Antibacterial hydrogel dressing for promoting healing of diabetic wounds and preparation method thereof

Abstract

The invention provides an antibacterial hydrogel dressing for promoting healing of diabetic wounds and a preparation method of the antibacterial hydrogel dressing. The hydrogel is prepared by taking a combination of a specific amount of nano-silver and antibacterial peptide conjugate and adenovirus as an active component, the toxicity of silver ions is eliminated, the stability of antibacterial peptide is enhanced, meanwhile, the advantages of high replication capacity, stability, long-term expression and the like of the adenovirus are achieved, and the obtained hydrogel can remarkably promote microangiogenesis and has a good application prospect. The collagen regeneration is promoted, the cell proportion of new granulation tissues is improved, and the effect of repairing the diabetic wound is good. The preparation method is simple and suitable for industrial production.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to the treatment of diabetic wound healing, in particular to an antibacterial hydrogel dressing for promoting diabetic wound healing and a preparation method thereof. Background Art

[0002] Diabetes is a common metabolic disease with more than 400 million patients worldwide, which imposes a huge burden on society, economy and healthcare systems. Deaths in diabetic patients are mainly due to chronic complications caused by severe hyperglycemia. Among them, diabetic wounds, especially diabetic foot ulcers, are common complications, which cause 15-25% of diabetic patients to require amputation and suffer disability throughout their lives. Normal wound healing is a dynamic and complex biological process, including four typical stages: hemostasis, inflammation, proliferation and remodeling, which involve multiple types of cells, cytokines and extracellular matrix. However, wounds with a microenvironment with high blood sugar levels are more susceptible to bacterial infection, and macrophages produce more reactive oxygen species to resist foreign pathogens. Excessive ROS can damage normal cells and tissues, leading to nutrient deficiency, impaired angiogenesis, hypoxia and neuropathy. Ultimately, it leads to persistent inflammation and long-term non-healing chronic wounds. Therefore, clearing infection and reducing inflammation are crucial for the management of chronic wounds in diabetes, and subsequently providing other growth factors such as vascular endothelial growth factor to promote wound healing.

[0003] Traditional wound dressings are in the form of gauze made of cotton, but each dressing change will cause secondary damage and have no biological activity to promote wound healing and moisturize the wound. Wound hydrogel dressing is an emerging wound care material that has received widespread attention in the medical and biomedical fields in recent years. Compared with other types of new wound dressings, hydrogels have good flexibility, biocompatibility, moisture retention and high sensitivity to the physiological environment. Compared with other dressings, hydrogels can not only increase wound moisture, absorb wound exudate, and reduce wound temperature, but also are comfortable, non-irritating, easy to change, and more importantly, have analgesic effects on injured tissues. However, most of the hydrogels on the market still have problems such as a single antibacterial method, poor antibacterial effect, and slow wound healing during use, resulting in poor results in the treatment of diabetic foot with hydrogels. Summary of the invention

[0004] In order to solve the problems in the prior art, the present invention provides an antibacterial hydrogel dressing for promoting diabetic wound healing and a preparation method thereof. The present invention uses nanosilver@antimicrobial peptide conjugates and adenovirus as active ingredients to prepare hydrogels, which have the effects of reducing the toxicity of the complex, increasing stability, and preventing bacterial infection. Studies have found that the dosage of nanosilver@antimicrobial peptide conjugates and adenovirus is very critical. Excessive nanosilver@antimicrobial peptide conjugates will be toxic to cell tissues and inhibit cell proliferation, which is not conducive to wound recovery. Adenovirus concentration that is too low is not enough to promote cell proliferation, and a concentration that is too high will affect the cell state, resulting in slow wound healing.

[0005] The present invention adopts the following technical solution: In a first aspect, the present invention provides a pharmaceutical composition.

[0006] A pharmaceutical composition, characterized in that it comprises a silver nanoparticle @ antimicrobial peptide conjugate, adenovirus and water, wherein the concentration of the silver nanoparticle @ antimicrobial peptide conjugate is 4-16 μg / ml, and 10 7 —10 10 Adenovirus copies.

[0007] In a second aspect, the present invention provides a composite hydrogel.

[0008] A composite hydrogel, characterized in that the above-mentioned pharmaceutical composition is prepared into a hydrogel by adding chitosan and glutaraldehyde. Further, chitosan is first dissolved in an acetic acid solution, and then adenovirus and nanosilver@antimicrobial peptide are added, glutaraldehyde is added after mixing, and the mixture is mixed again and allowed to stand to form a gel. Chitosan is dissolved in pure water as a basic component of the hydrogel.

[0009] According to one embodiment of the present invention, the amount of chitosan used accounts for 1-5% of the amount of the hydrogel raw material used, and the amount of glutaraldehyde used accounts for 1-3% of the amount of the hydrogel raw material used.

[0010] According to one embodiment of the present invention, the hydrogel comprises silver nano@antimicrobial peptide conjugate, adenovirus, chitosan, glutaraldehyde and water; wherein the amount of chitosan accounts for 1-5% of the amount of the hydrogel raw material, the amount of glutaraldehyde accounts for 1-3% of the amount of the hydrogel raw material, the amount of silver nano@antimicrobial peptide conjugate accounts for 4-16 μg / ml of the amount of the hydrogel raw material, and 10 7 —10 10 Adenovirus copies.

[0011] According to one embodiment of the present invention, the hydrogel is prepared from silver nano@antimicrobial peptide conjugate, adenovirus, chitosan, glutaraldehyde and water; wherein the amount of chitosan accounts for 1-5% of the amount of the hydrogel raw material, the amount of glutaraldehyde accounts for 1-3% of the amount of the hydrogel raw material, the amount of silver nano@antimicrobial peptide conjugate accounts for 4-16 μg / ml of the amount of the hydrogel raw material, and 10 7 —10 10 The remaining amount is water.

[0012] In a third aspect, the present invention also provides a method for preparing the composite hydrogel.

[0013] The preparation method of the composite hydrogel comprises the following steps: Chitosan was dissolved in acetic acid solution, adenovirus and nanosilver@antimicrobial peptide were added, glutaraldehyde was added after mixing, and the mixture was mixed again and allowed to stand to form a gel.

[0014] The preparation method of nanosilver@antimicrobial peptide is as follows: sodium citrate and silver nitrate are dissolved in deionized water, sodium borohydride and polyvinyl pyrrolidone are added, and the reaction is allowed to stand for 20-40 minutes. After the reaction is allowed to stand, the nanosilver solution is filtered using a 0.22 μm filter, and the nanosilver solution is allowed to stand at -80°C for 4-6 hours to solidify. The nanosilver solution is then freeze-dried in a freeze dryer for 20-30 hours, and the nanosilver solution is prepared by coupling the solution with the antimicrobial peptide solution.

[0015] According to one embodiment of the present invention, the coupling ratio of silver nanoparticles to antimicrobial peptides is 1:1-1.5.

[0016] The adenovirus preparation method is as follows: Cμtsmart bμffer, plasmid and Pme I enzyme are mixed, incubated in a water bath at 35-40 ℃ for 1-2 hours, placed in a centrifuge tube, and deionized water, glycogen, ammonium acetate and anhydrous ethanol are added, the centrifuge tube is fully shaken at room temperature, centrifuged at 12000-15000 rpm for 4-6 minutes, ethanol is added to the centrifuge tube, and centrifuged under the same conditions for 2-3 minutes to obtain the enzyme cleavage product; the enzyme cleavage product, deionized water and AdEasy-1 BJ5183 electroporation competent state are mixed and electroporated at 2.5-3.0 kV; LB liquid without resistance is added to suspend the bacterial solution and incubated on a shaker at 35-40 ℃ and 200-250 rpm for 1-2 hours, a single small colony is picked and added to the kana+ resistant LB liquid, and incubated at 35-40 ℃ and 200-250 Shake overnight at rpm; centrifuge the bacterial solution at room temperature at 13000-15000 rpm, and add BD-Ⅰ, BD-Ⅱ, and BD-Ⅲ in turn to mix, and centrifuge for 8-10 minutes; add isopropanol and ethanol to the supernatant products, respectively, and centrifuge in turn to obtain the enzyme cleavage product; mix OPTI-MEM + Lipo8000 and add the enzyme cleavage product; add the mixed solution dropwise into the complete culture medium for culture, and obtain adenovirus after 10 days.

[0017] In a fourth aspect, the present invention provides the use of the above-mentioned composite hydrogel in the preparation of an antibacterial hydrogel dressing for promoting diabetic wound healing; the antibacterial hydrogel dressing for promoting diabetic wound healing is used for the antibacterial and repair of diabetic wounds.

[0018] According to one embodiment of the present invention, the diabetic wound is diabetic foot.

[0019] The beneficial effects of the present invention are as follows: The present invention prepares hydrogel by using a combination of a specific amount of nanosilver@antimicrobial peptide conjugate and adenovirus as active ingredients, while eliminating the toxicity of silver ions, enhancing the stability of antimicrobial peptides, and realizing the high replication ability, stability, long-term expression and other advantages of adenovirus. The obtained hydrogel can significantly promote microvascular regeneration, promote collagen regeneration, improve the cell ratio of new granulation tissue, and has a good effect in repairing diabetic wounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The figure shows the characterization of silver nanoparticles alone and after coupling, where (A) shows the appearance properties of silver nanoparticles coupled with antimicrobial peptides at different concentrations, (B) shows the change in Zeta potential after coupling silver nanoparticles with antimicrobial peptides at different concentrations, and (C) shows the change in particle size after coupling silver nanoparticles with antimicrobial peptides. Figure 2TEM images of silver nanoparticles alone and after coupling, where (A) is the morphology of silver nanoparticles under transmission electron microscopy, and (B) is the morphology of silver nanoparticles after coupling with antimicrobial peptides under transmission electron microscopy; Figure 3 It is the minimum bactericidal concentration result diagram before and after the coupling of silver nanoparticles and antimicrobial peptides, where A is the minimum bactericidal concentration of antimicrobial peptides alone against three kinds of bacteria, B is the minimum bactericidal concentration of silver nanoparticles alone against three kinds of bacteria, and C is the minimum bactericidal concentration of silver nanoparticle antimicrobial peptide conjugates against three kinds of bacteria; Figure 4 It is a graph showing the killing effect of different substances on bacteria, wherein A is the time killing curve of different substances on Pseudomonas aeruginosa, B is the time killing curve of different substances on Staphylococcus aureus, and C is the time killing curve of different substances on methoxy-resistant Staphylococcus aureus; Figure 5 is the result diagram of VEGF expression, wherein A is the VEGF expression level detected by Western blotting after adenovirus infection of cells, and B is the VEGF expression level detected by QPCR after adenovirus infection of cells; Figure 6 is the SEM image of the hydrogel; Figure 7 It is a characterization result diagram of the hydrogel, wherein A is the swelling rate of the simple hydrogel carrier CS composite hydrogel ALL, and B is the water retention of the simple hydrogel carrier CS composite hydrogel ALL; Figure 8 The results of the biocompatibility of the hydrogels are shown in Figure 1. A) is the hemolysis rate of the composite hydrogel, and B) is the hemolysis condition. Fig. 9 The results of the antioxidant efficiency of the hydrogels are shown in Figure 1, where A) is the result of 6h DPPH scavenging in all groups, and B) is the DPPH scavenging ability of the simple hydrogel carrier CS composite hydrogel ALL, n = 3; Fig.10 The results of the cell compatibility of the hydrogel are shown in Figure 1, where A) shows the effect of different concentrations of adenovirus copies on cells after infection, and B) shows the effect of different concentrations of silver nano antimicrobial peptide conjugates on cells after infection; Fig.11 This is a diagram showing the therapeutic effects of different treatment groups on chronic wounds in diabetic mice; Fig.12 This is a quantitative analysis result of wound closure. DETAILED DESCRIPTION

[0021] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention is described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0022] Unless otherwise specified, the parts described in the present invention are all parts by weight and the percentages described are all mass percentages.

[0023] The raw materials and reagents used in the present invention are all commercially available products. For example, the human umbilical vein endothelial cells and human renal epithelial cells used in the examples of the present invention were purchased from Prosai, and 6-8 week old C57 male mice (SPF grade) were purchased from Vital River. Example

[0024] Synthesis method of simple hydrogel carrier: Weigh the chitosan powder, dissolve 0.1 g of chitosan in 4.75 ml of 2% acetic acid solution, stir until fully dissolved, and place it in a 4 ℃ refrigerator to eliminate bubbles for later use; add 250 ml of 1.8% glutaraldehyde solution, stir quickly, and place it in a 4 ℃ refrigerator to wait for gelation.

[0025] Synthesis method of composite hydrogel: At 4 °C, dissolve 0.2 g of chitosan powder in 5 ml of 2% acetic acid solution in a scintillation vial and let stand for 1 hour to eliminate bubbles. Add 50 μl of adenovirus and stir slowly, 12 μl of nanosilver@antimicrobial peptide, rotate for one hour on a rotator, add 250 μl of 1.8% glutaraldehyde, mix the reactants quickly, and let stand to form a gel.

[0026] Preparation of nanosilver@antimicrobial peptide: Weigh 1.29 g sodium citrate powder, dissolve it in 200 ml deionized water, and stir it magnetically at room temperature for 30 min to obtain a 20 mMol / L sodium citrate solution for use; weigh 0.85 g silver nitrate powder, dissolve it in 200 ml deionized water, and stir it magnetically at room temperature for 30 min to obtain a 20 mMol / L silver nitrate solution for use; weigh 5.80 g sodium borohydride powder, dissolve it in 10 ml deionized water, and stir it magnetically at room temperature for 30 min to obtain a 20 mMol / L sodium borohydride solution for use; draw 0.5 ml of sodium citrate solution and 0.5 ml of silver nitrate solution into 19 ml deionized water, stir it at room temperature for 3 min, let it stand, then add 0.5 ml of sodium borohydride and 8.5 mg of polyvinyl pyrrolidone solution to the precursor solution, and let it stand for 30 min. After standing, filter it with a 0.22 μm filter, stand it at -80 °C for 5 h to solidify, and freeze-dry it for 24 h using a freeze dryer. Weigh the freeze-dried product and prepare it into a 1 mg / ml nanosilver solution, which is ultrasonically cleaned for 15 min. Pipette 0.5 ml of the 1 mg / ml antimicrobial peptide solution and 0.5 ml of the 1 mg / ml nanosilver solution into a centrifuge tube and couple them using a rotator at room temperature, in the dark, and under rotating conditions.

[0027] Adenovirus preparation: 100 μl Cμtsmart bμffer, plasmid and Pme I enzyme were mixed, incubated in a 37 ℃ water bath for one hour, placed in a centrifuge tube, and 100 μl deionized water, 5 μl glycogen, 100 μl 7.5 M ammonium acetate and 700 μl anhydrous ethanol were added. The centrifuge tube was fully shaken at room temperature, centrifuged at 14000 rpm for 5 minutes, 500 μl 75% ethanol was added to the centrifuge tube, and centrifuged for 2 minutes under the same conditions to obtain the enzyme digestion product. 7.5 μl of the enzyme digestion product was mixed with 10 μl deionized water and 10 μl AdEasy-1 BJ5183 electroporation competent medium and electroporated at 2.5 kV. Add 500 μl of LB liquid without resistance to suspend the bacterial solution and incubate it in a shaker at 37 ℃ and 220 rpm for one hour. Pick a single small colony and add it to 2 ml of kana+ resistant LB liquid, and shake it overnight at 37 ℃ and 200 rpm. Take 2 ml of bacterial solution and centrifuge it at room temperature and 14000 rpm, and add 200 μl BD-Ⅰ, 200 μl BD-Ⅱ, and 200 μl BD-Ⅲ in turn to mix, and centrifuge for 8 minutes. Add 500 μl of frozen isopropanol and 500 μl of 70% ethanol to the supernatant, centrifuge them in turn, and obtain the enzyme digestion product. Mix 250 μl OPTI-MEM+7.5 ΜL Lipo8000 and add 20 μl of the enzyme digestion product. Add the mixed solution dropwise to 5 ml of complete culture medium for culture, and adenovirus will be obtained after 10 days.

[0028] Plasmid sequence: MNFLLSWVHWSLALLLYLHHAKWSQAAPMAEGGGQNHHEVVKFMDVYQRSYCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEGLECVPTEESNITMQ IMRIKPHQGQHIGEMSFLQHNKCECRPKKDRARQEKKSVRGKGKGQKRKRKKSRYKSWSVPCGPCSERRKHLFVQDPQTCKCSCKNTDSRCKARQLELNERTCRCDKPRR.

[0029] 1) Characterization of silver nanoparticles and antimicrobial peptide conjugates (1) Particle size and its Zata potential 1) Characterization of silver nanoparticles and antimicrobial peptide conjugates (1) Particle size and its Zata potential NanoBrook 90PLUS PALS particle size analyzer was used to measure the particle size distribution, polydispersity index (PDI) and Zeta potential properties to verify the properties of nanosilver particles and their nanosilver antimicrobial peptide conjugates. The nanosilver particles and their nanosilver antimicrobial peptide conjugates with different coupling ratios were directly diluted 200 times with PBS, and the emission scattering intensity was adjusted to 10500 ± 1500 cps for measurement to verify whether the coupling was successful and to screen out the optimal coupling concentration. The results are shown in Figure 1 As shown. According to the Zeta potential, the potential of silver nanoparticles and antimicrobial peptides changed from negative to positive after coupling, and when the coupling ratio of silver nanoparticles to antimicrobial peptides was 1:1 and 1:1.5, the Zeta potential value no longer changed significantly, indicating that the coupling was saturated. Considering the actual economic use, silver nanoparticles and antimicrobial peptides were selected for use in a 1:1 ratio. Subsequently, the particle size was measured, and the particle size after coupling changed from the original 10nm to 13nm. All proved that the coupling of silver nanoparticles and antimicrobial peptides was successful, and the optimal coupling ratio was 1:1.

[0030] (2) Morphological characteristics The microscopic features of silver nanoparticles and their silver nanoparticle antimicrobial peptide conjugates were observed using a transmission electron microscope. A drop of nanoparticle solution was added to a copper mesh covered with a carbon film. After 5 minutes of contact, it was negatively stained with a 2% phosphotungstic acid solution for 2 minutes. After the copper mesh was dried, the surface morphology of Pue-NPs was observed and photographed at an accelerating voltage of 120 kV. The transmission electron microscopy results are shown in Figure 2. Figure 2 As shown, the morphology of the silver nanoparticles and the silver nanopeptide conjugates is a regular sphere, and there is no adhesion between the particles. The silver nanoparticles are about 10 nanometers, and the silver nanopeptide conjugates are slightly larger than the silver nanoparticles.

[0031] 2) Antibacterial test The antibacterial activity of silver nanoparticles before and after coupling with antimicrobial peptides was evaluated by colony forming units (CFU). The specific operation method is as follows: (1) Nanosilver antibacterial experiment 1. For methicillin-resistant Staphylococcus aureus, Staphylococcus aureus, and Pseudomonas aeruginosa, single colonies were selected from the third and fourth zones respectively. The colonies were picked with a cotton swab and dissolved in a 2.5 ml saline colorimetric tube. The McFarland turbidity was determined to be about 0.55 (1.5*10 8 ).

[0032] 2. The culture environment of the experimental group and the control group was formed in a 96-well plate. 80 μl of inorganic culture medium was added to each well of the control group, and inorganic culture medium and nanosilver solution were added to each well of the experimental group to form an experimental concentration gradient of 0.25 μg / ml-128 μg / ml.

[0033] 3. Add 20 μl of bacterial solution (1*10 6 ).

[0034] 4. Incubate at 37°C and 200 rpm on a rotator for 2 hours.

[0035] 5. Use a dispenser to pipette 1 μl of the bacterial solution onto the antibiotic-free plate and evaluate the antibacterial effect of each group.

[0036] (2) Antimicrobial peptide antibacterial experiment 1. For methicillin-resistant Staphylococcus aureus, Staphylococcus aureus, and Pseudomonas aeruginosa, single colonies were selected from the third and fourth zones respectively. The colonies were picked with a cotton swab and dissolved in a 2.5 ml saline colorimetric tube. The McFarland turbidity was determined to be about 0.55 (1.5*10 8 ).

[0037] 2. The culture environment of the experimental group and the control group was formed in a 96-well plate. 80 μl of inorganic culture medium was added to each well of the control group, and inorganic culture medium and antimicrobial peptide solution were added to each well of the experimental group to form an experimental concentration gradient of 0.25 μl / ml-128 μl / ml.

[0038] 3. Add 20 μl of bacterial solution (1*10 6 ).

[0039] 4. Incubate at 37°C and 200 rpm on a rotator for 2 hours.

[0040] 5. Use a dispenser to pipette 1 μl of the bacterial solution onto the antibiotic-free plate and evaluate the antibacterial effect of each group.

[0041] (3) Nanosilver@antimicrobial peptide coupling antibacterial experiment 1. For methicillin-resistant Staphylococcus aureus, Staphylococcus aureus, and Pseudomonas aeruginosa, single colonies were selected from the third and fourth zones respectively. The colonies were picked with a cotton swab and dissolved in a 2.5 ml saline colorimetric tube. The McFarland turbidity was determined to be about 0.55 (1.5*10 8 ).

[0042] 2. The culture environment of the experimental group and the control group was formed in a 96-well plate. 80 μl of inorganic culture medium was added to each well of the control group, and inorganic culture medium and nanosilver@antimicrobial peptide solution were added to each well of the experimental group to form an experimental concentration gradient of 0.25 μl / ml-128 μl / ml.

[0043] 3. Add 20 μl of bacterial solution (1*10 6 ).

[0044] 4. Incubate at 37°C and 200 rpm on a rotator for 2 hours.

[0045] 5. Use a dispenser to pipette 1 μl of the bacterial solution onto the antibiotic-free plate and evaluate the antibacterial effect of each group.

[0046] The experimental results are as follows Figure 3 The minimum bactericidal concentrations of silver nanoparticles before and after coupling with antimicrobial peptides for Pseudomonas aeruginosa, Staphylococcus aureus, and MRSA are summarized in Table 1.

[0047] Table 1 Minimum bactericidal concentration of silver nanoparticles coupled with antimicrobial peptides The experimental results show that the minimum bactericidal concentrations of antimicrobial peptides against these three bacteria are 4μg / ml, 16μg / ml, and 8μg / ml, respectively, the minimum bactericidal concentrations of silver nanoparticles against these three bacteria are 8μg / ml, 64μg / ml, and 32μg / ml, respectively, and the minimum bactericidal concentrations of silver nanoparticle antimicrobial peptide conjugates are 4μg / ml, 4μg / ml, and 4μg / ml, respectively. It can be clearly seen that AgNPs exhibit a more excellent antibacterial effect after coupling with antimicrobial peptides.

[0048] (4) Time-killing experiment 1. For three types of bacteria, methicillin-resistant Staphylococcus aureus, Staphylococcus aureus, and Pseudomonas aeruginosa, four-zone streak culture was performed, and a single colony was picked and shaken overnight. The next day, 3 ml of antibiotic-free culture medium was added with 50 μl of overnight bacterial solution and incubated at 37°C for 1-2 hours.

[0049] 2. Use antibiotic-free culture medium to adjust the bacterial solution concentration to 0.55. A total of 40 ml of bacterial solution with a McFarland turbidity of 0.55 is required.

[0050] 3. Ultrasonicate 1 mg / ml AgNps and couple SAAP-148 with AgNPs in a 1:1 ratio.

[0051] 4. Add three milliliters of the bacterial solution with a McFarland turbidity of 0.55 into each 15-ml screw-top tube.

[0052] 5. Add silver nanoparticles, antimicrobial peptides, and silver nano-antimicrobial peptide conjugates into the screw-mouth tube according to the minimum bactericidal concentration.

[0053] 6. Place in a shaker and start coating the plate at 0h without adding drugs (3970μl M9 medium, add 30μl of the test bacterial solution).

[0054] 7. Add 90μl of PBS to each 1.5ml ep tube, invert the tube to mix, aspirate 10μl from the tube to add to the first ep tube, put the tube back into the shaker, blow and mix, aspirate 10μl and add to the second tube, and repeat this process.

[0055] 8. Take the dilution factor as 10 2 , 10 4 , 10 6 Take 50 μl of each and plate it.

[0056] The experimental results are as follows Figure 4 shown.

[0057] 2) Validation of adenovirus (1) Verification of VEGF secretion after adenovirus infection of cells by WB and QPCR 1. Infect HUVEC cells with the packaged adenovirus carrying VEGF. Use a six-well plate and add 20ul of 10 12 The adenovirus stock solution with a copy / mL was used as the experimental group. The cells were treated after 24 hours of culture.

[0058] 2. Digest the cells and centrifuge them. Divide the cells in the control group and the experimental group into two equal parts.

[0059] 3. Two samples of cells were used for protein extraction and mRNA extraction respectively.

[0060] 4. Prepare the lysis system: add 5ul of protease inhibitor PMSF into 500ul of cell lysis buffer.

[0061] 5. Add the lysis system to the cells and lyse on ice for 30 minutes.

[0062] 6. Centrifuge the cells for 15 min at 12,000 rpm and 4°C.

[0063] 7. After centrifugation, take 20ul for BCA protein concentration test, add 6*loading to the remaining supernatant, and place in metal bath for 10min at 100℃.

[0064] 8. Extract mRNA from the remaining two tubes of cells and reverse transcribe them into DNA, and use QPCR to detect gene expression levels.

[0065] The experimental results are as follows Figure 5 As shown in the figure, under the condition of equal internal reference, at the position of 25KD, the target band of the experimental group was significantly deepened and widened compared with the control group, indicating that the expression level of VEGF in the experimental group was significantly higher than that in the control group. When QPCR detection was performed, the expression level of VEGF gene in the experimental group was about 244 times that of the control group, indicating that after infection with adenovirus packaging VEGF, the expression level of VEGF in cells would be significantly increased.

[0066] 3) Characterization and biocompatibility of hydrogels (1) Gelation: Dissolve 0.2 g of chitosan powder in 5 ml of 2% acetic acid solution in a scintillation vial at 4°C and let stand for 1 hour to eliminate bubbles. Add 50 μl of adenovirus and stir slowly, 12 μl of nanosilver antimicrobial peptide conjugate, rotate for one hour on a rotator, add 250 μl of 1.8% glutaraldehyde, mix the reactants quickly, and let stand to form a gel.

[0067] (2) Morphological characteristics The internal structure of SANH was characterized under a scanning electron microscope (SEM, Gemini SEM300, Carl Zeiss, Germany). After the hydrogel was synthesized, the water was first drained by a vacuum freeze dryer and then dehydrated with liquid nitrogen and broken to obtain a cross section. The sample was placed on the sample stage, gold was sprayed on the holder for 60 seconds, and then a scanning image was obtained by scanning with a current of 10 mA ( Figure 6 ). The porous structure of the hydrogel facilitates the transport of nutrients and waste during skin regeneration. The internal morphology of the hydrogel shows a honeycomb porous structure, which may endow the hydrogel with sufficient air permeability.

[0068] (3) Swelling rate: Place the hydrogel in 20 mL of phosphate buffered saline (PBS) in a sealed bottle at 37°C. When the preset time interval is reached, remove the hydrogel from the solution and absorb the surface moisture with filter paper. After that, weigh the hydrogel. The test is completed until the weight of all hydrogels remains constant. Each group is measured in parallel 3 times. The swelling rate is calculated as: Swelling rate = (Wt-W0) / W0 × 100%, where W0 and Wt are the initial weight of the hydrogel and the weight after the preset swelling time, respectively.

[0069] (3) Water retention: The initial weight of the hydrogel (W0) was accurately weighed, and then placed in an incubator at 37°C for three days. The weight of the sample (Wt) was accurately measured every day. The water retention rate of the hydrogel was calculated as follows: Water retention rate (%) = Wt / W0 × 100%, where Wt and W0 are the weights of the composite hydrogel on day t and day 0, respectively. Each group was measured three times in parallel.

[0070] The results showed that when all the components were integrated into the hydrogel, the swelling rate was greater and the overall water retention of the hydrogel was better than that of a simple hydrogel carrier, indicating that the collective hydrogel can absorb more wound exudate and ensure good wettability, making it more suitable for diabetic wounds.

[0071] (4) Concentrated red blood cells (RBCs) were obtained by centrifuging citrated whole blood at 1000 rpm for 5 min and washing repeatedly. Then, 100 μL of RBCs were added to every 2 mL of PBS to prepare an RBC suspension with a hematocrit of 2%. The hydrogel extract was then incubated at 37°C for 24 h to obtain the hydrogel extract. The hydrogel extract (100%) and its dilutions in PBS (75%, 50%, 25%) were then mixed with the RBC suspension in a volume ratio of 1:1 and then incubated at 37°C for 1 h. Physiological saline and deionized water were used instead of the hydrogel extract as negative and positive controls, respectively. After centrifuging the mixture at 2000 rpm for 10 min, 200 μL of the supernatant was transferred to a 96-well plate. The absorbance of the supernatant was measured at a wavelength of 540 nm using a microplate reader.

[0072] The hemolysis rate (%) was calculated according to the following formula: Hemolysis rate (%) = (A sample - A negative) / (A positive - A negative) × 100%, where A represents the absorbance of each group at 540 nm. Here, each group was tested five times.

[0073] The results are as follows Figure 8 As shown: It can be observed that except for the slight hemolysis of 100% hydrogel leaching solution, all groups showed that the supernatant was almost clear and transparent like the saline group. In contrast, the positive control deionized water group showed a bright red color, indicating complete hemolysis. The data were quantitatively analyzed, and the data showed that the hemolysis rate of all groups was less than 5% of the hemolysis limit. The hemolysis rates of 75% leaching solution, 50% leaching solution and 25% leaching solution were 0.72 ± 0.46%, 0.16 ± 0.72% and 0.08 ± 0.58%, respectively.

[0074] (5) DPPH clearance rate The present invention investigates the antioxidant activity of the hydrogel using 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) free radical as a free radical. The hydrogel simple carrier and the integrated hydrogel (0.2 g) were added to 2 mL of DPPH ethanol solution, and the ethanol solution was used as a negative control. The mixture was incubated in the dark at room temperature. The absorbance of the supernatant at 517 nm was measured using an ultraviolet spectrophotometer every 2 hours. The DPPH free radical scavenging rate was calculated as follows: DPPH scavenging rate = [A blank - (A determination - A control)] / A blank * 100.

[0075] The experimental results are as follows Fig. 9 As shown in the figure, when 0.2 g of gel pure carrier and integrated hydrogel were added to DPPH solution, their antioxidant activity gradually increased with the increase of time.

[0076] 4) Cell viability assay CCK-8 was used to determine the effects of different concentrations of silver nanopeptide conjugates and different concentrations of adenovirus on cell viability. HUVEC human umbilical vein endothelial cells were plated in 96-well plates and cultured for 12 hours. 70-80% confluent cells were treated with different concentrations of silver nanopeptide conjugates and different concentrations of adenovirus for 24 hours. Then 10 μL of CCK-8 solution was added to each well and incubated at 37 °C for 2 h. The absorbance value at 450 nm was measured using an enzyme reader.

[0077] The experimental results are as follows Fig.10 When the number of adenovirus copies added to the infection is greater than 10 per ml 7 The cells showed obvious proliferation. The amount added was 10 per ml. 7 The number of cell copies did not change significantly compared with the control group, but the number of adenovirus copies added to the infection was greater than 10 per ml. 10 Copy, under the fluorescence microscope, the fluorescence is too bright to see the cell morphology. In subsequent experiments, 10 10 When the concentration of silver nano-antimicrobial peptide conjugate added was greater than 4MIC, the cell viability was less than 80%, which affected the cell state. The maximum concentration added to the hydrogel was 4MIC, i.e. 16μg / ml. The silver nano-antimicrobial peptide conjugate showed excellent antibacterial properties and good biocompatibility. In subsequent experiments, 4 times the minimum inhibitory concentration of silver nano-antimicrobial peptide conjugate, i.e. 16μg / ml, was selected to prepare drug-loaded hydrogel.

[0078] 5) Mouse experiments (1) Thirty male mice weighing 18-22 g were divided into four groups: a diabetic mouse control group, a diabetic-bacterial-infected mouse control group, a diabetic-bacterial-infected mouse treatment group 1, and a diabetic-bacterial-infected mouse treatment group 2.

[0079] (2) Modeling.

[0080] 1. Modeling of diabetic mice: On days 1-3, mice in the diabetic group were continuously injected with 100 mg / kg streptozotocin. On day 8, random blood glucose was tested. If the blood glucose was greater than 16.2 mM / L for three consecutive weeks and there was polyphagia, polyuria, polydipsia, and weight loss, the modeling was considered successful.

[0081] 2. Modeling of diabetic mice infected with bacteria: On days 1-3, mice in the diabetic group were continuously injected with 100 mg / kg streptozotocin. On day 8, random blood sugar was tested. If the blood sugar was greater than 16.2 mM / L for three consecutive weeks and polyphagia, polyuria and polydipsia occurred, the model was considered successful on day 29 by using a trephine with a diameter of 8 mm to make a skin wound model on the back of the mouse and applying 30 μl of methicillin-resistant Staphylococcus aureus with a McFarland turbidity of 0.55.

[0082] 3. Diabetes-bacteria-infected mice treatment group 1: The wounds of the successfully established diabetic-bacteria-infected mice were treated with a simple hydrogel carrier.

[0083] 4. Diabetes-bacteria-infected mice treatment group 2: The wounds of the successfully modeled diabetic-bacteria-infected mice were treated with the composite hydrogel.

[0084] (3) On day 29, a trephine with a diameter of 8 mm was used to make skin wounds on the backs of all mice. The diabetic-bacteria-infected mice group was treated with a simple hydrogel carrier and a composite hydrogel, respectively.

[0085] (4) On the 32nd day, the wound healing was observed. On the 36th day, samples were taken from the wound to observe the bacterial infection. On the 43rd day, samples were taken from the wound to observe the bacterial infection. The therapeutic effects of different treatment groups on chronic wounds in diabetic mice are shown in Figure 2. Fig.11 Quantitative analysis of wound closure is shown in Fig.12 As shown in the figure, the wound area of ​​all groups gradually decreased by naked eye observation. According to the wound healing curves of different treatment methods at different time points, at each specific time interval, the healing speed of the composite hydrogel group was faster than that of the hydrogel carrier group and the bacterial infection group, and was comparable to the wound healing speed of the control group 1. This indicates that the silver nano antimicrobial peptide conjugate in the composite hydrogel effectively inhibited and cleared bacterial infection, and the addition of adenovirus played a role in promoting wound healing.

Claims

1. A pharmaceutical composition, characterized in that The invention comprises a silver nanoparticle @ antimicrobial peptide conjugate, adenovirus and water, wherein the concentration of the silver nanoparticle @ antimicrobial peptide conjugate is 4-16 μg / ml, and 10 7 —10 10 Adenovirus copies.

2. A composite hydrogel, characterized in that: The pharmaceutical composition of claim 1 is prepared into a hydrogel by adding chitosan and glutaraldehyde.

3. The hydrogel according to claim 2, characterized in that First, chitosan was dissolved with acetic acid solution, and then adenovirus and nanosilver@antimicrobial peptide were added, glutaraldehyde was added after mixing, and the mixture was mixed again and allowed to stand to form a gel.

4. The hydrogel according to claim 3, characterized in that The amount of chitosan used accounts for 1-5% of the amount of the hydrogel raw material used, and the amount of glutaraldehyde used accounts for 1-3% of the amount of the hydrogel raw material used.

5. The hydrogel according to claim 3, characterized in that The hydrogel is prepared from silver nano@antimicrobial peptide conjugate, adenovirus, chitosan, glutaraldehyde and water; wherein the amount of chitosan accounts for 1-5% of the amount of the hydrogel raw material, the amount of glutaraldehyde accounts for 1-3% of the amount of the hydrogel raw material, the amount of silver nano@antimicrobial peptide conjugate accounts for 4-16 μg / ml of the amount of the hydrogel raw material, and 10 7 —10 10 The remaining amount is water.

6. The method for preparing the composite hydrogel according to any one of claims 2 to 5, comprising the following steps: dissolving chitosan with an acetic acid solution, adding adenovirus and nanosilver@antimicrobial peptide, adding glutaraldehyde after mixing, mixing again and standing to form a gel; the method for preparing nanosilver@antimicrobial peptide is: dissolving sodium citrate and silver nitrate with deionized water, adding sodium borohydride and polyvinyl pyrrolidone, standing to react for 20-40 min, filtering it with a 0.22 μm filter after standing, standing it at -80°C for 4-6 h to solidify it, freeze-drying it in a freeze dryer for 20-30 h, configuring the freeze-dried product into a nanosilver solution, and coupling it with an antimicrobial peptide solution to obtain the obtained product.

7. The method according to claim 6, characterized in that The coupling ratio of silver nanoparticles to antimicrobial peptides is 1:1-1.

5.

8. The method according to claim 6 or 7, characterized in that The adenovirus preparation method is as follows: Cμtsmart bμffer, plasmid and Pme I enzyme are mixed, incubated in a water bath at 35-40 ℃ for 1-2 hours, placed in a centrifuge tube, and deionized water, glycogen, ammonium acetate and anhydrous ethanol are added, the centrifuge tube is fully shaken at room temperature, centrifuged at 12000-15000 rpm for 4-6 minutes, ethanol is added to the centrifuge tube, and centrifuged under the same conditions for 2-3 minutes to obtain the enzyme cleavage product; the enzyme cleavage product, deionized water and AdEasy-1 BJ5183 electroporation competent state are mixed and electroporated at 2.5-3.0 kV; LB liquid without resistance is added to suspend the bacterial solution and incubated in a shaker at 35-40 ℃ and 200-250 rpm for 1-2 hours, a single small colony is picked and added to the kana+ resistant LB liquid, and incubated at 35-40 ℃ and 200-250 Shake overnight at rpm; centrifuge the bacterial solution at room temperature at 13000-15000 rpm, add BD-Ⅰ, BD-Ⅱ, and BD-Ⅲ in turn, mix well, and centrifuge for 8-10 minutes; add isopropanol and ethanol to the supernatant products, centrifuge in turn, and obtain the enzyme digestion products; mix OPTI-MEM + Lipo8000 and add the enzyme digestion products; The mixed solution was added dropwise into complete culture medium for cultivation, and adenovirus was obtained after 10 days.

9. Use of the composite hydrogel according to any one of claims 2 to 5 in the preparation of an antibacterial hydrogel dressing for promoting diabetic wound healing.

10. The use according to claim 9, characterized in that The diabetic wound is diabetic foot.

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