Application of anti-infection biological patch in temporary abdominal closure material for abdominal cavity opening

Abstract

The invention relates to the field of biomedical materials, in particular to application of an anti-infection biological patch in temporary abdominal closure materials for abdominal cavity opening. The anti-infection biological patch provided by the invention has good biodegradability and biocompatibility, can effectively control short-term large-scale infection, has good active antibacterial ability and drug sustained release effect, and also has good mechanical strength and tissue compliance; the anti-infection biological patch disclosed by the invention is soft in texture, and does not cause damage to contacted intestinal canals and other visceral sac tissues; when the material is applied to temporary abdominal closure materials for opening the abdominal cavity, the occurrence rate of intestinal air fistula can be reduced to 2% or below, meanwhile, the risk of incisional hernia and abdominal cavity bleeding is reduced, fascia retraction can be reduced, and therefore adhesion between the intestine and the abdominal wall and adhesion between the intestine and abdominal cavity dressing are further avoided, and closure repair of abdominal wall fascia tissue is effectively promoted.

Description

Technical Field

[0001] The invention relates to the field of biomedical materials, and more specifically to the application of an anti-infection biological patch in temporary abdominal closure materials for abdominal cavity opening. Background Art

[0002] Open abdominal therapy is an effective means of treating severe abdominal infections, abdominal hypertension and other critical abdominal diseases. After open abdominal therapy, temporary abdominal closure is required, and then the full-thickness closure of the incision can be delayed (definitive abdominal closure) at an appropriate time. Patent No. CN115006592B provides a solution to the material for temporary abdominal closure of open abdominal cavity. The decellularized dermis is impregnated in a placental tissue slurry containing nanosilver to obtain a composite material, which is covered on the open abdominal intestinal tract to reduce the occurrence of air fistula. However, the document does not mention to what extent the occurrence of air fistula can be reduced. In addition, the drug release of this composite material obtained by immersing the biomaterial in the drug solution is very fast. Wei Chao et al. have conducted related research ("Research on Mechanical Properties and Antibacterial Functionalization of Calfskin Bioremediation Membrane", May 2022). The bovine acellular dermal biofilm was immersed in 30-50 mg / mL gentamicin sulfate solution for 24 hours, and then taken out and freeze-dried. The results showed that the drug release rate exceeded 90% within 24 hours, and the drug release rate was close to 100% after 42 hours. Gentamycin sulfate is an antibiotic with a low albumin binding rate. In theory, if drugs with a high albumin binding rate are used, the drug release rate will be faster.

[0003] At the same time, the material impregnation method described in the patent No. CN115006592B is equivalent to attaching an antibacterial coating on the surface of the decellularized dermis, and the biological patch formed by this coating method usually shows low drug loading in clinical practice, which may further lead to the situation where the antibacterial treatment effect cannot be achieved. Similar to the impregnation method in the patent No. CN115006592B, the patent No. CN101623518B provides a body wall repair material for long-term closure of the abdominal cavity after laparotomy, in which SIS is immersed in a nanosilver working solution to implant nanosilver particles in the SIS, with a silver content of 1±0.048μg / cm 2 . Summary of the invention

[0004] In order to overcome the above problems and expand the application of composite products of extracellular matrix loaded with functional active substances so that it is not only used as a prosthesis to induce healing, the present invention provides an anti-infection biological patch for use in temporary abdominal closure materials for abdominal cavity opening, wherein the anti-infection biological patch is obtained by loading at least two antibiotics on an uncross-linked extracellular matrix; the antibiotics are antibiotics with a high protein binding rate.

[0005] In the early stage of abdominal opening, especially within 3-5 days of abdominal opening, the intestine is exposed and is prone to serious infection, so some temporary abdominal closure measures need to be taken. Medical gauze is often used as a temporary abdominal closure material, but the isolation effect of gauze is very limited; the "Chinese Expert Consensus on Open Abdominal Therapy" points out that synthetic patches are prone to adhesion and wear during abdominal reconstruction, so synthetic patches are not recommended in open abdominal treatment; although non-cross-linked biological patches have high biocompatibility, they are easy to degrade and have unstable mechanical properties, and cannot achieve the desired effect, while cross-linked biological patches may cause certain stimulation to the intestine due to the residue of cross-linking agents; the above-mentioned existing temporary abdominal closure technologies are mostly mechanical protection and only play the role of isolating the intestine. In order to obtain a patch with both mechanical protection and chemical protection, the inventor wants to load antibiotics on the non-cross-linked biological patch to make it have an antibacterial effect, but existing literature and practice have found that the antibiotics on the non-cross-linked patch not only have a low drug loading, but also have a too fast release rate, which cannot achieve an antibacterial effect, making it difficult to further apply.

[0006] Generally speaking, a high drug-protein binding rate is conducive to drug transport, and the efficacy will be correspondingly improved, which also means that the drug release rate will be relatively faster.

[0007] The high protein binding rate antibiotics are selected from at least two of tetracycline, rifamycin and β-lactam antibiotics.

[0008] The tetracycline is selected from one or more of minocycline, minocycline hydrochloride, chlortetracycline, and doxycycline hydrochloride.

[0009] The tetracycline is minocycline and / or minocycline hydrochloride.

[0010] The rifamycin is selected from one or more of 3-[[(4-methyl-1-piperazinyl)imino]methyl]-rifamycin, cyclopentylpiperazine rifamycin and 4-N-isobutylspiperidinyl rifamycin S.

[0011] Rifamycin is 3-[[(4-methyl-1-piperazinyl)imino]methyl]-rifamycin.

[0012] The β-lactam antibiotics are selected from one or more of ceftriaxone, ertapenem, imipenem-cilastatin sodium, amoxicillin, and penicillin.

[0013] β-lactam antibiotics are ceftriaxone and / or amoxicillin.

[0014] Preferably, the highly protein-bound antibiotics are tetracycline and rifamycin.

[0015] The anti-infective biological patch is obtained by loading at least tetracycline and rifamycin on the non-cross-linked extracellular matrix.

[0016] Uncross-linked extracellular matrix was purchased from Zhuoruan Medical Technology (Suzhou) Co., Ltd., and was prepared from small intestinal submucosa, peritoneum, dermis, bladder basement membrane or pericardium according to the perfusion-pressure difference method disclosed in patent CN106075583B.

[0017] Preferably, the non-cross-linked extracellular matrix originates from at least two of the following: small intestinal submucosa, peritoneum, dermis, bladder basement membrane, and pericardium.

[0018] More preferably, the non-cross-linked extracellular matrix is ​​derived from the small intestinal submucosa and the bladder basement membrane.

[0019] The drug release rate of each antibiotic in the anti-infection biological patch within 24 hours is 15-85wt%, and the drug release rate of each antibiotic in the anti-infection biological patch within 120 hours is 85-100wt%.

[0020] Preferably, the drug release rate of each antibiotic in the anti-infection biological patch within 24 hours is 20.3-85wt%, and the drug release rate of each antibiotic in the anti-infection biological patch within 120 hours is 85-100wt%.

[0021] In the present invention, the anti-infection biological patch is loaded with at least two antibiotics, and the antibiotics are high protein binding rate antibiotics. The drug release rate of each antibiotic in the anti-infection biological patch in 24 hours is 15-85wt%, and the drug release rate of each antibiotic in the anti-infection biological patch in 120 hours is 85-100wt%, which overcomes the technical resistance of low antibiotic loading and too fast release on the non-cross-linked patch, and the inability to achieve antibacterial effect. The anti-infection biological patch is used in the field of temporary abdominal closure materials for abdominal cavity opening, which not only plays a mechanical protective role on the intestinal tract while ensuring the mechanical stability of the material, but also plays a chemical protective role, has a good active antibacterial function, can not only reduce the incidence of intestinal air fistula caused by abdominal cavity opening to less than 2%, but also reduce the risk of abdominal bleeding, protect the intestinal tract from the influence of blood hypoperfusion, in addition, it can also improve the fascia closure rate and shorten the definitive abdominal closure time, especially when the uncross-linked extracellular matrix is ​​derived from the submucosal layer of the small intestine and the basement membrane of the bladder, the effect is particularly obvious.

[0022] Preferably, the drug release rate of tetracycline loaded in the anti-infection biological patch is 20.3-64.5wt% in 24 hours.

[0023] Further preferably, the drug release rate of tetracycline loaded in the anti-infection biological patch is 21.6-63.9wt% in 24 hours.

[0024] The drug release rate of rifamycin loaded in the anti-infection biological patch was 70.8-85wt% within 24 hours.

[0025] Preferably, the drug release rate of each antibiotic in the anti-infection biological patch is 86-100wt% in 120h.

[0026] Preferably, the drug release rate of tetracycline loaded in the anti-infection biological patch is 86.2-98.7 wt % in 120 h.

[0027] The drug release rate of rifamycin loaded in the anti-infection biological patch was 95.1-100wt% within 120h.

[0028] As a preferred embodiment, the drug release rate of each antibiotic in the anti-infection biological patch is less than 40wt% within 4 hours.

[0029] The drug release rate of tetracycline loaded in the anti-infection biological patch was 22-33wt% within 4 hours.

[0030] The drug release rate of rifamycin loaded in the anti-infection biological patch was 30-39wt% within 4 hours.

[0031] As a preferred embodiment, the drug loading of each antibiotic in the anti-infection biological patch is 155-580 μg / cm 2 .

[0032] The tetracycline loading in the anti-infective biological patch is 155-580 μg / cm 2 .

[0033] The drug loading of rifamycin in the anti-infective biological patch is 155-580 μg / cm 2 .

[0034] The drug loading of each antibiotic in the anti-infective biological patch is 179-540μg / cm 2 .

[0035] The tetracycline loading in the anti-infective biological patch is 179-459 μg / cm 2 .

[0036] The drug loading of rifamycin in the anti-infective biological patch is 213-540 μg / cm 2 .

[0037] Furthermore, the drug loading ratio of tetracycline and rifamycin loaded in the anti-infection biological patch is 1:(0.9-1.4).

[0038] If the drug loading is too low, the antibacterial effect cannot be achieved. If the drug loading is too high, it may cause antibiotic resistance and produce side effects. Therefore, under the premise of achieving the ideal antibacterial effect, the higher the drug loading, the better. In order to explore the drug loading range of the anti-infection biological patch used for temporary abdominal closure materials, the inventor unexpectedly found that when the drug loading of each antibiotic in the anti-infection biological patch is 155-580μg / cm 2 , which can improve the mechanical properties of the material, ensure the durability and safety of the material, and reduce the impact of peritoneal contents such as peritoneal effusion on the material. When the drug loading ratio of tetracycline and rifamycin in the anti-infection biological patch is 1: (0.9-1.4), the bursting strength of the anti-infection biological patch is significantly improved.

[0039] The antibiotics loaded on the anti-infection biological patch are non-coated.

[0040] As a preferred embodiment, the method for preparing the anti-infection biological patch at least comprises: (1) Preparation of continuous layers: laying down uncross-linked extracellular matrix; The continuous layer of uncross-linked extracellular matrix is ​​derived from the bladder basement membrane or the small intestine submucosa; (2) Preparation of discontinuous layers: Laying down uncross-linked extracellular matrix, repeating the laying down for 2-10 times, and then forming and cutting the layers to form a discontinuous layer; The discontinuous layers of uncross-linked extracellular matrix are derived from the small intestinal submucosa; The cutting process specifically includes cutting into a plurality of rectangular strips and / or tic-tac-toe strips and / or U-shaped strips.

[0041] (3) After the continuous layer and the discontinuous layer are superimposed and subjected to a molding process, the result is obtained.

[0042] Preferably, in step (2), the discontinuous layer comprises a plurality of rectangular strips, and the width of the rectangular strips is 5-40 mm.

[0043] The length of the rectangular strip is ≤ the length of the largest diagonal of the consecutive layers.

[0044] In step (2), there is a gap between every two rectangular strips, and the gap between the rectangular strips is 0.2-1.9 cm.

[0045] In step (2), the rectangular strips are spaced apart and placed in parallel to form a discontinuous layer.

[0046] The number of rectangular strips in each discontinuous layer is at least 2.

[0047] Preferably, in step (2), the discontinuous layer comprises tic-tac-toe-shaped stripes.

[0048] Preferably, in step (2), the discontinuous layer comprises U-shaped strips.

[0049] As a preferred embodiment, in the step (1), when preparing the continuous layer, water and / or a suspension of antibiotics are used for laying.

[0050] Preferably, in step (1), when preparing the continuous layer, water or a suspension of antibiotics is used for laying.

[0051] The antibiotic suspension is a tetracycline suspension.

[0052] The tetracycline suspension is prepared as follows: 0.01-0.2 g of tetracycline is dissolved in 5-20 ml of water, and then 50-600 μL of 0.2-0.8 g / ml calcium chloride solution is added to adjust the pH to 7-7.7.

[0053] As a preferred embodiment, in the step (2), when preparing the discontinuous layer, an antibiotic solution is used for laying.

[0054] The antibiotic solution is an organic solution of rifamycin.

[0055] The organic solution of rifamycin is prepared by adding rifamycin into 80wt%-95wt% ethanol aqueous solution; in the organic solution of rifamycin, the concentration of rifamycin is 0.01-0.1g / mL.

[0056] The specific method of laying with water and / or antibiotic suspension is as follows: placing the substrate horizontally, wetting the substrate with water and / or antibiotic suspension, laying the uncross-linked extracellular matrix on the substrate, flattening the decellularized matrix material on the substrate, and then scraping off excess water and / or antibiotic suspension on the surface of the decellularized matrix material with a scraper.

[0057] The purpose of wetting the substrate is to exclude air between the decellularized matrix material and the substrate, and to utilize the adhesion of water and / or antibiotics to the decellularized matrix material to make the decellularized matrix material flatter on the substrate.

[0058] The material of the substrate is not limited. The substrate is only used to provide a horizontal operating plane for the base layer. It can be a commercially available glass substrate, steel plate, copper plate, etc.

[0059] The operation of paving with a solution of antibiotics is similar to the above-mentioned paving with a suspension of water and / or antibiotics, and the principle is the same.

[0060] In step (3) of the method for preparing an anti-infection biological patch, the number of continuous layers and discontinuous layers and the stacking order are not limited. For example, from bottom to top, the sequential combination is 1 continuous layer, 1 discontinuous layer and 1 continuous layer; for example, from bottom to top, the sequential combination is 8 continuous layers, 2 discontinuous layers and 6 continuous layers; for example, from bottom to top, the sequential combination is 1 continuous layer, 1 discontinuous layer, 4 continuous layers, 1 discontinuous layer and 1 continuous layer; for example, from bottom to top, the sequential combination is 2 continuous layers, 2 discontinuous layers, 4 continuous layers, 1 discontinuous layer and 1 continuous layer, etc.

[0061] Although the stacking order of the continuous layer and the discontinuous layer is not limited, in order to ensure that the upper and lower surfaces of the discontinuous layer have contact areas with the continuous layer, the outermost layer of the anti-infection biological patch is the continuous layer.

[0062] It is known that a suspension is a mixture containing solid particles observable by the naked eye, and a solution is a uniform mixture of particles that cannot be observed by the naked eye. Through a lot of creative work, the inventors found that using an antibiotic suspension for laying when preparing a continuous layer and using an antibiotic solution for laying when preparing a discontinuous layer is not only beneficial to increasing the drug loading of the anti-infection biological patch, but also improves the suture strength and enhances the structural stability of the anti-infection biological patch. The inventors speculate that this may be because the antibiotic solute particles loaded when preparing the discontinuous layer are small, and the antibiotic particles loaded when preparing the continuous layer are larger. Compared with small particles, the distribution of large particles is looser. The combination of the two particle dispersion effects makes the distribution of force in the overall structure more balanced, which is beneficial to weakening the structural instability of the discontinuous layer caused by the existence of intervals.

[0063] As a preferred embodiment, the molding process is selected from one or more of lamination, freeze-drying, bonding and suturing.

[0064] Preferably, the forming process is lamination or freeze-drying.

[0065] Preferably, the forming process is lamination.

[0066] As a preferred embodiment, the antibacterial biological patch has an inhibition rate of >95% against Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis and Pseudomonas aeruginosa within 4 hours.

[0067] As a preferred embodiment, the anti-infection biological patch is used in conjunction with a negative pressure auxiliary system during application.

[0068] The negative pressure assist system is currently an effective measure for protecting open abdominal wounds. It is generally composed of medical foam materials, adhesive films and negative pressure pipelines. Its main purpose is to drain waste fluid from the abdominal cavity.

[0069] Beneficial effects: The anti-infection biological patch of the present invention has good biodegradability and biocompatibility, can effectively control short-term large-scale infection, has good active antibacterial ability and drug sustained-release effect, and also has good mechanical strength and tissue compliance; the obtained anti-infection biological patch is soft in texture and will not cause damage to the intestinal tract and other visceral sac tissues in contact; when the anti-infection biological patch of the present invention is used in the temporary abdominal closure material for open abdominal cavity, it can not only reduce the incidence of intestinal air fistula to less than 2%, but also reduce the risk of incisional hernia and abdominal bleeding, and can also reduce fascia retraction, thereby further avoiding adhesion between the intestine and the abdominal wall and between the intestine and the abdominal dressing, and effectively promote the closure and repair of the abdominal wall fascia tissue; when the anti-infection biological patch is used, it is combined with the open abdomen negative pressure vacuum assisted closure to protect the intestine from the influence of blood hypoperfusion. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 This is a scanning electron microscope image of the surface of the anti-infection biological patch obtained in Example 1; Figure 2 is a scanning electron microscope image of a cross section of the anti-infection biological patch obtained in Example 1; Figure 3 for Figure 2 Magnified image of . DETAILED DESCRIPTION

[0071] Example 1

[0072] This example provides an anti-infection biological patch, which is prepared as follows: (1) Preparation of continuous layer 1: Uncross-linked extracellular matrix (derived from the small intestinal submucosa, Zhuoruan Medical Technology (Suzhou) Co., Ltd.) was laid with a suspension of tetracycline (specifically minocycline hydrochloride); the suspension of minocycline hydrochloride was prepared as follows: 0.12 g of minocycline hydrochloride was dissolved in 15 ml of water, and 300 μL of 0.4 g / ml calcium chloride solution was added to adjust the pH to 7.4.

[0073] Preparation of continuous layer 2: Uncrosslinked extracellular matrix (derived from bladder basement membrane, Zhuoruan Medical Technology (Suzhou) Co., Ltd.) was laid with water; Preparation of continuous layer 3: Uncross-linked extracellular matrix (derived from small intestinal submucosa, Zhuoruan Medical Technology (Suzhou) Co., Ltd.) was laid with water; (2) Preparation of discontinuous layer: An organic solution of rifamycin (specifically 3-[[(4-methyl-1-piperazinyl)imino]methyl]-rifamycin) was used to lay down an uncross-linked extracellular matrix (derived from the submucosal layer of the small intestine, Zhuoruan Medical Technology (Suzhou) Co., Ltd.), and the stacking and laying were repeated for 5 times. After lamination, the matrix was cut into rectangular strips with a width of 10 mm. The rectangular strips were placed in parallel to form a discontinuous layer. There was a gap between every two rectangular strips. The gap between the rectangular strips was 1.8 cm. The rectangular strips were spaced and placed in parallel to form a discontinuous layer. The organic solution of 3-[[(4-methyl-1-piperazinyl)imino]methyl]-rifamycin is prepared by adding 3-[[(4-methyl-1-piperazinyl)imino]methyl]-rifamycin to a 92wt% ethanol aqueous solution; in the organic solution of rifamycin, the concentration of rifamycin is 0.06g / mL.

[0074] (3) From bottom to top, the layers are stacked in the order of 1 continuous layer 2, 4 continuous layers 1, 1 discontinuous layer, 2 continuous layers 3 and 1 continuous layer 2, and then laminated to obtain the result.

[0075] Example 2

[0076] This example provides an anti-infection biological patch. The difference from Example 1 is that step (3) is to stack from bottom to top in the order of 1 continuous layer 2, 6 continuous layers 1, 1 discontinuous layer, 2 continuous layers 1, 1 discontinuous layer, 4 continuous layers 3, 1 discontinuous layer and 4 continuous layers 3, and then laminate to obtain the patch.

[0077] Comparative Example 1 This example provides an anti-infection biological patch, referring to the impregnation preparation method described in patent number CN115006592B, and the specific preparation method is as follows: Preparation of uncross-linked extracellular matrix slurry: Use a tissue grinder homogenizer to granulate the uncross-linked extracellular matrix (derived from the submucosa of the small intestine, Zhuoruan Medical Technology (Suzhou) Co., Ltd.) using ultrasonic homogenization technology at low temperature. Take 25 g of the treated particles and add 20 mL of 0.01 g / mL 3-[[(4-methyl-1-piperazinyl)imino]methyl]-rifamycin 90 wt% ethanol aqueous solution and 20 mL of 0.01 g / mL minocycline hydrochloride aqueous solution.

[0078] Uncross-linked extracellular matrix (derived from bladder basement membrane, Zhuoruan Medical Technology (Suzhou) Co., Ltd.) was immersed in uncross-linked extracellular matrix slurry for 24 h, and a composite material was obtained after vacuum freeze drying and sterilization.

[0079] Comparative Example 2 This example provides an anti-infection biological patch, and the preparation method is as follows: A layer of uncross-linked extracellular matrix (derived from porcine bladder submucosa, purchased from Zhuoruan Medical Technology (Suzhou) Co., Ltd.), 7 layers of uncross-linked extracellular matrix (derived from porcine small intestine submucosa, purchased from Zhuoruan Medical Technology (Suzhou) Co., Ltd.) and a layer of uncross-linked extracellular matrix (derived from porcine bladder submucosa, purchased from Zhuoruan Medical Technology (Suzhou) Co., Ltd.) were stacked in order from bottom to top, laminated for 24 hours, taken out, immersed in a tetracycline suspension for 1 hour, taken out, immersed in an organic solution of rifamycin for 1 hour, taken out, and repeated the above-mentioned immersion operation twice (equivalent to the laminated material being immersed in a tetracycline suspension three times and in an organic solution of rifamycin three times), with an interval of 1 hour between each immersion, and finally dried at room temperature for 24 hours, and placed in a 40°C oven for further drying for 24 hours.

[0080] Wherein, the tetracycline suspension and the rifamycin organic solution are prepared according to the method of Example 1.

[0081] Comparative Example 3 This example provides an anti-infection biological patch. Different from Example 1, the non-cross-linked extracellular matrix of the anti-infection biological patch has only one source. The specific method is as follows: (1) Preparation of continuous layer 1: Uncross-linked extracellular matrix (derived from the small intestinal submucosa, Zhuoruan Medical Technology (Suzhou) Co., Ltd.) was laid with a suspension of tetracycline (specifically minocycline hydrochloride); the suspension of minocycline hydrochloride was prepared as follows: 0.12 g of minocycline hydrochloride was dissolved in 15 ml of water, and 200 μL of 0.5 g / ml calcium chloride solution was added to adjust the pH to 7.5.

[0082] Preparation of continuous layer 2: Uncross-linked extracellular matrix (derived from small intestinal submucosa, Zhuoruan Medical Technology (Suzhou) Co., Ltd.) was laid with water; (2) Preparation of discontinuous layer: An organic solution of rifamycin (specifically 3-[[(4-methyl-1-piperazinyl)imino]methyl]-rifamycin) was used to lay down an uncross-linked extracellular matrix (derived from the submucosal layer of the small intestine, Zhuoruan Medical Technology (Suzhou) Co., Ltd.), and the stacking and laying was repeated for 4 times. After lamination, the matrix was cut into rectangular strips with a width of 10 mm. The rectangular strips were placed in parallel to form a discontinuous layer. There was a gap between every two rectangular strips. The gap between the rectangular strips was 1.8 cm. The rectangular strips were spaced and placed in parallel to form a discontinuous layer. The organic solution of 3-[[(4-methyl-1-piperazinyl)imino]methyl]-rifamycin is prepared by adding 3-[[(4-methyl-1-piperazinyl)imino]methyl]-rifamycin to 89wt% ethanol aqueous solution; in the organic solution of rifamycin, the concentration of rifamycin is 0.04g / mL.

[0083] (3) From bottom to top, one continuous layer 2, four continuous layers 1, one discontinuous layer and three continuous layers 2 are stacked in this order, and then laminated to obtain the result.

[0084] Comparative Example 4 This example provides an anti-infection biological patch. Different from Example 1, the non-cross-linked extracellular matrix of the anti-infection biological patch is derived from the small intestinal submucosa and porcine pericardium. The specific method is as follows: (1) Preparation of continuous layer 1: Uncross-linked extracellular matrix (derived from the small intestinal submucosa, Zhuoruan Medical Technology (Suzhou) Co., Ltd.) was laid with a suspension of tetracycline (specifically minocycline hydrochloride); the suspension of minocycline hydrochloride was prepared as follows: 0.1 g of minocycline hydrochloride was dissolved in 20 ml of water, and 250 μL of 0.3 g / ml calcium chloride solution was added to adjust the pH to 7.3.

[0085] Preparation of continuous layer 2: Uncross-linked extracellular matrix (derived from porcine pericardium, Zhuoruan Medical Technology (Suzhou) Co., Ltd.) was laid with water; Preparation of continuous layer 3: Uncross-linked extracellular matrix (derived from small intestinal submucosa, Zhuoruan Medical Technology (Suzhou) Co., Ltd.) was laid with water; (2) Preparation of discontinuous layer: An organic solution of rifamycin (specifically 3-[[(4-methyl-1-piperazinyl)imino]methyl]-rifamycin) was used to lay down an uncross-linked extracellular matrix (derived from the submucosal layer of the small intestine, Zhuoruan Medical Technology (Suzhou) Co., Ltd.), and the stacking and laying was repeated for 6 times. After lamination, the matrix was cut into rectangular strips with a width of 8 mm. The rectangular strips were placed in parallel to form a discontinuous layer. There was a gap between every two rectangular strips. The gap between the rectangular strips was 1.4 cm. The rectangular strips were spaced and placed in parallel to form a discontinuous layer. The organic solution of 3-[[(4-methyl-1-piperazinyl)imino]methyl]-rifamycin is prepared by adding 3-[[(4-methyl-1-piperazinyl)imino]methyl]-rifamycin to a 93wt% ethanol aqueous solution; in the organic solution of rifamycin, the concentration of rifamycin is 0.04g / mL.

[0086] (3) From bottom to top, the layers are stacked in the order of 1 continuous layer 2, 4 continuous layers 1, 1 discontinuous layer, 2 continuous layers 3 and 1 continuous layer 2, and then laminated to obtain the result.

[0087] Comparative Example 5 This example provides an anti-infection biological patch. The difference from Example 1 is that when preparing a continuous layer, an antibiotic solution is used for laying, and when preparing a discontinuous layer, an antibiotic suspension is used for laying. The specific preparation is as follows: (1) Preparation of continuous layer 1: Uncrosslinked extracellular matrix (derived from the small intestinal submucosa, Zhuoruan Medical Technology (Suzhou) Co., Ltd.) was laid with an organic solution of rifamycin (specifically 3-[[(4-methyl-1-piperazinyl)imino]methyl]-rifamycin); the organic solution of 3-[[(4-methyl-1-piperazinyl)imino]methyl]-rifamycin was prepared by adding 3-[[(4-methyl-1-piperazinyl)imino]methyl]-rifamycin to 85 wt % ethanol aqueous solution; in the organic solution of rifamycin, the concentration of rifamycin was 0.03 g / mL.

[0088] Preparation of continuous layer 2: Uncrosslinked extracellular matrix (derived from bladder basement membrane, Zhuoruan Medical Technology (Suzhou) Co., Ltd.) was laid with water; Preparation of continuous layer 3: Uncross-linked extracellular matrix (derived from small intestinal submucosa, Zhuoruan Medical Technology (Suzhou) Co., Ltd.) was laid with water; (2) Preparation of discontinuous layer: Use a tetracycline (specifically minocycline hydrochloride) suspension to lay uncross-linked extracellular matrix (derived from the submucosal layer of the small intestine, Zhuoruan Medical Technology (Suzhou) Co., Ltd.), and continue to repeat the stacking and laying for 6 times. After lamination, cut into rectangular strips with a width of 5 mm. The rectangular strips are placed in parallel to form a discontinuous layer. There is a gap between every two rectangular strips. The gap between the rectangular strips is 1 cm. The rectangular strips are spaced and placed in parallel to form a discontinuous layer. The suspension of minocycline hydrochloride was prepared as follows: 0.08 g of minocycline hydrochloride was dissolved in 15 ml of water, and 300 μL of 0.2 g / ml calcium chloride solution was added to adjust the pH to 7.4.

[0089] (3) From bottom to top, the layers are stacked in the order of 1 continuous layer 2, 4 continuous layers 1, 1 discontinuous layer, 2 continuous layers 3 and 1 continuous layer 2, and then laminated to obtain the result.

[0090] Performance Testing 1. Scanning electron microscopy (SEM) characterization: The anti-infection biological patch obtained in Example 1 was characterized by SEM scanning electron microscopy. The results are as follows: Figure 1-3 It can be seen that antibiotics are loaded and distributed on the surface and inside of the anti-infection biological patch, and the antibiotics are not distributed only on the surface of the patch in a coating manner.

[0091] 2. Drug loading test: The anti-infection biological patches obtained in Examples 1-2 and Comparative Examples 1-5 were cut into 5 cm × 5 cm samples, and extracted in methanol. The extract was taken every 24 hours and tested by liquid chromatography injection until the extract was colorless. Sampling and testing were stopped. The drug content of the sample was obtained by referring to the chromatogram of each drug standard. The results are shown in Table 1.

[0092] Table 1

[0093] As can be seen from Table 1, the drug loading of the biological patches of Comparative Examples 1-2 is very low, all within 100 μg / cm 2 the following.

[0094] The drug loading of the biological patch of Comparative Example 4 is close to that of Example 1.

[0095] 3. Drug release rate test: 21 rats were given gas anesthesia, and the abdominal hair was shaved. A 5 cm long incision was made in the middle of the abdomen to fully expose the abdominal cavity. The anti-infection biological patches obtained in Examples 1-2 and Comparative Examples 1-5 were cut into 2 cm × 2 cm samples, and placed in the peritoneum of the rats on both sides of the abdominal cavity. The same samples were placed in the peritoneum and fixed with sutures. The wound was closed after implantation. Samples were taken at 4h, 1 day, and 5 days after surgery. After euthanasia, the samples were taken out. After the samples were taken out, they were extracted with PBS buffer solution for 24h. The drug loading was calculated according to the drug standard curve, which was recorded as W (the standard samples of rifamycin and tetracycline were dissolved in PBS buffer solution, diluted into a series of standard solutions of 1-500μg / mL, and the standard curve was obtained by liquid chromatography injection test). In vivo release rate = (drug loading of each sample in performance test 2-W) / drug loading of each sample in performance test 2 × 100%; the results are shown in Table 2: Table 2

[0096] In Table 2, the corresponding antibiotics in Comparative Examples 1-3 and Comparative Example 5 could not be detected at 120 h.

[0097] It can be seen from Table 2 that the drug release rates of the biological patches of Comparative Examples 1-3 and Comparative Example 5 are too fast.

[0098] The drug release rate of the biological patch of Comparative Example 4 is close to that of Example 1.

[0099] Antibacterial rate test: Prepare Staphylococcus aureus, Escherichia coli, Staphylococcus epidermidis, and Pseudomonas aeruginosa as test strains. After activating the strains, pick a single colony and mix it in sterile saline to make 10 6CFU / mL bacterial suspension, take the anti-infection biological patches obtained in Examples 1-2 and Comparative Examples 3-5, 2 cm×3 cm in size, and place them in a test tube, add 9.9 mL of MH culture medium and 0.1 mL of bacterial suspension, and do not add any patches to the blank control group. After the test tube is placed in a shaker at 37°C and 180 rpm for 1, 2, 3, and 4 h, take the culture for 10-fold serial dilution, take 0.1 mL of each dilution culture and spread it on a TSA plate, and culture it at 35°C for 24 h to count the colonies. The same dilution test is repeated 3 times, and the time is used as the horizontal axis and the logarithm of the colony forming unit (CFU) is used as the vertical axis to draw a sterilization curve, and the inhibition rate at 4 h is recorded. The inhibition rate (%) = (the number of colonies in the blank control group-the number of colonies on the patch group plate) / the number of colonies in the blank control group × 100%. The results are shown in Table 3: Table 3

[0100] 5. Mechanical property test: The anti-infection biological patches obtained in Examples 1-2 and Comparative Examples 3-5 were made into samples of 50 mm×50 mm and 25 mm in width, respectively. After being immersed in water for 5 min, the bursting strength (tested in accordance with GB / T 19976-2005, using a spherical probe with a diameter of 20 mm) and suture strength were tested respectively. The suture strength test method is: suture with surgical suture at a distance of 5 mm from the edge of the sample, fix the other end of the suture and the product on a tensile tester, and stretch at a speed of 100 mm / min until the suture point is torn, and record the tension when the suture point is torn, which is the suture strength. The results are shown in 4: Table 4 Example Bursting strength (N) Suture strength (N) Example 1 65 10 Example 2 88 11 Comparative Example 3 26 4 Comparative Example 4 61 5 Comparative Example 5 14 2 6. Animal model verification: The anti-infection biological patch obtained in Example 1 was used for animal experiment verification. Forty 6-month-old sows weighing about 25 kg were taken to construct an animal model of abdominal infection caused by cecal ligation and puncture (CLP) combined with abdominal restraint device method and abdominal hypertension (IAH). After modeling, the abdominal cavity was opened again along the original incision, decompressed, and the stump was buried after the cecum was removed to expose the intestinal mucosa with intestinal fistula eversion. After debridement and drainage, open abdominal treatment with negative pressure-assisted temporary abdominal closure technology was performed. During the treatment, the anti-infection biological patch obtained in Example 1 was used to cover the intestinal mucosa for temporary abdominal closure. It was observed that no abdominal bleeding and adhesion occurred during the period, and definitive abdominal closure was performed three days later. The anti-infection biological patch previously placed was not removed during the definitive abdominal closure, and the fascia closure rate was 100%. The animals were kept in breeding after the operation. After 10 days, only one case of intestinal air fistula was observed, and no incisional hernia occurred in the postoperative follow-up. That is to say, the anti-infection biological patch of the present invention is used as a temporary abdominal closure material for abdominal cavity opening, and the incidence of intestinal air fistula is 2.5% (1 / 40).

Claims

1. An application of an anti-infective biological patch in temporary abdominal closure material for abdominal cavity opening, characterized in that: The anti-infection biological patch is obtained by loading at least two antibiotics on an uncross-linked extracellular matrix; the drug release rate of each antibiotic in the anti-infection biological patch is 15-85wt% in 24 hours, and the drug release rate of each antibiotic in the anti-infection biological patch is 85-100wt% in 120 hours; the antibiotics are high protein binding rate antibiotics.

2. The use of an anti-infection biological patch as a temporary abdominal closure material for abdominal cavity opening according to claim 1, characterized in that: The drug release rate of each antibiotic in the anti-infection biological patch is less than 40wt% within 4 hours.

3. The use of an anti-infection biological patch as a temporary abdominal closure material for abdominal cavity opening according to claim 1, characterized in that: The high protein binding rate antibiotics are selected from at least two of tetracycline, rifamycin and β-lactam antibiotics.

4. The use of an anti-infection biological patch as a temporary abdominal closure material for abdominal cavity opening according to claim 1, characterized in that: The drug loading of each antibiotic in the anti-infection biological patch is 155-580 μg / cm 2 .

5. The use of an anti-infection biological patch as a temporary abdominal closure material for abdominal cavity opening according to claim 1, characterized in that: The preparation method of the anti-infection biological patch at least comprises: (1) Preparation of continuous layers: laying down uncross-linked extracellular matrix; (2) Preparation of discontinuous layers: Laying down the uncross-linked extracellular matrix, repeating the laying down for 2-10 times, and then forming and cutting the layers to form a discontinuous layer; (3) After the continuous layer and the discontinuous layer are superimposed and subjected to a molding process, the result is obtained.

6. The use of an anti-infection biological patch as a temporary abdominal closure material for abdominal cavity opening according to claim 5, characterized in that: In the step (1), when preparing the continuous layer, water and / or a suspension of antibiotics are used for laying.

7. The use of an anti-infective biological patch as a temporary abdominal closure material for abdominal cavity opening according to claim 5, characterized in that: In the step (2), when preparing the discontinuous layer, an antibiotic solution is used for laying.

8. Use of an anti-infective biological patch according to any one of claims 5 to 7 as a temporary abdominal closure material for abdominal cavity opening, characterized in that: The molding process is selected from one or more of lamination, freeze-drying, bonding and suturing.

9. Use of the anti-infection biological patch according to any one of claims 1 to 5 as a temporary abdominal closure material for abdominal cavity opening, characterized in that: The antibacterial biological patch has an inhibition rate of more than 95% on Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis and Pseudomonas aeruginosa at 4 hours.

10. Use of the anti-infective biological patch according to any one of claims 1 to 5 as a temporary abdominal closure material for abdominal cavity opening, characterized in that: The anti-infection biological patch is used in conjunction with a negative pressure auxiliary system when in use.

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