DNA tetrahedron complex antibacterial hydrogel for promoting wound healing and preparation method and use thereof

Abstract

The application provides a DNA tetrahedron complex antibacterial hydrogel for promoting wound healing and a preparation method and application thereof, and belongs to the field of biomedical materials. The application first provides a DNA tetrahedron complex antibacterial hydrogel precursor for promoting wound healing, which is obtained by dissolving DNA tetrahedron framework nucleic acid containing antibacterial peptide, hyaluronic acid methacrylic acid and a photoinitiator in a solvent. The hydrogel precursor can be cured by ultraviolet light to obtain the hydrogel. The hydrogel can be used as a dressing for treating infected skin wounds. The hydrogel has strong adhesion and deformation capacity, and excellent antibacterial and anti-inflammatory effects, and can provide a slightly moist microenvironment for the wound, promote wound healing and reduce scar formation. The strategy provides a new scheme for treating infected skin wounds.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biomedical materials, and particularly relates to a DNA tetrahedron complex antibacterial hydrogel for promoting wound healing and a preparation method and application thereof. BACKGROUND

[0002] Skin, as a barrier between the internal and external environment, is not only the core of host immune defense, but also the first line of defense against external threats. When the skin is damaged by physical and chemical factors such as trauma, burns or surgical incision, the integrity of the skin will be destroyed, and pathogenic bacteria infection can occur. Surgical infection is very common in surgical diseases, accounting for 1 / 3-1 / 2 of all surgical cases, among which Staphylococcus aureus and Escherichia coli are the most common sources of infection. When infection is severe, bacteria can invade lymphatic vessels and blood, and even cause fatal sepsis and severe metastatic infection. Staphylococcus aureus and Escherichia coli can also produce various toxins and enzymes, thereby slowing down wound healing and reducing immune function. At present, antibiotics are still the main choice for clinical treatment of infection, but their frequent use can promote bacterial drug resistance. In addition, traditional wound dressings often lack biocompatibility, have certain irritability to the skin, or cause healthcare-associated infections. There is still a lack of wound dressings that can effectively resist bacteria, reduce inflammation, promote healing and have good biocompatibility.

[0003] Antibacterial peptides (AMP) are a kind of polypeptides with potential to replace traditional antibiotics, which have broad-spectrum antibacterial ability against gram-positive and gram-negative bacteria. Antibacterial peptides bind to the inner membrane of bacteria, causing peptide penetration, which leads to leakage of bacterial cell contents and cell death. However, because antibacterial peptides need relatively high concentrations to achieve effective antibacterial effect, but high-concentration antibacterial peptides exhibit certain cytotoxicity, so direct use of natural AMP for treatment is not ideal. At the same time, some biological factors, such as changes in environmental pH and the presence of proteases, can affect the stability and bioavailability of AMP. Antibacterial peptide GL13K is a polypeptide derived from salivary protein BPIFA2, which is obtained by replacing three amino acid residues in GL13NH2 with lysine residues, and has effective bactericidal effect on gram-negative and gram-positive bacteria, and also has certain effect on bacterial biofilm and lipopolysaccharide. However, GL13K is also susceptible to the above-mentioned common problems. Therefore, it is an urgent problem to be solved to retain the antibacterial performance of AMP while improving its stability and biological safety.

[0004] Tetrahedral framework nucleic acid (tFNA) is a kind of self-assembled nucleic acid material, which is simple to synthesize and widely used due to the biological properties of nucleic acid. tFNA can be used as a drug delivery material through embedding, electrostatic interaction and other mechanisms, and also has a variety of biological properties. tFNA has the ability to scavenge ROS, thereby achieving anti-inflammatory and antioxidant activity. Liu Yuhao et al. used tFNA as a nano delivery material for GL13K and found that it could improve the stability and antibacterial efficacy against E. coli and P. gingivalis, which indicates that tFNA-based AMP delivery has a wide range of potential applications.

[0005] Hydrogel has good hydrophilicity, excellent biocompatibility and adjustable physical properties, and is one of the most promising wound dressing materials. Methacrylated hyaluronic acid hydrogel (HAMA) has a loose porous network structure, which is conducive to cell adhesion and migration, maintains a moist environment and absorbs wound exudate. Therefore, it has been widely used in the treatment of skin, joint and nervous system diseases.

[0006] However, the effect of hydrogel carrying DNA tetrahedral framework nucleic acid to deliver antibacterial peptide GL13K for the treatment of infected wounds has not been reported. SUMMARY

[0007] The purpose of the present application is to provide a DNA tetrahedral complex antibacterial hydrogel for promoting wound healing and a preparation method and use thereof.

[0008] The present application provides a DNA tetrahedral complex antibacterial hydrogel precursor for promoting wound healing, which is obtained by dissolving antibacterial peptide-containing DNA tetrahedral framework nucleic acid, methacrylic hyaluronic acid and photoinitiator in a solvent.

[0009] The antibacterial peptide-containing DNA tetrahedral framework nucleic acid is obtained by mixing and incubating DNA tetrahedral framework nucleic acid and antibacterial peptide.

[0010] Further, after being dissolved in the solvent, the concentration of the antibacterial peptide-containing DNA tetrahedral framework nucleic acid is 200-300 nM; the concentration of the methacrylic hyaluronic acid is 10-15 mg / mL, and the concentration of the photoinitiator is 1-3 mg / mL.

[0011] Preferably, after being dissolved in the solvent, the concentration of the antibacterial peptide-containing DNA tetrahedral framework nucleic acid is 250 nM; the concentration of the methacrylic hyaluronic acid is 10 mg / mL, and the concentration of the photoinitiator is 1 mg / mL.

[0012] Further, the solvent is PBS or physiological saline; and / or, the photoinitiator is LAP.

[0013] Further, the molar ratio of the DNA tetrahedron framework nucleic acid and the antibacterial peptide is 1:100-500 when the DNA tetrahedron framework nucleic acid and the antibacterial peptide are mixed and incubated, and preferably 1:500.

[0014] Further, the preparation method of the DNA tetrahedron framework nucleic acid containing the antibacterial peptide comprises the following steps: mixing the DNA tetrahedron framework nucleic acid and the antibacterial peptide, and incubating at 20-40℃ for 30-40 min to obtain the DNA tetrahedron framework nucleic acid containing the antibacterial peptide.

[0015] Further, the DNA tetrahedron framework nucleic acid is a single-stranded DNA with a nucleotide sequence as shown in SEQ NO. 1-4, which is self-assembled into a tetrahedron structure through base complementary pairing.

[0016] The antibacterial peptide is antibacterial peptide GL13K.

[0017] Further, the DNA tetrahedron framework nucleic acid is prepared by the following method: adding four DNA single strands in an equimolar ratio into a TM buffer solution with pH=8.0, maintaining at 95℃ for 10 min, and rapidly cooling to 4℃ for more than 20 min to obtain the DNA tetrahedron framework nucleic acid.

[0018] The application further provides a DNA tetrahedron complex antibacterial hydrogel for promoting wound healing, which is obtained by curing the aforementioned hydrogel precursor by ultraviolet light; preferably, the wavelength of the ultraviolet light is 365 nm.

[0019] The application further provides a method for preparing the aforementioned hydrogel, which comprises the following steps: taking the aforementioned hydrogel precursor, and curing by ultraviolet light to obtain the hydrogel; preferably, the wavelength of the ultraviolet light is 365 nm.

[0020] The application further provides the use of the aforementioned hydrogel precursor or the aforementioned hydrogel in the preparation of a drug for promoting wound healing.

[0021] Preferably, the wound is an infected wound.

[0022] Compared with the prior art, the application has the following beneficial effects:

[0023] The application provides a DNA tetrahedron complex antibacterial hydrogel for promoting the healing of infected wounds, which can be used as a dressing for treating infected skin wounds. The antibacterial peptide and tFNA are self-assembled into a controllable complex whole (tFNA-GL13K), and then an injectable hydrogel is prepared. The hydrogel has strong adhesion and deformation capacity, and excellent antibacterial and anti-inflammatory effects, and provides a micro-wet microenvironment for the wound, promotes wound healing and reduces scar formation. This strategy provides a new solution for treating infected skin wounds.

[0024] Obviously, according to the above content of the present application, other various forms of modification, replacement or change can be made according to the ordinary technical knowledge and common practice in the art without departing from the above basic technical idea of the present application.

[0025] The above content of the present application will be further explained in detail by a specific embodiment in the form of examples. However, it should not be understood that the scope of the above subject matter of the present application is limited to the following examples. Any technology realized based on the above content of the present application belongs to the scope of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 Preparation and characterization results of HAMA / tFNA-GL13K hydrogel: a is a schematic diagram of the preparation of HAMA / tFNA-GL13K hydrogel; b is the result of polyacrylamide gel electrophoresis (PAGE); c is the result of particle size measurement of tFNA and tFNA-GL13K by DLS and zeta potential measurement by ELS; d is the result of atomic force microscope evaluation of tFNA and tFNA-GL13K, scale = 500 nm; e is the TEM image of tFNA and tFNA-GL13K, scale = 100 nm; f is the optical photograph of each hydrogel before and after light curing; g is the SEM image of each hydrogel, the scale of the upper image = 1 mm, and the scale of the lower image = 300 μm; h is the attenuated total reflection Fourier transform infrared spectrum (ATR-FTIR) of HAMA and HAMA / tFNA-GL13K hydrogel; i is the result graph of the change of storage modulus (G') and loss modulus (G") with the oscillation frequency of the hydrogel.

[0027] Figure 2 Injectability of HAMA / tFNA-GL13K hydrogel: a is the injectability and light curing ability of HAMA / tFNA-GL13K hydrogel; b is the elasticity and viscosity of HAMA / tFNA-GL13K hydrogel.

[0028] Figure 3 In vitro antibacterial activity of HAMA / tFNA-GL13K hydrogel on E. coli and S. aureus: a is the image of E. coli and S. aureus incubated on agar plates containing different hydrogels; b is the CFU counting of E. coli and S. aureus, and the superscript a-d indicates that there is a significant difference in CFU between different treatment groups (p < 0.05); c is the growth curve of E. coli and S. aureus under the treatment of different hydrogels; n = 3 independent samples; the data is represented by mean ± standard deviation; the error bar represents SD.

[0029] Figure 4Results of the influence of HAMA / tFNA-GL13K hydrogel on the biological behavior of HaCaT cells: a is the distribution of Cy5-tFNA, FITC-GL13K and Cy5-FITC-t-GL13K complexes in HaCaT cells (Cy5: tFNA: red; FITC: yellow; nucleus: blue; cytoskeleton: green), scale bar = 20 pm; b is the flow cytometry analysis of the distribution of Cy5-tFNA, FITC-GL13K and Cy5-FITC-t-GL13K complexes in HaCaT cells, superscripts a-d indicate significant differences, p < 0.05; c is the CCK-8 assay to detect the cytotoxicity of hydrogel on HaCaT cells, superscripts a-d indicate significant differences, p < 0.05; d is the scratch test image of HaCaT cells treated with different hydrogels at 0 h, 4 h, 8 h, 12 h and 24 h, scale bar = 200 pm, the statistical analysis of the scratch test is marked with superscripts a-e, indicating significant differences, p < 0.05; e is the quantification of ROS in HaCaT cells; f is the quantification of inflammatory factor (IL-1β, IL-G, TNF-α and p65) expression in HaCaT cells, superscripts a-e indicate significant differences, p < 0.05; data is represented as mean ± SD (n = 3).

[0030] Figure 5 Results of the in vivo evaluation of HAMA / tFNA-GL13K hydrogel on skin repair and wound healing: a is the flowchart of in vivo experiment; b is the representative image of 5 groups of wound healing process at the specified time point; c is the wound area of 5 groups at 7 days and 14 days, superscripts a-c indicate significant differences, p < 0.05; d is the wound healing rate of different groups, p < 0.0001, ****; e is the image of wound tissue after H&E and Masson staining at 7 days, scale bar = 200 pm; f is the image of wound tissue after H&E and Masson staining at 14 days, scale bar = 4 mm (black) and 200 pm (white) (unhealed area is indicated by black and white dotted line); g is the quantitative analysis result of unhealed dermis area of five groups; h is the quantitative analysis result of epidermis thickness of five groups; i is the quantitative analysis result of collagen volume fraction; superscripts a-d indicate significant differences, p < 0.05, data is represented as mean ± SD (n = 3).

[0031] Figure 6 HE staining images of internal organs sections using HAMA / tFNA-GL13K hydrogel for 7 days and 14 days. DETAILED DESCRIPTION

[0032] The raw materials and equipment used in the specific embodiments of the present application are known products, which are obtained by purchasing commercially available products.

[0033] Example 1, synthesis and identification of HAMA / tFNA-GL13K hydrogel of the present application

[0034] The synthesis of the HAMA / tFNA-GL13K hydrogel of the present application is shown in the schematic diagram Figure 1 a.

[0035] 1. Synthesis of tFNA-GL13K

[0036] Four DNA single strands (ssDNA: S1, S2, S3, S4) with DNA sticky ends as shown in Table 1 were dissolved in TM buffer (10 mM Tris-HCl, 50 mM MgCl2, pH = 8.0) to make the final concentration of the four ssDNA 1000 nM, and then mixed thoroughly, heated to 95℃ for 10 minutes, and then quickly cooled to 4℃ and maintained for 20 minutes or more, to obtain the tetrahedral framework nucleic acid tFNA. Then the tFNA was mixed with the positively charged antibacterial peptide GL13K at a molar ratio of 1:500, incubated at room temperature for 30 min, to obtain the tFNA-GL13K complex.

[0037] Table 1. Nucleotide sequence of DNA single strand

[0038]

[0039] The amino acid sequence (from N to C) of the antibacterial peptide GL13K is: GKIIKLKASLKLL-CONH2 (SEQ. ID. NO 5), which is synthesized by a biological company.

[0040] 2. Synthesis of HAMA / tFNA-GL13K hydrogel

[0041] Hyaluronic acid methacrylate (HAMA) and photoinitiator LAP were weighed and dissolved in PBS to prepare a solution in the dark, then mixed with the above tFNA-GL13K complex, to obtain a mixed solution with a final concentration of HAMA of 10 mg / mL, a final concentration of LAP of 1 mg / mL, and a final concentration of tFNA-GL13K complex of 250 nM. The solution was stored at 4℃ after ultrasonic shaking and vortexing, and then injected into the wound site when used, and cured by UV 365 nm light for 30 s to obtain the HAMA / tFNA-GL13K (HAMA-t-G) hydrogel.

[0042] Hyaluronic acid methacrylate (HAMA) and photoinitiator LAP were weighed and dissolved in PBS to prepare a solution in the dark, then mixed with the tFNA prepared above, to obtain a mixed solution with a final concentration of 10 mg / mL HAMA, 1 mg / mL LAP and 250 nM tFNA. The solution was stored at 4°C after ultrasonic oscillation and vortex, and then injected into the wound site for UV 365 nm light curing for 30 s to obtain HAMA / tFNA (HAMA-t) hydrogel.

[0043] Hyaluronic acid methacrylate (HAMA) and photoinitiator LAP were weighed and dissolved in PBS to prepare a solution in the dark, then mixed with the tFNA prepared above, to obtain a mixed solution with a final concentration of 10 mg / mL HAMA, 1 mg / mL LAP and 250 nM tFNA. The solution was stored at 4°C after ultrasonic oscillation and vortex, and then injected into the wound site for UV 365 nm light curing for 30 s to obtain HAMA / tFNA (HAMA-t) hydrogel.

[0044] Hyaluronic acid methacrylate (HAMA) and photoinitiator LAP were weighed and dissolved in PBS to prepare a solution in the dark, then mixed with the tFNA prepared above, to obtain a mixed solution with a final concentration of 10 mg / mL HAMA, 1 mg / mL LAP and 250 nM tFNA. The solution was stored at 4°C after ultrasonic oscillation and vortex, and then injected into the wound site for UV 365 nm light curing for 30 s to obtain HAMA / tFNA (HAMA-t) hydrogel.

[0045] 3. Characterization of HAMA / tFNA-GL13K hydrogel and characterization results

[0046] Polyacrylamide gel electrophoresis (PAGE) was used to characterize the tFNA prepared and the tFNA-GL13K complex prepared according to the above method using different molar ratios of tFNA and GL13K (1:100, 1:250, 1:500, 1:750 and 1:1000). The tFNA-GL13K complex was abbreviated as t-G100, t-G250, t-G500, t-G750 and t-G1000, respectively, according to the molar ratio. According to the results of 8% PAGE gel, ssDNA was gradually loaded into tFNA, and finally GL13K was successfully loaded onto tFNA. Figure 1 b), the more GL13K loaded, the slower the movement speed of the complex, and the less clear the image, which more directly indicates that GL13K is successfully loaded onto tFNA.

[0047] The particle size measurement by DLS and zeta potential measurement by ELS of tFNA and tFNA-GL13K (t-G100 and t-G500) show that the particle size increases and the negative charge value decreases with the increase of GL13K loading, which proves the successful loading of GL13K Figure 1 c).

[0048] The shape of the material is determined by transmission electron microscopy and atomic force microscopy, and it can be seen that the shape and size of tFNA-GL13K (t-GL13K) and tFNA are consistent, and the particles are uniformly distributed in the form of triangles Figure 1 d and Figure 1 e), where t-GL13K is t-G500. According to the loading efficiency, the subsequent preparation of hydrogel is selected as the complex of tFNA and antibacterial peptide GL13K in a molar ratio of 1:500.

[0049] The optical photographs of HAMA, HAMA-t, HAMA-G and HAMA-t-G hydrogels before and after light curing are shown in Figure 1 f, and each hydrogel has flowability before light curing, and no flowability after light curing.

[0050] The porous structure of the hydrogel is scanned by scanning electron microscopy, and it can be seen that the surface pore size of HAMA-t-G hydrogel is about 120-150 μm, which is suitable for material and oxygen exchange of nutrients Figure 1 g).

[0051] Using attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) to detect HAMA and HAMA-t-G hydrogels, it can be seen that tFNA-GL13K is combined with HAMA hydrogel in the form of non-covalent bond to form HAMA-t-G hydrogel Figure 1 h).

[0052] Through the rheological experiment, the change of storage modulus (G') and loss modulus (G'') with the oscillation frequency can be seen that HAMA-t-G hydrogel has the strongest elasticity and moderate viscosity among the three hydrogels Figure 1 i).

[0053] HAMA-t-G hydrogel has injectability before light curing Figure 2 a), so that it is suitable for various shape wounds, and has adhesion and deformation ability after light curing Figure 2 b).

[0054] The beneficial effects of the present application are proved by the following specific test examples.

[0055] Test Example 1, antibacterial performance detection of HAMA / tFNA-GL13K hydrogel of the present application

[0056] 1. Plate count

[0057] HAMA / tFNA-GL13K (HAMA-t-G) hydrogels were prepared according to the method described in Example 1 using a molar ratio of tFNA and GL13K of 1 :500. 200 μL of the hydrogel was added to a 12-well plate and cured by UV light at 365 nm for 30 s. 1 mL of a suspension of S. aureus and E. coli (1 x 10 5 CFUs / mL) was added to the hydrogel-coated 12-well plate and incubated at 37 °C for 12 h. The treated bacterial suspension was diluted 1000-fold and 20 μL was added to LB agar plates. After incubation at 37 °C for 24 h, the colony forming units (CFUs) on the plates were counted. HAMA, HAMA-t and HAMA-G hydrogels prepared according to the method described in Example 1 were used as experimental controls. Plates containing bacteria only and no hydrogel were used as a control.

[0058] 2. Growth curve determination

[0059] HAMA-t-G, HAMA, HAMA-t and HAMA-G hydrogels prepared according to the method described in Example 1 were each taken 500 μL and soaked in 2 mL PBS for 12 h. The liquid after soaking was collected and used for the experiment.

[0060] S. aureus and E. coli, which had been incubated overnight (16 h), were adjusted to 1 x 10 5 CFUs / mL. The bacteria were harvested by centrifugation at 4000 g for 10 min at 4 °C and added to 200 μL LB broth (1 / 4 of the volume of the LB broth was the hydrogel soak) and incubated at 37 °C for 10 h. The OD 600nm values were measured every hour for 10 h using a spectrophotometer (UV1601, Shimadzu, Japan). The experiment was performed in triplicate at each time point and repeated at least three times. Plates containing bacteria only and no hydrogel soak were used as a control.

[0061] 3. Results

[0062] As shown in Figures Figure 3 a and 3b: there was no significant difference in the number of colonies between the HAMA hydrogel group and the control group, indicating that the HAMA hydrogel had no antibacterial effect. The HAMA-t hydrogel had a poor antibacterial effect. The HAMA-G and HAMA-t-G hydrogels both showed significant antibacterial properties and the number of colonies in the HAMA-t-G group was significantly lower than in the HAMA-G group. As shown in Figures Figure 3It can be seen that both S. aureus and E. coli showed growth inhibition in the presence of HAMA-t-G and HAMA-G hydrogels, and HAMA-t-G showed the strongest inhibition. The above results indicate that HAMA-t-G hydrogel has effective antibacterial properties for both gram-positive and gram-negative bacteria, and the presence of tFNA enhances the antibacterial ability of GL13K.

[0063] Test Example 2, Effect of HAMA / tFNA-GL13K Hydrogel of the Invention on the Biological Behavior of Keratinocytes (HaCaT)

[0064] HAMA-t-G hydrogel (HAMA-t-GL13K), HAMA-t hydrogel (HAMA-tFNAs) and HAMA-G hydrogel (HAMA-GL13K) were prepared according to the method described in Example 1, except that tFNA was replaced by Cy5-tFNA when preparing the HAMA-t hydrogel, GL13K was replaced by FITC-GL13K when preparing the HAMA-G hydrogel, and t-GL13K was t-GL13K loaded with both Cy5 and FITC when preparing the HAMA-t-G hydrogel.

[0065] 500 μL each of the prepared HAMA-t-G hydrogel, HAMA hydrogel, HAMA-t hydrogel and HAMA-G hydrogel were soaked in 2 mL of PBS for 12 hours, and the soaked liquid was collected to obtain each hydrogel soaking liquid for use in experiments.

[0066] 1. Distribution of Cy5 and FITC-loaded t-GL13K in HaCaT cells

[0067] Flow cytometry and immunofluorescence analysis were used to detect the distribution of tFNA, GL13K and t-GL13K complexes in HaCaT cells.

[0068] First, HaCaT cells were seeded in a six-well plate (2 x 10 5 After 24 hours of culture, the standard culture medium was replaced with 2% FBS RPMI culture medium, and LPS (10 μg / ml) was added to simulate the microenvironment of bacterial infection. After 1 hour, 500 μl of hydrogel soaking liquid containing FITC-GL13K (125 μM), Cy5-tFNA (250 nM) or Cy5 and FITC-loaded t-GL13K (250 nM) was added, and after 6 hours of incubation, the cells were extracted. Flow cytometry was used to evaluate the cellular distribution of tFNA, GL13K and t-GL13K complexes.

[0069] HaCat cells were seeded in a cell culture dish (2 x 10 5After 24 hours of incubation, the standard medium was replaced with 2% FBS RPMI medium and LPS (10 pg / ml) was added to mimic the bacterial infected microenvironment. After 1 hour, 500 mI of hydrogel soaking solution containing FITC-GL13K (125 pM), Cy5-tFNA (250 nM) or Cy5 and FITC loaded t-GL13K (250 nM) was added and the cells were incubated for 6 hours. The cells in the wells were washed with PBS for 3 times and fixed with 4% (w / v) paraformaldehyde solution (Boster, Wuhan, China) for 15 minutes. After washing again, the cells were stained with red ghost pen for 20 minutes and DAPI for 10 minutes, and washed with PBS for 3 times after each step. Finally, the cells were observed under an ultrahigh-resolution two-photon laser confocal microscope (N-SIM, Nikon, Tokyo, Japan).

[0070] The cell distribution was observed using confocal fluorescence microscopy, with the FITC-GL13K channel marked in yellow and the Cy5-tFNA channel marked in red Figure 4 a) GL13K was mainly found in the extracellular matrix, and tFNA and GL13K potentially influenced each other in the distribution of cells. Further flow cytometry analysis was used to quantify the cell distribution of t-GL13K. It was found that both GL13K and t-GL13K had high cell uptake and fluorescence intensity after 6 hours of treatment Figure 4 b).

[0071] 2. CCK8 cytotoxicity experiment

[0072] CCK-8 assay (KeyGEN, Jiangsu, China) was used to evaluate the cytotoxicity of the samples. HaCat cells were cultured in 96-well plates (5 x 10 3 / well) overnight, and the medium was replaced with RPMI containing 2% (v / v) FBS and 10 pg / ml LPS, and 50 mI of hydrogel soaking solution was added after 1 hour. For the control group, no hydrogel soaking solution was added to the cell culture medium. The cytotoxicity was checked after 6 hours, 12 hours and 24 hours of CCK-8 solution treatment. RPMI medium was used as a blank group, and the absorbance of each well was measured at a wavelength of 450 nm.

[0073] There was no significant difference in cell proliferation of HaCaT cells between HAMA-tFNAs, HAMA-t-GL13K and the control group after 6, 12 and 24 hours of treatment Figure 4c). The cytotoxicity of the LPS and HAMA-GL13K groups was significantly higher than that of other groups, indicating that HAMA-tFNAs and HAMA-t-GL13K can alleviate the inhibition of cell proliferation by LPS, and further demonstrating that the presence of tFNA can reduce the cytotoxicity of GL13K.

[0074] 3. Cell scratch assay

[0075] Cell migration was observed using a scratch assay. HaCaT cells (2×10⁻⁶) were used. 5 Cells were seeded in 12-well plates and cultured overnight. After washing with PBS, each well was cross-streaked using a 200 μL pipette tip. After washing three times, cells were cultured in RPMI medium containing 2% (v / v) FBS and 10 μg / ml LPS. One hour later, 250 μL of hydrogel soaking medium was added for each group. Images were taken under a light microscope after 4, 8, 12, and 24 hours of treatment. The scratch area was measured and recorded, and compared with the original scratch at baseline using Image-J software.

[0076] In the presence of LPS, both LPS and HAMA-GL13K significantly inhibited HaCaT cell migration, while HAMA-tFNAs and HAMA-t-GL13K promoted the closure of monolayer wounds on scratched HaCaT cells, with the HAMA-t-GL13K group showing a significantly better effect on promoting wound closure than the HAMA-tFNAs group. Figure 4 d). The results showed that tFNAs and GL13K could synergistically promote cell migration injury and further promote wound closure.

[0077] 4. Detection of reactive oxygen species (ROS) and RNA isolation and quantitative reverse transcription polymerase chain reaction (qRT-PCR)

[0078] The presence of reactive oxygen species (ROS) and inflammatory factors can exacerbate the inflammatory response, thereby affecting wound healing.

[0079] Intracellular ROS levels were determined by DCF staining and flow cytometry. DCF is a fluorescent material derived from DCFH-DA that is permeable to cells. HaCat cells were cultured in groups in 96-well plates (5 × 10⁻⁶). 3 Cells were cultured overnight in wells ( / well). The medium was replaced with RPMI containing 2% (v / v) FBS and 10 μg / ml LPS. One hour later, 50 μl of hydrogel soaking buffer was added to each group. After treatment with hydrogel soaking buffer for 6 hours, DCFH-DA was diluted with serum-free medium (1:1000 v / v) and added to the cell suspension. The cells were incubated at 37°C in the dark for 30 minutes. Cells were collected and washed three times with PBS, and DCF levels in the cells were measured by flow cytometry.

[0080] HaCaT cells (2×10) 5 HaCaT cells were seeded in 6-well plates and cultured overnight. Cells were cultured in RPMI medium containing 2% (v / v) FBS and 10 μg / ml LPS. After 1 hour, 500 μl of hydrogel soaking buffer was added to each group. After 6 hours of treatment, RNA from HaCaT cells was isolated and purified using TriZol reagent and chloroform. RNA was reverse transcribed into cDNA using a cDNA synthesis kit, and genes encoding inflammation-related factors, including TNF-α, IL-1β, IL-6, and p65, were expressed. PCR was performed using a SYBR Green qPCR kit, with GADPH as an internal control. Primer sequences are shown in Table 2. Relative quantification was performed using the 2-ΔΔCT method.

[0081] LPS is a common inflammatory stimulant in bacterial infections; therefore, this invention uses LPS to simulate the overactive inflammatory response process at the site of bacterial infection. Flow cytometry analysis of intracellular ROS levels revealed that HAMA-tFNAs and HAMA-t-GL13K reduced ROS generated by LPS stimulation in HaCaT cells. Figure 4 e). q-PCR results showed ( Figure 4 f) HAMA-t-GL13K can downregulate the expression of LPS-induced inflammatory factors IL-1β, TNF-α, and IL-6, while HAMA-GL13K has no regulatory effect on the expression of the above inflammatory factors.

[0082] Table 2. Primer Sequences

[0083]

[0084]

[0085] Experimental Example 3: The effect of the HAMA / tFNA-GL13K hydrogel of the present invention on the treatment prognosis of an animal model of infected skin wounds.

[0086] Mice were anesthetized and placed on an operating table. After removing the hair from their backs using an electric shaver and wax, a circular incision penetrating the full thickness of the skin was made on the back of each mouse using a 10 mm diameter biopsy puncturist. 10 μL each of Staphylococcus aureus and Escherichia coli (CFU = 10) were then injected. 8ml) was instilled on the exposed wound to establish the infected wound. All mice were randomly divided into 5 groups (n=6), from the second day, the wound sites were treated according to the grouping by topical application of hydrogels (HAMA hydrogel, HAMA-t hydrogel, HAMA-G hydrogel and HAMA-t-G hydrogel prepared according to the method described in Example 1). And the wound was observed and photographed on postoperative days 0, 3, 5, 7, 10, 14, half of the mice were euthanized on postoperative days 7 and 14, then the wound area and surrounding tissues were immediately collected for further analysis, and the heart, liver, spleen, lung and kidney were dissected for biosafety test.

[0087] After the skin and internal organs were fixed with 4% paraformaldehyde for 1 hour, they were embedded in paraffin, cut into 4 pm sections, and stained with Masson staining kit or hematoxylin-eosin (HE) staining kit. The stained tissue sections were imaged using FSX100 microscope (Olympus, Tokyo, Japan). The unhealed wound area, epidermis thickness and fibrosis percentage of each sample were obtained by measuring three different fields of each slide and taking the average value.

[0088] The in vivo healing effect of the hydrogel was evaluated by analyzing the wound area by gross observation. Figure 5 b shows representative images of the wounds after S. aureus and E. coli infection on days 0, 3, 5, 7, 10 and 14. The wound area was analyzed on postoperative days 7 and 14, there was no difference between the HAMA group and the control group, the area of the HAMA-t and HAMA-G groups was smaller than that of the control group, and the area of the HAMA-t-G group was the smallest Figure 5 c), the relevant wound healing rate of the infected mice is shown in Figure 5 d, the Two-way ANOVA analysis showed that there was a significant difference between each group. The wound healing rate of the HAMA-t-G group after 14 days of treatment was 97.83±1.566%, which was the best treatment effect in each group.

[0089] Histological analysis was used to evaluate the quality of regenerated tissue in each group Figure 5 e-5i). On day 7, HE and MASSON staining sections showed that the HAMA-t-G group had the least inflammatory cell infiltration and the most collagen fiber deposition in the dermal tissue Figure 5 e). On day 14, the unhealed wound area of the HAMA-t-G group was significantly reduced compared with the control group and other groups Figure 5 g, p<0.05). In addition, on day 14, the epidermis thickness of the HAMA-t and HAMA-t-G groups had no significant difference with healthy skin, while the epidermis thickness of the other groups was still thin Figure 5h) On day 14, Masson staining showed that there was collagen fiber deposition in the dermal tissue of the HAMA-t-G group, while there was almost no deposition in the wound site of the HAMA-G group, which was not conducive to wound healing. However, a large amount of collagen fibers were deposited in the control group and the HAMA group, leading to scar formation Figure 5 i, p<0.05). Although the control group and the HAMA group both formed basic epithelial and dermal structures, significant inflammatory reactions could be observed during the dermal repair process. In addition, it was found that Figure 6 It can be known that the HAMA-t-G hydrogel has no obvious side effects on various organs.

[0090] The above experimental results show that the HAMA-t-G hydrogel prepared by the present application has excellent antibacterial and anti-inflammatory effects, can significantly promote the healing of infected wounds, and inhibit scar formation.

[0091] The HAMA-t-G hydrogel can improve the inflammatory microenvironment of bacterial infected skin wounds by inhibiting bacteria in the skin lesion site, eliminating ROS, and inhibiting the expression of inflammatory factors. The HAMA-t-G hydrogel can significantly reduce the area of unhealed wounds and the degree of fibrosis, and the effect of HAMA-t-G is much higher than that of the control group. In addition, HAMA-t-G promotes the proliferation of keratinocytes under LPS stimulation, thereby promoting wound closure.

[0092] Compared with the hydrogel prepared by using tFNA and GL13K alone or the HAMA group, the HAMA / tFNA-GL13K hydrogel has excellent effect on improving the inflammatory environment of bacterial infected skin wounds, has excellent therapeutic effect, and plays a synergistic effect.

[0093] In summary, the present application provides a DNA tetrahedron complex antibacterial hydrogel for promoting the healing of infected wounds, which can be used as a dressing for treating infected skin wounds. The antibacterial peptide and tFNA are first assembled into a controllable complex whole (tFNA-GL13K), and then an injectable hydrogel is prepared. The hydrogel has strong adhesion and deformation ability, and excellent antibacterial and anti-inflammatory effects, providing a micro-wet microenvironment for the wound, promoting wound healing and reducing scar formation. This strategy provides a new solution for the treatment of infected skin wounds.

Claims

1. A DNA tetrahedral complex antibacterial hydrogel precursor to promote wound healing, characterized in that: It is obtained by dissolving the DNA tetrahedral framework nucleic acid containing antibacterial peptide, hyaluronic acid methacrylate and photoinitiator in a solvent; The DNA tetrahedral framework nucleic acid containing antibacterial peptide is obtained by mixing and incubating DNA tetrahedral framework nucleic acid and antibacterial peptide; The DNA tetrahedral framework nucleic acid is a tetrahedral structure formed by self-assembly of single-stranded DNA with nucleotide sequences such as SEQ NO. 1-4 through base complementary pairing; The antibacterial peptide is antibacterial peptide GL13K.

2. The hydrogel precursor of claim 1, wherein: After being dissolved in the solvent, the concentration of the DNA tetrahedral framework nucleic acid containing antibacterial peptide is 200-300 nM; the concentration of hyaluronic acid methacrylate is 10-15 mg / mL, and the concentration of photoinitiator is 1-3 mg / mL.

3. The hydrogel precursor of claim 2, wherein: After being dissolved in the solvent, the concentration of the DNA tetrahedral framework nucleic acid containing antibacterial peptide is 250 nM; the concentration of hyaluronic acid methacrylate is 10 mg / mL, and the concentration of photoinitiator is 1 mg / mL.

4. The hydrogel precursor of any one of claims 1-3, wherein: The solvent is PBS or physiological saline; and / or, the photoinitiator is LAP.

5. The hydrogel precursor of any one of claims 1-3, wherein: When the DNA tetrahedral framework nucleic acid and antibacterial peptide are mixed and incubated, the molar ratio of the DNA tetrahedral framework nucleic acid to antibacterial peptide is 1:100-500.

6. The hydrogel precursor of claim 5, wherein: The molar ratio of the DNA tetrahedral framework nucleic acid to antibacterial peptide is 1:

500.

7. The hydrogel precursor of any one of claims 1-3, wherein: The preparation method of the DNA tetrahedral framework nucleic acid containing antibacterial peptide comprises the following steps: mixing the DNA tetrahedral framework nucleic acid and antibacterial peptide, and incubating at 20-40℃ for 30-40 min to obtain the DNA tetrahedral framework nucleic acid containing antibacterial peptide.

8. The hydrogel precursor of any one of claims 1-3, wherein: The DNA tetrahedral framework nucleic acid is prepared by the following method: adding four DNA single strands in equal molar ratio into TM buffer solution with pH = 8.0, maintaining at 95℃ for 10 min, rapidly cooling to 4℃ for more than 20 min to obtain the DNA tetrahedral framework nucleic acid.

9. A DNA tetrahedron complex antibacterial hydrogel to promote wound healing, characterized in that: It is obtained by subjecting the hydrogel precursor of any one of claims 1-8 to ultraviolet light curing.

10. The DNA tetrahedral complex-antibacterial hydrogel according to claim 9, wherein: The wavelength of the ultraviolet light is 365 nm.

11. A method of preparing a hydrogel according to claim 9 or 10, characterized in that: It comprises the following steps: taking the hydrogel precursor of any one of claims 1-8, and obtaining the hydrogel after ultraviolet light curing.

12. The method of claim 11, wherein: The wavelength of the ultraviolet light is 365 nm.

13. Use of the hydrogel precursor of any one of claims 1-8 or the hydrogel of claim 9 or 10 in the preparation of a drug for promoting wound healing.

14. Use according to claim 13, characterized in that: The wound is an infected wound.

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