{"paper_id":"009288da-df31-4d96-ac54-ae478fcf9481","body_text":"Co-delivery of Tacrolimus and Thymoquinone topically by Nanostructured lipid carriers gel for enhanced efficacy against imiquimod-induced psoriasis in Balb/c mice | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Co-delivery of Tacrolimus and Thymoquinone topically by Nanostructured lipid carriers gel for enhanced efficacy against imiquimod-induced psoriasis in Balb/c mice Meraj Alam, Md. Rizwanullah, Shahnawaz Ahmad, Ashif Iqubal, Showkat R. Mir, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4448132/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The primary objective of this current investigation is to evaluate the antipsoriatic potential of a novel nanogel delivery system that co-encapsulates tacrolimus and thymoquinone within nanostructured lipid carriers (NLCs). Therefore, TAC-THQ-NLCs-gel was formulated by emulsification solvent-evaporation technique and evaluated for their potential in improving skin permeation, skin bioavailability, skin safety, and therapeutic efficacy in imiquimod-induced psoriasis in mice plaque model. The ex-vivo skin permeation study shows 2.51- and 2.12-folds higher permeation for TAC-THQ-NLCs-gel as compared to TAC-THQ-suspension-gel, also the permeation enhancement mechanism of NLCs gel was confirmed using FTIR and DSC. Further skin retention study shows 2.87- and 2.36-fold improvement in retention of the drug as compared to free drug gel formulation. Further, the dermatokinetic study shows 2.78 and 2.37 folds higher C max and 2.93- and 2.40-fold higher AUC (area under the curve) for TAC and THQ respectively as compared to free drugs gel. The results of the in-vivo skin compliance study suggested that the fabricated TAC-THQ-NLCs-gel was safe for skin delivery. Furthermore, TAC-THQ-NLCS-gel represented much better amelioration of psoriasis in Balb/c mice, with a cumulative PASI score reduction of 83.80% as compared to 57.14% for free drugs gel after the end of treatment. In addition, the insignificant changes in the histology of the skin, spleen, and liver further confirm the efficacy and safety of the developed TAC-THQ-NLCs-gel. Based on these observations, it can be inferred that TAC-THQ-NLCs-gel holds promise as a combined treatment approach for managing psoriasis topically. NLCs Tacrolimus Psoriasis Thymoquinone Imiquimod Dermatokinetic Skin permeation retention Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction Psoriasis is a chronic autoimmune inflammatory skin disease that affects millions of individuals worldwide, leading to significant adverse impacts on patients' physical, social, and psychological well-being [ 1 ]. Psoriasis is characterized by skin thickening, scaling, and epidermal changes, which include an inflammatory infiltrate presence in both the epidermal and dermal layers [ 2 ]. This condition involves a series of interconnected changes in skin cells, including the excessive growth of epidermal keratinocytes, increased blood vessel formation, and the infiltration of T-lymphocytes, neutrophils, and various other types of cells into the affected skin [ 3 ]. The development of psoriasis is thought to be the result of a complex interaction between genetic elements and external influences like smoking, alcohol consumption, oxidative stress, physical injury, and bacterial infections [ 4 ]. As psoriasis remains an incurable disease, current approaches aim to alleviate its symptoms by administering various drugs. These treatment options include topical therapy, phototherapy, and systemic therapy involving oral medications and newer biological agents delivered through injections. While oral and intravenous drugs can be effective, they often come with the risk of severe adverse effects that can affect different organs [ 5 , 6 ]. Among these options, topical therapy is often favored due to its ease of application, localized effects, and reduced systemic impact [ 7 , 8 ]. However, a significant challenge like limited drug penetration into the psoriatic skin limits the application of topical therapy. Furthermore, the altered condition of psoriatic skin like increased rigidity, lack of moisture, and lipid imbalance significantly hinders the permeation of conventional topical products [ 9 ]. Tacrolimus (TAC) is a potent immunosuppressant that belongs to the 'second generation' macrolides, originally derived from Streptomyces tsukubaensis . Upon binding to the immunophilin FKBP-12 protein, they form a complex that hinders the dephosphorylation of NF-AT (nuclear factor of activated T-cells) by calcineurin. This, in turn, leads to anti-psoriatic effects as it suppresses T-cell reactions and diminishes the expression of IL-2 and other immune response-related cytokines [ 10 ]. However, its high lipophilicity and molecular weight hinder its ability to penetrate the skin effectively, limiting its efficacy. Additionally, commercially available TAC ointments have shown variable and poor dermal bioavailability, often leading to local adverse reactions such as skin burning, itching, pain, and redness [ 11 ]. Thymoquinone (THQ) is a lipid-soluble benzoquinone and the principal bioactive compound found in black cumin ( Nigella sativa ) seeds. THQ has demonstrated remarkable antioxidant, anti-inflammatory, and anticancer properties in both in vitro and in vivo studies [ 12 ]. Moreover, several studies have explored its potential in treating psoriasis, yielding promising results [ 13 – 16 ]. Despite its therapeutic advantages, the pharmaceutical application of THQ is constrained by its limited solubility, high lipophilicity, and limited skin penetration [ 17 ]. Hence, there is an utmost need for the development of innovative and effective topical delivery systems for both TAC and THQ. As the pathogenesis of psoriasis involves multiple factors, the existing topical single drug formulations on the market are often unable to provide a complete cure for this condition. Consequently, the use of combination therapy has proven beneficial for the improved management of psoriasis [ 18 ]. The rationale behind employing a combination of drugs involves the potential to act through distinct pathways, offering a multi-targeted approach that enhances efficacy while minimizing dose-related adverse effects [ 19 ]. Thus, combining synthetic drugs with natural bioactives has become a valuable strategy in recent times, aiming to achieve multifaceted advantages compared to monotherapy. To enhance therapeutic efficacy while minimizing side effects, nanocarrier-based approaches have emerged as a promising strategy to ensure effective drug delivery into the deeper layers of psoriatic skin. Over the past many years, extensive research has been conducted on lipid nanoparticles, aimed at achieving improved and safer topical drug delivery of various drugs [ 20 ]. These carriers exhibit significant potential for addressing various skin conditions by interacting with the skin at the molecular level [ 21 ]. Among different lipid-based nanoparticles, nanostructured lipid carriers (NLCs) are recognized as highly efficient options for delivering topical anti-psoriatic drugs [ 22 ]. NLCs offer additional advantages, including enhanced drug loading, improved release profiles, and greater stability of the encapsulated compounds compared to other lipid nanoparticles [ 23 , 24 ]. Following topical application, NLCs adhere to the lipid film of the Stratum corneum (SC) due to their small particle size. This phenomenon facilitates increased or controlled drug penetration into the skin while also protecting the drug from degradation and extending its release duration [ 7 ] [ 25 ]. In our previous investigation, we developed TAC and THQ co-loaded NLCs to achieve better cytotoxicity against keratinocyte cell lines (HaCaT cells) [ 26 ]. The TAC-THQ-NLCs was prepared by emulsification solvent-evaporation technique and then optimized by 3 3 Box–Behnken Design. The optimized TAC-THQ-NLCs revealed excellent drug release profiles and stability of the formulation. The particle size (PS), polydispersity (PDI), zeta potential (ZP), and encapsulation efficiency (%EE) of the optimized TAC-THQ-NLCs was observed to be < 150 nm, < 0.2, > 25 mV, and > 70% respectively. Further, the cell culture study showed much better cytotoxicity of optimized TAC-THQ-NLCs against HaCaT cells in comparison with the conventional TAC-THQ-suspension. Therefore, the main objective of this study is to evaluate the anti-psoriatic potential of TAC and THQ co-loaded NLCs-gel in imiquimod induced Balb/c mice model. The ex-vivo skin permeation and permeation dynamics of the optimized formulation was evaluated in mice skin. Further, the skin dermatokinetics of TAC and THQ from the optimized formulation were evaluated and compared with the results from the free gels prepared from combined drugs. In addition, in vivo therapeutic efficacy of the developed TAC-THQ-NLCs gels was also conducted in imiquimod-induced psoriatic Balb/c mice. 2. Experimental 2.1. Materials and Chemicals Tacrolimus (TAC) was provided as a gift sample by Concord Biotech, Gujarat, India. Thymoquinone (THQ) and Rhodamine B were purchased from Sigma Aldrich, Saint Louis, MO, USA. Capryol 90, Labrasol, Gelot 64, Apifil CG, Precirol ATO 5, Labrafil M 1944 CS, Lauroglycol 90, Peceol, Masine 35 − 1, and Plurol Diisostearique were provided as a gift sample by Gattefose India Pvt. Ltd., Mumbai, India. Glyceryl monostearate (GMS), triethanolamine, and stearic acid were procured from CDH Fine Chemicals (New Delhi, India). Tween 80, Span 20 and Mannitol were purchased from SD Fine Chemicals, New Delhi, India. Carbopol Ultrez10 was gifted from Lubrizol India (Mumbai, India). Imiquimod cream (5%) was purchased from Glenmark Pharmaceuticals, Mumbai, India. All additional chemicals and reagents employed in this study met the criteria of analytical-grade quality. 2.2 Experimental Animals To conduct both the ex-vivo and in-vivo studies, Balb/c mice (either male or female) was duly approved by the Institutional Animal Ethics Committee (IAEC; 173/GO/Re/S/2000), Jamia Hamdard (Approval number: 1894). The animals were kept in polypropylene enclosures within typical laboratory settings, with a controlled temperature of 24 ± 2°C and humidity levels maintained at 55 ± 5%. They were provided with a standardized pellet diet and had unrestricted access to water. 2.3. Ex-Vivo Studies The dorsal hair of the mice was removed using a depilatory cream, and subcutaneous fat was excised through surgical means. Subsequently, the dermal side of the skin was cleansed with phosphate-buffered saline to eliminate any adhering particles and then wrapped in aluminum foil before being stored in a freezer maintained at − 20°C. On the day of the study, the skin that had been frozen was allowed to reach room temperature and then it was placed into Franz diffusion cell apparatus. The SC side was oriented upwards, facing the donor compartment, while the dermis side was placed facing downwards against the receiver compartment [ 27 ]. 2.3.1. Skin Permeation Study Skin permeation experiments for TAC-THQ-NLCs-gel abbreviated as “TAC-THQ-NG” and TAC-THQ-Suspension-gel abbreviated as “TAC-THQ-SG” were conducted using a Franz diffusion cell with excised mice skin. A skin sample was sandwiched between the donor and acceptor compartments of a Franz diffusion cell, which was positioned on a magnetic stirrer set at 100 rpm for a period of 24 h. The temperature was maintained at 37 ± 1°C throughout the experiment. In the acceptor compartment of the Franz diffusion cell, 13 mL of diffusion medium (PBS, pH 7.4, 0.5% Tween 80) was added. In two separate Franz diffusion cells, 1 g each of TAC-THQ-NG and TAC-THQ-SG was uniformly applied in a non-occlusive manner inside the donor compartment, which had an exposed skin surface area of 0.785 cm 2 . At predefined time points (0.5, 1, 2, 4, 6, 8, 12, and 24 h), 1 mL sample was withdrawn from the receiver cell and promptly substituted with a same volume of fresh dissolution medium. The gathered samples were then passed through a 0.2 µm membrane filter and subsequently subjected to analysis using our previously reported RP-HPLC method for drug quantification. For both formulations, the cumulative percentage of drugs (TAC and THQ) permeated through the skin membrane was graphed against time. Parameters like the Flux i.e. steady-state permeation rate (µg.cm − 2 .h − 1 ) was determined by analyzing the slope of the linear segment of graph, and permeability coefficient (cm.h − 1 ) was then computed by dividing the flux by the amount of drug taken initially in the Franz diffusion cells. Additionally, an enhancement factor was calculated by dividing the flux of the TAC-THQ-NG formulation with respect to that of the TAC-THQ-SG formulation [ 28 ]. 2.3.2. Skin Retention Study For skin retention study, the procedure was analogous to skin permeation study. Briefly, the mounted skin was discharged from the donor compartment of Franz diffusion cells after 24 h of study. Then, the skin samples were rinsed thoroughly with normal saline three times to eliminate any residual formulations. To ensure the complete extraction of drugs retained in the skin, the skin was finely minced, and the drugs were extracted through maceration in methanol for a period of 24 h, followed by an additional 1 h of bath sonication [ 29 ] Finally, the methanolic extract containing both drugs was subsequently filtered using a 0.2 µm filter, and the drug content was quantified using our previously established RP-HPLC method. 2.3.3. Dermatokinetics The dermatokinetic study was conducted to measure the concentration of drugs in the skin at different time intervals as per previously reported method with modification. The dermatokinetic experiments mirrored the skin permeation study, except that, for each time point, a single Franz diffusion cell was utilized. The experiments were performed on mouse skin mounted in a Franz diffusion cell, with the skin being treated using the optimized TAC-THQ-NG and TAC-THQ-SG formulations, similar to the procedure followed in skin permeation study. However, in the dermatokinetic investigation, the complete skin sample was removed at predefined time points (0.5, 1, 2, 3, 4, 6, 12, and 24 h) from the Franz diffusion cell setup. Following that, the skin samples that had been taken off underwent a series of three rinses with normal saline solution in order to eliminate any residual formulations adhering to them. At each time point, the skin samples were macerated in methanol for 24 h, followed by sonication (1 h) to ensure complete extraction of both drugs (TAC and THQ). After extraction of drugs the methanolic extract was filtered with 0.2 µm filter before quantification of drugs by our previously developed RP-HPLC technique. The concentration of drugs (TAC and THQ) at each specified time point was assessed through non-compartmental dermatokinetics modeling. The software PK solver was utilized to calculate different dermatokinetic variables, like C max , AUC 0 − 24h , K e , and T max [ 30 , 31 ]. 2.3.4. Dynamics of Skin Permeation The dynamics of skin permeation of NLC-gel was conducted on excised mice skin using two distinct analytical techniques i.e., DSC and FTIR. The mouse skin was positioned between the donor and acceptor compartments of two separate Franz diffusion cells, both filled with a diffusion medium (PBS, pH 7.4, 0.5% Tween 80) in the acceptor compartment. These cell assemblies were then placed above magnetic stirrers set at a constant speed of 100 rpm for duration of 24 h, while the temperature was maintained at 37 ± 1°C. One of the Franz diffusion cells was exposed to the NLCs-gel formulation, while the other cell remained untreated and served as a control. After the completion of the permeation experiment, the skin samples from both diffusion cells were carefully removed, rinsed with normal saline, and subsequently cut into small pieces before being dried for subsequent DSC and FTIR analysis. The DSC analysis involved placing a suitable amount of dried samples into aluminum pans. The examination was conducted within the temperature range of 30 to 300°C, with a heating rate of 10°C per minute. The remaining skin samples underwent FTIR spectral analysis, encompassing a spectral range from 500 to 4000 cm⁻¹ [ 32 ]. 2.4. In-Vivo Study 2.4.1. In-Vivo Antipsoriatic Activity This study was conducted on imiquimod (IMQ) induced psoriatic Balb/c mice to assess the efficacy of TAC-THQ-NG as per the reported method [ 33 , 34 ]. Initially, the hair from the dorsal skin of animals was removed using depilatory cream and examined the skin for any signs of inflammation. Psoriasis-like symptoms were induced in mice by applying 5% IMQ cream at a daily dose of 62.5 mg per mouse for a continuous period of seven days. Mice were monitored daily to visualize the development of psoriatic lesion. Animals were randomly divided into 6 groups, each group containing 4 mice: Where, Group 1, serving as the normal control; Group 2 was subjected to psoriasis induction but left untreated (Toxic); Group 3 was treated with TAC-THQ-SG; Group 4 was treated with TAC-NG; Group 5 was treated with THQ-NG; and Group 6 was treated with TAC-THQ-NG. Then, different formulations at a dosage of 40 mg/cm 2 (containing 0.1% TAC and 0.3% THQ) were applied to the psoriatic skin using a spatula, and this treatment continued for next seven days. After the seventh day of treatment, animals of different groups were assessed visually for signs of erythema, scaling and thickness and then sacrificed. Thereafter skin, spleen, and liver were removed and preserved in formalin (10% v/v) for histological examinations. 2.4.1.1. Psoriasis Area and Severity Index (PASI) Scoring The psoriasis-inflammation evaluation criteria were assessed according to the PASI scoring. Evaluation was conducted to assess and assign scores on a scale from 0 to 4 for erythema, scaling, and thickening. This scale ranges from absence (0) to various levels of severity (1: slight, 2: moderate, 3: marked, 4: severe) for each of these skin characteristics. Then the cumulative PASI score was calculated, which combines the individual scores for erythema, scaling, and thickening. It serves as the definitive indicator of the severity of inflammation, providing a measure of the extent of psoriasis [ 35 ]. 2.4.1.2. Body Weight, Spleen Dimension & weight Body weights of all the groups were measured initially, after induction of disease and after the end of treatment period. In inflammatory diseases such as psoriasis, the spleen often undergoes an enlargement in size, a response associated with high cytokine production. Hence, the dimension and weight of the spleen were measured [ 36 ]. 2.4.1.3. Enzyme-Linked Immunosorbent Assay for TNF-α & IL-6 ELISA experiments were done on skin samples obtained from various treatment groups to quantitatively assess the levels of TNF-α and IL-6 as inflammatory cytokine markers. The skin tissues were chopped and homogenized in phosphate saline buffer solution (pH 7.4) at 3000 rpm using a tissue homogenizer (Remi Elektrotechnik Ltd., Mumbai, India), followed by centrifugation at 3000 rpm for a period of 15 min. The resulting supernatants were separated and stored at a temperature of − 80°C until analysis. Finally, the concentrations of TNF-α and IL-6 was measured using an corresponding kits from Mouse ELISA (Krishgen biosystems, CA, USA) as per the protocol provided by manufacturer [ 37 ]. 2.4.1.4. Histological Assessment of Skin, Spleen, and Liver In the end, mice were euthanized by CO 2 inhalation and the skin excised from their dorsal area, as well as spleen and liver were collected from all experimental groups. These samples were subsequently preserved in formalin (10% v/v), embedded in paraffin, and subjected to Hematoxylin and Eosin staining (H&E). Sections measuring 5 µm in thickness were prepared and used for histological analysis. The histological examination of the skin, spleen, and liver sections was conducted under a light microscope [ 38 ]. 2.4.2. In-Vivo Skin Compliance Study Firstly, the Balb/c mice were divided into three groups (n = 4). Group 1 served as the control, where no formulation was applied. Group 2 received the application of TAC-THQ-NG, while Group 3 was subjected to the application of TAC-THQ-SG formulations over a span of 7 days. The animals underwent daily visual inspections to check for the presence of erythema or edema 24 h after the application of a treatment. Once the treatment period was completed, their observations were recorded and evaluated using a scoring system that ranged from 0 to 4. This scale encompasses categories for evaluating erythema, where 0 signifies the absence of erythema, whereas 1, 2, 3, 4 denoted slight, moderate, marked and severe erythema respectively. Additionally, the scale also included categories for edema, with 0 representing the absence of edema, whereas 1, 2, 3, 4 signifies slight, moderate, marked and severe edema respectively [ 39 ]. 2.5. Data Analysis The findings were presented in the form of mean values accompanied by their respective standard deviations (SD). The statistical analysis was carried out using GraphPad Prism V.8.3.0 software from San Diego, CA, USA. To compare the data statistically, a one-way ANOVA and two-way ANOVA were initially employed, followed by the Bonferroni multiple comparison test. A significance threshold of p < 0.05 was set for all analysis. 3. Results and Discussion In our previous research, we have successfully prepared the TAC-THQ-NLCs by emulsification solvent-evaporation technique [ 26 ]. The PS, PDI, and ZP of optimized NLCs was obtained to be 144.95 ± 2.80 nm, 0.160 ± 0.021, and − 29.47 ± 1.9 mV, respectively with excellent texture profile, homogeneity and spreadability. The developed formulation exhibited sustained release of the drugs over a period of 24 h from the optimized TAC-THQ-NG. Furthermore, the TAC-THQ-NG demonstrated notably greater, dose-related cytotoxicity towards the HaCaT cell line (a keratinocyte cell line) in comparison to TAC-THQ-SG. These outcomes underscore the efficacy of the optimized TAC-THQ-NLCs to enhance psoriasis treatment while concurrently mitigating dose-related toxicity, a concept that warrants validation through in vivo studies. In this research, our aim was to evaluate the effectiveness of TAC-THQ-NG in enhancing skin delivery and to assess its ability in treating psoriasis in-vivo using a mouse model induced with imiquimod. Additionally, skin compliance studies were conducted to evaluate the safety profile of the developed TAC-THQ-NG formulation. 3.1. Ex-Vivo Studies 3.1.1. Skin Permeation Study The Stratum corneum (SC), which is a primary impediment to drug penetration and not the place of the drugs to exercise their antipsoriatic effects. Therefore, in order to treat the disease at its target site that is epidermis and dermis, the drugs must therefore cross the SC and release the drug in a sustained manner to minimize its systemic absorption [ 40 ]. In ex vivo permeation investigation, the cumulative quantity of permeated drug at each time point is measured. The ex vivo permeation profile of TAC-THQ-NG formulation across the skin was compared with the permeation profile of TAC-THQ-SG. The permeation profiles of all the samples are depicted in ( Fig. 1 A ) and other skin transport characteristics like flux, permeability coefficient and enhancement factors are summarized in Table 1 . The % cumulative amount of drugs permeated from TAC-THQ-NG is 20.27 ± 1.01 and 26.03 ± 1.32 for TAC and THQ respectively, whereas from TAC-THQ-SG; 8.02 ± 0.45 and 12.10 ± 0.64 for TAC and THQ respectively. The % cumulative skin permeation of THQ (MW: 164 g/mol) was higher than that of TAC (MW: 822 g/mol), due to its low molecular weight as compared to TAC [ 41 ]. All the transport factors of TAC-THQ-NG were on the higher side as compared to TAC-THQ-SG for both the drugs. The TAC-THQ-NG showed 2.51- and 2.12-times enhanced permeation of TAC and THQ respectively in contrast to TAC-THQ-SG. The TAC-THQ-NG revealed a significant enhancement in drug permeation than TAC-THQ-SG. The enhanced drug permeation with TAC-THQ-NG is ascribed to the nanosized particles. In addition, the lipid nanoparticles (i.e., NLCs) exhibit an occlusive effect that can alter and perturb the lipid structures within the SC. Additionally; they contribute to improved skin hydration, which, in turn, enhances drug permeation [ 42 ]. Moreover, we incorporated Tween 80, a well-recognized non-ionic surfactant with a well-established reputation for its ability to alter the organization of SC lipids, thereby enhancing the permeation of drugs across the skin [ 43 ]. Table 1 Skin transport parameters of TAC-THQ-NG and TAC-THQ-SG in ex-vivo skin permeation study. Formulations Flux (µg cm − 2 h − 1 ) Permeability coefficient x 10 − 2 (cm/h − 1 ) Enhancement factor TAC-THQ-NG (TAC) 8.05 ± 0.41 0.80 ± 0.041 2.51 TAC-THQ-NG (THQ) 30.51 ± 1.73 1.02 ± 0.060 2.12 TAC-THQ-SG (TAC) 3.21 ± 0.20 0.32 ± 0.020 - TAC-THQ-SG (THQ) 14.40 ± 0.75 0.48 ± 0.025 - 3.1.2. Skin Retention Study The topical delivery of TAC and THQ by encapsulating in the NLCs opted to improve dermal localization for enhancing the localized psoriasis treatment while reducing systemic toxicity [ 44 ]. Hence, the ex-vivo experiment was conducted by using mice skin to assess the skin retention of TAC-THQ-NG and TAC-THQ-SG, and the findings are presented in ( Fig. 1 B ). The % drug deposition of drugs; TAC and THQ was found to be 57.85 ± 2.55% and 60.66 ± 2.83% respectively from TAC-THQ-NG while only 20.12 ± 1.19% and 25.68 ± 1.48% deposited TAC and THQ was obtained from TAC-THQ-SG. The drug retention study revealed that TAC-THQ-NG exhibited higher drug retention in the skin compared to TAC-THQ-SG. This enhanced retention can be attributed to the interaction between the nanosized formulation and keratinocytes, leading to increased drug retention. Nevertheless, the increased drug retention in the skin is advantageous, as these skin layers are primarily affected by psoriasis. 3.1.3. Dermatokinetics The dermatokinetic profiles of TAC-THQ-NG and TAC-THQ-SG are depicted in ( Fig. 2 ) and dermatokinetic parameters are summarized in Table 2 . As depicted in the figure, the TAC-THQ-NG exhibited substantially higher concentrations of drugs in the skin compared to TAC-THQ-SG at all-time points. Achieving significantly elevated drug concentrations in the skin, especially for poorly absorbed drugs like TAC and THQ, represents a noteworthy accomplishment. This favorable outcome can be attributed to the presence of nanosized particles and the occlusive effects of the TAC-THQ-NG at the application site [ 45 ]. In the case of TAC-THQ-NG, there was approximately a 2.78- and 2.37-folds increase in the C max and a 2.93- and 2.40-folds increase in the area under the curve (AUC) for TAC and THQ, respectively, compared to TAC-THQ-SG. Furthermore, the values of T max and K e are on the lower side for TAC-THQ-NG in contrast with the TAC-THQ-SG formulation in the skin. This study clearly highlights the enhanced topical bioavailability potential of TAC and THQ, as they effectively traverse the skin barrier when administered through the TAC-THQ-NG formulation. Table 2 Dermatokinetic parameters obtained from the drug concentration–time profile of TAC-THQ-NG and TAC-THQ-SG in animal skin. Dermatokinetic parameters TAC-THQ-NG TAC-THQ-SG TAC THQ TAC THQ C skin−max (µg.cm ‒2 ) 789.52 ± 28.15 2648.02 ± 91.03 284.39 ± 14.34 1115.26 ± 48.14 T skin−max (h) 2 2 3 3 AUC 0→24h (µg.cm ‒2 h ‒1 ) 15985.92 ± 585.31 51193.32 ± 1543.98 5448.63 ± 290.49 21113.62 ± 998.17 Ke (h ‒1 ) 0.0117 ± 0.0007 0.0123 ± 0.0008 0.0139 ± 0.0013 0.0131 ± 0.0011 3.1.4. Skin Permeation Dynamics The permeation-enhancing mechanism of NLCs-gel was validated through the application of DSC and FTIR analyses. The DSC thermogram and FTIR spectrum of NLCs-gel treated skin and skin without any treatment are presented in ( Fig. 3 ). The DSC analysis revealed changes in the properties of the skin, as indicated by alterations in the melting point, as shown in ( Fig. 3 A ). The untreated skin, serving as a control, exhibited three endothermic melting peaks at temperatures of 103.696°C (Peak-I), 126.162°C (Peak-II), and 133.379°C (Peak-III). Peak-I and Peak-II were indicative of lipid fluidization, while Peak-III signaled the denaturation of proteins within the skin. In contrast, the skin treated with NLCs-gel displayed the disappearance of Peak-I, with Peak-II and Peak-III appearing at temperatures of 116.186°C and 121.502°C, respectively. This represented a reduction in the melting point transitions of approximately 10–12°C when compared to untreated skin. The disappearance of Peak-I and the shifting of endothermic transitions to lower temperatures were attributed to the enhanced lipid fluidization and the interference with the tight junctions in the SC [ 40 , 46 ]. The characteristic FTIR peaks observed for both treated and untreated skin samples, as displayed in ( Fig. 3 B ) , revealed various specific peaks associated with different stretching modes, such as C-H stretching, C = O stretching, and N-H bending vibrations in the case of skin treated with NLCs and untreated skin. These spectral features can be utilized to assess permeation dynamics [ 47 ]. These peaks correspond to the molecular vibrations of proteins and lipids present in the SC. In the FTIR analysis of untreated skin, characteristic peaks at 2918 cm − 1 due to asymmetric C–H stretching and 2850 cm − 1 were observed, arising from the symmetric C–H stretching, attributed to lipid alkyl chains [ 48 ]. In the NLCs-treated skin, similar asymmetric and symmetric C–H stretching peaks were observed at 2924 cm − 1 and 2853 cm − 1 , respectively with reduced peak intensity. The reduction in peak area may be attributed to the altered lipid composition of the SC. Furthermore, the distortion of lipid bilayers and lipid fluidization in the SC is evidenced by the shifting of CH2 stretching peaks to higher wavenumbers. This aids in surpassing the barrier characteristics of the SC [ 49 ]. Additionally, in untreated skin, the proteins within the SC displayed distinctive amide bands I and II in their infrared spectrum. These bands, occurring at 1641 cm⁻¹ (for amide I, linked to C = O stretching) and 1548 cm⁻¹ (for amide II, related to N–H stretching), serve as evidence affirming the existence of keratin in the SC. Notably, changes in the intensity as well as shifting of the amide I and II peaks were detected in skin treated with NLCs at frequencies of 1645 cm⁻¹ and 1544 cm⁻¹, respectively. These alterations indicate that the NLCs facilitate permeation by interacting with keratin, a significant protein component of the skin [ 50 ]. Therefore, the FTIR analysis provided evidence supporting the idea that the disruption and fluidization of lipids, along with the denaturation of keratin, are the fundamental mechanisms contributing to the improved permeability of the skin. 3.2. In-Vivo Studies 3.2.1. In-Vivo Antipsoriatic Activity Psoriasis, an inflammatory skin condition, typically presents as plaques characterized by prominent erythema, scaling, and thickening of skin. An ideal animal model for evaluating anti-psoriatic treatments should replicate both the phenotypic features of the disease and its biochemical context. In this particular investigation, psoriasis was induced by applying IMQ topically for a continuous period of seven days. IMQ, a ligand for Toll-Like receptors (TLR), can induce psoriasis-like lesions on the skin of mice by affecting the IL-23/IL-17 axis [ 51 ]. The daily application of IMQ on the dorsal area of mice resulted in the emergence of skin abnormalities in the form of inflamed, scaly skin lesion characterized by increased epidermal growth, aberrant cell maturation, the accumulation of neutrophils, the formation of new blood vessels, and the infiltration of cells that mediate immune responses. These manifestations closely mirrored the characteristics of plaque-type psoriasis [ 52 ]. The therapeutic effectiveness of TAC-THQ-NG, was assessed in a research model that induced psoriatic plaques through IMQ application and subsequently the treatment was compared to other treatment groups employing different drugs treatments. The visual representation of animals in various experimental groups after the end of the treatment period is illustrated in ( Fig. 4 ). 3.2.2.1. PASI Scoring The IMQ-induced psoriasis-like inflammation was evaluated using the PASI scoring, an extensively utilized clinical tool to gauge the severity of psoriasis and evaluate the effectiveness of anti-psoriatic therapies. In our in vivo experiments, we scored the severity of the lesion by visually evaluating the redness, thickness, and scaling of the skin inflammation. Following the initiation of IMQ treatment, mice exhibited initial signs of mild thickening, erythema, and scaling on their dorsal skin, which became apparent on days 2–3. The PASI score reached its peak on day 7. The cumulative PASI score was determined by summing the scores for erythema, thickness, and scaling of inflammation, each rated on a scale from 0 to 4. The PASI score for erythema, thickness and scaling of inflammation as wells as cumulative score on a scale from (0–4), for all treated groups are shown in ( Fig. 5 ). On the 7th day of IMQ application, all treatment groups displayed scaling, thickness, and erythema with a PASI score falling in the range of 3–4, indicating the development of severe inflammation. In contrast to the toxic control group, all drug treatment groups showed significant reduction in PASI score of scaling, thickness, and erythema but maximum reduction was observed with TAC-THQ-NG (p < 0.0001). Also as compared to TAC-THQ-SG, TAC-THQ-NG showed 2.0-, 4.5- and 1.67-fold higher reduction in PASI score of thickness, erythema, and scaling respectively. Furthermore, Table 3 presents the comparison between the percentage reduction in the cumulative PASI score after the completion of drug treatment and the cumulative PASI score observed following the onset of the disease. Reduction in the cumulative PASI score represented the healing potential of different formulations. In IMQ treated group, slight increase (2.34%) in cumulative PASI score was observed after induction of disease. In contrast to IMQ treated group, drug treated groups (TAC-THQ-SG, TAC-NG, THQ-NG, TAC-THQ-NG) showed significant reduction in cumulative PASI score (expressed in %) with maximum reduction with TAC-THQ-NG. After the end of treatment, TAC-THQ-SG (57.14%), TAC-NG (46.15%), THQ-NG (39.18%) and TAC-THQ-NG (83.80%) showed reduction in cumulative PASI score. The higher % cumulative PASI score reduction of TAC-THQ-NG as compared to single drug nanogel formulation (TAC-NG and THQ-NG), confirms the enhanced efficacy of TAC and THQ, when used in combination. The enhanced efficacy of TAC-THQ-NG is attributed to the higher skin penetration and drug retention due to the small size and occlusive characteristics of the formulation. Based on our analysis of PASI scores across all groups, we can infer that the developed formulation, TAC-THQ-NG, shows greater promise in terms of both efficacy and safety when compared to TAC-THQ-SG. Table 3 % Reduction in cumulative PASI of different animal groups at the end of treatments. Treatments Cumulative PASI score % Reduction in PASI score After induction of disease After end of treatment TOXIC 3.42 ± 0.17 3.50 ± 0.43 -2.34 TAC-THQ-SG 3.50 ± 0.19 1.50 ± 0.33 57.14 TAC-NG 3.25 ± 0.32 1.75 ± 0.32 46.15 THQ-NG 3.42 ± 0.16 2.08 ± 0.32 39.18 TAC-THQ-NG 3.58 ± 0.17 0.58 ± 0.17 83.80 3.2.2.2. Body weight, Spleen Dimension & Weight) Body weight measurements were recorded for all groups during the treatment period, and changes in body weight relative to the initial body weight were plotted over time, as illustrated in ( Fig. 6 A ). The control group exhibited no significant alterations in body weight throughout the study. While the toxic group displayed a noticeable change in the average body weight of the animals. During the study, there was a decrease in body weight among the drug treatment groups, which included TAC-THQ-SG, TAC-NG, THQ-NG, and TAC-THQ-NG. However, this decrease in body weight was not statistically significant. Notably, the group labeled TAC-THQ-NG showed the least amount of body weight reduction. Spleen enlargement serves as a significant indicator of immunological disorders. The application of IMQ led to the development of an inflammatory condition, resulting in a substantial increase in spleen size and weight due to the release of inflammatory cytokines [ 53 ]. The results concerning spleen dimensions and weight are presented in ( Fig. 6 B ) , while the specific spleen weights are displayed in ( Fig. 6 C ). In the toxic group, an increase in spleen weight was observed, indicative of the induction of psoriasis following IMQ application. The toxic group exhibited an average spleen weight of 380 ± 17.05 mg. In the drug-treated groups (TAC-THQ-SG, TAC-NG, THQ-NG, and TAC-THQ-NG), the average spleen weights were measured at 225.5 ± 11.47, 260 ± 12.36, 296.75 ± 14.45, and 184 ± 10.20 mg, respectively. Notably, the spleen weight of the TAC-THQ-NG group closely resembled that of the normal control (p > 0.05), with an average weight of 166.75 ± 9.71 mg. Moreover, the induction of psoriasis following IMQ application was confirmed by evaluating the spleen to body weight ratio (SBWR) in mice [ 54 ]. The SBWR values for various treatment groups were assessed at the conclusion of the experiment, and depicted in ( Fig. 6 D ). In the toxic group, a substantial increase in SBWR was observed compared to the normal group, signifying the successful development of disease. Conversely, the different drug treatment groups yielded a reduction in SBWR with maximum reduction observed for TAC-THQ-NG. 3.2.2.3. Enzyme-Linked Immunosorbent Assay for TNF-α & IL-6 Among the array of immune cytokines, TNF-α and IL-6 are pivotal pro-inflammatory cytokines known for their significant roles in triggering the onset of psoriasis when stimulated by the application of IMQ [ 55 ]. Therefore, a comparative anti-inflammatory efficacy of different drug treated groups was determined. The changes in levels of TNF-α and IL-6 observed in skin homogenate of treated animals belonging to different groups are illustrated in ( Fig. 7 ). As compared to normal group, 3.25 and 3.74-folds increase in levels of TNF-α and IL-6 respectively was observed in case of IMQ treated group (toxic) indicating induction of psoriasis. Following drug treatment, all drug treatment groups revealed a marked decrease in levels of TNF- α and IL-6 levels in contrast with the IMQ-treated group. Further, the level of TNF-α and IL-6 in the TAC-THQ-NG treated group was non-significant, when compared to the normal group (without any treatment). There was 2.76 and 2.99 times decrease in TNF-α and IL-6 levels for the TAC-THQ-NG treated animals in comparison to the IMQ-treated group. Furthermore, the TAC-THQ-SG group showed 1.84 and 1.97 times decrease in TNF-α and IL-6 levels in comparison to the IMQ-treated group. The higher reduction in cytokines levels on topical treatment with TAC-THQ-NG was attributed to higher skin penetration and drug retention. The higher reduction potential of TAC-THQ-NG as compared to single drug NLC-gel for TNF-α and IL-6 level as compared to IMQ group, confirmed the enhanced efficacy of TAC and THQ when used in combination. Thus, the study substantiated the enhanced efficacy of nanogel combination (TAC-THQ-NG) as compared to their conventional gel (TAC-THQ-SG) and single drug-containing nanogel (TAC-NG and THQ-NG). 3.2.2.4. Histological Assessment of Skin, Spleen, and Liver The H&E-stained histological micrographs of the skin, spleen, and the liver of different treatment groups of Balb/c mice are represented in ( Fig. 8 ). Histologically, normal mouse skin typically displays a well-structured epidermis and dermis, featuring thin layers of the epidermis and normal SC. While, IMQ treated (toxic group) skin displays noticeable epidermal thickening, elongation of epidermal rete ridges, disrupted epidermal differentiation, leukocyte infiltration, and a lack of a granular layer when compared to normal skin [ 56 ]. All the drug treatment groups exhibited a reduction in epidermal thickness and normalized keratinocyte differentiation, with an intact granular layer. However, the group treated with TAC-THQ-NG demonstrated the highest efficacy in combating the inflammation induced by IMQ, when compared to the other groups receiving different treatments. In the histological examination of the spleen, it was evident that the IMQ treatment group represents the rupture of spleen cells due to spleen enlargement, leading to an unclear distinction between the red pulp (appearing pink) and white pulp (appearing blue) regions in the spleen. Conversely, in the normal group (without IMQ treatment), both red pulp and white pulp were clearly distinguishable. Notably, treatment with TAC-THQ-NG exhibited the most significant improvement in the restoration of both red pulp and white pulp areas of the spleen in contrast to other drug treatment groups [ 57 ]. The histological examination of the liver revealed that the group treated with IMQ experienced damage to liver cells (hepatocyte degeneration), fibrotic changes, pyknosis and cellular disintegration. In contrast, the normal group (without IMQ treatment) exhibited no alterations in hepatocyte density. All the groups treated with drugs demonstrated a reduction in hepatocyte degeneration. Notably, animals treated with TAC-THQ-NG experienced the most significant improvement in hepatocyte regeneration compared to other drug treatment groups [ 58 ]. 3.2.2. In-Vivo Skin Compliance Study Topical therapy associated with adverse effect like skin irritation, specifically erythema, considerably restricts its clinical application. This challenge is exacerbated by the fact that many conventional dosage forms, including creams, lotions, and gels, often fail to mitigate the irritation caused by topical application. A hypothesis was formulated that the encapsulation of drugs within NLCs could avoid drug contact with the SC, potentially leading to a reduction in erythema episodes [ 59 ]. Also, TAC marketed formulation is associated with skin irritation (skin burning, itching) when used topically [ 60 ]. Therefore, the skin compliance study of TAC-THQ-NG and TAC-THQ-SG on healthy mice skin was done to analyze the compatibility of the developed formulation with skin. The irritation potential was assessed on the basis of scores attained for erythema and edema for both formulations as represented in Table 4 . The application of TAC-THQ-NG did not show any sign of erythema and edema while in the case of TAC-THQ-SG, slight erythema was observed which was due to TAC in its free form. This suggests that the excipients utilized in the NLCs-gel, as well as the encapsulation of drugs within the NLCs, do not pose an irritation risk. The individual primary irritancy index (PII) for TAC-THQ-NG and TAC-THQ-SG was 0.0 and 1.25 ± 0.50 respectively. The skin compliance study indicated that TAC-THQ-NG exhibited no signs of erythema and edema, even after repeated application for 7 days as compared to TAC-THQ-SG which showed a sign of slight erythema. Therefore, it can be inferred that TAC-THQ-NG is safe for topical application. Table 4 Skin compliance score of mice treated with TAC-THQ-NG and TAC-THQ-SG. Animals (Balb/c) Control TAC-THQ-SG TAC-THQ-NG Erythema Edema Erythema Edema Erythema Edema 1 0 0 0 0 0 0 0 0 1 2 1 1 0 0 0 0 0 0 2 0 0 3 0 0 4 0 0 Mean score ± SD 0 ± 0.0 0 ± 0.0 1.25 ± 0.50 0 ± 0.0 0 ± 0.0 0 ± 0.0 PII 0 1.25 0 Erythema or edema scale: 0, No erythema/edema; 1, slight; 2, moderate; 3, marked; 4, severe. 4. Conclusion In this research, we developed TAC-THQ-NG formulation for the topical treatment of psoriasis. The developed formulation demonstrated superior effectiveness in ex-vivo and in-vivo studies compared to conventional gel. Ex-vivo permeation experiments and CLSM study indicated enhanced skin permeation and retention of drugs as TAC-THQ-NG compared to the TAC-THQ-SG. Dermatokinetic studies clearly highlighted the enhanced topical bioavailability potential of drugs, as they effectively traverse the skin barrier when administered through the developed formulation. Moreover, in-vivo studies on IMQ-induced BALB/c mice showed that the TAC-THQ-NG reduced psoriatic-like skin inflammation, lowered PASI scores of (Thickness, erythema and scaling) and decreased inflammatory markers (TNF-α and IL-6) more effectively than TAC-THQ-SG and individual drug formulation. The enhanced delivery of TAC and THQ to the skin via NLCs resulted in increased efficacy against psoriatic-like plaques, effectively alleviating the symptoms of IMQ-induced psoriasis. The formulation TAC-THQ-NG was also found to be safe, with no toxicity or irritation observed in skin compliance tests and minimal changes in histological examinations (skin, spleen and liver) were observed compared to control group. These findings suggest that the TAC-THQ-NG could be a promising platform for the topical treatment of psoriasis. However, further research is needed to explore the clinical benefits of the developed formulation in treating psoriasis. Declarations Declaration of Interest: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Competing Interests: Authors declare no conflict of interest. Ethical Approval: The Institutional Animal Ethics Committee, Jamia Hamdard, New Delhi, India, approved protocol No. 1894 for Animal Studies (registration No. 173/GO/Re/S/ 2000/CPCSEA, 8 April 2021). Author Contribution Conceptualization Authors NameData curation Meraj Alam,Formal analysis Md. RizwanullahFunding acquisition of the financial support for the project leading to this publication.Investigation. Writing and Reviewing Prof. Saima AminMethodology Prof. Saima AminProject administration Prof. Showkat R MirSoftware use and validation of analysis Ashif Iqubal and Shahnawaz AhmadSupervision Prof. Tae-Geum Kim Acknowledgement The first author also thanks Jamia Hamdard for their generous financial assistance through the Hamdard National Fellowship (HNF). Additionally, the authors want to acknowledge Concord Biotech Limited, Gujarat, India, for gifting tacrolimus. Availability of Data and Materials: Data will be made available on request. References J. Nowowiejska, A. Baran, P. 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Chitkara, Multi-Component Clobetasol-Loaded Monolithic Lipid-Polymer Hybrid Nanoparticles Ameliorate Imiquimod-Induced Psoriasis-like Skin Inflammation in Swiss Albino Mice, Acta Biomater. 115 (2020) 393–409. P. Tripathi, A. Kumar, P.K. Jain, J.R. Patel, Carbomer Gel Bearing Methotrexate Loaded Lipid Nanocontainers Shows Improved Topical Delivery Intended for Effective Management of Psoriasis, Int. J. Biol. Macromol. 120 (2018) 1322–1334. S. Jain, R. Addan, V. Kushwah, H. Harde, R.R. Mahajan, Comparative Assessment of Efficacy and Safety Potential of Multifarious Lipid Based Tacrolimus Loaded Nanoformulations, Int. J. Pharm. 562 (2019) 96–104. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {\"props\":{\"pageProps\":{\"initialData\":{\"identity\":\"rs-4448132\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":true,\"archivedVersions\":[],\"articleType\":\"Research Article\",\"associatedPublications\":[],\"authors\":[{\"id\":309569181,\"identity\":\"5ebfca95-a043-4d93-ab4b-318d154031e3\",\"order_by\":0,\"name\":\"Meraj Alam\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Jamia Hamdard\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Meraj\",\"middleName\":\"\",\"lastName\":\"Alam\",\"suffix\":\"\"},{\"id\":309569185,\"identity\":\"7d836599-a576-48f9-a378-6b38f870f39e\",\"order_by\":1,\"name\":\"Md. Rizwanullah\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Saveetha Medical College \\u0026 Hospital, Saveetha Institute of Medical and Technical Sciences (SIMATS)\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Md.\",\"middleName\":\"\",\"lastName\":\"Rizwanullah\",\"suffix\":\"\"},{\"id\":309569186,\"identity\":\"1323a7e6-7e9c-44db-a5e2-f6b5553664ed\",\"order_by\":2,\"name\":\"Shahnawaz Ahmad\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Jamia Hamdard\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Shahnawaz\",\"middleName\":\"\",\"lastName\":\"Ahmad\",\"suffix\":\"\"},{\"id\":309569187,\"identity\":\"0d6ecefa-e26a-4eb6-9724-d0889e06b6a8\",\"order_by\":3,\"name\":\"Ashif Iqubal\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Jamia Hamdard\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Ashif\",\"middleName\":\"\",\"lastName\":\"Iqubal\",\"suffix\":\"\"},{\"id\":309569189,\"identity\":\"f532528d-5b36-4a39-887b-12a3ee98f4d7\",\"order_by\":4,\"name\":\"Showkat R. Mir\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Jamia Hamdard\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Showkat\",\"middleName\":\"R.\",\"lastName\":\"Mir\",\"suffix\":\"\"},{\"id\":309569190,\"identity\":\"bd51f9d3-99b3-4d03-9acd-df3594e56966\",\"order_by\":5,\"name\":\"Tae-Geum Kim\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Jeonbuk National University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Tae-Geum\",\"middleName\":\"\",\"lastName\":\"Kim\",\"suffix\":\"\"},{\"id\":309569191,\"identity\":\"7fcfbf15-8adb-47ce-8b47-38b09c707fae\",\"order_by\":6,\"name\":\"Saima Amin\",\"email\":\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABAUlEQVRIiWNgGAWjYJCCAwwVbAwMzAxAAkgyMCSABCUIaDlDqhYGxjYwhaIFN5BvP514uHAen7xuO/OzBz9zrOXN2xMYP/xgsMjDpcXgTO6GwzO3sRluO8xmbti7Ld1wzpkHzJI9DBLFOLUwALXwbmNj3HaYwUyCd9thxhkSCQzSQL8kNuByWP9boJY5bPbbDrN/k/y77bA9UAvzb3xaGG6AbGlgS9x2mMdMGmhLIlALG15bDG4AbeE5xpYM1FImLbstPXkGz8M2yx4DfA7L3fyZp+aY7bbzx7dJvt1mbTuDPfnwjR8VdbgdBgHHkDmMDaBgIQRqCKoYBaNgFIyCEQwATHFXeI3L12oAAAAASUVORK5CYII=\",\"orcid\":\"\",\"institution\":\"Jamia Hamdard\",\"correspondingAuthor\":true,\"prefix\":\"\",\"firstName\":\"Saima\",\"middleName\":\"\",\"lastName\":\"Amin\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2024-05-20 09:33:04\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-4448132/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-4448132/v1\",\"draftVersion\":[],\"editorialEvents\":[],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":57649425,\"identity\":\"ec5abf79-3081-4114-8223-ebb3a75523cb\",\"added_by\":\"auto\",\"created_at\":\"2024-06-03 21:43:15\",\"extension\":\"jpg\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":253279,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eComparative ex-vivo [A] skin permeation and [B] retention profiles of TAC and THQ from the optimized TACTHQ-NG and TAC-THQ-SG.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage1.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4448132/v1/2b3b396e28647b9d91f75b9e.jpg\"},{\"id\":57649308,\"identity\":\"b10d6443-9a69-4b1f-8ede-ad7ab1c0fc7e\",\"added_by\":\"auto\",\"created_at\":\"2024-06-03 21:35:15\",\"extension\":\"jpg\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":125411,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eComparative concentration of drugs [A] TAC and [B]\\u003cstrong\\u003e \\u003c/strong\\u003eTHQ found in skin at different time points from the TACTHQ-NG and TAC-THQ-SG.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage2.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4448132/v1/6ff696f93b9d058eb11e4a76.jpg\"},{\"id\":57649310,\"identity\":\"1d800090-4079-4800-a641-5138e888d02e\",\"added_by\":\"auto\",\"created_at\":\"2024-06-03 21:35:15\",\"extension\":\"jpg\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":295847,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eImage showing [A]\\u003cstrong\\u003e \\u003c/strong\\u003eDSC thermogram and [B] FTIR spectra of (I) untreated and (II) NLCs treated animal skin.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage3.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4448132/v1/4e7d77e10b189a225a8cb5d2.jpg\"},{\"id\":57649426,\"identity\":\"f5bc7419-fd1d-47b3-a585-bb33f19bb4d4\",\"added_by\":\"auto\",\"created_at\":\"2024-06-03 21:43:15\",\"extension\":\"jpg\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":218648,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eThe visual representation of animals in various experimental groups after the end of the treatment period.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage4.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4448132/v1/0d7340f6120b854d6f7d33f1.jpg\"},{\"id\":57649316,\"identity\":\"276463cd-9381-44d6-a953-b45570de62ca\",\"added_by\":\"auto\",\"created_at\":\"2024-06-03 21:35:16\",\"extension\":\"jpg\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":290590,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eImages showing effectiveness of different treatment groups on IMQ induced psoriasis based on thickness, erythema, and scaling of skin recorded after induction of disease and end of treatment, on a PASI scale of 0 to 4. [A]Thickness; [B] Erythema; [C] Scaling and [D] Cumulative PASI score.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage5.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4448132/v1/55c0acb2d5c0b34df50ba6ad.jpg\"},{\"id\":57649313,\"identity\":\"173c5b36-7192-4ec1-abe2-04dcaa2f6d25\",\"added_by\":\"auto\",\"created_at\":\"2024-06-03 21:35:15\",\"extension\":\"jpg\",\"order_by\":6,\"title\":\"Figure 6\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":455116,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eImage showing effect of different formulations on [A]Body weight [B]; spleen dimension [C]; Spleen weight and [D] Spleen: body weight ration of different treatment groups.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage6.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4448132/v1/217ead37aa9d1f4645d8cf72.jpg\"},{\"id\":57649311,\"identity\":\"e632401c-b803-48cd-a3e0-d2ec86df92d8\",\"added_by\":\"auto\",\"created_at\":\"2024-06-03 21:35:15\",\"extension\":\"jpg\",\"order_by\":7,\"title\":\"Figure 7\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":240021,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eImage showing effect of different treatment groups on inflammatory cytokines levels [A]\\u003cstrong\\u003e \\u003c/strong\\u003eTNF and [B] IL-6 on IMQ induced psoriasis on mice skin.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage7.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4448132/v1/de9359cbba66c12245c9ac2b.jpg\"},{\"id\":57649312,\"identity\":\"fc45777d-1e22-4b07-9080-2a205422c69b\",\"added_by\":\"auto\",\"created_at\":\"2024-06-03 21:35:15\",\"extension\":\"jpg\",\"order_by\":8,\"title\":\"Figure 8\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":852560,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eThe histological visual representation of skin, spleen and liver of different treatment groups of Balb/c mice after end of treatment period.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage8.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4448132/v1/6be57f6740942c9d4292be71.jpg\"},{\"id\":63688730,\"identity\":\"b25a4b90-2271-4270-9902-81649284b8e0\",\"added_by\":\"auto\",\"created_at\":\"2024-08-31 09:46:43\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":3601015,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4448132/v1/bd095459-7a5f-4511-b199-28447b8bcdb0.pdf\"}],\"financialInterests\":\"No competing interests reported.\",\"formattedTitle\":\"Co-delivery of Tacrolimus and Thymoquinone topically by Nanostructured lipid carriers gel for enhanced efficacy against imiquimod-induced psoriasis in Balb/c mice\",\"fulltext\":[{\"header\":\"1. Introduction\",\"content\":\"\\u003cp\\u003ePsoriasis is a chronic autoimmune inflammatory skin disease that affects millions of individuals worldwide, leading to significant adverse impacts on patients' physical, social, and psychological well-being [\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e]. Psoriasis is characterized by skin thickening, scaling, and epidermal changes, which include an inflammatory infiltrate presence in both the epidermal and dermal layers [\\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e]. This condition involves a series of interconnected changes in skin cells, including the excessive growth of epidermal keratinocytes, increased blood vessel formation, and the infiltration of T-lymphocytes, neutrophils, and various other types of cells into the affected skin [\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e]. The development of psoriasis is thought to be the result of a complex interaction between genetic elements and external influences like smoking, alcohol consumption, oxidative stress, physical injury, and bacterial infections [\\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e]. As psoriasis remains an incurable disease, current approaches aim to alleviate its symptoms by administering various drugs. These treatment options include topical therapy, phototherapy, and systemic therapy involving oral medications and newer biological agents delivered through injections. While oral and intravenous drugs can be effective, they often come with the risk of severe adverse effects that can affect different organs [\\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e]. Among these options, topical therapy is often favored due to its ease of application, localized effects, and reduced systemic impact [\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e]. However, a significant challenge like limited drug penetration into the psoriatic skin limits the application of topical therapy. Furthermore, the altered condition of psoriatic skin like increased rigidity, lack of moisture, and lipid imbalance significantly hinders the permeation of conventional topical products [\\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eTacrolimus (TAC) is a potent immunosuppressant that belongs to the 'second generation' macrolides, originally derived from \\u003cem\\u003eStreptomyces tsukubaensis\\u003c/em\\u003e. Upon binding to the immunophilin FKBP-12 protein, they form a complex that hinders the dephosphorylation of NF-AT (nuclear factor of activated T-cells) by calcineurin. This, in turn, leads to anti-psoriatic effects as it suppresses T-cell reactions and diminishes the expression of IL-2 and other immune response-related cytokines [\\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e]. However, its high lipophilicity and molecular weight hinder its ability to penetrate the skin effectively, limiting its efficacy. Additionally, commercially available TAC ointments have shown variable and poor dermal bioavailability, often leading to local adverse reactions such as skin burning, itching, pain, and redness [\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e]. Thymoquinone (THQ) is a lipid-soluble benzoquinone and the principal bioactive compound found in black cumin (\\u003cem\\u003eNigella sativa\\u003c/em\\u003e) seeds. THQ has demonstrated remarkable antioxidant, anti-inflammatory, and anticancer properties in both in vitro and in vivo studies [\\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e]. Moreover, several studies have explored its potential in treating psoriasis, yielding promising results [\\u003cspan additionalcitationids=\\\"CR14 CR15\\\" citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e]. Despite its therapeutic advantages, the pharmaceutical application of THQ is constrained by its limited solubility, high lipophilicity, and limited skin penetration [\\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e]. Hence, there is an utmost need for the development of innovative and effective topical delivery systems for both TAC and THQ. As the pathogenesis of psoriasis involves multiple factors, the existing topical single drug formulations on the market are often unable to provide a complete cure for this condition. Consequently, the use of combination therapy has proven beneficial for the improved management of psoriasis [\\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e]. The rationale behind employing a combination of drugs involves the potential to act through distinct pathways, offering a multi-targeted approach that enhances efficacy while minimizing dose-related adverse effects [\\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e]. Thus, combining synthetic drugs with natural bioactives has become a valuable strategy in recent times, aiming to achieve multifaceted advantages compared to monotherapy.\\u003c/p\\u003e \\u003cp\\u003eTo enhance therapeutic efficacy while minimizing side effects, nanocarrier-based approaches have emerged as a promising strategy to ensure effective drug delivery into the deeper layers of psoriatic skin. Over the past many years, extensive research has been conducted on lipid nanoparticles, aimed at achieving improved and safer topical drug delivery of various drugs [\\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e]. These carriers exhibit significant potential for addressing various skin conditions by interacting with the skin at the molecular level [\\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e]. Among different lipid-based nanoparticles, nanostructured lipid carriers (NLCs) are recognized as highly efficient options for delivering topical anti-psoriatic drugs [\\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e22\\u003c/span\\u003e]. NLCs offer additional advantages, including enhanced drug loading, improved release profiles, and greater stability of the encapsulated compounds compared to other lipid nanoparticles [\\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e23\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e]. Following topical application, NLCs adhere to the lipid film of the \\u003cem\\u003eStratum corneum\\u003c/em\\u003e (SC) due to their small particle size. This phenomenon facilitates increased or controlled drug penetration into the skin while also protecting the drug from degradation and extending its release duration [\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e] [\\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e25\\u003c/span\\u003e]. In our previous investigation, we developed TAC and THQ co-loaded NLCs to achieve better cytotoxicity against keratinocyte cell lines (HaCaT cells) [\\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e26\\u003c/span\\u003e]. The TAC-THQ-NLCs was prepared by emulsification solvent-evaporation technique and then optimized by 3\\u003csup\\u003e3\\u003c/sup\\u003e Box\\u0026ndash;Behnken Design. The optimized TAC-THQ-NLCs revealed excellent drug release profiles and stability of the formulation. The particle size (PS), polydispersity (PDI), zeta potential (ZP), and encapsulation efficiency (%EE) of the optimized TAC-THQ-NLCs was observed to be \\u0026lt;\\u0026thinsp;150 nm, \\u0026lt; 0.2, \\u0026gt; 25 mV, and \\u0026gt;\\u0026thinsp;70% respectively. Further, the cell culture study showed much better cytotoxicity of optimized TAC-THQ-NLCs against HaCaT cells in comparison with the conventional TAC-THQ-suspension.\\u003c/p\\u003e \\u003cp\\u003eTherefore, the main objective of this study is to evaluate the anti-psoriatic potential of TAC and THQ co-loaded NLCs-gel in imiquimod induced Balb/c mice model. The ex-vivo skin permeation and permeation dynamics of the optimized formulation was evaluated in mice skin. Further, the skin dermatokinetics of TAC and THQ from the optimized formulation were evaluated and compared with the results from the free gels prepared from combined drugs. In addition, in vivo therapeutic efficacy of the developed TAC-THQ-NLCs gels was also conducted in imiquimod-induced psoriatic Balb/c mice.\\u003c/p\\u003e\"},{\"header\":\"2. Experimental\",\"content\":\"\\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.1. Materials and Chemicals\\u003c/h2\\u003e \\u003cp\\u003eTacrolimus (TAC) was provided as a gift sample by Concord Biotech, Gujarat, India. Thymoquinone (THQ) and Rhodamine B were purchased from Sigma Aldrich, Saint Louis, MO, USA. Capryol 90, Labrasol, Gelot 64, Apifil CG, Precirol ATO 5, Labrafil M 1944 CS, Lauroglycol 90, Peceol, Masine 35\\u0026thinsp;\\u0026minus;\\u0026thinsp;1, and Plurol Diisostearique were provided as a gift sample by Gattefose India Pvt. Ltd., Mumbai, India. Glyceryl monostearate (GMS), triethanolamine, and stearic acid were procured from CDH Fine Chemicals (New Delhi, India). Tween 80, Span 20 and Mannitol were purchased from SD Fine Chemicals, New Delhi, India. Carbopol Ultrez10 was gifted from Lubrizol India (Mumbai, India). Imiquimod cream (5%) was purchased from Glenmark Pharmaceuticals, Mumbai, India. All additional chemicals and reagents employed in this study met the criteria of analytical-grade quality.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec4\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.2 Experimental Animals\\u003c/h2\\u003e \\u003cp\\u003eTo conduct both the ex-vivo and in-vivo studies, \\u003cem\\u003eBalb/c\\u003c/em\\u003e mice (either male or female) was duly approved by the Institutional Animal Ethics Committee (IAEC; 173/GO/Re/S/2000), Jamia Hamdard (Approval number: 1894). The animals were kept in polypropylene enclosures within typical laboratory settings, with a controlled temperature of 24\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;2\\u0026deg;C and humidity levels maintained at 55\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;5%. They were provided with a standardized pellet diet and had unrestricted access to water.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec5\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.3. Ex-Vivo Studies\\u003c/h2\\u003e \\u003cp\\u003eThe dorsal hair of the mice was removed using a depilatory cream, and subcutaneous fat was excised through surgical means. Subsequently, the dermal side of the skin was cleansed with phosphate-buffered saline to eliminate any adhering particles and then wrapped in aluminum foil before being stored in a freezer maintained at \\u0026minus;\\u0026thinsp;20\\u0026deg;C. On the day of the study, the skin that had been frozen was allowed to reach room temperature and then it was placed into Franz diffusion cell apparatus. The SC side was oriented upwards, facing the donor compartment, while the dermis side was placed facing downwards against the receiver compartment [\\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e27\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cdiv id=\\\"Sec6\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e2.3.1. Skin Permeation Study\\u003c/h2\\u003e \\u003cp\\u003eSkin permeation experiments for TAC-THQ-NLCs-gel abbreviated as \\u0026ldquo;TAC-THQ-NG\\u0026rdquo; and TAC-THQ-Suspension-gel abbreviated as \\u0026ldquo;TAC-THQ-SG\\u0026rdquo; were conducted using a Franz diffusion cell with excised mice skin. A skin sample was sandwiched between the donor and acceptor compartments of a Franz diffusion cell, which was positioned on a magnetic stirrer set at 100 rpm for a period of 24 h. The temperature was maintained at 37\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1\\u0026deg;C throughout the experiment. In the acceptor compartment of the Franz diffusion cell, 13 mL of diffusion medium (PBS, pH 7.4, 0.5% Tween 80) was added. In two separate Franz diffusion cells, 1 g each of TAC-THQ-NG and TAC-THQ-SG was uniformly applied in a non-occlusive manner inside the donor compartment, which had an exposed skin surface area of 0.785 cm\\u003csup\\u003e2\\u003c/sup\\u003e. At predefined time points (0.5, 1, 2, 4, 6, 8, 12, and 24 h), 1 mL sample was withdrawn from the receiver cell and promptly substituted with a same volume of fresh dissolution medium. The gathered samples were then passed through a 0.2 \\u0026micro;m membrane filter and subsequently subjected to analysis using our previously reported RP-HPLC method for drug quantification. For both formulations, the cumulative percentage of drugs (TAC and THQ) permeated through the skin membrane was graphed against time. Parameters like the Flux i.e. steady-state permeation rate (\\u0026micro;g.cm\\u003csup\\u003e\\u0026minus;\\u0026thinsp;2\\u003c/sup\\u003e.h\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e) was determined by analyzing the slope of the linear segment of graph, and permeability coefficient (cm.h\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e) was then computed by dividing the flux by the amount of drug taken initially in the Franz diffusion cells. Additionally, an enhancement factor was calculated by dividing the flux of the TAC-THQ-NG formulation with respect to that of the TAC-THQ-SG formulation [\\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e28\\u003c/span\\u003e].\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec7\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e2.3.2. Skin Retention Study\\u003c/h2\\u003e \\u003cp\\u003eFor skin retention study, the procedure was analogous to skin permeation study. Briefly, the mounted skin was discharged from the donor compartment of Franz diffusion cells after 24 h of study. Then, the skin samples were rinsed thoroughly with normal saline three times to eliminate any residual formulations. To ensure the complete extraction of drugs retained in the skin, the skin was finely minced, and the drugs were extracted through maceration in methanol for a period of 24 h, followed by an additional 1 h of bath sonication [\\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e] Finally, the methanolic extract containing both drugs was subsequently filtered using a 0.2 \\u0026micro;m filter, and the drug content was quantified using our previously established RP-HPLC method.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec8\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e2.3.3. Dermatokinetics\\u003c/h2\\u003e \\u003cp\\u003eThe dermatokinetic study was conducted to measure the concentration of drugs in the skin at different time intervals as per previously reported method with modification. The dermatokinetic experiments mirrored the skin permeation study, except that, for each time point, a single Franz diffusion cell was utilized. The experiments were performed on mouse skin mounted in a Franz diffusion cell, with the skin being treated using the optimized TAC-THQ-NG and TAC-THQ-SG formulations, similar to the procedure followed in skin permeation study. However, in the dermatokinetic investigation, the complete skin sample was removed at predefined time points (0.5, 1, 2, 3, 4, 6, 12, and 24 h) from the Franz diffusion cell setup. Following that, the skin samples that had been taken off underwent a series of three rinses with normal saline solution in order to eliminate any residual formulations adhering to them. At each time point, the skin samples were macerated in methanol for 24 h, followed by sonication (1 h) to ensure complete extraction of both drugs (TAC and THQ). After extraction of drugs the methanolic extract was filtered with 0.2 \\u0026micro;m filter before quantification of drugs by our previously developed RP-HPLC technique. The concentration of drugs (TAC and THQ) at each specified time point was assessed through non-compartmental dermatokinetics modeling. The software PK solver was utilized to calculate different dermatokinetic variables, like C\\u003csub\\u003emax\\u003c/sub\\u003e, AUC\\u003csub\\u003e0\\u0026thinsp;\\u0026minus;\\u0026thinsp;24h\\u003c/sub\\u003e, K\\u003csub\\u003ee\\u003c/sub\\u003e, and T\\u003csub\\u003emax\\u003c/sub\\u003e [\\u003cspan citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e30\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e31\\u003c/span\\u003e].\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec9\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e2.3.4. Dynamics of Skin Permeation\\u003c/h2\\u003e \\u003cp\\u003eThe dynamics of skin permeation of NLC-gel was conducted on excised mice skin using two distinct analytical techniques i.e., DSC and FTIR. The mouse skin was positioned between the donor and acceptor compartments of two separate Franz diffusion cells, both filled with a diffusion medium (PBS, pH 7.4, 0.5% Tween 80) in the acceptor compartment. These cell assemblies were then placed above magnetic stirrers set at a constant speed of 100 rpm for duration of 24 h, while the temperature was maintained at 37\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1\\u0026deg;C. One of the Franz diffusion cells was exposed to the NLCs-gel formulation, while the other cell remained untreated and served as a control. After the completion of the permeation experiment, the skin samples from both diffusion cells were carefully removed, rinsed with normal saline, and subsequently cut into small pieces before being dried for subsequent DSC and FTIR analysis. The DSC analysis involved placing a suitable amount of dried samples into aluminum pans. The examination was conducted within the temperature range of 30 to 300\\u0026deg;C, with a heating rate of 10\\u0026deg;C per minute. The remaining skin samples underwent FTIR spectral analysis, encompassing a spectral range from 500 to 4000 cm⁻\\u0026sup1; [\\u003cspan citationid=\\\"CR32\\\" class=\\\"CitationRef\\\"\\u003e32\\u003c/span\\u003e].\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec10\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.4. In-Vivo Study\\u003c/h2\\u003e \\u003cdiv id=\\\"Sec11\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e2.4.1. In-Vivo Antipsoriatic Activity\\u003c/h2\\u003e \\u003cp\\u003eThis study was conducted on imiquimod (IMQ) induced psoriatic \\u003cem\\u003eBalb/c\\u003c/em\\u003e mice to assess the efficacy of TAC-THQ-NG as per the reported method [\\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e33\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR34\\\" class=\\\"CitationRef\\\"\\u003e34\\u003c/span\\u003e]. Initially, the hair from the dorsal skin of animals was removed using depilatory cream and examined the skin for any signs of inflammation. Psoriasis-like symptoms were induced in mice by applying 5% IMQ cream at a daily dose of 62.5 mg per mouse for a continuous period of seven days. Mice were monitored daily to visualize the development of psoriatic lesion. Animals were randomly divided into 6 groups, each group containing 4 mice: Where, Group 1, serving as the normal control; Group 2 was subjected to psoriasis induction but left untreated (Toxic); Group 3 was treated with TAC-THQ-SG; Group 4 was treated with TAC-NG; Group 5 was treated with THQ-NG; and Group 6 was treated with TAC-THQ-NG. Then, different formulations at a dosage of 40 mg/cm\\u003csup\\u003e2\\u003c/sup\\u003e (containing 0.1% TAC and 0.3% THQ) were applied to the psoriatic skin using a spatula, and this treatment continued for next seven days. After the seventh day of treatment, animals of different groups were assessed visually for signs of erythema, scaling and thickness and then sacrificed. Thereafter skin, spleen, and liver were removed and preserved in formalin (10% v/v) for histological examinations.\\u003c/p\\u003e \\u003cdiv id=\\\"Sec12\\\" class=\\\"Section4\\\"\\u003e \\u003ch2\\u003e2.4.1.1. Psoriasis Area and Severity Index (PASI) Scoring\\u003c/h2\\u003e \\u003cp\\u003e The psoriasis-inflammation evaluation criteria were assessed according to the PASI scoring. Evaluation was conducted to assess and assign scores on a scale from 0 to 4 for erythema, scaling, and thickening. This scale ranges from absence (0) to various levels of severity (1: slight, 2: moderate, 3: marked, 4: severe) for each of these skin characteristics. Then the cumulative PASI score was calculated, which combines the individual scores for erythema, scaling, and thickening. It serves as the definitive indicator of the severity of inflammation, providing a measure of the extent of psoriasis [\\u003cspan citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e35\\u003c/span\\u003e].\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec13\\\" class=\\\"Section4\\\"\\u003e \\u003ch2\\u003e2.4.1.2. Body Weight, Spleen Dimension \\u0026amp; weight\\u003c/h2\\u003e \\u003cp\\u003eBody weights of all the groups were measured initially, after induction of disease and after the end of treatment period. In inflammatory diseases such as psoriasis, the spleen often undergoes an enlargement in size, a response associated with high cytokine production. Hence, the dimension and weight of the spleen were measured [\\u003cspan citationid=\\\"CR36\\\" class=\\\"CitationRef\\\"\\u003e36\\u003c/span\\u003e].\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec14\\\" class=\\\"Section4\\\"\\u003e \\u003ch2\\u003e2.4.1.3. Enzyme-Linked Immunosorbent Assay for TNF-α \\u0026amp; IL-6\\u003c/h2\\u003e \\u003cp\\u003eELISA experiments were done on skin samples obtained from various treatment groups to quantitatively assess the levels of TNF-α and IL-6 as inflammatory cytokine markers. The skin tissues were chopped and homogenized in phosphate saline buffer solution (pH 7.4) at 3000 rpm using a tissue homogenizer (Remi Elektrotechnik Ltd., Mumbai, India), followed by centrifugation at 3000 rpm for a period of 15 min. The resulting supernatants were separated and stored at a temperature of \\u0026minus;\\u0026thinsp;80\\u0026deg;C until analysis. Finally, the concentrations of TNF-α and IL-6 was measured using an corresponding kits from Mouse ELISA (Krishgen biosystems, CA, USA) as per the protocol provided by manufacturer [\\u003cspan citationid=\\\"CR37\\\" class=\\\"CitationRef\\\"\\u003e37\\u003c/span\\u003e].\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec15\\\" class=\\\"Section4\\\"\\u003e \\u003ch2\\u003e2.4.1.4. Histological Assessment of Skin, Spleen, and Liver\\u003c/h2\\u003e \\u003cp\\u003eIn the end, mice were euthanized by CO\\u003csub\\u003e2\\u003c/sub\\u003e inhalation and the skin excised from their dorsal area, as well as spleen and liver were collected from all experimental groups. These samples were subsequently preserved in formalin (10% v/v), embedded in paraffin, and subjected to Hematoxylin and Eosin staining (H\\u0026amp;E). Sections measuring 5 \\u0026micro;m in thickness were prepared and used for histological analysis. The histological examination of the skin, spleen, and liver sections was conducted under a light microscope [\\u003cspan citationid=\\\"CR38\\\" class=\\\"CitationRef\\\"\\u003e38\\u003c/span\\u003e].\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec16\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e2.4.2. In-Vivo Skin Compliance Study\\u003c/h2\\u003e \\u003cp\\u003eFirstly, the Balb/c mice were divided into three groups (n\\u0026thinsp;=\\u0026thinsp;4). Group 1 served as the control, where no formulation was applied. Group 2 received the application of TAC-THQ-NG, while Group 3 was subjected to the application of TAC-THQ-SG formulations over a span of 7 days. The animals underwent daily visual inspections to check for the presence of erythema or edema 24 h after the application of a treatment. Once the treatment period was completed, their observations were recorded and evaluated using a scoring system that ranged from 0 to 4. This scale encompasses categories for evaluating erythema, where 0 signifies the absence of erythema, whereas 1, 2, 3, 4 denoted slight, moderate, marked and severe erythema respectively. Additionally, the scale also included categories for edema, with 0 representing the absence of edema, whereas 1, 2, 3, 4 signifies slight, moderate, marked and severe edema respectively [\\u003cspan citationid=\\\"CR39\\\" class=\\\"CitationRef\\\"\\u003e39\\u003c/span\\u003e].\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec17\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.5. Data Analysis\\u003c/h2\\u003e \\u003cp\\u003eThe findings were presented in the form of mean values accompanied by their respective standard deviations (SD). The statistical analysis was carried out using GraphPad Prism V.8.3.0 software from San Diego, CA, USA. To compare the data statistically, a one-way ANOVA and two-way ANOVA were initially employed, followed by the Bonferroni multiple comparison test. A significance threshold of p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05 was set for all analysis.\\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"3. Results and Discussion\",\"content\":\"\\u003cp\\u003eIn our previous research, we have successfully prepared the TAC-THQ-NLCs by emulsification solvent-evaporation technique [\\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e26\\u003c/span\\u003e]. The PS, PDI, and ZP of optimized NLCs was obtained to be 144.95\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;2.80 nm, 0.160\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.021, and \\u0026minus;\\u0026thinsp;29.47\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.9 mV, respectively with excellent texture profile, homogeneity and spreadability. The developed formulation exhibited sustained release of the drugs over a period of 24 h from the optimized TAC-THQ-NG. Furthermore, the TAC-THQ-NG demonstrated notably greater, dose-related cytotoxicity towards the HaCaT cell line (a keratinocyte cell line) in comparison to TAC-THQ-SG. These outcomes underscore the efficacy of the optimized TAC-THQ-NLCs to enhance psoriasis treatment while concurrently mitigating dose-related toxicity, a concept that warrants validation through in vivo studies. In this research, our aim was to evaluate the effectiveness of TAC-THQ-NG in enhancing skin delivery and to assess its ability in treating psoriasis in-vivo using a mouse model induced with imiquimod. Additionally, skin compliance studies were conducted to evaluate the safety profile of the developed TAC-THQ-NG formulation.\\u003c/p\\u003e \\u003cdiv id=\\\"Sec19\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e3.1. Ex-Vivo Studies\\u003c/h2\\u003e \\u003cdiv id=\\\"Sec20\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e3.1.1. Skin Permeation Study\\u003c/h2\\u003e \\u003cp\\u003eThe \\u003cem\\u003eStratum corneum\\u003c/em\\u003e (SC), which is a primary impediment to drug penetration and not the place of the drugs to exercise their antipsoriatic effects. Therefore, in order to treat the disease at its target site that is epidermis and dermis, the drugs must therefore cross the SC and release the drug in a sustained manner to minimize its systemic absorption [\\u003cspan citationid=\\\"CR40\\\" class=\\\"CitationRef\\\"\\u003e40\\u003c/span\\u003e]. In ex vivo permeation investigation, the cumulative quantity of permeated drug at each time point is measured. The ex vivo permeation profile of TAC-THQ-NG formulation across the skin was compared with the permeation profile of TAC-THQ-SG. The permeation profiles of all the samples are depicted in \\u003cb\\u003e(\\u003c/b\\u003eFig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eA\\u003cb\\u003e)\\u003c/b\\u003e and other skin transport characteristics like flux, permeability coefficient and enhancement factors are summarized in Table\\u0026nbsp;\\u003cspan refid=\\\"Tab1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e. The % cumulative amount of drugs permeated from TAC-THQ-NG is 20.27\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.01 and 26.03\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.32 for TAC and THQ respectively, whereas from TAC-THQ-SG; 8.02\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.45 and 12.10\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.64 for TAC and THQ respectively. The % cumulative skin permeation of THQ (MW: 164 g/mol) was higher than that of TAC (MW: 822 g/mol), due to its low molecular weight as compared to TAC [\\u003cspan citationid=\\\"CR41\\\" class=\\\"CitationRef\\\"\\u003e41\\u003c/span\\u003e]. All the transport factors of TAC-THQ-NG were on the higher side as compared to TAC-THQ-SG for both the drugs. The TAC-THQ-NG showed 2.51- and 2.12-times enhanced permeation of TAC and THQ respectively in contrast to TAC-THQ-SG. The TAC-THQ-NG revealed a significant enhancement in drug permeation than TAC-THQ-SG. The enhanced drug permeation with TAC-THQ-NG is ascribed to the nanosized particles. In addition, the lipid nanoparticles (i.e., NLCs) exhibit an occlusive effect that can alter and perturb the lipid structures within the SC. Additionally; they contribute to improved skin hydration, which, in turn, enhances drug permeation [\\u003cspan citationid=\\\"CR42\\\" class=\\\"CitationRef\\\"\\u003e42\\u003c/span\\u003e]. Moreover, we incorporated Tween 80, a well-recognized non-ionic surfactant with a well-established reputation for its ability to alter the organization of SC lipids, thereby enhancing the permeation of drugs across the skin [\\u003cspan citationid=\\\"CR43\\\" class=\\\"CitationRef\\\"\\u003e43\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003cdiv class=\\\"gridtable\\\"\\u003e\\u003ctable float=\\\"Yes\\\" id=\\\"Tab1\\\" border=\\\"1\\\"\\u003e \\u003ccaption language=\\\"En\\\"\\u003e \\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 1\\u003c/div\\u003e \\u003cdiv class=\\\"CaptionContent\\\"\\u003e \\u003cp\\u003eSkin transport parameters of TAC-THQ-NG and TAC-THQ-SG in ex-vivo skin permeation study.\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/caption\\u003e \\u003ccolgroup cols=\\\"4\\\"\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c1\\\" colnum=\\\"1\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" class=\\\"colspec\\\" colname=\\\"c2\\\" colnum=\\\"2\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" class=\\\"colspec\\\" colname=\\\"c3\\\" colnum=\\\"3\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c4\\\" colnum=\\\"4\\\"\\u003e\\u003c/div\\u003e \\u003cthead\\u003e \\u003ctr\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eFormulations\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eFlux (\\u0026micro;g cm\\u003csup\\u003e\\u0026minus;\\u0026thinsp;2\\u003c/sup\\u003e h\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e)\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003ePermeability\\u003c/p\\u003e \\u003cp\\u003ecoefficient x 10\\u003csup\\u003e\\u0026minus;\\u0026thinsp;2\\u003c/sup\\u003e (cm/h\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e)\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eEnhancement factor\\u003c/p\\u003e \\u003c/th\\u003e \\u003c/tr\\u003e \\u003c/thead\\u003e \\u003ctbody\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eTAC-THQ-NG (TAC)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e8.05\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.41\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e0.80\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.041\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e2.51\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eTAC-THQ-NG (THQ)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e30.51\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.73\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e1.02\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.060\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e2.12\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eTAC-THQ-SG (TAC)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e3.21\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.20\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e0.32\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.020\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e-\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eTAC-THQ-SG (THQ)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e14.40\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.75\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e0.48\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.025\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e-\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003c/tbody\\u003e \\u003c/colgroup\\u003e \\u003c/table\\u003e\\u003c/div\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec21\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e3.1.2. Skin Retention Study\\u003c/h2\\u003e \\u003cp\\u003eThe topical delivery of TAC and THQ by encapsulating in the NLCs opted to improve dermal localization for enhancing the localized psoriasis treatment while reducing systemic toxicity [\\u003cspan citationid=\\\"CR44\\\" class=\\\"CitationRef\\\"\\u003e44\\u003c/span\\u003e]. Hence, the ex-vivo experiment was conducted by using mice skin to assess the skin retention of TAC-THQ-NG and TAC-THQ-SG, and the findings are presented in \\u003cb\\u003e(\\u003c/b\\u003eFig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eB\\u003cb\\u003e).\\u003c/b\\u003e The % drug deposition of drugs; TAC and THQ was found to be 57.85\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;2.55% and 60.66\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;2.83% respectively from TAC-THQ-NG while only 20.12\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.19% and 25.68\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.48% deposited TAC and THQ was obtained from TAC-THQ-SG. The drug retention study revealed that TAC-THQ-NG exhibited higher drug retention in the skin compared to TAC-THQ-SG. This enhanced retention can be attributed to the interaction between the nanosized formulation and keratinocytes, leading to increased drug retention. Nevertheless, the increased drug retention in the skin is advantageous, as these skin layers are primarily affected by psoriasis.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec22\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e3.1.3. Dermatokinetics\\u003c/h2\\u003e \\u003cp\\u003eThe dermatokinetic profiles of TAC-THQ-NG and TAC-THQ-SG are depicted in \\u003cb\\u003e(\\u003c/b\\u003eFig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e\\u003cb\\u003e)\\u003c/b\\u003e and dermatokinetic parameters are summarized in Table\\u0026nbsp;\\u003cspan refid=\\\"Tab2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e. As depicted in the figure, the TAC-THQ-NG exhibited substantially higher concentrations of drugs in the skin compared to TAC-THQ-SG at all-time points. Achieving significantly elevated drug concentrations in the skin, especially for poorly absorbed drugs like TAC and THQ, represents a noteworthy accomplishment. This favorable outcome can be attributed to the presence of nanosized particles and the occlusive effects of the TAC-THQ-NG at the application site [\\u003cspan citationid=\\\"CR45\\\" class=\\\"CitationRef\\\"\\u003e45\\u003c/span\\u003e]. In the case of TAC-THQ-NG, there was approximately a 2.78- and 2.37-folds increase in the C\\u003csub\\u003emax\\u003c/sub\\u003e and a 2.93- and 2.40-folds increase in the area under the curve (AUC) for TAC and THQ, respectively, compared to TAC-THQ-SG. Furthermore, the values of T\\u003csub\\u003emax\\u003c/sub\\u003e and K\\u003csub\\u003ee\\u003c/sub\\u003e are on the lower side for TAC-THQ-NG in contrast with the TAC-THQ-SG formulation in the skin. This study clearly highlights the enhanced topical bioavailability potential of TAC and THQ, as they effectively traverse the skin barrier when administered through the TAC-THQ-NG formulation.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003cdiv class=\\\"gridtable\\\"\\u003e\\u003ctable float=\\\"Yes\\\" id=\\\"Tab2\\\" border=\\\"1\\\"\\u003e \\u003ccaption language=\\\"En\\\"\\u003e \\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 2\\u003c/div\\u003e \\u003cdiv class=\\\"CaptionContent\\\"\\u003e \\u003cp\\u003eDermatokinetic parameters obtained from the drug concentration\\u0026ndash;time profile of TAC-THQ-NG and TAC-THQ-SG in animal skin.\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/caption\\u003e \\u003ccolgroup cols=\\\"5\\\"\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c1\\\" colnum=\\\"1\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c2\\\" colnum=\\\"2\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c3\\\" colnum=\\\"3\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c4\\\" colnum=\\\"4\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c5\\\" colnum=\\\"5\\\"\\u003e\\u003c/div\\u003e \\u003cthead\\u003e \\u003ctr\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c1\\\" morerows=\\\"1\\\" rowspan=\\\"2\\\"\\u003e \\u003cp\\u003eDermatokinetic parameters\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colspan=\\\"2\\\" nameend=\\\"c3\\\" namest=\\\"c2\\\"\\u003e \\u003cp\\u003eTAC-THQ-NG\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colspan=\\\"2\\\" nameend=\\\"c5\\\" namest=\\\"c4\\\"\\u003e \\u003cp\\u003eTAC-THQ-SG\\u003c/p\\u003e \\u003c/th\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eTAC\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eTHQ\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eTAC\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003eTHQ\\u003c/p\\u003e \\u003c/th\\u003e \\u003c/tr\\u003e \\u003c/thead\\u003e \\u003ctbody\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eC\\u003csub\\u003eskin\\u0026minus;max\\u003c/sub\\u003e (\\u0026micro;g.cm\\u003csup\\u003e‒2\\u003c/sup\\u003e)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e789.52\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;28.15\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e2648.02\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;91.03\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e284.39\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;14.34\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e1115.26\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;48.14\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eT\\u003csub\\u003eskin\\u0026minus;max\\u003c/sub\\u003e (h)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e2\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e2\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e3\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e3\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eAUC\\u003csub\\u003e0\\u0026rarr;24h\\u003c/sub\\u003e (\\u0026micro;g.cm\\u003csup\\u003e‒2\\u003c/sup\\u003e h\\u003csup\\u003e‒1\\u003c/sup\\u003e)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e15985.92\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;585.31\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e51193.32\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1543.98\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e5448.63\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;290.49\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e21113.62\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;998.17\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eKe (h\\u003csup\\u003e‒1\\u003c/sup\\u003e)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e0.0117\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.0007\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e0.0123\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.0008\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e0.0139\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.0013\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e0.0131\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.0011\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003c/tbody\\u003e \\u003c/colgroup\\u003e \\u003c/table\\u003e\\u003c/div\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec23\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e3.1.4. Skin Permeation Dynamics\\u003c/h2\\u003e \\u003cp\\u003eThe permeation-enhancing mechanism of NLCs-gel was validated through the application of DSC and FTIR analyses. The DSC thermogram and FTIR spectrum of NLCs-gel treated skin and skin without any treatment are presented in \\u003cb\\u003e(\\u003c/b\\u003eFig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e\\u003cb\\u003e).\\u003c/b\\u003e The DSC analysis revealed changes in the properties of the skin, as indicated by alterations in the melting point, as shown in \\u003cb\\u003e(\\u003c/b\\u003eFig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eA\\u003cb\\u003e).\\u003c/b\\u003e The untreated skin, serving as a control, exhibited three endothermic melting peaks at temperatures of 103.696\\u0026deg;C (Peak-I), 126.162\\u0026deg;C (Peak-II), and 133.379\\u0026deg;C (Peak-III). Peak-I and Peak-II were indicative of lipid fluidization, while Peak-III signaled the denaturation of proteins within the skin. In contrast, the skin treated with NLCs-gel displayed the disappearance of Peak-I, with Peak-II and Peak-III appearing at temperatures of 116.186\\u0026deg;C and 121.502\\u0026deg;C, respectively. This represented a reduction in the melting point transitions of approximately 10\\u0026ndash;12\\u0026deg;C when compared to untreated skin. The disappearance of Peak-I and the shifting of endothermic transitions to lower temperatures were attributed to the enhanced lipid fluidization and the interference with the tight junctions in the SC [\\u003cspan citationid=\\\"CR40\\\" class=\\\"CitationRef\\\"\\u003e40\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR46\\\" class=\\\"CitationRef\\\"\\u003e46\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eThe characteristic FTIR peaks observed for both treated and untreated skin samples, as displayed in \\u003cb\\u003e(\\u003c/b\\u003eFig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eB\\u003cb\\u003e)\\u003c/b\\u003e, revealed various specific peaks associated with different stretching modes, such as C-H stretching, C\\u0026thinsp;=\\u0026thinsp;O stretching, and N-H bending vibrations in the case of skin treated with NLCs and untreated skin. These spectral features can be utilized to assess permeation dynamics [\\u003cspan citationid=\\\"CR47\\\" class=\\\"CitationRef\\\"\\u003e47\\u003c/span\\u003e]. These peaks correspond to the molecular vibrations of proteins and lipids present in the SC. In the FTIR analysis of untreated skin, characteristic peaks at 2918 cm\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e due to asymmetric C\\u0026ndash;H stretching and 2850 cm\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e were observed, arising from the symmetric C\\u0026ndash;H stretching, attributed to lipid alkyl chains [\\u003cspan citationid=\\\"CR48\\\" class=\\\"CitationRef\\\"\\u003e48\\u003c/span\\u003e]. In the NLCs-treated skin, similar asymmetric and symmetric C\\u0026ndash;H stretching peaks were observed at 2924 cm\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e and 2853 cm\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e, respectively with reduced peak intensity. The reduction in peak area may be attributed to the altered lipid composition of the SC. Furthermore, the distortion of lipid bilayers and lipid fluidization in the SC is evidenced by the shifting of CH2 stretching peaks to higher wavenumbers. This aids in surpassing the barrier characteristics of the SC [\\u003cspan citationid=\\\"CR49\\\" class=\\\"CitationRef\\\"\\u003e49\\u003c/span\\u003e]. Additionally, in untreated skin, the proteins within the SC displayed distinctive amide bands I and II in their infrared spectrum. These bands, occurring at 1641 cm⁻\\u0026sup1; (for amide I, linked to C\\u0026thinsp;=\\u0026thinsp;O stretching) and 1548 cm⁻\\u0026sup1; (for amide II, related to N\\u0026ndash;H stretching), serve as evidence affirming the existence of keratin in the SC. Notably, changes in the intensity as well as shifting of the amide I and II peaks were detected in skin treated with NLCs at frequencies of 1645 cm⁻\\u0026sup1; and 1544 cm⁻\\u0026sup1;, respectively. These alterations indicate that the NLCs facilitate permeation by interacting with keratin, a significant protein component of the skin [\\u003cspan citationid=\\\"CR50\\\" class=\\\"CitationRef\\\"\\u003e50\\u003c/span\\u003e]. Therefore, the FTIR analysis provided evidence supporting the idea that the disruption and fluidization of lipids, along with the denaturation of keratin, are the fundamental mechanisms contributing to the improved permeability of the skin.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec24\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e3.2. In-Vivo Studies\\u003c/h2\\u003e \\u003cdiv id=\\\"Sec25\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e3.2.1. In-Vivo Antipsoriatic Activity\\u003c/h2\\u003e \\u003cp\\u003ePsoriasis, an inflammatory skin condition, typically presents as plaques characterized by prominent erythema, scaling, and thickening of skin. An ideal animal model for evaluating anti-psoriatic treatments should replicate both the phenotypic features of the disease and its biochemical context. In this particular investigation, psoriasis was induced by applying IMQ topically for a continuous period of seven days. IMQ, a ligand for Toll-Like receptors (TLR), can induce psoriasis-like lesions on the skin of mice by affecting the IL-23/IL-17 axis [\\u003cspan citationid=\\\"CR51\\\" class=\\\"CitationRef\\\"\\u003e51\\u003c/span\\u003e]. The daily application of IMQ on the dorsal area of mice resulted in the emergence of skin abnormalities in the form of inflamed, scaly skin lesion characterized by increased epidermal growth, aberrant cell maturation, the accumulation of neutrophils, the formation of new blood vessels, and the infiltration of cells that mediate immune responses. These manifestations closely mirrored the characteristics of plaque-type psoriasis [\\u003cspan citationid=\\\"CR52\\\" class=\\\"CitationRef\\\"\\u003e52\\u003c/span\\u003e]. The therapeutic effectiveness of TAC-THQ-NG, was assessed in a research model that induced psoriatic plaques through IMQ application and subsequently the treatment was compared to other treatment groups employing different drugs treatments. The visual representation of animals in various experimental groups after the end of the treatment period is illustrated in \\u003cb\\u003e(\\u003c/b\\u003eFig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e\\u003cb\\u003e).\\u003c/b\\u003e\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cdiv id=\\\"Sec26\\\" class=\\\"Section4\\\"\\u003e \\u003ch2\\u003e3.2.2.1. PASI Scoring\\u003c/h2\\u003e \\u003cp\\u003eThe IMQ-induced psoriasis-like inflammation was evaluated using the PASI scoring, an extensively utilized clinical tool to gauge the severity of psoriasis and evaluate the effectiveness of anti-psoriatic therapies. In our in vivo experiments, we scored the severity of the lesion by visually evaluating the redness, thickness, and scaling of the skin inflammation. Following the initiation of IMQ treatment, mice exhibited initial signs of mild thickening, erythema, and scaling on their dorsal skin, which became apparent on days 2\\u0026ndash;3. The PASI score reached its peak on day 7. The cumulative PASI score was determined by summing the scores for erythema, thickness, and scaling of inflammation, each rated on a scale from 0 to 4. The PASI score for erythema, thickness and scaling of inflammation as wells as cumulative score on a scale from (0\\u0026ndash;4), for all treated groups are shown in \\u003cb\\u003e(\\u003c/b\\u003eFig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003e\\u003cb\\u003e).\\u003c/b\\u003e On the 7th day of IMQ application, all treatment groups displayed scaling, thickness, and erythema with a PASI score falling in the range of 3\\u0026ndash;4, indicating the development of severe inflammation. In contrast to the toxic control group, all drug treatment groups showed significant reduction in PASI score of scaling, thickness, and erythema but maximum reduction was observed with TAC-THQ-NG (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.0001). Also as compared to TAC-THQ-SG, TAC-THQ-NG showed 2.0-, 4.5- and 1.67-fold higher reduction in PASI score of thickness, erythema, and scaling respectively.\\u003c/p\\u003e \\u003cp\\u003eFurthermore, Table\\u0026nbsp;\\u003cspan refid=\\\"Tab3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e presents the comparison between the percentage reduction in the cumulative PASI score after the completion of drug treatment and the cumulative PASI score observed following the onset of the disease. Reduction in the cumulative PASI score represented the healing potential of different formulations. In IMQ treated group, slight increase (2.34%) in cumulative PASI score was observed after induction of disease. In contrast to IMQ treated group, drug treated groups (TAC-THQ-SG, TAC-NG, THQ-NG, TAC-THQ-NG) showed significant reduction in cumulative PASI score (expressed in %) with maximum reduction with TAC-THQ-NG. After the end of treatment, TAC-THQ-SG (57.14%), TAC-NG (46.15%), THQ-NG (39.18%) and TAC-THQ-NG (83.80%) showed reduction in cumulative PASI score. The higher % cumulative PASI score reduction of TAC-THQ-NG as compared to single drug nanogel formulation (TAC-NG and THQ-NG), confirms the enhanced efficacy of TAC and THQ, when used in combination. The enhanced efficacy of TAC-THQ-NG is attributed to the higher skin penetration and drug retention due to the small size and occlusive characteristics of the formulation. Based on our analysis of PASI scores across all groups, we can infer that the developed formulation, TAC-THQ-NG, shows greater promise in terms of both efficacy and safety when compared to TAC-THQ-SG.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003cdiv class=\\\"gridtable\\\"\\u003e\\u003ctable float=\\\"Yes\\\" id=\\\"Tab3\\\" border=\\\"1\\\"\\u003e \\u003ccaption language=\\\"En\\\"\\u003e \\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 3\\u003c/div\\u003e \\u003cdiv class=\\\"CaptionContent\\\"\\u003e \\u003cp\\u003e% Reduction in cumulative PASI of different animal groups at the end of treatments.\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/caption\\u003e \\u003ccolgroup cols=\\\"4\\\"\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c1\\\" colnum=\\\"1\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" class=\\\"colspec\\\" colname=\\\"c2\\\" colnum=\\\"2\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" class=\\\"colspec\\\" colname=\\\"c3\\\" colnum=\\\"3\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"char\\\" char=\\\".\\\" class=\\\"colspec\\\" colname=\\\"c4\\\" colnum=\\\"4\\\"\\u003e\\u003c/div\\u003e \\u003cthead\\u003e \\u003ctr\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eTreatments\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colspan=\\\"2\\\" nameend=\\\"c3\\\" namest=\\\"c2\\\"\\u003e \\u003cp\\u003eCumulative PASI score\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e% Reduction in PASI score\\u003c/p\\u003e \\u003c/th\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u0026nbsp;\\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eAfter induction of disease\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eAfter end of treatment\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u0026nbsp;\\u003c/th\\u003e \\u003c/tr\\u003e \\u003c/thead\\u003e \\u003ctbody\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cb\\u003eTOXIC\\u003c/b\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e3.42\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.17\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e3.50\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.43\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e-2.34\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cb\\u003eTAC-THQ-SG\\u003c/b\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e3.50\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.19\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e1.50\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.33\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e57.14\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cb\\u003eTAC-NG\\u003c/b\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e3.25\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.32\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e1.75\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.32\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e46.15\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cb\\u003eTHQ-NG\\u003c/b\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e3.42\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.16\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e2.08\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.32\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e39.18\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cb\\u003eTAC-THQ-NG\\u003c/b\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e3.58\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.17\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e0.58\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.17\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e83.80\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003c/tbody\\u003e \\u003c/colgroup\\u003e \\u003c/table\\u003e\\u003c/div\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec27\\\" class=\\\"Section4\\\"\\u003e \\u003ch2\\u003e3.2.2.2. Body weight, Spleen Dimension \\u0026amp; Weight)\\u003c/h2\\u003e \\u003cp\\u003eBody weight measurements were recorded for all groups during the treatment period, and changes in body weight relative to the initial body weight were plotted over time, as illustrated in \\u003cb\\u003e(\\u003c/b\\u003eFig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eA\\u003cb\\u003e).\\u003c/b\\u003e The control group exhibited no significant alterations in body weight throughout the study. While the toxic group displayed a noticeable change in the average body weight of the animals. During the study, there was a decrease in body weight among the drug treatment groups, which included TAC-THQ-SG, TAC-NG, THQ-NG, and TAC-THQ-NG. However, this decrease in body weight was not statistically significant. Notably, the group labeled TAC-THQ-NG showed the least amount of body weight reduction. Spleen enlargement serves as a significant indicator of immunological disorders. The application of IMQ led to the development of an inflammatory condition, resulting in a substantial increase in spleen size and weight due to the release of inflammatory cytokines [\\u003cspan citationid=\\\"CR53\\\" class=\\\"CitationRef\\\"\\u003e53\\u003c/span\\u003e]. The results concerning spleen dimensions and weight are presented in \\u003cb\\u003e(\\u003c/b\\u003eFig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eB\\u003cb\\u003e)\\u003c/b\\u003e, while the specific spleen weights are displayed in \\u003cb\\u003e(\\u003c/b\\u003eFig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eC\\u003cb\\u003e).\\u003c/b\\u003e In the toxic group, an increase in spleen weight was observed, indicative of the induction of psoriasis following IMQ application. The toxic group exhibited an average spleen weight of 380\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;17.05 mg. In the drug-treated groups (TAC-THQ-SG, TAC-NG, THQ-NG, and TAC-THQ-NG), the average spleen weights were measured at 225.5\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;11.47, 260\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;12.36, 296.75\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;14.45, and 184\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;10.20 mg, respectively. Notably, the spleen weight of the TAC-THQ-NG group closely resembled that of the normal control (p\\u0026thinsp;\\u0026gt;\\u0026thinsp;0.05), with an average weight of 166.75\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;9.71 mg. Moreover, the induction of psoriasis following IMQ application was confirmed by evaluating the spleen to body weight ratio (SBWR) in mice [\\u003cspan citationid=\\\"CR54\\\" class=\\\"CitationRef\\\"\\u003e54\\u003c/span\\u003e]. The SBWR values for various treatment groups were assessed at the conclusion of the experiment, and depicted in \\u003cb\\u003e(\\u003c/b\\u003eFig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eD\\u003cb\\u003e).\\u003c/b\\u003e In the toxic group, a substantial increase in SBWR was observed compared to the normal group, signifying the successful development of disease. Conversely, the different drug treatment groups yielded a reduction in SBWR with maximum reduction observed for TAC-THQ-NG.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec28\\\" class=\\\"Section4\\\"\\u003e \\u003ch2\\u003e3.2.2.3. Enzyme-Linked Immunosorbent Assay for TNF-α \\u0026amp; IL-6\\u003c/h2\\u003e \\u003cp\\u003eAmong the array of immune cytokines, TNF-α and IL-6 are pivotal pro-inflammatory cytokines known for their significant roles in triggering the onset of psoriasis when stimulated by the application of IMQ [\\u003cspan citationid=\\\"CR55\\\" class=\\\"CitationRef\\\"\\u003e55\\u003c/span\\u003e]. Therefore, a comparative anti-inflammatory efficacy of different drug treated groups was determined. The changes in levels of TNF-α and IL-6 observed in skin homogenate of treated animals belonging to different groups are illustrated in \\u003cb\\u003e(\\u003c/b\\u003eFig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003e\\u003cb\\u003e).\\u003c/b\\u003e As compared to normal group, 3.25 and 3.74-folds increase in levels of TNF-α and IL-6 respectively was observed in case of IMQ treated group (toxic) indicating induction of psoriasis. Following drug treatment, all drug treatment groups revealed a marked decrease in levels of TNF- α and IL-6 levels in contrast with the IMQ-treated group. Further, the level of TNF-α and IL-6 in the TAC-THQ-NG treated group was non-significant, when compared to the normal group (without any treatment). There was 2.76 and 2.99 times decrease in TNF-α and IL-6 levels for the TAC-THQ-NG treated animals in comparison to the IMQ-treated group. Furthermore, the TAC-THQ-SG group showed 1.84 and 1.97 times decrease in TNF-α and IL-6 levels in comparison to the IMQ-treated group. The higher reduction in cytokines levels on topical treatment with TAC-THQ-NG was attributed to higher skin penetration and drug retention. The higher reduction potential of TAC-THQ-NG as compared to single drug NLC-gel for TNF-α and IL-6 level as compared to IMQ group, confirmed the enhanced efficacy of TAC and THQ when used in combination. Thus, the study substantiated the enhanced efficacy of nanogel combination (TAC-THQ-NG) as compared to their conventional gel (TAC-THQ-SG) and single drug-containing nanogel (TAC-NG and THQ-NG).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec29\\\" class=\\\"Section4\\\"\\u003e \\u003ch2\\u003e3.2.2.4. Histological Assessment of Skin, Spleen, and Liver\\u003c/h2\\u003e \\u003cp\\u003eThe H\\u0026amp;E-stained histological micrographs of the skin, spleen, and the liver of different treatment groups of Balb/c mice are represented in \\u003cb\\u003e(\\u003c/b\\u003eFig.\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e8\\u003c/span\\u003e\\u003cb\\u003e).\\u003c/b\\u003e Histologically, normal mouse skin typically displays a well-structured epidermis and dermis, featuring thin layers of the epidermis and normal SC. While, IMQ treated (toxic group) skin displays noticeable epidermal thickening, elongation of epidermal rete ridges, disrupted epidermal differentiation, leukocyte infiltration, and a lack of a granular layer when compared to normal skin [\\u003cspan citationid=\\\"CR56\\\" class=\\\"CitationRef\\\"\\u003e56\\u003c/span\\u003e]. All the drug treatment groups exhibited a reduction in epidermal thickness and normalized keratinocyte differentiation, with an intact granular layer. However, the group treated with TAC-THQ-NG demonstrated the highest efficacy in combating the inflammation induced by IMQ, when compared to the other groups receiving different treatments. In the histological examination of the spleen, it was evident that the IMQ treatment group represents the rupture of spleen cells due to spleen enlargement, leading to an unclear distinction between the red pulp (appearing pink) and white pulp (appearing blue) regions in the spleen. Conversely, in the normal group (without IMQ treatment), both red pulp and white pulp were clearly distinguishable. Notably, treatment with TAC-THQ-NG exhibited the most significant improvement in the restoration of both red pulp and white pulp areas of the spleen in contrast to other drug treatment groups [\\u003cspan citationid=\\\"CR57\\\" class=\\\"CitationRef\\\"\\u003e57\\u003c/span\\u003e]. The histological examination of the liver revealed that the group treated with IMQ experienced damage to liver cells (hepatocyte degeneration), fibrotic changes, pyknosis and cellular disintegration. In contrast, the normal group (without IMQ treatment) exhibited no alterations in hepatocyte density. All the groups treated with drugs demonstrated a reduction in hepatocyte degeneration. Notably, animals treated with TAC-THQ-NG experienced the most significant improvement in hepatocyte regeneration compared to other drug treatment groups [\\u003cspan citationid=\\\"CR58\\\" class=\\\"CitationRef\\\"\\u003e58\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec30\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e3.2.2. In-Vivo Skin Compliance Study\\u003c/h2\\u003e \\u003cp\\u003eTopical therapy associated with adverse effect like skin irritation, specifically erythema, considerably restricts its clinical application. This challenge is exacerbated by the fact that many conventional dosage forms, including creams, lotions, and gels, often fail to mitigate the irritation caused by topical application. A hypothesis was formulated that the encapsulation of drugs within NLCs could avoid drug contact with the SC, potentially leading to a reduction in erythema episodes [\\u003cspan citationid=\\\"CR59\\\" class=\\\"CitationRef\\\"\\u003e59\\u003c/span\\u003e]. Also, TAC marketed formulation is associated with skin irritation (skin burning, itching) when used topically [\\u003cspan citationid=\\\"CR60\\\" class=\\\"CitationRef\\\"\\u003e60\\u003c/span\\u003e]. Therefore, the skin compliance study of TAC-THQ-NG and TAC-THQ-SG on healthy mice skin was done to analyze the compatibility of the developed formulation with skin. The irritation potential was assessed on the basis of scores attained for erythema and edema for both formulations as represented in Table\\u0026nbsp;\\u003cspan refid=\\\"Tab4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e. The application of TAC-THQ-NG did not show any sign of erythema and edema while in the case of TAC-THQ-SG, slight erythema was observed which was due to TAC in its free form. This suggests that the excipients utilized in the NLCs-gel, as well as the encapsulation of drugs within the NLCs, do not pose an irritation risk. The individual primary irritancy index (PII) for TAC-THQ-NG and TAC-THQ-SG was 0.0 and 1.25\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.50 respectively. The skin compliance study indicated that TAC-THQ-NG exhibited no signs of erythema and edema, even after repeated application for 7 days as compared to TAC-THQ-SG which showed a sign of slight erythema. Therefore, it can be inferred that TAC-THQ-NG is safe for topical application.\\u003c/p\\u003e \\u003cp\\u003e \\u003cdiv class=\\\"gridtable\\\"\\u003e\\u003ctable float=\\\"Yes\\\" id=\\\"Tab4\\\" border=\\\"1\\\"\\u003e \\u003ccaption language=\\\"En\\\"\\u003e \\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 4\\u003c/div\\u003e \\u003cdiv class=\\\"CaptionContent\\\"\\u003e \\u003cp\\u003eSkin compliance score of mice treated with TAC-THQ-NG and TAC-THQ-SG.\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/caption\\u003e \\u003ccolgroup cols=\\\"7\\\"\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c1\\\" colnum=\\\"1\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c2\\\" colnum=\\\"2\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c3\\\" colnum=\\\"3\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c4\\\" colnum=\\\"4\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c5\\\" colnum=\\\"5\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c6\\\" colnum=\\\"6\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c7\\\" colnum=\\\"7\\\"\\u003e\\u003c/div\\u003e \\u003cthead\\u003e \\u003ctr\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c1\\\" morerows=\\\"1\\\" rowspan=\\\"2\\\"\\u003e \\u003cp\\u003eAnimals (Balb/c)\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colspan=\\\"2\\\" nameend=\\\"c3\\\" namest=\\\"c2\\\"\\u003e \\u003cp\\u003eControl\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colspan=\\\"2\\\" nameend=\\\"c5\\\" namest=\\\"c4\\\"\\u003e \\u003cp\\u003eTAC-THQ-SG\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colspan=\\\"2\\\" nameend=\\\"c7\\\" namest=\\\"c6\\\"\\u003e \\u003cp\\u003eTAC-THQ-NG\\u003c/p\\u003e \\u003c/th\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eErythema\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEdema\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eErythema\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003eEdema\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003eErythema\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003eEdema\\u003c/p\\u003e \\u003c/th\\u003e \\u003c/tr\\u003e \\u003c/thead\\u003e \\u003ctbody\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cb\\u003e1\\u003c/b\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\" morerows=\\\"3\\\" rowspan=\\\"4\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\" morerows=\\\"3\\\" rowspan=\\\"4\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\" morerows=\\\"3\\\" rowspan=\\\"4\\\"\\u003e \\u003cp\\u003e1\\u003c/p\\u003e \\u003cp\\u003e2\\u003c/p\\u003e \\u003cp\\u003e1\\u003c/p\\u003e \\u003cp\\u003e1\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\" morerows=\\\"3\\\" rowspan=\\\"4\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cb\\u003e2\\u003c/b\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cb\\u003e3\\u003c/b\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cb\\u003e4\\u003c/b\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cb\\u003eMean score\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;SD\\u003c/b\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e0\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.0\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e0\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.0\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e1.25\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.50\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e0\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.0\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e0\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.0\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003e0\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.0\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cb\\u003ePII\\u003c/b\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colspan=\\\"2\\\" nameend=\\\"c3\\\" namest=\\\"c2\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colspan=\\\"2\\\" nameend=\\\"c5\\\" namest=\\\"c4\\\"\\u003e \\u003cp\\u003e1.25\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colspan=\\\"2\\\" nameend=\\\"c7\\\" namest=\\\"c6\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003c/tbody\\u003e \\u003c/colgroup\\u003e \\u003c/table\\u003e\\u003c/div\\u003e \\u003c/p\\u003e \\u003cp\\u003eErythema or edema scale: 0, No erythema/edema; 1, slight; 2, moderate; 3, marked; 4, severe.\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/div\\u003e\"},{\"header\":\"4. Conclusion\",\"content\":\"\\u003cp\\u003eIn this research, we developed TAC-THQ-NG formulation for the topical treatment of psoriasis. The developed formulation demonstrated superior effectiveness in \\u003cem\\u003eex-vivo\\u003c/em\\u003e and \\u003cem\\u003ein-vivo\\u003c/em\\u003e studies compared to conventional gel. \\u003cem\\u003eEx-vivo\\u003c/em\\u003e permeation experiments and CLSM study indicated enhanced skin permeation and retention of drugs as TAC-THQ-NG compared to the TAC-THQ-SG. Dermatokinetic studies clearly highlighted the enhanced topical bioavailability potential of drugs, as they effectively traverse the skin barrier when administered through the developed formulation. Moreover, \\u003cem\\u003ein-vivo\\u003c/em\\u003e studies on IMQ-induced BALB/c mice showed that the TAC-THQ-NG reduced psoriatic-like skin inflammation, lowered PASI scores of (Thickness, erythema and scaling) and decreased inflammatory markers (TNF-α and IL-6) more effectively than TAC-THQ-SG and individual drug formulation. The enhanced delivery of TAC and THQ to the skin via NLCs resulted in increased efficacy against psoriatic-like plaques, effectively alleviating the symptoms of IMQ-induced psoriasis. The formulation TAC-THQ-NG was also found to be safe, with no toxicity or irritation observed in skin compliance tests and minimal changes in histological examinations (skin, spleen and liver) were observed compared to control group. These findings suggest that the TAC-THQ-NG could be a promising platform for the topical treatment of psoriasis. However, further research is needed to explore the clinical benefits of the developed formulation in treating psoriasis.\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e \\u003cstrong\\u003eDeclaration of Interest:\\u003c/strong\\u003e \\u003cp\\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\\u003c/p\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003cstrong\\u003eCompeting Interests:\\u003c/strong\\u003e \\u003cp\\u003eAuthors declare no conflict of interest.\\u003c/p\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003cstrong\\u003eEthical Approval:\\u003c/strong\\u003e \\u003cp\\u003e The Institutional Animal Ethics Committee, Jamia Hamdard, New Delhi, India, approved protocol No. 1894 for Animal Studies (registration No. 173/GO/Re/S/ 2000/CPCSEA, 8 April 2021).\\u003c/p\\u003e \\u003c/p\\u003e\\u003ch2\\u003eAuthor Contribution\\u003c/h2\\u003e\\u003cp\\u003eConceptualization Authors NameData curation Meraj Alam,Formal analysis Md. RizwanullahFunding acquisition of the financial support for the project leading to this publication.Investigation. Writing and Reviewing Prof. Saima AminMethodology Prof. Saima AminProject administration Prof. Showkat R MirSoftware use and validation of analysis Ashif Iqubal and Shahnawaz AhmadSupervision Prof. Tae-Geum Kim\\u003c/p\\u003e\\u003ch2\\u003eAcknowledgement\\u003c/h2\\u003e\\u003cp\\u003eThe first author also thanks Jamia Hamdard for their generous financial assistance through the Hamdard National Fellowship (HNF). Additionally, the authors want to acknowledge Concord Biotech Limited, Gujarat, India, for gifting tacrolimus.\\u003c/p\\u003e\\u003ch2\\u003eAvailability of Data and Materials:\\u003c/h2\\u003e \\u003cp\\u003eData will be made available on request.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\u003cli\\u003e\\u003cspan\\u003eJ. Nowowiejska, A. Baran, P. Grabowska, M. Lewoc, T.W. Kaminski, I. 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Pharm. 562 (2019) 96\\u0026ndash;104.\\u003c/span\\u003e\\u003c/li\\u003e\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":true,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":false,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true},\"keywords\":\"NLCs, Tacrolimus, Psoriasis, Thymoquinone, Imiquimod, Dermatokinetic, Skin permeation, retention\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-4448132/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-4448132/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003eThe primary objective of this current investigation is to evaluate the antipsoriatic potential of a novel nanogel delivery system that co-encapsulates tacrolimus and thymoquinone within nanostructured lipid carriers (NLCs). Therefore, TAC-THQ-NLCs-gel was formulated by emulsification solvent-evaporation technique and evaluated for their potential in improving skin permeation, skin bioavailability, skin safety, and therapeutic efficacy in imiquimod-induced psoriasis in mice plaque model. The ex-vivo skin permeation study shows 2.51- and 2.12-folds higher permeation for TAC-THQ-NLCs-gel as compared to TAC-THQ-suspension-gel, also the permeation enhancement mechanism of NLCs gel was confirmed using FTIR and DSC. Further skin retention study shows 2.87- and 2.36-fold improvement in retention of the drug as compared to free drug gel formulation. Further, the dermatokinetic study shows 2.78 and 2.37 folds higher C\\u003csub\\u003emax\\u003c/sub\\u003e and 2.93- and 2.40-fold higher AUC (area under the curve) for TAC and THQ respectively as compared to free drugs gel. The results of the in-vivo skin compliance study suggested that the fabricated TAC-THQ-NLCs-gel was safe for skin delivery. Furthermore, TAC-THQ-NLCS-gel represented much better amelioration of psoriasis in Balb/c mice, with a cumulative PASI score reduction of 83.80% as compared to 57.14% for free drugs gel after the end of treatment. In addition, the insignificant changes in the histology of the skin, spleen, and liver further confirm the efficacy and safety of the developed TAC-THQ-NLCs-gel. Based on these observations, it can be inferred that TAC-THQ-NLCs-gel holds promise as a combined treatment approach for managing psoriasis topically.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Co-delivery of Tacrolimus and Thymoquinone topically by Nanostructured lipid carriers gel for enhanced efficacy against imiquimod-induced psoriasis in Balb/c mice\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2024-06-03 21:35:10\",\"doi\":\"10.21203/rs.3.rs-4448132/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"ee26e96e-b3cf-493e-9dce-08ee6edf51e7\",\"owner\":[],\"postedDate\":\"June 3rd, 2024\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"posted\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2024-08-31T09:38:35+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2024-06-03 21:35:10\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-4448132\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-4448132\",\"identity\":\"rs-4448132\",\"version\":[\"v1\"]},\"buildId\":\"qtupq5eGEP_6zYnWcrvyt\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}