Zn-MOF hydrogel: regulation of ROS-mediated inflammatory microenvironment for treatment of atopic dermatitis

Preparation and characterization of ROS-scavenging PVA-TSPBA hydrogel

Firstly, ROS responsive linkers N1–(4–boronobenzyl)–N3–(4-boronophenyl)–N1, N1, N3, N3–tetramethylpropane–1,3–diaminium (TSPBA) are synthesized by quaternization reaction from N, N, N', N'-Tetramethyl-1,3-propanediamine and 4–(bromomethyl) phenylboronic acid. The structure of TSPBA is confirmed with 1H NMR (400 MHz, DMSO) (Additional file 1: Fig. S1).

Then, we study the effect of the concentration of polyvinyl alcohol (PVA) and TSPBA linker on the gel formation (Fig. 1A). Through simple mixing of wt% (3%, 6% and 9%) PVA aqueous solution and wt% (3%, 6% and 9%) TSPBA aqueous solution, ROS responsive hydrogel (Gel) can be rapidly formed by phenylboric acid and alcohol hydroxyl. When TSPBA concentration is high (more than 6%), solid gel can be formed regardless of PVA concentration. The PVA concentration may be relatively low (3%), and the local concentration of TSPBA is high during the reaction, the colloid formed is easy to disperse into blocks, and there is no good integrity after freeze-drying. For this reason, we chose four groups of hydrogels with PVA and TSPBA ratios of Gel-1 (PVA: TSPBA = 6%:9%), Gel-2 (PVA: TSPBA = 9%:9%), Gel-3 (PVA: TSPBA = 6%:6%) and Gel-4 (PVA: TSPBA = 9%:6%) for subsequent experiments.

After the prepared gel is freeze-dried, through the scanning electron microscope (SEM), we can clearly observe that the surface of polyvinyl alcohol-based antioxidant hydrogel (Gel-3) is rough, and the hydrogel presents a porous microstructure with interpenetrating networks, and the pore distribution is dense, with the pore size ranging from 20 μm to 60 μm (Fig. 1B).

Rheological tests further confirm the formation of hydrogel. The rheological properties of PVA based hydrogels are characterized by monitoring their storage modulus (G ') and loss modulus (G ") as a function of frequency and stress. For frequency scanning (Fig. 1C), all tested gel show similar nonlinear rheological behavior, and their values increase with the increase of frequency, which means that there is similar microstructure. In addition, the tangent value of the loss angle represents the ratio between the viscous and elastic properties, and is a sensitive indicator of the motion of various molecules within the material, with tan δ = G "/G'. The lower the value of tanδ, the more elastic the material, where a value of tanδ < 1 usually indicates that the sample is elastic, while a value of tanδ > 1 corresponds to a viscous sample. The loss angle tangent values of Gel-1 (6–9%), Gel-2 (9–9%), Gel-3 (6–6%) and Gel-4 (9–6%) were 0.409, 0.301, 0.417 and 0.373, respectively. All of these values are less than 1, indicating that all samples are elastic. For strain scanning (Fig. 1D), the G' and G" values of the four groups of hydrogels decrease with the increase of strain, indicating the dissociation of chemical bond crosslinking and the collapse of network structure. In the linear viscoelastic region, the value of G' is higher than G", indicating that the hydrogel is a viscoelastic solid.

Swelling property

Swelling rate (SR) is an important parameter of hydrogel. High SR is conducive to maintaining a moist wound environment and improving inflammation, so hydrogel is required to have good swelling performance. As shown in the Fig. 1E, in the first 10 min of the swelling test, the swelling ratio of all hydrogels with different concentration ratios has increased. Among them, the maximum swelling ratio of Gel-3 is close to 750%, and with the increase of the concentration of PVA or TSPBA, the swelling ratio of the other three groups of hydrogel gel decreases, of which Gel-2 hydrogel has the lowest swelling ratio. According to the above experimental results, Gel-3 (6%PVA: 6%TSPBA = 1:1) is selected for subsequent experiments.

The prepared hydrogel can quickly reach the expansion equilibrium, which may be caused by the wicking effect of PVA-TSPBA hydrogel pores, and the existence of a large number of hydroxyl groups (–OH) in the molecular chain of PVA, which can form hydrogen bonds with water molecules, locking a large number of water molecules in three-dimensional porous structure. As the concentration ratio increases, the hydrogel crosslinks become a more compact structure, the pore size of the three-dimensional porous structure decreases, and water molecules are not easy to diffuse in the narrow pores, resulting in a lower swelling behavior, which further limits the network to absorb more water. Therefore, the prepared hydrogel can maintain the moist microenvironment of the injured part, and can help reduce the dryness of atopic dermatitis.

ROS-scavenging ability of gel

Because aryl borate esters and their derivatives are common materials for building functional polymers and conjugated molecules, the B-C bond in their structures can be broken to generate phenol under the action of H2O2, and can become one of the biodegradable materials. In order to explore the response degradation ability of the hydrogel prepared under H2O2 environment, the hydrogel is incubated with H2O2 to observe the changes at different times. As shown in the Fig. 2A, with the increase of H2O2 concentration and reaction time, the hydrogel response speed increases and the time required for degradation decreases. The morphology of the hydrogel incubated with H2O2 is further observed by SEM (Fig. 2B). Under the action of 10 mM H2O2, the porous structure of the hydrogel is destroyed after 1 h. This shows that the hydrogel prepared has good response to ROS.

Fig. 2figure 2

A The changes of Gel in H2O2 (0 mM, 0.5 mM and 1 mM) at different times. B Scanning electron microscopy (SEM) visualization of Gel in 10 mM after 1 h (scale bar = 60 μm). C The photo of MB in Fenton reaction solution incubates with or without the hydrogel (I: MB + Fe, II: MB + Fe + H2O2, III: MB + Fe + H2O2 + Hydrogel). The place pointed by the black arrow is the hydrogel. D The relative absorbance value of MB triggered by Fenton reaction with or without the hydrogel. (n = 3). *** indicates p < 0.001

The scavenging activity of hydrogel against hydroxyl radical (•OH) is studied through using methylene blue (MB) as the •OH indicator probe. As shown in the Fig. 2C, ferrous ion (Fe2+) and 1 mM H2O2 solution are used to generate •OH through Fenton reaction. The color of MB solution quickly changes from dark blue to light blue, indicating the generation of •OH. However, after adding hydrogel, the color change of MB in •OH solution is small. As shown in the Fig. 2D, with the increase of time, the relative absorbance gradually decreases, showing a dependence on time. After 240 min, the relative absorbance value of the hydrogel group decreases by about 25%, and that of the anhydrous gel group decreases by about 65%. It can be seen that the hydrogel prepared has a good response to • OH, and it also shows that our hydrogel has a strong ability to scavenge • OH.

Synthesis and characterization of ZIF-8 nanoparticles

The ZIF-8 nanoparticles consist of 2-methylimidazole (2-melm) and zinc nitrate hexahydrate (Zn (NO3) 2) via covalent bonds. The average particle size and dispersion index PDI of ZIF-8 are detected by the particle size analyzer. The particle size of ZIF-8 is normally distributed, with an average particle size of 98.72 nm and a PDI of 0.096, showing a good control over the size of nanoparticles (Fig. 3A). The morphology and size of ZIF-8 nanoparticles verified by SEM show that they have a landmark hexagonal structure (Fig. 3B). The X-ray diffraction test results are shown in Additional file 1: Fig. S2. There are obvious strong peaks at 2θ = 7.26°, 10.33°, 12.68°, 14.63°, 16.88° and 18.11°, corresponding to crystal plane (011), (022), (112), (022), (013) and (222) respectively. The characteristic peaks of the prepared ZIF-8 are consistent with the simulated ZIF-8XRD pattern (JCPDS No: 10–0454), indicating that ZIF-8 has been successfully synthesized and has high crystallinity. When ZIF-8 is decomposed, the released Zn2+ coordinates with the hydroxyl group on the hydrogel molecule, which reduces the voids in the hydrogel network and makes the pore distribution and shape more compact and regular (Fig. 3C).

Fig. 3figure 3

A Particle size distribution of ZIF-8. B The morphology and size of ZIF-8 nanoparticles verified by SEM. C Scanning electron microscopy (SEM) visualization of Gel@ZIF-8 (scale bar = 60 μm). D Cumulative release profile of Zn2+ from Gel@ZIF-8 (n = 3). ** and *** indicate p < 0.01 and p < 0.001, respectively

Zinc ion release curve of the Gel@ZIF-8

Under physiological conditions, Zn2+ is usually used as a booster for human growth and immunity. However, high concentration of Zn2+ has potential toxicity to cell growth. In order to evaluate the release kinetics of Zn2+ after decomposition of Zn MOF, a series of standard curves (Additional file 1: Fig. S3) of Zn2+ concentration gradient are established by zinc spectrophotometry method, color blocks represent the colors of solutions with different concentrations. The release amount of Zn2+ is positively correlated with the concentration of ZIF-8 nanoparticles, showing a steady upward trend within a week (Fig. 3D).

In vitro cytotoxicity test and cytoprotective test with Gel and Gel@ZIF-8 under highly oxidative conditions

The presence of ZIF-8 and the release of zinc ions may cause toxicity to cells. Therefore, we use different concentrations of Gel@ZIF-8 to coculture with L929 cells to evaluate the cytotoxicity of the Gel@ZIF-8. MTT assay and live/dead cell staining are used to detect cell viability. As shown in the Fig. 4A, the OD value is measured by MTT method at 490 nm. When the concentration of ZIF-8 nanoparticles exceeds 500 μg/mL, the proportion of living cells decreases, which inhibit the cell growth. In addition, at concentrations of 0, 80, 500 and 5000 μg/mL, the number of living cells assessed by live/dead cell staining is basically the same (Fig. 4B and C).

Fig. 4figure 4

A Cell viability of different Gel@ZIF-8 on L929 cells at 24 h and 48 h. B The photo of live/dead cell staining at 48 h. (scale bar = 100 μm). C Fluorescence intensity of the live/dead cell staining at 48 h. D Cell viability of L929 cells with various H2O2 concentrations. E Cytoprotective effect of Gel@ZIF-8 under a highly oxidative medium (H2O2) (n = 3). *, ** and *** indicate p < 0.05, p < 0.01 and p < 0.001, respectively

In order to test the protective ability of Gel@ZIF-8 on cells, L929 cells are placed in H2O2 (Fig. 4D). Gel or Gel@ZIF-8(the diameter is 5 mm, the thickness is 1 mm) are added in 1000 μM H2O2 at the same time (Fig. 4E). Compared with the control group, the addition of Gel and Gel@ZIF-8 significantly improve the cell viability, indicating that Gel@ZIF-8 can prevent cell damage by scavenging excessive H2O2 from the culture medium.

Antibacterial ability

Atopic dermatitis is easy to be infected by malignant bacteria such as Staphylococcus aureus, which aggravates inflammation and even presents life-threatening complications. Therefore, preventing bacterial infection is the key to the treatment of dermatitis. We evaluate the bactericidal activity of Gel@ZIF-8. ZIF-8 can remove microorganisms and pathogens harmful to inflammatory skin lesions. ZIF-8 can inhibit microorganisms and pathogens harmful to inflammatory skin lesions. We study the bactericidal properties of 0、80、500 and 5000 μg/mL Gel@ZIF-8. As shown in the Fig. 5A, after 24 h of incubation, the higher the concentration of ZIF-8 nanoparticles, the fewer colonies of S. aureus, MRSA and E. coli. The bacteriostatic rate increases significantly (Fig. 5B–D), According to the cell experiment results, Gel has no direct effect on microorganisms and pathogens, and the appropriate concentration of ZIF-8 nanoparticles is 500 μg/mL, which has good cell biocompatibility and antibacterial effect.

Fig. 5figure 5

A The statistical graph of the bacteriostatic rate of the Gel at different ZIF-8-laden concentrations. The bacteriostatic rate of B S. aureus C MRSA D E. coli (scale bar = 100 μm, n = 3). *, ** and *** indicate p < 0.05, p < 0.01 and p < 0.001, respectively

In vivo therapeutic effect of Gel@ZIF-8 DNCB-induced AD mouse model

To investigate the therapeutic potential of the Gel@ZIF-8 (500 μg/mL) for the treatment of AD, a mouse AD model is induced with DNCB. DNCB is one of the chemicals used to prepare AD animal models. When applied to skin, DNCB interacts with skin protein to form a complex, which is absorbed by antigen presenting cells, and then activated Th2 cells and mast cells. Figure 6A shows that the skin of mice treated with DNCB contains a compound of blood and pus, indicating that AD is well induced in the skin in the first week.

Fig. 6figure 6

A Representative photographs of dorsal skin of each group for monitoring the change in the lesion. B Histology of mouse skin sections stained with H&E. The space between red lines denotes the epidermal thickness. C Histology of mouse skin sections stained with toluidine blue for dermal mast cells. The red arrow heads indicate the mast cells (scale bar = 100 μm). D Dermatitis score measurements conducted over three weeks. E Comparison of epidermal thickness. F Measurement of the number of mast cells for each group. G The concentrations of IgE in blood serum retrieved from each group at W3. (n = 6). *, ** and *** indicate p < 0.05, p < 0.01 and p < 0.001, respectively

After 14 days of treatment, the skin of untreated group, dexamethasone group and Gel group still have wounds, while the skin of ZIF-8 hydrogel group has smaller wounds. The thickness of epidermal layers is a representative indicator of AD. The untreated groups, DXMS and Gel groups show epidermal layers 5.3, 3.5 and 3.0-fold thicker than the healthy group, respectively. The Gel@ZIF-8 groups recover a thinner epidermal thickness, which is twofold smaller than that in the untreated group (Fig. 6B, E). A large number of mast cells is a characteristic feature of AD, so mast cells are visualized by toluidine blue staining. The results reveal that the lowest infiltration of mast cells in the dermis is in the Gel@ZIF-8 group (Fig. 6C, F). The dermatitis scores show that the diseases are provoked with similar severity in all mice at week 1, and the score decreases to different degrees depending on the treatment (Fig. 6D). The score changes of untreated group, dexamethasone group and blank hydrogel group are similar, while the dermatitis score of ZIF-8 hydrogel group is the lowest, with a statistically significant difference.

We further evaluate the changes of AD related immune protein levels after hydrogel treatment. IgE is a representative biomarker of AD, which can enhance mast cell activation, allergen internalization and other immune responses. It can be seen from the Fig. 6G that the IgE level in the blood of AD mice in Gel group decreases, and the Gel@ZIF-8 continues to produce therapeutic effect.

Thus, after treating the skin of AD mice, Gel@ZIF-8 group can reduce the size of AD skin wound, restore the thickness of epidermis, and inhibit AD related immune factors, including mast cell infiltration and IgE.

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