Lactoferrin and antibacterial peptide bifunctional hydrogel dressing as well as preparation method and application thereof

Abstract

The invention discloses a lactoferrin and antibacterial peptide bifunctional hydrogel dressing as well as a preparation method and application thereof. The hydrogel dressing is prepared by mixing lactoferrin, antibacterial peptide NZ2114, a magnesium lithium silicate gel matrix and water. The hydrogel dressing provided by the invention has the following advantages: 1) the hydrogel dressing is simple to prepare, low in cost and suitable for batch production; 2) the gel is convenient to apply, has a moisturizing effect, is long in residence time, good in biocompatibility, small in irritation, safe and reliable, or is accurately injected to the wound, moisturizes, slowly releases and efficiently improves the compliance of the body, and 3) the gel is good in antibacterial and anti-inflammatory effects, good in biocompatibility, free of irritation and capable of promoting wound healing, and only needs to be smeared on the surface of the wound.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and specifically to a lactoferrin and antimicrobial peptide dual-functional hydrogel dressing and a preparation method and application thereof. Background Art

[0002] Wound infection is a common disease in humans and animals (including pets). Some purulent bacteria such as Staphylococcus aureus invade the skin through mechanical, thermal, and electrical damage, causing local inflammation and suppuration, delaying wound healing, and even endangering life and health (S. Raffaele, 2015). Currently, antibiotics are the main drugs used to treat wound infections, but the tolerance of multiple drug-resistant bacteria to them greatly reduces the efficacy of the drugs. Some antibiotic substitutes have been studied. Some nanomaterials such as silica, nanogold, and nanosilver have better antibacterial effects and biological penetration ability of drug-resistant bacteria than antibiotics, but their synthesis methods are complicated and most materials have unresolved toxicity problems (S. Kaul, 2022). Wound dressings are considered to be one of the most effective strategies to protect wounds from bacterial infection and water loss. Among wound dressings, hydrogels have the advantages of good moisture retention and biocompatibility, and can be used as carrier delivery systems, which have attracted widespread attention from researchers. The moist environment they provide can promote wound healing (Y. Huang, 2022). However, traditional hydrogels exist in a specific form and need to be fixed to the wound site, which may cause a prolonged healing period and discomfort to the body. Therefore, it is of great significance to prepare an antibacterial and highly adaptable hydrogel dressing.

[0003] Compared with the original hydrogel, injectable hydrogel can be injected into specific sites and maintain low toxicity. It is widely used in drug, bioactive small molecules and cell delivery systems. Injectable hydrogels are mainly formed by physical cross-linking and chemical cross-linking. Compared with chemical cross-linking, physical cross-linking has the advantages of simple operation, no need for cross-linking catalysts and no heat generation, so as to achieve efficient absorption and utilization of specific sites. Antimicrobial peptides are small peptide molecules. As an important component of the body's defense, they have cationic and amphiphilic characteristics and can interact with the negative charge on the surface of bacteria to exert efficient antibacterial activity. Their unique bactericidal mechanism makes it difficult for them to induce drug resistance in bacteria. As the next generation of antibiotic substitutes, they have attracted widespread attention from researchers (Z. Yang, 2021). Lactoferrin (LF) is an iron-binding glycoprotein with a molecular weight of 70-80 kDa, an isoelectric point of 8.4-9.0, and a positive charge in a wide pH range. LF is one of the multifunctional proteins in mammals, and plays an important role in anti-inflammatory, immunomodulatory, anticancer, antiviral and antibacterial functions (B. Wang, 2022). LF has antibacterial and anti-inflammatory functions and has the potential to be a candidate for wound infection treatment. However, it does not form a hydrogel by itself. It can form an injectable hydrogel by cross-linking with anionic gel matrix using its surface positive charge. At present, there are no reports on the combination of LF and antimicrobial peptides for the preparation of wound dressings that promote healing. Summary of the invention

[0004] The purpose of the present invention is to provide a lactoferrin and antimicrobial peptide dual-functional hydrogel dressing and a preparation method and application thereof.

[0005] The concept of the present invention is as follows: through the anti-inflammatory, immunomodulatory and surface positive charge properties of lactoferrin, the high antibacterial activity and surface positive charge characteristics of the antimicrobial peptide NZ2114 (Y. Zhang, 2014), and the surface negative charge and good biocompatibility characteristics of lithium magnesium silicate, electrostatic force is used to prepare an antibacterial and anti-inflammatory dual-functional injectable hydrogel Lf / NZ2114 wound dressing.

[0006] In order to achieve the purpose of the present invention, in a first aspect, the present invention provides a lactoferrin and antimicrobial peptide dual-functional (having antibacterial and anti-inflammatory functions) hydrogel dressing, which is prepared by mixing lactoferrin, antimicrobial peptide NZ2114, a gel matrix and water (sterile deionized water).

[0007] The amino acid sequence of the antimicrobial peptide NZ2114 of the present invention has been disclosed in CN103045636A.

[0008] Preferably, the lactoferrin is bovine lactoferrin.

[0009] Furthermore, in the hydrogel dressing, the concentration of bovine lactoferrin is 20-320 mg / mL, the concentration of antimicrobial peptide NZ2114 is 200-1600 µg / mL, and the concentration of lithium magnesium silicate is 0.25-1.5%.

[0010] Preferably, the concentration of bovine lactoferrin is 20 mg / mL, 40 mg / mL, 80 mg / mL, 160 mg / mL or 320 mg / mL, the concentration of antimicrobial peptide NZ2114 is 200 µg / mL, 400 µg / mL, 800 µg / mL or 1600 µg / mL, and the concentration of lithium magnesium silicate is 0.25%, 0.5%, 1%, or 1.5%.

[0011] More preferably, the concentration of bovine lactoferrin is 40 mg / mL, the concentration of antimicrobial peptide NZ2114 is 400 µg / mL, and the concentration of lithium magnesium silicate is 1% or 1.5%.

[0012] In a second aspect, the present invention provides a method for preparing the hydrogel dressing, comprising the following steps: (1) Using sterile water for injection, prepare lactoferrin solution, antimicrobial peptide solution and lithium magnesium silicate gel matrix solution respectively according to the mass ratio; (2) Mix the lactoferrin solution, antimicrobial peptide solution and lithium magnesium silicate gel matrix solution in proportion, stir thoroughly, and perform ultrasonic treatment to obtain the product.

[0013] In a third aspect, the present invention provides any of the following applications of the hydrogel dressing: 1) Used for preparing trauma medical supplies; 2) Used to prepare drugs for promoting the healing of trauma, burns or infected wounds.

[0014] Among them, the infection described in 2) is caused by Gram-positive bacteria.

[0015] The Gram-positive bacteria include but are not limited to Staphylococcus aureus ( Staphylococcus aureus ).

[0016] In a fourth aspect, the present invention provides a composition, the active ingredients of which are bovine lactoferrin and antimicrobial peptide NZ2114.

[0017] Among them, the concentration of bovine lactoferrin is 20-320 mg / mL, and the concentration of antimicrobial peptide NZ2114 is 200-1600 µg / mL.

[0018] Preferably, the concentration of bovine lactoferrin is 20 mg / mL, 40 mg / mL, 80 mg / mL, 160 mg / mL or 320 mg / mL, and the concentration of antimicrobial peptide NZ2114 is 200 µg / mL, 400 µg / mL, 800 µg / mL or 1600 µg / mL.

[0019] More preferably, the concentration of bovine lactoferrin is 40 mg / mL, and the concentration of antimicrobial peptide NZ2114 is 400 µg / mL.

[0020] In a fifth aspect, the present invention provides any of the following applications of the composition: a) Used for preparing anti-inflammatory and immunomodulatory adjuvants; b) for the preparation of antibacterial products; c) Used for preparing a healing promoting agent.

[0021] Wherein, the bacteria described in b) are Gram-positive bacteria, such as Staphylococcus aureus and the like.

[0022] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects: (i) The present invention provides a lactoferrin and antimicrobial peptide hydrogel wound dressing, which forms a porous mesh gel through simple electrostatic interaction between a positively charged lactoferrin solution and a negatively charged lithium magnesium silicate. The preparation cost is low and the wound dressing is suitable for mass production.

[0023] (ii) It is simple to use and easy to administer. Simply apply the gel to the wound surface. It has a moisturizing effect, a long retention time, good biocompatibility, little irritation, and is safe and reliable. Alternatively, it can be precisely injected into the wound to moisturize, release slowly and efficiently, and increase the body's compliance.

[0024] (III) It has good antibacterial and anti-inflammatory effects, good biocompatibility and is non-irritating, and promotes wound healing. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the hydrogel form prepared in the preferred embodiment of the present invention.

[0026] Figure 2 The results of the in vitro inhibition zone test of the hydrogel in the preferred embodiment of the present invention are shown.

[0027] Figure 3 The results of Fourier transform infrared spectroscopy test of hydrogel in the preferred embodiment of the present invention are shown.

[0028] Figure 4 The following are the results of rheological properties analysis of the hydrogel in the preferred embodiment of the present invention.

[0029]

[0030] Figure 6 The in vitro safety evaluation of the hydrogel in the preferred embodiment of the present invention. ab: hemolytic test; c: epithelial cell toxicity test results.

[0031] Figure 7 Evaluation of the in vivo therapeutic effect of the hydrogel in the preferred embodiment of the present invention. a: Changes in mouse body weight; b: Bacterial load in epithelial tissue; c: Pictures of mouse wound recovery; d: Mouse wound recovery rate.

[0032] Figure 8 This is a HE-stained section of mouse skin tissue in a preferred embodiment of the present invention.

[0033] Fig. 9 It is the expression level of mouse inflammatory factor (IL-6), vascular endothelial growth factor (VECF), epidermal growth factor (EGFR) and platelet endothelial cell adhesion molecule (CD31) in the preferred embodiment of the present invention.

[0034] Fig.10 The results of immunohistochemistry (CD31 and IL-6) analysis of mouse skin tissue in a preferred embodiment of the present invention are shown. DETAILED DESCRIPTION

[0035] The present invention aims to provide a novel gel wound dressing and a preparation method and application thereof.

[0036] The present invention adopts the following technical solution: The invention provides a lactoferrin and antimicrobial peptide hydrogel wound dressing, which comprises an active ingredient, a gel matrix and a solvent. The active ingredient comprises bovine lactoferrin and the antimicrobial peptide NZ2114, the gel matrix is ​​lithium magnesium silicate, and the solvent is sterile deionized water.

[0037] Preferably, the hydrogel comprises the following components by mass fraction: lactoferrin concentration is 20 mg / mL, 40 mg / mL, 80 mg / mL, 160 mg / mL, 320 mg / mL, antimicrobial peptide NZ2114 concentration is 200 µg / mL, 400 µg / mL, 800 µg / mL, 1600 µg / mL, lithium magnesium silicate concentration is 0.25%, 0.5%, 1%, 1.5%, and the rest is sterile deionized water.

[0038] Preferably, the hydrogel comprises the following ingredients: lactoferrin at a concentration of 40 mg / mL, antimicrobial peptide NZ2114 at a concentration of 400 µg / mL, lithium magnesium silicate at a concentration of 1% or 1.5%, and the remainder being deionized water.

[0039] The present invention also provides any of the following applications of the lactoferrin and antimicrobial peptide NZ2114: 1) Used for the preparation of anti-inflammatory and immunomodulatory adjuvants; 2) Used to prepare antibacterial products; 3) Used to prepare healing agents.

[0040] The bacteria are Staphylococcus aureus and the like.

[0041] The present invention also provides a method for preparing a lactoferrin and antimicrobial peptide hydrogel wound dressing (injectable hydrogel Lf / NZ2114), comprising the following steps: 1) Take an appropriate amount of sterile water for injection according to the mass ratio, add lactoferrin and antimicrobial peptide respectively, stir and mix thoroughly to prepare the solution.

[0042] 2) Prepare the lithium magnesium silicate gel matrix solution according to the mass ratio and stir thoroughly to mix.

[0043] 3) The lactoferrin and antimicrobial peptide solution and the gel matrix solution are fully stirred and sonicated to obtain the lactoferrin and antimicrobial peptide hydrogel.

[0044] The present invention also provides any of the following applications of a lactoferrin and antimicrobial peptide hydrogel wound dressing: 1) Application in the preparation of trauma medical supplies; 2) Application in the preparation of drugs for promoting the healing of trauma, burns or infected wounds; Among them, the infection described in 2) is caused by Staphylococcus aureus.

[0045] Lactoferrin is an iron-binding glycoprotein with a molecular weight of 70-80 kDa, an isoelectric point of 8.4-9.0, and a positive charge in a wide pH range. LF is one of the multifunctional proteins in mammals, and plays an important role in anti-inflammatory, immunomodulatory, anticancer, antiviral and antibacterial functions. Anti-inflammatory and immunomodulatory functions are the main functions of lactoferrin. It has also been reported to have antibacterial activity, but requires a higher concentration of more than mg / mL, which increases the cost of the drug and makes it difficult to apply. Reducing the amount of lactoferrin used and using it in combination with other drugs is a better choice. LF's antibacterial and anti-inflammatory functions have the potential to be a candidate for the treatment of wound infections, but it does not form a hydrogel itself. It can form an injectable hydrogel by cross-linking with an anionic gel matrix using surface positive charges.

[0046] The antimicrobial peptide NZ2114 is a relatively excellent derivative peptide of plectasin, with a molecular weight of 4400Da and an isoelectric point PI of 8.4. It behaves as a cation in an environment below the isoelectric point. It has a strong inhibitory effect on Gram-positive bacteria, especially Staphylococcus aureus, and has good biocompatibility. It can be used as a candidate drug for wound treatment, but it cannot form a hydrogel by itself and is unstable when used in vivo. It can form a hydrogel by using the interaction between its surface positive and negative charges.

[0047] Lithium magnesium silicate is an excellent hydrogel matrix material. Its sheet surface is negatively charged, while its edge is positively charged. The unique positive and negative charges attract each other to form a hydrogel with a three-dimensional network structure. It is also found to have antibacterial, anti-inflammatory, anti-tumor, antioxidant and immunomodulatory functions. Lithium magnesium silicate also exhibits good biocompatibility, promotes cell adhesion and proliferation, promotes platelet aggregation, and exerts a hemostatic effect. The negative charge on the surface of lithium magnesium silicate can interact with the positive charge on the surface of lactoferrin and antimicrobial peptide NZ2114 to form an injectable hydrogel, which meets the requirements of antibacterial and healing-promoting high-quality wound dressings.

[0048] The present invention also conducts a comprehensive and systematic analysis of the physicochemical properties (antibacterial, anti-inflammatory, and biocompatibility) of the injectable hydrogel Lf / NZ2114 wound dressing.

[0049] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0050] The lactoferrin used in the following examples is bovine lactoferrin purchased from Shanghai Yuanye Company.

[0051] Lithium magnesium silicate was purchased from Shanghai Yuanye Company.

[0052] Chitosan (HACC) and sodium alginate (SA) were purchased from Shanghai McLean Company.

[0053] Example 1 Preparation of injectable hydrogel Lf / NZ2114 / LMSH

[0054] The concentrations of lithium magnesium silicate (LMSH) were 0.25%, 0.5%, 1%, and 1.5%, and the final concentrations of 20 mg / mL, 40 mg / mL, 80 mg / mL, 160 mg / mL, and 320 mg / mL lactoferrin (Lf) were fully stirred and ultrasonically mixed to determine the conditions for gel formation; at the same time, the final concentrations of 200 µg / mL, 400 µg / mL, 800 µg / mL, and 1600 µg / mL antimicrobial peptide NZ2114 were fully stirred and ultrasonically mixed to determine the conditions for gel formation. The concentrations of the components in the Lf / NZ2114 / LMSH hydrogel were finally determined to be 1.5% or 1% LMSH, 40 mg / mL Lf, and 400 µg / mL antimicrobial peptide NZ2114. According to previous laboratory reports, 3% chitosan hydrogel can be coated with NZ2114 to form a hydrogel. In this experiment, 3% HACC hydrogel was coated with the above concentrations of Lf and NZ2114 to prepare the hydrogel Lf / NZ2114 / HACC as a control. Figure 1It shows that Lf and NZ2114 can form milky yellowish solutions with lithium magnesium silicate at room temperature, among which 0.25% LMSH and 0.5% LMSH cannot form gels, while 1% LMSH and 1.5% LMSH undergo a rapid sol-gel transition and can form hydrogels within seconds.

[0055] Example 2 Determination of the Minimum Inhibitory Concentration of Lactoferrin and Antimicrobial Peptide NZ2114 against Staphylococcus aureus

[0056] The minimum inhibitory concentration (MIC) was determined according to the method established by the Clinical and Laboratory Standards Institute (CLSI, https: / / clsi.org / ). A single colony of Staphylococcus aureus ATCC 25923 was picked into MHB liquid medium, cultured overnight at 37°C and 250 rpm for activation, and then transferred to MHB liquid medium for culture until the logarithmic growth phase, and then prepared into 10 5 CFU / mL of bacterial solution was added to a 96-well sterile cell culture plate, 90 μL per well. Lactoferrin and antimicrobial peptide NZ2114 were diluted with PBS by a 2-fold dilution method, 10 μL of succinate per well, so that the final concentrations were 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.0625, 0.0312 and 0.0156 μg / mL, respectively. The negative control group was PBS instead of the test bacterial solution of antimicrobial peptides, and the blank control group was sterile MHB medium. Three parallel samples were prepared for each treatment. The culture plate was placed in a 37°C constant temperature incubator and incubated for 16 to 18 hours until the negative control well showed obvious turbid bacterial solution visible to the naked eye. The lowest concentration that could completely inhibit bacterial growth was the MIC value of the antimicrobial peptide for the test strain. If skipping or inconsistent results between parallel samples occurred, the test was repeated. The results are shown in Table 1. The antimicrobial peptide NZ2114 has a strong inhibitory effect on Staphylococcus aureus with a MIC of 4 μg / mL. Lactoferrin has a weak inhibitory effect on Staphylococcus aureus and mainly plays an immunomodulatory and anti-inflammatory function.

[0057] Table 1 MIC of lactoferrin and antimicrobial peptides against Staphylococcus aureus

[0058] Example 3 Performance testing and effect evaluation of injectable hydrogel

[0059] 1. Hydrogel characterization

[0060] The inhibition zone test was used to preliminarily screen the effect of hydrogels on the antibacterial activity of lactoferrin and NZ2114. Specifically, the hydrogels prepared in Example 1 (Lf / NZ2114 / LMSH, Lf / NZ2114 / HACC) and the control groups (CK, Lf, NZ2114, Lf+NZ2114, 3%SA, Lf / NZ2114 / 3%SA) were incubated with 30 μL of the suspension droplets on MHA plates (containing logarithmic phase Staphylococcus aureus, 1×10 6 CFU / mL), cultured in a 37℃ constant temperature incubator for 16 h and observed the inhibition zone. Figure 2 As shown, the combination of antimicrobial peptide NZ2114 with lithium magnesium silicate has almost no effect on its inhibitory effect on Staphylococcus aureus.

[0061] German BRUKER Fourier transform near infrared spectrometer TENSOR 27 (FT-IR, wavelength range 400-4000cm-1) was used to determine the chemical structure of Lf, NZ2114, LMSH, HACC Lf + NZ2114, 1.5% Lf / NZ2114 / LMSH, 1% Lf / NZ2114 / LMSH, 3% Lf / NZ2114 / HACC (sample dry powder was ground and mixed with potassium bromide, pressed into tablets and put into the instrument for detection), and scanning electron microscopy was used to observe the surface morphology of the gel (freeze-dried samples were fixed on silicon wafers, sprayed with gold and put into the instrument for observation). All samples were freeze-dried. The results are shown in Figure 3 As shown, the synthesized hydrogel Lf / NZ2114 / LMSH simultaneously showed the characteristic peaks of Lf, NZ2114 and LMSH, indicating that the three materials were successfully compounded by physical means without chemical reaction. The preparation is simple, the cost is low, and it is suitable for mass production.

[0062] The shear thinning characteristics and modulus of the gels 1% Lf / NZ2114 / LMSH, 1.5% Lf / NZ2114 / LMSH, and 3% Lf / NZ2114 / HACC were tested using a Physica MCR 301 rotational rheometer. 2-3 ml of gel was evenly spread on the rheometer disk, and a small amount of silicone oil was added to prevent water evaporation. The relationship between the shear force and viscosity of the gel was measured at different frequencies (0.01-100 s-1). At the same time, the shear thinning ability of the gel was studied at a fixed frequency (1 Hz) and a strain scanning range of low (0.1%)-high (1000%)-low (0.1%), and the storage modulus (G') and loss modulus (G") of the samples in the 1% Lf / NZ2114 / LMSH, 1.5% Lf / NZ2114 / LMSH, and 3% Lf / NZ2114 / HACC groups were measured. The results are shown in Figure 4 As shown, the storage modulus (G') of 1% Lf / NZ2114 / LMSH and 1.5% Lf / NZ2114 / LMSH groups was initially greater than the loss modulus (G''), and the gel was more inclined to the elastic solid state, indicating that the hydrogel phase was more biased. G' decreased with the increase of shear force in the region of 0.1-1, while G" increased with the increase of shear force. A sol-gel transition occurred at a shear stress of about 20%, and the gel structure was severely damaged, transforming from a viscoelastic solid to a liquid. Later, both G' and G" showed a downward trend, further indicating that 1% Lf / NZ2114 / LMSH had significant shear thinning properties and could be injected into specific parts to exert its effect. In addition, 1.5% Lf / NZ2114 / LMSH showed a similar consistent trend, indicating that the prepared lithium magnesium silicate gel was uniform and consistent, and did not affect the sol-gel transition behavior.

[0063] 2. In vitro antibacterial activity and biocompatibility determination of hydrogel wound dressings

[0064] The hydrogel was sterilized by cobalt 60 radiation and set aside. Staphylococcus aureus was cultured to logarithmic phase and diluted to 1 × 10 6 CFU / mL, incubate with sterile gel for 16-18 h, then dilute each sample in a gradient manner, take 100 µL and evenly spread it on the MHA plate, place it in a 37 ℃ constant temperature incubator for 16 h, and record the number of colonies. Figure 5 As shown in the figure, 1% Lf / NZ2114 / LMSH has a significant inhibitory effect on Staphylococcus aureus, with an inhibition rate of 99.99%, while the inhibition rates of lithium magnesium silicate and lactoferrin alone on Staphylococcus aureus are approximately 42% and 53%, respectively. This indicates that the introduction of trace antimicrobial peptide NZ2114 can significantly enhance the ability to inhibit Staphylococcus aureus and successfully prepare highly antibacterial hydrogels.

[0065] The biocompatibility of the synthesized gel was mainly tested for hemolysis and cytotoxicity. The hemolysis test has been described previously. Briefly, anticoagulated whole blood was collected from healthy mice, and red blood cells were collected and diluted to 8% concentration. They were incubated with sterile prepared hydrogels, and PBS and 0.1% Trix-100 blank and positive control groups were set up, with three replicates. The MTT method was used to determine the toxicity of the gel to HaCaT cells, with three replicates per group, which has been described previously. The optimal HACAT cell dilution was 2×10 5After incubation with gel for 1 d, 2 d, and 4 d, the cells were washed with PBS, and the Calcein-AM / PI live / dead cell double staining kit was used to detect cell survival. 1 μL Calcein-AM and 3 μL PI were added to 1 mL of cells, washed three times with PBS, and observed under a fluorescence microscope (excitation wavelength 490 nm, emission wavelength 545 nm). The results are shown in Figure 6 As shown in (a and b), each group had almost no hemolytic activity against mouse erythrocytes, and the hemolytic rate was less than 5%, showing good biocompatibility. Cytotoxicity tests were performed using the MTT method and Calcein-AM / PI double staining method to detect the biocompatibility of the prepared hydrogels. The results are shown in Figure 6 As shown in Figure c, on the first day, the cell survival rate of the 1% Lf / NZ2114 / LMSH group reached a maximum of 95%, which was higher than that of Lf, NZ2114 and LMSH alone. Its sheet-like reticular structure may help cell growth and proliferation. On the second day, the trend of cell survival rate change was similar to that on the first day. Among them, the cell survival rates of the 1% Lf / NZ2114 / LMSH group, Lf + NZ2114 group and Lf group increased significantly, all higher than 100%. The cell survival rates of the NZ2114 and LMSH groups alone showed a downward trend, but were higher than 80%. On the fourth day, the change trends of each group were consistent with those on the first and second days. It had good biocompatibility with human immortalized epidermal cells HACAT cells and promoted growth, indicating that the prepared hydrogel had the dual functions of antibacterial and promoting proliferation.

[0066] 3. Evaluation of the therapeutic effect of lactoferrin and NA2114 hydrogel preparations on wounds infected with Staphylococcus aureus

[0067] Forty-eight female BALB / c mice were randomly divided into four groups and shaved before the experiment. The mice were anesthetized by isoflurane breathing, and the full-thickness skin of appropriate size was cut. The uninfected group served as the negative control group, and the infected group was inoculated with 100 µL logarithmic phase Staphylococcus aureus (2 × 10 8 CFU / mL) was evenly applied to the wound positive control group, and 100 µL of 1% Lf / NZ2114 / LMSH and 3% Lf / NZ2114 / HACC hydrogels were applied to the infected wounds in the treatment groups, respectively. The wound healing conditions were observed and recorded 4, 8, 12 and 14 days after treatment. The wound area was analyzed and calculated using ImageJ software, and the wound healing rate was calculated according to the following formula: Wound healing rate = (A0-At) / A0×100%

[0068] Among them, A0 is the initial wound area and At is the area after healing.

[0069] The results are as follows Figure 7As shown in the figure, skin homogenate was collected on the 4th day after treatment, diluted and coated on MHA solid plate, and the number of colonies was determined to calculate the antibacterial rate of 99.92%. At the same time, wound images were recorded 4 days, 8 days, 12 days and 14 days after treatment, and the wound area of ​​each group was calculated by imagJ, and the wound healing rate was calculated. Among all groups, 1% Lf / NZ2114 / LMSH had the best healing effect after treatment, with a healing rate close to 100%, indicating that it has good antibacterial and healing ability and has the characteristics of a good wound dressing. The healing rates of CK (uninfected group), Positive (infected treatment group) and 3% Lf / NZ2114 / HACC groups after 14 days of treatment were 92%, 78% and 96%, respectively. The results show that the dual-functional injectable hydrogel was successfully prepared, which can effectively treat (antibacterial and anti-inflammatory) wound infections and promote rapid wound healing.

[0070] Skin tissues were collected at different times and fixed with 4% paraformaldehyde, and HE staining was performed to observe tissue sections. At the same time, the expression levels of inflammatory factors IL-6, TNF-α, and angiogenic factor CD31 in skin tissues were determined at the RNA level, and the expression of inflammatory factors IL-6, TNF-α, and angiogenic factor CD31 at the protein level was determined by immunohistochemistry. Figure 8 As shown in the figure, after 4 days of treatment with 1% Lf / NZ2114 / LMSH, NZ2114 played an excellent antibacterial function and Lf played an important anti-inflammatory function, the number of tissue inflammatory cells decreased, and the inflammatory response was greatly reduced. On the 8th day after treatment, there were a large number of inflammatory cells in the blank control group wound, and the tissue structure was irregular. After treatment with 1% Lf / NZ2114 / LMSH, the number of inflammatory cells decreased significantly, the tissue structure gradually became clear and regular, thickened, and a large number of new blood vessels and glands recovered, and basically returned to normal after 14 days of treatment. The results of RNA level and protein expression level are shown in the figure. Fig. 9 and Fig.10 As shown in the figure, after 14 days of treatment with 1% Lf / NZ2114 / LMSH hydrogel, the expression of EGFR, CD31 and VEGF factors increased with the increase of treatment days, and the number of new blood vessels in the tissue increased. Angiogenesis can increase cell proliferation and new tissue generation, and promote wound healing. At the same time, the hydrogel treatment can significantly reduce the inflammatory factor IL-6, restore the normal inflammatory level of the tissue, and promote wound healing. Therefore, 1% Lf / NZ2114 / LMSH can be used as a good wound dressing to promote angiogenesis and anti-inflammatory functions and promote wound healing.

[0071] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

[0072] References

[0073] 1. S. Raffaele, R. Alessio, G. Luca, A. Bruno, S. Francesco. Chronicwound infections: the role of pseudomonas aeruginosa and Staphylococcus aureus ,Expert Rev. Anti-Infect. Ther. 13(5) (2015) 605-613..

[0074] 2. S. Kaul, P. Sagar, R. Gupta, P. Garg, N. Priyadarshi, NKSinghal, Mechanobactericidal, gold nanostar hydrogel-based bandage for bacteria-infected skin wound healing, Acs Appl. Mater. Interfaces 14 (39) (2022) 44084-44097.

[0075] 3. Y. Huang, N. Yang, D. Teng, R. Mao, Y. Hao, X. Ma, L. Wei, J.Wang, Antibacterial peptide nz2114-loaded hydrogel accelerates staphylococcus aureus -infected wound healing, Appl. Microbiol. Biotechnol. 106 (9) (2022)3639-3656.

[0076] 4. Z. Yang, R. Huang, B. Zheng, W. Guo, H. Wang, Highly stretchable,adhesive, biocompatible, and antibacterial hydrogel dressings for woundhealing, Adv. Sci. 8 (8) (2021) 2003627.

[0077] 5. B. Wang, J. Zhao, W. Lu, Y. Ma, X. Wang, X. An, Z. Fan, Thepreparation of lactoferrin / magnesium silicate lithium injectable hydrogel andapplication in promoting wound healing, Int. J. Biol. Macromol. 220 (2022)1501-1511.

[0078] 6. Y. Zhang, D. Teng, R. Mao, X. Wang, D. Xi, X. Hu, J. Wang, Highexpression of a plectasin-derived peptide nz2114 in pichia pastoris and itspharmacodynamics, postantibiotic and synergy against staphylococcus aureus, Appl. Microbiol. Biotechnol. 98 (2) (2014) 681-694。

Claims

1. Lactoferrin and antimicrobial peptide dual-functional hydrogel dressing, characterized in that: It is made of lactoferrin, antimicrobial peptide NZ2114 and gel matrix mixed with water; Preferably, the gel matrix is ​​lithium magnesium silicate.

2. The hydrogel dressing according to claim 1, characterized in that The lactoferrin is bovine lactoferrin.

3. The hydrogel dressing according to claim 2, characterized in that: The concentration of bovine lactoferrin is 20-320 mg / mL, the concentration of antimicrobial peptide NZ2114 is 200-1600 µg / mL, and the concentration of lithium magnesium silicate is 0.25-1.5%.

4. The hydrogel dressing according to claim 3, characterized in that The concentration of bovine lactoferrin was 20 mg / mL, 40 mg / mL, 80 mg / mL, 160 mg / mL or 320 mg / mL, the concentration of antimicrobial peptide NZ2114 was 200 µg / mL, 400 µg / mL, 800 µg / mL or 1600 µg / mL, and the concentration of lithium magnesium silicate was 0.25%, 0.5%, 1%, 1.5%.

5. The hydrogel dressing according to claim 4, characterized in that The concentration of bovine lactoferrin was 40 mg / mL, the concentration of antimicrobial peptide NZ2114 was 400 µg / mL, and the concentration of lithium magnesium silicate was 1% or 1.5%.

6. The method for preparing the hydrogel dressing according to any one of claims 1 to 5, characterized in that: The steps include: (1) Using sterile water for injection, prepare lactoferrin solution, antimicrobial peptide solution and lithium magnesium silicate gel matrix solution respectively according to the mass ratio; (2) Mix the lactoferrin solution, antimicrobial peptide solution and lithium magnesium silicate gel matrix solution in proportion, stir thoroughly, and perform ultrasonic treatment to obtain the product.

7. Any of the following uses of the hydrogel dressing according to any one of claims 1 to 5: 1) Used for preparing trauma medical supplies; 2) Used to prepare drugs for promoting the healing of trauma, burns or infected wounds.

8. The use according to claim 7, characterized in that: 2) The infection is caused by Gram-positive bacteria; The Gram-positive bacteria include Staphylococcus aureus ( Staphylococcus aureus ).

9. A composition, characterized in that The active ingredients are bovine lactoferrin and antimicrobial peptide NZ2114; Among them, the concentration of bovine lactoferrin was 20-320 mg / mL, and the concentration of antimicrobial peptide NZ2114 was 200-1600 µg / mL; Preferably, the concentration of bovine lactoferrin is 20 mg / mL, 40 mg / mL, 80 mg / mL, 160 mg / mL or 320 mg / mL, and the concentration of antimicrobial peptide NZ2114 is 200 µg / mL, 400 µg / mL, 800 µg / mL or 1600 µg / mL; More preferably, the concentration of bovine lactoferrin is 40 mg / mL, and the concentration of antimicrobial peptide NZ2114 is 400 µg / mL.

10. Any of the following uses of the composition according to claim 9: a) Used for preparing anti-inflammatory and immunomodulatory adjuvants; b) for the preparation of antibacterial products; c) used for preparing a healing promoting agent; in, b) The bacteria described in is Gram-positive bacteria; The Gram-positive bacteria include Staphylococcus aureus.

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