Antisense nucleic acid antibacterial hydrogel for wound healing of diabetic foot and preparation method of antisense nucleic acid antibacterial hydrogel

Abstract

The invention discloses a preparation method of antisense nucleic acid antibacterial hydrogel for diabetic foot wound healing, and belongs to the technical field of biological materials.The preparation method comprises the steps that 12 nt antisense nucleic acid is designed according to a yycF promoter-10 region sequence, and a 24 nt double-chain nucleic acid structure is formed through base complementation of a 5'end sequence and a 3 'end sequence; loading a 24nt double-chain nucleic acid structure onto the graphene oxide to form a stable long-chain structure; the obtained stable long-chain structure is combined with SIS biological hydrogel, and functional gel with antibacterial antisense nucleic acid is prepared; basic fibroblast growth factors are added into the functional gel, and the antisense nucleic acid antibacterial hydrogel for diabetic foot wound healing is prepared. The problem of difficult wound healing of the diabetic foot is solved from the microscopic perspective by designing antisense nucleic acid.

Description

Technical Field

[0001] The invention belongs to the technical field of biomaterials, and in particular relates to an antisense nucleic acid antibacterial hydrogel for healing diabetic foot wounds and a preparation method thereof. Background Art

[0002] Diabetes is one of the global public health issues. Up to 34% of patients have foot ulcers, which increases the disability rate of patients and aggravates the social disease burden. The high-sugar microenvironment inhibits the repair of ulcer wounds, reduces the defense capacity of the skin barrier, and easily leads to wound infection and even osteomyelitis. In my country, the annual prevalence of diabetic foot is as high as 8.1%, and 28% of ulcer patients have lower limb amputations of varying degrees, which seriously affects their quality of life.

[0003] The dynamic balance between host and microorganism is one of the most critical factors in the development of diabetic wound infection. Among them, Staphylococcus aureus is an important pathogen. The formation of biofilm significantly enhances the resistance of Staphylococcus aureus, aggravates the severity of infection, and makes traditional treatment methods (debridement combined with anti-infection) ineffective. Therefore, exploring new strategies for the treatment of diabetic foot infection wounds is an urgent need to improve the level of public health. From the above content, it can be seen that in order to effectively control the impact of the test results and reduce the batch differences of reference products. At present, this field urgently needs to develop a diluent that can effectively disperse the bacterial solution and reduce the batch differences for bacterial solution dispersion. Summary of the invention

[0004] One of the purposes of the present invention is to provide a method for preparing an antisense nucleic acid antibacterial hydrogel for diabetic foot wound healing to solve the shortcoming that the prior art cannot effectively treat diabetic foot ulcers.

[0005] The present invention is achieved through the following technical scheme, which is a method for preparing an antisense nucleic acid antibacterial hydrogel for healing diabetic foot wounds, comprising: designing a 12nt antisense nucleic acid according to the -10 region sequence of the yycF promoter, forming a 24nt double-stranded nucleic acid structure through base complementarity between the 5' end sequence and the 3' end sequence; loading the 24nt double-stranded nucleic acid structure onto graphene oxide to form a stable long-chain structure; combining the obtained stable long-chain structure with an SIS biohydrogel to prepare a functional gel with antibacterial antisense nucleic acid; and adding basic fibroblast growth factor to the functional gel to prepare an antisense nucleic acid antibacterial hydrogel for healing diabetic foot wounds.

[0006] Furthermore, the preparation method may also include preparing a supporting gel for carrying the functional gel and forming a composite biological dressing, wherein the preparation of the supporting gel includes adding calcium chloride (CaCl2) to sodium alginate for cross-linking, then adding carboxymethyl chitosan, adding fibroblast growth factor after mixing evenly, and evenly stirring and mixing to prepare the supporting gel.

[0007] Furthermore, the concentration of sodium alginate is 1-3% w / v, the concentration of calcium chloride is 0.1-2% w / v, and the concentration of carboxymethyl chitosan is 15-20% w / v.

[0008] Furthermore, the supporting gel may include, first, slowly adding a 2% sodium alginate solution to a 0.1% calcium chloride solution through a pipette, the adding time is 10 to 15 minutes, and the dropping speed is 1 to 3 mL / min; after the adding is completed, standing for 20 to 30 minutes, then taking out the initially formed sodium alginate gel from the 0.1% calcium chloride solution, transferring it to a calcium chloride solution with a concentration of 2% w / v and soaking it for 20 to 30 minutes; breaking the sodium alginate gel after cross-linking into particles, and then adding carboxymethyl chitosan with a concentration of 20% w / v, stirring for 3 to 5 minutes using a homogenizer, the stirring speed is 1000 to 5000 rpm, and standing for 10 to 20 minutes to obtain a preliminary mixture; placing the preliminary mixture in an ultrasonic processor, and using intermittent ultrasonic treatment for 2 to 5 minutes, the frequency of the intermittent ultrasonic treatment is 20 to 40KHz, and the interval is 10 seconds per treatment and 5 seconds of pause.

[0009] Furthermore, the sequence of the -10 region of the yycF promoter is 5'-ATTTTACTATAT-3'; the bases of the 5' end sequence and the 3' end sequence are 5'-TATA-3'.

[0010] Furthermore, loading the 24nt double-stranded nucleic acid structure onto graphene oxide can be achieved through π-π stacking or hydrogen bonding.

[0011] Furthermore, the method of loading the 24nt double-stranded nucleic acid structure onto graphene oxide by π-π stacking specifically includes preparing a graphene oxide suspension, adding 10 mg of graphene oxide powder to 10 mL of deionized water, stirring with a glass rod to initially wet the graphene oxide powder and disperse it in the water, and then placing it in an ultrasonic processor for ultrasonic dispersion, the ultrasonic frequency is 20~40KHz, the ultrasonic dispersion time is 30~60min, and the solution temperature is kept below 25°C during the ultrasonic treatment; 100 µL of a 1 µM 24nt double-stranded nucleic acid solution is added to 900 µL of a 1 mg / mL graphene oxide suspension and mixed to obtain a mixed solution; the mixed solution is placed in a rotating mixer and maintained at a temperature of 20~25°C, incubated for 30~60min, and the rotation speed of the mixing rotator is 20~60 rpm, and the tilt angle is 30~45°; after the mixed solution after the incubation is stood at 4~16°C for 10 minutes, the mixed solution is heated to 95°C, maintained for 3~5 minutes, cooled to room temperature and centrifuged to obtain a stable long-chain structure of graphene oxide loaded with a 24nt double-stranded nucleic acid structure, wherein the centrifuge speed is 10,000~11,000 rpm, and the centrifugation time is 10 minutes.

[0012] Furthermore, the method of loading the 24nt double-stranded nucleic acid structure onto graphene oxide by hydrogen bonding specifically includes preparing a graphene oxide suspension, adding 10 mg of graphene oxide powder to 10 mL of phosphate buffer, stirring with a glass rod to initially wet the graphene oxide powder and disperse it in the phosphate buffer, and then placing it in an ultrasonic processor for ultrasonic dispersion, the ultrasonic frequency is 20~40KHz, the ultrasonic dispersion time is 30~40min, and the solution temperature is kept below 25°C during the ultrasonic treatment, and then the pH value of the graphene oxide suspension is adjusted to 6~7; 100 µL of 1 µM 24nt double-stranded nucleic acid solution is added to the graphene oxide suspension, mixed to obtain a mixed solution, and 1 mM magnesium ions are added to the mixed solution; the mixed solution is placed in a rotating mixer and maintained at a temperature of 20~25°C, incubated for 30~60min, and the rotation speed of the mixing rotator is 20~60 rpm, and the tilt angle is 30~45°; after the mixed solution after the incubation is allowed to stand at 4~16°C for 10 minutes, the mixed solution is heated to 95°C, maintained for 3~5 minutes, cooled to room temperature and then centrifuged to obtain a stable long-chain structure of graphene oxide loaded with a 24nt double-stranded nucleic acid structure, wherein the centrifuge speed is 10,000~11,000 rpm, and the centrifugation time is 10 minutes.

[0013] Furthermore, combining the obtained stable long-chain structure with the SIS biohydrogel may include weighing 3 grams of SIS powder and adding it to 100 mL of 0.9% physiological saline, placing the solution in a 37°C water bath for stirring and dispersion, measuring the pH value of the solution using a pH meter, adjusting the pH of the solution to between 7.0 and 7.4 to ensure biocompatibility, and preparing the SIS biohydrogel; then dripping the prepared stable long-chain structure into the SIS biohydrogel, wherein the dripping includes slowly dripping through a pipette, the dripping time is 10 to 15 minutes, and the dripping speed is 1 to 4 mL / min; after the dripping is completed, placing the SIS biohydrogel in a rotating mixer, maintaining the temperature at 20 to 25°C, mixing for 5 to 15 minutes, the rotation speed of the mixing rotor is 20 to 60 rpm, and the tilt angle is 30 to 45°.

[0014] Furthermore, the added amount of the basic fibroblast growth factor is 500 ng / mL.

[0015] To achieve the technical purpose of the present invention, another aspect of the present invention further provides an antisense nucleic acid antibacterial hydrogel for healing diabetic foot wounds, wherein the antisense nucleic acid antibacterial hydrogel is prepared according to the preparation method described in any one of the above items.

[0016] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0017] 1. The present invention solves the problem of difficult wound healing in diabetic foot from a microscopic perspective by designing antisense nucleic acids. Taking the transformation application of the post-transcriptional regulatory mechanism of pathogenic bacteria antisense nucleic acids as the starting point, the antisense nucleic acid transformation system is designed according to the only necessary two-component signal pathway yycF sequence, and the effect of antibacterial nucleic acid hydrogels on the biofilm formation and pathogenicity of Staphylococcus aureus is explored. From the perspective of controlling biofilms, new strategies for the treatment of pathogenic microorganism infections are explored. At the same time, the SIS matrix and the loaded basic fibroblast growth factor are used to jointly construct a hydrogel that promotes wound repair, which improves the effect of wound healing.

[0018] 2. The present invention adopts graphene oxide to load antisense nucleic acid to form a stable long-chain structure. At the same time, through the mutual adsorption of the surface charges of the hydrogel materials, the positively charged carboxymethyl chitosan and the negatively charged sodium alginate gel are combined to promote the formation of a moisturizing microenvironment on the wound surface. The antibacterial hydrogel prepared by the present invention can exert a dual effect of antibacterial and repair, and has the potential of high antibacterial efficiency and strong repair performance. It provides a new antibacterial hydrogel dressing for the treatment of diabetic foot infection wounds and provides a new strategy for the treatment of diabetic foot wound infections. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The drawings described herein are used to provide a further understanding of the embodiments of the present invention, constitute a part of this application, and do not constitute a limitation of the embodiments of the present invention. In the drawings:

[0020] Figure 1 The results of each group of antibacterial ring test provided in Example 3 of the present invention are shown.

[0021] Figure 2 The results of each group of E-Test experiments provided in Example 3 of the present invention are shown.

[0022] Figure 3 This is the live and dead cell staining diagram of each group after SYTO9 staining provided in Example 3 of the present invention.

[0023] Figure 4 This is a statistical graph of the fluorescence intensity of live and dead cells in each group after SYTO9 staining provided in Example 3 of the present invention.

[0024] Figure 5 This is a statistical graph of the experimental results of each PCR group provided in Example 3 of the present invention. DETAILED DESCRIPTION

[0025] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments.

[0026] Unless otherwise specified, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art in the art to which the present invention relates. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe methods and / or materials related to the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail. Regarding the "comprising", "including", "having", "containing", etc. used herein, they are all open-ended terms, that is, they mean including but not limited to. Unless the context clearly indicates otherwise, the expressions "a" and "one" used herein include plural references. For example, referring to "a cell" includes a plurality of such cells and equivalents known to those skilled in the art, etc. The term "about" used herein means a range of ±20% of the value thereafter. In some embodiments, the term "about" means a range of ±10% of the value thereafter. In some embodiments, the term "about" means a range of ±5% of the value thereafter.

[0027] Embodiment 1,

[0028] This exemplary embodiment provides a method for preparing an antisense nucleic acid antibacterial hydrogel for diabetic foot wound healing. Staphylococcus aureus, especially drug-resistant strains, plays an important role in diabetic foot infection. The biofilm and drug resistance formed by it increase the complexity of infection treatment. The preparation method in this exemplary embodiment inhibits yycF transcription and translation by designing a 24nt double-stranded nucleic acid structure, thereby reducing bacterial biofilm formation and bacterial pathogenicity, and ultimately achieving the effect of promoting diabetic foot wound healing, and specifically includes the following steps:

[0029] Step 1: Based on the sequence of the -10 region of the yycF promoter, a 12nt antisense nucleic acid was designed to form a 24nt double-stranded nucleic acid structure through base complementarity between the 5' end sequence and the 3' end sequence.

[0030] The sequence of the -10 region of the yycF promoter is 5'-ATTTTACTATAT-3'; the bases of the 5' end sequence and the 3' end sequence are 5'-TATA-3'.

[0031] It should be noted that the YycFG two-component signaling pathway is the only necessary two-component signaling pathway for the adaptive response of Staphylococcus aureus. It positively regulates the metabolism of biofilm extracellular polysaccharide matrix and enhances bacterial resistance. After the system recognizes the extracellular environmental pressure, the transmembrane receptor (histidine protein kinase YycG) receives the environmental stimulus signal and activates the YycF transcriptional regulator to bind to the specific target gene promoter sequence, thereby regulating the biological characteristics of the bacteria.

[0032] YycF used in this exemplary embodiment is a transcription factor necessary for the survival of Staphylococcus aureus. It can promote the transcription of ica, thereby affecting biofilm formation and reducing pathogenicity, providing an important target for the treatment of Staphylococcus aureus infection. Specifically, in this embodiment, the antisense nucleic acid is optimized and designed based on the sequence of the yycF promoter-10 region, and complementary binding to the yycF-10 region inhibits the transcription initiation of yycF, thereby reducing the biofilm formation and pathogenicity of Staphylococcus aureus.

[0033] Step 2: Load the 24nt double-stranded nucleic acid structure onto graphene oxide to form a stable long-chain structure.

[0034] Specifically, loading the 24nt double-stranded nucleic acid structure onto graphene oxide can be achieved through π-π stacking or hydrogen bonding.

[0035] The loading of 24nt double-stranded nucleic acid structure onto graphene oxide by π-π stacking specifically includes the following:

[0036] 1) Prepare graphene oxide suspension: add 10 mg of graphene oxide powder into 10 mL of deionized water, stir with a glass rod to make the graphene oxide powder initially wet and dispersed in the water, and then put it into an ultrasonic processor for ultrasonic dispersion. The ultrasonic frequency is 20-40 kHz and the ultrasonic dispersion time is 30-60 min. Keep the solution temperature below 25 °C during the ultrasonic treatment.

[0037] Ultrasonic dispersion can ensure that graphene oxide powder is evenly distributed in water, and the temperature is controlled below 25°C, thereby avoiding high temperature damage to the material structure and ensuring uniformity of dispersion.

[0038] 2) Add 100 µL of 1 µM 24nt double-stranded nucleic acid solution to 900 µL of 1 mg / mL graphene oxide suspension and mix well to obtain a mixed solution.

[0039] 3) Place the mixed solution in a rotating mixer at a temperature of 20-25°C and incubate for 30-60 min. The rotation speed of the mixing rotator is 20-60 rpm and the tilt angle is 30-45°.

[0040] By incubating the mixed solution in a rotating mixer and mixing it at a specific temperature, rotation speed and tilt angle, it is ensured that the 24nt double-stranded nucleic acid structure can fully contact and load onto the graphene oxide to form a stable long-chain structure, while optimizing the π-π stacking effect between nucleic acid and graphene oxide.

[0041] 4) After the incubation, the mixed solution was allowed to stand at 4-16°C for 10 minutes, then heated to 95°C, maintained for 3-5 minutes, and then centrifuged to obtain a stable long-chain structure of graphene oxide loaded with a 24nt double-stranded nucleic acid structure, wherein the centrifuge speed was 10,000-11,000 rpm and the centrifugation time was 10 minutes.

[0042] The mixed solution after incubation is heated to 95°C and then cooled to promote the cross-linking of nucleic acid and graphene oxide. It should be noted that nucleic acid (including DNA and RNA) may denature (i.e., double-stranded DNA is melted into single strands) or degrade at high temperatures, but in certain cases, this treatment is controllable. For example, the temperature of 95°C selected in this exemplary embodiment is indeed high enough to denature nucleic acid molecules. However, for the short-chain oligonucleotides (12nt antisense nucleic acids) selected in the present invention, the denaturation process caused by the temperature of 95°C may be reversible. When the temperature returns to room temperature, these single-stranded nucleic acid molecules can be re-cross-linked with graphene oxide to form a stable long-chain structure.

[0043] The above content describes the specific operation steps for loading the 24nt double-stranded nucleic acid structure onto graphene oxide through π-π stacking. π-π stacking, as a powerful interaction, can effectively adsorb double-stranded nucleic acids onto the surface of graphene oxide. Since the planar structure of graphene oxide can interact with the aromatic rings of nucleic acid bases in π-π, this adsorption process is very efficient and stable, and can achieve the loading of nucleic acids without the need for additional chemical modification. And π-π stacking, as a non-covalent interaction, does not involve the formation or breaking of chemical bonds, so it will not destroy the double-stranded structure of nucleic acids. Compared with covalent modification, this method can effectively maintain the original function and biological activity of nucleic acids, avoiding structural denaturation or functional loss. At the same time, the coating effect of graphene oxide can prevent the influence of the external environment on nucleic acids, thereby improving the stability of nucleic acids.

[0044] The method of loading the 24nt double-stranded nucleic acid structure onto graphene oxide through hydrogen bonding specifically includes:

[0045] 1) Prepare graphene oxide suspension. Add 10 mg of graphene oxide powder into 10 mL of phosphate buffer and stir with a glass rod to make the graphene oxide powder initially wet and dispersed in the phosphate buffer. Then put it into an ultrasonic processor for ultrasonic dispersion. The ultrasonic frequency is 20-40 kHz and the ultrasonic dispersion time is 30-40 min. Keep the solution temperature below 25 °C during the ultrasonic treatment. Then adjust the pH value of the graphene oxide suspension to 6-7.

[0046] 2) Add 100 µL of 1 µM 24nt double-stranded nucleic acid solution to the graphene oxide suspension, mix well to obtain a mixed solution, and add 1 mM magnesium ions to the mixed solution.

[0047] 3) Place the mixed solution in a rotating mixer at a temperature of 20-25°C and incubate for 30-60 min. The rotation speed of the mixing rotator is 20-60 rpm and the tilt angle is 30-45°.

[0048] 4) After the incubation, the mixed solution was allowed to stand at 4-16°C for 10 minutes, then heated to 95°C, maintained for 3-5 minutes, cooled to room temperature, and centrifuged to obtain a stable long-chain structure of graphene oxide loaded with a 24nt double-stranded nucleic acid structure, wherein the centrifuge speed was 10,000-11,000 rpm and the centrifugation time was 10 minutes.

[0049] In the above steps, a process of loading the promoter antisense nucleic acid sequence by using layered graphene oxide (through non-covalent bonds such as hydrogen bonds) is disclosed. This step can significantly improve the conversion efficiency of antisense nucleic acids and enhance their antibacterial efficacy. At the same time, single-stranded nucleic acids are used to form a hydrophilic loose porous structure through the principle of base complementarity (hydrogen bonding), which, combined with the modification and regulation of polymer compounds, helps to enhance the physicochemical properties and biocompatibility; antisense nucleic acids are loaded on graphene oxide (nucleic acid-graphene oxide) by means of base hydrogen bonds, and extended by base complementarity (nucleic acid-nucleic acid-graphene oxide), and finally form a long chain structure of -[graphene oxide-nucleic acid-nucleic acid-graphene oxide]-.

[0050] Step 3: Combine the obtained stable long-chain structure with SIS biohydrogel to prepare a functional gel with antibacterial antisense nucleic acid, and add basic fibroblast growth factor to the functional gel to prepare an antisense nucleic acid antibacterial hydrogel for diabetic foot wound healing. The addition amount of the basic fibroblast growth factor is 500 ng / mL.

[0051] The obtained stable long-chain structure is combined with SIS biohydrogel, specifically including:

[0052] 1) Weigh 3 g of SIS powder and add it to 100 mL of 0.9% saline. Place the solution in a 37°C water bath for stirring and dispersion. Use a pH meter to measure the pH of the solution and adjust the pH of the solution to between 7.0 and 7.4 to ensure biocompatibility. The SIS biohydrogel was prepared.

[0053] Specifically, the SIS powder can be a natural extracellular matrix obtained by freeze-drying - porcine small intestinal submucosa (SIS) tissue. Generally speaking, SIS is mainly composed of type I and type III collagen, and contains a variety of bioactive factors that can promote fibroblast proliferation and endothelial angiogenesis. By encapsulating the nucleic acid-graphene oxide composite structure in the SIS hydrogel through the SIS biohydrogel, the compatibility of the composite structure in the organism can be significantly improved, and the SIS biohydrogel can provide a physical barrier to protect the 24nt double-stranded nucleic acid structure from damage by the external environment (such as enzymatic hydrolysis, degradation, etc.). This encapsulation method not only increases the stability of the nucleic acid structure, but also prolongs its functional time on the wound surface, thereby achieving a sustained release effect and prolonging the action time of the nucleic acid. Finally, the nucleic acid structure encapsulated in the SIS biohydrogel is easier to pass through the biological barrier, improving its transfer efficiency in the body. SIS hydrogel can promote the transmembrane transport of nucleic acids and enhance their uptake rate in target cells, thereby improving the effectiveness of nucleic acid therapy.

[0054] 2) Then, the prepared stable long-chain structure is added dropwise to the SIS biohydrogel, wherein the adding includes slowly adding dropwise through a pipette, the adding time is 10 to 15 minutes, and the adding speed is 1 to 4 mL / min.

[0055] By combining the long-chain structure with the SIS biohydrogel in a slow dripping manner, it is possible to ensure that the long-chain structure is evenly distributed in the SIS hydrogel, reduce local concentration differences, and ensure the functional uniformity of the entire hydrogel.

[0056] 3) After the addition is completed, place the SIS biohydrogel in a rotating mixer, maintain the temperature at 20-25°C, mix for 5-15 minutes, and rotate the mixer at a speed of 20-60 rpm and a tilt angle of 30-45°. During the rotational mixing, the temperature and rotation speed are controlled within a relatively mild range to avoid damage to the SIS hydrogel and the long-chain structure, while ensuring that the long-chain structure can be evenly distributed in the SIS hydrogel. This helps maintain the integrity and functionality of the material.

[0057] It should be noted that the antisense nucleic acid antibacterial hydrogel prepared by steps 1 to 3 can achieve the purpose of promoting the healing of diabetic foot wounds. However, during use, a single antisense nucleic acid antibacterial hydrogel is coated on the wound surface, and the gel film is exposed to the air and dries up quickly, which leads to loss of activity, and cannot play a good sustained release effect, and can not continuously ensure the healing of the wound for a long time, which is poor for promoting wound healing. Therefore, in step 4, the antisense nucleic acid antibacterial hydrogel prepared by supporting the supporting gel is coated on the supporting gel, and then the antisense nucleic acid antibacterial hydrogel surface is covered on the wound, and one side of the supporting gel is exposed to the air, thereby ensuring that the antisense nucleic acid antibacterial hydrogel can be long-lastingly moisturizing and ensuring that the effective ingredients therein can be sustained-released.

[0058] Step 4: Prepare a supporting gel for carrying the functional gel and forming a composite biological dressing.

[0059] The preparation of the support gel specifically includes adding calcium chloride (CaCl2) to sodium alginate for cross-linking, then adding carboxymethyl chitosan, adding fibroblast growth factor after mixing evenly and stirring evenly to prepare the support gel. The concentration of sodium alginate is 1-3% w / v, the concentration of calcium chloride is 0.1-2% w / v, and the concentration of carboxymethyl chitosan is 15-20% w / v. The specific steps are:

[0060] 1) First, slowly add 2% sodium alginate solution into 0.1% calcium chloride solution through a pipette. The addition time is 10-15 minutes and the addition speed is 1-3 mL / min.

[0061] 2) After the addition is completed, let it stand for 20 to 30 minutes, then take out the initially formed sodium alginate gel from the 0.1% calcium chloride solution and transfer it to a 2% w / v calcium chloride solution and soak it for 20 to 30 minutes.

[0062] 3) The cross-linked sodium alginate gel was broken into particles, and then carboxymethyl chitosan with a concentration of 20% w / v was added. The mixture was stirred with a homogenizer for 3 to 5 minutes at a stirring speed of 1000 to 5000 rpm, and then allowed to stand for 10 to 20 minutes to obtain a preliminary mixture.

[0063] 4) placing the preliminary mixture in an ultrasonic processor and subjecting it to intermittent ultrasonic treatment for 2 to 5 minutes, wherein the frequency of the intermittent ultrasonic treatment is 20 to 40 kHz, and the intermittent treatment is performed for 10 seconds and then paused for 5 seconds.

[0064] Embodiment 2,

[0065] This exemplary embodiment provides an antisense nucleic acid antibacterial hydrogel for diabetic foot wound healing. The antisense nucleic acid antibacterial hydrogel is prepared by steps 1 to 3 as described in exemplary embodiment 1 above.

[0066] The supporting gel is prepared by step 4 as described in the above exemplary embodiment 1, and the antisense nucleic acid antibacterial hydrogel is coated on the supporting gel to obtain the composite biological dressing.

[0067] In order to better understand the present invention, the present invention is further explained below in conjunction with the embodiments, but the present invention is not limited to the following embodiments.

[0068] Embodiment 3,

[0069] In this example, experiments were performed to verify the killing effect of the antisense nucleic acid antibacterial hydrogel provided by the method of the present invention on Staphylococcus aureus. The antisense nucleic acid antibacterial hydrogel used in this example was prepared according to the steps described in Example 1. The Staphylococcus aureus used in this example was a standard strain of Staphylococcus aureus (ATCC29213).

[0070] In this example, in order to determine the minimum inhibitory concentration (MIC) of the antisense nucleic acid antibacterial hydrogel prepared according to the steps described in Example 1, Staphylococcus aureus was co-cultured with antisense nucleic acid antibacterial hydrogels of different concentrations, and its antibacterial effect was evaluated by a series of experimental methods.

[0071] A standard strain of Staphylococcus aureus (ATCC 29213) was cultured to the logarithmic growth phase, which means that the bacterial population increases rapidly and the optical density (OD600 nm) reaches 0.5.

[0072] A total of 5 experimental groups were set up, namely, S. aureus, H / C, H / C@bFGF, H / C@GO-AS, and H / C@GO-AS@bFGF. Wherein, S.aureus represents Staphylococcus aureus, which is the control group of Staphylococcus aureus samples in this embodiment; H / C (Hydrogel / Control) represents the control group using ordinary hydrogel, and the hydrogel in this experimental group is not treated in other ways; "@bFGF" in H / C@bFGF represents that basic Fibroblast Growth Factor (bFGF) is loaded through SIS matrix, that is, basic Fibroblast Growth Factor is added to the SIS hydrogel in this experimental group; "@GO-AS" in H / C@GO-AS represents the addition of graphene oxide (GO) and the 24nt double-stranded nucleic acid structure (Antibiotic Substance, AS) formed by the complementary bases of the 5' end sequence and the 3' end sequence according to the sequence of the -10 region of the yycF promoter in Example 1 by designing a 12nt antisense nucleic acid; and finally, H / C@GO-AS@bFGF is a 24nt double-stranded nucleic acid structure (Antibiotic Substance, AS) formed by the addition of graphene oxide (GO) and the 12nt antisense nucleic acid designed according to the sequence of the -10 region of the yycF promoter in Example 1; GO), 24nt double-stranded nucleic acid structure (Antibiotic Substance, AS) and basic fibroblast growth factor (basic Fibroblast Growth Factor, bFGF) hydrogel, that is, the antisense nucleic acid antibacterial hydrogel prepared by the steps described in Example 1 of the present invention.

[0073] The antibacterial activity of different hydrogels against Staphylococcus aureus (S. aureus) was tested by the inhibition ring test. The preserved Staphylococcus aureus stock solution was cultured into 5×10 5 cfu / ml concentration suspension, dip the bacterial solution with a sterile cotton swab and evenly apply it on the surface of the LB solid culture medium plate for 3 times. Each time it is applied, the plate should be rotated 60°, and finally the cotton swab should be applied around the edge of the plate for a week. Cover the plate, incubate for 24 hours, cut the antisense nucleic acid antibacterial hydrogel samples of different concentrations into samples with a diameter of about 5 mm, and place them in the center of the culture dish on the surface of the culture medium. After co-cultivation in a constant temperature incubator at 37°C for 24 hours, take out the culture dish and measure the diameter of the antibacterial ring to evaluate the antibacterial effect of the antisense nucleic acid antibacterial hydrogel. Through the comparison of biomass, it can be found that at a working concentration of 5μL / mL, the bacterial biomass of the H / C@GO-AS@bFGF group is the lowest among all groups, almost reduced to half of the biomass of the control group.

[0074] Figure 1The figure shows the antibacterial effect of the microneedle further tested by the inhibition zone determination of each group in the antibacterial ring test in this embodiment. It can be seen from the figure that the inhibition zone diameter of the H / C@GO-AS@bFGF and H / C@GO-AS groups is 0.35 cm, which is significantly larger than the 0.2 cm of the H / C and H / C@bFGF groups.

[0075] The E-Test test was used to further test the antibacterial ability of the hydrogel against Staphylococcus aureus (S. aureus). The preserved Staphylococcus aureus stock solution was prepared to a McFarland turbidity of OD600nm=0.5, and was evenly smeared on the surface of 5 LB solid culture media with a sterile cotton swab and incubated at room temperature for 15min. The first well was the negative control group, and 20uL LB liquid culture medium was added. The other 4 wells were added with different concentrations of antisense nucleic acid antibacterial hydrogel extracts, and incubated in a 37℃ constant temperature incubator for 24h. Five sterile drug sensitivity test strips were soaked in vancomycin solution. After being completely soaked, the test strips were clamped with sterile tweezers, dried slightly, and gently pressed on the center of the LB culture medium surface to fit closely to the surface of the culture medium. Place in a 37℃ constant temperature incubator and incubate for 24h. Take out the culture medium, take pictures, record, and measure the value of antibiotic inhibition intersection.

[0076] Figure 2 The figure shows the sensitivity of the microneedle hydrogel to vancomycin in each group of the E-Test test in this embodiment when the biofilm is down-regulated. Figure 2 It can be seen that the H / C@GO-AS@bFGF and H / C@GO-AS groups showed higher sensitivity to vancomycin at concentrations of 2 mg / L and 1.5 mg / L.

[0077] 20uL of the prepared Staphylococcus aureus stock solution was extracted with a pipette and added to the first 5 wells of the 6-well plate. The first well was used as the control group, and 20uL of the pre-made LB liquid culture medium was added. 20uL of antisense nucleic acid antibacterial hydrogel extracts of different concentrations were added to the subsequent wells. The well plate was placed in a 37°C constant temperature incubator for 24 hours. The well plate was taken out and observed under a high power microscope to show that Staphylococcus aureus grew well. The well plates were stained with SYTO9 in the live cell / dead cell staining box (Invitrogen, USA). The Nikon TE300 fluorescence microscope was used for detection. Among them, the live cells of Staphylococcus aureus showed green fluorescence, and the dead cells did not emit light. The growth of Staphylococcus aureus can be understood according to the fluorescence intensity.

[0078] Figure 3 The live and dead cell staining images of each group after the biofilm was stained green using SYTO9 in this embodiment are shown. It can be seen from the figure that the fluorescence intensity of the H / C@GO-AS@bFGF group is around 20 AU, which is the lowest, and the fluorescence intensity is significantly reduced compared with other groups. Figure 4The fluorescence intensity statistics of live and dead cells in each group are shown after the biofilm was stained green using SYTO9. Figure 4 It can be seen that the fluorescence intensity of H / C@GO-AS@bFGF is lowest at around 20 AU, and the fluorescence intensity is significantly decreased compared with other groups.

[0079] Finally, PCR experiments were used to verify the expression of biofilm-related genes of Staphylococcus aureus in each group. Total RNA of Staphylococcus aureus was extracted and purified using the MasterPureTM RNA Purification Kit (Epicenter Technologies, USA) and reverse transcribed into cDNA using the Reverse Transcription First Strand cDNA Synthesis Kit (Thermo, USA). Afterwards, qRT-PCR was performed using the LightCycler 480 system (Roche, Switzerland). The data were analyzed using the 2-ΔΔCt method, with 16S rRNA as the internal reference. The primer list is shown in Table 1.

[0080] Table 1 Primer sequences used for qRT-PCR analysis

[0081]

[0082] The specific operation of PCR experiment is as follows:

[0083] 1) After thoroughly aspirating the culture liquid, add 350 μl of Lysis Buffer RLT Plus directly and repeatedly pipette to lyse the cells. Add the entire lysis mixture to the DNA cleanup column.

[0084] 2) Immediately centrifuge at 13000 rpm for 1 minute and retain the filtrate.

[0085] 3) Estimate the volume of the filtrate more accurately, add an equal volume of 70% ethanol, and mix immediately by pipetting.

[0086] 4) Immediately add the mixture (less than 720 μL each time) to an adsorption column RA, centrifuge at 13,000 rpm for 30 seconds, and discard the waste liquid.

[0087] 5) Add 700 μL of deproteinized solution RW1, let stand at room temperature for 30 seconds, centrifuge at 13,000 rpm for 30 seconds, and discard the waste liquid.

[0088] 6) Add 500μL of Rinse Buffer RW, centrifuge at 13000 rpm for 30 seconds, discard the waste liquid, add 500μL of Rinse Buffer RW, and repeat.

[0089] 7) Place the adsorption column RA back into the empty collection tube and centrifuge at 13,000 rpm for 2 minutes to remove as much of the rinse solution as possible to prevent residual ethanol in the rinse solution from inhibiting downstream reactions.

[0090] 8) Take out the adsorption column RA and put it into a clean 1.5 ml centrifuge tube. Add 30-50 μl RNase free water to the middle part of the adsorption membrane according to the expected RNA yield. Place it at room temperature for 1 minute. Centrifuge at 13,000 rpm for 1 minute to obtain the RNA solution. Detect the RNA concentration on the machine.

[0091] 8) Reverse transcription: According to the RNA concentration of each group, prepare a 20μl volume of reverse transcription system (Enzyme 2μl Buffer 4μl Nuclear-free H2O 4μl RNA X μl Nuclear-free H2O 10-Xμl) and set up the reverse transcription instrument.

[0092] 9) PCR: Add samples for testing as required.

[0093] The final result is Figure 5 As shown, Figure 5 The result graph of the PCR experiment in this embodiment is shown, from which it can be seen that YycFG and biofilm-related pathways were significantly downregulated in the H / C@GO-AS@bFGF and H / C@GO-AS groups, especially the expression of yycF / yycG / icaA genes decreased to less than half of the control group. In addition, the negative regulatory system of codY was significantly upregulated in the H / C@GO-AS@bFGF and H / C@GO-AS groups. The synergistic effect of increased negative regulation and decreased positive regulation may lead to the dispersion and low vitality of bacterial biofilms.

[0094] It can be seen from the drawings in this embodiment that the antisense nucleic acid antibacterial hydrogel prepared according to the steps of Example 1 of the present invention has a good killing effect on Staphylococcus aureus.

[0095] The specific implementation methods described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific implementation method of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing an antisense nucleic acid antibacterial hydrogel for diabetic foot wound healing, characterized in that: The preparation method comprises: According to the sequence of the -10 region of the yycF promoter, a 12nt antisense nucleic acid was designed, and a 24nt double-stranded nucleic acid structure was formed through base complementarity between the 5' end sequence and the 3' end sequence; Loading 24nt double-stranded nucleic acid structure onto graphene oxide to form a stable long-chain structure; The obtained stable long-chain structure is combined with SIS biohydrogel to prepare a functional gel with antibacterial antisense nucleic acid; Basic fibroblast growth factor is added to the functional gel to prepare an antisense nucleic acid antibacterial hydrogel for healing diabetic foot wounds.

2. The method for preparing the antisense nucleic acid antibacterial hydrogel for diabetic foot wound healing according to claim 1, characterized in that: The preparation method further comprises preparing a supporting gel for carrying the functional gel and forming a composite biological dressing. The preparation of the supporting gel comprises adding calcium chloride to sodium alginate for cross-linking, then adding carboxymethyl chitosan, adding fibroblast growth factor after mixing evenly, and evenly stirring and mixing to prepare the supporting gel.

3. The method for preparing the antisense nucleic acid antibacterial hydrogel for diabetic foot wound healing according to claim 2, characterized in that: The concentration of the sodium alginate is 1-3% w / v, the concentration of the calcium chloride is 0.1-2% w / v, and the concentration of the carboxymethyl chitosan is 15-20% w / v.

4. The method for preparing the antisense nucleic acid antibacterial hydrogel for diabetic foot wound healing according to claim 2, characterized in that: The supporting gel comprises: First, slowly add 2% sodium alginate solution to 0.1% calcium chloride solution through a pipette. The addition time is 10-15 minutes and the drop speed is 1-3 mL / min. After the addition is completed, the solution is allowed to stand for 20 to 30 minutes, and then the initially formed sodium alginate gel is taken out from the 0.1% calcium chloride solution and transferred to a 2% w / v calcium chloride solution to be immersed for 20 to 30 minutes. The cross-linked sodium alginate gel was broken into particles, and then carboxymethyl chitosan with a concentration of 20% w / v was added, and the mixture was stirred for 3 to 5 minutes using a homogenizer at a stirring speed of 1000 to 5000 rpm, and then allowed to stand for 10 to 20 minutes to obtain a preliminary mixture; The preliminary mixture is placed in an ultrasonic processor and treated with intermittent ultrasonic waves for 2 to 5 minutes. The frequency of the intermittent ultrasonic waves is 20 to 40 kHz, and the interval is 10 seconds for each treatment and 5 seconds for each pause.

5. The method for preparing the antisense nucleic acid antibacterial hydrogel for diabetic foot wound healing according to claim 1, characterized in that: The sequence of the -10 region of the yycF promoter is 5'-ATTTTACTATAT-3'; The bases of the 5' end sequence and the 3' end sequence are 5'-TATA-3'.

6. The method for preparing the antisense nucleic acid antibacterial hydrogel for diabetic foot wound healing according to claim 1, characterized in that: The 24nt double-stranded nucleic acid structure is loaded onto graphene oxide through π-π stacking or hydrogen bonding.

7. The method for preparing the antisense nucleic acid antibacterial hydrogel for diabetic foot wound healing according to claim 6, characterized in that: The method of loading the 24nt double-stranded nucleic acid structure onto graphene oxide by π-π stacking specifically includes: Prepare graphene oxide suspension, add 10 mg of graphene oxide powder into 10 mL of deionized water, stir with a glass rod to make the graphene oxide powder initially wet and dispersed in the water, then put it into an ultrasonic processor for ultrasonic dispersion, the ultrasonic frequency is 20~40KHz, the ultrasonic dispersion time is 30~60min, and the solution temperature is kept below 25℃ during the ultrasonic treatment; 100 µL of 1 µM 24nt double-stranded nucleic acid solution was added to 900 µL of 1 mg / mL graphene oxide suspension and mixed to obtain a mixed solution; Place the mixed solution in a rotating mixer and maintain the temperature at 20-25°C for 30-60 minutes. The rotation speed of the mixing rotator is 20-60 rpm and the tilt angle is 30-45°. After the incubation, the mixed solution was allowed to stand at 4-16°C for 10 minutes, then heated to 95°C, maintained for 3-5 minutes, cooled to room temperature, and centrifuged to obtain a stable long-chain structure of graphene oxide loaded with a 24nt double-stranded nucleic acid structure, wherein the centrifuge speed was 10,000-11,000 rpm and the centrifugation time was 10 minutes.

8. The method for preparing the antisense nucleic acid antibacterial hydrogel for diabetic foot wound healing according to claim 6, characterized in that: The method of loading the 24nt double-stranded nucleic acid structure onto graphene oxide by hydrogen bonding specifically includes: Prepare a graphene oxide suspension, add 10 mg of graphene oxide powder into 10 mL of phosphate buffer, stir with a glass rod to make the graphene oxide powder initially wet and dispersed in the phosphate buffer, then put it into an ultrasonic processor for ultrasonic dispersion, the ultrasonic frequency is 20~40KHz, the ultrasonic dispersion time is 30~40min, keep the solution temperature below 25℃ during the ultrasonic treatment, and then adjust the pH value of the graphene oxide suspension to 6~7; Add 100 µL of 1 µM 24nt double-stranded nucleic acid solution to the graphene oxide suspension, mix well to obtain a mixed solution, and add 1 mM magnesium ions to the mixed solution; Place the mixed solution in a rotating mixer and maintain the temperature at 20-25°C for 30-60 minutes. The rotation speed of the mixing rotator is 20-60 rpm and the tilt angle is 30-45°. After the incubation, the mixed solution was allowed to stand at 4-16°C for 10 minutes, then heated to 95°C, maintained for 3-5 minutes, cooled to room temperature, and centrifuged to obtain a stable long-chain structure of graphene oxide loaded with a 24nt double-stranded nucleic acid structure, wherein the centrifuge speed was 10,000-11,000 rpm and the centrifugation time was 10 minutes.

9. The method for preparing an antisense nucleic acid antibacterial hydrogel for diabetic foot wound healing according to claim 1, wherein the obtained stable long-chain structure is combined with the SIS biohydrogel, comprising: Weigh 3 g of SIS powder and add it to 100 mL of 0.9% saline. Place the solution in a 37°C water bath for stirring and dispersion. Use a pH meter to measure the pH of the solution and adjust the pH of the solution to between 7.0 and 7.4 to ensure biocompatibility. SIS biohydrogel is prepared. Then, the prepared stable long-chain structure is dripped into the SIS biohydrogel, wherein the dripping includes slowly dripping through a pipette, the dripping time is 10 to 15 minutes, and the dripping speed is 1 to 4 mL / min; After the dropwise addition is completed, the SIS biohydrogel is placed in a rotating mixer, the temperature is maintained at 20-25°C, and the mixture is mixed for 5-15 minutes. The rotation speed of the mixing rotator is 20-60 rpm, and the tilt angle is 30-45°.

10. An antisense nucleic acid antibacterial hydrogel for diabetic foot wound healing, characterized in that: The antisense nucleic acid antibacterial hydrogel for diabetic foot wound healing is prepared according to the method as described in any one of claims 1 to 9.

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