Nanoemulsion loaded gel for topical co-delivery of ...

NANO-01531; No of Pages 10

Nanomedicine: Nanotechnology, Biology, and Medicine xx (2017) xxx – xxx nanomedjournal.com

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Amanpreet Kaur, Sameer S. Katiyar, Varun Kushwah, Sanyog Jain⁎

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Centre for Pharmaceutical Nanotechnology, Department of Pharmaceutics, National Institute of Pharmaceutical Education and Research, Sector 67, SAS Nagar, Punjab, India Received 8 July 2016; accepted 9 February 2017

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Nanoemulsion loaded gel for topical co-delivery of clobitasol propionate and calcipotriol in psoriasis

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Abstract

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Current work reports the development and optimization of clobitasol propionate (CP) and calcipotriol (CT) loaded nanoemulsion based gel for topical treatment of psoriasis. Components of nanoemulsion viz., oil and surfactant/co-surfactant were selected depending upon solubility and emulsification potential respectively. The optimized ratio of 5:3:2 of Capmul MCM C8 EP, Cremophor RH 40 and Labrafil 1944 CS was selected. Carbopol 980 was used as gelling agent to achieve final drug concentration of 0.05% w/w and 0.005% w/w respectively for CP and CT. HaCaT cell lines showed higher uptake of drug from nanoemulsion in correlation with the enhancement in penetration of both drugs in stratum corneum (SC) and viable layer from nanoemulsion and gel as compared to free drugs. Imiquimod induced psoriatic BALB/c mice revealed significantly higher anti-psoriatic activity of nanoemulsion gel as compared to free drugs and marketed formulation. The developed formulation showed negligible skin irritation despite increased penetration into the skin. © 2017 Elsevier Inc. All rights reserved.

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Key words: Nanoemulsion; Psoriasis; Higher permeation; Efficacy

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Psoriasis is a chronic autoimmune inflammatory disease, affecting 2%–5% of world population, characterized by macules and plaques at the skin due to hyperproliferation and abnormal keratinocyte differentiation. 1–4 The disease mainly occurs in late childhood or early adulthood (b40 years of age) and persists for lifetime. 5 The disease sententiously affects the quality of life of patients with marked depression and suicidal contemplations leading to high mortality rates. 5,6 Topical therapy is the principal treatment for psoriasis, being employed as monotherapy in mild psoriasis and in adjunct to phototherapy, systemic therapy and biological agents in moderate to severe cases. 7 Topical treatment strategies including corticosteroids like clobitasol propionate (CP) (0.05% w/w) and vitamin D analogues like calcipotriol (CT) (0.005% w/w) are

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well reported in the literature, wherein their combination is even reported as the first choice of treatment for plaque psoriasis requiring once daily application which makes it better than individual therapy of both the drugs. 8–10 CP is a highly potent steroid with rapid and prolonged anti-inflammatory action as it inhibits hyperproliferation in skin by suppressing DNA synthesizing cells. 11–13 The efficacy of CT involves suppression of epidermal proliferation and differentiation due to inhibition of dendritic cells as well as IL-2 and IL-6 production by epidermal T cells in psoriatic lesions. 14–16 CT is often associated with high incidence of skin irritation that can be masked by the use of topical corticosteroids. 16 The psoriatic skin being rough and embedded with plaques paves the major barrier in topical delivery of drugs in psoriasis. 17

Conflict of interest and disclosure: The authors report no financial interest that might pose a potential, perceived, or real conflict. All sources of support for research: The authors are thankful to the Department of Pharmaceuticals, Ministry of Chemicals and Fertilizers, Govt. of India and Director NIPER S.A.S. Nagar for providing fellowship and funding for the proposed work. The authors also want to express their thanks to NIPER S.A.S. Nagar for providing necessary benefits. Varun Kushwah is thankful to CSIR, Govt. of India for providing fellowship. The authors also thank Symbiotec Pharmalab, India for providing gift sample of CP and Abitech Corporation, Janesville, USA and Gattefosse, France for providing gift sample of lipids utilized in development of formulation. ⁎Corresponding author. E-mail addresses: [email protected], [email protected] (S. Jain). http://dx.doi.org/10.1016/j.nano.2017.02.009 1549-9634/© 2017 Elsevier Inc. All rights reserved. Please cite this article as: Kaur A., et al., Nanoemulsion loaded gel for topical co-delivery of clobitasol propionate and calcipotriol in psoriasis. Nanomedicine: NBM 2017;xx:1-9, http://dx.doi.org/10.1016/j.nano.2017.02.009

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Topical delivery systems offer numerous advantageous due to localized action and minimization of systemic side effects. 18 Novel colloidal carriers are potential cargoes for delivering drugs to desired site in the skin. 17 The various carriers majorly used for topical delivery include liposomes, niosomes, transferosomes, ethosomes, SLNs and NLCs. 19–22 However, nanoemulsion as topical delivery vehicles is a potential approach as they are simplest transparent kinetically stable systems with globule size in the range of 20-200 nm. 17 They offer high drug loading and retention into the skin owing to their small globule size and lipidic nature. 23 Nanoemulsion is hypothesized to penetrate the rough plaques of psoriatic skin by extracting and swelling of skin lipids to achieve enhanced penetration through the pores. 24,25 Further, it can be easily loaded into gel form, 26 and it aids in the deliverability of drugs into the skin by hydration mechanism 27 and enhancing retention of drugs in the skin. 26,28,29 In the present work, a nanoemulsion (NE) with the combination of CP and CT was prepared and characterized for release profile, ex vivo skin permeation, ex vivo efficacy studies using HaCaT cell line and in vivo anti-psoriatic efficacy and was assessed by Imiquimod induced psoriasis in mice.

solubility of drug in these components and their emulsification capacity. Capmul MCM C8 EP, Cremophor RH 40 and Labrafil 1944 CS in the weight ratio 5:3:2 were selected as oil, surfactant and co-surfactant. NE was formed by spontaneous emulsification method. Briefly, 5 mg CP and 0.5 mg CT were dissolved in 500 mg oil followed by addition of 300 mg surfactant and 200 mg co-surfactant. The mixture was then subjected to vortexing for 15 min and gently heated at 40 °C for 5 min and then diluted to 10 ml with water to attain final dose of 0.05% w/v CP and 0.005% w/v CT. The resultant NE was allowed to stand for 2 h for equilibration before characterization which is also depicted in supplementary data. Similarly, NE-gel preparation and optimization could be retrieved through electronic supplementary data. Characterization of NE-gel for spreadability, stability and rheological behavior is also supplemented in electronic database.

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Morphology of NE

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The shape and morphology of NE was examined using TEM, (FEI, Tecnai, G2). Sample was stained with 1% w/v aqueous solution of phosphotungstic acid and then viewed under the microscope.

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A. Kaur et al / Nanomedicine: Nanotechnology, Biology, and Medicine xx (2017) xxx–xxx

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Methods

Drug release and cell line studies

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Materials

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CP was provided as a generous gift by Symbiotec Pharmalab, India. CT was purchased from Chem Express, China. Pluronic F-68, Pluronic F-127, TWEEN® 20, TWEEN® 80, trypsin– EDTA, coumarin-6 (C-6), sodium dodecyl sulfate (SDS), triton X-100, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), Dulbecco's modified Eagle's medium (DMEM), and 10% fetal bovine serum were purchased from Sigma, USA. Capmul® MCM EP, Captex® 355, Capmul® MCM-C8, and Captex® 200P were procured as generous gifts from Abitech Corporation, Janesville, USA. Isopropyl myristate (IPM) was procured from Loba Chemie Pvt. Ltd., India. Plurol® Oleique, Caproyl PGMC, Maisine 35-I and Labrafil® 1944 CS were obtained as gift samples from Gattefosse, France. Cremophor RH 40 was purchased from BASF Chemical Company, USA. Carbopol® 980 was purchased from Lubrizol India Pvt. Ltd., India. Penicillin and streptomycin were purchased from PAA Laboratories GmbH, Austria. HaCaT cell line was procured from National Centre for Cell Science (NCCS), Pune, India. ELISA kits, i.e., RayBio® ELISA Kit TNF-alpha and RayBio® ELISA Kit IL-6 were purchased from RayBiotech, Inc. GA, USA. Imiquimod cream 5% (Imiquad™) and betamethasone dipropionte gel 0.05% w/w (Betagel®) was purchased from local chemist. Ultra-pure deionized water was used throughout all the experiments. All other reagents were of analytical grades or higher.

Drug release studies Release of CP–CT from NE and NE-gel was determined by dialysis method using cellophane membrane (molecular weight cut-off 12,000 Da). 30 Release media consisted of phosphate buffer saline (pH 5.8) and methanol in the proportion 70:30 with maintenance of sink conditions. The dialysis bag was filled separately with free CP–CT dispersed in release media and CP– CT loaded NE as well as NE-gel, which were then immersed individually in 30 ml glass vial containing 20 ml release medium. These vials were placed in shaker bath (50 rpm and 37 °C) and 200 μl samples were withdrawn from receptor compartment and replaced with an equal volume of release medium. The samples were then analyzed for cumulative amount of drug release by validated HPLC method. Ex vivo HaCaT cell line studies The ex vivo efficacy studies were carried out on the HaCaT cell lines. 31 HaCaT cell lines were grown in tissue culture flasks (75 cm 2) at 37 °C under 5% CO2 atmosphere. The cell growth medium comprised Dulbecco's modified Eagle's medium (DMEM, Sigma) supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin (PAA Laboratories GmbH, Austria). For qualitative uptake and MTT assay, HaCaT cells were seeded at a density of 50,000 cells/well and 10,000 cells/well in 6-well culture plate and 96-well plate (Costars, Corning Inc., NY, USA). For cellular uptake studies, cells were incubated for 3 h with free C6 and C6 loaded nanoformulation (equivalent to 1 μg/mL of free C6). After incubation, extracellular particles were removed by washing with Hanks' Balanced Salt Solution (HBSS) and were observed under a confocal laser microscope (CLSM) (Olympus FV1000). Further for MTT assay, HaCaT cells were treated with fresh media containing 1% Triton-X 100 (positive control), free drugs

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Preparation, optimization and characterization of CP–CT NE and NE-gel Preparation and optimization of drug loaded NE using design of Expert® is described in supplementary data. Selection of oils, surfactants and co-surfactants was accomplished based on

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In vivo studies

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In vivo anti-psoriatic activity The in vivo efficacy studies were carried out using BALB/c mice, which were duly approved by Institutional Animal Ethics Committee, NIPER (IAEC Project Number: IAEC/14/70-R). Animals were housed at 25 ± 2 °C, 45% relative humidity and 12 h light/dark cycle and provided with food and water ad libitum. The animals were divided into 5 groups (4 animals per group) as follows:

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Dermal pharmacokinetic study by tape stripping Dermal pharmacokinetics was determined by tape stripping technique. 34,35 The pig skin samples were exposed to free drug, CP–CT loaded NE and NE-gel for 8 h in Franz diffusion cell. After 8 h, the exposed skin was washed thrice with phosphate buffer saline (PBS, pH 5.8) and stratum corneum (SC) was completely removed by detaching 15 strips (19 mm Scotch™ 3M USA) from the exposed surface. The tapes and the remaining skin samples, after removal of SC, were chopped into small pieces and soaked in methanol overnight followed by sonication for 2 h to extract the drugs. The extracted samples were analyzed by validated HPLC method. The amount detected in receptor compartment is the index of transdermal delivery, while the amount in SC, viable layers (epidermis and dermis) is the index of topical delivery.

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Skin permeation The ex vivo skin permeation was studied on pig ear skin 23 using static Franz diffusion cells (PermeGear Inc. USA) having contact surface area of 0.64 cm 2. The assembly was maintained at 37 °C with speed of rotation of magnetic bead in the receptor compartment at 75 rpm. The pig ears were collected from slaughterhouse. For permeation studies, firstly pig ears were processed and dorsal portion of pig ear skin was shaved using Sterling-2 animal hair clipper (Wahl, USA). The subcutaneous fat was removed using blunt forceps and the skin was washed thrice with phosphate buffer saline of pH 5.8. The skin samples of 15 mm diameter were punched and mounted between donor and receptor compartment with the help of clamp. The receptor compartment was filled with 5 ml of phosphate buffer saline of pH 5.8 and methanol (70:30). Then the formulations viz. free CP and CT dissolved in receptor methanol, CP–CT loaded NE and CP–CT loaded NE-gel were applied in the donor compartment. Aliquots of 250 μl were periodically withdrawn from receptor compartment and equal volume of receptor medium was replaced. The samples collected were analyzed by developed and validated HPLC based bioanalytical method.

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Group I: Healthy animals (sham group) Group II: Negative control (disease induced) Group III: Positive control (marketed formulation) Group IV: Free drug (CP–CT) in gel Group V: CP–CT loaded NE-gel

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Mechanistic understanding of skin distribution using confocal laser scanning microscopy (CLSM) The skin samples were prepared as per similar procedure followed for ex vivo skin permeation. The excised skin was mounted with a clamp between donor compartment exposed to C6 aqueous dispersion, C6 loaded into optimized NE as well as NE-gel and receptor compartment filled with phosphate buffer saline pH 5.8. The skin samples were collected after 24 h of application and the transverse sections of skin samples (15-20

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The hair on the back of animals was shaved using Sterling-2 animal hair clipper (Wahl, USA) a day prior to initiation of the study and psoriasis was induced by once daily application of Imiquimod cream 5%. 36 The cream (0.125 g) was applied in the morning, consecutively for 6 days, onto the shaved back and left ear of each mouse in all the groups except the sham group, which was the control normal mice group (Figure 4, A shows model development). For treatment, 0.05% w/w Betamethasone Dipropionate gel (Betagel®, Micro Labs Pvt. Ltd.) was applied once daily in the evening as positive control (marketed preparation) group and similarly the other two groups, i.e., Group IV and V were treated with once daily dose of free CP– CT in gel and NE-gel respectively for 5 consecutive days in the evening. The negative control group was not provided with any treatment and kept as induced control group. The morning once daily application of Imiquimod was continued during the treatment period also. After 5 days, the animals were sacrificed and blood, skin and spleen were collected from each animal.

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Measurement of ear thickness and inflammation severity scoring

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The thickness of the ear (right ear as control and left ear for extent of inflammation) was measured using digital caliper. The severity of redness and inflammation of ear skin and back skin of mice was measured based on the presence of erythema, scaling, thickening and lesions on the skin. The scoring was reported individually for all groups from 0 to 4 as: 0, none; 1, slight; 2, moderate; 3, marked; and 4, very marked.

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Spleen and skin histology

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Samples of the spleen and skin were fixed in 10% neutral buffered formalin. The samples were fixed onto the slides after staining and observed under microscope. The skin samples were also visualized under scanning electron microscope and compared for ceding of the lesions.

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Ex vivo studies

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μm thickness) were taken using Freeze Microtome (MICROM), which were observed under IX 51 microscope combined with Flu view 1000 confocal laser unit (OLYMPUS, Japan). The fluorescent portion images were captured at optical zoom of 10X keeping excitation and emission wavelengths as 459 nm and 505 nm respectively.

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and nanoformulation at a concentration of 20, 50 and 100 μg/mL. After 24 h incubation time, the media was aspirated and cells were washed three times with HBSS. Subsequently, 150 μL of MTT solution (500 μg/mL in PBS) was added to each well and reincubated for 4 h. The developed formazan crystals were then dissolved in 200 μL of DMSO. The optical density (OD) of the resultant solution was then measured at 570 nm using an ELISA plate reader (BioTek, USA). 32,33 IC50 was calculated using GraphPad Prism 7.0 GraphPad Software (San Diego, CA, USA).

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Analysis of TNF-α and IL-6 levels in serum

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Blood collected from each animal was allowed to clot for 30 min at room temperature and subjected to centrifugation at 2500 rpm for 5 min at 25 °C. Serum was collected in the separate set of plastic vials and stored in freezer at −80 °C and elevated levels of IL-6 and TNF-α were measured. The procedure followed was according to the manual specified with the ELISA kits for TNF-α (RayBio® ELISA Kit TNF-alpha, RayBiotech, Inc. GA, USA) and IL-6 (RayBio® ELISA Kit IL-6, RayBiotech, Inc. GA, USA).

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In vivo skin irritation The in vivo skin irritation study was performed on dorsal shaved (using 1 mm animal hair clipper, Sterling) skin of SD rats (weighing 200-250 g). The rats were grouped as: Group I: Negative control (capsaicin) Group II: Free CP–CT loaded gel Group III: CP–CT loaded NE-gel

Figure 1. TEM image of CP–CT loaded NE.

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Statistical analysis

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The statistical analysis of all the result was accomplished utilizing GraphPad Prism 7.0 GraphPad Software (San Diego, CA, USA). One way ANOVA followed by Bonferroni multiple comparison procedure was applied throughout the manuscript and P value of b0.01 was considered significant. All results are depicted as value ± SD.

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Results

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Preparation, optimization and characterization of CP–CT NE and NE-gel

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Samples weighing about 200 mg were applied on the shaved region using a neat cotton swab and kept for 2 h on the shaved region. The trans-epidermal water loss (TEWL) was measured 30 using vapometer (Delfin, Finland) before treatment and subsequently for 4 days during treatment so as to assess skin integrity. The skin irritation was assessed on the basis of redness or erythema based on visual observations such as follows: 0, no erythema; 1, slight erythema (light pink); 2, moderate erythema (dark pink); 3, moderate to severe erythema (light red); and 4, severe erythema (extreme redness).

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Morphology of NE

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Figure 1 shows the TEM image of the resultant o/w NE. The spherical oil droplets were distributed uniformly and homogeneously throughout the formulation with no aggregates present in the system.

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CP–CT NE and NE-gel were prepared wherein, optimized CP–CT loaded NE depicted the average droplet size of 35.45 ± 2.68 nm and lower polydispersity values of 0.080 ± 0.024 with uniform and homogenous distribution. Similarly, by incorporation of NE into gel, the properties of gel were unaltered and led to the formation of stable NE-gel formulation. Detailed results for the section could be retrieved through electronic supplementary data attached with the article.

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Drug release and cell line studies

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Drug release studies The in vitro release of CP and CT from NE and NE-gel compared to free drugs solution is shown in Figure 2. The figure clearly indicates complete release of free drugs within 6 h; whereas CP–CT loaded NE showed slower release up to 10 h. The CP–CT loaded NE-gel showed controlled release of both the drugs for up to 36 h.

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Ex vivo HaCaT cell line studies The % growth inhibition of cells was measured as shown in Figure 3, A. The IC50 value of the groups is depicted in Table 1. On the other hand, 1% Triton-X 100 solution (utilized as positive control) showed N90% growth inhibition. Further, the results depict relatively higher efficacy of formulation as compared to free drug(s). The evidence of higher uptake in HaCaT cell line was generated from microscopic images using C6, as shown in Figure 3, B, where aqueous dispersion of coumarin-6 showed less fluorescence than C6 loaded into NE as seen in Figure 3, A(a).

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Ex vivo studies

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Skin permeation Non-detectable amounts of CP and CT permeated through the pig ear skin during the period of exposure of skin to the various formulations.

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Dermal pharmacokinetic The penetration of drugs from various formulations into the pig ear skin is shown in Figure 3. The free CP–CT solution and gel showed limited drug penetration. As can be seen in the tape strip profiles in Figure 4, A-D, the penetration of NE-gel is slightly lower than NE due to controlled release of drug from the gel. The penetration of CP from NE increased by 5.48 and 8.08 in SC and viable layers respectively, as compared to free drugs, whereas the NE-gel increased 4.18 times and 5.20 times the penetration of CP in SC and viable layers respectively. Similarly, for CT, NE showed 2.6 and 3.1 times increased penetration into SC and viable layers respectively. The NE-gel showed 1.88 and 2.86 times enhanced penetration of CT into SC and viable layers respectively.

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Figure 2. In vitro drug release profile of free drug mixture CP-NE and CP–CT loaded NE-gel. Each value is represented as mean ± SD (n = 3).

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Figure 3. HaCaT cell lines studies with graph showing % growth inhibition of cells by CP–CT NE compared with free drugs (CP/CT) and their mixture (CP– CT). Each value is represented as mean ± SD (n = 3). Optical microscopic images of coumarin-6 loaded NE- (A) nanoemulsion globules upon incubation at 1 μg/ml for 24 h. In all images, panel (a) images under green fluorescence channel; (b) corresponding differential interference contrast images of HaCaT cells; (c) superimposition of panels (a) and (b). (d) Image showing horizontal lines series analysis of fluorescence along the white line. (B) Free coumarin-6 treated cells (** signifies P b 0.01 in comparison to free drugs).

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Mechanistic understanding of skin distribution using confocal laser scanning microscopy (CLSM) The results of sections of pig ear skin treated with aqueous dispersion of C6, C6 loaded into optimized NE and NE-gel are show in Figure 4, E. As evident from Figure 4, E(a), the aqueous dispersion of C6 did not show any penetration into the skin but was majorly confined to the upper layer of skin. Figure 4, E(b), shows the results of C6 loaded NE, wherein the dye showed equal distribution

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Table 1 IC50 value of different study groups in MTT assay. Study group

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Free CP Free CT Free CP–CT CP–CT NE

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In vivo studies

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In vivo anti-psoriatic activity As shown in Figure 5, B, there was a reduction in skin thickness, redness and ear inflammation for different groups. The NE-gel showed maximum reduction in skin inflammation and scaly lesions when compared to the positive control group, negative control group and groups treated with free CP–CT loaded gel. The augmented action could be attributed to higher uptake of CP–CT from NE and NE-gel into epidermal and dermal layers of the skin as evident from the skin penetration studies.

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Measurement of ear thickness

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Figure 5, G depicts the measurements of ear thickness. The negative group showed left ear thickness of N400 μm in comparison to the control right ear thickness (≈250 μm). All treated groups showed a significant decrease in left ear thickness, but it was lowest for the NE-gel treated group.

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Scoring severity of skin inflammation

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The psoriatic scaling of all the groups is mentioned in Table 2 and the value for the negative control group is ≥2, due to marked development of psoriatic lesions. The severity of psoriatic

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into SC and viable layers. Similarly, Figure 4, E(c) shows the penetration of dye loaded into NE-gel. The gel base also enhanced the retention of dye into the deeper layers of the skin.

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Figure 4. Skin distribution of CP and CT from NE and NE-Gel (A). Comparison of penetration profiles of CP in skin layers. (B) Comparison of penetration profiles of CT in skin layers. (C) Depth of penetration of CP. (D) Depth of penetration of CT. (E) CLSM images of skin sections treated with (a) coumarin-6 aqueous dispersion, (b) coumarin-6 loaded NE, and (c) coumarin-6 NE loaded gel (** signifies P b 0.01 in comparison to free drugs).

lesions is significantly less (≤2) for the NE-gel, positive and free CP–CT loaded gel treated groups.

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Histology and SEM analysis of skin

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The SEM images of skin for all groups are shown in Figure 5, C. The SEM images of the sham group shows the presence of intact skin with hair follicles. The negative control group showed complete disruption of skin with dispersed psoriatic lesions, scales and no visible hair follicles. The positive control group and free CP–CT loaded gel treated group depicted signs of disrupted skin lipids compared to the sham group after treatment. However, the NE-gel treated group resembled the normal physiology of skin with revival of hair follicles as well. It can be clearly seen in the images that the NE-Gel treated group showed disappearance of psoriatic lesions. The histological images of skin of mice from different groups are shown in Figure 5, E, which also depicts similar results, wherein the NE-gel treated group restored normal physiology of skin and it resembled to that of the sham group.

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Histology of the spleen

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The histology of the spleen, as depicted in Figure 5, D, showed complete remission of spleen tissue in the case of the NE-gel treated group with reappearance of white pulp (blue in color) and red pulp (pink in color) areas of the spleen as in the case of normal spleen tissue. The red pulp and white pulp areas cannot be distinguished in the negative control group. Similar, but insignificant restoration of splenic tissue could be seen in the

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Psoriatic scaling

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Table 2 Score chart of severity of psoriatic inflammation for all the animals in different groups.

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Figure 5. (A) Progressive scaling of mice skin over the period of 7 days after application of 5% Imiquimod cream. (B) Visual analysis of improvement in psoriatic lesions after 6 days of treatment for different groups. (C) SEM images of skin samples of different groups. (D) Histological images of skin for different groups. (E) Histology of spleen for different groups. (F) Dimensions of the spleen for various treated groups after 5 days of treatment. (G) Comparison of ear thickness (right and left ear) for different treated groups. Each value is represented as mean ± SD. (** signifies P b 0.01 in comparison to the sham group [healthy control] and “ns” as non-significant change).

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case of marketed formulation and free CP–CT loaded gel. The dimensions of the spleen after treatment were also compared as shown in Figure 5, F. The size of the spleen for the negative control group was the largest followed by a decreasing order for the positive control group, free CP–CT gel treated group, sham group and NE-gel treated group being nearly equal.

Analysis of TNF-α and IL-6 levels in serum

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The levels of TNF-α and IL-6 found in serum of animals of different groups are depicted in Figure 6. The marked increase in levels of IL-6 and TNF-α (with concentration ≈ 1000 pg/ml and 90 pg/ml respectively) in the case of the negative control group shows a good model development. Following treatment, there was an approximately 95% reduction in IL-6 levels for the NE-gel treated group compared to negative control. Similarly, the NE-gel treated group showed a 77.77% reduction in TNF-α concentration compared to the negative control group. The analyzed levels of IL-6 and TNF-α are depicted in Figure 6.

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In vivo skin irritation

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As can be seen in Figure 7, capsaicin showed higher values of percentage increase in TEWL and maximum irritation potential. Free CP–CT when loaded into gel had higher irritation potential as compared to NE-Gel, which was better tolerated. The scoring

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Figure 6. IL-6 and TNF-α levels in blood serum of animals from the different study groups (** signifies P b 0.01 in comparison to the sham group [healthy control] and “ns” as non-significant change).

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Table 3 Score chart of severity of skin irritation potential for all the animals in different groups.

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Discussion

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In view of the high dose of CP, selection of oil was based on its maximum solubility. CP showed high solubility in almost all the oils, with maximum solubility in Caproyl 90 (Supplementary data Figure S1). The latter, however, is hard to emulsify and required a large amount of surfactant for its emulsification, which is undesirable for topical delivery in psoriasis due to irritation tendency of surfactants. 37 Similarly, CP had high solubility in Caproyl PGMC, which possesses surfactant properties of its own and resulted in nanoemulsion with no

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of skin irritation potential of the various formulations is depicted in Table 3 with highest values in capsaicin followed by the free CP–CT gel and NE-gel treated group.

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Figure 7. Comparison of % increase in TEWL for NE-gel with free drug mixtures (CP–CT) loaded gel and capsaicin (** signifies P b 0.01 in comparison to negative control).

Psoriatic scaling

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trend in globule size, and PDI due to interactions with the surfactant. Thus, Capmul MCM C8 EP, though had lower solubility of CP (40.68 ± 2.83 mg/g), was selected for formulation development. Capmul MCM C8 EP was able to solubilize low dose of CT (0.005% w/w) as well, hence, it served as common oil for both the drugs. The two drugs were found to be compatible in oil as revealed by HPLC analysis (data not shown here). The mechanism of solubilization is due to the interaction of the hydrophobic groups of both the drugs with the hydrophobic chains of the oil. Similar mechanism has been reported for solubilization of Tmx and QT by Capmul MCM EP in the literature. 38 Cremophor RH 40 was selected as surfactant due to formation of small droplets in the emulsion system coupled with narrow PDI (Supplementary data Table S1A). Co-surfactants were also added to the nanoemulsion, which further lowered the globule size and PDI. The co-surfactants are reported to act by imparting flexibility to the interfacial film formed by surfactants and further reducing the interfacial tension. 39,40 Labrafil 1944 CS was selected as co-surfactant since it produced sub-micron emulsion (Supplementary data Table S1B). In in vitro release study (Figure 2), it is evident that the NE and NE-gel follows sustained release pattern for both the drugs in comparison to free drug. This can be explained on the basis that the drug is encapsulated into the

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formulations have limited skin access. The combination therapy approach in the present work causes good healing process in psoriasis. The present approach has reduction in skin irritation as compared to the free drug in gel. Also, the local topical concentration improved compared to the other groups mentioned in the study. This approach would offer selective as well as satisfactory safety profile. With less irritation potential of formulation, long term treatment is possible with high patient compliance. This nurtures the future application and commercialization of the developed formulation.

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lipidic system in the NE and thus release is hindered as the drug is more hydrophobic and tends to retain in the lipidic system. Further, NE-Gel retarded the release even more since the NE is retained more inside the 3-D network structure of the gel system and takes a lag time in release of the system. The developed nanoemulsion showed higher growth inhibition in HaCaT cell line and penetration of drugs in SC and viable layers (Figure 3). The above benefits are probably due to enhanced penetration and controlled release of drugs from the nanoemulsion system (Figure 5). The first basic component of nanoemulsion, i.e., oil, carries the drug in solubilized form, could be involved in breaking the thermodynamic and kinetic barrier for diffusion of drug molecules. 41 The other basic components are surfactants and co-surfactants which act by solubilizing and disrupting the lipidic structure of skin. 42 The water in the NE also contributes toward enhanced penetration by increasing hydration of the skin leading to swelling of lipids, paving a way for drugs to diffuse through the pores in the skin. 43 The small globules of size b100 nm for nanoemulsion also contribute to penetration by exploiting conventional routes of intracellular, intercellular and transappendageal transport. 44 Another mechanism reported in literature is that nanoemulsion penetrates into the skin by extracting the lipids and disrupting the H-bonds of lipid bilayers. 45 The results of retention of nanoemulsion locally into the viable layers of skin was desirable as pathogenesis of psoriasis involves epidermal keratinocytes and angiogenesis of deeper layers. 24 The higher penetration into deeper layers is desirable, as the drug can be expected to penetrate efficiently through the scaly psoriatic skin. Since psoriasis is an autoimmune disorder, generation of auto-antibodies takes place further increasing the levels of the cytokines (IL-6 and TNF-α). The process takes place in the spleen being the major organ related to immunity. This leads to higher burden on the spleen and increased cytokine production, which lead to swelling of the spleen or spleenomegaly. 39–42,46 The higher uptake of drugs, i.e., CT had a marked effect on reduction in levels of IL-6. 15 Furthermore, CT inhibits keratinocytes in the SC thereby, suppressing the production of cytokines in the psoriatic skin. 47 CP, an anti-inflammatory compound, acts on inflammatory cells like macrophages and neutrophils and inhibits the maturation of dendritic cells ultimately leading to suppression of the production of various pro-inflammatory cytokines. 7,13 This leads to decreased spleenomegaly condition which is validation for higher activity of formulation in diseased condition. The higher penetration of drugs was anticipated to cause irritation on the rat skin, however, negligible irritation was observed in the case of the NE-gel treated group (Figure 7). CT has tendency to cause skin irritation, but higher dose of CP penetrated into the skin could mask the irritation caused by CT. The other reasons for minimum irritation could be sustained release and controlled exposure of drugs to the skin. Additionally, NE-gel contains drugs in encapsulated form with minimal direct contact with the skin. The developed formulation improved the local concentration of the two drugs reducing their systemic side effects, in specific higher penetration into the skin layers where conventional

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