Cu–Fe bimetallic nanozyme@halloysite–sodium alginate composite hydrogels for the treatment of atopic dermatitis

Abstract

Atopic dermatitis is a typical chronic inflammatory disease with pathological characteristics of persistent immune activation and oxidative stress. Combined anti-inflammatory and antioxidant treatment can effectively block the inflammatory cascade while reducing oxidative damage. Halloysite nanotubes (HNTs) are the main components of the traditional Chinese medicine “Chishizhi”, which shows the medicinal functions of hemostasis and astringency. However, the efficacy of HNTs alone in treating diseases is relatively weak, and their therapeutic effect can be improved by surface modification and drug loading. Herein, CuO–Fe₃O₄ nanoparticles were synthesized on the outer surfaces of HNTs by a hydrothermal reaction. CuO–Fe₃O₄@HNTs have high SOD and CAT enzyme activities under neutral conditions. Then, the nanozyme-modified HNT powder was prepared into sprayable hydrogels by introduction of sodium alginate (SA) and aloe vera extracts. Cell experiments confirmed that the hydrogel can promote HacaT cell proliferation within 0–200 μg mL⁻¹ concentration. Through the mouse dermatitis model, it was seen that a CuO–Fe₃O₄@HNTs–SA composite hydrogel has a good therapeutic effect on atopic dermatitis. Compared with the positive drug halcinonide solution, the CuO–Fe₃O₄ nanozyme-incorporated hydrogel showed an enhanced therapeutic effect, which shows promising prospects for the clinical treatment of atopic dermatitis.

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1. Introduction

As the largest organ in the human body, the skin protects the body from external damage. Atopic dermatitis is an inflammatory skin disease with an early onset and a lifetime prevalence of approximately 20%. As a heterogeneous skin disease, its pathogenesis is complex and little is known at present.¹ In addition to genetic susceptibility, the development and maintenance of atopic dermatitis are believed to be related to environmental and psychological triggers and skin barrier defects.² Atopic dermatitis is generally an acute or chronic dermatitis with no characteristic features, which mainly leads to epidermal barrier dysfunction, skin and systemic immune disorders, and skin microbiome disorders, usually manifested as local skin itching. Atopic dermatitis cannot be cured and needs to go through a chronic course. Prevention can reduce the number of exacerbations of the disease.³ The best way to prevent it is to minimize skin dryness and to apply oily products such as moisturizers to the skin every day to act as a closure layer to maintain epidermal moisture and reduce irritation to the epidermis. Due to the damage to the skin barrier caused by atopic dermatitis, several studies have suggested that the use of mild emollients without potential allergens is effective for the treatment of atopic dermatitis.⁴

Increased oxidative stress is associated with the development of atopic dermatitis. Therefore, inhibiting reactive oxygen species (ROS)-induced oxidative stress in atopic dermatitis lesions may be one of the feasible strategies for treating atopic dermatitis.⁵ Oxidative stress refers to the formation of oxidants in human cells, which leads to oxidative stress when acute or chronic levels exceed the antioxidant defense capacity.⁶ ROS include H₂O₂, superoxide anions, and hydroxyl radicals, which are by-products of cellular metabolism. Oxidative stress can cause fatal oxidative damage to the DNA and proteins and lipid peroxidation of membranes. For example, studies have found that the level of antioxidants in the sera of atopic dermatitis patients is low but the level of lipid peroxides is very high. Therefore, it is possible to treat atopic dermatitis by regulating the local oxidative stress of patients with atopic dermatitis.⁷

Halloysite nanotubes (HNTs) are natural, bio-compatible, inexpensive, and high-performance nanomaterials.⁸ HNTs have a large aspect ratio and a tubular structure with silicon oxide tetrahedra outside and an aluminum oxide octahedron inside the tubes.^9–11 The length of HNTs ranges from 0.2 to 2.0 μm, the outer diameter is 50–100 nm, and the inner diameter is 10–30 nm. The size of HNTs varies slightly depending on the deposition site.¹² The outer surface of HNTs is rich in silicon–oxygen bonds, which makes its surface rich in negative charges.¹³ Its zeta potential in water is about −40 mV (pH = 7.4).¹⁴ Its interior is positively charged due to the presence of an alumina octahedron. This property makes HNTs a drug carrier for drug adsorption and controlled release, and therefore HNTs have broad application prospects in the biomedical field.¹⁵

Nanozymes can treat inflammatory diseases by removing ROS, reducing oxidative stress levels, and downregulating pro-inflammatory factors. Among them, Fe₃O₄, as a representative of peroxidase, has an obvious effect of removing H₂O₂ and ˙OH.¹⁶ However, unlike single-metal nanozymes, bimetallic nanozymes have unique multifunctional properties due to the synergistic effect between the two metal components.^17–19 Mao et al.²⁰ explored the relationship between the activity of Au–Cu bimetallic nanoparticles and their composition, and showed that the uniform Au₃Cu bimetallic nanozymes had the highest catalytic activity, while pure Cu nanoparticles had the lowest catalytic activity. In 2022, Li et al.²¹ prepared ZnMn₂O₄–Mt using the properties of montmorillonite natural clay minerals to inhibit the agglomeration of ZnMn₂O₄ and increase the active sites of ZnMn₂O₄ nanoparticles, which led to a significant enhancement of its oxidase-like activity. In summary, compared with single-metal nanozymes, the design of bimetallic nanozymes greatly optimizes their catalytic performance. However, for the complex atopic dermatitis microenvironment involving excessive oxidative stress, materials are required with good anti-inflammatory and antioxidant capabilities.

In recent years, researchers have combined nanozymes with HNTs, which not only allows them to utilize the drug carrier properties of HNTs, but also enhances the therapeutic effect through the catalytic activity of nanozymes.²² Hydrogels can maintain the moisture of skin or wounds due to their excellent water retention properties. Huang et al.²³ demonstrated that nanoparticles composed of chitosan and alginate can enhance the transdermal delivery of tacrolimus, thereby effectively improving the symptoms of atopic dermatitis. This sodium alginate (SA)-based hydrogel not only improves the skin permeability of the drug, but also reduces the systemic absorption of the drug, thereby reducing potential side effects. These studies provide new ideas and methods for the topical treatment of atopic dermatitis. Alginate, a naturally sourced biopolymer, has been widely studied in the biomedical area due to its abundant resources, simple preparation process, excellent biocompatibility and low immunogenicity.²⁴

In this study, the CuO–Fe₃O₄ bimetallic nanozyme was anchored on naturally occurring HNTs via hydrothermal synthesis. Then, an injectable and adhesive SA composite hydrogel – was prepared by incorporation of CuO–Fe₃O₄@HNTs. HNTs act as carriers for the CuO–Fe₃O₄ nanozyme, which can decrease the size of the nanozyme and increase the activity of the catalyst. HNTs are selected for their superior inherent procoagulant and wound-healing properties, demonstrating a superior performance in superficial skin wound and inflammation management. The blood clotting properties and biocompatibility of the composite hydrogel are assessed in detail. The therapeutic effect of the CuO–Fe₃O₄@HNT composite hydrogel with good anti-inflammatory and antioxidant capabilities towards chemically induced atopic dermatitis was investigated, and the mechanism of the materials in the treatment of atopic dermatitis was discussed.

2. Results and discussion

2.1. Preparation and characterization of CuO–Fe₃O₄@HNTs

As shown in Fig. 1a, HNTs were dispersed in ethylene glycol, ultrasonicated and stirred vigorously, and then CuSO₄·5H₂O and FeCl₃·6H₂O were added. After they were completely mixed, the mixture became a dark green color. NaAc was then added and stirred, and the solution turned brown. After adding polyvinyl pyrrolidone (PVP) as a stabilizer, the mixture was further stirred for 12 h and transferred to a polytetrafluoroethylene reactor. After reacting at 200 °C for 10 h, the product was cooled naturally and washed with deionized water and anhydrous ethanol several times, and CuO_x–Fe₃O₄@HNTs were obtained by freeze-drying.

Fig. 1 Basic characterization of the CuO–Fe₃O₄@HNT nanozymes. (a) Preparation process of CuO–Fe₃O₄@HNTs. (b) TEM images of HNTs, CuO–Fe₃O₄, and CuO–Fe₃O₄@HNTs. (c) Energy spectrum scan of the CuO–Fe₃O₄@HNTs. (d) XRD patterns. (e) TGA curves. (f) Zeta potential.

In the classic Fenton reaction, H₂O₂ can react with Fe under acidic conditions to generate highly oxidizing ˙OH.²⁵ Bimetallic catalysts can overcome the limitations of linear scaling relationships observed in single-metal catalysts. The combination of two different transition metals provides an opportunity to reduce the number of metal sites and plays a key role in promoting the reduction of Fe(III) to Fe(II).²⁶ In addition, the redox cycle between the two different metals significantly enhances the interfacial electron transfer, thereby increasing the generation of ˙OH. Cu is the most abundant and inexpensive transition metal. Cu-containing catalysts have attracted widespread attention, and Cu(II)/Cu(I) and Fe(III)/Fe(II) have similar redox properties. Cu doping can achieve fast electron transfer and redox cycling. Also, the introduction of oxygen vacancies can significantly promote the generation of H₂O and ˙OH by reacting Cu ions with H₂O₂.

The large specific surface area of HNTs and the stable cluster structure of the nanoenzyme can provide abundant absorption sites. These characteristics significantly enhance the Fenton-like activity of CuO_x–Fe₃O₄@HNTs. PVP, the most commonly used stabilizer, was used for assembling transition metal nanomaterials in this study.²⁷ PVP prevents metal nanomaterial aggregation through repulsive forces generated by its hydrophobic carbon chains. These repulsive forces extend into the solvent and interact with each other (steric hindrance). The pyrrolidone functional groups of PVP readily block Fe₃O₄, promoting the formation of small nanoparticles and hindering their aggregation.²⁸ The amide carbonyl (C=O) and pyrrolidone nitrogen atoms on the PVP molecular chain carry lone pairs of electrons, which can adsorb onto the CuO_x surface through hydrogen bonding and electrostatic interactions.²⁷ Subsequently, Cu and Fe were connected by PVP-mediated coordination. Transmission electron microscopy (TEM) results (Fig. 1b) show that CuO_x–Fe₃O₄ is evenly distributed outside the HNTs and the diameter is about 10 nm, which is smaller than that of pure CuO_x–Fe₃O₄ (30 nm). This is because HNTs as nanoconfined substrates effectively disperse and stabilize the nucleating nanoparticles, thereby suppressing their agglomeration and controlling their growth.²⁹ Analysis of the element distribution via the electron energy spectrum (Fig. 1c) shows that Fe and Cu elements are evenly distributed on the surface of the HNTs, and X-ray diffraction (XRD) analysis results show a mixed phase of CuO_x and Fe₃O₄ (Fig. 1d). In order to further determine the loading rate of CuO_x–Fe₃O₄ on the HNTs, a TGA test (Fig. 1e) was conducted. The CuO_x–Fe₃O₄ sample exhibited a slight mass increase. This was due to Fe₃O₄ undergoing oxidation to Fe₂O₃ upon heating under an oxygen-containing gas atmosphere.³⁰ Ong et al. found that Fe₃O₄ was stable in pure N₂ and only underwent oxidation and weight gain in an oxygen-containing environment.³¹ From the TGA results, it was calculated that the loading rate of CuO_x–Fe₃O₄ on the HNTs was approximately 47.6%. Like pure HNTs, the CuO_x–Fe₃O₄@HNTs still maintained a negative potential of −7.8 mV (Fig. 1f).

Fig. 2 shows the full spectrum and fine scanning of X-ray photoelectron spectra (XPS) of the CuO_x–Fe₃O₄ and CuO_x–Fe₃O₄@HNTs. The fine spectra of Cu 2p and Fe 2p further show the changes in the elemental valence state of CuO_x–Fe₃O₄. In the Cu 2p fine spectrum, the peaks at 953.83 eV and 934.13 eV can be attributed to Cu 2p1/2 and Cu 2p3/2, which are the ionic characteristics of Cu²⁺.¹⁶ The existence of CuO in the two composite materials can be confirmed by three satellite peaks with the binding energies 962.2 eV, 943.6 eV, and 941.1 eV,³² further verifying the formation of CuO. In the Fe 2p fine spectrum, the peaks at 723.8 eV and 725.4 eV correspond to the satellite peaks of Fe²⁺ 2p1/2 and Fe³⁺ 2p1/2, respectively. The Cu and Fe species in the CuO_x–Fe₃O₄@HNTs nanozyme exist in two states, i.e., CuO and Fe²⁺/Fe³⁺. Therefore, the XPS results indicate that the CuO–Fe₃O₄@HNTs composite nanozyme was successfully constructed.

Fig. 2 XPS full spectrum and Cu 2p and Fe 2p fine spectra of the samples. (a) CuO_x–Fe₃O₄@HNTs. (b) CuO_x–Fe₃O₄.

2.2. Characterization of the enzyme activity of CuO–Fe₃O₄@HNTs

The enzyme-like activity of the CuO–Fe₃O₄@HNTs composite (CF@H) was then studied and compared with those of CuO–Fe₃O₄ and HNTs. In 2007, an Fe₃O₄ nanoparticle was found to have peroxidase-like activity, which could be used to perform intrinsic enzyme-like activities.³³ Peroxidases belong to a class of reductases that can catalyze many reactions. They are enzymes that catalyze substrate oxidation using H₂O₂ as an electron acceptor. Peroxidase activity can usually be detected using the Fenton reaction. In addition, a superoxide anion (˙O₂⁻) is a highly oxidizing and destructive ROS, and superoxide dismutase (SOD) is a key ˙O₂⁻ scavenger that catalyzes the dismutation of ˙O₂⁻ into H₂O₂ and O₂. Although SOD can protect cells from ˙O₂⁻ damage, the H₂O₂ produced by SOD can also damage DNA.³⁴ The removal of H₂O₂ from cells depends largely on chloramphenicol acetyltransferase (CAT) enzymes.³⁵

Fig. 3a shows the scavenging effect of the CF@H nanozyme towards a superoxide anion (˙O₂⁻) detected using a SOD detection kit. It was found that the scavenging rate for ˙O₂⁻ can reach 80% at 100 µg mL⁻¹. In order to further investigate its ability to eliminate ROS, EPR was used to detect its ability to scavenge ˙OH (Fig. 3b). The EPR of ˙OH presents an isometric quartet with a peak ratio of 1 : 2 : 2 : 1. This work also found that at the same concentration, the scavenging effect of CF@H is better than that of CuO–Fe₃O₄. Therefore, CF@H has a good scavenging effect on ROS (˙OH and ˙O₂⁻). The enzyme-like activity of CF@H was qualitatively measured using a CAT detection kit. The results showed that CF@H has an activity equivalent to that of a natural CAT enzyme and can effectively scavenge H₂O₂. TEMPO is a nitric oxide free radical that acts as a selective scavenger of ROS in mitochondria.³⁶ As shown in Fig. 3c, the EPR electron spin resonance spectrometer can detect the type and intensity of free radicals generated. CF@H has a certain scavenging effect on NO free radicals, indicating that CF@H has a certain scavenging effect on both ROS and reactive nitrogen species (RNS).

Fig. 3 The ROS scavenging effect of the CuO–Fe₃O₄@HNTs nanozymes and the enzyme-like activity. (a) Scavenging rate of ˙O₂⁻. (b) Scavenging effect on ˙OH by EPR detection. (c) TEMPO (NO free radical) scavenging. (d) GPx-like enzyme activity. (e and h) DPPH detection of antioxidant capacity. (g) Degradation of methylene blue. (f and i) ABTS total antioxidant capacity detection.

Glutathione peroxidase (GPx), as a type of peroxidase, is one of the most important enzymes in the human redox system and the main enzyme for maintaining the content of reduced glutathione in cells.³⁷ The activity of GPx of CF@H was evaluated using glutathione. Fig. 3d shows that the concentration of CF@H is positively correlated with the GPx activity. In addition to achieving anti-inflammatory effects by scavenging ROS, the antioxidant effect of nanozymes is also important. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) can be used to measure the free radical scavenging activity of antioxidants.³⁸Fig. 3e and h show an evaluation of the antioxidant capacity of the CF@H nanozyme through the UV spectra and DPPH free radical scavenging rate (the discontinuity around 500 nm is an instrument artifact due to a grating/detector switch). It can be seen that CF@H has a certain scavenging ability for DPPH at 30 min, and the scavenging of DPPH reaches the highest level at 60 min. Combined with Fig. 3h, it can be seen that CF@H can scavenge around 20% of free radicals within 30 min. Fig. 3f and i also show the testing of the total antioxidant performance of the CF@H nanozyme on the basis of ABTS. Since the maximum absorption peak of the cation radical of ABTS is at 415 nm, its absorption peak change is usually measured at 420 nm. Compared with HNTs and PBS, CF@H and CuO–Fe₃O₄ have no obvious absorption peak at 420–415 nm. So, CF@H has good antioxidant capacity as a composite nanozyme. These results suggest that the CF@H composite nanozyme has an SOD enzyme, a GPx enzyme, a CAT enzyme and good antioxidant capacity, which shows good potential in terms of anti-inflammatory and antioxidant capabilities.

2.3. Characterization of CuO–Fe₃O₄@HNTs–SA hydrogel properties

Fig. 4a shows the appearance of the SA gel, HNTs–SA gel and CuO–Fe₃O₄@HNTs-SA (CF@H–SA) gel. It can be seen that the fluidity of SA is slightly affected by the incorporation of HNTs and CF@H. CF@H–SA could be used by spraying onto the wound tissue, as it has a high coverage and ability to adhere to the skin. As shown in Video S1, it can be clearly seen that the CF@H–SA pre-gel solution is sprayed from the spray bottle, and compared with a saline solution, the CF@H–SA hydrogel is able to form a stable adherent hydrogel in the culture dish. Fig. 4b and c shows the rheological properties of CF@H–SA. The storage modulus of the gel at different time periods is greater than the loss modulus, and the storage modulus gradually becomes smaller than the loss modulus as the frequency increases. This indicates that the hydrogel changes from solid to liquid with an increase in the frequency. The CF@H–SA gel can be expressed as a viscous liquid, which has good application prospects as a skin dressing. Atopic dermatitis is a chronic disease. After long-term damage to the skin barrier, the skin becomes fragile and prone to ulceration.³⁹ As a hydrogel dressing, it should have anti-inflammatory and antioxidant effects as well as certain hemostatic properties and blood compatibility. The coagulation time and blood coagulation index (BCI) of CF@H and the hydrogel were also evaluated (Fig. 4d and e). CF@H has a short coagulation time of ∼230 s and the CF@H–SA gel has a lowBCI at different time periods. This suggests that the CF@H–SA sprayable gel can be used as a wound dressing with good adaptability to wounds.

Fig. 4 Characterization of CF@H–SA sprayable gel physical properties. (a) Gel appearance. (b and c) Rheological properties. (d) Coagulation time. (e) Blood coagulation index.

2.4. In vitro cytocompatibility evaluation of CF@H–SA

In order to systematically evaluate the biosafety of CF@H–SA and its protective effect on keratinocytes, human immortalized keratinocytes (HacaT) were used as a model. The cell compatibility of SA, HNTs–SA and CF@H–SA was compared through gradient concentration experiments. The MTT experiment results showed (Fig. 5c) that the cell survival rate of the CF@H–SA treatment group was maintained at more than 80% (Fig. 5b). No significant cytotoxicity was observed, confirming that the CF@H–SA material has good biosafety. When HacaT cells were stimulated by DNCB (5,5-dithiodi(2-nitrobenzoic acid), significant oxidative stress damage was induced (Fig. 5a), simulating the cell damage pattern under the pathological state of atopic dermatitis. CF@H–SA pretreatment can significantly alleviate DNCB-induced cell damage, indicating that it can not only maintain keratinocyte viability but also protect skin barrier cells from oxidative damage through antioxidant mechanisms. The in vitro experimental results suggest the potential of CF@H–SA in the treatment of atopic dermatitis.

Fig. 5 In vitro cell safety evaluation of the CF@H–SA sprayable gel. (a) Scavenging ROS mechanism diagram. (b) Cell live–dead staining images. (c) Cell viability at different time.

2.5. Therapeutic effect of the CF@H–SA hydrogel on DNCB-induced atopic dermatitis mice

In order to confirm the therapeutic effect of the CF@H–SA hydrogel on atopic dermatitis, DNCB was used to establish a chemically induced atopic dermatitis mouse model (Fig. 6a). Different gels reduced the severity index scores of drug-induced dry skin, redness, swelling, and epidermal thickening in mice (Fig. 6f). Among them, the positive drug halcinonide solution (HS) had the best effect. Compared with the positive drug, CF@H–SA had a better therapeutic effect on the dry skin of the mice, but its relief in terms of epidermal thickening was weaker. The images of mouse ear inflammation in Fig. 6b and d show that the redness and swelling of the ears of the halcinonide solution group mice are significantly improved compared with the CF@H–SA  group, and the ear thickness of the mice in the CF@H–SA and halcinonide solution treatment groups is significantly decreased. Spleen enlargement may reflect disorder of the lymphocyte immune response in the body.⁴⁰ The spleens of mice in each group were weighed (Fig. S1 and Fig. 6c). The spleens of mice in the halcinonide solution group and CF@H–SA group were significantly smaller than those of the mice with atopic dermatitis and untreated mice, indicating that halcinonide solution and CF@H–SA have certain anti-inflammatory therapeutic effects on atopic dermatitis. The therapeutic effect of CF@H–SA on atopic dermatitis in mice was also evaluated through the weights and the disease activity index scores of the mice (Fig. 6e and f). It can be seen that atopic dermatitis did not have a significant effect on the weights of the mice from the different groups. The disease activity index score results show that the CF@H–SA and halcinonide solutions had a good therapeutic effect.

Fig. 6 Therapeutic effect of the CF@H–SA sprayable gel on DNCB-induced atopic dermatitis mice. (a) Flow chart of mouse atopic dermatitis modeling and treatment. (b) Representative images of the lesions on the left ear of mice stimulated by DNCB on day 7, n = 5 samples per group. (c) Statistics of the spleen weight of the sampled mice, n = 5 samples per group. (d) Statistics of abnormal thickening of the left ear of mice on day 7 of the treatment. (e) Mouse weight. (f) Disease activity index scores of the mice.

2.6. CF@H–SA hydrogel reduces skin thickness and mast cell production in atopic dermatitis mice

In order to further illustrate the use of CF@H–SA hydrogel as skin dressings for the treatment of atopic dermatitis, the therapeutic mechanism of CF@H–SA for atopic dermatitis was analyzed through gene expression, protein expression and histopathological analysis. First, Masson staining of the skin of each group showed the skin thickness of mice in the different groups (Fig. 7a and b). Except for the treatment group CF@H–SA, halcinonide solution and healthy group mice, the other groups showed obvious epidermal hypertrophy, enlarged glands, inflammatory cell infiltration in the dermis, and obvious dermal–epidermal separation. The anti-inflammatory effect of CF@H–SA was verified by Masson staining, dyeing the mast cells in the mouse skin tissue dark red, combined with H&E staining (Fig. 7c).

Fig. 7 Skin sections of the dermatitis mouse model. (a) Masson staining – whole scan. (b) Mouse skin thickness statistics, n = 5 samples per group. (c) Mast cell staining.

The histological fluorescence staining images (Fig. S2) show that CF@H–SA can effectively penetrate the stratum corneum barrier and is significantly enriched in the dermis, which enables it to target the core area of atopic dermatitis lesions. The uniform distribution of fluorescence signals in the dermis indicates that CF@H–SA has good tissue permeability, and can directly reach the subcutaneous inflammatory site for treatment. Based on this finding, this study considers that CF@H–SA may alleviate atopic dermatitis symptoms through the following mechanisms: (1) directly regulating the immune microenvironment of the dermis and inhibiting inflammatory cell infiltration; (2) significantly reducing the local recruitment and activation of mast cells, thereby reducing the release of inflammatory mediators such as histamine; and (3) neutralizing local ROS through its nanozyme properties and blocking the positive feedback loop of oxidative stress inflammation. To verify this hypothesis, real-time fluorescence quantitative PCR technology was used to quantitatively analyze the expression levels of key inflammatory factors (such as IL-4, IL-13, etc.) and antioxidant genes (SOD2, CAT and GPx).

2.7. The expression of inflammatory factors and the antioxidant capacity of the skin

Atopic dermatitis is a complex chronic inflammatory skin disease, and its pathogenesis involves the expression and interaction of multiple interleukins. Studies have shown that cytokines represented by IL-4, IL-13 and thymic stromal lymphopoietin (TSLP) play a core regulatory role in the pathogenesis of atopic dermatitis, promoting the development of the disease through multiple pathways such as affecting the Th2 immune response, destroying the skin barrier function and mediating the chronic inflammatory response.⁴¹ In order to evaluate the anti-inflammatory and antioxidant effects of CF@H–SA at the nucleic acid level, this work used qPCR to detect key inflammation-related factors and analyze their gene expression differences. Among them, IL-4 and IL-13 are anti-inflammatory cytokines in immune response. IL-13 seems to play a role in skin fibrosis remodeling.⁴² It is mainly produced by mast cells, Th2 cells and other cells, and plays an important role in regulating immune balance and inhibiting excessive inflammatory response. The results in Fig. 8a and b show that combined with the difference analysis, the levels of IL-4 and IL-13 anti-inflammatory cytokines are significantly different from those in the model group, the control group, the CF@H–SA group and the positive drug group (*P < 0.05), indicating that CF@H–SA can treat atopic dermatitis by activating immune cells to release anti-inflammatory cytokines.

Fig. 8 The antioxidative stress effects of the CF@H–SA sprayable gel. (a–c) RT-qPCR results of inflammatory factors IL-4, IL-13, and TSLP. (d–f) RT-qPCR results of the antioxidant factors GPX4, CAT, and SOD2. All statistical data are expressed as mean ± SD (n = 5). *P < 0.05, **P < 0.01 and ***P < 0.001.

TSLP is a cytokine related to the pathogenesis of atopic dermatitis, asthma and other eosinophil-related diseases.⁴³ TSLP is one of the initiators of the inflammatory cascade reaction, so inhibiting TSLP can intervene in the early stages of inflammation and prevent immune cells from releasing pro-inflammatory factors.⁴⁴ In addition, TSLP can destroy the skin barrier by downregulating the expression of filaggrin, forming a vicious cycle of “barrier defect–inflammation”. Therefore, inhibiting the expression of TSLP may become an effective strategy for early intervention in atopic dermatitis. As shown in Fig. 8c, the mRNA expression of TSLP in atopic dermatitis mouse cells was significantly reduced under treatment with CF@H–SA (*P < 0.05), which further illustrates that CF@H–SA has an inhibitory effect on the release of pro-inflammatory factors by immune cells.

The above-mentioned qPCR analysis results of anti-inflammatory and pro-inflammatory cytokines related to atopic dermatitis show that CF@H–SA can maintain immune balance, reduce inflammatory response and improve skin barrier by upregulating anti-inflammatory factors and downregulating pro-inflammatory factors. This finding provides a theoretical basis at the molecular level for the application of CF@H–SA in the treatment of atopic dermatitis.

Cells regulate the redox state in cells by maintaining a balance between the generation and elimination of ROS (Fig. 8d–f). CF@H–SA can promote the restoration of redox balance in fibroblasts by increasing the expression of antioxidant genes (SOD2, GPX4 and CAT), thereby achieving an antioxidant purpose and treating oxidative stress diseases.⁴⁵ The results show that the expression levels of the antioxidant factors GPX4, CAT, and SOD2 in the CF@H–SA group were significantly upregulated, indicating that in addition to activating immune cells to secrete anti-inflammatory factors to treat atopic dermatitis, CF@H–SA can also upregulate antioxidant genes to clear ROS and maintain redox balance, achieving an effective dual therapeutic effect on atopic dermatitis. Based on these results, CF@H–SA can regulate immune response and enhance antioxidant capacity at the genetic level, and has good application prospects for the treatment of atopic dermatitis.

2.8. CF@H–SA hydrogel reduces the expression of inflammatory proteins

Although qPCR has high sensitivity, it cannot locate the location of proteins. Immunohistochemistry can further screen protein expression and provide relevant expression information at the protein level. Immunohistochemistry is used to visualize the expression of pro-inflammatory and anti-inflammatory proteins associated with atopic dermatitis in a morphological context to provide more comprehensive evidence for the therapeutic effect of CF@H–SA and further verify the therapeutic effect of CF@H–SA on atopic dermatitis.

IL-33 is an “alarmin” that is mainly released by epithelial cells, endothelial cells and fibroblasts when tissues are damaged or stressed, and participates in the occurrence and development of atopic dermatitis through multiple pathways.⁴⁶ When the skin barrier function is damaged, keratinocytes will release IL-33. Studies have shown that when the skin barrier function is damaged, keratinocytes will significantly upregulate the expression of IL-33. Clinical data further confirm that the expression level of IL-33 in the skin lesions of atopic dermatitis patients is significantly higher than that in healthy people.⁴⁷ According to the immunohistochemical staining and quantitative statistical analyses shown in Fig. 9a and c, the IL-33 level after CF@H–SA treatment was significantly decreased, which was significantly different from the model group (*P < 0.05), indicating that the IL-33 level was significantly downregulated compared with atopic dermatitis mice.

Fig. 9 The expression of inflammatory proteins in the mice treated by the CF@H–SA sprayable gel. (a and b) Immunohistochemical staining of IL-33 and IgE. (c and d) Statistics of IL-33 and IgE protein expression. All statistical data are expressed as mean ± SD (n = 3). *P < 0.05, **P < 0.01 and ***P < 0.001.

The development of atopic dermatitis is partly due to excessive autoimmunity. IgE (the immunoglobulin with the lowest content but the highest activity) is a marker for diagnosing atopic dermatitis. About 80% of atopic dermatitis patients have elevated total IgE levels in their serum. It participates in the pathogenesis of atopic dermatitis through multiple levels of action such as sensitization.⁴⁸ So by inhibiting the production of IgE, the development of atopic dermatitis can be effectively inhibited. Fig. 9b and d show a quantitative analysis of the expression of IgE in mouse skin sections by immunohistochemical staining. The statistical results show that the level of IgE decreased significantly under treatment with CF@H–SA, and there was no significant difference from the control group. To further clarify the mechanism of action of CF@H–SA, this work analyzed the anti-oxidative stress effect of CF@H–SA nanozymes by protein expression.

To systematically evaluate the therapeutic effect of the CF@H–SA gel on atopic dermatitis, immunofluorescence was employed to quantitatively analyze the related oxidative stress and keratin expression characteristics in mice. Oxidative stress plays a key role in the pathogenesis of atopic dermatitis; 8-OHdG is a specific biomarker of DNA oxidative damage, which is produced by ROS attacking guanine bases.⁴⁹ In atopic dermatitis, the level of 8-OHdG can reflect the degree of oxidative stress, inflammation-related DNA damage, etc. The immunofluorescence quantitative results in Fig. 10a and b show that compared with the model group, the expression level of 8-OHdG in the skin of mice in the CF@H–SA gel treatment group was significantly reduced (*P < 0.05), and it was superior to that of the positive drug in terms of anti-oxidative stress and closer to the level in healthy mice. This result confirms that the CF@H–SA gel can efficiently remove ROS through its nanozyme activity, thereby alleviating oxidative stress-induced skin damage, providing a molecular basis for atopic dermatitis treatment.

Fig. 10 The oxidative stress and keratin expression of mice treated by the CF@H–SA sprayable gel. (a) 8-OHdG, K14 immunofluorescence, and (b and c) statistical analysis of the average fluorescence intensity of immunofluorescence. All statistical data are expressed as mean ± SD (n = 3). *P < 0.05, **P < 0.01 and ***P < 0.001.

Besides 8-OHdG, epidermal barrier dysfunction is one of the core pathological characteristics of atopic dermatitis. The intact structure of the epidermis can not only prevent the invasion of external allergens and pathogens and resist physical/chemical damage from the outside world, but also maintain skin hydration and reduce the release of inflammatory factors. Keratin 14 (K14), as an intermediate filament protein specifically expressed by epidermal basal cells, is essential for maintaining the integrity and mechanical stability of the skin barrier⁵⁰ and participates in intercellular connections to ensure the adhesion of the epidermis to the dermis.⁵¹ A quantitative analysis of K14 expression was performed and is shown in Fig. 10a and c. The expression of K14 in the skin of mice in the atopic dermatitis model group was significantly downregulated, indicating that the epidermal barrier structure was damaged; the expression of K14 in the skin of mice treated with the CF@H–SA gel was upregulated and significantly different from that in the model group mice (*P < 0.05), indicating that CF@H–SA gel had a significant therapeutic effect on atopic dermatitis, promoting the expression of keratin K14 and the recovery of the skin barrier, thereby enhancing the skin's defense against external stimuli.

In summary, the CF@H–SA gel can treat acute atopic dermatitis mainly through two pathways: inhibiting the release of inflammatory factors and promoting the expression of antioxidant genes. The therapeutic effect of the CF@H–SA gel is comparable to that of halcinonide solution and the CF@H–SA gel is superior to halcinonide solution in relieving the symptoms of dry skin in atopic dermatitis and resisting oxidative stress. Furthermore, the CF@H–SA gel is evenly distributed in the dermis upon application to the skin (Fig. S2), and in vitro cell experiments (Fig. 5) show that the gel mitigates DTMB-induced cell damage and maintains keratinocyte viability. H&E staining of the heart, liver, spleen, lungs, and kidneys revealed no obvious signs of disease in any of the five internal organs of the mice examined in each group (Fig. S3). The cardiac myocardium was normal in shape, with neatly arranged muscle fibers. The liver sections showed no evidence of hepatocyte necrosis or nanoparticle invasion, and no signs of inflammation. The lungs of all mice in each group were clear, with normal alveoli. The spleens and kidneys were also normal, with no signs of inflammation. These findings show the good biosafety of the CF@H–SA hydrogel. This not only enhances skin permeability and maintains a moist wound environment, but also localizes the nanozyme to the lesion site, minimizing systemic absorption and potential side effects. The CF@H–SA gel has good application prospects for the treatment of acute atopic dermatitis.

3. Conclusion

In summary, CuO–Fe₃O₄ bimetallic nanozymes with a small size of ∼10 nm were synthesized on the outer walls of medicinal clay HNTs through a hydrothermal reaction. Utilizing SA as the matrix, an sprayable and adhesive gel material (CF@H–SA gel) with excellent anti-inflammatory and antioxidant stress properties was designed for the treatment of chemically induced atopic dermatitis. The therapeutic efficacy of the CF@H–SA gel on chemically induced atopic dermatitis was investigated, and the underlying mechanisms of the CuO–Fe₃O₄@HNTs nanozyme in treating atopic dermatitis were explored. The enzyme-like activity results show that the CuO–Fe₃O₄@HNTs nanozyme exhibits SOD, CAT, and GPx enzyme activities, as well as robust antioxidant capabilities. Analysis of the expression levels of inflammatory factors (IL-4, IL-13, TSLP and IL-33), antioxidant factors (SOD2, CAT and GPx), and inflammatory proteins (lgE and K14) in mouse skin tissues revealed that the CF@H–SA gel effectively scavenges ROS and reduces oxidative damage. Compared to the commercial halcinonide solution, the CF@H–SA gel exhibits superior moisturizing properties and is more effective in alleviating skin dryness. This work has developed a novel bimetallic nanozyme gel system of CF@H–SA for the high-efficiency treatment of atopic dermatitis diseases.

4. Materials and methods

4.1. Materials

High-purity HNTs (purity >99%) were obtained from Guangzhou Runwo Materials Technology Co., Ltd, China. Sodium alginate, copper sulfate pentahydrate (CuSO₄·5H₂O, AR 99%) and methiazolyl tetrazolium (MTT) were purchased from Shanghai Macklin Biochemical Co., Ltd, China. Hydrogen peroxide (H₂O₂, 30%) and iron chloride hexahydrate (FeCl₃·6H₂O, AR 99%) were purchased from Tianjin Yongda Chemical Reagent Co., Ltd, China. Polyvinylpyrrolidone (AR), sodium acetate (AR), 5,5-dimethyl-1-pyrroline N-oxide (DMPO, AR 99%), glutathione GSH (99%) and 1-chloro-2,4-dinitrobenzene (AR, 99%) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd, China. The calcein/PI (AM/PI) staining kit, the CAT detection kit and the 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) were purchased from Shanghai Beyotime Biotechnology Co., Ltd, China. Crystal violet staining solution was obtained from Beijing Lanjieke Technology Co., Ltd, China. 1,3,5-Trimethylbenzene (TMB AR 98%) was purchased from RHAWN, China. A total SOD activity detection kit was purchased from Beijing Labgic Technology Co., Ltd, China. All cell culture reagents were obtained from Thermo Fisher Scientific Inc., USA. Deionized water was supplied by a Milli-Q Integral Water Purification System, Germany.

4.2. Synthesis of the CuO_x–Fe₃O₄@HNTs@SA hydrogel

1 mmol of CuSO₄·5H₂O, 2 mmol of FeCl₃·6H₂O and 20 mmol of NaAc were dissolved in 25 mL of ethylene glycol HNT dispersion and mixed into a green reaction mixture. The solution was transferred to a stainless steel reactor and subjected to a hydrothermal reaction at 200 °C for 10 h. After the reaction was complete, it was cooled naturally, washed several times with ethanol and deionized water, and freeze-dried to obtain the CuO–Fe₃O₄@HNTs composite nanozymes. 1 mg of CuO–Fe₃O₄@HNTs was dissolved in 10 mL of ultrapure water and dispersed completely by ultrasound; 3 wt% sodium alginate was added to completely dissolve it, and then 800–1000 µL of 0.25 wt% CaCl₂ solution was slowly dripped in, and the CuO–Fe₃O₄@HNTs-SA hydrogel was obtained.

4.3. Characterization

The HNTs, CuO–Fe₃O₄, and CuO–Fe₃O₄@HNTs powders were dispersed in an ethanol solution by ultrasonic treatment to prepare a suspension with a mass fraction of 0.5–1%. The dispersion was dropped onto a copper mesh and observed under a TEM microscope after drying. Sufficient amounts of HNTs and CuO–Fe₃O₄ and CuO–Fe₃O₄@HNTs were placed in a test tank. The diffractometer was scanned at a speed of 10° min⁻¹ and a scanning angle range of 5–80°. After the powder sample was fully dried, the sample was scanned in the entire spectrum, the Fe and Cu elements were finely scanned and the valence spectrum was also scanned. The TG curves of HNTs and CuO–Fe₃O₄ and CuO–Fe₃O₄@HNTs were tested at a heating ratio of 10 °C min⁻¹ at a temperature of 50–850 °C using a thermogravimetric (TG) analyzer under a nitrogen atmosphere. The mass ratio of CuO–Fe₃O₄ to HNTs was calculated based on the TGA data at 800 °C. The calculation formula was as follows:
where W_{CuO–Fe3O4@HNTs} represents the weight loss of CuO–Fe₃O₄@HNTs at 800 °C, W_HNTs represents the weight loss of HNTs at 800 °C, and W_CuO–Fe3O4 represents the weight loss of CuO–Fe₃O₄ at 800 °C.

4.4. Radical scavenging assay

A SOD activity detection kit (WST-1 method) was used to detect the SOD-like enzyme activity. First, 20 μL of CuO–Fe₃O₄@HNTs aqueous dispersion (200 μg mL⁻¹) was added to 160 μL of WST-1 working solution, and then 20 μL of enzyme was added to the working solution. The reaction produced a superoxide anion color reaction. After incubation at 37 °C for 30 minutes, the absorbance of the sample was measured at 450 nm using a microplate reader. The CAT-like enzyme activity of CuO–Fe₃O₄@HNTs (200 µg mL⁻¹) was detected using a hydrogen peroxide content detection kit. H₂O₂ (5 mM) and titanium sulfate formed a yellow titanium peroxide complex with characteristic absorption at 415 nm. The ˙OH-rich solution was produced by the Fenton reaction of a mixed aqueous solution of Fe²⁺/H₂O₂ (1.8 mM FeSO₄ and 5 mM H₂O₂).

Glutathione (GSH) (10 mM, 30 µL), CuO–Fe₃O₄@HNTs material dispersion (1, 2, 4, 6 and 8 µL) and H₂O₂ (40 mM, 10 µL) were mixed in PBS to a total volume of 400 µL. After 10 min of reaction, 150 µL of the mixture was aspirated and DNTB (5 mM, 10 µL) was added to measure its absorbance at 412 nm. ABTS (3.2 mg) and potassium persulfate (1 mg) were dissolved in 10 mL of ultrapure water and incubated in the dark for 12 h to obtain the ABTS working solution. The material (50 µL) was reacted with the working solution (950 µL) at room temperature for 5 min, and the absorbance at 734 nm was measured using a UV spectrophotometer. DPPH (0.1 mM) was dissolved in methanol to obtain a stock solution, the material (25 mg mL⁻¹) was reacted with DPPH (0.5 mL) in the dark for 30 min, and the absorbance at 517 nm was measured using a UV spectrophotometer.

4.5. Determination of scavenging of ˙OH by EPR spectra analysis

The ˙OH-rich solution was generated by the Fenton reaction of the Fe²⁺/H₂O₂ system (1.8 mM FeSO₄ and 5 mM H₂O₂). The solution rich in ˙OH was mixed with the aqueous dispersion of HNTs and CuO–Fe₃O₄ and CuO–Fe₃O₄@HNT materials and incubated for 30 min. The hydroxyl radical trapping agent DMPO was added to 90 μL of the mixed solution. The volume ratio of the mixed solution to DMPO was 9 : 1, and the test was conducted by electron paramagnetic resonance (EPR, A300, Bruker, USA) within 10 min.

4.6. Cell culture

The intestinal epithelial cell line HacaT was selected for testing. The cells were purchased from the ATCC in the United States and cultured in DMEM containing 10% fetal calf serum, 50 U mL⁻¹ streptomycin and 100 U mL⁻¹ penicillin. Culture conditions: 37 °C, 5% CO₂; relative humidity: 95%.

4.7. In vitro cytotoxicity assay

HacaT cells in the logarithmic growth phase were seeded in a 96-well plate at a density of 2 × 10⁴ cells per mL and allowed to adhere and grow. At the end of the test, 25 µL of MTT detection solution was added to each well and incubated for 2–4 h. The culture medium in the 96-well plate was discarded, and 150 µL of DMSO was added to fully dissolve the purple crystals. The absorbance value of each well was tested at 570 nm using a multifunctional microplate reader and the cell survival rate was calculated.

AO/EB staining was used to analyze the morphological evidence of cell viability. HacaT cells in the logarithmic growth phase were seeded into a 24-well plate at a density of 3 × 10⁴ cells per mL, cultured and allowed to adhere to the wall, and treated with SA, HNTs@SA, and CuO–Fe₃O₄@HNTs–SA hydrogel immersion solutions. After 24 h or 48 h, they were stained according to the AO/EB staining procedure, and a fluorescence microscope was used to observe and photograph the cell morphology. Each group was shot in at least 5 independent areas.

4.8. Animals for the experiments

SPF-grade KM mice were used as the experimental animals, aged 6–8 weeks and purchased from Guangdong Huawei Testing Co., Ltd, China. All animal care and experimental procedures were conducted in accordance with the animal ethical standards. Mice were housed in SPF-grade animal facilities, maintained at a constant temperature of 24 ± 1 °C and a constant humidity of 51 ± 5%, with free access to food and water. The mice were adaptively raised for one week before being divided into groups for the experiments. All animal procedures were performed in accordance with the Guidelines for Care and Use of Laboratory Animals of Jinan University and approved by the Animal Ethics Committee of Guangdong Huawei Testing Co., Ltd, China (approval number: 202412005).

4.9. In vivo animal experiments

For construction of the mouse atopic dermatitis model, 1% DNCB was prepared with acetone as a solvent and applied to the backs and ears of shaved mice. Five days later, the back and ear skin of the mice showed symptoms such as redness, swelling, dryness, itching, and dandruff, indicating that the dermatitis model had been established. During the first three days of treatment, 0.5% DNCB continued to be used to stimulate the mouse skin to maintain the dermatitis state. The spray volume for the experimental group was 200 μL.

The mice were sprayed with the drug daily for 6 consecutive days. The weights of the mice were recorded every day during the treatment. At the end of the treatment, the epidermal tissues, hearts, livers, lungs, spleens, and kidneys of the mice were obtained, rinsed with saline, and fixed with 4% paraformaldehyde, and pathological sections were made according to the requirements of H&E staining.

4.10. Q-PCR

Cells (5 × 10⁶ cells) or colon tissues were lysed by TRIzol (G3013, Servicebio, China) for 10 min, and supernatants were harvested by centrifugation at 12 000 rpm for 10 min. The total RNA was extracted, and cDNA was synthesized with the SweScript All-in-One RT SuperMix for qPCR (G3337, Servicebio, China). Q-PCR was performed with 2× Universal Blue SYBR Green qPCR Master Mix (G3326, Servicebio, China) and run on a CFX Connect Real-Time System (Bio-Rad Laboratories, USA).

4.11. Blood compatibility

1 mL of whole sheep blood was dispersed in 10 mL of PBS (pH = 7.4) and centrifuged at 500 g min⁻¹ for 10 min three times. Red blood cells were collected from the serum and diluted in PBS (10 mL). 500 µL of the sample extract was incubated at 37 °C for 2 h. The suspension was centrifuged at 1000 g min⁻¹ for 10 min, and 100 µL of the supernatant was transferred after centrifugation to a 96-well plate. The absorbance at 540 nm was measured using a microplate reader.

4.12. Mouse skin immunofluorescence staining analysis

Immunofluorescence staining was used to assess the expression of oxidative stress indicators (8-OHdG and K14). Briefly, the preprocessing procedure for the tissue sections was similar to that mentioned previously. The treated sections were incubated with the primary antibody anti-4-HNE (1 : 100, ab46545, Abcam, USA) overnight at 4 °C. Next, the samples were incubated with the corresponding Alexa 488-labeled or Cy3-labeled fluorescent secondary antibody for 30 min, followed by incubation with DAPI for 5 min. Stained slides were visualized using a confocal laser scanning microscope.

4.13. Immunohistochemical analysis of colon tissue

Immunohistochemistry (IHC) staining was performed to investigate the expression levels of several inflammatory cytokines (IL-33 and IgE) in the colonic tissues. Antigen retrieval was performed on the sections using microwave heat retrieval, and endogenous peroxidase activity was blocked with 3% H₂O₂ at room temperature for 30 min. Subsequently, the sections were blocked with 5% bovine serum albumin (BSA) for 30 min and then incubated with primary antibodies overnight at 4 °C. Slides were then incubated with HRP-labeled secondary antibodies for 1 h at room temperature, followed by treatment with a DAB detection kit and counterstaining with hematoxylin. Finally, slides were visualized using a light microscope.

4.14. CF@H–SA hydrogel transdermal test

The CF@H–SA gel labeled with FITC was applied to the skin of healthy mice. After 6 h, the mice were euthanized and their skin tissues were taken. The skin tissues were spread on tin foil and quickly cooled with liquid nitrogen, and then sliced and photographed to observe the penetration effect of the material in the mouse skin.

Author contributions

Di Zhang: investigation, validation, methodology, data curation, formal analysis, and writing – original draft. Luying Zeng: methodology, validation, and writing – original draft. Xiangyu Chen: methodology and validation. Shuiqing Zhou: validation and investigation. Binghong Luo: writing – review & editing. Mingxian Liu: validation, supervision, writing – review & editing, project administration, and funding acquisition.

Conflicts of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

The data supporting this article have been included as part of the supplementary information (SI). Photos of mouse spleen of different groups; Histological fluorescence staining of mouse skin sections, and H&E staining of major mouse organs. Supplementary information is available. See DOI: https://doi.org/10.1039/d5bm01372k.

Acknowledgements

This work acknowledges the financial support received from the National Natural Science Foundation of China (52573297), the Natural Science Foundation of Guangdong Province (2025A1515011860), and the Fundamental Research Funds for the Central Universities (21624115).

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Footnote

† These authors contributed equally to this work.

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