Enhanced skin penetration of curcumin by a nanoemulsion-embedded oligopeptide hydrogel for psoriasis topical therapy

Abstract

Topical delivery of therapeutics on the skin can effectively alleviate skin symptoms of psoriasis and reduce systemic toxicity. However, the low delivery efficiency caused by the stratum corneum barrier limits the therapeutic impact. Here, we reported an oligopeptide hydrogel that encapsulates cell-penetrating-peptide (CPP)-decorated curcumin-loaded nanoemulsions (Cur-CNEs) to enhance the skin penetration of curcumin for topical treatment of psoriasis. After being applied to the skin of psoriatic mice, the Cur-CNE embedded oligopeptide hydrogel (Cur-CNEs/Gel) provided a prolonged residue time of Cur-CNEs on the skin lesion. The fluidic and elastic properties of the nanoemulsions enabled them to effectively pass through the interstitial spaces of the stratum corneum, while the CPP decoration further enhanced skin penetration and cellular uptake of Cur-CNEs. The Cur-CNEs/Gel exhibits effective alleviation of the symptoms of psoriasis in mice and provides a promising strategy for topical treatment of psoriasis.

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Introduction

Psoriasis is a complex chronic inflammatory systemic disease that affects approximately 3% of the global population.^1,2 The skin, as the organ most frequently affected by psoriasis, is characterized by erythema, papules and plaques due to the hyperproliferation of keratinocytes and infiltration of inflammatory cells into the epidermis.^3–5 For skin lesions, topical administration can directly act on the inflammatory skin and relieve the symptoms of patients, which has the advantages of avoiding the first pass effect, rapid onset of action, reducing systemic toxicity, and improving patient compliance. However, the development of a topical preparation for psoriasis suffers from limited skin penetration, due to the existence of a natural stratum corneum (SC) barrier.^6–8 The SC is composed of dense dead keratinocytes, which makes it difficult for drug molecules with a molecular weight over 500 Da or not meeting the appropriate partition coefficient (log P 1–3) to penetrate the skin.^9–11 Therefore, developing a drug carrier with enhanced skin permeability for the local treatment of psoriasis is of great significance.

A nanoemulsion is a homogeneous dispersion system composed of oil, water, surfactant, and cosurfactant, with a particle size range of 10–100 nm. Due to their high stability, low irritation, fast absorption, high skin permeability, and good bioavailability, nanoemulsions are considered to be promising pharmaceutical carriers for topical therapy.^12–16 The composition of oil, surfactant and cosurfactant endows a nanoemulsion with elasticity, fluidity, and a deformable structure, which facilitates skin penetration of the nanoemulsion through intercellular gap junctions.^17,18 The small droplet size of the nanoemulsion is also favorable for it to pass through the skin barrier.¹⁹

In this work, we developed an enhanced penetrating nanoemulsion for the topical delivery of curcumin, which was embedded in an oligopeptide hydrogel to ease psoriasis (Fig. 1). The nanoemulsion is composed of isopropyl myristate (IPM) as the oil phase, polyoxyethylene sorbitan monolaurate (Tween80) as the surfactant, and polyethyleneglycol 400 (PEG400) as the cosurfactant, which helps to distribute the nanoemulsion into deeper layers of the skin. Meanwhile, R8H3, a cell-penetrating peptide (CPP) developed by our group, was decorated on the surface of nanoemulsion particles to further intensify the penetration of the nanoemulsion.²⁰ Curcumin has garnered substantial interest due to its well-documented anti-inflammatory and antioxidant properties, making it a suitable model bioactive compound for investigating innovative delivery systems. Among them, a topical delivery strategy can effectively avoid problems such as poor systemic pharmacokinetics and bioavailability of curcumin.^21–25 Herein, we loaded it as a model drug into CPP-decorated nanoemulsions (CNEs). To prolong the skin retention of nanoemulsions, Phe-Phe (FF) and Fmoc-Phe (Fmoc-F) were used as precursors to form the gelators, which transform into the fiber net framework as a reservoir to entrap the nanoemulsion when exposed to the protease WQ9-2.

Fig. 1 Schematic representation of the preparation of the Cur-CNEs/Gel and their application in the psoriasis therapy. (a) Cur-CNEs are loaded into protease-triggered assembly Fmoc-FFF hydrogels to form the coatable patch Cur-CNEs/Gel. (b) After being applied to the skin, the Cur-CNEs/Gel can enhance curcumin transdermal efficiency and reduce inflammation.

Compared with the Cur-CNE solution, the Cur-CNE embedded hydrogels (Cur-CNEs/Gel) showed prolonged retention time on the skin. Meanwhile, the Cur-CNEs can pass through the stratum corneum to reach the deep layers of the skin, which is attributed to the efficient penetration of the nanoemulsion and the membrane penetration enhancement by the R8H3 peptide. After being taken up by the cells, the Cur-CNEs escaped from the endosomes with the assistance of the R8H3 peptide and released curcumin in the cytoplasm to achieve anti-inflammatory and anti-proliferative effects.

Materials and methods

Materials

Curcumin was obtained from Shanghai Aladdin Biochemical Technology Co., Ltd. IPM was purchased from Shanghai Macklin Biochemical Co., Ltd. Tween80 and PEG400 were obtained from Sinopharm Chemical Reagent Co., Ltd. Fmoc-F and FF with purity higher than 98% were purchased from GL Biochem Co., Ltd. Imiquimod (IMQ) ointment was purchased from Sichuan Mingxin Pharmaceutical Co., Ltd.

Cell lines and animals

Human keratinocytes (HaCaT) and mouse mononuclear macrophages (RAW264.7) were cultured in Dulbecco's modified Eagle medium (DMEM) containing 10% fetal bovine serum and 1% penicillin and streptomycin at 37 °C. Male ICR mice (6–8 weeks old) were provided by the Animal Center of Yangzhou University. All animal procedures were performed in accordance with the Guidelines for Care and Use of Laboratory Animals of Nanjing Tech University and approved by the Animal Ethics Committee of Nanjing Tech University.

Preparation and characterization of Cur-CNEs

The water titration method was used to prepare Cur-CNEs. Tween80, PEG400, and IPM were used as the surfactant, co-surfactant, and oil phase, respectively. In brief, a mixture of Tween80, PEG400, and IPM at a ratio of 4.5 : 4.5 : 1 was prepared by magnetic stirring. Curcumin was dissolved in the mixture solution, followed by titration with pure water to obtain Cur-NEs. Subsequently, R8H3-C18 (2.5 mol% of surfactant) was added to the Cur-NEs and incubated at 4 °C for 30 min to acquire Cur-CNEs with a curcumin concentration of 16 mg mL⁻¹. For the preparation of fluorescent labeled nanoemulsions, coumarin 6 (Cou6) was added to the mixed solution instead of curcumin, and Cou6-CNEs were prepared using the same method.

The diameter and zeta potential of the nanoemulsions were measured with a nanometer particle size potentiometer (Malvern, Nano ZS90). Transmission electron microscopy (TEM) (Hitachi, H7800) was used to observe the morphology of Cur-CNEs. The curcumin concentrations were detected using an HPLC instrument (Ultimate 3000) with a C18 ODS column. The mobile phase was acetonitrile : 0.5% acetic acid (65 : 35, v/v) with a flow rate of 1 mL min⁻¹. The detection wavelength was set at 425 nm. The entrapment efficiency was calculated according to the equation EE = C/C₀ × 100%, where C₀ is the total amount of curcumin in the nanoemulsion solution, and C is the amount of curcumin in the nanoemulsions after separation using a column packed with Sephadex G-50 resin. The fluorescence intensity of Cou6 was assayed with a microplate reader (466 nm/504 nm, Tecan M1000 Pro).

Skin penetration

The penetration of Cur-CNEs into the abdominal skin of mice was assessed by the Franz diffusion cell method. The depilated skin was placed between the donor chamber and the receptor chamber. 0.5 mL of Cur-CNEs was dripped into the donor chamber. The receptor chamber was filled with a mixed solution of ethanol and phosphate-buffered saline (1 : 3, v/v). The diffusion cell was placed in a 37 °C water bath and stirred at a rate of 500 rpm. At predetermined time points, the solution in the receptor chamber was sampled to determine the permeated amount of curcumin.

At 12 h, the skin was harvested to analyze the intradermal amount of curcumin. After the residual curcumin was washed away by 50% alcohol solution, the skin was homogenized in the hydro-alcoholic solution, followed by 1 h of sonication. After centrifugation at 5000 rpm for 30 min, the concentration of curcumin in the supernatant was analyzed.

The skin penetration capability of Cou6-CNEs was also assessed by the same procedure. For qualitative observation, after 4 h of treatment with Cou6-CNEs, the skin was fixed with 4% paraformaldehyde, stained with DAPI, and imaged by a digital slide scanner (3DHISTECH, Pannoramic 250 FLASH).

In vitro cell uptake

To track the uptake of nanoemulsions in cells, HaCaT and RAW264.7 cells were seeded in a 6-well plate (1 × 10⁶ cells per well) and cultured with TNF-α (20 ng mL⁻¹) and LPS (100 ng mL⁻¹) for 12 h separately. Cou6-NEs and Cou6-CNEs were added to each well and incubated for 2 h. The intracellular fluorescent intensities were visualized by a fluorescence microscope (Nikon, Eclipse Ti2) and quantified by ImageJ software. The cellular uptake of Cou6-NEs and Cou6-CNEs was also analyzed using a flow cytometer (Agilent, ACEA NovoCyte).

In vitro anti-inflammation

HaCaT cells were seeded in a 96-well plate (1 × 10⁴ cells per well), and TNF-α (20 ng mL⁻¹) was added to each well. After incubation for 12 h, different formulations (Cur Sol, Cur-NEs, and Cur-CNEs) were added to each well with 1 μg mL⁻¹ curcumin. After incubation for 48 h, 20 μL MTT solution (5 mg mL⁻¹) was added and the incubation continued for another 4 h. After the incubation, the culture medium was carefully removed and 150 μL dimethyl sulfoxide (DMSO) was added to each well. The absorbance was measured at 490 nm to calculate the cell viability.

RAW264.7 cells were seeded in a 6-well plate (1 × 10⁶ cells per well), and incubated for 12 h. Then lipopolysaccharide (LPS) with a final concentration of 1 μg mL⁻¹ was added. After incubation for 2 h, different curcumin formulations (1 μg mL⁻¹) were added to each well. Following a 6 h incubation at 37 °C, the mRNA levels of TNF-a, IL-6, and IL-23 were determined using kits (Beyotime, China). Table S1† shows the primer sequences.

Cytotoxicity

HaCaT and RAW264.7 cells were seeded in a 96-well plate (1 × 10⁴ cells per well). After incubation for 12 h, blank nanoemulsions at concentrations of 250, 100, 50, and 10 μg mL⁻¹ were added to each well. Then the cell viability was tested by MTT assay after further incubation for 24 h.

Construction and characterization of Cur-CNEs/Gel

0.1 mL of Cur-CNEs was mixed with 0.9 mL of PBS containing 20 mM Fmoc-F and FF, and then WQ9-2 was added to trigger gelation. After incubation at 37 °C for 2 h, the Cur-CNEs/Gel was obtained (curcumin concentration: 1.6 mg mL⁻¹). TEM (Hitachi, H7800) and scanning electron microscopy (SEM) (Hitachi, Regulus-8100) were used to observe the morphology of the Cur-CNEs/Gel.

To investigate the efficiency of the conversion of precursors into hydrogels, the content of precursor Fmoc-F was examined at different time points by HPLC. The conversion rate was calculated according to the conversion of Fmoc-F. The mobile phase consisted of methanol and water (75 : 25, v/v) with a flow rate of 1 mL min⁻¹. The wavelength of detection was 256 nm.

In vivo skin retention

The psoriatic skin model of mice was established according to the method described by Punit.²⁶ The hair on the back of the mice was shaved, and imiquimod (IMQ) cream was applied to the back at a dose of 25 mg per day for 7 days.

100 μL of Cou6-CNEs solution or Cou6-CNEs/Gel (Cou6: 100 ng mL⁻¹) were applied to the dorsal skin of normal and IMQ-induced psoriasis mice. Images of the mice were captured using an in vivo imaging system (IVIS, PerkinElmer) at different time points.

Effect on imiquimod-induced psoriasiform inflammation in mice

Psoriasis area and severity index of the tested formulations. Forty IMQ-induced psoriatic mice were randomly divided into 5 groups: the normal group, model group, Cur-Sol treatment group, Cur-NEs/Gel treatment group and Cur-CNEs/Gel treatment group. Curcumin formulations (5 mg kg⁻¹) were applied topically once a day for 10 days to treat the inflammatory skin of psoriasis mice. The psoriasis area and severity index (PASI) was used to evaluate the severity of the lesions, rating erythema and scaling on a scale of 0–4 points (0 for no symptoms, 1 for mild, 2 for moderate, 3 for severe, and 4 for very severe). Meanwhile, we applied the Cur-CNEs/Gel to the back skin of normal mice and observed the skin condition for 7 consecutive days to test the dermal safety of our formulation.

Histopathological and immunohistochemical study. Skin lesions and main organs were sampled for hematoxylin and eosin (H&E) staining. The expression of TNF-α and IL-17 in the skin after different treatments was evaluated by immunohistochemistry staining. The stained tissue slices were visualized using a microscope (Nikon, Eclipse Ti2).

Detection of cytokine mRNA transcription in skin tissues. To determine the mRNA transcription of TNF-α, IL-6, and IL-17, the skin samples were collected, homogenized with 2% β-mercaptoethanol, and centrifuged to obtain the supernatant. RNA extraction and mRNA detection were carried out according to the protocol.

Statistical analysis

GraphPad Prism software was used for statistical analysis. All data were presented as mean ± standard deviation (mean ± SD), one-way ANOVA was used for comparison among multiple groups, while independent samples t-test was employed for comparison between two groups.

Results and discussion

Preparation and characterization of Cur-CNEs

The curcumin-loaded nanoemulsions (Cur-NEs) were prepared by a low energy method.^27–30 Isopropyl myristate (IPM),^31,32 Tween80, and PEG400 (ref. 33) were used as the oil phase, surfactant, and co-surfactant, respectively. The obtained Cur-NEs presented a diameter of approximately 15 nm and a negative charge of −4.1 mV. R8H3, a CPP containing octaarginine and trihistidine, was conjugated with stearic acid C18 and anchored on the surface of the NEs through electrostatic force and hydrophobic interactions. The obtained Cur-CNEs had a particle size of 16 nm and a charge inversion of +10.1 mV. TEM images showed that Cur-CNEs have a uniformly spherical nano-structure (Fig. 2a and S1 and S2†). Curcumin was efficiently encapsulated in the nanoemulsions with an entrapment efficiency of approximately 98%.

Fig. 2 (a) Particle sizes and TEM images of Cur-CNEs. Scale bars: 50 nm. (b) Comparison of cumulative amounts of various curcumin formulations (Cur-Sol, Cur-NEs, and Cur-CNEs) in the skin over time. (c) Comparison of transdermal permeation and intradermal retention amounts of various curcumin formulations at 12 h. *p < 0.05, ***p < 0.001. Fluorescence images of the skin tissues (d) and fluorescence intensity of Cou6 (e) after incubation with Cou6-NEs and Cou6-CNEs for 4 h. ***p < 0.001. Scale bar: 500 μm.

The Franz cell approach was adopted to perform the ex vivo drug permeation and deposition studies on excised abdominal mouse skin. The curcumin formulations were mounted in the donor chamber, and the curcumin permeated through the skin into the receptor chamber medium was detected at specified time points. After 12 h, the cumulative permeated and intradermal amounts of Cur-NEs were 15.2 and 19.0 μg cm⁻², respectively (Fig. 2b and c), notably higher than 2.5 and 6.9 μg cm⁻² of Cur-Sol, indicating that the nanoemulsions enhance the skin penetration ability of curcumin, which is attributed to the contained surfactants altering the orientation of lipid molecules in the stratum corneum and promoting their mobility, as well as the oil phase IPM integrating the stratum corneum matrix and perturbing the multilamellar lipid arrangement,³⁴ thus improving transdermal delivery of the curcumin. Notably, Cur-CNEs exhibited more preferable skin penetration efficiency in comparison with both Cur-Sol and Cur-NEs, owing to the decoration of the penetration enhancer R8H3. It is speculated that the R8H3 peptide disrupts the ordered arrangement of the lipid matrix in the skin and generates channels for the skin penetration of nanoemulsion.

Furthermore, the hydrophobic fluorescent probe Cou6 was loaded into CNEs to further evaluate its skin penetration ability. The quantitative results of the Franz cell experiments showed that the intradermal and transdermal amounts of Cou6 in the Cou6-CNE treated group were significantly increased compared with the Cou6-NE treated group, reconfirming the superior skin penetration capability of CNEs (Fig. S3†). Confocal microscopic observation revealed that the signal intensity of Cou6 in the skin of the Cou6-CNE group was stronger than that of the Cou6-NE group, especially in the deep layer, suggesting that CNEs can efficiently pass through the SC to reach the deep layers of skin (Fig. 2d and e).

In vitro cell uptake of Cur-CNEs

Following the demonstration of CNEs' efficient skin penetration, their cellular uptake efficiency was further investigated. As shown in Fig. 3a and S5a,† HaCaT and RAW264.7 cells incubated with Cou6-CNEs both presented a stronger intensity of green fluorescence compared to the cells treated with Cou6-NEs. The quantitative data assayed by Image J software indicated that the fluorescence intensity of the Cou6-CNE group was significantly higher than that of the Cou6-NE group (Fig. S4a and S5b†). The results of flow cytometry corroborated the same trend (Fig. S4b†), with the Cou6-CNE group exhibiting the highest fluorescence intensity, confirming that the R8H3 peptide could significantly enhance the cellular uptake of NEs. It has been demonstrated by our group that the R8H3 peptide can enhance transcytosis and it is speculated that the guanidinium groups in the arginine residues strongly bind to anionic cell membranes, contributing to the internalization of nanoemulsions. Moreover, the imidazole group in histidine can facilitate the endosomal escape of nanoemulsions through the proton sponge effect, thereby improving intracellular delivery.²⁰

Fig. 3 (a) Intracellular uptake of Cou6-loaded nanoemulsions by TNF-α-treated HaCaT cells after coculture for 2 h. Scale bar: 200 μm. (b) Growth inhibition of TNF-α-treated HaCaT cells by various curcumin-loaded formulations (Cur-Sol, Cur-NEs, and Cur-CNEs). ***p < 0.001. The mRNA levels of cytokines TNF-α (c), IL-6 (d), and IL-23 (e) in RAW264.7 cells treated by various curcumin-loaded formulations.

In vitro anti-inflammatory effect of nanoemulsions

Psoriasis is characterized by the excessive proliferation and abnormal differentiation of keratinocytes and macrophage infiltration.^35,36 Initially, we investigated the anti-proliferation effects of the Cur-CNEs towards HaCaT cells. As shown in Fig. 3b, the addition of TNF-α successfully induced an abnormal proliferation of HaCaT cells,^37,38 which was inhibited by the curcumin formulations (Cur-Sol, Cur-NEs, and Cur-CNEs) to a different extent. Among them, Cur-CNEs exhibited the strongest inhibition, which is ascribed to the increased uptake of curcumin to HaCaT cells mediated by CNEs.

We further evaluated the anti-inflammatory effects of Cur-CNEs on macrophage RAW264.7 cells. The mRNA levels of cytokines, including TNF-α, IL-6, and IL-23 were detected in RAW264.7 cells treated with different formulations. As shown in Fig. 3c–e, LPS treatment resulted in the significant up-regulation of mRNA levels of various inflammatory factors. The curcumin formulations (Cur-Sol, Cur-NEs, and Cur-CNEs) all alleviated the up-regulated mRNA of different inflammatory factors (Fig. 3c–e). The Cur-CNE group displayed superior anti-inflammatory effects compared to the Cur-Sol and Cur-NE groups. In addition, the cytotoxicity of blank CNEs towards RAW264.7 and HaCaT cells was assessed by MTT assay. As shown in Fig. S6,† the viabilities of HaCaT and RAW264.7 cells incubated with blank CNEs were above 80% in different concentrations, indicating the high cytocompatibility of CNEs.

Preparation and characterization of the Cur-CNEs/Gel

WQ9-2, a protease,^39,40 can catalyze the formation of an amide bond between Fmoc-F and FF to form the gel factor Fmoc-FFF, which then self-assembles into hydrogels.^41,42 In order to prolong the skin retention time of the drug, we formulated the Cur-CNEs/Gel by loading Cur-CNEs using WQ9-2-triggered oligopeptide hydrogels (Fig. 4a). The TEM image displayed uniform nanoparticles were dispersed in the fiber bundles of the hydrogel, indicating that the nanoemulsions were successfully loaded into the hydrogel (Fig. 4b). The SEM image showed that the interwoven fibers in the hydrogel formed a reticulated porous structure, indicating the stability of the hydrogel (Fig. 4c).

Fig. 4 (a) Images of the gelation of the Cur-CNEs/Gel. (b)TEM image of the Cur-CNEs/Gel. Scale bar: 200 nm. (c) SEM image of the Cur-CNEs/Gel. Scale bar: 100 nm. The profile of modulus to time (d) and frequency (e) of the blank hydrogel and Cur-CNEs/Gel complex. (f) The fluorescence images of free Cou6-CNEs or Cou6-CNEs/Gel retained on the back skin of the mice (healthy group and psoriasis group) following topical treatment (I: healthy mice with Cou6-CNEs; II: healthy mice with the Cou6-CNEs/Gel; III: psoriasis mice with Cou-6-CNEs; IV: psoriasis mice with the Cou-6-CNEs/Gel).

The efficiency of enzyme catalysis was calculated by detecting the conversion of the precursor Fmoc-F. As shown in Fig. S7,† after calculation, more than 40% of Fmoc-F had participated in the synthesis of the gelators at 15 min and the accumulative rate of conversion was approximately 62% in 4 h.

The rheological properties of the Cur-CNEs/Gel were measured with an Antonpa rotational rheometer. Both of the storage modulus (G′) and loss modulus (G′′) increased over time after the addition of WQ9-2 (Fig. 4d). The storage modulus value G′ was over 1000 Pa, and outweighed G′′, indicating the formation of a hydrogel with robust mechanical properties (Fig. 4e). The short peptide hydrogel is formed by the assembly of gelators through non-covalent bonds, so it is spreadable and paintable, and can be applied on the skin. The Cur-CNEs/Gel could recover its gel morphology after extrusion or injection onto the slide and adhere well to the surface of the painted object (Fig. S8a†). Meanwhile, we performed a rheological analysis of the extruded Cur-CNEs/Gel. As shown in Fig. S8b,† the storage modulus and loss modulus recovered within 300 s, indicating that the hydrogel formed rapidly after extrusion, indicating the paintability of the Cur-CNEs/Gel.

Skin retention of the Cou6-CNEs/Gel

We further monitored the retention of the Cou6-CNEs/Gel on the dorsal skin of psoriasis mice using an in vivo imaging system (Fig. 4f). Imiquimod cream was applied on the back skin of ICR mice continuously for 7 days to induce stable psoriasis-like symptoms, including erythema, desquamation, and skin thickening. The Cou6-CNEs/Gel and Cou6-CNEs solution were topically applied to the back skin of healthy and psoriatic mice. At all the time points, the Cou6-CNEs/Gel groups exhibited a stronger fluorescence signal intensity than the Cou6-CNE solution groups, suggesting that the hydrogel as a carrier facilitated the skin retention of the nanoemulsions.

Notably, the retention time of the Cou6-CNEs/Gel on psoriatic mice was longer than on healthy mice. Even at a longer time of 24 h, a strong fluorescence signal remained visible in the dorsal skin of the psoriatic mice, which may be due to the rough surface of psoriatic mice being beneficial to the retention of the Cou6-CNEs/Gel.

In vivo anti-psoriatic inflammation effects of curcumin in mice

The in vivo anti-inflammation activity of the Cur-CNEs/Gel was evaluated on the psoriatic mice. After the successful establishment of the IMQ-induced mouse model, various treatments were administered to the skin once a day for 10 days (Fig. 5a). The images of the backs of the mice after treatment are shown in Fig. 5b. The severity of the lesions was assessed according to the psoriasis area and severity index (PASI), including erythema and desquamation scores. As shown in Fig. 5c and d, it can be seen that the PASI scores of all curcumin-treated groups gradually decreased during the treatment period. At the end of the experiment, the erythema and desquamation scores of the mice in all groups were significantly lower than those of the model group. Compared to the Cur-Sol group, the Cur-NEs/Gel group showed reduced skin inflammation and damage, due to the semi-solid hydrogel extending the retention time of curcumin on the skin and the good transdermal ability of the nanoemulsion. In addition, the nanoemulsion preparation avoids the introduction of DMSO to dissolve free curcumin, which may be accompanied by skin irritation, itching, tingling, and other side effects. Among all groups, the Cur-CNEs/Gel group exhibited the best therapeutic effects, which was mainly attributed to the improved penetration achieved by the decoration of CPP on the surface of nanoemulsions. During the treatment period, there was no dramatic change in the body weight of mice (Fig. 5e). Furthermore, no significant pathological damage was observed in the major organs of mice in all groups, indicating the safety of the formulations (Fig. S9†). The potential allergy risk of curcumin has always been a consideration for us. As shown in Fig. S10,† no allergic reactions such as redness, swelling, rash, and increased secretion were observed on the skin of mice after 7 days of treatment, both under normal light and UVB light conditions, which to some extent verified the dermal safety of our Cur-CNEs.

Fig. 5 (a) Protocol scheme of the mice psoriatic model and treatment. (b) Representative images of mouse back after treatments with different formulations for 7 days. PASI scores of mice skin treated with different dosage forms: (c) erythema and (d) desquamation (n = 8). **p < 0.01 ***p < 0.001. (e) The body weight changes in different groups.

Histopathological analysis of the skin tissues was further performed. Previous studies have reported that the main feature of psoriasis is epidermal hyperplasia, which is the result of an abnormal proliferation of keratinocytes and a marked infiltration of inflammatory immune cells.¹ H&E staining revealed significant symptoms of psoriasis in the IMQ-treated skin (Fig. 6a), with scaly breaks, abnormal skin thickening, and a massive neutrophilic infiltrate. Meanwhile, skin proliferation was significantly inhibited and the original skin structure was restored in the Cur-CNEs/Gel group. In addition, the expression of TNF-α and IL-17 was examined by the immunohistochemistry (IHC) experiment (Fig. 6b), which showed that IMQ increased the expression of the above indicators, suggesting that IMQ treatment resulted in infiltration of pro-inflammatory immune cells in skin tissues. The Cur-NEs/Gel and Cur-CNEs/Gel, in contrast, showed significant inhibitory effects on the expressions of TNF-α and IL-17 and reduced inflammatory response. The results suggest that the hydrogels of curcumin-loaded nanoemulsions have favorable anti-inflammatory capacity as well as therapeutic efficacy.

Fig. 6 (a) H&E stained back-skin tissues from the normal and the psoriasis mice receiving various treatments. Scale bars: 200 μm. (b) Immunohistochemical stained back skin sections of normal mice and IMQ-induced psoriasis mice receiving various treatments for TNF-α and IL-17 staining. Scale bars: 200 μm. The mRNA levels of cytokines TNF-α (c), IL-6 (d), and IL-23 (e) from back-skin tissues in different groups.

Subsequently, we assessed the mRNA levels of pro-inflammatory cytokines, TNF-α, IL-6, and IL-23 in the skin tissue by the real-time fluorescence PCR experiment. As shown in Fig. 6c–e, compared to the normal group, IMQ-induced psoriasis mice displayed a significant up-regulation of the mRNA levels. This up-regulation in the mRNA levels of pro-inflammatory cytokines was inhibited to a different extent in the group of various curcumin formulations. Among them, Cur-CNEs/Gel treatment showed the strongest inhibition.

In summary, despite the controversy surrounding curcumin due to its confounding bioactivity and poor pharmacokinetics, curcumin delivered locally still holds promise for a wide range of therapeutic applications. It is on this basis that we justified the use of curcumin as a model bioactive that has genuine potential as being repurposed in the context of a topically-applied anti-inflammatory compound, and where we provided data validating its biosafety after application.

Conclusions

In summary, we developed a Cur-NEs/Gel as a paintable patch to enhance skin penetration for the topical treatment of psoriasis. As the drug carrier, this protease-triggered self-assembling hydrogel, which embeds nanoemulsions provides a new strategy for the treatment of psoriasis. The Cur-CNEs/Gel has been experimentally confirmed to prolong the retention of drugs on the skin surface. This nanoparticle/Gel complex can effectively penetrate the stratum corneum barrier and reach the deeper layers of the skin, owing to the properties of the nanoemulsions and the introduction of the cell-penetrating peptide. We have demonstrated that the topical delivery of the Cur-CNEs/Gel effectively alleviated the development of psoriatic skin inflammation and aids in restoring the skin's original structure. We believe that this enhanced retention and penetration strategy provides a valuable reference for the treatment of psoriasis and potentially other skin diseases.

Data availability

The data supporting this article have been included as part of the ESI.†

Author contributions

Kehan Chen: writing – original draft, methodology, visualization, investigation, and data curation. Hui Yang: writing – review & editing, investigation, and validation. Guo Xu: writing – original draft, visualization, and investigation. Yunhan Hu: investigation and validation. Xue Tian: investigation and validation. Song Qin: resources, supervision, and visualization. Tianyue Jiang: writing – review & editing, resources, project administration, and conceptualization.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (82072045).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4md00781f

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