Development of perilla essential oil-based nanoemulsions as anti-psoriatic and dermal delivery platforms for topical therapy

Abstract

Psoriasis is a chronic inflammatory skin disorder characterized by keratinocyte hyperproliferation, immune dysregulation, and impaired barrier regeneration, highlighting the need for safe and effective topical therapies. Perilla essential oil (PO) contains a rich profile of long-chain fatty acids and lipid-related metabolites with reported anti-inflammatory and barrier-modulating activities. However, its clinical utility is limited by its volatility and poor stability. In this study, we developed PO-based nanoemulsions (PO-NEs) to preserve bioactive components while enabling dermal delivery. GC-MS analysis confirmed that the oil was dominated by fatty acids and related lipid metabolites relevant to cutaneous inflammation and epidermal repair. PO-NEs significantly inhibited keratinocyte proliferation in vitro, reduced the expression of key proinflammatory cytokines, and lowered intracellular ROS accumulation. In a mouse model of imiquimod-induced psoriasis, topical application of 5% PO-NEs markedly improved erythema, scaling scores, epidermal hyperplasia, and inflammatory cell infiltration. RNA sequencing demonstrated that PO-NEs broadly reprogrammed lesional gene expression, with enrichment of pathways related to extracellular matrix organization, epidermal differentiation, and immune regulation and pronounced suppression of aberrantly activated Wnt/β-catenin signaling, which was further supported by targeted qRT-PCR validation. Building on this bioactive carrier, we next encapsulated curcumin into PO-NEs (Cur@PO-NEs) and compared with curcumin suspensions and curcumin-loaded medium-chain triglycerides (MCT) nanoemulsions. Cur@PO-NEs exhibited superior skin permeation and intradermal deposition, and produced the most pronounced improvements in clinical scores, histopathology, and inflammatory readouts. Comprehensive local and systemic safety evaluations indicate good tolerability and biocompatibility. Together, these findings identify PO-NEs as a natural bioactive nanoemulsion capable of modulating epidermal signaling and the psoriatic microenvironment and establish Cur@PO-NEs as a synergistic and safe topical strategy for psoriasis therapy that combines carrier-mediated bioactivity with enhanced curcumin delivery.

Graphical abstract

1. Introduction

Psoriasis is a chronic, immune-mediated inflammatory skin disorder characterized by sustained keratinocyte hyperproliferation, aberrant immune activation, and compromised barrier function (Shen et al., 2025; S.-Y. To et al., 2025). The standard of care for mild-to-moderate psoriasis primarily relies on topical corticosteroids and vitamin D analogs (Svendsen et al., 2019; Deng and Lu, 2025; Artusa and White, 2025). However, the long-term application of these agents is frequently associated with cutaneous adverse effects, such as atrophy and telangiectasia, as well as potential systemic risks (Kresch et al., 2023; Thein et al., 2022). Furthermore, the stratum corneum serves as a formidable physical barrier that restricts the penetration of many therapeutics, leading to insufficient intradermal drug accumulation and suboptimal clinical outcomes (Sparr et al., 2023; Wang et al., 2024a; Chen et al., 2025). Consequently, there is a compelling need to develop advanced topical delivery strategies that not only enhance skin permeation but also utilize safe, natural bioactive components to simultaneously target cutaneous inflammation and restore barrier integrity (Safta et al., 2024; Wojcieszak et al., 2025).

Perilla frutescens, a medicinal and edible plant that is widely cultivated in Asia, possesses a broad spectrum of pharmacological activities (Liu et al., 2025a; Bhaswant et al., 2024). Perilla essential oil (PO), extracted from the leaves, is rich in polyunsaturated fatty acids (PUFAs), particularly α-linolenic acid, and specific lipid metabolites with established anti-inflammatory potential (Zhong et al., 2024; Xia et al., 2025). Although preliminary studies have indicated that simple PO treatment exerts certain anti-psoriatic effects, a systematic investigation to explicitly elucidate its therapeutic role and underlying mechanisms is lacking (Xu et al., 2022). Furthermore, the translation of PO into topical formulations is severely restricted by inherent physicochemical limitations including low oxidative stability, high volatility, and poor water solubility (Zhao et al., 2023; Li et al., 2024). Consequently, direct application often fails to achieve therapeutic concentrations in the viable epidermis owing to rapid evaporation and limited permeation (Bo et al., 2025; Soleiman-Dehkordi et al., 2024). Notably, recent advances in food science have demonstrated that establishing nanoemulsion systems can effectively preserve the stability and bioavailability of PO, suggesting that this strategy is an ideal solution for overcoming delivery barriers in psoriasis therapy (Wang et al., 2024b; Wang et al., 2023).

Nanoemulsions are kinetically stable colloidal systems characterized by ultrafine droplet sizes that provide a large surface area for adhesion and facilitate deep skin penetration via follicular pathways and hydration effects (Lin et al., 2025; Parseghian et al., 2025). While conventional NEs typically utilize inert oils (e.g., medium-chain triglycerides) solely as vehicles, we propose using PO as the oil phase to engineer a functional bioactive carrier (Liu et al., 2022; Cui et al., 2025). This strategy aims to stabilize the volatile components of PO while leveraging the inherent permeation-enhancing properties of the nano system. Although the anti-inflammatory potential of bulk PO has been recognized whether its nanoemulsions (PO-NEs) retain these activities and effectively treats psoriasis remains to be verified. Furthermore, the specific therapeutic efficacy and precise molecular mechanisms by which PO-NEs modulate psoriatic keratinocytes and the tissue microenvironment have not been elucidated.

Building on the establishment of PO-NEs as pharmacologically active formulations, we further sought to exploit their potential as bioactive delivery platforms for other hydrophobic anti-psoriatic agents. Curcumin (Cur), a polyphenol with well-documented efficacy against psoriasis, is clinically restricted by its extremely low aqueous solubility and poor skin permeability (Lu et al., 2024; Wu et al., 2025; Liu et al., 2025b). We hypothesized that encapsulating curcumin in PO-NEs (Cur@PO-NEs) would establish a synergistic therapeutic strategy. In this rational design, PO-NEs not only solubilize and facilitate the transdermal transport of curcumin, but also exert their own intrinsic therapeutic activities. This approach aims to achieve vehicle-active synergy, offering superior efficacy compared with conventional non-bioactive carriers.

Here, we developed a stable PO-NE formulation and investigated its therapeutic efficacy in a mouse model of imiquimod (IMQ)-induced psoriasis. Transcriptomic profiling coupled with molecular validation was used to elucidate the specific signaling pathways modulated by PO-NEs. Subsequently, to verify the bioactive carrier concept, we encapsulated curcumin to generate Cur@PO-NEs and performed comparative studies to assess the skin permeation and therapeutic synergy. Collectively, this study presents PO-NEs as a dual-function platform that integrates intrinsic bioactivity with enhanced topical delivery for effective psoriasis management.

2. Materials and methods

2.1. Materials, animals and cell lines

PO (the content of α-linolenic acid was 60%), curcumin (Cur), carbomer 940, and medium-chain triglycerides (MCT) were purchased from Shanghai Yuanye Biotechnology Co. Ltd. (Shanghai, China). PEG-40 stearyl ether (S40) was obtained from Croda GmbH (Nett et al., Germany). DAPI staining solution, RIPA buffer, hematoxylin and eosin (H&E) staining kit, and bicinchoninic acid (BCA) protein assay kits were obtained from Beyotime Biotechnology (Shanghai, China). Antibodies against Ki67, TNF-α, IL-6, IL-1β, IL-17 and a one-step TUNEL assay kit were from Servicebio (Wuhan, China). A 5% active IMQ topical cream was produced by Hubei Keyi Pharmaceutical, Ltd. (Wuhan, China). Tacrolimus cream (0.03%) was purchased from Leo Laboratories, Ltd. (Shanghai, China). Analytical-grade sodium chloride, disodium hydrogen phosphate, potassium dihydrogen phosphate, and potassium chloride were of analytical grade. All the aqueous solutions were prepared using ultrapure water (Elga Maxima, High Wycombe, UK).

BALB/c mice (18-22 g, 4-6 weeks) were purchased from GemPharmatech (Nanjing, China) and used in this study to establish a psoriasis model and evaluate the therapeutic efficacy of the targeted formulations. All mice were randomly assigned to experimental groups and maintained in a new environment for one week before proceeding with the study. All animal tests and experimental procedures were conducted in accordance with the Administration Committee of Experimental Animals in Jiangsu Province and the Ethics Committee of Nanjing University of Chinese Medicine (AEWC-20220329-198).

The human epidermal keratinocyte HaCaT cells were purchased from the Cell Bank of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (China). The HaCaT cells were maintained at 37 °C with 5% CO2 and cultured in Dulbecco’s modified Eagles medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin.

2.2. Preparation of perilla essential oil-based nanoemulsions

Nanoemulsions were prepared using PO as the designated oil phase in the formulation. The emulsifier S40 was used based on previous studies, denoting it as a non-irritative PEGylated emulsifier (Liu and Jasmin Lunter, 2021). PO-based nanoemulsions were fabricated according to previously reported procedures with modifications (Liu et al., 2022). First, S40 was completely dissolved in water via magnetic stirring at room temperature. The oil phase of PO was slowly added to the emulsifier solution and subjected to Ultra-Turrax (T25-digital; IKA, Staufen, Germany) at a constant speed of 9000 rpm for 2 min to obtain coarse emulsions. Then, the nanoemulsions were processed by a high-pressure homogenization (AH1500, ATS Engineering Inc., Canada) at 1000 bar for eight cycles at a condenser temperature of 4 °C. Afterwards, the carbomer was added, and the pH was adjusted with 5% NaOH to achieve a suitable viscosity for the final emulsions. The Cur@PO-NEs were manufactured by pre-dissolving curcumin in the oil phase. To mark the functions of PO in nanoemulsions, MCT was also applied as an oil phase to determine the penetration enhancement and therapeutic efficacy compared with PO (Liu et al., 2022). Unless otherwise specified, all physicochemical characterizations were performed on the final nanoemulsions after carbomer addition and pH adjustment.

2.3. Characterization of perilla essential oil-based nanoemulsions

2.3.1. Rheological properties

Rheological properties of the PO-based nanoemulsions were assessed using a rotational viscometer (Physica MCR501 rheometer, Anton Paar, Graz, Austria) equipped with a plate/plate geometry (diameter: 25 mm; gap size: 0.2 mm). The samples were placed on a measuring plate for 2 minutes for equilibration. Subsequently, flow curves were generated to elucidate the flow behavior. The dynamic viscosity η (Pa s) was recorded as a function of shear rate within the range of 1–1000 s⁻¹. Amplitude sweep tests were performed under oscillatory conditions. The viscoelastic linear region, indicative of structural strength, was identified within a strain range of 0.01–1000%. Viscoelastic parameters, including storage modulus (G') and loss modulus (G”), were determined throughout the measurements.

2.3.2. Confocal Raman spectroscopy

The molecular fingerprints of PO and PO-NEs were determined by confocal Raman microscopy (LabRAM HR, Horiba France SAS, Villeneuve d'Ascq, France). Aliquots of the samples were deposited on glass slides and immediately sealed with coverslips to prevent evaporation. Prior to measurement, the samples were allowed to equilibrate for a brief period to minimize laser-induced thermal effects and stabilize the nanoparticles. The spectra were acquired using a 532 nm excitation laser focused through a 100× objective lens. Data acquisition was performed in the spectral range–400-3800 cm^-1 using a point-scan mode with an integration time of 10 s per accumulation.

2.3.3. Transmission electron microscopy (TEM)

The shape and morphology of the produced nanoemulsions were characterized with an H-7650 transmission electron microscopy (TEM) (H-7650, Hitachi, Japan). Briefly, all the samples were diluted, transferred onto a carbon-supported copper grid, and dried under reduced pressure at room temperature. The grids were stained with 2% uranyl acetate prior to the TEM observations.

2.3.4. Particle size and zeta potential

The average particle size, polydispersity index (PDI), and zeta potential of all the prepared samples were evaluated by dynamic light scattering (DLS) using a Zetasizer Nano ZS (Malvern Instruments, Malvern, UK). Prior to the analysis, the samples were all diluted 1000 times freshly with distilled water. All measurements were performed in triplicates.

2.3.5. GC-TOF-MS metabolomics analysis

Metabolomic profiling was performed using a standardized GC-TOF-MS workflow. Briefly, 50 μL of each sample was extracted with 1 mL of pre-chilled acetonitrile: isopropanol: water (3:3:2, v/v/v), sonicated, and centrifuged at 12,000 rpm. The resulting supernatant was vacuum-dried and derivatized sequentially with methoxyamine hydrochloride (60 °C, 60 min) and BSTFA-TMCS (70 °C, 90 min). GC–TOF–MS analysis was conducted on an Agilent 7890B GC system coupled with a LECO Pegasus BT TOF-MS equipped with a DB-5MS capillary column using helium as the carrier gas (1 mL/min), a split ratio of 1:10, and an oven program ramping from 50 °C to 320 °C. Mass spectra were acquired using electron ionization at 70 eV. Raw data were processed following the established pipeline of the facility, including peak extraction, deconvolution, retention time alignment, and library matching, resulting in 90 reliably identified metabolites for downstream statistical analysis.

2.3.6. Stability studies

The physicochemical stability of the developed PO-NEs was evaluated under storage and culture-medium conditions. For long-term storage stability, freshly prepared PO-NEs were stored in sealed glass containers at room temperature (23 ± 2 °C) for 3 and 6 months. To assess colloidal stability in biologically relevant medium, PO-NEs were diluted in complete cell culture medium and incubated at 37 °C for 48 and 72 h. At each predetermined time point, samples were collected and analyzed for mean droplet size, PDI, and zeta potential.

2.4. In vitro cellular evaluation of PO-NEs in HaCaT cells

2.4.1. Cell cytotoxicity assay

The cytotoxicity of the PO-NEs toward HaCaT cells was evaluated using the CCK-8 assay. Briefly, cells were seeded in 96-well plates at a density of 6 × 10³ cells/well and allowed to adhere overnight. Cells were then treated with a concentration gradient of PO-NEs (0, 5, 10, 25, and 50 μg/mL) for 48 h. Cells were cultivated at 37 °C in a humidified incubator containing 5% CO₂, and passaging occurred upon achieving 70-80% confluence.

2.4.2. Anti-proliferation assay

To assess the effect of PO-NEs on IL-6-induced hyperproliferation, HaCaT cells were seeded in 96-well plates at 3 × 10³ cells per well. After overnight adhesion, cells were stimulated with IL-6 (25 ng/mL) to induce a proliferative inflammatory phenotype. Subsequently, cells were treated with different concentrations of PO-NEs (0, 5, 10, 25, and 50 μg/mL) and incubated for 24, 48, and 72 h. At each time point, the CCK-8 reagent was added for 30 min, and the absorbance was measured at 450 nm. The relative growth rate was calculated by normalization to the control group at the corresponding time point.

2.4.3. Intracellular ROS detection

Intracellular ROS levels were quantified using DCFH-DA probe. IL-6-stimulated HaCaT cells were treated with PO-NEs for 24 h followed by incubation with DCFH-DA diluted in serum-free medium for 30 min at 37 °C in the dark. After washing, the cells were visualized using CLSM.

2.4.4. Evaluation of inflammatory cytokines

The secretion levels of inflammatory cytokines IL-6 and IL-1β were quantified using commercial ELISA kits ((Hangzhou Lianke Biotechnology Co., Ltd., Hangzhou, China). HaCaT cells were stimulated with IL-6 and treated with PO-NEs for 24 hours. The supernatant was collected and centrifuged to remove debris. The ELISA was performed according to the manufacturer’s instructions. Absorbance values at 450 nm were measured using a microplate reader, and cytokine concentrations were calculated based on standard curves.

2.4.5. Immunofluorescence staining

HaCaT cells were seeded in 6-well plates and cultured at 37 °C with 5% CO₂. After IL-6 stimulation, the cells were treated with 25 μg/mL PO-NEs for 24 h and then fixed with 4% paraformaldehyde. Immunofluorescence staining was performed to assess the expression of IL-6 and IL-1β. Nuclei were counterstained with DAPI and fluorescence images were acquired to evaluate the distribution of inflammatory markers.

2.4.6. Quantitative real-time PCR (q-PCR) assay

HaCaT cells were collected after treatment and stored at −80 °C for RNA preservation. Total RNA was extracted using the YALEPIC Animal Cell & Tissue Total RNA Fast Isolation Kit (Life Technologies; Suzhou, China). cDNA synthesis was performed using the HiScript III RT SuperMix for qPCR (Vazyme, Nanjing, China). Gene expression profiles were analyzed using a custom human inflammatory mRNA PCR array (Wcgene Biotech, Shanghai, China), with GAPDH serving as the endogenous control. Relative expression changes were calculated using the 2ΔCt method and normalized to the reference gene based on the Ct values.

2.5. Characterization and transdermal delivery evaluation of Cur@PO-NEs

2.5.1. Encapsulation efficiency and drug loading

The encapsulation efficiency (EE%) and drug loading (DL%) of curcumin in Cur@PO-NEs were determined using a photometric microplate reader (Varioskan LUX, Thermo Fischer Scientific, USA) at 422 nm to quantify curcumin content. A calibration curve was generated by diluting a stock solution of curcumin in a mixture of ethanol and PBS (50:50, v/v). The concentration of the standard solutions ranged from 0.025-25 to μg/ml (R² = 0.9992).

To determine the EE% and DL%, the Cur@PO-NEs sample was dissolved in a mixture of ethanol and PBS (50:50, v/v), sonicated for 15 min, and filtered through a 0.22 μm filter. The filtrate was collected and quantified to determine the total curcumin content. Another aliquot was placed inside a dialysis tube that contained a mixture of ethanol and PBS (50:50, v/v). After centrifugation at 3000 rpm for 15 min, the amount of free curcumin was determined. The EE% and DL% of curcumin inside Cur@PO-NEs were calculated using the following equations (Niu et al., 2020).

$$\mathit{EE}\%=\frac{\mathit{Total\; amount\; of\; curcumin}-\mathit{Free\; curcumin}}{\mathit{Total\; amount\; of\; curcumin}}\#$$ (1)

$$\begin{array}{c}\mathit{DL}\%=\frac{\mathit{Total\; amount\; of\; curcumin}-\mathit{Free\; curcumin}}{\mathit{Added\; amount\; of}\mathit{PO}}\#\end{array}$$ (2)

2.5.2. In vitro drug release study

The drug release behavior of different Cur-loaded samples was evaluated using the dialysis method (Hamza et al., 2025). Briefly, 5 g of Cur suspension, Cur@PO-NEs, and Cur@MCT-NEs with known curcumin concentrations were transferred into dialysis bags (MWCO cutoff of 3 kDa) and placed into 50 mL centrifuge tubes. Next, 25 mL of PBS containing 0.5% of Tween 80 (w/v) was added respectively and maintained at 37°C in the dark. At different preset time points, 1 mL of the buffer solution was removed and replaced with 1 mL of fresh buffer solution. The release of curcumin was analyzed using a photometric microplate reader at 422 nm, as described above. Each experiment was repeated in triplicate, and the release profiles were subsequently generated.

2.5.3. In vitro hemolysis study

Whole blood was obtained from mice and centrifuged at 3000 rpm for 15 min to obtain red blood cells. PBS was then added to the original whole blood volume to obtain a saline suspension. Owing to the higher viscosity of the NEs samples, the PO-NEs, Cur@PO-NEs, and Cur@MCT-NEs were diluted (1:1) with PBS. The red blood cell suspension (20 μL) was then added and gently mixed with PBS and deionized water to serve as negative and positive controls, respectively. After incubation at 37 °C for 4 h, all samples were centrifuged at 3000 rpm for 15 min. The absorbance of the supernatant was measured at 540 nm wavelength using a photometric microplate reader. Hemolysis was analyzed using the following equation (Shahriar et al., 2025):

$$\begin{array}{c}\mathit{Relative\; hemolysis}\%=\frac{\mathit{Sample\; absorbance}-\mathit{PBS}\mathit{absorbance}}{\mathit{dd}{H}_{2}O\mathit{absorbance}-\mathit{PBS}\mathit{absorbance}}\times 100\#\end{array}$$ (3)

2.5.4. Establishment of Imiquimod-induced psoriasis-like dermatitis model

The psoriasis skin model was induced by consecutively applying IMQ cream to BALB/c mice following established protocols (Liu et al., 2025c; Li et al., 2025). Briefly, the dorsal back skin of BALB/c mice (male, 6-8 weeks, 18-22 g) was shaved and depilated 24 h prior to experiment. The animals were then randomly assigned to seven groups (n = 6 per group): control (saline), model (IMQ only), Cur suspension, Cur@MCT-NEs, PO-NEs, Cur@PO-NEs, and a positive control (tacrolimus). To induce psoriasis-like skin lesions, mice in all groups except the control received a daily topical application of IMQ cream (5%, 62.5 mg) on the shaved dorsal skin for seven consecutive days. From day 3 to day 7, 100 μL of the respective formulations or 100 mg of tacrolimus cream (0.1%) were topically applied to the lesional skin 4 h after the IMQ application. The control and model groups received equivalent volumes of normal saline. On day 8, all animals were euthanized for subsequent evaluations.

2.5.5. Franz diffusion cells

Skin samples were freshly excised from both healthy and IMQ-induced psoriatic mice immediately after the sacrifice. The subcutaneous fatty tissue was carefully removed and the skin was washed with physiological saline. The prepared skin samples were then mounted between the donor and receptor compartments of TP-6 Franz diffusion cells (Boyuweiye Tech, Tianjin, China), with the stratum corneum facing the donor chamber. The receptor compartment was filled with 12 mL of degassed, pre-warmed (32 °C) phosphate-buffered saline (PBS, pH 7.4), and maintained under constant stirring at 500 rpm. Following an equilibration period of 30 min, 0.04 g/cm² of the nanoemulsions was applied to the skin surface. After 4 h of permeation, the skin samples were removed and thoroughly cleaned with isotonic saline-soaked cotton swabs (wiped 30 times) to remove unabsorbed formulations. Finally, the effective permeation areas (diameter: 15 mm) were punched out and dried for further analysis.

2.5.6. Confocal laser scanning microscopy

To visualize the permeation depth and distribution of curcumin in the skin layers, the cleaned skin tissues obtained from the permeation study were immediately embedded in Optimal Cutting Temperature (OCT) compound and frozen at -80 °C. Frozen tissue blocks were sectioned into 10 μm-thick sections and carefully mounted onto glass slides. Finally, the nuclei in the skin sections were stained with DAPI for 10 min in the dark. Curcumin penetration was imaged using a fluorescence microscope (FV10i, Olympus, Japan) at excitation and emission wavelengths of 422 and 530 nm, respectively, whereas DAPI was individually viewed at 380 and 420 nm. The fluorescence area of each skin section was analyzed using the ImageJ software (NIH, Bethesda, Maryland).

2.6. Skin penetration and therapeutic efficacy of Cur@PO-NEs in psoriasis model

2.6.1. Psoriatic skin penetration studies in-vivo

In vivo skin penetration studies were performed using an IMQ-induced psoriatic skin model. Following the application of IMQ cream to the dorsal skin for seven days, 100 μL of Cur suspension, Cur@MCT-NEs, and Cur@PO-NEs were administered. To determine curcumin penetration into the dorsal skin, mice were imaged using an In Vivo Imaging System Lumina XR (Perkin Elmer, UK) at different time points (5, 30, 60, and 120 min). An excitation wavelength of 495 nm and an emission wavelength of 525 nm were used. To eliminate the effects of the remaining formulations applied to the skin surface, the dorsal skin was wiped clean before measurements. Afterwards, the mouse skin was subjected to multiphoton microscopy (Leica STELLARIS 8 system, Leica, Germany) with the dermal side down in PBS solution to maintain skin hydration. The scan started from above the skin surface and ended at a depth of 50 μm inside the skin, with a scan step of 5 μm.

2.6.2. Assessment of in vivo therapeutic efficacy

The in vivo therapeutic efficacy of the formulations was evaluated using the IMQ-induced psoriasis BALB/c mouse model, as established and detailed in Section 2.5.4. During the treatment period, changes in body weight, dorsal skin thickness, and macroscopic skin lesions were monitored and recorded daily for all the experimental groups. The severity of skin inflammation was evaluated using the Psoriasis Area and Severity Index (PASI), based on three parameters: erythema (redness), induration (thickness), and desquamation (scaling). Each parameter was graded on a scale of 0 to 4 (0, none; 1, mild; 2, moderate; 3, marked; 4, severe), yielding a cumulative score ranging from 0 to 12 (Li et al., 2025). At the end of the experiment, the mice were sacrificed and dorsal skin lesions and spleens were collected. The spleens were photographed and weighed to calculate the spleen index (spleen weight-to-body weight ratio) as an indicator of systemic inflammation (Ou et al., 2025). For skin tissue processing, a portion of the dorsal skin was fixed in 4% paraformaldehyde for histological, immunohistochemical (IHC), and TUNEL assays. The remaining skin tissues were snap-frozen in liquid nitrogen and stored at −80 °C for subsequent inflammatory cytokine analysis.

2.6.3. Immunohistochemical study

For immunohistochemical staining, primary antibodies against Ki67 (1:1000), TNF-α (1:300), IL-6 (1:300), and IL-17 (1:500) were used. The dorsal skin tissues were sectioned and incubated with these antibodies at 4 °C overnight. The following day, the sections were thoroughly washed with saline and incubated with secondary antibodies for 1 h. Afterwards, the skin sections were counterstained with 3,3'-diaminobenzidine (DAB) and hematoxylin. After dehydration, the sections were imaged and the stained areas were analyzed using ImageJ software (NIH, Bethesda, Maryland).

2.6.4. Histological study

Dorsal skin, spleen, and liver tissues were fixed with 4% paraformaldehyde, dehydrated, and embedded in paraffin. The resulting paraffin blocks were sectioned into 10 μm-thick slices and stained with H&E. The slices were then examined with a microscope, digitally scanned using a slide scanner, and analyzed using the CaseViewer software.

2.6.5. TUNEL staining

TUNEL staining was performed to detect apoptosis in psoriasis skin samples. Briefly, fixed dorsal skin tissues were embedded, sectioned (10 μm), and evaluated using a TUNEL Assay kit (Servicebio, Wuhan, China) according to the manufacturer’s instructions. Apoptosis of the skin cells was imaged, scanned, and analyzed using the CaseViewer software.

2.6.6. Determination of inflammatory cytokines

Inflammatory cytokine levels in the dorsal skin and blood serum samples were determined. Freshly frozen dorsal skin was weighed, cut into pieces, mixed with RIPA buffer containing protease inhibitors, and homogenized using a tissue grinder (JXFSTPRP-CL, Jingxin Indrustrial Co., Ltd, Shanghai, China). After centrifugation, supernatants were collected for further analysis. Whole blood samples were collected from the mice and centrifuged to obtain blood serum. The concentrations of IL-6, IL-23, IL-17a, IL-1β, and TNF-α in both skin and blood samples were measured using ELISA kits, according to the manufacturer’s instructions.

2.7. In-vivo safety evaluation

The safety assessments of Cur@PO-NEs in healthy mice involved the administration of the formulations for 1 and 7 days, with untreated mice serving as the control group. The skin condition was photographed and changes in body weight and full skin thickness were recorded. Subsequently, the dorsal skin, spleen, and liver were harvested from mice for H&E staining. Whole blood serum samples were collected to evaluate the inflammatory cytokine levels. Subsequently, comprehensive hematological and biochemical analyses were performed. Automated hematology analysis was employed to determine the counts of white blood cells (WBC), red blood cells (RBC), and lymphocytes (lymph) in the whole blood samples. An autobiochemical analyzer was used to quantify enzymatic activity, including aspartate aminotransferase (AST), alanine aminotransferase (ALT), γ-glutamyltransferase (γ-GT), blood urea nitrogen (BUN), creatinine (CREA), and uric acid (UA), using blood serum samples.

2.8. Statistics

The graphs are presented as mean ± standard deviation (mean ± SD). Significant differences were analyzed using one-way analysis of variance (ANOVA) followed by the Student-Newman-Keuls (SNK) test, which was performed using GraphPad Prism 9.0 (GraphPad Software Inc., La Jolla, CA, USA). Significance is marked with different numbers of asterisks: * P < 0.05, ** P< 0.01, and *** P < 0.001.

3. Results

3.1. Preparation and characterization of PO-NEs

To elucidate the molecular basis underlying the dermatological potential of PO, its chemical composition was comprehensively profiled by GC-MS (Table 1). Among the fifteen most abundant constituents, hexadecanoic acid and octadecanoic acid were key contributors to barrier repair. Gamma-tocopherol provides potent antioxidant protection by suppressing oxidative stress, whereas organic acids such as lactic acid regulate epidermal turnover and hydration. In addition, glyceryl monooleate, a membrane-compatible lipid, enhances interfacial fluidity and improves dermal adaptability (Carlson and Ford, 2025; Darimi et al., 2024; Wei et al., 2025; Joichi et al., 2025). These components accounted for more than 80% of the total chromatographic peak area. Overall, this compositional profile provided a biochemical foundation for the intrinsic dermatological bioactivity of PO.

Table 1.

Gas chromatography for the bioactive compounds of PO (Mean ± SD, n=3)

No.	Compound	Retention time (min)	Area (%)
1	Hexadecanoic acid	11.67	20.64 ± 0.42
2	Octadecanoic acid	12.58	14.10 ± 0.32
3	Lactic acid	5.48	8.83 ± 0.27
4	(9Z)-Octadecenoic acid	12.48	6.59 ± 0.22
5	gamma-Tocopherol	15.93	4.15 ± 0.18
6	Linoleate	12.46	3.68 ± 0.21
7	3-Hydroxypropanoate	6.04	3.52 ± 0.15
8	Glyceryl monooleate	14.66	3.16 ± 0.11
9	α-D-Glucopyranoside, β-D-fructofuranosyl	14.13	2.97 ± 0.07
10	6-Aminohexanoate	7.27	2.75 ± 0.04
11	Dodecane	8.91	2.20 ± 0.02
12	(9Z,12Z,15Z)-Octadecatrienoic acid	12.49	2.10 ± 0.05
13	2,3-Diaminopropionic acid	9.23	1.82 ± 0.08
14	Isobutylamine	5.81	1.37 ± 0.11
15	4-Hydroxypyridine	6.26	1.32 ± 0.03

Despite its intrinsic bioactivity, the combination of poor solubility, high volatility, and weak transdermal penetration renders PO unsuitable for topical applications. Nanoscale encapsulation improves the colloidal stability, enhances skin spreading, facilitates transdermal transport, and protects volatile constituents from degradation. Thus, PO-NEs containing 2%, 5%, and 10% PO were successfully generated by high-pressure homogenization. Macroscopically, the prepared PO-NEs with varying oil concentrations (2%, 5%, and 10%) appeared as uniform, milky-white dispersions without visible phase separation or oil floating (Fig. 1A). During the high-pressure homogenization process, the droplet size exhibited a progressive decrease with increasing homogenization cycles (Figur 1C). To ensure the optimal nanosizing for different oil loadings, the following specific pressures were optimized: 1200 bar for 2% PO, 1000 bar for 5% PO, and 800 bar for 10% PO. Under these optimized conditions, all formulations reached a stable size plateau after approximately 8 homogenization cycles, indicating that the emulsification process had approached a steady state and that additional cycles provided no meaningful further size reduction. In specific, dynamic light scattering (DLS) analysis revealed that the optimized PO-NEs had a narrow unimodal size distribution (Fig. 1B). The average hydrodynamic diameters were approximately 175, 180, and 178 nm for the 2%, 5%, and 10% PO-NEs, respectively, with polydispersity indices (PDI) consistently below 0.2, indicating excellent homogeneity (Fig. 1E). Transmission electron microscopy (TEM) images confirmed the formation of well-dispersed spherical droplets with smooth surfaces (Fig. 1D), whose morphologies and sizes correlated well with DLS measurements. Furthermore, the zeta potential of all PO-NE formulations was negative, ranging from -20 to -25 mV (Fig. 1G). This negative surface charge is crucial for electrostatic stabilization, preventing droplet aggregation, and contributing to the colloidal stability of the system. The detailed physicochemical parameters are summarized in Table S1.

Fig. 1.

Formation and physicochemical characterization of PO-based nanoemulsions (PO-NEs). (A) Macroscopic appearance of PO-NEs containing 2%, 5%, and 10% PO, prepared by high-pressure homogenization. (B) Dynamic light scattering (DLS) size distribution profiles of 2%, 5%, and 10% PO-NEs. (C) Droplet size recorded over successive homogenization cycles for different PO concentrations. (D) TEM micrographs of 2%, 5%, and 10% PO-NEs (Scale bar, 200 nm). (E) Particle size and polydispersity index (PDI) of PO-NEs across formulations. (F) Rheological measurements: viscosity as a function of shear rate (left) and oscillatory shear profiles showing storage (G’) and loss (G”) moduli (right). (G) Zeta potential values of PO-NEs with different PO concentrations. (H) Raman spectra of PO and PO-NEs, with characteristic bands annotated for comparison.

The rheological behavior of PO-NEs was investigated to assess their structural integrity and applicability for topical administration. As shown in Fig. 1F, all formulations exhibited shear-thinning behavior, where the viscosity decreased with increasing shear rate, which is desirable for easy spreading on the skin. The rheological behavior of the optimized PO-NEs was further analyzed to assess their structural integrity and suitability for topical application. In the linear viscoelastic region at low strains, the storage modulus (G') exceeded the loss modulus (G”), indicating predominantly elastic behavior. This elastic network reflects a stable internal droplet structure and effective interfacial interactions, which resist coalescence and phase separation, thereby enhancing long-term physical stability. Such viscoelastic properties are particularly advantageous for topical applications, as they ensure the nanoemulsion maintains its integrity during handling and spreading(Kim et al., 2023). Finally, Raman spectroscopy was performed to verify the chemical integrity of the encapsulated oil (Fig. 1H). The spectrum of the PO-NEs retained the characteristic peaks of pure PO, including the C=C stretching vibration at 1660 cm^-1 (amide I region) and 3013 cm^-1, as well as CH scissoring at 1440 cm^-1. The preservation of these molecular fingerprints confirmed that the high-pressure homogenization process did not alter the chemical structure of bioactive PO.

In addition, the storage stability of PO-NEs was further evaluated under ambient conditions. As summarized in Table S2, only a modest increase in droplet size was observed after 3 and 6 months of storage (195.2 ± 1.7 and 201.9 ± 2.5 nm, respectively), while the PDI remained below 0.25. The zeta potential became slightly less negative over time, but the formulation still maintained acceptable colloidal characteristics. These results suggest that the nanoemulsion system retained good physicochemical stability during storage.

3.2. PO-NEs attenuate keratinocyte hyperproliferation and inflammatory activation

Since the in vitro assays were performed in complete culture medium, the colloidal stability of PO-NEs under these conditions was first examined. As summarized in Table S2, PO-NEs showed only a modest increase in droplet size after incubation in culture medium for 48 and 72 h, while the PDI remained within an acceptable range. The zeta potential became less negative over time, likely due to the ionic strength of the medium and adsorption of medium components onto the droplet surface. Overall, these results indicate that PO-NEs maintained acceptable colloidal stability during the time window of in vitro experiments.

Afterwards, the biocompatibility of PO-NEs was assessed in HaCaT cells using the CCK-8 assay. As shown in Fig. 2A, PO-NEs exhibited negligible cytotoxicity at concentrations up to 25 μg/mL, maintaining cell viability above 90%. Although a slight reduction in viability was observed at 50 μg/mL, it remained within an acceptable range. Thus, a concentration gradient of 5-50 μg/mL was selected for subsequent efficacy studies. As keratinocyte hyperproliferation is critical for psoriasis, we investigated the antiproliferative effect of PO-NEs on IL-6-stimulated HaCaT cells (Fig. 2B). As shown in Fig. 2C, IL-6 significantly enhanced HaCaT proliferation over time, whereas PO-NEs effectively suppressed this response in a dose-dependent manner. The most effective suppression of abnormal cell proliferation was observed at 25 μg/mL, indicating that this concentration had an optimal anti-proliferative effect. The subsequent examination of intracellular oxidative stress was facilitated by DCF-DA staining (Fig. 2D). The stimulation of IL-6 resulted in a significant increase in ROS levels as evidenced by the robust fluorescence signals obtained. PO-NEs treatment significantly suppressed ROS accumulation (Fig. 2E). Notably, the 25 μg/mL group exhibited the lowest fluorescence intensity, indicating that this concentration was most effective in alleviating oxidative stress. This finding is consistent with the anti-proliferative results, suggesting a coordinated improvement in cellular inflammatory status. To further validate the anti-inflammatory mechanism, the expression of key psoriatic cytokines was quantified. ELISA results demonstrated that the secretion levels of IL-6 and IL-1β were markedly upregulated in the model group but were significantly downregulated by PO-NEs treatment in a dose-dependent manner (Fig. 2F). This result was consistent with the immunofluorescence staining results (Fig. 2G). While the model group exhibited strong fluorescence for IL-6 and IL-1β (green), PO-NEs treatment substantially reduced fluorescence intensity, confirming the ability of PO-NEs to inhibit the inflammatory cascade in psoriatic keratinocytes.

Fig. 2.

In vitro biocompatibility and anti-inflammatory effects of PO-NEs in IL-6–stimulated HaCaT cells. (A) Cell viability of HaCaT cells treated with increasing concentrations of PO-NEs for 48 h. (B) Schematic illustration of CCK-8 assay workflow evaluating PO-NEs–mediated modulation of IL-6-induced proliferation. (C) Relative cell growth rates at 24, 48, and 72 h after PO-NEs treatment. (D) Schematic of ROS assessment in IL-6–activated HaCaT cells using DCF-DA staining following PO-NEs treatment. (E) Representative fluorescence images showing intracellular ROS levels across PO-NEs concentrations. (F) ELISA quantification of IL-6 and IL-1β secretion in HaCaT cells. Mean ± SD (n = 3). (G) Immunofluorescence staining of IL-6 and IL-1β in control, PO-NEs–treated, and model groups (Scale bar, 50 μm).

In summary, PO-NEs attenuated IL-6-induced hyperproliferation, reduced oxidative stress, and decreased the expression of inflammatory cytokines. Across all assessments, 25 μg/mL consistently demonstrated the most prominent effects, presenting a favorable balance between biological activity and cellular compatibility.

3.3. Topical application of PO-NEs significantly ameliorates IMQ-induced psoriasis-like dermatitis

An IMQ-induced psoriasis-like dermatitis model was used to evaluate the therapeutic efficacy of the PO-NEs. As shown in Fig. 3A, mice received daily IMQ application followed by topical administration of PO-NEs at varying concentrations. Macroscopic assessment demonstrated that PO-NEs markedly diminished erythema, scaling, and epidermal thickening relative to the model group (Fig. 3B; Fig. S1), indicating substantial alleviation of cutaneous inflammation and barrier disruption. Among the treated groups, the 5% PO-NEs group exhibited the most pronounced improvement in skin appearance. The histological analysis confirmed these observations. The skin tissue in the IMQ-treated group displayed typical pathological features of psoriasis, characterized by significant epidermal thickening, abnormal keratinization, and extensive infiltration of inflammatory cells in the dermis (Fig. 3C). These changes reflect abnormal proliferation of keratinocytes and local immune activation. However, treatment with PO-NEs significantly reversed these pathological changes in a dose-dependent manner, resulting in a thinner epidermis and reduced immune cell infiltration, consistent with the antiproliferative and anti-inflammatory activities observed in vitro.

Fig. 3.

Evaluations of therapeutic efficacy of 2%, 5% and 10% PO-NEs on IMQ-induced psoriasis mice. (A) IMQ-induced psoriasis model with PO-NEs treatment. (B) Photographs of back skin from healthy mice and IMQ-induced psoriatic mice as well as the psoriatic mice treated with formulations including 2%, 5% and 10% PO-NEs after IMQ treatments. The ruler indicates length in centimeters (cm). (C) H&E staining images of the skin sections showing the effects PO-NEs relieving psoriasis symptoms to some extents and the 2% and 5% PO-NEs had better therapeutic efficacy compared with 10% PO-Nes (Scale bar, 100 μm). (D) Percentages of spleen/body weight. (E) Dynamic changes in PASI scores and full-thickness skin measurements during PO-NEs treatment. Mean ± SD (n ≥ 6).

Spleen index was measured to assess the systemic immune response associated with the disease. As shown in Fig. 3D, the model group showed a significant increase in spleen index. In contrast, PO-NEs treatment prevented spleen enlargement in a dose-dependent manner, with the 5% formulation showing the optimal effect. These results suggest that PO-NEs can not only alleviate local skin inflammation but also regulate systemic immune status. Disease progression was monitored using PASI scoring and dorsal skin thickness measurement. Both indicators increased rapidly in the model group, indicating continuous development of inflammation. Conversely, PO-NEs treatment significantly suppressed this trend (Fig. 3E), demonstrating its ability to control psoriasis-like symptom progression.

Collectively, these findings demonstrated that PO-NEs effectively alleviated macroscopic skin symptoms, attenuated spleen enlargement, and improved epidermal histopathology by reducing hyperplasia and inflammatory infiltration, thereby confirming their therapeutic efficacy against IMQ-induced psoriasis-like dermatitis.".

3.4. PO-NEs reprogram psoriatic gene expression by suppressing Wnt signaling and inflammatory pathways

Transcriptomic profiling revealed that PO-NEs treatment triggered substantial reprogramming of gene expression in psoriatic skin, indicating broad restoration of dysregulated epidermal and immune pathways. As shown in Fig. 4A, 308 differentially expressed genes (DEGs) were identified, of which 91 were upregulated and 217 downregulated following PO-NEs intervention. The volcano plot highlighted the prominent upregulation of genes, such as Dlx2, Acan, Hes2, Cps1, and Rspo2, which are associated with tissue remodeling and epithelial homeostasis. In contrast, genes linked to hyperkeratosis and inflammatory signaling, including Krtap15, Cst6, Sprr2k, Cxcl5, and numerous keratin-associated structural genes, were markedly downregulated (Fig. 4B). These transcriptional patterns are consistent with reduced keratinocyte hyperproliferation and attenuated chemokine-driven inflammation. Hierarchical clustering further revealed a clear separation between PO-NEs and model samples, indicating an obvious shift in psoriatic transcriptional state following treatment (Fig. 4C). GO enrichment analysis showed that differentially expressed genes were preferentially enriched in pathways related to extracellular matrix organization, basement membrane integrity, plasma membrane regulation, and chemotaxis (Fig. 4D), suggesting that PO-NEs not only modulate inflammatory signaling but also contribute to the structural normalization of the epidermal microenvironment. Notably, the GO term positive regulation of the Wnt signaling pathway was significantly enriched among altered genes, pointing to the central role of Wnt pathway dysregulation in psoriatic pathology and its responsiveness to PO-NEs intervention. KEGG pathway enrichment further identified Wnt signaling, ECM-receptor interaction, and neuroactive ligand-receptor interaction as the major regulated pathways (Fig. 4E) that collectively govern epidermal differentiation, extracellular microenvironment remodeling, and inflammatory communication. This finding highlights the multilevel impact of PO-NEs on psoriatic skin biology. Gene set enrichment analysis (GSEA) provided direct pathway-level validation, demonstrating significant suppression of aberrantly activated Wnt signaling in the PO-NEs group (NES = −2.26, P < 0.001, FDR < 0.001) (Fig. 4F). This suppression aligns with the observed reduction in epidermal hyperplasia, and supports the mechanistic role of Wnt inhibition in alleviating psoriatic pathology. Chord diagram visualization further revealed that the regulated genes participated in a broad array of biological processes, including the extracellular region, keratinocyte differentiation, inflammatory response, and immune regulation (Fig. 4G). Taken together, these data provide robust transcriptomic evidence that PO-NEs exert therapeutic effects by coordinately inhibiting Wnt signaling and suppressing inflammation.

Fig. 4.

Transcriptomic analysis framework for PO-NE–treated psoriatic skin. (A) Strategy for identifying differentially expressed genes between PO-NEs and the model group. (B) Volcano plot visualizing global transcriptional alterations. (C) Heatmap depicting overall transcriptional pattern shifts. (D) GO enrichment analysis of gene functional categories. (E) KEGG pathway enrichment analysis outlining major regulated pathways. (F) GSEA workflow applied to assess pathway-level enrichment trends. (G) Chord diagram illustrating the relationships between key genes and associated biological processes.

To validate the transcriptomic results, qRT-PCR was performed to identify key genes involved in the Wnt pathway. As shown in Fig. 5A and Fig. S2, the model group showed clear activation of the Wnt cascade. Specifically, the expression of upstream ligands and receptors (Wnt10b, Fzd7, Lrp6) and intracellular signal mediators (Dvl1, Dvl2) was significantly increased. This upstream activation leads to the accumulation of Ctnnb1 (β-catenin), the core component of the Wnt/β-catenin pathway, which subsequently triggers the upregulation of downstream target genes (Lef1, Axin2, Dkk1). Treatment with PO-NEs effectively downregulated all these key genes, indicating successful blockade of the Wnt/β-catenin pathway signaling flow. This inhibition was consistent with a reduction in Krt16, a marker of cell proliferation, confirming that PO-NEs suppressed abnormal skin growth. Parallel to Wnt inhibition, the immune-modulating effects of PO-NEs were validated by assessing the levels of proinflammatory cytokines (Fig. 5B). The mRNA levels of key inflammatory drivers, such as Il1b, Il6, Tnf, and the transcriptional regulator Nfkb1, were significantly increased in the model group but were effectively inhibited by PO-NEs administration. Moreover, the expression of Ptgs2 (which encodes the proinflammatory enzyme COX-2) was significantly downregulated. Taken together, these data provide robust molecular evidence that PO-NEs exert anti-psoriatic efficacy by dual-targeting the aberrant Wnt/β-catenin axis and the local inflammatory cascade.

Fig. 5.

qRT-PCR validation of transcriptomic changes in psoriatic skin following PO-NEs treatment. (A) Relative mRNA expression levels of representative Wnt signaling-related genes, including Wnt10b, Ctnnb1, Fzd7, Gsk3b, Znrf3, Lef1, Lrp6, Rnf43, Tcf7l2, and Krt16, in the Control, Model, and PO-NEs groups. (B) Relative mRNA expression levels of key inflammatory response–related genes, including Il1b, Il6, Nfkb1, Tnf, and Ptgs2, in the indicated groups.

3.5. PO-NEs serve as an effective nanocarrier for curcumin with enhanced skin permeation

To evaluate the feasibility of PO-based nanoemulsions as dual-functional topical delivery systems that can simultaneously enable efficient drug loading and enhance transdermal penetration, we first prepared a formulation of Cur@PO-NEs by incorporating curcumin into the PO oil phase according to the compositions shown in Table 2. Benefiting from the strong solubilizing capacity and lipid-compatible microenvironment of the PO-NEs, the resulting Cur@PO-NEs readily formed a uniform, yellow, translucent dispersion with no visible precipitation, particularly in contrast to the coarse curcumin suspension (Fig. 6A and B). DLS analysis showed that both Cur@PO-NEs and Cur@MCT-NEs possessed narrow particle size distributions within the nanoscale range (Fig. 6C and D; Fig. S3). Although the Cur@PO-NEs exhibited a slightly lower PDI than the Cur@MCT-NEs, both formulations displayed high homogeneity and excellent dispersion, making them suitable for topical and transdermal drug delivery. These physicochemical parameters can be also found in Table S1. TEM imaging further confirmed the formation of spherical, well-dispersed nanodroplets with well-defined boundaries (Fig. 6E). Zeta potential measurements confirmed that both Cur@PO-NEs and Cur@MCT-NEs exhibited stable surface charges (Fig. 6F), indicating the favorable colloidal stability required for topical and transdermal delivery applications. The hemolysis assays further demonstrated excellent blood compatibility. Neither blank nanoemulsions nor curcumin-loaded formulations induced detectable hemolysis compared to the complete erythrocyte lysis observed with hypotonic ddH₂O (Fig. 6G). Visual inspection of red blood cell suspensions was also in line with these results, with all nanoemulsion-treated samples retaining a characteristic intact-cell appearance comparable to that of the PBS control (Fig. 6H).

Table 2.

Composition of different PO-based nanoemulsions in weight percentages.

Excipients	Nanoemulsions
	2% PO-NEs	5% PO-NEs	10% PO-NEs	Cur@PO-NEs	Cur@MCT-NEs
Oil phase
PO	2.0	5.0	10.0	5.0	-
MCT	-	-	-	-	5.0
Cur	-	-	-	0.05	0.05
Water phase
S40	5.0	5.0	5.0	5.0	5.0
Carbomer	0.2	0.2	0.2	0.2	0.2
5%(w/w) NaOH	1.2	1.2	1.2	1.2	1.2
Water to	100	100	100	100	100

Fig. 6.

Preparation, characterization, and skin permeation evaluation of Cur@PO-NEs. (A) Schematic illustration of the fabrication process for Cur@PO-NEs. (B) Macroscopic appearance of Cur suspension, Cur@MCT-NEs, PO-NEs, and Cur@PO-NEs. (C) Particle size distribution of Cur@PO-NEs determined by DLS. (D) Average hydrodynamic diameter and PDI of Cur@MCT-NEs and Cur@PO-NEs. (E) Representative TEM images showing the morphology of Cur@MCT-NEs and Cur@PO-Nes (Scale bar, 100 nm). (F) Zeta potential values of the indicated formulations. (G) Quantitative relative hemolysis percentage and (H) photographic images of erythrocyte suspensions incubated with PBS (negative control), ddH₂O (positive control), and different nanoemulsions. (I) In vitro cumulative drug release profiles of free curcumin, Cur@MCT-NEs, and Cur@PO-NEs over 60 h. (J) In vivo fluorescence imaging of mice dorsal skin following topical application of different curcumin formulations at indicated time points. (K) Representative CLSM images of skin cryosections showing the penetration depth and distribution of curcumin (green) in vitro (Scale bar, 100 μm). Nuclei were stained with DAPI (blue).

Drug release behavior was evaluated using the dialysis method. As shown in Fig. 6I, the free curcumin suspension exhibited poor release kinetics (< 20%), owing to its low aqueous solubility. Interestingly, Cur@PO-NEs demonstrated a significantly faster and more complete release profile (∼95% at 60 h) than that of Cur@MCT-NEs (∼50%). This suggests that the PO lipid matrix may provide a superior microenvironment for drug solubilization and diffusion. To investigate the transdermal efficiency in vivo, fluorescence imaging was performed on live mice following topical application. Fig. 6J shows that the Cur@PO-NEs group displayed the strongest fluorescence signals on the dorsal skin at all time points. Quantitative analysis confirmed that the fluorescence intensity of Cur@PO-NEs was consistently higher than that of the Cur@MCT-NEs and suspension groups, indicating an enhanced skin retention capability (Fig. S4). The penetration depth was further characterized using Z-stack scanning. As illustrated in Fig. S5, Cur@PO-NEs facilitated deep drug delivery up to 40 μm into the skin layers, whereas the fluorescence signals of the control groups were mostly confined to the superficial stratum corneum. Permeation behavior was assessed in both psoriatic and normal skin using Franz diffusion cells. CLSM images of skin cryosections revealed that Cur@PO-NEs effectively penetrated the viable epidermis and reached the dermis in both psoriatic (Fig. 6K) and normal (Fig. S6) skin. In contrast, Cur@MCT-NEs showed limited penetration and the suspension remained on the skin surface. Statistical quantification of the fluorescence area further demonstrated that Cur@PO-NEs achieved the highest drug deposition in both the skin models (Fig. S7). Collectively, these results demonstrate that PO-NEs act as efficient transdermal delivery vehicles, achieving markedly improved skin accumulation and deeper penetration of hydrophobic compounds compared with conventional carriers.

3.6. Cur@PO-NEs achieve superior anti-psoriatic efficacy via vehicle-drug synergy

To evaluate the in vivo therapeutic performance of the co-delivery system, an IMQ-induced psoriasis mouse model was used. As shown in Fig. 7A, daily IMQ application induced severe lesions characterized by erythema and scaling. Consistent with our earlier findings, the PO-NEs group exhibited evident therapeutic effects, alleviating skin symptoms, compared to the model group. However, the incorporation of curcumin (Cur@PO-NEs) further amplified this benefit, resulting in a remarkably smooth skin appearance comparable to that of the positive control (tacrolimus). Quantitative monitoring via PASI scores and skin thickness (Fig. 7D and E) confirmed this trend; while PO-NEs and Cur@MCT-NEs provided moderate relief, Cur@PO-NEs achieved the most significant inhibition of disease progression. Notably, Cur@PO-NEs statistically outperformed the inert carrier group (Cur@MCT-NEs, P < 0.05), validating the superior therapeutic potential of the bioactive PO vehicle compared with conventional oils. H&E staining further corroborated these findings (Fig. 7B). The model group displayed typical psoriatic features including pronounced epidermal hyperplasia and inflammatory infiltration. Cur@PO-NEs treatment effectively reversed these pathological changes, restoring the normalized epidermal structure more effectively than PO-NEs or Cur@MCT-NEs alone. Systemically, Cur@PO-NEs also significantly suppressed IMQ-induced splenomegaly (Fig. 7C), indicating a potent regulation of the systemic immune response.

Fig. 7.

Evaluation of Cur@PO-NEs in an IMQ-induced psoriasis-like dermatitis model. (A) Representative macroscopic images of dorsal skin and excised spleens from each treatment group following IMQ induction and topical administration of Cur suspension, Cur@MCT-NEs, PO-NEs, Cur@PO-NEs, or tacrolimus. (B) H&E-stained dorsal skin sections illustrating epidermal morphology and dermal immune-cell distribution across treatment groups (Scale bar, 100 μm). (C) Quantification of spleen index (spleen weight/body weight) for each group. (D) Time-course measurements of full-thickness dorsal skin throughout the 7-day treatment period. (E) Daily PASI scoring recorded during the experimental course. (F) ELISA measurements of IL-23, IL-17A, TNF-α, and IL-1β levels in skin homogenates. Data are presented as mean ± SD (n = 6). Statistical significance is indicated as *P < 0.05, **P < 0.01, ***P < 0.001.

We then quantified inflammatory cytokines using ELISA results (Fig. 7F), showing that key drivers of psoriasis (IL-23, IL-17A, TNF-α, IL-6, and IL-1β) were drastically elevated in the model group. Although PO-NEs alone reduced these levels, Cur@PO-NEs showed the most substantial reduction, demonstrating a synergistic anti-inflammatory effect. This was further confirmed by immunohistochemical (IHC) staining (Fig. 8A and Fig. S8), where IMQ markedly increased the epidermal expression of IL-1β, IL-6, IL-17A, and TNF-α, accompanied by abundant positive cells within the epidermis. In contrast, Cur@PO-NEs treatment substantially reduced the staining of all proinflammatory cytokines, comparable to tacrolimus. Quantification of the positive areas (Fig. 8B) confirmed consistent and significant reductions in all cytokines.

Fig. 8.

Immunohistochemical and TUNEL staining analyses of inflammatory markers, proliferation, and apoptosis in psoriasiform skin. (A) Representative immunohistochemical (IHC) staining images of dorsal skin sections from each treatment group, showing the localization and expression patterns of TNF-α, IL-17, Ki-67, and IL-6. TUNEL staining is presented to visualize apoptotic cells within epidermal and dermal layers. Scale bars: 100 μm. (B) Quantitative analysis of the percentage of positively stained area for TNF-α, IL-17, Ki-67, IL-6, and IL-1β across treatment groups. Data are presented as mean ± SD. Statistical analysis was performed using one-way ANOVA with post-hoc comparisons (*P < 0.05, **P < 0.01, ***P < 0.001).

Finally, we evaluated the balance between keratinocyte proliferation and apoptosis. As shown in Fig. 8A and 8B, the model group exhibited a high density of Ki67-positive cells. Cur@PO-NEs treatment significantly reduced Ki67 expression, while concurrently increasing TUNEL-positive apoptotic cells and effectively correcting aberrant epidermal hyperproliferation. Collectively, these results demonstrated that Cur@PO-NEs exert superior anti-psoriatic efficacy by integrating the intrinsic bioactivity of the PO carrier with the pharmacological action of curcumin to synergistically suppress inflammation and normalize epidermal differentiation.

3.7. Comprehensive safety assessment demonstrates favorable biocompatibility of Cur@PO-NEs

The in vivo safety of the nanoemulsion system was evaluated to ensure its suitability for long-term topical applications. First, we examined the local and systemic effects of a blank vehicle (PO-NEs). As shown in Fig. S9, the topical administration of PO-NEs did not induce any visible skin irritation, erythema, or abnormalities. Furthermore, monitoring of body weight and full-thickness skin measurements revealed no significant differences between PO-NEs treated mice and healthy controls (Fig. S10), suggesting that the PO-based matrix is non-toxic and well tolerated by the skin barrier.

Building on the favorable safety profile of the vehicle, a multidimensional evaluation of Cur@PO-NEs was conducted. Histopathological examination of major organs (spleen, liver, and skin) following repeated application showed no detectable structural disruption, inflammatory infiltration, or cellular damage, with the tissue architecture remaining comparable to that of the untreated controls (Fig. 9A). The systemic inflammatory status was examined by quantifying serum cytokines (TNF-α, IL-1β, IL-17A, and IL-6). As shown in Fig. 9B, cytokine concentrations remained within a comparable range across all groups, indicating that topical Cur@PO-NEs did not induce systemic immune activation after repeated exposure. This was further supported by the steady body weight curves of the mice throughout the treatment period, which showed no signs of systemic burden or metabolic distress (Fig. 9C). To further assess hematological and biochemical safety, routine blood parameters (WBC, lymphocytes, and RBC) and serum biomarkers associated with hepatic and renal function (ALT, AST, γ-GT, BUN, CREA, and UA) were analyzed. As shown in Fig. 9D, all measured indicators remained within the normal ranges and displayed no significant differences among treatment durations. These results indicate preserved liver and kidney function, as well as stable hematologic profiles following Cur@PO-NEs application. Collectively, the multidimensional safety assessment demonstrated that Cur@PO-NEs exhibited excellent biocompatibility, did not induce local irritation or systemic toxicity, and maintained stable physiological, hematological, and biochemical profiles during short-term and extended topical administrations.

Fig. 9.

In vivo safety evaluations of healthy mice and Cur@PO-NEs treated mice for 1 and 8 days. (A) H&E staining images of main organs including spleen, skin, and liver to reflect toxicity signs of Cur@PO-Nes (Scale bar, 100 μm). (B) Representative photographs of dorsal mice back skin, and the profiles of body weight and full skin thickness recorded daily without or with treatment of Cur@PO-NEs for 8 days. (C) Comparative analysis of inflammatory cytokine levels (TNF-α, IL-1β, IL-17a and IL-6) from blood serum of different skin-treated mice groups. (D) Acute toxic evaluations using blood serum through levels of indicators including AST, ALT, γ-GT, BUN, CREA, and UA, as well as the whole blood through blood routine parameters including WBC, RBC, and lymphocytes. Mean ± SD (n ≥ 3).

4. Discussion

In the present study, we successfully developed a stable PO-NE system and systematically validated its dual function as both a therapeutic agent and a nanocarrier for psoriasis management. Our results demonstrated that PO-NEs alone significantly alleviated the psoriatic symptoms. Furthermore, transcriptomic profiling revealed that these therapeutic effects are largely mediated through suppression of the aberrant Wnt/β-catenin signaling pathway. Building on this intrinsic bioactivity, we further confirmed that PO-NEs can serve as efficient delivery vehicles for curcumin. The engineered Cur@PO-NEs exhibited superior skin permeation and retention compared to conventional formulations, resulting in a synergistic enhancement of therapeutic efficacy (Scheme 1).

Scheme 1.

Schematic illustration of the therapeutic mechanisms and bioactive carrier functions of PO-NEs for the treatment of psoriasis-like skin inflammation. The schematic depicts the topical application of PO-NEs and their deep penetration into the viable epidermis. As illustrated, PO-NEs play a dual role. First, as an active therapeutic agent, they inhibit the aberrantly activated Wnt/β-catenin signaling pathway in keratinocytes, thereby suppressing TCF/LEF transcriptional activity, reducing ROS accumulation, and downregulating pro-inflammatory cytokines (IL-17a, IL-23, TNF-α). Second, PO-NEs serve as an efficient delivery vehicle for curcumin (Cur@PO-NEs), achieving enhanced transdermal permeation and synergistic anti-psoriatic efficacy.

Importantly, the biological performance of PO-NE-based formulations appears to be closely associated with their physicochemical properties. Their nanoscale droplet size, narrow size distribution, and good colloidal stability are favorable for intimate skin contact, efficient cutaneous partitioning, and reproducible local delivery, all of which are critical for topical psoriasis therapy. Accordingly, the enhanced efficacy of PO-NE and Cur@PO-NE likely arises from both the intrinsic bioactivity of PO and the optimized nanoemulsion structure of the formulation. By exploring the molecular mechanisms, we found that PO-NEs suppressed the Wnt/β-catenin axis, which may be attributed to the specific lipid composition of PO. α-Linolenic acid, the dominant constituent of PO, has been widely reported to act as a natural ligand for Peroxisome Proliferator-Activated Receptors (PPARs), particularly PPAR-γ (Briganti et al., 2024; Takić et al., 2024). Since PPAR-γ activation is known to functionally antagonize Wnt/β-catenin signaling and suppress inflammation, it is plausible that the high α-linolenic acid content in PO-NEs exerts its effects by activating PPAR-γ, which subsequently inhibits β-catenin nuclear translocation and downstream transcriptional activity(Zuo et al., 2021; Luo et al., 2022). Moreover, other lipid metabolites present in the essential oil may also contribute to this inhibitory effect, collectively exerting a multi-target blockade of the proliferative signaling cascade.

Beyond signaling regulation, our transcriptomic analysis also highlighted the significant enrichment of genes associated with extracellular matrix (ECM) organization and epidermal differentiation. Psoriasis is typically characterized by a compromised barrier function and a disorganized dermal structure. The observed promotion of ECM remodeling is likely driven by the nutritional role of PO-derived lipids. PO is rich in polyunsaturated fatty acids (PUFAs), which are essential precursors in the synthesis of stratum corneum ceramides and structural proteins (Andrew et al., 2025). These bioactive lipids can modulate keratinocyte differentiation and facilitate restoration of the physical skin barrier. This aligns with previous findings suggesting that topical lipid application can accelerate skin repair and restore barrier integrity, providing a biochemical basis for the structural improvements observed in the PO-NEs group (Mijaljica et al., 2024).

Furthermore, PO-NEs also demonstrated significant potential as transdermal delivery platforms. The encapsulation of curcumin (Cur@PO-NEs) resulted in a markedly higher intradermal drug deposition than curcumin suspensions or MCT-based nanoemulsions. This enhancement can be explained by the permeation-promoting properties of the essential oil components, which have been widely demonstrated in previous studies (Scuteri et al., 2022; Yihan et al., 2025). In addition, essential oil components and unsaturated lipids in PO may interact with the stratum corneum lipid matrix, increase lipid fluidity, and facilitate diffusion of hydrophobic molecules into viable skin layers (Liu and Lunter, 2020). At the same time, the nanoemulsion structure may prolong skin contact and improve local partitioning of the encapsulated drug. For topical psoriasis treatment, such a delivery profile is particularly advantageous because enhanced intradermal retention is generally more desirable than unrestricted transdermal permeation, as it maximizes local drug exposure at the lesion site while potentially limiting systemic distribution. Thus, the greater skin deposition of Cur@PO-NEs provides a direct physicochemical basis for its effective therapeutic efficacy in vivo.

Another important point is that the enhanced efficacy of Cur@PO-NEs is likely attributable to both improved delivery efficiency and pathophysiological complementarity between the carrier and the cargo. On the one hand, PO-NEs themselves can normalize aspects of the diseased skin microenvironment through suppression of proliferative signaling, attenuation of inflammation, and support of barrier repair. On the other hand, curcumin provides additional anti-inflammatory and antioxidative effects once effectively delivered into the lesion. The combined system therefore does not merely increase drug loading or skin permeation in a passive sense; rather, it establishes a cooperative therapeutic mode in which the carrier actively participates in treatment while simultaneously enhancing local availability of the loaded compound. This dual contribution may explain why Cur@PO-NEs achieved better anti-psoriatic performance than conventional formulations lacking either the bioactive oil phase or the optimized nanoemulsion structure.

Despite these promising findings, the current study had several limitations that require further investigation. First, although we hypothesized the involvement of the α-linolenic acid/PPAR axis, the precise molecular interactions and specific bioactive ligands within the complex oil mixture remain unclear. Second, although short-term biocompatibility was confirmed, the long-term systemic safety of repeated topical application of these bioactive lipids requires a more comprehensive toxicological evaluation. Nevertheless, this study established PO-NEs as promising bioactive nanocarriers, offering a strategy that integrates natural product bioactivity with nanotechnology for the effective and safe management of psoriasis.

5. Conclusion

In summary, we have successfully developed a dual-functional nanoemulsion system based on PO, moving beyond the use of inactive pharmaceutical ingredients to create functional delivery systems. We demonstrated that PO-NEs exert intrinsic therapeutic effects primarily by inhibiting the Wnt/β-catenin signaling pathway, thereby reducing keratinocyte hyperproliferation and inflammation. Transcriptomic analysis indicated the potential benefits of restoring the structural integrity of the skin. Furthermore, the formulation served as a superior delivery vehicle for curcumin, achieving enhanced skin permeation and improved therapeutic outcomes compared with standard formulations. Collectively, our findings highlight the potential of transforming natural medicinal oils into advanced nanomedicines, providing a promising strategy for effective management of psoriasis and other inflammatory skin disorders.

CRediT authorship contribution statement

Zhengwei Zhang: Writing – original draft, Visualization, Methodology, Investigation, Conceptualization. Ji Mu: Writing – review & editing, Visualization, Investigation. Shanmin Tao: Visualization, Investigation. Tianjiao Li: Visualization, Investigation. Dominique Jasmin Lunter: Supervision, Methodology. Zhenhan Bao: Visualization, Investigation. Yan Chen: Visualization, Investigation. Liangyu Cai: Visualization, Investigation. Peng Cao: Supervision, Funding acquisition. Yali Liu: Writing – review & editing, Supervision, Methodology, Funding acquisition, Conceptualization.

Declaration of competing 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.

Appendix A. Supplementary data

Supplementary material

Data availability

Data will be made available on request.