart were collected and
designed to investigate the effective drug distribution and weighed carefully after dissection. The preserved tissue
probe rhodamine 123 in the skin by confocal laser scanning samples in formalin (10% v/v) were preserved and processed
microscopy (CLSM) where 0.1% w/v rhodamine 123-loaded for the gross histopathological study by sectioning of 5 mm
dye nanoformulations were prepared. Rats were randomly thickness. Then, these individual organ specimen was stained
grouped, and each group has three rats. The skin of the with hematoxylin and eosin. The excised skin and isolated
dorsal region was shaved. NE containing probe (100 ml), organs were preserved in 10% formalin, and sections were
RA-AmB-NE gel (100 mg) and 0.1% w/v solution (100 ml) fixed as well as blocks were made using the procedure as
solely as marker rhodamine 123 were applied in defined area reported (Chen-Yu et al., 2012).
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 Results and discussion
gel and NE formulation, the dye was carefully dressed to
Physicochemical characteristics studies
remove the applied formulation with cotton bud at 24 h. Now,
rats were subjected to sacrifice ethically. The rat’s excised In the present investigation, various NE formulations were
skin was blotted and washed thrice using ethanol on inactive developed and screened out for further characterization and
paper. The applied area was then removed into the pieces of evaluation as listed in Table 1. The highest AmB solubility
DOI: 10.3109/10717544.2014.933284 Nanoemulsion gel for topical delivery of AmB 647
Table 1. Selected nanoemulsions with their globular size, zeta potential, polydispersity index and viscosity values.

Formulation Oil Smix Water Mean droplet Polydispersity Zeta potential Viscosity
code (% w/w) ratioa (% w/w) size (nm) index (mV) (cP) pH
b
F-I 10 (25) 2:1 65 112.3 ± 1.8 0.235 42.05 26.38 ± 1.2 6.5
F-II 13 (22) 2:1c 65 167.7 ± 2.6 0.614 36.38 32.52 ± 1.8 6.6
F-III 10 (30) 1:2b 70 106.0 ± 1.5 0.351 35.03 38.7 ± 1.3 6.8
F-IV 12 (30) 1:3c 58 86.6 ± 2.0 0.741 34.94 41.51 ± 1.5 6.0
F-V 10 (25) 1:3a 65 76.2 ± 1.4 0.303 31.48 39.01 ± 1.4 7.4
F-VI 10 (25) 1:2c 65 172.6 ± 2.1 0.435 30.14 33.11 ± 1.7 6.6

Value represented as mean ± SD (n ¼ 3).

a
Transcutol-P as cosurfactant.
b
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.

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

Amphotericin B Rhodamine 123 Nanoemulsion Carbopol 980 gel Final ratio

Formulations (mg) (RA) (mg) (ml)a (g)b (a:b)
Placebo-NE gel – – 5 5 1:1
AmB-NE gel 10 mg – 5 5 1:1
RA AmB-NE gel 10 mg 5 5 5 1:1
AmB solution in DMSO 0.1 mg/ml – – – –
Rhodamine 123 solution – 0.1%w/v – – –

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 combin- Table 3. Characterization of nanoemulsion gel (AmB-NE gel).
ation with DMSO was 55.82 ± 0.6 mg/ml than other excipi-
ents. In the Table 1, it has been depicted that formulation F-V Parameters Placebo NE gel AmB-NE gel
was the most suitable among all selected formulation for Particle size (nm) 93.17 ± 8.5 97.04 ± 7.4
further characterization. It was evaluated for their globular PI 0.17 ± 0.02 0.19 ± 0.01
size (76.2 ± 1.4 nm), zeta potential (–31.48 mV), minimum Zeta potential (mV) 34.84 ± 0.8 39.27 ± 0.25
Drug entrapment efficiency (%) – 85.92 ± 3.96
polydispersity index (0.303), optimum viscosity (39.01 ± 1.4 pH 7.4 7.4
cP) and skin physiological pH (7.4). Pseudoternary phase % Spread by weight 67.93 ± 1.7 68.73 ± 3.2
diagram dictated the selection of TweenÕ 80 as surfactant and Viscosity (cP) 2388 ± 12.83 892 ± 9.64
Steady state flux (Jss, mg/cm2/h) Nil 18.09 ± 0.6
Transcutol-P as co-surfactant in development of NE. Placebo
carbopol gel, NE loaded with AmB, AmB-NE gel incorpo- The reported are mean ± SD (n ¼ 3).
rated 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, co-
Rheological study
surfactant and gel-forming gent, respectively (Table 2). The
globular size (mean) of placebo-NE gel and AmB-NE gel The rheology is a significant parameter for evaluation of the
were found to be 93.17 ± 8.5 nm and 97.04 ± 7.4 nm, respect- gel when applied topically. Spreading and adherence behavior
ively, with minimum polydispersity indices value as indicated of topical formulations to the skin surface need to be
in the Table 3. Moreover, the globular surface potential charge evaluated. Finally, the gel was again held for seven days to
of placebo-NE gel and AmB-NE gel was found to be reach pH equilibrium. After seven days, corresponding shear
–34.84 ± 0.8 mV and –39.27 ± 0.25 mV, respectively rate and shear stress values were made. The relationship
(Table 3). Based on these results, it can be suggested that between the stress (related to the force applied) and the shear
there is no significant variation (p40.05) between placebo- rate on the samples were measured to study flow behavior and
NE gel and AmB-NE gel in terms of their globular size and to determine the viscosity (Weyenberg et al., 2004a,b). A
zeta potential values. The percentage drug entrapment rotary viscometer equipped with coaxial cone and plat
efficiency of AmB-NE gel was found to be 85.92 ± 3.25. (Bohlin Visco 88) was used to determine viscosity at shear
The photograph of transmission electron microscopy in rates between 12.28 and 120.5 s1. Experimental values of
Figure 2(A and B) also revealed the morphological charac- _ s1), are
viscosity (Z, Pa s) is a function of the shear rate (y,
teristics of spherical globular oil droplets in nanoscale in both graphically represented in Figure 3, for a 0.5% (w/w)
Placebo gel as well as AmB-NE gel. These droplets are still concentration gel at pH 7.4 neutralized with triethanolamine
non-aggregated in both formulations revealing its stability (0.1 ml). The rheogram of the placebo-NE gel and AmB-NE
against Oswald ripening owing to globular collapse in gel displayed thixotropic characteristics as indicated by the
heterogeneous globular sizes. The pH of both placebo gel differences observed in ascending and descending curves of
and AmB-NE gel formulations was obtained 7.4 as depicted the rheogram (Figure 3). The viscosity of Placebo-NE gel
in the Table 3. (0.5% w/w) and AmB-NE gel (0.5% w/w) was found to be
648 A. Hussain et al. Drug Deliv, 2016; 23(2): 642–657

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 There were no significant differences (p40.05) between
892 ± 9.64 cP (coefficient of correlation ¼ 0.9922), respect- the spreadability of placebo-NE gel and AmB-NE gel
ively. Thus, the viscosity of AmB-NE gel was reduced 2.677- (0.5% w/w).
fold as compared to placebo-NE gel owing to incorporation of
NE containing AmB. However, the curves were insignificant In vitro drug release assessment
in both curves (p40.05) by the presence of AmB indicating The release of the drug from the NE and AmB-NE gel
constant consistency at same pH (7.4) and temperature formulations was absolutely significant (p50.001) in com-
(25.0 ± 1  C). It should also be noted that the correlation parison to the DS (0.01% w/w). The AmB NE gel provided
coefficient was excellent; the intercept and the slope was not higher drug release rate over period of 24 h than DS
statistically different from zero and one, respectively. Data for (Figure 4). The in vitro drug release profile showed that
evaluation was fitted in Newtonian model using Bohlin 10.96 ± 1.5% w/w drug of the formulation from the AmB-NE
software: Visco 88 to get the above Newtonian viscosity value gel, whereas 49.12 ± 1.8% w/w and 99.97 ± 2.3% w/w drug
and coefficient of correlation. were released from the NE and AmB solution, respectively,
within initial two hours. This revealed that rapid release of the
Spreadability study
drug from DS indicated absence any interaction of the drug
Spreadability is a pivot for transdermal formulation (Chow with dialysis membrane in given set of experimental condi-
et al., 2008). In this study, the optimized semisolid AmB-NE tion. Higher release rate of AmB from the NE could be
gel exhibited maximum percentage of spread by weight attributed to the smaller globular size for larger surface area
(68.73 ± 3.2) assuring and suggesting practically excellent and permitting drug release rate. Although, the drug release
spreadability behavior to the skin as shown in Table 3. rate from the AmB-NE gel and NE (pH 7.4) were slow as
DOI: 10.3109/10717544.2014.933284 Nanoemulsion gel for topical delivery of AmB 649

compared to DS, which was statistically significant (p50.05). AmB-NE gel as shown in Figure 5 and compared with DS. It
Also among formulations, possible reason for this signifi- was observed that the cumulative amount of drug permeated
cantly different release rate of NE is its lower value of at the end of 24 h was found to be 254.161 ± 1.45 mg,
viscosity as well as its smaller globular size. Thus, NE 870.42 ± 4.2 mg and 999.81 ± 7.3 mg for AmB DS, NE (pH
(42.12%) and AmB NE gel (10.96%) formulation had shown 7.4) and AmB-NE gel, respectively (Figure 5). The NEs are
2.0- and 9.12-fold slower drug release, respectively, as known to provide increase permeation rate and decreased lag
compared to DS (99.97%) in first 2 h suggesting controlled times by altering both the lipophilic and the polar pathway by
and extended release profile. The release profile data as synergistic interaction of vehicle component with the SC
treated in zero-order, first-order, Higuchi and Korsmeyer– (Godwin et al., 1997; Shin & Choi 2005). Moreover, the
Peppas mathematical models to evaluate the release pattern permeation flux rate of AmB had been enhanced in AmB-NE
from carrier systems and the drug release mechanism. The gel (18.09 ± 0.6 mg/cm2/h), NE (15.74 ± 0.4 mg/cm2/h) as
values of correlation coefficient of Korsmeyer–Peppas model compared to DS (4.59 ± 0.01 mg/cm2/h) as listed in the
for the obtained release data were greater than 0.96 in NE and Table 4. This might be due to greater extraction efficiency
AmB-NE gel except DS. The drug release pattern from AmB- of skin lipid by Transcutol-P. The penetration enhancement of
NE gel, NE and AmB NE-gel showed zero-order release lipophilic drug by alcohol is due to higher solubilization
kinetics with a best fit r2 value 0.9965, 0.9886 and 0.9815, power of the drug substance in the lipophilic area of the SC
respectively, whereas DS followed first order release kinetics because of the presence of DMSO enhancer. Significant
(r2 ¼ 0.9997) during first initial 2 h. enhancement of the permeation was achieved by the use of
5% DMSO (Reddy & Ghosh 2001). The permeability
In vitro skin permeation study potential of AmB-NE gel was determined and results clearly
The in vitro skin permeation parameters of the test formu- displayed that the flux rate of AmB-NE gel and NE were
lations and DS were determined as the results depicted in found to be 3.9- and 3.5-fold higher, respectively, with respect
Figure 5. Permeation parameters are listed in the Table 4. The to the DS. The flux values obtained for NE and AmB-NE
permeation profiles of AmB through rat skin from NE and 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 compos-
ition 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 Jss1 (mg/cm2/h) ER1

AmB-NE gel 18.09 ± 0.6 10.42
Nanoemulsion 15.74 ± 0.4 8.97
Amphotericin B (drug) solution 4.59 ± 0.01 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.
Figure 4. In vitro cumulative amount of amphotericin B release through ER1 ¼ Enhancement ratio; it is ratio of transdermal flux from formula-
dialysis membrane. tion to drug solution.

Figure 5. Cumulative amount of amphotericin

B permeated through albino rat skin.
650 A. Hussain et al. Drug Deliv, 2016; 23(2): 642–657

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.

in the DSC report (Figure 6A). The SC lipid transition at

Ex vivo globule-skin interaction
59  C (T1) and 65  C (T2) were not appeared from the DSC
Ex vivo globule–skin interaction using the differential thermogram of negative control SC samples (Figure 6B) with
scanning calorimetry (DSC) was studied. To elucidate the a broad new transition at 77.52  C. DSC results demonstrated
mechanism of the drug permeation across the highly hydro- that efficient conformational changes had been developed by
phobic strata of the skin AmB gel and NE-treated formulation the AmB gel formulation on SC lipids leading new respon-
were subjected to DSC study. Furthermore, the SC intercel- sible characteristics endothermic transition peaks at T1
lular lipids were also studied in term of thermal properties. (59  C) and T2 (65  C). The absence of peaks T1 and T2 in
The most common SC acts as the highly crystalline potential both the negative control skin samples and AmB-NE gel
barrier to the diffusion of the drugs. It essentially consists of treated verified the intercellular lipid transition associated
flattened keratinocytes embedded in a matrix of multi with the skin (Zellmer et al., 1995; Carelli et al, 1998;
lamellar lipid bilayers (Elias, 1996). Furthermore, it was Changez, et al., 2006). Hence, our results suggested that
suggested that cholesterol and lipids with long saturated acyl AmB-NE gel caused significant modification in the lipid
chains (e.g. free fatty acids and ceramides) predominate in the architectural structures of SC by lipids dissolution.
barrier layer (Bowser & White, 1985). The DSC profile of the
control, AmB-NE gel treated and Placebo (negative control)
The erythrocyte hemolytic study
rat skin SC, respectively, are shown in Figure 6(A–C). In this
investigation, it has been demonstrated that Figure 6(C) shows To find out the hemobiocompatibility, NE, AmB-NE gel, DS,
(control) the three typical endothermic transition temperatures sefsol-218 and others formulation components were subjected
at 59  C, 65  C and 114.38  C with the first peak being to hemolytic study. The extent of lysis induced by different
attributed to either lipid lamella phase transition from a formulations and its components after incubation with RBCs
crystalline to a gel-like phase or melting of sebaceous lipids, is shown in Figure 7. On incubating human erythrocytes with
the second peak corresponding to the transformation from a Triton X-100 (as positive control) led to 100% hemolysis over
lamellar to disordered state in the lipid structure and protein- the period of 12 h. In case of negative control, normal saline
associated lipid transition from gel to liquid state, respect- caused no significant (p50.05) hemolysis. After incubation
ively, the third peak being allied with the conformation with 5 lg ml1 concentration of AmB-NE gel led to
change of protein (Golden et al., 1986). In the rat skin treated insignificant RBC lysis (19.3 ± 0.95%) compared to normal
with AmB-NE gel, it has been found that the SC lipid saline (negative control, 18.04 ± 0.1%) after 24 h. But, the
transition at 59  C (T1) and 65  C (T2) extremely reduced and erythrocytes lysis in the presence 5 lg/ml of NE
broadened new characteristic transition at 101.24  C observed (16.14 ± 0.07%) and sefsol-218 oil (12.6 ± 0.5%) was
DOI: 10.3109/10717544.2014.933284 Nanoemulsion gel for topical delivery of AmB 651
Figure 7. In vitro RBC lysis following
incubation of RBCs with nanoemulsion and 100
excipients.

80

% Hemolysis
60

40

20

n
8

0
E
el
tio

eb

tio
21

10
N
-g
lu

lu
ac

X-
ol

E
so

so
Pl
N
fs

n
Sa

ito
g

e
B
ru

lin
Am

Tr
D

Sa
Components and formulations

significantly (p50.01) low (Figure 6). Moreover, hemoglobin anionic RBCs membranes. Non-hemolytic characteristic of
release in the placebo NE after 24 h incubation with the the NE system is to be considered as very attractive,
erythrocytes was found to be insignificant as compared to especially when administered transdermally and topically.
normal saline solution group. This suggested that NE
components are unable to interact with human erythrocytes In vitro antifungal activity studies of
at physiological level. The controlled and extended release of nanoformulation containing AmB
AmB from NE and AmB-NE gel rationalized the minimum The in vitro antifungal activities of AmB formulations were
erythrocytes lysis as comparison to Triton X-100. This could assayed against three different fungal strains such as C.
be explained based on the fact that gel and NE formulation albicans, A. niger and A. fumigatus. Among these fungal
both are safe and biocompatible physiologically. Free drug strains, C. albicans was found to be more sensitive fungal
induced hemolysis itself. strain than others. All nanoformulations prepared in this study
exerted significantly (p  0.001) higher antifungal activity in
SEM of erythrocyte
comparison with reference drug AmB solution in DMSO as
All of the lipids in erythrocytes are present in the stroma. shown in Table 5. The NE, DS, Transcutol-P (5%v/v) and
These erythrocytes are analogous to large liposomes, and it AmB-NE gel formed uniform growth inhibition zones against
seems that peroxidation was the main factor observed in lysis A. niger strain (Figure 9C) as compared with that of other two
(Welles et al., 1977). To investigate, the capability of fungal strain (Figures 9A and B), thus showing the higher
formulation and its component to do so that it leads to sensitivity of the formulations. In the Figure 9(A), it has been
hemolysis was apparently determined by SEM. The morpho- seen that NE and AmB-NE gel caused extended growth
logical changes in RBCs cell dictates its stability in presence inhibition zone due to nanoscale size globule leading to
of formulations. Hence, to investigate erythrocytes changes profound diffusion as compared to other two strains. The zone
were exposed to Triton X-100 and NE (pH 7.4) at a of inhibition was found to be enhanced with increasing the
concentration of 5 mg/ml. SEM images for control RBCs, concentration of Amp-B. AmB-loaded NE and AmB-NE gel
Triton X-100 and NE are Figure 8(A, B and C), respectively. showed maximum antifungal activity than DS against
As shown in Figure 8(A), RBCs exposed to the normal saline C. albicans and A. fumigatus. Although least sensitivity was
was found to be normal biconcave disc shaped. In contrast, observed against A. niger as shown in the Figure 9(C) as
positive control Triton X-100 caused significant changes at compared to other strain. Moreover, in some literature, it has
this concentration leading to development of spiny outgrowth. been reported that liposomal AmB and free AmB had
The presence of discocytes and many abnormal cells with comparable antifungal activities against various fungi such
spiny protuberances have been seen as shown in Figure 8(B). as Candida spp., Aspergillus spp. and Fusarium spp [Fukui
The NE formulation observed with absence of such outgrowth et al, 2003a). The transfer mechanism of AmB from lipidic
as lysis indicator. The role of sefsol-218 in protecting the carriers to fungal cells was not fully understood. The similar
system from hemolysis is not clear at this moment. However, or higher anti-fungal activities of AmB-loaded NE and AmB-
sefsol-218 is thought to be non interacting component with NE gel compared to DMSO and DS might be explained by the
 652
A. Hussain et al.

Skin irritation studies

Triton X-100 (c) nanoemulsion concentration of 5 mg/mL for1 h.

than to cholesterol in LNPs (Fukui et al., 2003a,b and c).

due to its higher affinity to ergosterol in fungal cell membrane
readily released in the presence of ergosterol-rich fungal cells
the presence of fungal cells. Transcutol P showed very least

acceptability by the patients has been limited when any

been published in the previous literature. Hence, it has been

and results have been listed in Table 6. Its utility and

The irritation potential of topical formulations was evaluated,
hyphae, which might be the main potential cause of higher
are readily available to ergosterol composition of fungal
fumigatus and A. niger. Drug incorporated in oily phase of NE
ZOI against C. albicans and completely absent against A.

reported by the author that AmB loaded into LNPs could be

growth inhibition against fungal strain. Similar finding had
difference in AmB release from the liposomal lipid carriers in
Figure 8. SEM of red blood cells exposed to (a) untreated control (b)
Table 5. Comparison of inhibitory activities against different fungal species by the agar well diffusion method.

Growth inhibition zone (mm)a

Candida albicans (MTCC 4748) Aspergillus fumigatus (MTCC 6500) Aspergillus niger (MTCC 282)
Formulations 0.125 mg/ml 12.5 mg/ml 25.0 mg/ml 50.0 mg/ml 0.125 mg/ml 12.5 mg/ml 25.0 mg/ml 50.0 mg/ml 0.125 mg/ml 12.5 mg/ml 25.0 mg/ml 50.0 mg/ml
Nanoemulsion 0.7 ± 0.01 2.2 ± 0.2 3.2 ± 0.11 4.8 ± 0.24 0.3 ± 0.09 1.4 ± 0.13 2.6 ± 0.7 3.5 ± 0.2 0.0 ± 0.0 1.2 ± 0.1 2.8 ± 0.4 3.1 ± 0.7
DMSO (5% v/v) 0.3 ± 0.02 1.1 ± 0.23 2.5 ± 0.09 2.9 ± 0.17 0.4 ± 0.07 0.6 ± 0.6 2.3 ± 0.32 2.8 ± 0.8 0.7 ± 0.1 1.7 ± 0.8 2.3 ± 0.1 2.8 ± 0.22
Placebo 1.2 ± 0.08 1.2 ± 0.1 1.3.0 ± 0.6 1.4 ± 0.5 1.0 ± 0.05 1.2 ± 0.06 1.1 ± 0.08 1.2 ± 0.1 0.8 ± 0.02 1.0 ± 0.1 1.0 ± 0.2 1.40 ± 0.1
nanoemulsion
Transcutol Pb 0.2 ± 0.07 0.3 ± 0.05 1.3 ± 0.06 1.6 ± 0.2 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0
AmB solution 0.4 ± 0.1 1.3 ± 0.03 1.7 ± 0.05 2.8 ± 0.21 0.4 ± 0.03 1.4 ± 0.2 3.1 ± 0.1 3.2 ± 0.2 0.6 ± 0.08 1.2 ± 0.2 1.8 ± 0.2 2.2 ± 0.5
AmB-NE gel 0.5 ± 0.1 1.7 ± 0.01 2.9 ± 0.12 4.3 ± 0.28 0.4 ± 0.05 1.5 ± 0.8 2.7 ± 0.5 3.5 ± 0.3 0.7 ± 0.05 1.1 ± 0.6 2.3 ± 0.6 3.4 ± 0.12
Placebo gel 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0
a
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.
Drug Deliv, 2016; 23(2): 642–657
DOI: 10.3109/10717544.2014.933284 Nanoemulsion gel for topical delivery of AmB 653

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 24 h 48 h 72 h Visualization of formulations into the skin
Plain gel (negative control group I) 0 0 0 penetrated in vivo
Drug solution (group II) 0 0 1
Aqueous formalin solution 0.8% v/v (group III) 2 3 3 Presently, CLSM has become a well established technique to
AmB-NE gel (group IV) 0 0 0 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
irritation or erythema is observed on topical application. parameters, CLSM was performed. Figure 10 reveals the
Hence, any topical delivery system of AmB should be free of fluorescence images of sections (vertical) of albino rat skin
these erythematic reactions. In this study, the results showed after being treated with rhodamine-loaded NE, RA-AmB-NE
that no severe irritation symptoms such as erythema (redness) gel and rhodamine 123 solution at 24 h (Figures 10A–C).
and edema (swelling) during 72 h except reference positive Apparently, it was observed that dye content in sefsol-218 oil
control group III. After application, all the scores were zero of the NE depended on dye distribution in the skin. After 24 h,
for placebo/plain gel (group I), DS (group II) and AmB-NE the fluorescence images of dye distribution from NE and
gel (group IV) (Table 6). The reference formalin aqueous RA-AmB-NE gel was significantly high in the SC, viable
solution triggered itching and redness at the applied area, epidermis and deeper area of dermis. On the other hand,
resulting in visible skin irritation and inflammation with the dye solution was merely confined to the SC and upper
group III, calculated to be 1 score. Hence, skin irritation study viable epidermis as shown in Figures 10(A–C). The amount
revealed that neither polymeric plain gel alone nor NE of drug released from NE and its gel formulation mainly
containing drug incorporated in gel formulation (AmB-NE depends on the organic phase content in the NE. The higher
gel) exhibited any noticeable irritation or inflammation on or the oil loading, the higher the drug released is obtained
654 A. Hussain et al. Drug Deliv, 2016; 23(2): 642–657

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 nephrotoxicity. Plasma drug concentration should be mini-
drug release into the deeper area of rat skin could be mized and topical formulation fabricated to enhance epider-
explained that the dye solution application on the skin is mal drug permeation. However, alternative novel carrier
insufficient to form any film or creamy texture. Feasibility of systems like particulate carrier are rapidly removed from the
fabricating NE and RA-AmB-NE gel of AmB was improved systemic circulation by the cells of reticulate endothelial
drug penetration into the skin. This was achieved by phagocyte system (Lemke et al., 2005). On acute and
formation of a monolayer film on the skin and simultaneously repeated application of the NE, AmB-NE gel and Placebo
the loss of water induced the lipid modification leading to the gel did not reveal any abnormalities in the test animal groups
drug expulsion from the formulation. In this study, drug at the end of 28 d of study. The test groups were free from
penetration was significant. The results are also consistent any reaction at the application site and mortality. Moreover,
with the in vitro drug permeation study through the albino rat the histopathological examination results of major organs
skin for the same formulations. (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
In vivo acute and repeated-dose dermal toxicity study
owing to very small quantity of drug being absorbed into the
The most important aim in designing of NE based topical blood circulation after topical application of AmB from NE
delivery of AmB formulation was to resolve its potential and NE gel, and major proportion of drug being remained into
DOI: 10.3109/10717544.2014.933284 Nanoemulsion gel for topical delivery of AmB 655

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.
656 A. Hussain et al. Drug Deliv, 2016; 23(2): 642–657

the skin as evident of confocal laser microscopy results of the Dubey V, Mishra D, Dutta T, et al. (2007). Dermal and transdermal
delivery of an anti-psoriatic agent via ethanolic liposomes. J Contr Rel
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Elias PM. (1996). The stratum corneum revisited. J. Dermatol 23:756–8.
Conclusion Espuelas MS, Legrand P, Irache JM, et al. (1997). Poly(e-caprolacton)
nanospheres as an alternative way to reduce amphotericin B toxicity.
AmB is gold standard antifungal drug and used in the Int J Pharm 158:19–27.
Formariz TP, Sarmento VHV, Silva-Junior AA, et al. (2006).
treatment of wide variety of dermatological fungal infection.
Doxorubicin biocompatible O/W microemulsion stabilized by mixed
Higher molecular weight, least aqueous solubility and poten- surfactant containing soya phosphatidylcholine. Colloids Surf B
tial nephrotoxicity like problems have limited its clinical Biointerfaces 51:54–61.
application in systemic treatment. The objective of the Formariz TP, Urban MCC, Silva-Júnior AA, et al. (2005).
investigation was achieved by adopting alternative topical Microemulsões e fases lı́quidas cristalinas como sistemas de liberação
de fármacos. Braz J Pharm Sci 41:301–13.
route of administration of AmB and loading in the nanoscale Fukui H, Koike T, Nakagawa T, et al. (2003a). Comparison of LNS-
NE carrier system for enhanced skin permeation. The in vitro AmB, a novel low-dose formulation of amphotericin B with lipid
drug release profile results showed sustained release of AmB nano-sphere (LNSÕ ) with commercial lipid-based formulations. Int J
effectively as compared to free DS. Ex vivo skin permeation Pharm 267:101–12.
Fukui H, Koike T, Saheki A, et al. (2003b). Evaluation of the efficacy
improved enhanced skin permeation of NE and AmB NE-gel and toxicity of amphotericin B incorporated in lipid nano-sphere
to facilitate drug permeation after fabricating AmB NE gel of (LNSÕ ). Int J Pharm 263:51–60.
drug using Transcutol-P and TweenÕ 80 combination. This Fukui H, Koike T, Saheki A, et al. (2003c). A novel delivery system for
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Declaration of interest topical drug delivery for an antifungal drug. Drug Dev Ind Pharm
The authors report no conflicts of interest. The authors alone 40:527–41.
Hussain A, Samad A, Singh SK, et al. (2014). Enhanced stability and
are responsible for the content and writing of this article. permeation potential of nanoemulsion containing sefsol-218 oil for
topical delivery of amphotericin. B Drug Dev Ind Pharm. [Epub ahead
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