Characterization of HAMA/tFNA-GL13K hydrogel

As shown in Fig. ​Fig.1f,1f, the HAMA, HAMA/tFNA, HAMA/GL13K, and HAMA/tFNA-GL13K hydrogel were initially in a liquid state and appeared transparent before photocuring. After photocuring (UV light, 365nm), the hydrogel underwent a transformation into a semisolid gel. SEM was used to examine the multiporous structure of hydrogels’ surface (Fig. ​(Fig.1g).1g). As displayed, the pore sizes of HAMA/tFNA, HAMA/ GL13K, and HAMA/tFNA-GL13K hydrogels ranged from 120 to 200μm. This demonstrated that the HAMA/tFNA-GL13K hydrogels possess suitable pore sizes that facilitate nutrient exchange and cell migration.

The composition of composite hydrogels was analyzed using attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR). By comparing the characteristic peaks of HAMA and HAMA/tFNA-GL13K hydrogels, the presence of each polymer can be confirmed. Figure ​Figure1h1h shows the ATR-FTIR spectra of the initial materials and the identification of characteristic peaks. The wide peak near 3 380cm⁻¹ corresponds to HAMA, and the sharp peak at 1 600–1 700cm⁻¹ corresponds to GL13K (the C═O of the amide functional group). The peak at 1 100cm⁻¹ is associated with tFNA, as the infrared absorption bands of DNA include the phosphate symmetric stretching vibration, as shown in Fig. ​Fig.1h1h.

Rheological analysis was carried out at various frequencies to determine the mechanical properties of hydrogels (Fig. ​(Fig.1i).1i). Notably, the storage modulus (G′) was found to be larger than the loss modulus (G″), which is a remarkable feature of gels. Compared to other hydrogels, HAMA/tFNA-GL13K hydrogel exhibited a higher storage modulus (G’), indicating a stronger capacity for shape retention. The loss modulus (G″) of the HAMA/tFNA-GL13K hydrogel was midway between the two other hydrogels, indicating a medium strength of viscosity.

The combination of HAMA/tFNA-GL13K appears an effective antibacterial impact

HAMA and HAMA/tFNA hydrogel had virtually no inhibiting impact on the growth of S. aureus and E. coli, as demonstrated in Fig. ​Fig.2a,2a, b. However, both the HAMA/GL13K and HAMA/tFNA-GL13K hydrogels exhibited bacteriostatic properties, with the colony count of HAMA/tFNA-GL13K group being lower than that of HAMA/GL13K. To investigate the hydrogels’ antibacterial activity against common infectious bacteria, growth curves were measured. Both S. aureus and E. coli showed inhibited growth in the presence of HAMA/GL13K and HAMA/tFNA-GL13K, with HAMA/tFNA-GL13K having the strongest inhibitory ability (Fig. ​(Fig.2c).2c). In summary, the HAMA/tFNA-GL13K hydrogel revealed potent antibacterial properties against both Gram-positive and Gram-negative bacteria, and the presence of tFNAs enhanced the antibacterial ability of GL13K.

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Fig. 2

Antimicrobial activity of HAMA/tFNA-GL13K hydrogel on E. coli and S. aureus in vitro. a Images of E. coli and S. aureus incubated on agar plates with different hydrogels. b Count bacteria of CFU of E. coli and S. aureus. Superscripts a–d indicate significant differences for CFU between different treatment groups (P<0.05). c Growth curves of E. coli and S. aureus in different hydrogels treatment. n=3 independent samples. Data are presented as mean±SD. Error bars represent SD. T-GL13K is the abbreviation of tFNA-GL13K

Distribution of t-GL13K in HaCaT cells

Confocal fluorescence microscopy was used to observe the cellular distribution. It was observed that GL13K was primarily found in the extracellular mesenchyme. The phalloidin-stained cytoskeleton was distinguished from GL13K labeled with fluorescein isothiocyanate (FITC) by coloring the GL13K-FITC channel yellow after confocal fluorescence image capture (Fig. ​(Fig.3a).3a). Fluorescently labeled tFNAs and GL13K were used to investigate the potential effects of GL13K adsorption on the distribution of tFNAs and vice versa. Flow cytometric analysis was applied to ascertain the cellular distribution of t-GL13K. After 6h of treatment, both simplex GL13K and t-GL13K had high cellular uptake and fluorescence intensity. The cellular distribution of tFNAs was significantly altered by GL13K adsorption at 6h (Fig. ​(Fig.3b3b).

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Fig. 3

The distribution of tFNA-GL13K in HaCaT cells. a Distribution of Cy5-tFNAs, FITC-GL13K and Cy5- FITC-t-GL13K complex in HaCaT cells (Cy5: tFNAs: red; FITC: yellow; nucleus: blue; cytoskeleton: green). Scale bars=20μm. b Flow cytometric analysis of distribution of Cy5-tFNAs, FITC-GL13K and Cy5- FITC-t-GL13K complex in HaCaT cells. Superscripts a–d indicating significant differences (P<0.05)

tFNA-GL13K promoted the proliferation and migration of HaCaTs

There was no statistical difference in cell proliferation of HaCaT cells after 6, 12, and 24h of treatment between the HAMA/tFNA, HAMA/tFNA-GL13K and control groups (Fig. ​(Fig.4a).4a). The cytotoxicity of the LPS and HAMA/GL13K groups was significantly higher than the other groups, indicating that HAMA/tFNA and HAMA/tFNA-GL13K can alleviate the inhibition of cell proliferation caused by LPS, and the presence of tFNA can reduce cytotoxicity of GL13K. The wound-healing assay was applied to evaluate the effects of t-GL13K on the migratory activities of HaCaT cells. With the presence of LPS, the LPS and HAMA/GL13K groups significantly inhibited HACAT cell migration, whereas the HAMA/tFNA and HAMA/tFNA-GL13K groups promoted single-layer wound closure of scratched HACAT cells (Fig. ​(Fig.4b).4b). It was demonstrated that the presence of tFNAs could promote cell migration and reduce inhibition of LPS and GL13K.

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Fig. 4

The effect of HAMA/tFNA-GL13K hydrogel on the biological behavior of HaCaT cells. a CCK-8 assays to detect the cytotoxicity of hydrogels on HaCaT cells. Superscripts a–d indicating significant differences (P<0.05). b Images of scratch tests on HaCaT cells treated with different hydrogels at 0h, 4h, 8h, 12h and 24h. Scale bars=200μm. Statistical analysis of scratch tests was marked with superscripts a–e indicating significant differences (P<0.05). c Quantification of ROS in HaCaT cells. d Quantification of expression of inflammatory factors (IL-1β, IL-G, TNF-α, and p65) in HaCaT cells. Superscripts a–e indicating significant differences (P<0.05). Data are presented as the mean±SD (n=3). T-GL13K is the abbreviation of tFNA-GL13K

tFNA-GL13K inhibits the production of ROS and inflammatory factors

LPS is a well-known stimulator of ROS production that leads to inflammation. We investigated the potential influence of HAMA/tFNA-GL13K on LPS-stimulated ROS generation in HaCaT cells. Flow cytometric analysis of ROS levels revealed that HAMA/tFNA and HAMA/tFNA-GL13K reduced ROS generation stimulated by LPS in HaCaT cells (Fig. ​(Fig.4c).4c). LPS was used in this study to simulate the acidic stress caused by the bacterial infection’s local overactive inflammatory response. According to q-PCR results, HAMA/tFNA-GL13K could reduce the expression of inflammatory factors IL-1b, TNF-a, and IL-6 induced by LPS, whereas HAMA/GL13K had no regulatory effect on the expression of the forementioned inflammatory factors under the same conditions (Fig. ​(Fig.4d4d).

Cell culture

Mingjing Biology (Shanghai, China) provided the HaCaT cell lines, and the cells were cultured in Roswell Park Memorial Institute (RPMI) medium containing 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin-streptomycin in an incubator under controlled conditions (37°C, 5% CO₂).

Preparation and characterization of HAMA/tFNA-GL13K hydrogel

Equimolar concentrations of 4 DNA single strands were added in the TM buffer containing 10mmol/L Tris-HCl and 50mM MgCl₂•6H₂O (pH 8.0). The mixture was then denatured at 95°C (10min) and maintained at 4°C (20min) to synthesize tFNA as previously reported. GL13K and tFNA were mixed, and incubated for 30min at room temperature, then kept at 4°C overnight to form t-GL13K.²⁵

To verify tFNA and t-GL13K, 8% PAGE was performed to measure the approximate relative molecular weights of four ssDNAs, tFNA, and t-GL13K. TEM (Tecnai G2 F20, FEI, America) and AFM (Shimadzu, Kyoto, Japan) were used for morphological observation of tFNA and t-GL13K. The hydrodynamic diameters and zeta potentials of the particles were quantified applying DLS on a Zeta sizer Nano (ZS90, Malvern, UK).

The HAMA/tFNA-GL13K hydrogel was prepared based on HAMA (Aladdin, H398341), photo-initiator LAP, and t-GL13K. In brief, tFNA, GL13K, and t-GL13K (250nmol/L) were added after a predefined amount of HAMA (1wt% of the solvent mass) and LAP (0.1wt% of the solvent mass) were dissolved in the PBS. The hydrogel was cured below the UV light source (wavelength 365nm, intensity 18mW/cm²) via cross-linking for 30s to 1min before use. Hydrogels were soaked in phosphate-buffered saline (PBS), and the soak solution was collected and filtered through 0.22-μm Minisart® NML syringe filters as test agents for bacteriologic and cytological tests.

The hydrogels were swiftly frozen in a refrigerator set to −80°C, and after that, they were vacuum-dried for 24h at −50°C. Following their freeze-drying, the hydrogels were sprayed with gold after being quenched in liquid nitrogen along a cross-section. The rheological properties of hydrogels were measured by using Anton Paar MCR102 rheometer (Anton Paar, MCR102, Germany) with a cone plate system (diameter, 35mm; angle, 2°). The range of angular velocity was from 0.01 to 100rad/s under room temperature. The chemical composition of the hydrogels was determined using attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR, Nicolet iS50, Thermo Scientific, Waltham, MA, USA) analysis. Freeze-dried samples were placed on the attenuated total reflectance (ATR) crystal of a Bruker Alpha-P, 64 scans were recorded for each spectrum at a resolution of 4.0cm⁻¹, and the data between 4000 and 400cm^–1 were saved.

In vitro antimicrobial activity assay

200μL of hydrogel was loaded onto a 12-well plate and exposed to UV light for 30s. 1mL of S. aureus and E. coli suspension (1×10⁵ CFU per mL) was added to the hydrogel-coated 12-well plate and cultured for 12h. The bacterial suspension is diluted 1 000 times, LB agar plates were filled with 20μL of the forementioned bacterial solution and left at 37 °C for 24hours. The colony forming unit (CFU) of the plates was measured and determined.⁶⁷

S. aureus and E. coli cultures were adjusted to 1×10⁵ CFU per mL after overnight (16h) incubation. Bacteria were collected by centrifugation (4 000×g, 4°C, 10min) and incubated in LB culture containing test agents at 37°C for 10h. Spectrophotometer (UV1601, Shimadzu, Japan) was applied to measure the OD_{600 nm} value per hour for 10hours. This experiment was repeated at least three times using triplicate samples at each time point.

Distribution of Cy5 and FITC-loaded t-GL13K in HaCaT cells

Flow cytometry and immunofluorescence assays were used to examine the distribution of tFNA, GL13K, and t-GL13K complex in HaCaT cells. Firstly, HaCaT cells were seeded in the six-well plates (2×10⁵ per well). After 24h of culture in standard medium, RPMI culture with 2% FBS replaced the standard medium, and LPS (10μg/mL) was added to simulate the microenvironment of bacterial infection. After 1h, soak solution of hydrogels with FITC-loaded GL13K, Cy5-loaded tFNA (250nmol/L), or FITC&Cy5-loaded t-GL13K (250nmol/L) was added. Following a 6-hour incubation period, the cells were extracted. To evaluate the distribution of tFNA, GL13K, and t-GL13K complex’s cell, flow cytometry was used.⁶⁸ Following 6h of treatment, the cell samples were fixed for at least 30min in 4% (w/v) paraformaldehyde solution (Boster, Wuhan, China). TRITC- Phalloidin and DAPI (4′6-diamidino-2-phenylindole) (Sigma, St. Louis, MO) were applied for the cytoskeleton and nucleus staining respectively. Cytoskeleton staining was performed for 30min and the nucleus staining for 10min. Following every phase, all cell samples were washed three times with PBS. Ultimately, the stained samples were observed via a confocal laser microscope (Nikon N-SIM, Tokyo, Japan).³⁶

In vitro cytotoxicity assay

The cytotoxicity of the samples was measured by applying the CCK-8 assay (KeyGEN, Jiangsu, China). A 96-well plate containing roughly 8 000 Hacat cells per well was utilized for CCK-8. Cultured the cells overnight, the medium was shifted to RPMI containing 2% (v/v) FBS and 10μg/mL LPS, and tFNA with GL13K of different concentrations was added 1h later. The cell medium of control group was devoid of tFNA. The assessment of cytotoxicity was conducted following treatments with CCK-8 solution for 6h, 12h, and 24h. The absorbance of each well was quantified at a wavelength of 450nm relative to a blank sample consisting of RPMI medium.

Cell wound scratch assay

The behavior of cell migration was examined through scratch experiments. HaCaT cells (2×10⁵) were seeded in 12-well plate and left to culture overnight. Each well was cross-hatched with a pipette tip after being cleaned with PBS. After rewashing three times, the cells were cultured in RPMI medium containing 2% (v/v) FBS and 10μg/mL LPS, and varying soak solution mentioned above was added 1h later. After 4, 8, 12, and 24h of treatment, pictures of the samples were captured. The measurements of the scratch areas were conducted, documented, and subsequently compared to the initial scratch sizes at 0hours using Image-J software.

Measurement of ROS

The extent of intracellular reactive oxygen species (ROS) was detected using DCF staining and conducted by flow cytometry. DCFH-DA exhibits cell permeability, and DCF is a fluorescent material decomposed from intracellular DCFH-DA. A 96-well plate containing roughly 8 000 HaCaT cells per well was utilized for measurement of ROS. Cultured the cells overnight, the medium was altered to RPMI containing 2%(v/v) FBS and 10μg/ml LPS, and soak solution of different hydrogels was added 1h later. After treated with soak solution of hydrogels for 6h, DCFH-DA was diluted with serum-free medium and added to the cell suspension and incubated for 30min at 37°C in the dark. Then the cells were collected and washed three times with PBS, and flow cytometry was used to measure the DCF in cells.⁶⁹

RNA isolation and quantitative reverse transcription-polymerase chain reaction (qRT-PCR)

HaCaT cells (2×10⁵) were seeded in 6-well plate and left to culture overnight. The cells were cultured in RPMI medium containing 2% (v/v) FBS and 10μg/mL LPS, and varying soak solution mentioned above was added 1hour later. After being treated for 6h, RNA of HaCaT cells was isolated and purified with TriZol Reagent (Invitrogen) and chloroform. Reverse transcription of RNA into cDNA was performed with the cDNA synthesis kit (PrimeScript RT reagent kit, Takara, Japan), and then expression of genes encoding inflammatory-related factors, including TNF-α, IL-1β, IL-6, and p65(encoding genes were named as TNF-α, IL-1β, IL-6, and p65, respectively) was determined using a SYBR Green qPCR kit (Takara, Dalian, China) according to the manufacturer’s instructions and a previously described method,⁷⁰ with the GADPH as internal control. (The primer sequences are displayed in Appendix Table 1). The 2^−ΔΔCT method was applied for relative quantitative analysis.

Establishment of in vivo infected skin model

The modeling of full-thickness skin infected wounds in mice was executed following established protocols.⁷¹ Briefly, the mice were anesthetized and positioned on the surgical plate. After using electric scissors and depilatory wax to shave the back hair, each mouse had a full-thickness and round wound (diameter 10mm) made on its back with a biopsy punch. 10μLS. aureus and E. coli of each was instilled on the exposed wound to establish an infected wound. The mice were allocated into 5 groups in a randomized method, with 6 mice in each group. From the next day, all hydrogels were applied locally to the site of the wound every two days. The wound was tracked and photographed on days 0, 3, 5, 7, 10, and 14 following surgery. Half of the mice were subjected to euthanasia on the 7^th and 14^th days following the surgical procedure, and the wound area along with surrounding tissues were promptly gathered for subsequent analysis. And the heart, liver, spleen, lung, and kidney were dissected for biosafety test.

Histological analysis

Following fixation for 1h in 4% paraformaldehyde, the skin and internal organ tissues were embedded in paraffin, and then sliced into 4μm sections and stained with either the Masson Staining Kit or the Hematoxylin-Eosin (HE) Staining Kit. Subsequently, the stained tissue sections were imaged by applying an FSX100 microscope (Olympus, Tokyo, Japan). The area of the unhealed wound, the thickness of the epithelium, and the percentage of fibration of each sample were determined by measuring and averaging three distinct visual fields of each section.

This study was supported by National Key R&D Program of China (2019YFA0110600), National Natural Science Foundation of China (82370929, 81970916), Sichuan Science and Technology Program (2022NSFSC0002), Sichuan Province Youth Science and Technology Innovation Team (2022JDTD0021), Research and Develop Program, West China Hospital of Stomatology Sichuan University (RD03202302). We would like to thank Liying Hao (State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University) for help in characterizing AFM. We acknowledge the support of the State Key Laboratory of Oral Diseases, National Clinical Research Center for Oral Diseases, Department of Oral & Maxillofacial Surgery, West China Hospital of Stomatology, Sichuan University.