Colloids and Surfaces B: Biointerfaces 181 (2019) 623–631

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Colloids and Surfaces B: Biointerfaces

journal homepage: www.elsevier.com/locate/colsurfb

Itraconazole lipid nanocapsules gel for dermatological applications: In vitro T

characteristics and treatment of induced cutaneous candidiasis
Nabila A. El-Sheridya, Alyaa A. Ramadanb, Amira A. Eidc, Labiba K. El-Khordaguib,
⁎

a
Research and Development Section, European Egyptian Pharmaceutical Industries, Alexandria, Egypt
b
Department of Pharmaceutics, Faculty of Pharmacy, Alexandria University, Alexandria, 21521, Egypt
c
Department of Dermatology, Venereology and Andrology, Faculty of Medicine, Alexandria University, Alexandria, 21521, Egypt

ARTICLE INFO ABSTRACT

Keywords: There is a growing clinical demand for topical itraconazole (ITC) delivery systems because of the expanding
Itraconazole potential of the drug for topical fungal and non-fungal applications. Lipid-based nanocarriers offer great promise
Lipid nanocapsules in this respect. In the present study, a new topical ITC gel based on lipid nanocapsules (LNC) was developed. ITC-
Nanostructured lipid carriers LNC were compared to ITC-loaded nanostructured lipid carriers (ITC-NLC) with more established benefits as
Candidiasis
topical vectors. Both nanocarriers showed high entrapment efficiency (EE > 98%). Compared to ITC-NLC, the
Rat model
ITC-LNC showed a significantly smaller particle size (∼50 vs 155 nm), narrower size distribution (0.09 vs 0.38),
faster initial release rate under sink conditions and greater in vitro antifungal activity against Candida albicans (C.
albicans) (inhibition zone 29.4 vs 26.4 mm). ITC-LNC and ITC-NLC-based gels significantly enhanced the dermal
retention of ITC in excised human skin relative to a conventional ITC gel. Histopathological assessment of a 14-
day treatment of induced cutaneous candidiasis in a rat model indicated efficacy of the gel preparations. Fungal
elements developed in the superficial epidermal skin layer were cleared by the end of treatment. Equally im-
portant, no histopathological changes in the epidermal and dermal layers of rat skin were observed. Findings of
this study verified efficacy of topical ITC in the treatment of superficial fungal infections as well as effectiveness
of LNC as biomimetic nanocarrier for dermal drug delivery. Combining ITC and LNC would present a bioactive
nanocarrier system with good potentials for fungal infections and other skin applications.

1. Introduction administration of antifungal drugs [12,13]. Moreover, dispersing lipid

nanocarriers encapsulating hydrophobic antifungal drugs in non-greasy
The clinical interest in topical itraconazole (ITC) for long-term hydrogel vehicles generate formulations that combine the benefits of
therapy is increasing, a direct result of the rapidly extending fungal and both nano- and gel technologies [14]. These may eventually enhance
non-fungal uses of the drug in addition to the pharmacodynamic and antifungal activity and improve patient compliance [15,16]. In this
pharmacokinetic interactions associated with oral therapy [1]. Aside respect, lipid-based nanocarriers including microemulsions [4,17], li-
from cutaneous infections [2,3], topical ITC has been investigated for posomes [18] and solid lipid nanoparticles [19,20] were shown to en-
onychomycosis [4], fungal infections of the cornea [5,6] and the vagina hance the topical antifungal activity of ITC.
[7], as well as for the management of ocular neovascularization in Lipid nanocapsules (LNC) are a relatively new lipid-based biomi-
diabetic retinopathy [8]. Furthermore, there is developing proof af- metic nanocarrier consisting of a medium chain triglycerides oil core
firming anticancer activity of ITZ with expanding opportunity for re- that is stealth-coated with a relatively rigid phospholipid/ethoxylated
purposing the drug as anticancer agent. In fact, topical ITC is currently surfactant shell [21]. LNC are prepared by a low energy phase-inversion
under active investigation for the treatment of skin cancer [9,10]. method using approved ingredients. Small size (< 100 nm), narrow size
In topical drug delivery, advanced nanoformulations, particularly of distribution, high interfacial stability and deformability confer great
the lipid-based type, have consistently shown clinically relevant bene- stability to their dispersions [22,23]. LNC have been generally explored
fits because of their versatility, biocompatibility and ability to target a as effective nanocarrier for parenteral [24] and oral drug delivery
drug payload to deep skin layers [11]. A variety of lipid nanocarriers [25–27]. Promising outcomes have also been achieved in topical ap-
has attracted attention as novel delivery systems for the topical plications [28,29]. However, the potentials of LNC for the dermal

⁎
Corresponding author.
E-mail address: [email protected] (L.K. El-Khordagui).

https://doi.org/10.1016/j.colsurfb.2019.05.057
Received 27 March 2019; Received in revised form 20 May 2019; Accepted 23 May 2019
Available online 24 May 2019
0927-7765/ © 2019 Elsevier B.V. All rights reserved.
N.A. El-Sheridy, et al. Colloids and Surfaces B: Biointerfaces 181 (2019) 623–631

delivery of antifungal agents have not yet been explored. 2.3. Characterization of the lipid nanocarriers
In the present study, it was of interest to investigate the dermal
delivery of ITC formulated as a new LNC-based gel. A nanostructured 2.3.1. Colloidal properties
lipid carriers (NLC) gel formulation [30] was used for comparison. The z-average particle size (PS), polydispersity index (PdI) and zeta
Compared to LNC, NLC are lipid-based nanocarriers composed of both potential (ZP) of the test formulations (LNC, ITC-LNC, NLC and ITC-
solid and liquid lipids as a core matrix and are known to enhance the NLC) were measured by photon correlation spectroscopy (PCS
dermal targeting and antifungal activity of azole drugs including oxi- Zetasizer® Nano ZS Series DTS 1060, Malvern Instruments S.A.,
conazole and voriconazole [31,32]. ITC-loaded LNC and NLC were as- Worcestershire, UK) at a fixed angle (173°) at 25 °C using a 4 mW
sessed for physicochemical properties, in vitro antifungal activity He–Ne laser at 633 nm. Samples were diluted 1:40 v/v with filtered
against C. albicans, ex-vivo permeation of ITC through abdominal deionized water prior to measurements. Results are the average of three
human skin and efficacy of treating induced cutaneous C. albicans in- determinations.
fection in a rat model.
2.3.2. Transmission Electron microscopy (TEM)
2. Materials and methods The morphology of ITC-LNC and ITC-NLC was examined by trans-
mission electron microscopy (TEM) using JEOL, JEM-100 CX Electron
2.1. Materials Microscope, Tokyo, Japan. Samples of the diluted (1: 40) test for-
mulations were placed on a cupper grid, air-dried and negatively
Itraconazole was purchased from Neuland laboratories LTD India. stained with a drop of phosphomolybdate solution (1%). Shots were
Labrafac™ lipophile WL 1349 (Labrafac, caprylic-capric acid triglycer- taken at X 7500 at 80 kV.
ides, European Pharmacopeia, IVth, 2002), Compritol 888 ATO® and
Transcutol P® were a generous gift of Gattefossé S.A (Saint-Priest,
2.3.3. Itraconazole entrapment efficiency (EE%)
France). Lipoid ® S75-3 (soybean lecithin, 69% phosphatidylcholine and
ITC assay: An isocratic reverse phase HPLC method with UV de-
10% phosphatidyl ethanolamine, MW 800) was provided by Lipoid
tection at 225 nm was used for the determination of ITC [34]. Separa-
(Ludwigshafen, Germany). Kolliphor® HS 15 (Solutol® HS 15), a mixture
tion was carried out on Inertsil C-18 column (ODS, 250 × 4.6 mm, 5μ).
of free polyethylene glycol 660 and polyethylene glycol 660 hydro-
The mobile phase was a mixture of tetrabutylammonium hydrogen
xystearate, European Pharmacopeia, IVth, 2002 and Poloxamer 407 ®
sulphate buffer (2.72% w/v in purified water) and acetonitrile (40: 60)
(Lutrol F146) were provided by BASF (Ludwigshafen, Germany).
flowing at a rate of 10 mL/min. The injection volume was 20 μL and
Hydroxyethyl cellulose (Natrosol® Pharma 250 HX-PH) (Ashland
retention time 4 min. A calibration graph was constructed over the ITC
Covington, USA), Carbopol® 974 (Goodrich Chemicals - co., Ceveland,
concentration range 4–15 μg/mL in methanol using a standard ITC so-
Ohio, USA), triethanolamine (Merck, Darmstadt, Germany), methanol
lution (1 mg/100 mL) in methanol.
and acetonitrile (Fisher Scientific, Waltham, USA) and tetrabutyl am-
Determination of EE%: An accurately weighed amount (5 g) of LNC
monium hydrogen sulphate (Sigma-Aldrich, Missouri, USA) were used
and NLC formulations was ultracentrifuged at 6000 rpm at 5 °C for
in the study. All other chemicals were of analytical grade.
10 min using Vivaspin6® centrifugation tubes, MW membrane cut off
100.000 Da, Sartorius, GmbH. The free drug in the filtrate was analyzed
2.2. Preparation of colloidal nanocarriers by HPLC. The total drug content was determined by treating LNC and
NLC formulations with methanol followed by sonication for 60 min and
2.2.1. Preparation of lipid nanocapsules (LNC) heating in a water bath at 70 °C to dissolve the lipids. Samples were
Blank lipid nanocapsules (LNC) were prepared by the phase inver- centrifuged for 10 min at 6000 rpm and filtered through 0.45 μm filter
sion and temperature cycling method [21]. Briefly, Kolliphor® HS 15 before injection into the chromatographic system. All measurements
(18.8% w/w), Labrafac (20.4% w/w), sodium chloride (1.68% w/w), were carried out in triplicate under ambient conditions. The EE% was
Lipoid® S-75 (2.8% w/w) and demineralized water (56.16% w/w) were calculated as follows:
weighed and mixed in a closed container under magnetic stirring. The
mixture was subjected to three cycles of progressive heating and Total drug content (mg)–unentrapped drug amount (mg)
EE % = x 100
cooling between 65 and 85 °C at 4 °C/minute. An irreversible shock was Total drug content (mg)
induced by two-fold dilution with cold deionized water (0–2 °C) added
to the o/w emulsion at a temperature 1–3 °C from the beginning of the
phase inversion zone (PIZ). This was followed by slow magnetic stirring 2.3.4. Differential scanning calorimetry (DSC)
of the LNC dispersion for 5 min. To prepare itraconazole-loaded LNC The thermal behavior of pure ITC, components of the lipid carriers
(ITC-LNC), 0.02% w/w ITC was added to the oil phase and the proce- and lyophilized ITC-LNC and ITC-NLC was investigated under an at-
dure completed as outlined. The LNC dispersions obtained were kept at mosphere of nitrogen purged at flow rate of 20 mL/min. DSC traces
4 °C. were recorded between 20–200 °C at a constant 20 °C/min rate fol-
lowing 1 min stabilization at 25 °C. The temperature was held at 200 °C
for 15 min. Indium was used as standard and an empty pan as reference.
2.2.2. Preparation of nanostructured lipid carriers (NLC)
For lyophilization, samples of ITC-LNC and ITC-NLC dispersions were
Blank NLC were prepared by a microemulsion method [33]. The
frozen at −80 °C in an ultra-freezer and then placed in Telstar lyo-
lipid phase of the primary microemulsion consisting of Compritol 888
philizer at −80 °C and 0.1 mbar pressure. This was decreased to
ATO, (mp ∼70 °C, 8% w/w), Labrafac (2% w/w) and Transcutol P
0.01 mbar for 24 h. Lyophilized nanocarriers were stored at room
(27% w/w), was heated to 85 °C (1–15 °C above the mp of Compritol
temperature.
888 ATO). An aqueous phase, containing Poloxamer 407 (5% w/w) was
heated to the same temperature and added to the molten lipid phase
under magnetic stirring for 30 min. The optically transparent o/w mi- 2.3.5. Fourier transform-infrared spectroscopy (FT-IR)
croemulsion obtained was diluted 20-fold with cold water (2–8 °C) Samples of pure ITC, components of the lipid carriers and lyophi-
dropwise using a glass syringe and mixed for 10 min. ITC-loaded NLC lized ITC-LNC and ITC-NLC were individually mixed with KBr in a
were prepared by dissolving the drug (0.02% w/w) in the lipid phase mortar and compressed into discs. Samples were scanned from 500 to
prior to emulsification. The drug: total lipid ratio was 1: 25. NLC dis- 4000 cm−1 using FT-IR Spectrometer (Perkin Elmer FT-IR
persions were kept at 4 °C. Spectrometer, Spectrum BX, USA).

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2.4. In vitro drug release containing ITC (0.02% w/w) were further diluted in DMSO to give a
final concentration of 20 μg/mL ITC. A 0.3 mL aliquot of C. albicans
In vitro release of ITC from ITC-LNC and ITC-NLC dispersions was ATCC 10,231 culture containing ∼10 × 107 colony forming units
determined at 37 ± 0.5 °C in a thermostatically controlled shaking (CFU/mL) was added to 100 mL of Sabouraud Dextrose Agar (SDA,
water bath at 100 rpm. A release medium consisting of PBS pH 7.4 Oxoid, UK) at 45–50 °C. The inoculated SDA was poured into
containing 30% methanol and 3% Transcutol P was developed to en- 100 mm x 20 mm Petri-dishes with flat bottoms, 25 mL each, and al-
sure sink conditions. A 0.5 mL aliquot of the dispersions was added to lowed to harden. A total of 3 cups were made uniformly in each plate
35 mL of the release medium in screw-capped bottles. Samples (5 mL) using a sterile cork borer (8 mm diameter). A 100 μL-sample of ITC
were withdrawn at different time intervals (1, 6 and 24 h) and com- solution, blank and test nanoformulations was introduced individually
pensated with a similar volume of fresh medium at the same tem- in the cups in quadruplicate. The plates were incubated at 37 °C for 24 h
perature. Free ITC was separated by ultracentrifugation using and the diameters of inhibition zones measured.
Vivaspin®6 centrifugation tubes with a MW membrane cut off
100.000 Da (Sartorius, GmbH) at 12.000 rpm for 10 min and the filtrate 2.7. Ex vivo skin permeation through human skin
was injected into the HPLC column for ITC analysis. Results are the
average of three determinations. The ITC release kinetics were de- Full-thickness human abdominal skin of patients (40–50 years old)
termined using DDsolver software. was obtained from the Department of Plastic Surgery of the Faculty of
Medicine, Alexandria University according to institutional ethical
2.5. Preparation and characterization of lipid nanocarrier-based ITC gels guidelines. The ex vivo permeation of ITC from LNC and NLC-based gels
through human skin was assessed at 32 ± 2 °C using modified vertical
2.5.1. Preparation of itraconazole LNC and NLC-based gels Franz diffusion cells [35] having a receptor compartment volume of
Gels based on ITC-LNC and ITC-NLC containing 0.02% w/w of ITC 8 mL and a donor compartment with an effective diffusion area of 3.14
were prepared by incorporating the gelling agent into the lipid nano- cm2. Subcutaneous fat was excised; the skin was washed with normal
carrier dispersions. For ITC-LNC-based gel, 3% w/w of hydroxyl saline and blotted dry between two filter papers. Skin was cut into
ethylcellulose was added portion wise to the LNC dispersion and al- pieces and the thickness measured using a micrometer. Skin samples
lowed to hydrate. The gel was kept overnight for dissipation of en- were wrapped in aluminum foil and stored in polyethylene bags at
trapped air. For ITC-NLC, 0.7% w/w of Carbopol 974 P was dispersed −20 °C in a deep freezer pending further use (within 2–3 weeks). Be-
portion wise in the NLC aqueous dispersion with gentle stirring at fore the experiment, skin samples were allowed to thaw and were
300 rpm for 15 min. After hydration, the dispersion was neutralized by soaked in PBS for 1 h.
adding 0.2% w/w triethanolamine to adjust the pH within the range The prepared human skin was fastened carefully between the donor
5.5–6.5. A control ITC gel was prepared by dissolving 0.02% w/w ITC and receptor compartments of diffusion cells with the stratum corneum
in Carbopol gel 0.7% w/w containing methanol (50% w/w) to maintain side up and held in place with a clamp. The dermal side of the chamber
the drug solubility. contained a receptor solution consisting of PBS pH 7.4 containing 30%
methanol and 3% Transcutol P. Care was taken to minimize air bubbles
2.5.2. Characterization of itraconazole LNC and NLC-based gels under skin. Accurately weighed samples of the test gels (500 mg) were
2.5.2.1. Colloidal properties. The average hydrodynamic diameter and uniformly spread over skin surface. Diffusion cells were kept at
polydispersity index (PdI) of ITC-LNC and ITC-NLC in gel formulations 37 ± 2 °C in order to maintain skin surface at 32 °C throughout the
were determined by PCS as described in Section 2.3.1. Prior to size experiment. Cells were shaken at 100 rpm in a shaking water bath.
analysis, 0.5 g of the gel was diluted in 5 mL of deionized water with Following exposure for 6 h, the skin was removed and the receptor
magnetic stirring. Data are the average of three determinations. solution collected. The donor compartment and skin surface were wa-
shed with methanol. ITC concentration in the donor and receptor so-
2.5.2.2. Transmission electron microscopy (TEM). Samples were lutions was determined by HPLC. The percentage drug retained in the
prepared by placing a drop of diluted gel formulations on a cupper skin was calculated based on the mass balance assuming that the initial
grid. The air-dried samples were negatively stained with a drop of amount of ITC in the donor phase was the sum of the drug amounts
phosphomolybdate solution (1%) before TEM imaging. remaining in the donor phase, drug retained in skin and drug in the
receptor compartment. Data are the average of three determinations.
2.5.2.3. Rheological properties and pH. Viscosity measurements were
performed using a Brookfield viscometer DVII head with Helipath 2.8. Preclinical activity against cutaneous candidiasis
attachment and spindle 6 at a speed of 6 rpm at ˜25 °C. Viscosity
measurements were carried out at different levels from top, middle and The activity of ITC-LNC and ITC-NLC and their blank counterparts
bottom of beakers containing about 100 gm of the nanocarrier-based against induced cutaneous candidiasis was assessed in Wistar adult
gels. The pH of 5 % w/w dispersions of the gels in water was male rats using untreated skin and ITC gel as controls. The protocol was
determined using a Mettler Toledo pH meter. reviewed and approved by the Institutional Review Board (IRB) of the
Faculty of Medicine, Alexandria University (Approval No 00007555).
2.5.2.4. Itraconazole content. Itraconazole content of the nanocarrier-
based gels was assayed by the HPLC method described above. Briefly, 2.8.1. Study protocol
an accurately weighed amount (1.25 g) of the two gels was diluted with Thirty six male albino rats were housed under standard laboratory
methanol and sonicated for 60 min. The procedure was continued as conditions in 12 h light/dark cycles at 25 ± 2 °C in individual cages
described in Section 2.3.3. with free access to food and water. One week prior to the experiment,
the animals were allowed to acclimatize to their new habitat. Rats
2.6. In vitro antifungal activity against Candida albicans weighing 200–250 g were divided into 6 weight- and age- matched
groups as follows:
The antifungal activity of test ITC-LNC and ITC-NLC dispersions and Group 1: Infected untreated rats (Positive control)
their respective gels (0.02% w/w ITC) was determined using the agar Group 2: ITC control gel
cup diffusion method and C. albicans as test organism. ITC solution Group 3: Blank NLC gel
(0.02% w/w) in dimethylsulfoxide (DMSO) and blank nanocarriers Group 4: ITC-NLC gel
were used as controls. The ITC solution, blank and test nanocarriers Group 5: Blank LNC gel

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Group 6: ITC-LNC gel Table 1

The ITC concentration in all ITC-containing gels was 0.02% w/w Mean size, polydispersity index (PdI) and zeta potential (ZP) of the lipid na-
and 0.5 g of the gel was applied to infected skin. nocarriers.
Properties Blank LNC ITZ-LNC Blank NLC ITZ-NLCs
2.8.2. Preparation of Candida albicans suspension and induction of Mean size (nm) 46.2 ± 0.30 47.9 ± 4. 6 110.4 ± 5.7 154.7 ± 10.5
infection PdI 0.05 ± 0.01 0.09 ± 0.05 0.31 ± 0.06 0.38 ± 0.02
A fresh subculture of a reference C. albicans isolate (ATCC 90028) ZP (mV) −13.6 ± 0.70 −15.0 ± 0.07 −15.1 ± 4.6 −19.4 ± 4.1
was allowed to grow on SDA at 30 °C for 48 h. Subsequently, the cells of
fresh subculture were collected and washed with sterile saline. A fluid N.B. Data expressed as the mean ± standard deviation of at least three ex-
periments.
suspension in brain heart infusion broth (Oxoid, UK) was prepared to
reach a final concentration of 107 CFU/mL of C. albicans [36,37]. To
achieve a heavy cutaneous infection, animals were immunosuppressed 3.1. Characteristics of itraconazole LNC and NLC
by intraperitoneal injection of cyclophosphamide (100 mg/kg body
weight) administered three days prior to induction of fungal infection LNC were prepared by the phase inversion method using Labrafac,
[37]. Kolliphor® HS 15 and the lipophilic surfactant Lipoid as stabilizer. The
In all rats, a marked 3.0 cm2 area of dorsal hair was shaved with an total lipid and surfactant contents were 20.0% w/w and 21.6% w/w
electric hair clipper and infected with 100 μL of the C. albicans sus- respectively. NLC were prepared using a microemulsion method using
pension by direct inoculation and gentle rubbing onto the skin with a Compritol® 888 ATO as the solid lipid component, Labrafac as the liquid
sterile, cotton-tipped swab until no more fluid was visible. This was lipid component and Poloxamer 407 as stabilizer. The total lipid and
followed by injection of 50 μL of the C. albicans suspension in- surfactant contents were 10.0% w/w and 32% w/w respectively. Both
tradermally. An occlusive dressing held in place with an extra-adherent preparations contained 0.02% w/w ITC.
tape was applied to the infected area for 48 h before treatment. All
animals were observed for signs of cutaneous fungal infection including 3.1.1. Colloidal properties
erythema, papular or nodular lesions, white substance over affected The colloidal properties of nanocarriers are shown in Table 1. The
areas, crusting, maceration, ulceration and creamy satellite pustules at mean diameter of blank LNC and NLC was 46.2 ± 0.30 and
margins of affected areas [38]. 110.4 ± 5.7 nm respectively. Data obtained corroborated literature
data for size of LNC [41] and NLC [42] with similar composition and
prepared under comparable conditions. While ITC loading resulted in a
2.8.3. Mycological examination and beginning of treatment
slight increase in LNC size (47.9 ± 4.6 nm), a notable increase in ITC-
Following development of clinical signs of infection and prior to
NLC diameter (154.7 ± 10.5 nm) was observed. Both blank and ITC-
initiation of therapy, the infected skin was scrapped and the scrapped
LNC formulations showed a low PdI, 0.05 and 0.09 respectively. Rela-
material cultured on Sabouraud Dextrose Agar (SDA). Cultures were
tively small and controllable particle size (20 nm–100 nm) with
incubated at 30 °C and examined regularly for growth. A culture was
monomodal narrow size distribution is a documented advantage of LNC
considered negative when no growth of fungal colonies was detected
[41]. In general, the mean particle size of both test formulations was
after 4 weeks [39]. Treatment began 24 h after the infection was es-
appropriate for topical application. For dermal drug delivery, lipid
tablished and test gels (0.5 g) were applied topically twice daily for 14
nanocarriers smaller than 200 nm deposit smoothly and uniformly onto
consecutive days.
the skin, forming a dense monolayer and a transcutaneous hydration
gradient which facilitates drug penetration into deeper skin layers [11].
2.8.4. Assessment of antifungal efficacy The zeta potential (ZP) of ITC-LNC and ITC-NLC was -15 mV and
This was based on histopathological examination of treated skin for -19.4 mV respectively, corroborating literature data for LNC [43] and
the presence of the fungal elements, branching pseudohyphae, clusters NLC [44,45] formulations. Although surface charge greatly contributes
of budding yeast cells (blastoconidia) [40] and for cutaneous signs of to the stability of nanostructures by preventing aggregation, steric
fungal infection including epidermal hyperplasia and inflammatory stabilization of both LNC and NLC by their tensioactive surface layer is
cellular infiltrate [38]. At the end of the treatment protocol, all animals an additional effective determinant of their colloidal stability. For ex-
were sacrificed 48 h following application of the last treatment. The 3.0 ample, LNC with a relatively low ZP were reported to maintain their
cm2 area of infected skin was excised using a sterile scalpel and skin monodispersity upon storage for several months at refrigeration tem-
biopsies were fixed in 10% neutral buffered formalin, embedded in perature [43]. Maintenance of physical characteristics of NLC with a
paraffin blocks and processed for histopathological examination. The relatively low zeta potential upon storage has also been demonstrated
fungal elements were visualized using hematoxylin & eosin (H&E) and [45,46].
Periodic Acid-Schiff (PAS) staining.
3.1.2. Transmission electron microscopy (TEM)
2.9. Statistical analysis Fig. 1 shows TEM images of ITC-LNC (Fig. 1 A) and ITC-NLC
(Fig. 1B) test formulations. Both nanocarriers generally exhibited a
Statistical analysis was performed using IBM SPSS software package spherical shape, LNC showing smaller size and more uniform size dis-
version 20.0 (IBM Corp., Armonk, NY). Data were expressed as mean tribution. TEM imaging verified particle size analysis and PdI data
± standard deviation (S.D.). ANOVA test was used for comparison of obtained by PCS.
quantitative variables among the studied groups, and p < 0.05 in-
dicated significance.
3.1.3. Entrapment efficiency (EE%)
The EE% of LNC and NLC was determined using a reported HPLC
3. Results and discussion method [34] with slight modification after validation under the study
conditions. The calibration graph of ITC indicated linearity in the range
In the present work, an antifungal ITC nanoformulation based on of 5 - 15 μg/mL with a coefficient of correlation 0.9999. Data for LNC
the relatively new lipid nanocarrier, LNC, was prepared for dermato- and NLC, obtained by ultracentrifugation, indicated high EE%
logical applications. ITC-NLC with more documented topical antifungal (99.8 ± 0.005% and 99.0 ± 0.008% respectively), attributed to
application were included for comparison. compatibility of ITC and the nanocarrier matrices. In fact, LNC

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Fig. 1. TEM images of: A) ITC-LNC and B) ITC-NLC.

efficiently entrap lipophilic drugs such as quercetin and paclitaxel in disappearance of the peak corresponding to the C]O polar head
their oil core [47,48]. As to NLC, the many imperfections in the matrix stretching at 1697.74 cm-1, verified incorporation of ITC into the lipid
facilitate drug accommodation, promoting high loading efficiency of components in both nanocarriers.
lipophilic drugs. This has been reported for ITC-loaded NLC for pul-
monary [49] and ocular [8] delivery.
3.2. In vitro ITC release

3.1.4. Differential scanning calorimetry (DSC) Release data for ITC from LNC and NLC dispersions generated bi-
Fig. 2A and B show DSC thermograms for the ITC-LNC and ITC-NLC phasic sustained release profiles (Fig. 4). Initial faster release was fol-
systems respectively. ITC is characterized by an endothermic peak at lowed by sustained ITC release, approaching 100% drug delivery in
169.3 °C with 80.560 J/g enthalpy corresponding to the melting of its 24 h. Release of lipophilic drugs from LNC and NLC involves diffusion
crystalline form. Results indicated compatibility of ITC with the lipid of the drug molecules efficiently solubilized in the oil domain, followed
components and molecular dispersion of the drug in the lipid matrix in by partitioning into an aqueous surrounding medium across a ten-
both systems. sioactive interfacial barrier. The aqueous medium provides a barrier
that precludes drug transport due to the low drug solubility in water.
3.1.5. Fourier transform-infrared spectroscopy (FT-IR) This usually results in a relatively limited burst [51,52]. However, the
FT-IR spectra for the ITC-LNC and ITC-NLC systems are shown in rather fast release rate of ITC in the present study can be attributed to
Fig. 3A and B respectively. ITC spectrum showed characteristic peaks at the composition of the release medium (PBS pH 7.4 containing 30%
3127.38, 2864.08, 1688.31, 1581.84-1550.92, 1515.34-1384.21 and methanol and 3% Transcutol P), enhancing drug partitioning into to the
971.89-943.84 cm−1 as reported [50]. These were retained in spectra of surrounding aqueous phase. Almost 100% release of ITC from pul-
the physical mixtures ITC/Lipoid S-75 (Fig. 3A) and ITC/ Compritol monary NLC within 15 min was reported in a release medium con-
888 ATO (Fig. 3B). However, spectral changes of ITC, particularly taining 20% w/w 2-hydroxypropyl-beta cyclodextrin to achieve sink

Fig. 2. DSC thermograms of ITC, nanocarrier components and lyophilized ITC-loaded nanocarriers. (A) ITC-LNC: (a) ITC, (b) Lipoid S-75, (c) ITC/Lipoid S-75
physical mixture (1:1), and (d) lyophilized ITC-LNC. B) ITC-NLC: (a) ITC, (b) Compritol 888 ATO, (c) ITC/ Compritol 888 ATO physical mixture (1:1) and (d)
lyophilized ITC-NLC.

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Fig. 3. FT-IR spectra of ITC, nanocarrier components and lyophilized ITC-loaded nanocarriers. (A) ITC-LNC: (a) ITC, (b) Lipoid S-75, (c) ITZ/Lipoid S-75 physical
mixture (1:1), and (d) lyophilized ITC-LNC. B) ITC-NLC: (a) ITC, (b) Compritol 888 ATO, (c) ITC/Compritol 888 ATO physical mixture (1:1), and (d) lyophilized ITC-
NLC.

In this study, ITC-nanocarrier based gels were prepared by in-

corporating the gelling agent in the aqueous dispersion of LNC or NLC
to obtain nanocarrier gel combinations containing 0.02% w/w of ITC, a
method allowing for scale up [56]. Carbopol (0.7% w/w) was ade-
quately hydrated and swollen in the ITC-NLC dispersion because of the
relatively low lipid content (10% w/w) and higher water content. Hy-
droxyl ethylcellulose (3% w/w) was utilized as an alternative gelling
agent for ITC-LNC because of poor hydration of Carbopol in the LNC
dispersion (lipid content 20.4% w/w).
The two ITC nanocarrier-based gels were assessed for nanocarrier
size and homogenous distribution as well as gel properties, including
drug content, pH and viscosity. No significant change in the particle
size and size uniformity of ITC-LNC (50.5 ± 2.29 nm; PdI
0.221 ± 0.058) and ITC-NLC (156.3 ± 61.23 nm; PdI
0.320 ± 0.029) in the respective gels relative to the colloidal disper-
Fig. 4. in vitro release profile of ITC from lipid-nanocarrier dispersions, at sions could be observed. The pH values of ITC-LNC and ITC-NLC gels
37 °C ± 2 in phosphate buffer saline pH 7.4 containing 30% methanol and 3% were 4.4 and 5.4 respectively. Their respective viscosity was
Transcutol P. (▲) ITC-LNC and (■) ITC-NLC. Data are mean ± SD (n = 3). 113.25 Pa.s and 50.71 Pa.s (n = 3) respectively, within the range of
topical gels [57]. ITC content of ITC-LNC and ITC-NLC gels was
99.6 ± 0.55% and 98.0 ± 3.15% respectively.
conditions [53]. Data obtained in the present study showed that ITC
release from LNC and NLC would not present a rate-limiting step to the
penetration and/ or partitioning of ITC into skin upon application. Data 3.4. In vitro activity against Candida albicans
also implied that structural integrity of the ITC formulations could be
maintained for several hours to allow for skin permeation. Results for in vitro activity against C. albicans, expressed as fungal
ITC release data (0–24 h) were subjected to linear regression ana- inhibition zone diameters (IZ), are shown in Fig. 5. Images of re-
lysis using zero-order, first-order, Higuchi, and Korsmeyer-Peppas presentative plates are shown in Fig. 5A and values for IZ ± SD are
equations. Data analysis using DDsolver software according to the shown in Fig. 5B. Standard ITC solution in DMSO showed an IZ of
Korsmeyer-Peppas equation indicated a Fickian diffusion-based me- 28.41 ± 0.28 mm. DMSO included in all test formulations had IZ of
chanism for both nanocarriers. The calculated r2 value was 1 for both 12.5 ± 0.5 mm. Incorporation of ITC in 0.7% w/w Carbopol gel
ITC-LNC and ITC-NLC and the n value was 0.162 and 0.357 (n < 0.5) slightly decreased its IZ diameter, probably due to slower diffusion as
for ITC-LNC and ITC-NLC respectively. reported for antifungal ketoconazole gel [58]. Encapsulation of ITC into
LNC significantly enhanced antifungal activity (IZ = 29.37 ± 0.25
3.3. Characteristics of nanocarrier-based ITC gels mm) compared to ITC solution while a slight but significant reduction
in activity was observed for ITC-NLC (26.37 ± 0.25 mm). Incorpora-
Topical nanocarrier-based gel formulations provide pharmaceutical tion of these nanocarriers in a gel vehicle slightly reduced their activity,
combinations that allow for easier dermal application and prolonged though the difference was statistically significant (p < 0.05). The
skin contact, improving adherence and therapeutic outcomes [14]. greater antifungal activity of ITC-LNC gel despite higher viscosity of the
Different gelling agents are utilized for topical gels, Carbopol [16], gel (113.25 Pa.s) compared to the ITC-NLC gel (50.71 Pa.s), could be
cellulose polymers [54], and xanthan gum [55] being among the most attributed to their smaller size (Table 1) and faster initial release rate as
common. shown in Fig. 4. The antifungal activity of ITC was reported to depend

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Fig. 5. In-vitro antifungal activity of test ITC formulations against C. albicans expressed as inhibition zone diameter (mean ± SD, n = 4). (A) Images of representative
agar plates for (a) ITC-LNC and (b) ITC-NLC. (B) Mean inhibition zone diameters for test formulations.

on the particle size of the nanocarrier [59].

3.5. Ex-vivo skin permeation study

The skin permeability and retention of ITC formulated as conven-

tional and nanocarrier-based gels containing 0.02% w/w ITC were as-
sessed ex vivo using abdominal human skin (2.92 ± 0.34 mm) at
32 ± 2 °C. The ITC concentration in the donor and receptor compart-
ments was determined using HPLC with no interference from the skin
residues with the ITC peak. Results obtained at 6 h (Fig. 6) were used as
a surrogate indicator of skin permeation and retention of ITC. Treat-
ment of skin samples with control ITC gel (0.7% w/w Carbopol) re-
sulted in 46 ± 4.0% skin retention and minimal permeation through
full thickness skin (< 1%) at 6 h. Percutaneous absorption of ITC is
attributed to its lipophilic nature in addition to maintenance of the drug
in contact with the skin surface by the gel vehicle [29,60]. Application
of ITC as NLC in the same gel base significantly (p < 0.05) increased
skin permeation of the drug achieving 59.42 ± 0.645% retention at Fig. 6. Skin retention of itraconazole from the gel formulations under study at
6 h. Despite higher viscosity of the ITC-LNC gel, significantly greater 6 h at 32 ± 2 °C. Data are expressed as mean ± SD (n = 6).
(p < 0.05) skin retention (66.32 ± 2.46%) relative to ITC-NLC gel
was observed. Transdermal ITC delivery for both formulations was less present study has shown that LNC with a mean size of 50.5 nm in a gel
than 1%. Skin retention and dermal targeting of other azole drugs, such base significantly contributed to the skin retention of ITC, pointing to
as clotrimazole [61] and miconazole [62] was also achieved by en- factors other than the nanocarrier characteristics as determinants of
capsulation in lipid-based nanocarriers. skin permeation. ITC and other antifungal azole drugs have a great
Despite paucity of reports on LNC as topical nanocarriers, literature affinity for keratin with minimal percutaneous absorption into the
data indicate that LNC in the common size range 50–70 nm may pro- dermis or systemic circulation [65]. Thus, localized dermal drug de-
mote transdermal drug delivery [63,64]. Vectorization of hydrophobic livery or transdermal delivery would depend on the interplay of the
compounds by LNC to the keratinocytes without transdermal delivery drug molecular properties, the nanocarrier features, and the vehicle in
was reported to necessitate a size above 100 nm [29]. However, the

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Fig. 7. Periodic Acid-Schiff (PAS)-stained sec-

tions of dorsal rat skin: A: Infected untreated
rat skin (Positive control) showing pseudo hy-
phae and blastoconidia (black arrow); B: ITC
control gel-treated skin showing no evidence of
fungal infection; C: Blank NLC gel-treated skin
showing pseudo hyphae and blastoconidia
(black arrow); D: ITC-NLC gel-treated skin, E:
Blank LNC gel-treated skin and F: ITC-LNC gel-
treated skin. D – F sections showed no evidence
of fungal infection.

addition to interactions of the drug with specific skin components. Skin 4. Conclusion
permeation and retention data of ITC indicated potential of the test
lipid nanocarrier-based gels for dermal targeting of ITC with minimal Itraconazole-loaded lipid nanocapsules (ITC-LNC) presented in this
transdermal delivery. study show enhanced in vitro characteristics in terms of size, poly-
dispersity index, in vitro release profile and antifungal activity against
C. albicans compared to nanostructured lipid carriers (NLC). Topical
3.6. Preclinical activity against cutaneous candidiasis application of ITC-LNC in a gel vehicle increased dermal retention of
ITC in excised human skin relative to a conventional gel and ITC-NLC
Before induction of cutaneous candidiasis, all rats showed normal gel. In a preclinical study, both ITC-loaded nanocarrier-based gels and
skin with no sign of fungal infection. After induction of candidiasis, conventional ITC gel therapies proved qualitatively efficacious for the
animals showed clinical features of infection and culture results were treatment of induced cutaneous candidiasis in rats with no histo-
positive for C. albicans for all groups at day zero of the treatment, pathological changes in the epidermal and dermal layers of the skin
confirming establishment of infection. Histopathological changes of when applied for 14 consecutive days. The study outcomes provide
skin sections of animals sacrificed following a 14 day-treatment period evidence for the efficacy of ITC as a topical antifungal agent and en-
are shown in Fig. 7. Sections of untreated rats (group 1) clearly revealed hancement of its topical antifungal activity by lipid nanoencapsulation.
fungal elements including branching pseudohyphae and clusters of The study also points out the potential of LNC as nanocarriers with
budding yeast cells (blastoconidia) in the stratum corneum which also superior physicochemical characteristics for skin permeation enhance-
demonstrated evident hyperkeratosis. In addition, mild acanthosis and ment and dermal targeting of lipophilic drugs.
inflammatory cellular infiltrate in the upper dermis were noted. These
changes were cleared by the ITC control gel (group 2). As regards to References
nanocarrier-based gels, blank NLC gel-treated skin (group 3) was po-
sitive for fungal elements and exhibited changes similar to those de- [1] A.K. Gupta, S.G. Versteeg, N.H. Shear, Common drug-drug interactions in anti-
fungal treatments for superficial fungal infections, Expert Opin. Drug Metab.
tected in group 1. These signs of fungal infection were cleared from skin
Toxicol. 14 (2018) 387–398.
sections treated with their ITC-loaded counterparts (group 4). On the [2] N. Kumar, S. Goindi, Statistically designed nonionic surfactant vesicles for dermal
other hand, both LNC gels (Fig. 7) led to disappearance of fungal ele- delivery of itraconazole: characterization and in vivo evaluation using a standar-
dized Tinea pedis infection model, Int. J. Pharm. 472 (2014) 224–240.
ments and signs of fungal infection. Results suggested inherent anti- [3] J. Van Cutsem, F. Van Gerven, P.A. Janssen, Activity of orally, topically, and par-
fungal activity of the drug-free LNC, possibly attributed to their che- enterally administered itraconazole in the treatment of superficial and deep my-
mical composition. Although both blank LNC and NLC-based gels coses: animal models, Rev. Infect. Dis. 9 (Suppl. 1) (1987) S15–32.
[4] P. Pal, R.S. Thakur, S. Ray, B. Mazumder, Design and development of a safer non-
exerted weak in vitro antifungal activity, repeated application of the invasive transungual drug delivery system for topical treatment of onychomycosis,
gels twice daily for 14 consecutive days in the animal study revealed Drug Dev. Ind. Pharm. 41 (2015) 1095–1099.
[5] A.N. ElMeshad, A.M. Mohsen, Enhanced corneal permeation and antimycotic ac-
the anti-Candida effect of blank LNC. Except for blank NLC gel, quali- tivity of itraconazole against Candida albicans via a novel nanosystem vesicle, Drug
tative histopathological findings indicated antifungal efficacy of the gel Deliv. 23 (2016) 2115–2123.
formulations under study. [6] M. Jaiswal, M. Kumar, K. Pathak, Zero order delivery of itraconazole via polymeric
micelles incorporated in situ ocular gel for the management of fungal keratitis,
Histological examination of the skin layers at the end of the treat- Colloids Surf. B Biointerfaces 130 (2015) 23–30.
ment period revealed normal structure of the epidermis and the un- [7] P.A. Lucena, T.L. Nascimento, M.P. Gaeti, R.I. de Ávila, L.P. Mendes, M.S. Vieira,
derlying dermis with normal appendageal structures, an observation of D. Fabrini, A.C. Amaral, E.M. Lima, In vivo vaginal fungal load reduction after
treatment with itraconazole-loaded polycaprolactone-nanoparticles, J. Biomed.
importance regarding potential dermatological applications of the test Nanotechnol. 14 (2018) 1347–1358.
formulations. [8] K. Selvaraj, G. Kuppusamy, J. Krishnamurthy, R. Mahalingam, S.K. Singh, M. Gulati,

630
N.A. El-Sheridy, et al. Colloids and Surfaces B: Biointerfaces 181 (2019) 623–631

Repositioning of itraconazole for the management of ocular neovascularization [35] E.-A. Lee, P. Balakrishnan, C.-K. Song, J.-H. Choi, G.-Y. Noh, C.-G. Park, A.-J. Choi,
through surface-modified nanostructured lipid carriers, Assay Drug Dev. Technol. S.-J. Chung, C.-K. Shim, D.-D. Kim, Microemulsion-based hydrogel formulation of
(2019), https://doi.org/10.1089/adt.2018.898. itraconazole for topical delivery, J. Pharm. Invest. 40 (2010) 305–311.
[9] C. Carbone, C. Martins-Gomes, V. Pepe, A.M. Silva, T. Musumeci, G. Puglisi, [36] K. Maebashi, T. Itoyama, K. Uchida, N. Suegara, H. Yamaguchi, A novel model of
P.M. Furneri, E.B. Souto, Repurposing itraconazole to the benefit of skin cancer cutaneous candidiasis produced in prednisolone-treated guinea-pigs, J. Med. Vet.
treatment: a combined azole-DDAB nanoencapsulation strategy, Colloids Surf. B Mycol. 32 (1994) 349–359.
Biointerfaces 167 (2018) 337–344. [37] M. Gupta, S.P. Vyas, Development, characterization and in vivo assessment of ef-
[10] G. Kim, G. Kwon, I. Bailey-Healy, A. Mirza, R. Whitson, A. Oro, J. Tang, Pilot study fective lipidic nanoparticles for dermal delivery of fluconazole against cutaneous
of topical itraconazole for the treatment of basal cell carcinomas in gorlin syndrome candidiasis, Chem. Phys. Lipids 165 (2012) 454–461.
patients, J. Invest. Dermatol. 138 (2018) S77. [38] H.R. Conti, A.R. Huppler, N. Whibley, S.L. Gaffen, Animal models for candidiasis,
[11] M. Sala, R. Diab, A. Elaissari, H. Fessi, Lipid nanocarriers as skin drug delivery Curr. Protoc. Immunol. 105 (6) (2014) 1–17 19.
systems: Properties, mechanisms of skin interactions and medical applications, Int. [39] D.H. Ellis, Diagnosis of onychomycosis made simple, J. Am. Acad. Dermatol. 40
J. Pharm. 535 (2018) 1–17. (1999) S3–S8.
[12] R.S. Santos, K. Loureiro, P. Rezende, L. Nalone, R.M. Barbosa, A. Santini, [40] V. Veses, N.A. Gow, Pseudohypha budding patterns of Candida albicans, Med.
A.C. Santos, C.F. da Silva, E.B. Souto, D.P. de Souza, R.G. Amaral, P. Severino, Mycol. 47 (2009) 268–275.
Innovative nanocompounds for cutaneous administration of classical antifungal [41] N.T. Huynh, C. Passirani, P. Saulnier, J.P. Benoit, Lipid nanocapsules: a new plat-
drugs: a systematic review, J. Dermatol. Treat. (2019) 1–37, https://doi.org/10. form for nanomedicine, Int. J. Pharm. 379 (2009) 201–209.
1080/09546634.2018.1479726. [42] E.B. Souto, R.H. Muller, SLN and NLC for topical delivery of ketoconazole, J.
[13] G.M. Soliman, Nanoparticles as safe and effective delivery systems of antifungal Microencapsul. 22 (2005) 501–510.
agents: achievements and challenges, Int. J. Pharm. 523 (2017) 15–32. [43] E. Roger, F. Lagarce, J.P. Benoit, Development and characterization of a novel lipid
[14] M. Pitorre, H. Gonde, C. Haury, M. Messous, J. Poilane, D. Boudaud, E. Kanber, nanocapsule formulation of Sn38 for oral administration, Eur. J. Pharm. Biopharm.
G.A. Rossemond Ndombina, J.P. Benoit, G. Bastiat, Recent advances in nanocarrier- 79 (2011) 181–188.
loaded gels: Which drug delivery technologies against which diseases? J. Control. [44] S. Gade, K.K. Patel, C. Gupta, M.M. Anjum, D. Deepika, A.K. Agrawal, S. Singh, An
Release 266 (2017) 140–155. ex vivo evaluation of moxifloxacin nanostructured lipid carrier enriched in situ gel
[15] H. Choudhury, B. Gorain, M. Pandey, L.A. Chatterjee, P. Sengupta, A. Das, for transcorneal permeation on goat cornea, J. Pharm. Sci. (2019), https://doi.org/
N. Molugulu, P. Kesharwani, Recent update on nanoemulgel as topical drug de- 10.1016/j.xphs.2019.04.005.
livery system, J. Pharm. Sci. 106 (2017) 1736–1751. [45] S. Singh, M. Singh, C.B. Tripathi, M. Arya, S.A. Saraf, Development and evaluation
[16] S. El-Housiny, M.A. Shams Eldeen, Y.A. El-Attar, H.A. Salem, D. Attia, E.R. Bendas, of ultra-small nanostructured lipid carriers: novel topical delivery system for ath-
M.A. El-Nabarawi, Fluconazole-loaded solid lipid nanoparticles topical gel for lete’s foot, Drug Deliv. Transl. Res. 6 (2016) 38–47.
treatment of pityriasis versicolor: formulation and clinical study, Drug Deliv. 25 [46] N.M. Noor, K. Sheikh, S. Somavarapu, K.M.G. Taylor, Preparation and character-
(2018) 78–90. ization of dutasteride-loaded nanostructured lipid carriers coated with stearic acid-
[17] N. Kumar, Shishu, D-optimal experimental approach for designing topical micro- chitosan oligomer for topical delivery, Eur. J. Pharm. Biopharm. 117 (2017)
emulsion of itraconazole: characterization and evaluation of antifungal efficacy 372–384.
against a standardized Tinea pedis infection model in Wistar rats, Eur. J. Pharm. [47] T. Hatahet, M. Morille, A. Shamseddin, A. Aubert-Pouessel, J.M. Devoisselle,
Sci. 67 (2015) 97–112. S. Begu, Dermal quercetin lipid nanocapsules: Influence of the formulation on an-
[18] A.F. Leal, M.C. Leite, C.S. Medeiros, I.M. Cavalcanti, A.G. Wanderley, tioxidant activity and cellular protection against hydrogen peroxide, Int. J. Pharm.
N.S. Magalhaes, R.P. Neves, Antifungal activity of a liposomal itraconazole for- 518 (2017) 167–176.
mulation in experimental Aspergillus flavus keratitis with endophthalmitis, [48] J. Hureaux, F. Lacoeuille, F. Lagarce, M.C. Rousselet, A. Contini, P. Saulnier,
Mycopathologia 179 (2015) 225–229. J.P. Benoit, T. Urban, Absence of lung fibrosis after a single pulmonary delivery of
[19] B. Mohanty, D.K. Majumdar, S.K. Mishra, A.K. Panda, S. Patnaik, Development and lipid nanocapsules in rats, Int. J. Nanomed. 12 (2017) 8159–8170.
characterization of itraconazole-loaded solid lipid nanoparticles for ocular delivery, [49] J. Pardeike, S. Weber, H.P. Zarfl, M. Pagitz, A. Zimmer, Itraconazole-loaded na-
Pharm. Dev. Technol. 20 (2015) 458–464. nostructured lipid carriers (NLC) for pulmonary treatment of aspergillosis in fal-
[20] M.A. Mirza, A.K. Panda, S. Asif, D. Verma, S. Talegaonkar, N. Manzoor, A. Khan, cons, Eur. J. Pharm. Biopharm. 108 (2016) 269–276.
F.J. Ahmed, M. Dudeja, Z. Iqbal, A vaginal drug delivery model, Drug Deliv. 23 [50] D. Feng, T. Peng, Z. Huang, V. Singh, Y. Shi, T. Wen, M. Lu, G. Quan, X. Pan, C. Wu,
(2016) 3123–3134. Polymer-surfactant system based amorphous solid dispersion: Precipitation inhibi-
[21] B. Heurtault, P. Saulnier, B. Pech, J.E. Proust, J.P. Benoit, A novel phase inversion- tion and bioavailability enhancement of itraconazole, Pharmaceutics 10 (2018) 53.
based process for the preparation of lipid nanocarriers, Pharm. Res. 19 (2002) [51] M.M. Abdel-Mottaleb, D. Neumann, A. Lamprecht, In vitro drug release mechanism
875–880. from lipid nanocapsules (LNC), Int. J. Pharm. 390 (2010) 208–213.
[22] B. Heurtault, P. Saulnier, B. Pech, J. Benoı ̂t, J. Proust, Interfacial stability of lipid [52] F. Lacoeuille, E. Garcion, J.P. Benoit, A. Lamprecht, Lipid nanocapsules for in-
nanocapsules, Colloids Surf. B Biointerfaces 30 (2003) 225–235. tracellular drug delivery of anticancer drugs, J. Nanosci. Nanotechnol. 7 (2007)
[23] S. Hirsjarvi, G. Bastiat, P. Saulnier, J.P. Benoit, Evaluation of surface deformability 4612–4617.
of lipid nanocapsules by drop tensiometer technique, and its experimental assess- [53] J. Pardeike, S. Weber, T. Haber, J. Wagner, H.P. Zarfl, H. Plank, A. Zimmer,
ment by dialysis and tangential flow filtration, Int. J. Pharm. 434 (2012) 460–467. Development of an itraconazole-loaded nanostructured lipid carrier (NLC) for-
[24] A.C. Groo, M. Bossiere, L. Trichard, P. Legras, J.P. Benoit, F. Lagarce, In vivo mulation for pulmonary application, Int. J. Pharm. 419 (2011) 329–338.
evaluation of paclitaxel-loaded lipid nanocapsules after intravenous and oral ad- [54] J. Varshosaz, S. Taymouri, M. Minaiyan, F. Rastegarnasab, A. Baradaran,
ministration on resistant tumor, Nanomed. Lond. (Lond) 10 (2015) 589–601. Development and in vitro/in vivo evaluation of HPMC/chitosan gel containing
[25] R.O. Amara, A.A. Ramadan, R.M. El-Moslemany, M.M. Eissa, M.Z. El-Azzouni, simvastatin loaded self-assembled nanomicelles as a potent wound healing agent,
L.K. El-Khordagui, Praziquantel-lipid nanocapsules: an oral nanotherapeutic with Drug Dev. Ind. Pharm. 44 (2018) 276–288.
potential Schistosoma mansoni tegumental targeting, Int. J. Nanomed. 13 (2018) [55] U.A. Shinde, S.H. Modani, K.H. Singh, Design and development of repaglinide mi-
4493–4505. croemulsion gel for transdermal delivery, AAPS PharmSciTech. 19 (2018) 315–325.
[26] P. Bapat, R. Ghadi, D. Chaudhari, S.S. Katiyar, S. Jain, Tocophersolan stabilized [56] Kisak, E. and J. Singh, Diclofenac topical formulation, United States patent, US
lipid nanocapsules with high drug loading to improve the permeability and oral 20080300311A1 (2014).
bioavailability of curcumin, Int. J. Pharm. 560 (2019) 219–227. [57] D. Mundhey, P. Morris, G. Lohiya, J. Avari, Formulation and evaluation of mu-
[27] M.M. Eissa, R.M. El-Moslemany, A.A. Ramadan, E.I. Amer, M.Z. El-Azzouni, L.K. El- coadhesive vaginal gel containing novel combination of metronidazole and mico-
Khordagui, Miltefosine lipid nanocapsules for single dose oral treatment of schis- nazole nitrate for the treatment of vaginitis, World J. Pharmaceut. Sci. 3 (2015)
tosomiasis mansoni: a preclinical study, PLoS One 10 (2015) e0141788. 910–918.
[28] D. Abozaid, A. Ramadan, H. Barakat, N. Khalafallah, Acyclovir lipid nanocapsules [58] K. Winnicka, M. Wroblewska, P. Wieczorek, P.T. Sacha, E. Tryniszewska, Hydrogel
gel for oromucosal delivery: a preclinical evidence of efficacy in the chicken pouch of ketoconazole and PAMAM dendrimers: formulation and antifungal activity,
membrane model, Eur. J. Pharm. Sci. 121 (2018) 228–235. Molecules 17 (2012) 4612–4624.
[29] H.T.P. Nguyen, E. Munnier, X. Perse, F. Vial, F. Yvergnaux, T. Perrier, M. Souce, [59] N.R. Patel, K. Damann, C. Leonardi, C.M. Sabliov, Size dependency of PLGA-na-
I. Chourpa, Qualitative and quantitative study of the potential of lipid nanocapsules noparticle uptake and antifungal activity against Aspergillus flavus, Nanomedicine
of one hundred twenty nanometers for the topical administration of hydrophobic (Lond) 6 (2011) 1381–1395.
molecules, J. Pharm. Sci. 105 (2016) 3191–3198. [60] M. Gupta, A.K. Goyal, S.R. Paliwal, R. Paliwal, N. Mishra, B. Vaidya, D. Dube,
[30] A. Garces, M.H. Amaral, J.M. Sousa Lobo, A.C. Silva, Formulations based on solid S.K. Jain, S.P. Vyas, Development and characterization of effective topical lipo-
lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC) for cutaneous use: somal system for localized treatment of cutaneous candidiasis, J. Liposome Res. 20
A review, Eur. J. Pharm. Sci. 112 (2018) 159–167. (2010) 341–350.
[31] H.A. Ranpise, K.N. Gujar, D. Mathure, P.P. Satpute, R. Awasthi, K. Dua, J.R. Madan, [61] P.B. Shekhawat, Preparation and evaluation of clotrimazole nanostructured lipid
Skin Targeting of oxiconazole nitrate loaded nanostructured lipid-carrier gel for carrier for topical delivery, Int. J. Pharm. Biol. Sci. 4 (2013) 407–416.
fungal infections, Pharm. Nanotechnol. 6 (2018) 192–200. [62] A. Firooz, R. Namdar, S. Nafisi, H.I. Maibach, Nano-sized technologies for mico-
[32] B. Tian, Q. Yan, J. Wang, C. Ding, S. Sai, Enhanced antifungal activity of vor- nazole skin delivery, Curr. Pharm. Biotechnol. 17 (2016) 524–531.
iconazole-loaded nanostructured lipid carriers against Candida albicans with a di- [63] M.M. Abdel-Mottaleb, D. Neumann, A. Lamprecht, Lipid nanocapsules for dermal
morphic switching model, Int. J. Nanomed. 12 (2017) 7131–7141. application: a comparative study of lipid-based versus polymer-based nanocarriers,
[33] C. Zhang, F. Peng, W. Liu, J. Wan, C. Wan, H. Xu, C.W. Lam, X. Yang, Eur. J. Pharm. Biopharm. 79 (2011) 36–42.
Nanostructured lipid carriers as a novel oral delivery system for triptolide: induced [64] Y. Zhai, X. Yang, L. Zhao, Z. Wang, G. Zhai, Lipid nanocapsules for transdermal
changes in pharmacokinetics profile associated with reduced toxicity in male rats, delivery of ropivacaine: in vitro and in vivo evaluation, Int. J. Pharm. 471 (2014)
Int. J. Nanomed. 9 (2014) 1049–1063. 103–111.
[34] M. Kumudhavalli, Isocratic RP-HPLC, UV method development and validation of [65] G. Cauwenbergh, Skin kinetics of azole antifungal drugs, Curr. Top. Med. Mycol. 4
itraconazole in capsule dosage form, Int. J. Pharm. Sci. Res. 2 (2011) 3269–3271. (1992) 88–136.

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