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Introduction

The increasing incidences of skin associated superficial or deep seated fungal infections are rising, particularly in the case of immunocompromised AIDS and cancer patients due to their impaired immune function.[1,2] In general, cutaneous candidiasis is one of the most com-mon diseases in human beings and animals, caused by Candida sp. and has specific etiological dispositions entities such as folliculitis, intertrigo, paronynchia and onychomycosis.[3,4] The optimal treatment strategy for serious Candida infections yet remains a challenging and limited to a small armamentarium of compounds, mainly azoles.[5,6] Fluconazole (FLZ) is a synthetic triazole derivative of the third generation, used for skin fungal infections as well as serious systemic fungal infections.[5,7] The physicochemical parameter like log P value of FLZ to 0.5 suggesting it is slightly soluble in water.[8,9] There is renewed interest in exploiting FLZ to explore its potential benefits with an improved safety and tolerability profile, therefore, now it has become a first choice as an antifun-gal drug in clinically stable patients.[5,10] It is commercially

available in oral and parenteral dosage forms which are largely confronted with well-known adverse effects including irritation, GIT as well as taste disturbances. Serious hepatotoxicity has been also precipitated in patients suffering from AIDS or malignancy.[10] This fac-tor necessitates the development of an effective topical FLZ preparation that is supposed to avoid such type of problems.

The topical therapy is worth pursuing, as an attractive tool for localized delivery due to its non-invasiveness, direct drug application to the site of action, elimination of systemic adverse effects and drug interactions.[11] Marketed topical formulations in the form of creams, lotions or sprays are applied onto surface of the skin and readily penetrated into the stratum corneum (SC) to kill the fungi or at least render them unable to grow further. As a result, topical therapies work well to care and treat skin related fungal infections.[12] But these commercial formu-lations exhibited systemic absorption and skin irritation, insufficient residence time and therefore, fail to achieve the state of clinical and mycological eradication.[13,14] In

ReseaRch aRtIcle

Influence of various lipid core on characteristics of SLNs designed for topical delivery of fluconazole against cutaneous candidiasis

Madhu Gupta, Shailja Tiwari, and Suresh P. Vyas

abstractDermal delivery of fluconazole (FLZ) is still a major limitation due to problems relating to control drug release and achieving therapeutic efficacy. Recently, solid lipid nanoparticles (SLNs) were explored for their potential of topical delivery, possible skin compartments targeting and controlled release in the skin strata. The retention and accumulation of drug in skin is affected by composition of SLNs. Hence, the aim of this study was to develop FLZ nanoparticles consisted of various lipid cores in order to optimize the drug retention in skin. SLNs were prepared by solvent diffusion method and characterized for various in vitro and in vivo parameters. The results indicate that the SLNs composed of compritol 888 ATO (CA) have highest drug encapsulation efficiency (75.7 ± 4.94%) with lower particle size (178.9 ± 3.8 nm). The in vitro release and skin permeation data suggest that drug release followed sustained fashion over 24 h. The antifungal activity shows that SLNs made up of CA lipid could noticeably improve the dermal localization. In conclusion, CA lipid based SLNs are represents a promising carrier means for the topical treatment of skin fungal infection as an alternative to the systemic delivery of FLZ.

Keywords: Skin, fungal infection, dermal delivery, sustained effect, topical treatment

Address for Correspondence: Suresh P. Vyas, Drug Delivery Research Laboratory, Department of Pharmaceutical sciences, Dr. H. S. Gour Viswavidyalaya, Sagar-470003, MP, India. Tel.: 91–7582-265525, Fax: 91–7582-265525. E-mail: spvyas54@gmail.com

(Received 30 December 2010; revised 31 May 2011; accepted 09 June 2011)

Pharmaceutical Development and Technology, 2013; 18(3): 550–559© 2013 Informa Healthcare USA, Inc.ISSN 1083-7450 print/ISSN 1097-9867 onlineDOI: 10.3109/10837450.2011.598161

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addition, the emergence of resistance to traditional ther-apies is high, hence these factors collectively increased the demand for skin targeting which is so needed implies and thus for selective and effective localization of bio-actives at the site of where activity need.[15,16] The use of topical FLZ in the treatment of skin infection with various formulations including gel-microemulsion, gel-formula-tion, lecithin-based organogel and hydrogel have been reported.[7,9,17,18] Ideally, sustained drug release, cutane-ous accumulation for localized effect in different strata of skin and low extent of permeation of drug would be beneficial for topical delivery.[10,19] Mainly retention and affinity of agents for the SC are the significant criteria for achieving topical antifungal efficacy.[20]

The solid lipid nanoparticles (SLNs) have been reported as effective carriers for successful topical delivery, as an alternative to the liposomes and other systems due to obvious advantages such as improved physical stability and lower cost.[21,22] Moreover, their lipid cores are consisted of physiological lipids with appreciable biocompatibility and biodegradability. The potential of SLNs in epidermal targeting, follicular delivery and controlled delivery of active moiety have been very well documented and established.[14,23,24,25,26,27] The nano-sized particles and their narrow size distribu-tion allow them for site specific skin targeting favouring drug retention into the skin as shown in figure 1.[14,15] The enhanced penetration of drugs through the skin barrier seemingly occurs due to resultant thin film formation following topical application of SLNs.[28] The previous studies demonstrated that SLNs dispersion of ketoconazole and clotrimazole (antifungal drugs) were quite useful for skin targeting via topical route and notably offer localized effect.[29,30] Most of the stud-ies have established that SLNs as a promising carrier for topical delivery. Furthermore, previous published report have also well documented that the variation of lipid matrix (monoglyceride content, fatty acid chain length and saturated or unsaturated state of the lipids) may also alter the drug incorporation and load-ing and drug release pattern.[27] All these factors may contribute to alter the extent of drug incorporation or

loading amount as well as modified drug release pat-tern. Moreover, the lipidic core of SLNs may also modu-late the localization and skin accumulation pattern of drug in various strata of skin.

Therefore, the main aim of our study has been to develop and evaluate SLNs consisting of various lipid cores such as stearic acid (SA), monostearin (MS), tristearin (TS) and compritol 888 ATO (CA) and also to investigate the most potential lipidic core material for proportion of drug loaded nanoparticles as topical deliv-ery system, in order to improve the cutaneous delivery of FLZ. Furthermore, characterization of SLNs was car-ried out by using several parameters such as shape and size, encapsulation efficiency, release study and in vitro skin permeation. The potency of a developed system for antifungal activity was evaluated in using immunosup-pressed Sprague–Dawley (SD) rats.

Materials and method

MaterialsFLZ was obtained as a gift from Torrent Pharmaceuticals Ltd. (Ahmadabad, India). CA [Compritol 888 ATO is glyc-eryl behenate, a mixture of mono, di and triacylglycerols of behenic acid (C

22)] was provided by Colorcon Asia Ltd.

SA, MS, TS, L-α-egg phosphatidylcholine (PC), pluronic F-68 and sephadex G-50 were purchased from Sigma Chemicals Co. (St. Louis, MO). All other reagents and solvents were either of analytical or high-performance liquid chromatography (HPLC) grades.

Preparation of drug loaded SLNsFLZ-loaded SLNs were prepared by the solvent diffusion method in aqueous system as reported earlier with slight modifications.[31] Briefly, 100 mg selected lipid (SA, MS, TS and CA) were completely dissolved in 10 ml mixture of acetone and ethanol (1:1 v/v) in a water bath at 70°C. This lipid solution was poured into 100 ml of acidic aque-ous phase containing different ratio of PC and pluronic F-68 under continuous mechanical agitation (Remi Instruments,Mumbai, India) with 4000 rpm at room temperature (25–28°C) for 5 min. The pH of the acidic aqueous phase was adjusted to 1.10 by addition of 0.1 M hydrochloric acid. The SLNs dispersion was quickly pro-duced and centrifuged at 10,000 rpm for 10 min (Spinwin, Microcentrifuge, India) then re-suspended in distilled water. The different concentrations of PC and pluronic F-68 were used to optimize SLNs.

Entrapment efficiencyThe entrapment efficiency (EE %) was determined by measuring the concentration of unentrapped drug in the dispersion of SLNs as reported elsewhere.[32] In brief, the dispersion of prepared SLNs was subjected to centrifugation and the amount of FLZ in superna-tant was determined by HPLC method as described earlier.[33]The analytical column C

18 (150 × 4.6 mm) was

Figure 1. Schematic view of fungal infected rat skin and localized pattern of SLNs in various skin layers.

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protected by a C18

guard column (4.0 × 3.0 mm). The mobile phase used was a mixture of 10 mM of sodium acetate buffer (adjusted to pH 5.0 with glacial acetic acid) and methanol (65:35) and it was run at a flow rate of 1 ml/min. The detection was done at wavelength of 210 nm.

Shape, size and zeta potentialPrepared nanoparticles were characterized for shape by transmission electron microscopy (TEM; JEOL, Tokyo, Japan), using a copper grid coated with carbon film and phosphotungstic acid (1%; w/v) as a negative stain. The morphological study was carried out by scanning elec-tron microscopy (SEM). The average particle size and zeta potential were determined by photon correlation spectroscopy, using the Zetasizer nano ZS90 (Malvern Instruments, Ltd., Malvern, UK).

In vitro release studyThe in vitro release study was performed by using the dialysis tube method as reported earlier, with slight modification.[22] Briefly, 1 ml of the sample was taken into a dialysis tube (MWCO 10000, Sigma) and placed in a beaker containing 20 ml of PBS (pH 7.4). The beaker was placed over a magnetic stirrer, and the temperature of the assembly was maintained at 37 ± 1°C throughout the study. Samples were withdrawn at predetermined time intervals and assayed for drug content by measuring the absorbance at 261 nm against a reagent blank, using the UV spectrophotometer (Cintra 10, GBC Scientific). Sink conditions were maintained throughout the experi-ment. All the experiments were performed in triplicate (n = 3).

In vitro skin permeation studyThe rat was sacrificed by cervical dislocation following removal of skin hairs and the abdominal and dorsal skin was excised surgically. The full thickness skin was used after removing underlying fat and subcutaneous tis-sues. In vitro skin permeation studies were carried out by using a Franz diffusion cell, as described earlier with slight modification.[34] The skin was mounted between the donor and receptor compartments of the diffusion cell with SC facing upward (donor compartment). Each diffusion cell, with a diffusion area of 3.14 cm2 and the receptor medium was filled with 25 ml of PBS (pH 7.4), maintained at temperature 37 ± 1°C, and continuously stirred at 100 rpm throughout the experiment. 1ml formu-lation was placed in the donor compartment. At appro-priate time intervals up to 24 h, samples of 200 μl were withdrawn from the receptor compartment and replaced by an equal volume of fresh medium. The experiment was repeated in triplicate. The samples from the receptor compartment were analyzed by HPLC, using the method described in previous section.[33] The cumulative amounts of FLZ permeated through skin were plotted as a function of time. The permeation rate of FLZ at steady state (J, µg/cm2 h1) through skin was calculated from the slope of the

linear portion of the cumulative amount permeated per unit area versus time plot.

In vivo studiesIn vivo studies were carried out as per the guidelines compiled by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA, Ministry of Culture, Government of India). All the study protocols were approved by the Institutional Animal Ethical Committee of the Dr. H.S. Gour University, Sagar (India).

Skin-irritation testThe irritation potential of the FLZ formulations was eval-uated by carrying out the Draize patch test on rabbits.[21] The back of animals was clipped free of hair 24 h prior to the application of formulations and 0.5 ml of formulation was applied on the hair-free skin with uniform spread-ing within the area of 4 cm2. The skin was observed for any visible change, such as erythema (redness) at 24, 48 and 72 h after the application of formulations. The mean erythemal scores were recorded (ranging from 0 to 4), as follows: no erythema = 0; slight erythema (barely percep-tible-light pink) = 1; moderate erythema (dark pink) = 2; moderate to severe erythema (light red) = 3; and severe erythema (extreme redness) = 4 grade.

Antifungal activityPreparation of the animalsThe male albino rats (SD), each weighing 150–180 g, were selected, housed in individual cages and received food and water ad libitum. To achieve a heavy cutaneous infection, animals were immunosuppressed by adminis-tering cyclophosphamide (100 mg/kg body weight) given through an intraperitoneal injection for the first 3 days during the experiment prior to fungal infection.

Preparation of the microorganismsClinical isolate of Candida albicans (C. albicans, MTCC-Code*1637) (MTCC-Code*1637) was used to infect the animals. A working culture of the candida was grown for 48 h at 30°C on Sabouraud dextrose agar medium (SDA). The cells were then collected, washed and resuspended to a final concentration of 107 colony forming units/ml (cfu/ml, suspended in sterile saline) of C. albicans.[35]

Cutaneous infectionEach animal was shaved at back with an electric clip-per and an approximately 3.0 cm2 area was marked. The marked area was infected with 107cfu/ml suspension by gently rubbing onto the skin with the help of a ster-ile, cotton-tipped swab until no more visible fluid was observed.[35] Infection was produced under an occlusive dressing and the infected area was covered with a sterile adhesive bandage, held in place with extra-adherent tape for 48 h before the treatment began.[7,10] Control animals were infected in the same manner; however, they did not receive any FLZ formulation.

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Treatment of the infectionThe experimental animals were divided into six groups, each containing 6 animals. Group 1 was treated with plain drug solution, once-daily for 3 consecutive days, starting 24 h after challenge (day 0); group 2 was applied topi-cally with an equivalent dose of SA based SLNs (FLZ-SA); group 3 was applied topically with SLNs made up of MS (FLZ-MS); and in group 4 SLNs of TS (FLZ-TS), while group 5 was given FLZ-loaded CA cored SLNs (FLZ-CA), whereas the group 6 served as control. All animals were sacrificed 48 h following the last treatment, and 3.0 cm2 of skin from the infected sites was excised. The infected skin samples were collected, washed, and then plated onto culture media, incubated for 48 h at 37 ± 1°C and cfu values were recorded. For the colony counts in infected skin, an analysis of variance following Dunnet’s test was applied on the log10 colony counts for each day of infected skin culture samples from days 1 to 8.[36]

Statistical analysisAll the results are expressed as mean ± standard deviation. The treated groups were compared with control by apply-ing analysis of variance (ANOVA), following Dunnet’s test. The statistical analysis was carried out using Instat 2.1 software, Graph Pad Software Corp., San Diego, CA. The p value <0.05 was considered as significant.

Results and discussion

Preparation of SLNsThe SLNs were prepared by solvent diffusion method in an aqueous medium. It is a suitable method to prepare rela-tively smaller, homogenously sized lipid nanoparticulate dispersion, being quick and possible in a simple labora-tory setup. Reproducibility of the results and remarkably high loading of FLZ (75.7 ± 4.94%) in optimized CA lipid core based SLNs formulation were obtained. The sudden change in pH of SLNs dispersion to 1.10 followed by cen-trifugation provided a SLNs rich phase with compressed intermolecular geometry.[22]

Entrapment efficiencyThe lipid core material and their composition were also found to critically affect the extent of drug loading in SLNs. The results clearly showed the percent maximum entrapment was measured to be maximum in the case of formulations FLZ-SA, FLZ-MS, FLZ-TS and FLZ-CA, as 53.9 ± 3.23, 62.6 ± 4.12, 64.4 ± 3.78, 75.7 ± 4.94, respectively. Furthermore, it was observed that the formulations were with different lipids in the core however the amount of core forming lipid was kept constant. They were either in form of free fatty acid (SA) or in glycerides form (MS and TS), and thus the amount of loaded FLZ estimated was comparable (within ±10% variation of loaded drug). But, however in case of CA, triglycerides with high chain length, nearly ±20% higher percent encapsulation was recorded as compared to SA lipid based SLNs. The data represented that the percent drug loading was nearly

2.6–3.6% for all tested formulations. This may be ascribed to the more complex lipids such as CA, mixtures of mono, di and triglycerides and also containing fatty acids of dif-ferent chain length form less perfect crystals with many imperfections providing more space for occlusion the drugs thus, leading to an improved drug loading and entrapment efficiency.[27]

Indeed, it has been observed that SLNs with surfactant mixtures in optimized ratio (PC and pluronic F-68) have lower particle sizes and higher EE%. In general, binary mixtures of surfactant provided more efficient stabiliza-tion as compared to single surfactant alone. It was also previously reported that SLNs with PC and pluronic F-68 as surfactant/co-surfactant mixtures provide better physical stabilization and dermal localization.[37] A critical screening using different concentrations of binary mix-tures was performed to identify the optimum surfactant mixture that gives stable particles with all investigated lipids. The optimized mixture was composed of 0.9%w/w in 2:1 ratio of PC and pluronic F-68 and found to entrap maximum drug, while quantity of lipid was kept constant (Tables 1 and 2). This can be attributed to the concen-tration of surfactant that provides sufficient covering to the new generated surfaces since the agglomeration of uncovered lipid surfaces takes place.[27,38]

Shape, size and zeta potentialThe TEM photomicrographs (Figure 2a–d) confirm the spherical and regular particles with homogeneous populations for all formulations. Additionally, SLNs with CA core were more uniform in size and homoge-neous in comparison to other formulations. These pho-tographs also clearly reveal that the surfactant forms a monolayer core that surrounds the lipid core of the nanoparticles.[22,39] The morphological observation of the samples under in SEM revealed that the SLNs with CA core were the smooth surfaces and almost spheri-cal in shape (Figure 3a–d). The particle size analysis further showed a mean average size range of 178.9 nm

Table 1. Effect of concentration of binary surfactant mixtures on % EE of FLZ-loaded SLNs.Formulation code

%EE of FLZ-loaded SLNs at various concentration of binary surfactant mixture

0.6%w/w 0.9%w/w 1.2%w/wFLZ-SA 49.3 ± 2.67 53.9 ± 3.23 52.7 ± 3.43FLZ-MS 56.6 ± 3.29 62.6 ± 4.12 61.2 ± 3.19FLZ-TS 62.3 ± 3.98 64.4 ± 3.78 67.1 ± 2.81FLZ-CA 72.5 ± 2.65 75.7 ± 4.94 74.5 ± 4.84

Table 2. Effect of ratio of PC to pluronic F-68 on % EE of FLZ-loaded SLNs.Formulation code

% EE of drug loaded SLNs at various concentration of binary surfactant mixture

2:0.5 2:1 2:1.5FLZ-SA 48.3 ± 4.91 53.9 ± 3.23 51.8 ± 3.01FLZ-MS 59.4 ± 2.98 62.6 ± 4.12 60.2 ± 3.13FLZ-TS 61.2 ± 2.54 64.4 ± 3.78 62.4 ± 2.72FLZ-CA 70.8 ± 3.23 75.7 ± 4.94 73.6 ± 3.16

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to 279.0 nm with PDI value between 0.232 ± 0.023 and 0.289 ± 0.011 (Table 3).

Zeta potential is also an important criterion that affects the storage stability of lipid particles as well as their behavior in drug release. All the formulations, i.e. FLZ-SA, FLZ-MS, FLZ-TS and FLZ-CA possessed nega-tive zeta potential values viz; −33.8 ± 2.9 mV, −30.6 ± 3.1 mV, −23.4 ± 2.1 mV and −25.0 ± 3.7 mV, respectively. This

may be due to the chemical nature of pluronic F-68, that act as a steric stabilizer and decreases the zeta potential due to shift in the electrical shear plane of particles.[25] The observed result revealed that maximum negative zeta potential as recorded for FLZ-SA (–33.8 mV) formula-tion might be due to the high negative charge distributed on the surface of the SLNs. Interestingly a continuous decrease in the negative value of zeta potential was

Figure 2. TEM photographs of different FLZ-loaded SLNs formulation (a) FLZ-SA (b) FLZ-MS (c) FLZ-TS (d) FLZ-CA.

Figure 3. SEM photographs of different FLZ-loaded SLNs formulation (a) FLZ-SA (b) FLZ-MS (c) FLZ-TS (d) FLZ-CA.

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recorded as lipid core was changed, however the opti-mized formulation, i.e. FLZ-CA showed slightly lower value than FLZ-TS. The findings of this study are fully in accordance with the earlier reports.[22]

In vitro releaseThe amount of drug released from lipidic nanopar-ticles was determined in PBS (pH 7.4) for a period of 24 h. Figure 4, shows the cumulative percentage release of drug from SLNs formulations i.e. FLZ-SA, FLZ-MS, FLZ-TS and FLZ-CA. The results clearly suggest the release pattern was biphasic with an initial burst release followed by a slow prolonged release phase. The initial burst or faster release phase of drug was recorded at the initial 8 h followed by slow release. The diffusive release of drug seems, dependent on drug distribution in the carrier (nanoparticles) as well as erosion of nanopar-ticles in the dissolution media.[40] Factors which may contribute to the initial faster release rate are high diffusion coefficient of the drug and short diffusion distances as well as the presence of surfactants that may have put into accelerated burst release.[41]When FLZ was incorporated in SA nanoparticles, a 29% burst release of the drug was found conversely, the initial burst release for nanoparticles was reduced by using CA, as nanoparticles forming lipid.

The initial higher release rate from the SA nanopar-ticles may be due to the diffusion depended release of drug. The drug is predominantly localized near the sur-face owing to the use of solvent diffusion method in the preparation of SLNs.[42] The presence of surfactant may also accelerate the observed burst release. The small size of SLNs them excessively large surface area; furthermore the adsorbed surfactant may assist and facilitate the faster (burst) release. Afterwards, the release rate was slowed as 45.4%, 40.6%, 34.8% and 27.9% of drug release during from the nanoparticles prepared with SA, MS, TS and CA lipid, respectively. The prolonged drug release may be attributed to embedment of drug in the solid lipid matrix. Furthermore, the controlled release could have resulted due to chemical nature of lipids, drug-lipid interactions or surfactant-lipid molecules.[41,43] As indi-cated in Figure 4, both burst release as well as sustained release are desirable for effective dermal delivery. Burst release helps improved penetration of drug and faster onset of action, while sustained release supplies the drug over a prolonged period of time. It is reported that in case of SLNs dispersion, water loss and induced polymorphic transitions may be the key determinants of release phe-nomena. Most of the studies on SLNs report a good cor-relation between polymorphic transitions and increased

drug release. The sustain behavior may be well correlated with the metastable β’ polymorph of lipid. Drug expul-sion may be the part of polymorphic transition of β’→ β

i

and that can be controlled with combination of surfac-tant mixtures in SLNs. This phenomenon can be utilized for controlled release of drugs for topical delivery.[44] The drug release data from various formulations were fitted into different models. The value of r2 was found to be highest for the Higuchi model (r2 = 0.98). This indicates that the drug release from nanoparticles followed matrix diffusion based release kinetics.

In vitro skin permeation studyThe in vitro permeation study was carried on hairless rat skin with Franz diffusion cell. In this study, per-meation data obtained from various lipid cores based SLNs were compared with plain solution at the same drug concentration. The results revealed that the % cumulative drug permeated after 24 h from the FLZ-SA, FLZ-MS, FLZ-TS, and FLZ-CA were 39.84 ± 4.95%, 32.77 ± 4.18%, 25.34 ± 3.55% and 20.09 ± 3.31%, respec-tively, while the plain solution showed drug perme-ation of about 75.18 ± 4.12%, which was significantly higher permeation, in comparison to particulate dis-persions (Figure 5).

The % cumulative drug permeated after 24 h from the SLNs for different lipid core and flux values are reported in Table 4, where the flux value of CA core based SLNs was low compared to other formulations. This may be attributed to the slow permeation or owing to more amounts of drug encapsulated in the lipid core.

Table 3. Average size, PDI, zeta potential, %EE and % drug loading of drug loaded SLNs of different lipids.Formulation code Size (nm) PDI Zeta potential (mV) %EE % Drug loadingFLZ-SA 279.0 ± 3.2 0.289 ± 0.011 –33.8 ± 2.9 53.9 ± 3.23 2.62 ± 0.19FLZ-MS 238.7 ± 2.9 0.247 ± 0.064 –30.6 ± 3.1 62.6 ± 4.12 3.04 ± 0.23FLZ-TS 256.2 ± 4.7 0.287 ± 0.032 –23.4 ± 2.1 64.4 ± 3.78 3.12 ± 0.09FLZ-CA 178.9 ± 3.8 0.232 ± 0.023 –25.0 ± 3.7 75.7 ± 4.94 3.64 ± 0. 05

Figure 4. In vitro release of FLZ in PBS (pH 7.4) for various lipid core based SLNs formulations and plain drug solution. Values are expressed as mean ± standard deviation (n = 3).

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As already mentioned, the drug encapsulation is highly dependent on the type of lipid as more complex lipids may provide more space and environment to accom-modate the drug.[27]

Moreover, plot of the % cumulative drug permeated (from all the formulations) as a function of time, a lag time (15–30 min) was observed in invariably (Figure 5). Highest permeation was measured for FLZ-SA based formulation that can be ascribed to its high flux value (Table  4). As expected, CA lipid core formulated in FLZ-CA showed a decreased permeation through the skin which can be hypothesized as drug portioning was more favoured in the horny layer. In fact, lipidic nanopar-ticles have shown the peculiarity to reduce and/or sup-press the permeation (transdermal delivery) through the skin while they at large enhanced the penetration (dermal delivery) especially into the upper skin layers.[14,25,44,45,46,47] Therefore, these findings confirm the assumption that type of lipids in SLNs formulations promotes the accu-mulation of the embedded drug moiety into the upper skin layers, thus reducing an apparent drug flux by cre-ating a reservoir which also prolongs the skin residence time of the drug. The results are in good agreement with the earlier report where vitamin A loaded SLNs showed higher degree of penetration in horny layer with respect to nanoemulsion.[44] Similar findings were recorded by Liu et al.,[14] that SLNs loaded with isotretinoin, accumu-lated along with drug in the skin layers with respect to the control tincture. The data obtained from % cumulative

drug permeation was fitted into different models. The value of r2 was found to be highest for the Higuchi model (r2 = 0.99) in Table 5. This further indicates that the test product followed matrix diffusion based release kinetics.

Skin-irritation testConventional therapy is associated with noticeable skin irritation, which strongly restricts its applicability and acceptability by the patients. Ideally, the FLZ delivery system should be able to reduce irritation with local-ized effect in the skin strata. The skin-irritation studies indicated that SLNs based formulations resulted in con-siderably no or negligible skin irritation as compared to their plain drug solution even after 24 h of application. Nevertheless, in the case of SA and MS based SLNs, neg-ligible skin irritation was reported while in plain solution, it continually increased (Table 6). The primary irritation index (PII) was found to be 0.00 for SLNs based formula-tions (FLZ-TS and FLZ-CA). However, SA and MS lipid based SLNs (FLZ-SA and FLZ-MS) having PII of 0.33, were shown to be barely perceptible to the rabbit skin, respectively. Therefore, the developed SLNs formulations resulted in no erythema on the abraded rabbit skin and considered as a nonirritant products.

Antifungal activityThe antifungal activity of different formulations was determined by challenging the animals with C. albicans. Figure 6 convincingly suggested that C. albicans could established an infection in all the challenged animals, with a range of 3.89–3.98 log10 cfu/ml on day 0 prior to treatment, with slight variability. After 8 consecutive days of therapeutic treatment, in the control group C. albicans organisms were still detectable, having the signal for appropriate challenge inoculums. After the treatment

Figure 5. In vitro skin permeation of FLZ for various lipid core based SLNs formulations and plain drug solution. Values are expressed as mean ± standard deviation (n = 3).

Table 4. The cumulative FLZ permeated at the end of 24 h and flux in different lipid core based SLNs formulation.

Formulation code

Amount of FLZ permeated at the end of 24 h (% of

applied dose) Flux value (µg/cm2 h)FLZ-SA 39.84 ± 4.95 3.577 ± 0.58FLZ-MS 32.77 ± 4.18 2.866 ± 0.87FLZ-TS 25.34 ± 3.55 2.379 ± 0.52FLZ-CA 20.09 ± 3.31 2.007 ± 0.43

Table 5. Drug permeation kinetics from different FLZ-loaded formulations (according to Higuchi, zero order and first order release).Formulation code Higuchi(r2) Zero order(r2) First order(r2)FLZ-SA 0.9946 0.9320 0.9591FLZ-MS 0.9921 0.9243 0.9494FLZ-TS 0.9902 0.9255 0.9473FLZ-CA 0.9944 0.9258 0.9482Q

0 = drug to be released at zero time.

Qt= amount of drug released at time t.

t = time in h.

Table 6. Mean erythemal scores observed for various FLZ-loaded SLNs based formulations obtained at the end of 24, 48, and 72 h. (n = 3).

Formulation codeErythemal scores

24 h 48 h 72 hSLN-SA 0 0 1SLN-MS 0 1 0SLN-TS 0 0 0SLN-CA 0 0 0Plain drug solution 1 2 2Control (no formulation) 0 0 0

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with plain drug solution, out of 6 animals 5 remains infected. SA and MS based SLNs formulations could cure only 3 out of 6 animals, along with higher percent reduc-tion of cfu of C. albicans in the skin of remaining uncured animals. However, the TS and CA lipid cores SLNs based treated group showed significantly higher efficacy as 4 and 5 out of 6 animals, were successfully treated as nega-tive culture was seen. As it can be clearly stated that the animals treated with SLNs consisted of SA, MS, TS and CA as core forming lipids, demonstrated low fungal burden in skin, with a colony count significantly less as compared those treated with plain solution. The control group established the presence of challenging organ-isms at the surface of the epithelium with desquamation of superficial layers throughout the experiment. It was observed that initially (on day 3), plain solution reduced the number of cfu of C. albicans in skin and however this effect was not maintained throughout the experiment. In contrast to this, SLNs constantly reduced the number of cfu of the infecting organism and favour for longer term use for reduction of fungal infection in skin. Although the higher therapeutic efficacy in the case of CA lipid based SLNs may be expected due to penetration into the skin, followed by drug carrier accumulation resulting in to reservoir effect.[38] The latter may be accounted for more prominent sustained drug release and higher level of localization or accumulation of drug carrier in various layers of skin. These findings also confirmed that SLNs could promote the accumulation of the embedded drug into the upper skin layers, thus reducing the drug flux, across the skin. In particular, the more pronounced sus-tained release was recorded in the case of FLZ-CA based SLNs formulation which may ascribed to the drug diffu-sion from SC reservoir at slow rate in sustained manner.

This can be ascribed to the presence of analogies (i.e., C

22) in CA lipid, that reasonably play a role on the SLNs/

SC interaction and, consequently, on the adhesion and reservoir formation. Moreover, the presence of other fatty acids may also enhance SLNs components to get mixed with the SC and subsequently to retard the active per-meation.[48] Hence, the development of SLNs consisted of CA lipid, presents a set remarkable benefit over other formulations viz., provide long-term therapeutic con-centrations at the site of infection for eradication of the fungal burden, improved the skin tolerability and effec-tive topical delivery of FLZ.

conclusion

From the above preliminary studies it has been con-cluded that SLNs dispersion can serve as potential carri-ers especially with CA lipid core SLNs and enhanced the localized delivery of therapeutic molecules for the treat-ment of skin infections. They lead to the accumulation of the embedded drug into the upper skin layers, thus reducing the drug flux and creating a depot that prolongs the skin residence time. FLZ-loaded CA lipid based SLNs showed better antifungal activity due to localized drug-depot formation and subsequent controlled release of drug. This delivery system may also be responsible for the skin targeting and prolonged residence offering great potentials in dermal delivery to treat fungal cutaneous infection thus, it holds clinical applicability.

acknowledgements

The authors are thankful to Torrent Pharmaceuticals Ltd. (India) and Colorcon Asia Ltd. for providing the gift sample

Figure 6. Antifungal efficacy of different FLZ based SLNs formulations vs. time in days. Values are expressed as mean ± standard deviation (n = 6). Statistical significance was considered between control vs. plain drug solution, SA-FLZ, MS-FLZ, TS-FLZ, CA-FLZ at: *P < 0.05; **P < 0.001; ns, nonsignificant. [3rd day, plain drug solution (**), and other SLNs based formulation (ns); 5th day plain drug solution (ns) and other SLNs based formulation (**); 6th and 7th day plain drug solution (*) and other SLNs based formulation (**); 8th day plain drug solution (ns) and other SLNs based formulation (**)].

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Pharmaceutical Development and Technology

of FLZ and CA, respectively. The authors duly acknowledge the IMTECH (Chandigarh, India) for providing the fungal strain. The authors are also thankful to AIIMS, New Delhi (India) for carrying out the TEM and SEM of the formula-tions. Financial support provided by the AICTE in the form of a Junior Research Fellowship (JRF) to one of the authors (MG) is duly acknowledged.

Declaration of interest

The authors report no conflicts of interest. The first author alone is responsible for the content and writing of the paper.

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