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

Abstract

The present invention relates to the field of biomedical materials, and more specifically, to the use of an anti-infective biological patch in a temporary abdominal closure material for an open abdominal cavity. The anti-infective biological patch of the present invention has good biodegradability and biocompatibility, can effectively control short-term large-scale infections, has excellent active antibacterial ability and drug sustained-release effect, and also has good mechanical strength and tissue compliance. The anti-infective biological patch of the present invention is soft and will not cause damage to the intestinal tract and other visceral sac tissues with which it comes into contact. When used in a temporary abdominal closure material for an open abdominal cavity, it can not only reduce the incidence of intestinal air fistula to below 2%, while also reducing the risk of incisional hernia and abdominal bleeding, but 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.

Description

Technical Field

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

[0002] Open abdominal surgery is an effective treatment for severe abdominal infections, hypertension, and other critical abdominal conditions. After this procedure, temporary abdominal closure is required, followed by delayed full-thickness closure (definitive abdominal closure) at an appropriate time. Patent number CN115006592B provides a solution for temporary abdominal closure. The composite material is impregnated with a placental tissue slurry containing nanosilver and applied over the open abdominal intestinal tract to reduce the occurrence of air fistulas. However, the document does not specify the extent to which air fistulas can be reduced. In addition, the composite material obtained by immersing the biomaterial in the drug solution releases drugs very quickly. Wei Chao et al. have conducted related research ("Research on Mechanical Properties and Antibacterial Functionalization of Calfskin Biorepair 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 a drug with a high albumin binding rate is used, the drug release rate will be faster.

[0003] At the same time, the material impregnation method described in the above-mentioned patent number CN115006592B is equivalent to attaching a layer of antibacterial coating on the surface of the acellular dermis. However, the biological patch formed by this coating method usually shows low drug loading in clinical practice, which may further lead to the failure to achieve antibacterial therapeutic effects. Similar to the impregnation method in patent number CN115006592B, patent number CN101623518B provides a body wall repair material for long-term closure of the abdominal cavity after laparotomy. Nanosilver particles are implanted in the SIS by immersing the SIS in a nanosilver working solution. The silver content is 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 they are not only used as prostheses to induce healing, the present invention provides an anti-infection biological patch for use in temporary abdominal closure materials for open abdominal cavity. 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 stages of abdominal opening, especially within 3-5 days of abdominal opening, the intestine is exposed and is prone to serious infection, requiring some temporary abdominal closure measures. 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 for open abdominal treatment. Although non-cross-linked biological patches have high biocompatibility, they are easily degraded and have unstable mechanical properties, and cannot achieve the desired effect. Cross-linked biological patches may cause certain irritation 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 serve to isolate the intestine. In order to obtain a patch with both mechanical and chemical protection, the inventors want to load antibiotics on the non-cross-linked biological patch to make it have an antibacterial effect. However, 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 binding rate between drugs and proteins 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-infection biological patch is obtained by loading at least tetracycline and rifamycin on an uncross-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 differential method disclosed in patent CN106075583B.

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

[0018] More preferably, the uncross-linked extracellular matrix is ​​derived from the small intestine 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 20.3-85 wt %, and the drug release rate of each antibiotic in the anti-infection biological patch within 120 hours is 85-100 wt %.

[0021] In the present invention, the anti-infection biological patch is loaded with at least two antibiotics, and the antibiotics are high protein binding antibiotics. 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, overcoming the technical resistance of low antibiotic loading and too rapid release of antibiotics on non-cross-linked patches, which cannot achieve antibacterial effects. The anti-infection biological patch is used in the field of temporary abdominal closure materials for abdominal cavity opening. It not only plays a mechanical protective role for the intestinal tract while ensuring the mechanical stability of the material, but also plays a chemical protective role. It has 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 and 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 submucosa 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 20.3-64.5 wt % within 24 hours.

[0023] More preferably, the drug release rate of tetracycline loaded in the anti-infection biological patch is 21.6-63.9 wt % within 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 86-100 wt % in 120 h.

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

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

[0028] As a preferred embodiment, the drug release rate of each antibiotic in the anti-infection biological patch is less than 40 wt % 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 ranges from 179 to 540 μg / cm 2 .

[0035] The tetracycline drug 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 of open abdominal cavity, 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 ascites 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:

[0041] (1) Preparation of continuous layers: laying down uncross-linked extracellular matrix;

[0042] The continuous layer of uncross-linked extracellular matrix is ​​derived from the bladder basement membrane or the small intestine submucosa;

[0043] (2) Preparation of discontinuous layers: Laying uncross-linked extracellular matrix, repeating the laying process 2-10 times, and then forming and cutting the layers to form a discontinuous layer;

[0044] The discrete layers of uncross-linked extracellular matrix are derived from the small intestinal submucosa;

[0045] The cutting process specifically includes cutting into a plurality of rectangular strips and / or crisscross strips and / or U-shaped strips.

[0046] (3) After the continuous layer and the discontinuous layer are superimposed and subjected to a molding process, the result is obtained.

[0047] Preferably, in step (2), the discontinuous layer comprises a plurality of rectangular strips, and the width of the rectangular strips is 5-40 mm.

[0048] The length of the rectangular strip is ≤ the length of the largest diagonal of the consecutive layers.

[0049] 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.

[0050] In step (2), the rectangular strips are spaced apart and placed in parallel to form a discontinuous layer.

[0051] The number of rectangular strips in each discontinuous layer is at least 2.

[0052] Preferably, in step (2), the discontinuous layer comprises tic-tac-toe stripes.

[0053] Preferably, in step (2), the discontinuous layer comprises U-shaped strips.

[0054] As a preferred embodiment, in the step (1), when preparing the continuous layer, water and / or a suspension of antibiotics is used for laying.

[0055] Preferably, in step (1), when preparing the continuous layer, water or a suspension of antibiotics is used for laying.

[0056] The antibiotic suspension is a tetracycline suspension.

[0057] The tetracycline suspension is prepared as follows: 0.01-0.2 g of tetracycline is dissolved in 5-20 ml of water, 50-600 μL of 0.2-0.8 g / ml calcium chloride solution is added, and the pH is adjusted to 7-7.7.

[0058] As a preferred embodiment, in the step (2), when preparing the discontinuous layer, an antibiotic solution is used for laying.

[0059] The antibiotic solution is an organic solution of rifamycin.

[0060] The organic solution of rifamycin is prepared by adding rifamycin to 80wt%-95wt% ethanol aqueous solution; in the organic solution of rifamycin, the concentration of rifamycin is 0.01-0.1g / mL.

[0061] The specific steps of laying the decellularized matrix material with water and / or antibiotic suspension are as follows: placing the substrate horizontally, wetting the substrate with water and / or antibiotic suspension, laying the uncrosslinked 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.

[0062] The purpose of wetting the substrate is to exclude air between the decellularized matrix material and the substrate, and to utilize the adhesion between water and / or antibiotics and the decellularized matrix material to enable the decellularized matrix material to be better flattened on the substrate.

[0063] The material of the substrate is not limited. The substrate only serves to provide a horizontal operating surface for the base layer. It can be a commercially available glass substrate, steel plate, copper plate, etc.

[0064] The operation of paving with a solution of antibiotics is similar to the above-mentioned paving with water and / or a suspension of antibiotics, and the principle is the same.

[0065] In step (3) of the method for preparing an anti-infection biological patch, the number of layers and the stacking order of the continuous layers and the discontinuous layers are not limited. For example, the sequential combination from bottom to top is 1 continuous layer, 1 discontinuous layer and 1 continuous layer; for example, the sequential combination from bottom to top is 8 continuous layers, 2 discontinuous layers and 6 continuous layers; for example, the sequential combination from bottom to top is 1 continuous layer, 1 discontinuous layer, 4 continuous layers, 1 discontinuous layer and 1 continuous layer; for example, the sequential combination from bottom to top is 2 continuous layers, 2 discontinuous layers, 4 continuous layers, 1 discontinuous layer and 1 continuous layer, etc.

[0066] 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.

[0067] It is known that a suspension is a mixture containing solid particles observable with the naked eye, and a solution is a uniform mixture in which particles cannot be observed with the naked eye. Through a lot of creative work, the inventors found that using an antibiotic suspension for laying the continuous layer and using an antibiotic solution for laying the discontinuous layer not only helps to increase 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 types of particle dispersion makes the force distribution in the overall structure more balanced, which helps to weaken the structural instability of the discontinuous layer caused by the existence of gaps.

[0068] As a preferred embodiment, the molding process is selected from one or more of lamination, freeze-drying, bonding and suturing.

[0069] Preferably, the forming process is lamination or freeze-drying.

[0070] Preferably, the forming process is lamination.

[0071] 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.

[0072] As a preferred embodiment, the anti-infection biological patch is used in conjunction with a negative pressure auxiliary system during application.

[0073] 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.

[0074] Beneficial effects: The anti-infection biological patch of the present invention has good biodegradability and biocompatibility, can effectively control short-term large-scale infections, 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 temporary abdominal closure materials for open abdominal cavity, it can not only reduce the incidence of intestinal air fistula to below 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 abdominal dressing, and effectively promote the closure and repair of 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

[0075] Figure 1 This is a scanning electron microscope image of the surface of the anti-infection biological patch obtained in Example 1;

[0076] Figure 2 This is a scanning electron microscope image of the cross section of the anti-infection biological patch obtained in Example 1;

[0077] Figure 3 for Figure 2 Magnified image of . DETAILED DESCRIPTION

[0078] Example 1

[0079] This example provides an anti-infection biological patch, which is prepared as follows:

[0080] (1) Preparation of continuous layer 1: Uncross-linked extracellular matrix (derived from the submucosa of the small intestine, Zhuoran 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, and the pH was adjusted to 7.4.

[0081] Preparation of continuous layer 2: Uncrosslinked extracellular matrix (derived from bladder basement membrane, Zhuoruan Medical Technology (Suzhou) Co., Ltd.) was laid with water;

[0082] Preparation of continuous layer 3: Uncross-linked extracellular matrix (derived from small intestinal submucosa, Zhuoruan Medical Technology (Suzhou) Co., Ltd.) was laid with water;

[0083] (2) Preparation of discontinuous layer: An organic solution of rifamycin (specifically 3-[[(4-methyl-1-piperazinyl)imino]methyl]-rifamycin) was used to lay uncrosslinked extracellular matrix (derived from the submucosa of the small intestine, Zhuoran Medical Technology (Suzhou) Co., Ltd.), and the stacking was repeated 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.

[0084] An 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 92 wt % ethanol aqueous solution; the concentration of rifamycin in the organic solution of rifamycin is 0.06 g / mL.

[0085] (3) From bottom to top, the following order is stacked: 1 continuous layer 2, 4 continuous layers 1, 1 discontinuous layer, 2 continuous layers 3, and 1 continuous layer 2, and laminated to obtain the result.

[0086] Example 2

[0087] This example provides an anti-infection biological patch. Different from Example 1, step (3) is to stack the following 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.

[0088] Comparative Example 1

[0089] This example provides an anti-infection biological patch, which is prepared by dipping according to patent number CN115006592B. The specific preparation method is as follows:

[0090] Preparation of uncross-linked extracellular matrix slurry: Uncross-linked extracellular matrix (derived from the small intestinal submucosa, Zhuoruan Medical Technology (Suzhou) Co., Ltd.) was granulated using a tissue grinder homogenizer with low-temperature ultrasonic homogenization technology. 25 g of the treated granules were taken and added to 20 mL of 0.01 g / mL 90 wt% ethanol aqueous solution of 3-[[(4-methyl-1-piperazinyl)imino]methyl]-rifamycin and 20 mL of 0.01 g / mL minocycline hydrochloride aqueous solution.

[0091] 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 then vacuum freeze-dried and sterilized to obtain a composite material.

[0092] Comparative Example 2

[0093] This example provides an anti-infection biological patch, the preparation method of which is as follows:

[0094] 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 1 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, and immersed in a rifamycin organic solution for 1 hour, taken out, and repeated the above-mentioned immersion operation twice (equivalent to the laminated material being immersed in a tetracycline suspension 3 times and in a rifamycin organic solution 3 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.

[0095] The tetracycline suspension and the rifamycin organic solution were prepared according to the method of Example 1.

[0096] Comparative Example 3

[0097] This example provides an anti-infection biological patch. Unlike Example 1, the uncross-linked extracellular matrix of the anti-infection biological patch has only one source. The specific method is as follows:

[0098] (1) Preparation of continuous layer 1: Uncross-linked extracellular matrix (derived from the submucosa of the small intestine, Zhuoran 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, and the pH was adjusted to 7.5.

[0099] Preparation of continuous layer 2: Uncross-linked extracellular matrix (derived from small intestinal submucosa, Zhuoruan Medical Technology (Suzhou) Co., Ltd.) was laid with water;

[0100] (2) Preparation of discontinuous layer: An organic solution of rifamycin (specifically 3-[[(4-methyl-1-piperazinyl)imino]methyl]-rifamycin) was used to lay uncrosslinked extracellular matrix (derived from the submucosa of the small intestine, Zhuoran Medical Technology (Suzhou) Co., Ltd.), and the stacking was repeated 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.

[0101] An organic solution of 3-[[(4-methyl-1-piperazinyl)imino]methyl]-rifamycin is prepared by adding 3-[[(4-methyl-1-piperazinyl)imino]methyl]-rifamycin to an 89 wt % ethanol aqueous solution; the concentration of rifamycin in the organic solution of rifamycin is 0.04 g / mL.

[0102] (3) From bottom to top, the layers are stacked in the order of 1 continuous layer 2, 4 continuous layers 1, 1 discontinuous layer, and 3 continuous layers 2, and laminated to obtain the result.

[0103] Comparative Example 4

[0104] This example provides an anti-infection biological patch. Unlike Example 1, the uncross-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:

[0105] (1) Preparation of continuous layer 1: Non-cross-linked extracellular matrix (derived from the submucosa of the small intestine, Zhuoran Medical Technology (Suzhou) Co., Ltd.) was laid using 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, and the pH was adjusted to 7.3.

[0106] Preparation of continuous layer 2: Uncrosslinked extracellular matrix (derived from porcine pericardium, Zhuoruan Medical Technology (Suzhou) Co., Ltd.) was laid with water;

[0107] Preparation of continuous layer 3: Uncross-linked extracellular matrix (derived from small intestinal submucosa, Zhuoruan Medical Technology (Suzhou) Co., Ltd.) was laid with water;

[0108] (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 uncrosslinked extracellular matrix (derived from the submucosa of the small intestine, Zhuoran Medical Technology (Suzhou) Co., Ltd.), and the stacking was repeated 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.

[0109] An 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 93 wt % ethanol aqueous solution; the concentration of rifamycin in the organic solution of rifamycin is 0.04 g / mL.

[0110] (3) From bottom to top, the following order is stacked: 1 continuous layer 2, 4 continuous layers 1, 1 discontinuous layer, 2 continuous layers 3, and 1 continuous layer 2, and laminated to obtain the result.

[0111] Comparative Example 5

[0112] This example provides an anti-infection biological patch. The difference from Example 1 is that when preparing the continuous layer, an antibiotic solution is used for laying, and when preparing the discontinuous layer, an antibiotic suspension is used for laying. The specific preparation is as follows:

[0113] (1) Preparation of continuous layer 1: Uncrosslinked extracellular matrix (derived from the small intestinal submucosa, Zhuoruan Medical Technology (Suzhou) Co., Ltd.) was laid using 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; the concentration of rifamycin in the organic solution of rifamycin was 0.03 g / mL.

[0114] Preparation of continuous layer 2: Uncrosslinked extracellular matrix (derived from bladder basement membrane, Zhuoruan Medical Technology (Suzhou) Co., Ltd.) was laid with water;

[0115] Preparation of continuous layer 3: Uncross-linked extracellular matrix (derived from small intestinal submucosa, Zhuoruan Medical Technology (Suzhou) Co., Ltd.) was laid with water;

[0116] (2) Preparation of discontinuous layer: Tetracycline (specifically minocycline hydrochloride) suspension was used to lay uncrosslinked extracellular matrix (derived from the submucosa of the small intestine, Zhuoran Medical Technology (Suzhou) Co., Ltd.), and the stacking was repeated 6 times. After lamination, it was cut into rectangular strips with a width of 5 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 cm. The rectangular strips were spaced and placed in parallel to form a discontinuous layer.

[0117] 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.

[0118] (3) From bottom to top, the following order is stacked: 1 continuous layer 2, 4 continuous layers 1, 1 discontinuous layer, 2 continuous layers 3, and 1 continuous layer 2, and laminated to obtain the result.

[0119] Performance Testing

[0120] 1. Scanning electron microscopy (SEM) characterization: The anti-infection biological patch obtained in Example 1 was characterized by SEM. 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.

[0121] 2. Drug Loading Test: The anti-infection biopatches obtained in Examples 1-2 and Comparative Examples 1-5 were each cut into 5 cm × 5 cm samples and extracted in methanol. The extract was sampled every 24 hours and tested by liquid chromatography. Sampling was stopped until the extract became colorless. The drug content of the samples was determined by referring to the chromatograms of the drug standards. The results are shown in Table 1.

[0122] Table 1

[0123]

[0124] As can be seen from Table 1, the drug loading of the biopatch in Comparative Examples 1-2 is very low, both at 100 μg / cm 2 the following.

[0125] The drug loading of the biological patch of Comparative Example 4 is close to that of Example 1.

[0126] 3. Drug Release Rate Test: Twenty-one rats were anesthetized with gas, their abdominal hair shaved, and a 5 cm midline incision made to fully expose the abdominal cavity. The anti-infective biopatch obtained in Examples 1-2 and Comparative Examples 1-5 was cut into 2 cm × 2 cm samples. One identical sample was placed intraperitoneally on each side of the rat's abdominal cavity and secured with sutures. After implantation, the wounds were closed. Samples were collected 4 hours, 1 day, and 5 days after surgery. Following euthanasia, the samples were removed and extracted with PBS buffer for 24 hours. The drug loading, W, was calculated based on a drug standard curve (rifamycin and tetracycline standards were dissolved in PBS buffer, diluted to a series of standard solutions ranging from 1 to 500 µg / mL, and then tested by liquid chromatography injection). 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.

[0127] Table 2

[0128]

[0129] In Table 2, the corresponding antibiotics in Comparative Examples 1-3 and Comparative Example 5 could no longer be detected after 120 hours.

[0130] As can be seen from Table 2, the drug release rates of the biological patches of Comparative Examples 1-3 and Comparative Example 5 were too fast.

[0131] The drug release rate of the biological patch of Comparative Example 4 is close to that of Example 1.

[0132] Bacterial inhibition rate test: Prepare Staphylococcus aureus, Escherichia coli, Staphylococcus epidermidis, and Pseudomonas aeruginosa as test strains. After activating the strains, pick single colonies and mix them in sterile saline to make 10 6 CFU / mL bacterial suspension, the anti-infection biological patches obtained in Examples 1-2 and Comparative Examples 3-5, 2 cm × 3 cm in size, were placed in a test tube, 9.9 mL of MH medium and 0.1 mL of bacterial suspension were added, and no patch was added to the blank control group. The test tubes were incubated in a shaker at 37°C and 180 rpm for 1, 2, 3, and 4 h, and then the culture was serially diluted 10-fold. 0.1 mL of each dilution culture was spread on a TSA plate and incubated at 35°C for 24 h to count the colonies. The same dilution test was repeated 3 times, and a bactericidal curve was drawn with time as the horizontal axis and the logarithm of colony forming units (CFU) as the vertical axis. The bacteriostasis rate at 4 h was recorded. Bacteriostasis rate (%) = (number of colonies in the blank control group - number of colonies on the patch group plate) / number of colonies in the blank control group × 100%. The results are shown in Table 3:

[0133] Table 3

[0134]

[0135] 5. Mechanical Properties: The anti-infection biopatch obtained in Examples 1-2 and Comparative Examples 3-5 was prepared into 50 mm × 50 mm and 25 mm wide specimens. After soaking in water for 5 minutes, the specimens were tested for bursting strength (tested in accordance with GB / T 19976-2005, using a spherical probe with a diameter of 20 mm) and suture strength. The suture strength test method was as follows: surgical suture was used to suture the patch 5 mm from the edge of the sample. The suture and the other end of the patch were fixed to a tensile tester and stretched at a speed of 100 mm / min until the suture point tore. The tensile force at the suture point torn was recorded, which was the suture strength. The results are shown in Figure 4:

[0136] Table 4

[0137] 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

[0138] 6. Animal model validation: Animal experiments were conducted using the anti-infection biological patch obtained in Example 1. Forty six-month-old sows weighing approximately 25 kg were used 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 reopened along the original incision, decompression was performed, and the cecum was removed and the stump was buried to expose the intestinal mucosa with intestinal fistula eversion. After debridement and drainage, open abdominal treatment using 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 or adhesions occurred during the period, and definitive abdominal closure was performed three days later. The previously placed anti-infection biological patch was not removed during the definitive abdominal closure, and the fascia closure rate was 100%. After the operation, the animals were kept in the brooding room. Ten days later, only one case of intestinal air fistula was observed. No incisional hernia occurred during the postoperative follow-up. That is to say, when the anti-infection biological patch of the present invention is used as a temporary abdominal closure material for open abdominal cavity, the incidence of intestinal air fistula is 2.5% (1 / 40).

Claims

1. An application of an anti-infection biological patch in temporary abdominal closure material for open abdominal cavity, characterized in that: The anti-infection biological patch is obtained by loading at least two antibiotics onto an uncrosslinked extracellular matrix; the uncrosslinked extracellular matrix is ​​derived from at least two of the small intestinal submucosa, peritoneum, dermis, bladder basement membrane, and pericardium; the drug release rate of each antibiotic in the anti-infection biological patch is less than 40wt% in 4 hours; 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; and the antibiotics are highly protein-bound antibiotics; The preparation method of the anti-infection biological patch at least comprises: (1) Preparation of continuous layers: Laying down uncrosslinked extracellular matrix with a suspension of antibiotics; (2) Preparation of discontinuous layer: using antibiotic solution to lay non-crosslinked extracellular matrix, repeating the laying process 2-10 times, and then forming and cutting the layers to form a discontinuous layer; The cutting process is specifically to cut into a plurality of rectangular strips or / and tic-tac-toe strips and / or U-shaped strips, with a gap between every two rectangular strips; (3) After the continuous layer and the discontinuous layer are superimposed and subjected to a molding process, the result is obtained.

2. The use of an anti-infective biological patch as a temporary abdominal closure material for an open abdominal cavity 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.

3. The use of an anti-infective biological patch as a temporary abdominal closure material for an open abdominal cavity 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 .

4. The use of an anti-infective biological patch as a temporary abdominal closure material for an open abdominal cavity 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 uncrosslinked extracellular matrix with a suspension of antibiotics; (2) Preparation of discontinuous layer: using antibiotic solution to lay non-crosslinked extracellular matrix, repeating the laying process 2-10 times, and then forming and cutting the layers to form a discontinuous layer; The cutting process is specifically to cut into a plurality of rectangular strips or / and tic-tac-toe strips and / or U-shaped strips, with a gap between every two rectangular strips; (3) After the continuous layer and the discontinuous layer are superimposed and subjected to a molding process, the result is obtained; Uncross-linked extracellular matrix was derived from the small intestinal submucosa and bladder basement membrane.

5. Use of an anti-infective biological patch according to claim 1 or 4 as a temporary abdominal closure material for an open abdominal cavity, characterized in that: The forming process is selected from one or more of lamination, freeze-drying, bonding and suturing.

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

7. Use of the anti-infective biological patch according to any one of claims 1 to 4 as a temporary abdominal closure material for an open abdominal cavity, 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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