Solid lipid nanoparticles-loaded topical gel containing combination drugs: an approach to offset psoriasis

1. Introduction

2. Materials and methods

3. Results and discussion

4. Conclusions

Original Research

Solid lipid nanoparticles-loadedtopical gel containingcombination drugs: an approachto offset psoriasisRahul Sonawane, Harshad Harde, Mahesh Katariya, Satyam Agrawal &Sanyog Jain†

National Institute of Pharmaceutical Education and Research (NIPER), Centre for Pharmaceutical

Nanotechnology, Department of Pharmaceutics, Punjab, India

Aim: The primary aim of present work was to develop effective combination

drug therapy for topical treatment of psoriasis.

Methods: Betamethasone dipropionate and calcipotriol loaded solid lipid

nanoparticles (CT-BD-SLNs) were prepared by hot melt high shear homogeni-

zation technique, which were then incorporated in Carbopol gel matrix. The

anti-psoriatic potential was tested by sequential in vitro (skin permeation

and dermal distribution, anti-proliferative effect in HaCaT cells) and in vivo

(Draize patch irritation, transepidermal water loss (TEWL) and anti-psoriatic

mouse tail studies) experiments.

Results: A negligible amount in receptor compartment, yet confined distribu-

tion of drugs to epidermal and dermal region of skin was observed in case of

SLNs, which is essential for safe and effective anti-psoriatic therapy. Draize

patch test and TEWL demonstrated negligible skin irritation and better skin

tolerability of SLNs. The in vitro HaCaT cell line study demonstrated that

SLNs delayed the abrupt growth of keratinocytes, while in vivo mouse tail

model showed that SLNs gel significantly decreased the epidermal thickness

and increased melanocyte count in comparison to commercial Daivobet�

ointment.

Conclusion: The developed SLNs gel is expected to be potential strategies for

treatment of psoriasis and other topical diseases.

Keywords: betamethasone dipropionate, calcipotriol, mouse tail model, psoriasis,

solid lipid nanoparticles, topical delivery

Expert Opin. Drug Deliv. [Early Online]

1. Introduction

Psoriasis is a chronic immune-mediated skin disease characterized by keratinocytehyperproliferation and increased dermal vascularity, which affects ~ 2 -- 3% of theworld population [1]. It also makes patient feeble to live normal life. However,many patients suffering from the psoriasis fail to recognize or ignore to consultphysician, thereby creating enigma about prevalence of psoriasis [2]. A complex,multifactorial etiology of psoriasis and antigenic trigger of disease developmentare poorly understood that further obscure the antipsoriatic therapy [3,4].

In case of dermatological diseases whose triggers are located beneath the skin, it isalways preferable to administer drugs topically rather than systemically for moreefficient direct action. Topical administration also reduces the systemic bioburdenand therefore toxic effects of the drugs [5,6]. The first-line antipsoriatic therapycomprises safe and most accepted (75 -- 80%) topically administered drug(s) [7].Among these betamethasone dipropionate (BD) is an effective antiproliferative

10.1517/17425247.2014.938634 © 2014 Informa UK, Ltd. ISSN 1742-5247, e-ISSN 1744-7593 1All rights reserved: reproduction in whole or in part not permitted

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http://informahealthcare.com/journal/EDD

agent; however, it is strictly discouraged by patients due torisk of cutaneous atrophy and rebound of psoriasis. VitaminD derivative such as calcipotriol (CT) is another effectiveoption for antipsoriatic therapy, but associated with severeskin irritation potential.Combination therapy using two or more drugs acting

through different mechanism may result in synergistic effi-cacy [7]. Clinical efficacy and better skin tolerability of CTand BD combination such as Daivobet, Dovobet� andTaclonex� has already been demonstrated [8-11]. However,thick layers of keratinized stratum corneum (SC) act as a majorobstacle for topical therapy [6,12]. The SC is further thickenedand inflamed in case of psoriasis that may notably hamperthe antipsoriatic activity and efficacy of topically applied drugsin the form of gel, ointment and cream [13,14]. To overcome thelimitations of conventional topical dosage forms, the novelapproaches such as nanocarriers can be superlative options.The solid lipid nanoparticles (SLNs) have been proved for

efficient delivery of numerous therapeutic agents by variousadministration routes [15]. The ability to protect the labilecompounds against chemical degradation such as retinoid,coenzyme Q-10 and a-tocopherol [16,17]; modulation ofdrug release; excellent biocompatibility [18]; and less stringentregulatory requirements [19,20] are few silent features of SLNsas topical delivery vehicle. Therefore, SLNs appear to be aninteresting approach for effective delivery to basal epidermis,which is the site of action for psoriasis. Numerous researchershave already explored the potential of SLNs for topical deliv-ery of antipsoriatic drugs such as glucocorticoids [21], acitre-tin [22], tretinoin [23] and capsaicin. However, no study hasbeen performed that comprises investigation of combinationaldrugs co-encapsulated in SLNs to unfold their antipsoriaticefficacy following topical application.Therefore, the main objective of the present work was to

evaluate the potential of SLNs-loaded gel containing steroidsand vitamin D derivatives for effective treatment of psoriasis.The BD- and CT-loaded SLNs were prepared by hot highshear homogenization method and characterized for particlessize, entrapment efficiency (%EE) and drug release. The pre-pared SLNs were further incorporated into a gel for ease oftopical application. The in vitro skin permeation, dermal dis-tribution and dermal pharmacokinetic of the formulationwere evaluated in rat and human cadaver skin (HCS). Theskin irritation potential was checked in guinea pigs, whereasthe efficacy evaluation in HaCaT cells and ‘mouse tail model’was accomplished to rationalize the potential of SLNs gel astopical antipsoriatic vehicle.

2. Materials and methods

2.1 MaterialsBD was obtained as a gift sample from Glenmark Pharma-ceuticals Ltd., India. CT was purchased from Affine Chem,China. Compritol 888 ATO, glyceryl monostearate (GMS)and Precirol ATO 5 were procured from Gattefosse India

Pvt. Ltd. Stearic acid, tristearin, Pluronic F-68, Tween 80,Brij 78, Span 20, sodium chloride, methyl paraben, propylparaben, sodium metabisulphite and triethanolaminewere purchased from Sigma, USA. Carbopol 980 NF, Carbo-pol Ultrez 10 NF and Pemulen TR-1 were procured fromLubrizol, Belgium. Daivobet ointment (LEO Pharma,Denmark) was purchased from local pharmacy. HPLC gradeethanol, acetonitrile, acetone and ethyl acetate were purchasedfrom S.D. Fine Chemicals, India. Cryomatrix was obtainedfrom Thermo Shandon, USA. Dulbecco’s modified Eagle’smedium (DMEM), fetal bovine serum (FBS) and antibiotic--antimycotic solution were purchased from PAA laboratories,Austria. In-house ultrapure water was used for all experimen-tation. All other chemicals and reagents used were of HPLCor analytical grade.

2.2 Selection of solid lipids based on solubility studySolubility of drugs (CT and BD) in different solid lipidswas determined by lipid solubility studies [23]. A fixedamount of drug (5 mg BD and 1 mg CT) was added to100 mg of lipid melts, namely, Compritol 888 ATO, Pre-cirol ATO 5, GMS, tristearin and stearic acid maintainedat 80�C. The amount of lipid melt was increased by100 mg each time until the drugs were completely solubi-lized. A formation of clear transparent solution was theend point of the solubility study.

2.3 Preparation and optimization of BD-SLNsPrecirol ATO 5 was selected as solid lipid based on lipid solu-bility study, and hot melt high shear homogenization methodwas employed for the preparation of BD-loaded SLNs (BD-SLNs) [24]. Briefly, 1 g of Precirol ATO 5 was melted at80�C and 10 mg of BD was added. Simultaneously, aqueousPluronic F-68 solution was prepared at 80�C in anotherbeaker. The melted lipid containing dissolved BD was addedto 25 g of aqueous surfactant solution with continuous stirringusing high shear homogenizer at 80�C. The resultant hot o/wnanoemulsion was cooled at 4�C to obtain BD-SLNs.

The BD-SLNs were optimized for different formulationand process variables, including type of surfactant, surfactantconcentration, homogenization speed and homogenizationtime. The parameters were optimized using traditional ‘onevariable at a time’ technique keeping other parameter constant(Table 1). The optimum parameters were selected based oncritical quality attributes at each optimization level.

2.4 Preparation of CT-BD-SLNsThe optimized parameters used for the preparation of BD-SLNs were employed for co-encapsulation of CT and BD.In this method, 1 g of Precirol ATO 5 was melted at 80�Cand 10 mg of BD and 1 mg of CT were dissolved in it. Allother parameters such as 1%w/v Pluronic F-68, homogeniza-tion speed of 20,000 rpm for 10 min at 80�C were kept con-stant. The critical quality parameters of CT-BD-SLNs werealso determined.

R. Sonawane et al.

2 Expert Opin. Drug Deliv. (2014) 11(12)

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2.5 Characterization of SLNsThe SLNs were analyzed for particle size, polydispersity index(PDI) and %EE. The size and PDI of SLNs were determinedusing dynamic light scattering at an angle of 173� (Zeta Sizer,Nano ZS, Malvern Instruments, UK). All measurements wereperformed after proper dilution of SLNs dispersion withultrapure water.

The %EE corresponding to percentage of drugs encapsu-lated within nanoparticles was determined by indirectmethod. Briefly 100 µl of SLNs dispersion (5%w/v) wasdiluted up to 1 ml with 25%w/v sodium chloride solution.The resultant mixture was centrifuged at 21,000 rpm for30 min. Supernatant containing unentrapped CT and BDwas analyzed using validated HPLC method. The %EE wascalculated by the following formula:

(1)

%EEW W

W

initial drug free drug

initial drug= ×

−100

where Winitial is the initial amount of drug taken, andWfree drug is the amount of unentrapped free drug insupernatant.

The surface morphology of nanoparticles was determinedby scanning electron microscope (SEM). A drop of SLNs dis-persion was deposited on a glass cover slip that was previouslyadhered to carbon tape on metallic stub and allowed to airdry. The air-dried SLNs were then coated with conductingmaterials using gold sputter and visualized under SEM(S-3400N, Hitachi, Japan).

2.6 Preparation of CT-BD-SLNs-loaded gelA weighed quantity of gelling agent was dispersed in SLNsdispersion followed by mixing with the help of overheadstirrer at 1000 rpm for 3 h [23]. After complete mixing andhydration, preservatives viz. methyl paraben (0.2%w/v) andpropyl paraben (0.02%w/v) dissolved in propylene glycol(5%w/v) and sodium metabisulphite (0.02%w/v) were addedand stirred to obtain homogenous mixture. The pH wasadjusted to ~ 6.0 by drop-wise addition of 10%w/v trietha-nolamine solution. Gel was evaluated for viscosity and spread-ability. The type (Carbopol 980 NF, Carbopol Ultrez 10 NFand Pemulen TR-1) and concentration (0.5 and 1%w/v)of gelling agent were screened to attain optimal rheologicalcharacteristics of CT-BD-SLNs gel.

2.7 Characterization of CT-BD-SLNs-loaded gelThe viscosity and rheology was measured by cone and platerheometer (Bohlin Instrument Ltd., C-VOR) at 25�C.A plate having 20 mm diameter with an angle of 4� wasused for analysis. Rheological properties of gel were studiedby step shear stress investigations (80 to 250 to 80 Pa in60 steps with 10 s equilibration time at each point), whichwere performed in order to evaluate the shear rate (1/s) andviscosity as a function of shear stress (Pa) [23]. The suitablegrade of Carbopol was selected on the basis of single-pointviscosity measurement.

The spreadability was evaluated following already reportedmethod [23]. Briefly, 500 mg of gel sample was placed onacrylic plate at center and another plate was concentricallypositioned above it. The diameter of circle in which the gelwas spread was measured as initial diameter. A weight of500 g was then placed on the upper plate for 5 min. Thespreadability of gel as a function of applied weight wasmeasured on the basis of change in diameter.

2.8 Storage stability of SLNs and SLNs gelThe physicochemical stability of CT-BD-SLNs and CT-BD-SLNs gel were evaluated at different stability conditionsincluding 2 -- 8�C, 25�C/60% RH (relative humidity) and40�C/75% RH for 3 months. Formulations were evaluatedfor any change in particle size, PDI and %EE in case ofCT-BD-SLNs, whereas assay, viscosity and spreadabilitywere recorded in case of CT-BD-SLNs gel. The physicalinstability was also assessed after centrifugation of CT-BD-SLNs at 15000 rpm for 30 min [25].

2.9 In vitro drug releaseThe in vitro release of CT and BD from CT-BD-SLNs disper-sion and CT-BD-SLNs gel was determined by dialysis bagmethod using cellulose acetate membrane (molecular weightcut-off 12,000 Da) [26]. A dialysis bag filled with 1 ml ofCT-BD-SLNs dispersion and 1 g gel was immersed in vialcontaining 20 ml of release medium. Mixture of PBS(pH 7.4) and ethanol (70:30 v/v) was used as release mediain order to maintain sink condition [27]. Vials were placed inshaker bath at 80 rpm and 37�C. At each time interval,1 ml of aliquots was withdrawn from receptor compartmentand replaced with an equal volume of fresh release medium.Amount of drug released was estimated using validated

Table 1. Optimization of formulation and process variables for preparation of BD-SLNs.

Parameters Variables Parameters kept constant

Type of surfactant Type Surfactant concentration (1%w/v); homogenizationspeed of 20,000 rpm for 15 min

Concentration of surfactant 0.8 -- 1.4 %w/v Pluronic F-68; homogenization speed of 20,000 rpm for 15 minHomogenization speed 10,000 -- 25,000 rpm Pluronic F-68 (1%w/v); homogenization time of 15 minHomogenization time 5 -- 20 min Pluronic F-68 (1%w/v); homogenization speed of 20,000 rpm

BD: Betamethasone dipropionate; SLNs: Solid lipid nanoparticles.

Solid lipid nanoparticles loaded topical gel containing combination drugs

Expert Opin. Drug Deliv. (2014) 11(12) 3

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HPLC method. The curve-fitting method was utilized todetermine the mechanism of drug release from the SLNsand gel matrix.

2.10 In vitro skin permeation and dermal

pharmacokinetic using Sprague-Dawley rat skinThe study protocols for all animal studies were duly approvedby the Institutional Animal Ethics Committee of NationalInstitute of Pharmaceutical Education and Research(NIPER), SAS Nagar, India, before the start of study. Allanimal studies were performed according to Committee forthe Purpose of Control and Supervision of Experiments onAnimals, India, guidelines.The in vitro skin permeation was studied on shaved

excised dorsal skin of Sprague-Dawley rats using staticFranz diffusion cells (PermeGear, Inc., USA) with contactsurface area of 0.64 cm2. The dorsal skin of rats was shavedusing Sterling-2 animal hair clipper (Wahl, USA) and ani-mals were sacrificed after 24 h to excise the skin. The sub-cutaneous fat was removed and skin was washed thriceusing PBS. The skin samples of 15 mm diameter werepunched and mounted between donor and receptor com-partment. The receptor compartment was filled with 5 mlof receptor medium containing PBS and ethanol(70:30 v/v). The formulations namely free drug (CT andBD)-loaded Carbopol gel, CT-BD-SLNs dispersion, CT-BD-SLNs-loaded Carbopol gel and Daivobet ointmentequivalent to dose of 10 µg of CT and 100 µg of BDwere applied to donor compartment. The whole assemblywas maintained at 37�C. The aliquots were collected at dif-ferent time intervals and replaced with equal volume offresh receptor media [28]. The aliquots were analyzed by val-idated HPLC method.The in vitro dermal pharmacokinetics was accomplished

on same skin samples by using tape stripping technique [29].The skin samples were unclipped from Franz diffusion cellafter 24 h and washed with PBS. The 19 mm Scotch�(3M, USA) cellophane tape was used for tape stripping.

The first strip was discarded as it may contain the drugsadhered on skin surface after washing. Approximately10 strips were used for the removal of whole SC layer insuch a manner that the maximum area of tape wasutilized [30-32]. The treated skin samples and tape werechopped and incubated with ethanol for complete extractionof drugs. After incubation, samples were sonicated in bathsonicator (Elmasonic 560 H, Elma, Germany) for 15 minand centrifuged. The extracted samples were analyzed by val-idated HPLC method.

2.11 In vitro permeation and dermal

pharmacokinetics using HCSThe study protocol comprising the use of HCS was dulyapproved by the Human Ethics Committee of NIPER, India,before the start of the study. The permeation and dermalpharmacokinetics using HCS were studied following thesame protocol (use 15 strips instead of 10 strips) alreadydiscussed in detail in Section 2.9.

2.12 Mechanistic understanding of dermal

distributionThe dermal distribution of SLNs in different skin layers wasstudied using confocal laser scanning microscopy (CLSM)[33]. Fluorescent dye (coumarin-6)-loaded SLNs (Cou6-SLNs)were prepared by hot melt high shear homogenization andapplied on the SC region of mounted skin. Receptor compart-ment was filled with a mixture of PBS and ethanol (70:30 v/v). The skin samples were collected after 1, 2 and 4 h of appli-cation and sectioned (50 µm thickness) transversely in cryo-matrix using cryomicrotome (Microm HM505E�, ThermoScientific, USA). Sections were visualized under CLSM with10� objective without any gain (Olympus, Japan). The fluo-rescence images were captured at excitation and emissionwavelengths as 459 and 505 nm, respectively.

2.13 Draize patch test for skin irritationThe skin irritation was performed on adult guinea pigsweighing ~ 300 -- 350 g. The guinea pigs were maintainedin the controlled environment of 25�C and 45% RH. Thedorsal skin was shaved using Sterling hair clipper andobserved for any kind of irritation due to shaving. Guineapigs were randomly divided into four groups (n = 3), groupI and II served as negative and positive control and treatedwith water and 1 %w/v NaOH solution, respectively. Daivo-bet ointment and CT-BD-SLNs-loaded gel were applied togroup III and IV animals, respectively. A 200 mg of formula-tion was applied on the shaved region and kept for 2 h. Thetransepidermal water loss (TEWL) was measured usingVapoMeter (Delfin Technologies Ltd. USA) before and aftertreatment to assess the skin integrity. The skin irritation wasassessed on the basis of visual redness or erythema such asno erythema = 0; slight erythema (light pink) = 1; moderate

Table 2. Selection of solid lipid based on solubility of

BD and CT.

Lipid name Amount of lipid (mg)

required to solubilize

5 mg of BD

Amount of lipid (mg)

required to solubilize

1 mg of CT

PrecirolATO 5

500 100

GMS 900 100Tristearin 1500 200Stearic acid 1100 100Compritol888 ATO

1100 100

BD: Betamethasone dipropionate; CT: Calcipotriol; GMS: Glyceryl

monostearate.

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450

400

350

300

250

200

150

Surfactant type

A1.

B1.

D1.

Pluronic conc. (%w/w)

Par

ticl

e si

ze (

nm

)

450

400

350

300

250

200

150

Par

ticl

e si

ze (

nm

)

450

400

350

300

250

200

150

Par

ticl

e si

ze (

nm

)

450

400

350

300

250

200

150

Par

ticl

e si

ze (

nm

)

Pluronic F-68 Tween 80 Brij 78 Span 20

0.5 0.75 1 1.25 1.5

Homogenization speed (RPM)

10000 15000 20000 25000

Homogenization time (min)5 10 15 20

ppt.

Particle size PDI

PD

I

0.4

0.3

0.2

0.1

PD

I

0.4

0.3

0.2

0.1P

DI

0.4

0.3

0.2

0.1

PD

I

0.4

0.3

0.2

0.1

0

90

80

70

60

A2.

B2.

D2.

% E

E

90

80

70

60

% E

E

90

80

70

60

% E

E

90

80

70

60

% E

E

Surfactant type

Pluronic F-68 Tween 80 Brij 78 Span 20

Pluronic conc. (%w/w)

0.75 1 1.25 1.5 1.6

Homogenization speed (RPM)

10000 15000 20000 25000

Homogenization time (min)5 10 15 20

ppt.

C1. C2.

Figure 1. Effect of various formulation and process variables for BD-SLNs preparation, including (A) surfactant type, (B)

Pluronic F-68 concentration, (C) homogenization speed, and (D) homogenization time. ppt: precipitation; the z-average size

(d.nm) of particle is represented as ‘size distribution by intensity’.Values are represented as mean ± SD, n = 6.

BD: Betamethasone dipropionate; SLNs: Solid lipid nanoparticles.



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erythema (dark pink) = 2; moderate-to-severe erythema (lightred) = 3; and severe erythema (extreme redness) = 4 [23].

2.14 Antipsoriatic activity of CT-BD-SLNs2.14.1 In vitro antiproliferative activity using HaCaT

cell linesThe potency of SLNs was assessed by antiproliferative effect onhuman hyperproliferative keratinocyte cell line (HaCaT). Theinhibition of keratinocyte proliferation against homeostaticcontrol of keratinocyte growth and differentiation wasmeasured by in vitro sulforhodamine B (SRB) assay, which isthe predictive of antipsoriatic potential of the compound orformulation [34]. The HaCaT cells were cultured in DMEMsupplemented with 10% FBS and antibiotics at 37�C under5%CO2. The cells were treated with 5, 50 and 100 µg/ml con-centration of formulation and incubated for 24 h. After incu-bation, the cells were fixed using 50 µL/well trichloroaceticacid (50 %w/v) and incubated at 4�C for 1 h. The plateswere washed with distilled water and air dried. The cells werestained with 100 µl/well sulforhodamine B dye (0.4 %w/v in

1% acetic acid) followed by washing with 1% acetic acid.The adsorbed dye was then dissolved in 100 µl/well tris-buffer(0.01 M, pH 10.4) and optical density was measured at540 nm using a spectrophotometer (BioTek, USA).

2.14.2 In vivo antipsoriatic efficacy using mouse tail

modelThe mouse tail model using BALB/c mice was used to assessthe in vivo efficacy of antipsoriatic agents [35,36]. The animalswere divided into three groups comprising control (blankCarbopol gel), Daivobet and CT-BD-SLNs gel. The equiva-lent concentration of 0.05% BD and 0.005% CT was usedin each formulation. In each group, the formulation wasapplied to each animal once a daily for 2 weeks, and thenmice were sacrificed. The treated tail portions were sectionedhorizontally followed by staining with hematoxylin-eosin.The histological sections were analyzed for epidermal thick-ness and melanocyte count. In case of epidermal thicknessmeasurement, the thickness was measured from two different

Table 3. Critical formulation parameters and quality attributes of CT-BD-SLNs and CT-BD-SLNs gel.

Parameters CT-BD-SLNs Parameters CT-BD-SLNs gel

BD (mg) 10 Carbopol ultrez 10 NF (%w/v) 1CT (mg) 1 Methyl paraben (%w/v) 0.2GMS (mg) 1000 Propyl paraben (%w/v) 0.02Water (ml) 20 Propylene glycol (%w/v) 5Particle size (nm) 188 ± 16PDI 0.172 ± 0.014 Sodium metabisulphite (%w/v) 0.02%EE (BD) 85.10 ± 2.02%EE (CT) 97.87 ± 0.08 CT-BD-SLNs (ml) 100%w/w TL (BD) 1 Viscosity (Pa.s) 29.58 ± 0.25%w/w TL (CT) 0.1 Spreadability (cm2) 9.94 ± 0.91

z-average size (d.nm) of particle is denoted as ‘size distribution by intensity’.

Data are represented as mean ± SD (n = 6).

BD: Betamethasone dipropionate; CT: Calcipotriol; EE: Entrapment efficiency; GMS: Glyceryl monostearate; PDI: Polydispersity index; SLNs: Solid lipid nanoparticles;

TL: Theoretical loading.

A.

CPNNIPER 15.0 kV 10.7 mm × 21.0 k SE 10/7/2010 2.00 µm CPNNIPER 15.0 kV 9.5 mm × 20.0 k SE 12/2/2010 2.00 µm

B.

Figure 2. SEM images of (A) BD-SLNs and (B) CT-BD-SLNs.BD: Betamethasone dipropionate; CT: Calcipotriol; SEM: Scanning electron microscope; SLNs: Solid lipid nanoparticles.

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regions in each section per group (total 12 observations pergroup).

2.15 Statistical analysisThe statistical analysis was performed using SigmaStat(version 3.5, Systat Software, Inc.) utilizing one-way ANOVAfollowed by Tukey’s pairwise multiple comparison proce-dures. Significance was evaluated at p value of 0.05. DDsolver(Excel add in) was used to study the various release character-istics of formulations [37].

3. Results and discussion

3.1 Solubility of CT and BD in lipidsLipid selection for preparation of SLNs was accomplished byperforming the solubility of BD and CT in lipid melt. Thesolubility of CT and BD in different lipid melts was evaluatedand compared (Table 2). Both CT and BD showed higher sol-ubility in Precirol ATO 5 and thus selected as ‘lipid of choice’for the preparation of SLNs. Such solubility study was

important to achieve maximum drug entrapment, preventdrug crystallization or exudation and subsequent stability ofSLNs [38,39].

3.2 Preparation and optimization of BD-SLNs and

CT-BD SLNsSLNs were prepared by hot high shear homogenization tech-nique with some modifications. Optimum formulation wasdeveloped after studying influence of formulation and processvariables such as type of surfactant, surfactant concentration,homogenization speed and homogenization time on the parti-cle size, PDI and %EE of BD-SLNs (Figure 1).

The BD-SLNs were initially optimized for type of nonionicsurfactants including Brij 78, Pluronic F-68, Tween 80 andSpan 60, and their effect on critical quality attributes were stud-ied (Figure 1A). Pluronic F-68 produced smaller particles(~ 200 nm) compared with other surfactants (> 200 nm oraggregation) without any notable effect on %EE and PDI.It revealed that Pluronic F-68 had a pivotal role in the

30

25

20

15

10

5

12

10

8

6

4

0.5 1 0.5 1 0.5 1

980 NF

70 120 170 220 270

Vis

cosi

ty (

Pa.

s)

Vis

cosi

ty (

Pa.

s)S

pre

adab

ility

(cm

2 )

010

2030405060708090

100

Sh

ear

rate

(1/

S)

0

20

40

60

80

100

120

140

A. B.

C. D.

70 120 170 220

Shear stress (Pa)Shear stress (Pa)

Ultrez 10

Blank gel CT-BD-SLNs gel

Pemulen TR-1

0.5 1 0.5 1 0.5 1

980 NF Ultrez 10 Pemulen TR-1

Blank gel CT-BD-SLNs gel

Figure 3. Optimization of Carbopol grade on the basis of (A) single-point viscosity determination and (B) spreadability

evaluation. The rheological properties such as (C) shear stress versus shear rate and (D) shear stress versus viscosity evaluated

for blank and SLNs loaded 1%w/v Carbopol Ultrez 10 NF gel formulations.Values reported are mean ± SD, n = 6.

SLNs: Solid lipid nanoparticles.



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stabilization of SLNs by preventing the coalescence of highlyunstable surfaces. Additionally, it acts as permeation enhancereither by skin lipid extraction or by skin lipid bilayer fluidiza-tion [40]. Later, BD-SLNs were optimized using different con-centrations of Pluronic F-68 (0.5 -- 1.5 %w/w) (Figure 1B). Inthe process of SLNs production, concentration of surfactantshould be sufficient enough to cover the newly formed surfacearea of SLNs, otherwise leads to aggregation [39].Minimumpar-ticle size and PDI along with highest drug loading was obtainedat 1% w/w Pluronic F-68 concentration (Figure 1B). Formationof micelle-like nanostructures might have reduced the activesurfactant concentration that was necessary to stabilize the sur-face of newly formed nanostructures resulting in particlesagglomeration and larger size [38]. Formation of micelles mighthave also solubilized the drug molecules in aqueous phase caus-ing the reduction in encapsulation efficiency [15]. Therefore, 1%w/w of Pluronic F-68 was selected as optimum concentrationfor SLNs preparation.

The effect of homogenization speed was also studied by vary-ing homogenization speed from 10,000 to 25,000 rpm(Figure 1C). The particle size and PDI were exponentiallyreduced upon increasing homogenization speed up to 20,000rpm, which was further augmented upon increasing the speedup to 25,000 rpm. A very high input of energy in the form ofhomogenization might have led to higher collisions betweennewly formed nanoparticles and resulted in aggregation [39].The impact of homogenization speed on %EE was not signifi-cant (p > 0.05). Therefore, the speed of 20000 rpmwas used forthe production of SLNs. In case of homogenization time, par-ticle size and PDI decreased upon increasing time up to10min, although negative impact on%EE (Figure 1D). Beyondoptimum homogenization time (> 10 min), the particle sizeand PDI increased notably, whichmight be attributed to highercollisions between nascent particles with increase in time [39].

CT-BD-SLNs were prepared by optimized formulationand process parameters obtained from the preparation ofBD-SLNs. The final critical quality attributes of optimizedCT-BD-SLNs are presented in Table 3. The SEM analysisfurther confirmed the particle size and surface morphology.Particles prepared were spherical in shape with smooth surfacemorphology and had good correlation with particle sizeobtained using Zetasizer (Figure 2).

3.3 CT-BD-SLNs-loaded gelThe effect of different ionic preservatives, including benzalko-nium chloride and sodium salts of parabens, was screened.The CT-BD-SLNs precipitated with ionic preservativesowing to electrostatic interactions (data not shown). Thebest-suited combination of preservatives was 1:10 w/w mix-ture of propyl and methyl parabens. Methyl paraben remainssolubilized in aqueous part, whereas propyl paraben isrestrained in the vicinity of lipidic environment of SLNs, syn-ergistically preventing the microbial growth. Propylene glycolwas used to solubilize both preservatives that also act as per-meation enhancer contributing to enhance the efficacy ofT

able

4.StabilityofCT-BD-SLN

sandCT-BD-SLN

s-loadedgelatvariousstorageco

nditions.

Storage

conditions

2--8� C

25� C

/60%

RH

40� C

/75%

RH

Particle

size

(nm)

PDI

%EEBD

%EECT

Particle

size

(nm)

PDI

%EEBD

%EECT

Particle

size

(nm)

PDI

%EEBD

%EECT

CriticalqualityattributesofBD-CT-SLN

sInitial

188±16

0.172±0.01

85.10±2.02

97.87±0.08

188±16

0.172±0.01

85.10±2.02

97.87±0.08

188±16

0.172±0.014

85.10±2.02

97.87±0.08

1st

month

184±17

0.128±0.01

83.49±1.14

96.76±0.23

192±18

0.158±0.01

83.00±0.31

96.79±0.32

Aggregation

2ndmonth

191±18

0.202±0.01

82.27±1.11

95.79±0.73

196±12

0.150±0.01

82.36±0.80

96.03±0.44

Aggregation

3rd

month

195±23

0.206±0.01

81.80±0.52

94.18±0.97

201±21

0.172±0.02

82.05±1.62

95.05±1.37

Aggregation

AssayBD

AssayCT

Visco

sity

(Pa.s)

Spreadability

(cm

2)

AssayBD

AssayCT

Visco

sity

(Pa.s)

Spreadability

(cm

2)

AssayBD

AssayCT

Visco

sity

(Pa.s)

Spreadability

(cm

2)

CharacteristicsofCT-BD-SLN

s-loadedgel

Initial

100

100

34.58±0.38

5.6

±0.9

100

100

29.58±0.25

7.3

±0.7

100

100

26.58±0.41

9.1

±1.2

1st

month

99.74±1.32

98.87±0.24

36.67±0.46

5.3

±0.5

97.42±0.61

97.43±0.75

30.85±0.38

7.3

±0.4

99.02±0.13

96.18±0.82

23.14±0.26

8.7

±0.8

2ndmonth

98.23±1.12

96.45±0.72

35.71±0.66

5.4

±0.4

96.55±0.58

95.82±1.14

30.16±0.40

7.1

±0.8

98.26±0.94

95.77±0.45

21.09±0.56

8.9

±0.6

3rd

month

97.90±0.68

95.21±0.78

37.72±0.32

5.3

±0.5

95.73±0.24

94.12±1.02

29.36±0.37

7.1

±1.2

97.54±1.91

95.09±1.40

20.32±0.61

9.3

±1.0

Valuesare

reportedasmean±SD,n=6.

BD:Betamethasonedipropionate;CT:Calcipotriol;EE:Entrapmentefficiency;PDI:Polydispersityindex;

RH:Relative

humidity;

SLN

s:Solid

lipid

nanoparticles.

R. Sonawane et al.


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formulation. Sodium metabisulphite was used as an antioxi-dant to prevent the rancidification of lipids due to possibleoxidation upon storage.

The viscosity and spreadability of SLNs-loaded gel wasassessed to attain suitable pharmaceutical composition withoptimum consistency, stability and ease of applicability.Absence of trace solvent impurities in Carbopol 980 NF,Ultrez 10 NF and Pemulen TR-1 makes all these grades ofCarbopol acceptable by regulatory authority and were there-fore chosen for study [41]. The 1% w/v Ultrez 10 NF pro-duced highest viscosity (~ 30000 cP) and generated estheticformulation among all grades (Figure 3A). The spreadabilitystudy further demonstrated that 1% w/v Ultrez 10 NF hadsuperior spreading over larger surface area even at highest vis-cosity (Figure 3B). The characteristic difference in the viscosityand spreadability of various family of Carbopol was attributedto chain length and interpolymer structure [42].

The rheological curves depicted a thixotropic behaviorof SLNs gel (Figure 3C and D). The viscosity of Carbopolgel and thixotropic area was increased from 809 mm2 to1897 mm2 following the addition of SLNs. It may be attrib-uted to increased solid content of the gel after addition ofSLNs that hindered the recovery of gel microstructure owingto intermolecular hydrogen bonds. Conclusively, CarbopolUltrez 10 NF at 1% w/v concentration was selected as theoptimum gelling agent.

3.4 Storage stability of SLNs and SLNs gelNo significant change in quality attributes of SLNs wasrecorded upon 3 months storage at 2 -- 8�C and 25�C/60%;however, it was unstable at 40�C/75% (Table 4). The highertemperature conditions may trigger the thermodynamic insta-bility as well as partial melting of surface solid lipid leading toaggregation. The CT-BD-SLNs were also physically stableafter centrifugation at 15,000 rpm for 30 min.

The SLNs gel also retained physicochemical characteristicssuch as drug content, viscosity and spreadability at room tem-perature upon 3 months’ storage (Table 4). However, thespreadability was notably deviated at lower (2 -- 8�C) andhigher temperature conditions (40�C/75%), respectively.Therefore, CT-BD-SLNs or CT-BD-SLNs gel is recom-mended to be stored at room temperature.

3.5 In vitro drug releaseThe release behavior of CT and BD from CT-BD-SLNsdispersion and CT-BD-SLNs gel was controlled over a timeperiod of 48 h. Approximately 31% of CT and ~ 56% of BDrelease in case of SLNs dispersion, whereas ~ 25% of CTand~ 45%ofBD release after 48 h in case of SLNs gel (Figure 4).The difference in release behavior of BD and CTmay be attrib-uted to difference in concentration. The curve fitting evidencedHiguchi pattern of release of both drugs through lipid as well asgel matrix (Table 5). These results suggested that the drugs arehomogeneously distributed throughout the matrix and slowlydiffuse into receptor media [15]. Low dose of drugs and forma-tion of true solid solution with lipid might have prevented therapid release of drugs (DSC analysis, see supplementary infor-mation). DDsolver also confirmed significant differencebetween release of drugs from SLNs and SLNs gel on the basisof ANOVA, similarity factor (f2o [50,100]) and difference fac-tor (f1 o [0,15]) [37]. High viscosity of gel matrix furtherdecreased the release rate owing to lesser penetration of waterand thus hydration of gel matrix. The sustained release of drugsthrough lipid matrix of SLNsmight have helped in reducing theconcentration-dependent irritation of CT.

3.6 In vitro permeation and dermal pharmacokinetics

using rat skinA nondetectable amount of both CT and BD was permeatedthrough rat skin till 24 h (Table 6) confirming that SLNs werestrictly confined to the epidermal, dermal and SC of the skin.Negligible drug permeation further minimized the systemicside effects of drugs owing to insignificant absorption intosystemic circulation [43,44].

The dermal bioavailability of CT and BD following topicalapplication of formulations onto rat skin is presentedin Table 6. The dermal bioavailability of CT was 5.37- and4.12-fold higher for CT-BD-SLNs and CT-BD-SLNs gel,respectively, whereas the BD bioavailability was increased by27.80- and 10.47-fold for SLNs dispersion and SLNs gel,respectively, in comparison to Daivobet ointment. The

0

20

40

60

80

100%

rel

ease

0

20

40

60

80

100

% r

elea

se

Time (h)

0 10 20 30 40 50

Time (h)

0 10 20 30 40 50

A.

B.

CT BD BD-Higuchi CT-Higuchi

CT BD BD-Higuchi CT-Higuchi

Figure 4. Actual in vitro drug release profile and curve fitting

of Higuchi release mechanism for CT and BD release from

(A) CT-BD-SLNs and (B) CT-BD-SLNs gel.Values are reported as mean ± SD, n = 6.

BD: Betamethasone dipropionate; CT: Calcipotriol; SLNs: Solid

lipid nanoparticles.



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http://informahealthcare.com/doi/suppl/10.1517/17425247.2014.938634/suppl_file/10.1517/17425247.2014.938634_suppl.doc



penetration of SLNs through trans-follicular route of skin wasprominent mechanism of higher concentration of drugs in theskin, which was further supported by in vitro dermaldistribution [40,45]. Formation of thin occlusive film on thesurface of skin further improved dermal bioavailability. Rela-tively lower dermal bioavailability of SLNs gel than SLNsdispersion may be ascribed to additional barrier to diffusionof drugs imposed by gel matrix [46]. However, SLNs gel waspreferred over dispersion owing to ease of dispensing, betterpatient compliance and higher dermal bioavailability thanDaivobet ointment.

3.7 In vitro permeation and dermal pharmacokinetics

using HCSThe promising results of dermal pharmacokinetics using ratskin persuaded to study the permeation and dermal

Table 5. Curve-fitting method for determination of possible release mechanism for CT and BD from CT-BD-SLNs

dispersion based on correlation coefficient (R2).

Formulation Model Zero order First order Higuchi Hixson--Crowell cube root

Release

mechanism

Simple

diffusion

Simple diffusion

(concentration

dependent)

Matrix

system

Swelling type

CT-BD-SLNs CT 0.710 0.742 0.905 0.732BD 0.668 0.753 0.885 0.726

CT-BD-SLNs loaded gel CT 0.910 0.927 0.972 0.924BD 0.779 0.837 0.951 0.795

BD: Betamethasone dipropionate; CT: Calcipotriol; SLNs: Solid lipid nanoparticles.

Table 6. Dermal pharmacokinetics representing fold increase in concentration of CT and BD in SC and dermal

compartments of rat skin and human cadaver skin.

Parameters Rat skin Human cadaver skin

Daivobet CT-BD-SLNs CT-BD-SLNs gel Daivobet CT-BD-SLNs CT-BD-SLNs gel

BD CT BD CT BD CT BD CT BD CT BD CT

Drug concentration(%)

0.05 0.005 0.05 0.005 0.05 0.005 0.05 0.005 0.05 0.005 0.05 0.005

Actual doseapplied (µg)

102.50(3.54)

10.25(0.65)

101.88(2.97)

9.92(0.80)

99.88(1.66)

10.19(0.47)

100.45(3.21)

10.18(0.71)

101.28(2.54)

9.85(0.52)

101.3(1.97)

10.0(0.40)

SC (µg/cm2) 0.39(0.050)

0.08(0.013)

10.36(0.039)

0.26(0.033)

3.96(0.033)

0.12(0.013)

0.16(0.005)

0.07(0.002)

2.93(0.011)

0.19(0.011)

0.75(0.022)

0.17(0.002)

Epidermis anddermis (µg/cm2)

0.16(0.014)

0.08(0.006)

5.00(0.059)

0.59(0.092)

1.82(0.155)

0.53(0.009)

0.14(0.016)

0.12(0.003)

3.85(0.372)

1.22(0.088)

0.84(0.038)

0.37(0.005)

Dose permeated(µg)

* * * * * * * * * * * *

Total drug absorbed(µg/cm2)

0.55(0.064)

0.16(0.019)

15.36(0.098)

0.85(0.013)

5.78(0.188)

0.65(0.022)

0.30(0.021)

0.19(0.005)

6.78(0.383)

1.41(0.099)

1.59(0.060)

0.54(0.007)

Fold increase in BA NA NA 27.80 5.37 10.47 4.12 NA NA 22.48 7.33 5.28 2.80

Values in bracket represent standard deviation (n = 6).

*Nondetectable dose permeated.

BD: Betamethasone dipropionate; CT: Calcipotriol; NA: Not applicable; SC: Stratum corneum; SLNs: Solid lipid nanoparticles.

A. B.

C. D.

Figure 5. CLSM images of skin sections treated with (A) 4 h

control (free Cou-6), (B) 1 h, (C) 2 h, and (D) 4 h treatment

with Cou6-SLNs.CLSM: Confocal laser scanning microscopy; SLNs: Solid lipid nanoparticles.

R. Sonawane et al.


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bioavailability of CT-BD-SLNs formulation using HCS. Thepermeation study demonstrated same profile as observedusing rat skin, for example, nondetectable amount was perme-ated through the HCS (Table 6). The dermal bioavailability ofCT was augmented by 7.33- and 2.80-fold, whereas bioavail-ability of BD was increased by 22.48- and 5.28-fold for SLNsdispersion and SLNs gel, respectively, in comparison withDaivobet ointment. Although the results obtained in HCSand rat skin were in good correlation, HCS showed overallless drug concentration compared with rat skin. This mightbe attributed to difference in skin morphology and physiol-ogy. The thicker SC layer (15 -- 20 layers) in HCS comparedwith rat skin (only 2 -- 3 layers) and less density of hairfollicles are influential factors for relatively lower dermalconcentration of drugs in the HCS [47].

3.8 Mechanistic understanding of dermal distributionThe dermal distribution in excised rat skin was studied usingCou6-SLNs (Figure 5). Equivalent molecular weight andpoor aqueous solubility are physicochemical similaritiesbetween Cou6, BD and CT, which resulted in negligible per-meability and poor dermal bioavailability of molecules. Very

slow release of Cou6 from SLNs has additional advantagethat the observed fluorescence could be ascribed to SLNs thatassisted in mechanistic understanding of SLNs permeationthrough skin. After 1 h of application, SLNs were mostlyconfined to SC layer and could also penetrate to root of hairfollicles to some extent, which was significantly increased after2 h. At the end of the study (4 h), larger portion of the SLNswas concentrated at the root of hair follicles and also diffusedthrough the SC. It revealed that SLNs followed appendagealpathway as well as partial passive permeation via intercellularroute. Nanometer size of SLNs was a critical attribute for thepenetration into the deeper layers of the skin and accumulationin hair follicles, which can act as ‘long-term reservoirs’ for top-ically applied drug substance. Disruption of SC and occlusiveeffect of SLNs could be additional factors that led to higherpermeation [46]. Lipophilic drugs such as CT and BD have sim-ilar physicochemical properties as that of Cou6 and hence areexpected to follow a similar permeation mechanism.

3.9 Draize patch test for skin irritationThe Draize patch test for skin irritation in guinea pigswas accomplished to determine the topical tolerability of

A. B.

C.

TE

WL

(g

/m2 /

h)

Negativecontrol

Positivecontrol

2 h24 h

Daivobet® CT-BDSLNs gel

200

150

100

50

0

D.

E.

Figure 6. Draize patch test of different formulations, namely, (A) negative control (water), (B) positive control (1% w/v sodium

hydroxide solution), (C) CT-BD-SLNs-loaded gel, and (D) marketed formulation (Daivobet ointment). Left-side photograph in

each treatment group represents observation before treatment, whereas right-side photograph represents observation after

24 h of treatment. (E) Measurement of skin irritation as a function of TEWL.*Values reported are mean ± SD, n = 6.

BD: Betamethasone dipropionate; CT: Calcipotriol; SLNs: Solid lipid nanoparticles; TEWL: Transepidermal water loss.



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formulation. The guinea pigs are considered to be bestsuited for skin irritation owing to their high sensitivitytoward inflammatory substances due to high rate ofhistamine release [48]. Similar to negative control, bothCT-BD-SLNs gel and Daivobet ointment did not produceany erythema. In contrast, positive control producedsevere erythema equivalent to score 4 on irritation scale(Figure 6A -- D). The study suggested that CT-BD-SLNsgel formulation was nonirritant and safe to use even athigh permeated dose of CT and BD.The TEWL was also measured in same animal and the

results are depicted in Figure 6E. The principle behindTEWL measurement is augmentation of TEWL value dueto the disruption of the SC layer during the process of irrita-tion or damage to the skin. One percent sodium hydroxide(positive control) solution is a well-known irritant that causes

severe irritation on shaved skin of guinea pig, leading to ahigher TEWL value (150.23 g/m2h). On the contrary,Daivobet ointment and CT-BD-SLNs gel barely causedany irritation, resulting in low TEWL values of 4.23 and3.83 g/m2h, respectively. TWEL results are well correlatedwith the observations of the Draize patch test.

3.10 In vitro antiproliferative activity using HaCaT

cell linesDifferentiation and apoptosis of hyperproliferative epidermalcells is the key principle behind the HaCaT-SRB assay.Mechanistically CT induces the hydrolysis of sphingomyelinand increases formation of ceramidase [49], whereas BD actsas an anti-inflammatory as well as a mild antiproliferativeagent that forms synergistic combination [50]. CT-BD-SLNs

A.

Blank SLNs

5 µg/ml

50 µg/ml

100 µg/ml

100 µm

CT-BD-SLNs

CT-BD-SLNs gelDaivobet®Control CT-BD-SLNs gelDaivobet®Control

Free CT-BD

***

****

*****

***

B. C.

% g

row

th in

hib

itio

n100

80

60

40

20

0

E.

Ep

ider

mal

th

ickn

ess

(mm

)

F.N

um

ber

of

mel

on

ocy

tes/

sect

ion

0

10

20

30

40

50

0

10

20

30

40

D.

Figure 7. Antipsoriatic efficacy of the formulations: (A) comparative % growth inhibition of hyperproliferative keratinocytes

in HaCaT cell line as a function of antihyperproliferative activity of free combinational drugs and combination drug-loaded

SLNs. The histological examination of mouse tail treated with (B) control (blank Carbopol gel), (C) Daivobet and (D) CT-BD-

SLNs gel. The measurement of epidermal thickness (E) and melanocyte count (F) as a function of antipsoriatic efficacy of

formulations was obtained from histological samples.*p < 0.05, **p < 0.01, ***p < 0.001 represent different level of significance.

BD: Betamethasone dipropionate; CT: Calcipotriol; SLNs: Solid lipid nanoparticles.

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showed higher inhibition of hyperproliferative epidermal cellscompared with free CT-BD dispersion that was concentrationdependent (Figure 7A). It has been reported earlier that thenanoparticles taken up at a higher rate than free drugs maybe attributed to a special cellular uptake mechanism [35].A higher growth inhibition by SLNs could be attributed totheir higher uptake in comparison to free drugs dispersion.These results suggested that SLNs can be more effective forthe treatment of psoriasis than free drug therapy.

3.11 In vivo antipsoriatic efficacy using mouse tail

modelMouse tail model is an indirect method for the assessment ofefficacy of formulations. The change in epidermal thicknessand melanocyte count as a function of antipsoriatic activity isthe key principle of this study, which is clearly portrayed inFigure 7B -- D. A higher epidermal thickness, yet lowmelanocyte count was observed in control animals (Figure 7Eand F). On the contrary, the decrease in epidermal thickness(p < 0.05) and increase in melanocyte count (p < 0.01) wererecorded in case of SLNs gel and Daivobet ointment, andthe alteration was more significant in the case of SLNs gel(Figure 7E and F).

Increase in epidermal hyperproliferation and keratinizationare major distinguishing features of psoriatic skin that can be

explained by histological study. Histological section of psori-atic lesion showed an increase in thickness of epidermis,known as acanthosis [51]. Anti-inflammatory activity of potent

steroids leads to thinning of epidermal cells and thusdecreased epidermal thickness. In case of CT, the melanocyte

proliferation was increased due to the irritation and tyrosinasestimulatory ability leading to pigmentation. This effect hasbeen thoroughly studied in the case of vitiligo patients treated

with topical vitamin D therapy [11,21]. Similar histologicalobservations in case of CT-BD-SLNs gel proved its potential

as efficacious vehicle for topical treatment of psoriasis (Figure7B -- F).

4. Conclusions

The promising results of comprehensive in vitro and in vivoanalysis acclaimed the potential use of SLNs for topical

application of drugs targeting to skin diseases confined tothe dermal region. The SLNs gel restricted permeation ofCT and BD into systemic circulation, was nonirritant andmore effectively distributed to skin layers than commercialDaivobet ointment. The CT-BD-SLNs gel also exhibitedhigher in vitro and in vivo antipsoriatic efficacy. The resultsof this study can be a path forward for combinational drugtherapy using nanoparticles that is obligatory for effectiveand synergistic treatment of diseases. The promisingin vitro and in vivo outcomes can be transformed into poten-tial marketed product after successful clinical trials inhumans.

Acknowledgements

The authors acknowledge the Department of Science andTechnology, Government of India, New Delhi, India, forfinancial assistance and Director, NIPER, for providingnecessary infrastructure facilities. We are also grateful toDr. Kusum Joshi, Dr. B. D. Radotra and Dr. Pankaj fromPost Graduate Institute of Medical Education and Research,Chandigarh, for their help. Technical assistance provided byMr. Rahul Mahajan in SEM analysis is also duly acknowl-edged. The authors thank Dr Kusum Joshi for providingnecessary infrastructure facilities for cryosectioning, Dr BDRadotra for providing human cadaver skin, Dr Pankaj fortechnical assistance in cryosectioning and Mr Rahul Mahajan(NIPER) for technical assistance in SEM analysis.

Declaration of interest

This work is a part of Indian Patent Application No. 1394/DEL/2011 filed on May 11, 2011. The authors have no otherrelevant affiliations or financial involvement with any organi-zation or entity with a financial interest in or financial conflictwith the subject matter or materials discussed in the manu-script. This includes employment, consultancies, honoraria,stock ownership or options, expert testimony, grants orpatents received or pending, or royalties.



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J Dermatolog Treat 2014;25:196-9

AffiliationRahul Sonawane, Harshad Harde,

Mahesh Katariya, Satyam Agrawal &

Sanyog Jain†

†Author for correspondence

National Institute of Pharmaceutical Education

and Research (NIPER), Centre for

Pharmaceutical Nanotechnology, Department of

Pharmaceutics, S.A.S Nagar, Mohali-160062,

Punjab, India

Tel: +91 172 2292055;

Fax: +91 172 22914692;

E-mail: [email protected]

Supplementary materials available online

Supplementary information.



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Solid lipid nanoparticles-loaded topical gel containing combination drugs: an approach to offset psoriasis