In a typical experiment, FMA and acrylic acid (AA) were first mixed to obtain the pre-gel solution of the composite hydrogel. Colloidal crystal array (CCA) templates were prepared on the clean glass slides. During this procedure, silica nanoparticles with a diameter of 220 nm were self-assembled driven by capillary force, spontaneously formed into a periodically hexagonal packing arrangement (Fig. 2a). After the CCA template was put into a mold as the substrate, the pre-gel solution was then injected in the mold and fully infiltrated the nanoscale voids in CCAs, it was crosslinked to form a hydrogel under ultraviolet (UV) light-triggered polymerization (Fig. 2b). Finally, the silica nanoparticles were etching by hydrofluoric acid, and the hydrogel inverse opal film was obtained (Fig. 2c).

Fig. 2

The schematic, SEM image and photograph of a the CCA template, b the hydrogel penetrated composite material and c the resultant IOF. In each panel, the left is the schematic, the middle is the SEM image and the right is the photograph. In each panel, scale bars are 200 nm in the middle and 5 mm in the right

Because of the periodically ordered structure composed of the well assembled silica nanoparticles, the CCA templates on the glass slides showed vivid structural color. Scanning electron microscopy (SEM) was used to confirm its periodically ordered structure of the nanoparticles. The SEM images also suggested that the IOFs successfully replicated the ordered construction and performed interconnected porous structures. These periodic nanopores imparted the resultant films with the property of photonic bandgap (PBG). When incident light illuminated the material surface, the light propagation with a particular wavelength would be modulated and reflected by the nanopores. Typically, obvious structural colors were seen when the wavelength of incident light was in the visible range. The reflection wavelength value (λ) could be calculated approximately by Bragg’s equation:

$$lambda = 1.633{text{d}}{n_{average}}.$$

(1)

In this equation, d refers to the central distance between adjoining nanocrystals or nanopores, while naverage represents the material’s average reflectivity. Thus, according to the equation above, when the diameter of nanopores changes, the color of the IOFs would be adjusted.

We prepared a series of IOFs with various structural colors based on the FMA and PAA composited hydrogels and investigated their pH responsiveness. It has been confirmed that the PAA polymer networks could swell/shrink reversibly under the stimulus of pH variation [32, 33]. Under a low pH environment, the dissociation of –COOH groups in the hydrogel was inhibited. The PAA hydrogel shrunk because of the formation of hydrogen bonds between –COOH groups, thus leading to the decrease of volume at this time. As the pH value of the environment increased, –COOH groups gradually dissociated into –COO. The hydrogel would swell in volume because of the resulting increased electrostatic repulsion and osmotic pressure between networks. During these processes, the center distance between nanopores also change, resulting in the change of PBG and visual colors. The rise of reflection wavelength λ showed that increasing pH would lead to a red shift of structural color (Additional file 1: Fig. S1).

Previous researches have reported that the typical pH values are around 4.0–6.0 in the healed wound sites, and are elevated to 7.0 or above for unhealed wounds or infected burns. Thus, we focused on testing the responsiveness of IOFs within the pH range of 4.0–8.0. IOFs with lustrous structural colors of blue, green and red were then obtained by replicating CCA templates with the nanoparticle diameters of 220, 250, and 300 nm, respectively. Subsequently, the color changes of these IOFs under pH stimulation between 4 and 8 were measured. As shown in Additional file 1: Fig. S2, IOFs fabricated by templates with nanoparticle size of 220 nm had the most vivid and wide-range color variation in the mildly acidic and mildly alkaline environments. Thus, CCA templates with a nanoparticle size of 220 nm were selected for further research.

The color changes and the variation of reflection peaks of the IOFs fabricated by replicating templates under pH-stimulus were investigated (Fig. 3a). The IOF exhibited blue with a pH of 4.0, attributed to the hydrogel shrinkage caused by the formation of hydrogen bonds between –COOH groups. As the environmental pH progressively raised to 8.0, the IOF swelled, while the color gradually turned green. Meanwhile, the variation of the reflection peaks was detected by using optical spectrometer. During the procedure, with the hydrogel volume increasing, the spacing d between adjacent nanopores increased. As the Bragg’s equation shows, the reflection wavelength λ would also increase (Fig. 3b). More importantly, as shown in Fig. 3c, the hydrogel films still showed stable structural color variation even after several cycles, which could provide reliable results for wound sensing. Therefore, we tested its practical pH sensitive color change in a bacteria-infected wound. The pH values of normal skin and infected wound in animal models were first measured. The pH indicator paper demonstrated that the infected wound showed a significantly alkaline pH value, which resulted in the blue to green structure color change after the IOF was placed on the wound area (Additional file 1: Fig. S3). These results indicated that the intelligent pH-responsive structural color films had potential applications in wound healing monitoring.

Fig. 3
figure 3

The pH responsiveness of the IOF fabricated by replicating CCA templates with a nanoparticle size of 220 nm. a Optical images and variation of the reflection peaks during pH value increasing from 4.0 to 8.0. b The changes of reflection peak wavelength upon pH increase. c The switch value of the reflection peak of the IOF in the pH 5 and 8 buffers as a function of the pH cycle numbers

Sterilization is important for the repair of infected wounds. CS has been proved to have inherent antibacterial activity attributing to the positively charged amino groups [15]. Thus, we added CS solution into the pre-gel solution and polymerized under UV light. Subsequently, we verified the antibacterial properties of the IOFs loaded with CS by culturing Escherichia coli (E. coli, a type of Gram-negative bacterium) and Staphylococcus aureus (S. aureus, a type of Gram-positive bacterium) with the prepared films. The Live/Dead staining results indicated that the bacteria cultured in the PBS buffer almost survived. However, after incubated with the IOF loaded with 2% CS, SYTO staining result revealed that a small number of bacteria survived. Besides, with the increase of CS concentration, the antimicrobial activity increased dramatically. As CS could react with the bacterial cell wall due to the electrostatic adsorption, thereby changing bacterial nutrient intake and leading to bacterial death. Especially when treated with films containing 4% CS, the death rate of the two bacterial almost reached 100% (Fig. 4b, c), which is consistent with the reported result [34]. Similar antibacterial results could be verified by using the standard plate count method. As shown in Additional file 1: Fig. S4, little bacterial colones could be seen in the experimental group compared with the control group. As a consequence, 4% CS was chosen for the following research to impart the films with sufficient antibacterial functionality.

Fig. 4
figure 4

a Live/Dead staining of E. coli and S. aureus treated with PBS, IOF with 2% CS, IOF with 3% CS, and IOF with 4% CS for 24 h. The live and dead bacteria were stained in green and red by SYTO and propidium iodide (PI), respectively. Bacterial death rate of E. coli (b) and S. aureus (c) treated with PBS, IOF with 2% CS, IOF with 3% CS, and IOF with 4% CS for 24 h, respectively. Scale bars are 25 μm

In addition to antibacterial function, the interconnected nanopores have endowed the IOF with an outstanding scaffold for drug infiltrating. For the purpose of verifying the drug loading and releasing abilities of the IOFs loaded with 4% CS, the demo drug with fluorescence mark, fluorescein isothiocyanate-bovine serum albumin (FITC-BSA), was selected. After drug loading, the material was soaked into the phosphate-buffered saline (PBS) buffer. This liquid condition was used to simulate sustained drug release. For the first 12 h, we collected and replaced 100 µL of the buffer solution every 1 h, the time interval for collection would increase during the following 5 days. Microplate reader was used to detect the fluorescence intensity of FITC-BSA in the collected solution, and the amount of the released drug from the IOFs was determined by comparing to the standard curve. It was observed that approximately 30% of the demo drug was finally released in the solution over 144 h, which was less than the reported results, probably due to the different methods of drug loading. These results effectually proved that the IOFs loaded with 4% CS could provide practical and lasting drug treatment for promoting wound repair (Additional file 1: Fig. S5).

Subsequently, we carried in vitro cell experiments to evaluate the biocompatibility of composite film. After incubated with IOFs or the IOFs loaded with CS and VEGF, the cell staining results showed good survivability and normal morphological characteristics of the NIH-3T3 cells (Additional file 1: Fig. S6). Moreover, the hemocompatibility of the obtained films was further assessed by hemolysis tests, as wound dressings must come into contact with blood. In whole blood, IOFs induced nearly no hemolysis in contact with red blood cells (Additional file 1: Fig. S7). These results indicated that IOF was a safe material and suitable for biomedical applications.

The practical biomedical value of this multifunctional IOF was further validated in vivo. In a bacterial-infected full-thickness skin defect rat model, the wound with a diameter of 1 cm was established and the bacterial suspension was then injected into the wound area. The Sprague–Dawley (SD) rats were separated into three groups, including the IOF group, the IOF + CS + VEGF group, and PBS buffer group as control. During the wound healing processes, photographs of all the wound sites were recorded by using digital camera on day 0, 3, 5, 7 and 9 for the following detailed analysis (Fig. 5a). In addition, the new granulation tissues were shown by carrying out hematoxylin and eosin (H&E) staining (Fig. 5b). These photos clearly demonstrated that, compared with the control group, the wound in IOF patch-treated groups presented higher closure rate and smaller granulation tissue width. From the quantitative analysis of the wounds closure rate, it could be found that the wound healing efficiency was relatively enhanced benefiting from the function of VEGF (Fig. 5c). Meanwhile, the IOF + CS + VEGF group displayed minimal width of the granulation tissue (3.26 mm), while the PBS treated group demonstrated the maximum width of 4.77 mm. In addition to the contractible wound gap, compared with other groups, the IOF + CS + VEGF group also showed thickest granulation tissue (Fig. 5d). Furthermore, we compared the therapeutic effects of commercial dressings (CD) and the composite IOF. It has been found that the two groups had similar rates of wound closure, while the IOF + CS + VEGF owning a slightly better therapeutic effect (Additional file 1: Fig. S8). From these gross observation and HE staining results, it is believed that the composite film has potential practical application value.

Fig. 5
figure 5

Wound closure process and H&E staining. a Representative photographs of the skin wounds treated with PBS solution (control), IOF, the IOF loaded with both CS and VEGF. b H&E staining of wounds after 9 days. c The statistical graph of the wound closure situation (n = 4). d Quantitative analysis of granulation tissue width and thickness (n = 4). Scale bars are 5 mm in a and 1 mm in b

Wounds infected by bacteria may cause serious inflammatory response. At the early stage during wound healing procedure, interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were the two typical inflammatory factors whose expressions were selected as indicators to assess the wound infection level. The expressions were examined by immunohistochemical staining after day 9 (Fig. 6a, c). The stain results showed the highest expression quantity of IL-6 and TNF-α in control group due to the severe inflammatory response. In contrast, little inflammatory factors could be seen in IOF + CS + VEGF group benefiting from the antibacterial activity of CS, which could effectively block bacterial reproduction and protect the wound against bacterial infection. Statistical analysis also demonstrated that the IOF + CS + VEGF group showed the least infection among the three experimental groups (Fig. 6b, d). The in vivo antibacterial properties caused by CS was next assessed 24 h and 48 h after wound appearance by using the standard plate count method. It could be obviously found that compared with the control group, the IOF + CS + VEGF group had less bacterial colony formation, especially after 48 h (Additional file 1: Fig. S9). Additionally, the expression of proliferation cell nuclear antigen (PCNA, a DNA clamp essential for cell replication) and E-cadherin (a type of cell adhesion proteins), were also examined to reveal the cell proliferation and adhesion state, respectively. Additional file 1: Figure S10 presented that the IOF + CS + VEGF group owned the most positive area of PCNA and E-cadherin, indicating the best cell regeneration results. In addition, the IOF + CS + VEGF group showed the best collagen deposition and tissue remodeling result due to the combined treatment of CS and VEGF.

Fig. 6
figure 6

a Immunostaining of IL-6 of granulation tissues in different groups. c Immunostaining of TNF-α of granulation tissues in different groups. e Masson’s trichrome staining for collagen in different groups. Scale bars are all 50 μm. In each panel, (i) is the control group, (ii) is the IOF group, (iii) is the IOF + CS + VEGF group. Statistical analysis of b IL-6, d TNF-α and f collagen deposition in different groups after 9 days of wound repair. NS not significant, 0.01 < *p < 0.05, **p < 0.01 (n = 4)

At the last stage during wound healing process, the deposition of collagen in the wound bed is a necessary index that can reflect the tissue remodeling condition. The amount of the collagen deposition was examined by Masson’s trichrome staining (Fig. 6e). The results indicated that, the IOF + CS + VEGF group showed promoted collagen deposition in comparison with the control group, which might be attributed to the sterile environment created by CS. Angiogenesis is also a crucial index to evaluate the remodeling condition of tissue. Thus, we carried out double immunofluorescence staining of CD31 (a typical marker of the vascular endothelial cell) and α-smooth muscle actin (α-SMA, a typical marker of the vascular smooth muscle endothelial cell) to verify new blood vessels formation at the wound site (Additional file 1: Fig. S11). In the control group, a small quantity of new formed blood vessels could be observed, mainly attributed to the high inflammatory level resulted from the bacterial infection. Cell proliferation and differentiation was dramatically impeded. However, the density of new blood vessels at the wound area in the IOF + CS + VEGF group was obviously higher, profiting from the antibacterial property of CS and the stimulating angiogenesis property of VEGF. These features indicated that this synthetic film with multifunctions had great potential applications in the promotion of wound repair.

https://jnanobiotechnology.biomedcentral.com/articles/10.1186/s12951-022-01564-w

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