Biofilm for repairing abdominopelvic cavity wound surface and preparation method therefor

Abstract

The present invention pertains to the field of biomedical materials and specifically relates to a biofilm for repairing an abdominopelvic cavity wound surface and a preparation method therefor. The present invention provides a biofilm for repairing an abdominopelvic cavity wound surface. The biofilm comprises a first film layer and a second film layer. The thickness of the first film layer is 0.1-1 mm, and the thickness of the second film layer is 0.02-0.4 mm. The weight per unit area of the first film layer is 1.2-5 times that of the second film layer. The interlayer bonding ratio of the first film layer and the second film layer is 35%-75%. The biofilm has good mechanical-performance stability and good operability. The changes in tensile strength and peel strength after repeated soaking / drying cycles or two weeks of soaking remain within 13% compared to 4 h of soaking. The fat ratio of the two film layers is controlled again, thereby improving the ingrowth stability and the mechanical / dimensional stability of the implant material.

Description

A biofilm for repairing abdominal and pelvic wounds and its preparation method Technical Field

[0001] This invention belongs to the field of biomedical materials, specifically relating to a biomembrane for repairing abdominal and pelvic wounds and its preparation method. Background Technology

[0002] In the treatment of local recurrence after rectal cancer surgery, combined pelvic organ resection (PE) can result in extensive tissue defects, creating complex abdominopelvic wounds and anatomical cavities. These large wound defects not only increase the risk of massive intraoperative bleeding but also easily lead to postoperative complications such as infection, fluid accumulation, organ adhesions, and bowel dysfunction. Therefore, how to effectively repair these large pelvic and abdominal defects has become a key issue in clinical research. Current mainstream repair strategies include direct tissue suturing and implant material repair. The tissue ingrowth capacity and dimensional / mechanical stability of the implant material are among the core performance indicators determining the repair outcome.

[0003] In the functional improvement of implantable materials, drug-loaded patches have attracted widespread attention due to their potential for precision treatment. These patches achieve drug loading and controlled release through the structural design of the carrier material, forming an effective drug concentration locally, thereby achieving the dual goals of enhancing efficacy and reducing systemic toxicity. Based on clinical needs, anti-infective drugs have become the most commonly chosen type of loaded drug. Studies have shown that implantable device-related infections mainly originate from the biofilm barrier formed after bacteria adhere to the material surface—a symbiotic microenvironment constructed by bacteria through the secretion of extracellular matrix polymers. This not only significantly reduces antibiotic sensitivity but also easily leads to serious complications such as peritoneal infection, lacunar abscess, and even sepsis. Especially in open abdominal surgery, the occurrence of such infections significantly increases the difficulty of temporary abdominal closure, affecting the final outcome of definitive abdominal closure (delayed full-thickness suture closure).

[0004] Traditional coating-based drug delivery technologies have significant limitations in practical applications: on the one hand, surface coating limits the effective drug loading capacity, making it difficult to maintain long-lasting antibacterial concentrations; on the other hand, edge bonding processes easily lead to material structural loosening, resulting in decreased mechanical integrity and deterioration of intraoperative operability (the material easily becomes loose and difficult to handle). These shortcomings have prompted researchers to turn their attention to novel biomaterial systems. In the field of medical devices, biomaterials, especially decellularized matrix materials, exhibit unique advantages: their natural three-dimensional structure not only possesses excellent biocompatibility but also mimics the dynamic microenvironment of cells, promoting host tissue integration. However, it is worth noting that compared to synthetic polymer materials, uncrosslinked decellularized matrix materials degrade much faster than polymer materials, resulting in a significantly faster drug release rate. This degradation characteristic makes drug release kinetics difficult to control, leading to situations where the therapeutic effect has not yet been achieved, but the drug has already been released in clinical applications.

[0005] Therefore, developing biocomposite materials that combine stable mechanical support and sustained drug release properties (especially the development of sustained-release carrier technologies adapted to the ischemic microenvironment) has become one of the important research directions. Ideal biorepair materials need to achieve a triple functional balance: to achieve sustained controlled drug release by optimizing the material degradation rate, to promote in-situ tissue regeneration through biomimetic structural design, and to maintain sufficient mechanical strength to maintain the stability of anatomical structures. Summary of the Invention

[0006] Based on the aforementioned background technology and problems, firstly, in order to improve the stability of implantable materials, ensure the integrity of product implantation throughout the entire process of human wound healing, and avoid long-term loss of tensile strength, thereby solving the problems of tissue laxity and high recurrence rates, this invention provides a biomembrane for repairing abdominopelvic wounds. The biomembrane comprises a first membrane layer and a second membrane layer. The thickness of the first membrane layer is 0.1-1 mm, and the thickness of the second membrane layer is 0.02-0.4 mm. The weight per unit area of ​​the first membrane layer is 1.2-5 times that of the second membrane layer, and the interlayer bonding ratio between the first and second membrane layers is 35%-75%.

[0007] The first membrane layer includes decellularized matrix A and decellularized matrix B. Decellularized matrix A is derived from pig bladder, and decellularized matrix B is derived from at least one of pig small intestine, pig heart, goat lung, and goat kidney.

[0008] Preferably, the decellularized matrix B is derived from the pig small intestine.

[0009] In this invention, the first membrane layer is derived from Zhuoran Medical Technology (Suzhou) Co., Ltd. The first membrane layer includes decellularized matrix A and decellularized matrix B. Decellularized matrix A is derived from pig bladder and decellularized matrix B is derived from pig small intestine. The preparation of the first membrane layer can be found in patent number CN105920669B.

[0010] The second membrane layer was obtained by modifying the porcine bladder basement membrane.

[0011] The porcine bladder basement membrane was obtained from Zhuoran Medical Technology (Suzhou) Co., Ltd.

[0012] Preferably, the first and second membrane layers have the same length and width.

[0013] Preferably, the biomembrane used to repair abdominal and pelvic wounds comprises a first membrane layer and a second membrane layer, the thickness of the first membrane layer is 0.1-0.8 mm, the thickness of the second membrane layer is 0.02-0.3 mm, the weight per unit area of ​​the first membrane layer is 1.2-4.5 times that of the second membrane layer, and the interlayer bonding ratio of the first membrane layer and the second membrane layer is 40%-60%.

[0014] More preferably, the biomembrane used to repair abdominal and pelvic wounds includes a first membrane layer and a second membrane layer, the thickness of the first membrane layer is 0.1-0.6 mm, the thickness of the second membrane layer is 0.02-0.2 mm, the weight per unit area of ​​the first membrane layer is 1.7-3.9 times that of the second membrane layer, and the interlayer bonding ratio of the first membrane layer and the second membrane layer is 40%-60%.

[0015] Preferably, the interlayer bonding ratio of the first film layer and the second film layer is 45%-55%.

[0016] The first and second membrane layers are bonded together via vacuum lamination to form a biomembrane for repairing abdominal and pelvic wounds. In this invention, specific first and second membrane layers are used. After lamination, a "pore-like contact" is formed between the first and second membrane layers, meaning there are gaps in the contact between them, rather than a completely flat "surface contact." The method for testing the contact area between the first and second membrane layers is as follows: Take a biomembrane, randomly select 10 points for liquid nitrogen brittle fracture treatment, and observe the cross-section of the biomembrane patch. Divide the length of the complete bond between the first and second membrane layers observed in the cross-section by the overall length of the material's cross-section, and take the average of the 10 values ​​as the interlayer bonding ratio.

[0017] Generally, implant materials used for abdominopelvic floor repair should not be too thick. Excessive thickness not only affects the mechanical properties of the implant material, hindering repair and reducing patient comfort, but also makes it difficult to align the incision edges during suturing, increasing the likelihood of scarring. Conversely, implant materials should not be too thin. Excessive thinness affects puncture strength, reduces surgical operability, shortens surgical time, and may weaken the protective effect on organs, increasing the risk of adhesion. To address this, the inventors, through extensive experimentation and research, discovered that using a two-layer membrane-pressed biological patch, controlling the thickness and weight per unit area of ​​the first and second membrane layers, and ensuring that the first and second membrane layers are not completely in contact but partially in contact during pressing (specifically, the thickness of the first membrane layer is 0.1-1 mm, and the thickness of the second membrane layer is 0.02-0.4 mm; the weight per unit area of ​​the first membrane layer is 1.2-5 times that of the second membrane layer; and the interlayer bonding ratio of the first and second membrane layers is 35%-75%), results in a biological patch with good mechanical properties, good operability, and excellent post-implantation repair effects. It is precisely this "pore-like contact" formed between the first and second membrane layers that allows cells and tissues to grow well into the gaps after implantation, forming regeneration. Especially when the interlayer bonding ratio of the first and second membrane layers is 40%-60%, the resulting biomaterial exhibits excellent mechanical stability. After repeated soaking / drying or soaking for two weeks, the changes in tensile strength and peel strength remain within 13% compared to soaking for 4 hours. This significantly superior mechanical stability provides a prerequisite for the application of biological patches.

[0018] The ratio of fat content in the first membrane layer to fat content in the second membrane layer is 1-5.

[0019] Preferably, the ratio of fat content in the first membrane layer to fat content in the second membrane layer is 1-4.

[0020] More preferably, the ratio of fat content in the first membrane layer to fat content in the second membrane layer is 1-3.

[0021] During their investigation of the mechanical properties of biomembranes, the inventors unexpectedly discovered that controlling the fat content ratio of the first and second membrane layers could improve the ingrowth stability of the implant material. Specifically, based on the aforementioned technology, with the first membrane layer thickness at 0.1-1 mm and the second membrane layer thickness at 0.02-0.4 mm; the weight per unit area of ​​the first membrane layer being 1.2-5 times that of the second membrane layer; and the interlayer bonding ratio of the first and second membrane layers at 40-60%, and further controlling the fat content in the first membrane layer of the bio-patch to be 1-5 times its mass, good ingrowth of cells and tissues was achieved on the seventh day in peritoneal models with different fat layer distribution levels. The inventors speculate that this may be because the specific fat content influences the structure of the bio-patch to some extent, giving it both a dense basement membrane layer and a specific degree of looseness, allowing for self-buffering and improvement of the growth environment, thereby affecting the ingrowth pattern of tissue cells. This is beneficial for host tissue cells to form a continuous autologous tissue layer after implantation, providing a vascularized support surface. This effect is particularly pronounced when the ratio of fat content in the first membrane layer to fat content in the second membrane layer is further adjusted to 1-4.

[0022] As a preferred embodiment of the present invention, the second membrane layer is obtained by modifying the porcine bladder basement membrane. The modification process includes the following steps: S1. rinsing; S2. pretreatment; S3. soaking, to obtain the second membrane layer.

[0023] Preferably, step S1 specifically involves rinsing the porcine bladder basement membrane with PBS (phosphate buffer) for 5-15 minutes.

[0024] As a preferred technical solution of the present invention, a pretreatment agent is used in step S2. The pretreatment agent includes esterified amino acids and neutral liposomes, and the mass ratio of esterified amino acids to neutral liposomes is 1:(0.1-0.5).

[0025] Preferably, the pretreatment agent comprises esterified amino acids and neutral liposomes, with a mass ratio of esterified amino acids to neutral liposomes of 1:(0.15-0.35).

[0026] More preferably, the pretreatment agent comprises esterified amino acids and neutral liposomes, with a mass ratio of esterified amino acids to neutral liposomes of 1:(0.15-0.3).

[0027] Preferably, the esterified amino acid is phosphatidylserine.

[0028] As a preferred embodiment of the present invention, the neutral liposomes are selected from one or more of sphingomyelin, animal-derived phosphatidylcholine, and plant-derived phosphatidylcholine.

[0029] Preferably, the neutral liposomes are animal-derived phosphatidylcholine.

[0030] Preferably, step S2 specifically involves dissolving the pretreatment agent in PBS buffer to obtain a pretreatment agent solution. The porcine bladder basement membrane is placed in 100-1000 mL of the pretreatment agent solution and shaken at 40-70 r / min for 1-3 hours at 35-40°C in a shaker. The porcine bladder basement membrane is then removed and rinsed with deionized water for 3-10 minutes.

[0031] As a preferred technical solution of the present invention, step S3 specifically involves immersing the substance pretreated in step S2 in 0.15-2.2% of active substance for 130-150 minutes.

[0032] Preferably, in step S3, the active substance is selected from one of hyaluronic acid, polycystic acid, and chondroitin sulfate.

[0033] Further preferred active ingredient is chondroitin sulfate.

[0034] Phosphatidylcholine plays a role in regulating metabolism and can also provide some protection to the intestinal wall. It has been used in the functionalization research of implant materials. However, because phosphatidylcholine can accelerate blood circulation, it may cause elevated blood pressure, leading to inflammation and fever. The membrane material obtained in this invention overcomes these problems, resulting in stable blood pressure after implantation and reducing the likelihood of inflammation and fever. This may be because the porcine bladder basement membrane is a decellularized matrix with a large amount of protein adhering to it. The lipid groups of phosphatidylserine and neutral liposomes are embedded and adsorbed onto the proteins. Furthermore, since the polar head of phosphatidylserine carries a single negative charge, and chondroitin sulfate also carries a negative charge, these negative charges interact with the positively charged collagen in the decellularized matrix. This interaction improves the structure and performance of the biological patch, thereby reducing the impact of phosphatidylcholine on blood pressure and promoting metabolism, regulating cell and tissue ingrowth. Interestingly, the experiment also found that the use of chondroitin sulfate after the pretreatment in step S2 significantly improved the stability of the mechanical properties of the biological patch compared to treatment before the pretreatment in step S2. The mechanism behind this is still unclear and warrants further investigation.

[0035] The preparation method of the biological patch is as follows: freeze-dry the first film layer and the second film layer respectively, and then vacuum laminate them to obtain the patch.

[0036] As a preferred technical solution of the present invention, the vacuum lamination time is 2-18 hours.

[0037] In practice, freeze-drying lamination between the three membrane layers can also be performed to further expand the cell growth gap between the layers.

[0038] This invention also provides the application of biological patches in the preparation of materials for abdominal and pelvic cavity repair. It can be used in all scenarios involving peritoneal defects, such as perineal wound infections, bleeding, and other abdominal and pelvic wounds, reducing bleeding, preventing adhesions, shortening surgical time, and lowering the risk of internal hernias or infections.

[0039] The biological patch of the present invention is particularly suitable for use in environments with limited blood supply.

[0040] The biological patch of the present invention can be loaded with various drugs, active factors and other bioactive agents.

[0041] Furthermore, in order to expand the application of composite products containing functional active substances carried in the extracellular matrix, and to make biological patches not only used as prostheses for inducing healing, the second aspect of the present invention provides a high-encapsulation, high-dose, antibacterial, sustained-release biological patch.

[0042] In the early stages of abdominal cavity opening, especially within the first 3-5 days, the intestines are exposed and highly susceptible to severe infection, necessitating temporary abdominal closure measures. Medical gauze is commonly used as a temporary closure material, but its isolation effect is very limited. The "Chinese Expert Consensus on Open Abdominal Therapy" points out that synthetic patches are prone to adhesion and abrasion during abdominal cavity reconstruction, therefore their use is not recommended in open abdominal treatment. While non-crosslinked biological patches have high biocompatibility, they are easily degraded and have unstable mechanical properties, failing to achieve ideal results. Crosslinked biological patches may irritate the intestines due to residual crosslinking agents. Most existing temporary abdominal cavity closure techniques provide mechanical protection, only isolating the intestines. To obtain a patch that combines mechanical and chemical protection, the inventors attempted to load antibiotics onto non-crosslinked biological patches to give them antibacterial effects. However, existing literature and practice have shown that antibiotics on non-crosslinked patches not only have low loading capacity but also release too quickly, failing to achieve antibacterial effects, making further application difficult.

[0043] The high-capacity antibacterial sustained-release biological patch is obtained by encapsulating at least two antibiotics in a decellularized matrix material.

[0044] The antibiotic is a high protein binding rate antibiotic.

[0045] Generally speaking, a high binding rate between drugs and proteins is beneficial for drug transport and will improve drug efficacy, which also means that the drug release rate will be relatively faster.

[0046] The antibiotics with high protein binding rate are selected from at least two of the following: tetracycline, rifamycin, β-lactam antibiotics, and macrolide antibiotics.

[0047] The following are examples of tetracyclines: minocycline, minocycline hydrochloride, chlortetracycline, doxycycline hydrochloride, etc.; the following are examples of rifamycins: rifampin, rifapentine, rifabutin, etc.; the following are examples of β-lactam antibiotics: tazobactam, clindamycin, moxifloxacin, etc.; the following are examples of macrolide antibiotics: azithromycin and / or midecamycin.

[0048] Furthermore, the antibiotics include hydrophobic antibiotics and hydrophilic antibiotics, wherein the hydrophobic antibiotics are selected from one or more of β-lactam antibiotics, rifamycin, and macrolide antibiotics.

[0049] Furthermore, hydrophobic antibiotics include rifamycin; hydrophilic antibiotics include tetracycline antibiotics.

[0050] For example, the LogP value of the hydrophobic antibiotic is 0.8-2.1.

[0051] The LogP value is used to represent the distribution of a substance in the water and oil phases. The larger the LogP value, the higher the oleophilicity, which means the higher the hydrophobicity.

[0052] Preferably, the tetracycline antibiotics are selected from one or more of minocycline hydrochloride, doxycycline hydrochloride, and tigecycline.

[0053] Preferably, the tetracycline antibiotic is minocycline hydrochloride.

[0054] The antibiotics loaded in the high-capacity antibacterial sustained-release biological patch are uncoated.

[0055] The hydrophobic antibiotic encapsulation dosage in high-capsulation sustained-release antibacterial biological patches is 150-600 μg / cm³. 2 The encapsulation dosage of hydrophilic antibiotics in high-capsulation sustained-release antibacterial biological patches is 150-600 μg / cm³. 2 .

[0056] The encapsulation dosage of hydrophobic antibiotics in high-capsulation sustained-release biological patches is 150-580 μg / cm³. 2 The encapsulation dosage of hydrophilic antibiotics in high-capsulation sustained-release antibacterial biological patches is 150-580 μg / cm³. 2 .

[0057] Preferably, the hydrophobic antibiotic encapsulation amount in the high-capsulation-dosage antibacterial sustained-release biological patch is 150-570 μg / cm³. 2 The encapsulation dosage of hydrophilic antibiotics in high-capsulation sustained-release antibacterial biological patches is 150-570 μg / cm³. 2 .

[0058] The drug loading of each antibiotic in high-capacity antibacterial sustained-release biological patches is 175-540 μg / cm³. 2 .

[0059] The drug loading of each antibiotic in high-capacity antibacterial sustained-release biological patches is 179-540 μg / cm³. 2 .

[0060] For example, the hydrophilic antibiotic loading in high-capacity antibacterial sustained-release biological patches ranges from 155 to 580 μg / cm³. 2 The hydrophilic antibiotic loading in high-capacity antibacterial sustained-release biological patches is 179-459 μg / cm³. 2 .

[0061] For example, the hydrophobic antibiotic loading in high-capacity antibacterial sustained-release biological patches ranges from 155 to 580 μg / cm³. 2 The hydrophobic antibiotic loading in high-capacity antibacterial sustained-release biological patches ranges from 213 to 540 μg / cm³. 2 .

[0062] If the drug loading is too low, the antibacterial effect will not be achieved; if the drug loading is too high, it may cause antibiotic resistance, side effects, and potentially increase local drug toxicity. Therefore, achieving the desired antibacterial effect is not a case of the higher the drug loading, the better. The inventors discovered that when the hydrophilic antibiotic is a tetracycline, the encapsulation drug loading should be 150-600 μg / cm³. 2 The encapsulation dosage of the hydrophobic antibiotic is 150-600 μg / cm³. 2 This not only helps to avoid the phenomenon of increased toxicity caused by antibiotics, but also helps to overcome the technical resistance of tetracycline's rapid dissolution and release due to its strong hydrophilicity. It slows down the release rate of tetracycline in the patch of the present invention during application, so that the release rate of tetracycline in the patch within 24 hours is controlled at 20.3-64.5%, especially below 52%. This slowing effect is particularly obvious when the decellularized matrix material comes from the submucosa of the small intestine and the basement membrane of the bladder.

[0063] In high-capacity antibacterial sustained-release biological patches, the ratio of hydrophilic to hydrophobic antibiotic loading can be 1:(0.9-1.4). For example, the ratio of tetracycline to rifamycin loading in high-capacity antibacterial sustained-release biological patches can also be 1:(0.9-1.4). Subsequent optimization experiments revealed that when the ratio of tetracycline to rifamycin loading in the high-capacity antibacterial sustained-release biological patch was 1:(0.9-1.4), the burst strength of the patch significantly increased. This is presumably due to a series of non-covalent interactions between the drug molecules and the fibers in the patch, and their synergistic effect on the overall structure of the drug-loaded patch through fiber filling.

[0064] The ratio of the transverse tensile strength to the longitudinal tensile strength of the high-capacity antibacterial sustained-release biological patch is 1:(0.8-1.25).

[0065] The high-capacity antibacterial sustained-release biological patch has not been cross-linked.

[0066] High-capacity antibacterial sustained-release biopatch is obtained by encapsulating at least two antibiotics in an uncrosslinked decellularized matrix material.

[0067] In high-capacity antibacterial sustained-release biological patches, the decellularized matrix material is derived from at least two of the following: the submucosa of the small intestine, the peritoneum, the dermis, the bladder basement membrane, and the pericardium.

[0068] In high-capacity antibacterial sustained-release biological patches, the decellularized matrix material is derived from at least the bladder basement membrane.

[0069] Among the examples of high-capacity antibacterial sustained-release biological patches, the decellularized matrix material is derived from the submucosa of the small intestine and the basement membrane of the bladder.

[0070] For example, decellularized matrix materials can be purchased from Zhuoran Medical Technology (Suzhou) Co., Ltd., which are prepared from the submucosa of the small intestine, peritoneum, dermis, bladder basement membrane or pericardium according to the perfusion-pressure difference method disclosed in patent CN106075583B.

[0071] For example, decellularized matrix materials can be purchased from Zoruan Medical Technology (Suzhou) Co., Ltd., which are prepared from the submucosa of the small intestine and / or the basement membrane of the bladder according to the perfusion-pressure difference method disclosed in patent CN106075583B.

[0072] Examples of decellularized matrix materials may include the biological patch with a first membrane layer and a second membrane layer described above in this scheme.

[0073] As can be listed, decellularized matrix materials can be purchased from any commercially available biological patch.

[0074] The decellularized matrix materials of high-capacity antibacterial sustained-release biological patches include decellularized matrix material C, decellularized matrix material D, and decellularized matrix material E.

[0075] As a preferred embodiment, the high-capacity antibacterial sustained-release biological patch includes at least two continuous surface layers and at least one discontinuous surface layer.

[0076] The preparation method of high-capacity antibacterial sustained-release biological patch is as follows:

[0077] Preparation of the continuous surface layer: Laying decellularized matrix material C and / or decellularized matrix material D;

[0078] Preparation of discontinuous layers: Decellularized matrix material E is laid, and the layering is repeated 2-10 times. After molding, it is cut into rectangular strips. The rectangular strips are placed in parallel to form discontinuous layers.

[0079] After continuous and discontinuous surface layers are superimposed, they are formed through a molding process to obtain the final product.

[0080] The preparation method of high-capacity antibacterial sustained-release biological patch is as follows:

[0081] Preparation of the continuous surface layer: Decellularized matrix material C and / or decellularized matrix material D are laid with a medium;

[0082] Preparation of discontinuous layers: Decellularized matrix material E is laid with a medium and the layering is repeated 2-10 times. After molding, it is cut into rectangular strips and the rectangular strips are placed in parallel to form discontinuous layers.

[0083] After continuous and discontinuous surface layers are superimposed, they are formed through a molding process to obtain the final product.

[0084] The specific process of medium laying is as follows: wet the substrate with medium, lay the decellularized matrix material on the substrate, flatten the decellularized matrix material on the substrate, and then scrape off the excess medium on the surface of the decellularized matrix material with a scraper.

[0085] The purpose of wetting the substrate is to eliminate air between the decellularized matrix material and the substrate, and to utilize the adhesion between the wetting medium and the decellularized matrix material to allow the decellularized matrix material to be better flattened on the substrate.

[0086] The substrate material is not limited. The substrate is only used to provide a horizontal operating plane for the decellularized matrix material. It can be a commercially available glass substrate, steel plate, copper plate, etc.

[0087] The medium is selected from one or more of water, antibiotic solution X, and antibiotic solution Y.

[0088] Antibiotic solution X is a suspension of a hydrophilic antibiotic; antibiotic solution Y is an organic solution of a hydrophobic antibiotic.

[0089] In a preferred embodiment, the suspension of the hydrophilic antibiotic is obtained by adding a metal salt solution to an aqueous solution of the hydrophilic antibiotic and then adjusting the pH to 7.1-8.0 with an alkaline solution.

[0090] Preferably, the suspension of the hydrophilic antibiotic is obtained by adding a metal salt solution to an aqueous solution of the hydrophilic antibiotic and then adjusting the pH to 7.2-7.8 with an alkaline solution.

[0091] More preferably, the suspension of the hydrophilic antibiotic is obtained by adding a metal salt solution to an aqueous solution of the hydrophilic antibiotic and then adjusting the pH to 7.4-7.8 with an alkaline solution.

[0092] Preferably, the alkaline solution is a 0.01-0.1 mol / L aqueous solution of sodium hydroxide.

[0093] For example, the suspension of the hydrophilic antibiotic is obtained by adding 50-600 μL of a 0.18-0.95 g / mL metal salt solution to 3-15 mL of a 0.003-0.02 g / mL aqueous solution of the hydrophilic antibiotic, and then adjusting the pH to 7.1-8.1 with an alkaline solution.

[0094] Preferably, the suspension of the hydrophilic antibiotic is obtained by adding 50-600 μL of a 0.18-0.95 g / mL metal salt solution to 3-15 mL of a 0.005-0.015 g / mL aqueous solution of the hydrophilic antibiotic, and then adjusting the pH to 7.1-8.1 with an alkaline solution. In this process, the hydrophilic antibiotic and the metal salt form a chelate.

[0095] Preferably, the pH is 7.1-8.0.

[0096] Preferably, the pH is 7.2-8.1.

[0097] Preferably, the pH is 7.2-7.9.

[0098] More preferably, the pH is 7.2-7.7.

[0099] More preferably, the pH is 7.4.

[0100] In addition, the molar ratio of metal ions to hydrophilic antibiotics can be listed as (1.2-1.9):1 or (3-6.5):1.

[0101] The molar ratio of metal ions to hydrophilic antibiotics is (1.2-1.9):1 or (3.2-5.4):1.

[0102] The molar ratio of metal ions to hydrophilic antibiotics is (1.5-1.8):1 or (3.2-5.4):1.

[0103] The molar ratio of metal ions to hydrophilic antibiotics is (1.5-1.8):1 or (3.2-5.3):1.

[0104] The molar ratio of metal ions to hydrophilic antibiotics is (1.5-1.8):1 or (3.2-4.3):1.

[0105] When preparing a suspension of hydrophilic antibiotics, the molar mass ratio of the added hydrophilic antibiotic to the added metal salt is 1:(1.2-1.8).

[0106] Tetracycline is unstable in water and easily degrades due to epimerization. In this invention, tetracycline is complexed with metal ions. This complexation effect is a reversible reaction, which allows for the slow release of tetracycline from the patch. Theoretically, in the complexation reaction, one molecule of tetracycline will bind to two molecules of metal ions. In the experiment, when preparing the suspension of hydrophilic antibiotics, the inventors preferentially used a molar ratio of 1:(1.2-1.8) or 1:(3.2-5.4) for the added hydrophilic antibiotics and the added metal salts. They found that very few tetracycline epimers were generated at this ratio. Furthermore, the inventors controlled the molar ratio of 1:2 for the added hydrophilic antibiotics and the added metal salts. Unexpectedly, they found that compared with a molar ratio of 1:2, a molar ratio of 1:(1.2-1.8) or 1:(3.2-5.4) for the added hydrophilic antibiotics and the added metal salts, especially at a ratio of 1:(1.2-1.8), still resulted in very few tetracycline epimers. In addition, it unexpectedly helped to improve the stability of the hydrophobic antibiotic loading in the patch and improve the product stability. The inventors speculate that this may be because hydrophobic antibiotic molecules have strong intermolecular forces, making them prone to aggregation into clusters. These clusters become electron aggregation sites, and the metal ions in the tetracycline complex system interact with these clusters, thus affecting the distribution of the hydrophobic antibiotic and consequently the stability of the packaged drug dosage. Simultaneously, this coordination interaction may also affect the tetracycline complex system itself, meaning that the equilibrium between one tetracycline molecule and two metal ion molecules, which should ideally exist, is disrupted.

[0107] In a preferred embodiment, the metal salt solution contains metal ions, which are divalent metal cations that are diamagnetic.

[0108] Preferably, the metal ion is selected from Ca. 2+ Mg 2+ Ba 2+ Sr 2+ Zn 2+ One of them.

[0109] In a preferred embodiment, the organic solution of the hydrophobic antibiotic is prepared by adding the hydrophobic antibiotic to an 80wt%-95wt% aqueous ethanol solution; the concentration of the hydrophobic antibiotic in the organic solution is 0.01-0.1 g / mL.

[0110] For example, organic solutions of hydrophobic antibiotics are prepared by adding hydrophobic antibiotics to a 90wt%-95wt% aqueous ethanol solution.

[0111] For example, organic solutions of hydrophobic antibiotics are prepared by adding hydrophobic antibiotics to a 90 wt% aqueous ethanol solution.

[0112] Preferably, in the organic solution of the hydrophobic antibiotic, the concentration of the hydrophobic antibiotic is 0.01-0.07 g / mL.

[0113] Preferably, in the organic solution of the hydrophobic antibiotic, the concentration of the hydrophobic antibiotic is 0.01-0.04 g / mL.

[0114] In a preferred embodiment, in step (2), the width of the rectangular strip is 5-50 mm. Examples of rectangular strip widths include 5 mm, 5.1 mm, 5.2 mm, 5.3 mm, 5.4 mm, 5.5 mm, 5.6 mm, 5.7 mm, 5.8 mm, 5.9 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm, 11 mm, 12 mm, 13 mm, 14 mm, 15 mm, 16 mm, 17 mm, 19 mm, 20 mm, 21 mm, 23 mm, 25 mm, 28 mm, 32 mm, 35 mm, 36 mm, 38 mm, 42 mm, 48 mm, and 50 mm.

[0115] Preferably, in step (2), there is a gap between every two rectangular strips, and the gap between the rectangular strips is 0.2-2.2 cm.

[0116] The spacing between the bio-based strips is 0.2-1.9 cm.

[0117] The spacing between the bio-based strips is 0.7-2.2 cm.

[0118] The spacing between the bio-based strips is 0.7-2 cm.

[0119] Preferably, the spacing between the bio-based strips is 0.9-1.5 cm.

[0120] Further preferably, the spacing between the bio-based strips is 1-1.5 cm.

[0121] To obtain an antibacterial patch where antibiotics are distributed throughout the patch structure network, rather than just in a single layer, the inventors, through in-depth creative thinking, replaced the traditional lamination process (such as the pressing process described in patents CN105102009B or CN115054743B, which simply involves stacking dry film layers) with a medium-laying method. This not only ensures that the resulting patch has good mechanical properties and a structure that is not easily loosened when exposed to water, but also effectively achieves slow release of both hydrophobic and hydrophilic antibiotics while ensuring high drug loading, and further controls and regulates the release rate of hydrophobic and hydrophilic antibiotics (especially the sustained release of hydrophobic antibiotics). In the present invention, the above-mentioned sustained release effect is particularly significant, especially with further control of the spacing of the bio-based strips.

[0122] In step (2), the length of the rectangular strip is less than or equal to the length of the maximum diagonal of the continuous surface layer.

[0123] Preferably, in step (2), the rectangular strips are arranged at intervals and in parallel to form a discontinuous layer; the angle between the length direction and the horizontal direction of each rectangular strip in the discontinuous layer is 0-90°.

[0124] Preferably, the angle between the length direction and the horizontal direction of the bio-based strip is 15-75°.

[0125] More preferably, the angle between the length direction and the horizontal direction of the bio-based strip is 30-60°.

[0126] More preferably, the angle between the length direction and the horizontal direction of the bio-based strip is 35-45°.

[0127] For example, the angle between the length direction and the horizontal direction of the bio-based strip is 45°.

[0128] Based on the spacing of 0.2-2.2cm between the bio-based strips, the angle between the length direction of the bio-based strips and the length direction of the basal layer can be further adjusted to 15-75°. This improves to some extent the problem that tetracycline is unstable in water and is prone to epimerization, which leads to a decrease in the activity of tetracycline in antibiotic solution X and a reduction in the therapeutic effect.

[0129] Specifically, there is no particular limit to the number of bio-based strips; they can be laid at intervals of 0.2-2.2 cm.

[0130] Preferably, the molding process in steps (2) and (3) is selected from one or more of lamination, freeze drying, bonding and sewing.

[0131] The purpose of molding processes is to make laid or stacked materials into a whole.

[0132] Furthermore, the molding process involves lamination at 35-55℃.

[0133] In a preferred embodiment, in step (1), when preparing the continuous surface layer, decellularized matrix material C and / or decellularized matrix material D are laid with water and / or a suspension of hydrophilic antibiotics.

[0134] Preferably, in step (1), when preparing the continuous surface layer, the decellularized matrix material C or / and the decellularized matrix material C are laid with water or a suspension of hydrophilic antibiotics.

[0135] In a preferred embodiment, in step (2), when preparing the discontinuous layer, an organic solution of a hydrophobic antibiotic is used for application.

[0136] The specific steps for applying the substrate with water and / or a suspension of hydrophilic antibiotics are as follows: place the substrate flat, wet the substrate with water and / or a suspension of hydrophilic antibiotics, apply decellularized matrix material C and / or decellularized matrix material D onto the substrate, flatten the decellularized matrix material on the substrate, and then scrape off the excess water and / or suspension of hydrophilic antibiotics from the surface of the decellularized matrix material with a scraper.

[0137] The operation of laying with an organic solution of hydrophobic antibiotics is similar to that of laying with water and / or a suspension of hydrophilic antibiotics, and the principle is the same.

[0138] Furthermore, in step (1), water can be used to lay decellularized matrix material C to obtain a continuous surface layer, a suspension of hydrophilic antibiotics can be used to lay decellularized matrix material C to obtain a continuous surface layer, water can be used to lay decellularized matrix material D to obtain a continuous surface layer, a suspension of hydrophilic antibiotics can be used to lay decellularized matrix material D to obtain a continuous surface layer, etc.

[0139] In a preferred embodiment, both the upper and lower surfaces of the discontinuous layer have contact area with the continuous layer.

[0140] Preferably, the contact area is 50%-100% of the area of ​​the continuous surface layer.

[0141] Preferably, the contact area is 60%-100% of the area of ​​the continuous surface layer.

[0142] The arrangement of the intermittent layer in contact with the continuous layer on both sides not only improves the interlayer bonding force, making the final pressed product less prone to loosening, but also helps to increase the drug loading of hydrophilic antibiotics in antibiotic solution X and hydrophobic antibiotics in antibiotic solution Y. Furthermore, it promotes the distribution of hydrophilic antibiotics in antibiotic solution X and hydrophobic antibiotics in antibiotic solution Y throughout the final patch structure network (rather than the distribution trend of drugs only distributed in a single layer). In addition, the inventors unexpectedly discovered that the contact between the intermittent layer and the continuous layer on both sides also unexpectedly reduced the drug loss of tetracycline and hydrophobic antibiotics caused by subsequent lamination, allowing the drug loss to be as low as 15 wt%, controlled within the range of 15-50 wt%, greatly reducing drug waste and lowering production costs. The inventors speculate that this may be because the base, middle and top layers of the decellularized matrix material retain the complete triple helix structure of collagen fibers. In addition to some of the drug crystal nuclei being deposited in the porous pores of each layer, some of them can be embedded in the helical grooves on the triple helix chain. The discontinuous surface layer setting of the specific discontinuous surface layer has a significant impact on this deposition and embedding adsorption. Accompanied by the unique toughness effect between the fibers formed at this time, the binding force is increased.

[0143] In step (3) of the preparation method of high-capacity antibacterial sustained-release biological patch, the number of layers and the stacking order of continuous and discontinuous layers are not limited. For example, from bottom to top, the order is 1 continuous layer, 1 discontinuous layer and 1 continuous layer; from bottom to top, the order is 8 continuous layers, 2 discontinuous layers and 6 continuous layers; from bottom to top, the order is 1 continuous layer, 1 discontinuous layer, 4 continuous layers, 1 discontinuous layer and 1 continuous layer; from bottom to top, the order is 2 continuous layers, 2 discontinuous layers, 4 continuous layers, 1 discontinuous layer and 1 continuous layer, etc. Although the stacking order of continuous and discontinuous layers is not limited, in order to ensure that both the upper and lower surfaces of the discontinuous layer have contact area with the continuous layer, the outermost layer of the high-capsulation antibacterial sustained-release biological patch must be a continuous layer.

[0144] Preferably, the total number of continuous and discontinuous layers is ≤30.

[0145] Further preferred, the total number of continuous and discontinuous layers is ≤25.

[0146] Further preferred, the total number of continuous and discontinuous layers is ≤21.

[0147] Further preferred, the total number of continuous and discontinuous layers is ≤17.

[0148] Tetracycline has strong hydrophilicity, causing it to dissolve rapidly in water and be released when loaded onto a patch. If the amount of drug encapsulated in the patch is too low, the tetracycline cannot be continuously released during the high-incidence period of surgical infection, thus failing to achieve an antibacterial effect. Since the antibacterial sustained-release biological patch of this invention is used during the high-incidence period of surgical infection, i.e., the pre-operative period, a higher encapsulation amount is not necessarily better. Generally, the more layers stacked, the higher the encapsulation amount. At the same time, as the number of material layers increases, the manufacturability of the material also decreases. Therefore, in this invention, the inventors controlled the total number of continuous and discontinuous layers to within 30 layers. In experiments, the inventors unexpectedly discovered that this approach also benefits the mechanical uniformity (the transverse tensile strength and longitudinal tensile strength always approach 1:1) and mechanical stability of the antibacterial sustained-release biological patch, and does not pose an application risk due to instability caused by pressure, swelling, or other environmental factors. In particular, when the total number of continuous and discontinuous layers is ≤25, this mechanical uniformity and stability are even more pronounced.

[0149] Generally speaking, for synthetic polymer substrates and bio-based substrates, due to the inherent characteristics of synthetic polymer substrates, such as slow degradation, the drug release rate of drug-loaded bio-based substrates is usually much faster than that of drug-loaded synthetic polymer substrates, assuming the same drug loading and release environment (except for the type of substrate). If the drug is released too quickly, it cannot exert a sustained antibacterial effect, potentially failing to meet the needs of clinical antibacterial therapy. This necessitates controlling the drug release rate of drug-loaded bio-based substrates. Especially for the rifamycin antibiotics (specifically rifampin) used in this invention, their high hydrophilicity means that after being loaded onto the patch, they will rapidly dissolve in water and release, further accelerating the drug release rate. Therefore, it is crucial to control the drug release rate of drug-loaded bio-based substrates. The inventors of this invention have overcome the technical resistance to the rapid release of drugs on biological substrates, especially for rifamycin antibiotics and tetracycline metal chelate suspensions, by ensuring that the intermediate layer includes at least one interfacial layer (i.e., the drug-loaded biological patch includes at least one interfacial layer) and that the spacing and angle of the biological substrate strips are within a specific range. This overcoming effect is particularly significant. Furthermore, by controlling the number of interfacial layers and the width range of the biological substrate strips, the process stability of this sustained-release effect can be enhanced, and the sustained-release rate can be adjusted according to actual needs.

[0150] Although the stacking order of continuous and discontinuous layers is not limited, in order to ensure that both the upper and lower surfaces of the discontinuous layer have contact area with the continuous layer, the outermost layer of the anti-infection biological patch is a continuous layer.

[0151] It is known that a suspension is a mixture containing solid particles visible to the naked eye, while a solution is a homogeneous mixture containing particles that are not visible to the naked eye. Through extensive creative work, the inventors discovered that using an antibiotic suspension to lay the continuous surface layer and an antibiotic solution to lay the discontinuous surface layer not only increases the drug loading of the drug-loaded biological patch but also improves the suture strength and enhances the structural stability of the drug-loaded biological patch. The inventors speculate that this may be because the antibiotic solute particles loaded in the discontinuous surface layer are smaller, while the antibiotic particles loaded in the continuous surface layer are larger. Compared to the smaller particles, the larger particles are more loosely distributed. The combined effect of the two particle dispersions makes the force distribution in the overall structure more balanced, which helps to weaken the structural instability caused by the presence of gaps in the discontinuous surface layer.

[0152] Furthermore, the drug release rate of each antibiotic in the high-capsulation antibacterial sustained-release biological patch is 15-85 wt% in 24 hours and 85-100 wt% in 120 hours.

[0153] Furthermore, in high-capsulation antibacterial sustained-release biological patches, the drug release rate of each antibiotic is 20.3-85 wt% in 24 hours and 85-100 wt% in 120 hours.

[0154] Each antibiotic in the high-capacity antibacterial sustained-release biological patch has a drug release rate of 86-100 wt% over 120 hours.

[0155] The high-capacity antibacterial sustained-release biological patch exhibits a drug release rate of 15-85 wt% for each antibiotic within 24 hours and 85-100 wt% within 120 hours. This overcomes the technical obstacles of low antibiotic loading and rapid release on non-crosslinked patches, which prevent the achievement of antibacterial effects. Applied to the field of temporary abdominal closure materials, this high-capacity antibacterial sustained-release biological patch not only provides mechanical protection to the intestines while ensuring material mechanical stability but also provides chemical protection, exhibiting excellent active antibacterial function. It can reduce the incidence of intestinal air fistula caused by abdominal opening to below 2%, reduce the risk of abdominal bleeding, protect the intestines from low blood perfusion, improve fascial closure rate, and shorten definitive closure time, especially when the uncrosslinked extracellular matrix originates from the submucosa of the small intestine and the basement membrane of the bladder.

[0156] Furthermore, the tetracycline loaded in the high-capsulation antibacterial sustained-release biological patch had a drug release rate of 20.3-64.5 wt% at 24 h and 86.2-98.7 wt% at 120 h.

[0157] Furthermore, the tetracycline loaded in the high-capacity antibacterial sustained-release biological patch had a drug release rate of 21.6-63.9 wt% over 24 hours.

[0158] The drug release rate of rifamycin loaded in high-capsulation antibacterial sustained-release biological patches was 70.8-85 wt% at 24 h and 95.1-100 wt% at 120 h.

[0159] Furthermore, each antibiotic in the high-capsulation antibacterial sustained-release biological patch has a drug release rate of <40wt% over 4 hours.

[0160] The tetracycline loaded in the high-capacity antibacterial sustained-release biological patch has a drug release rate of 22-33 wt% in 4 hours.

[0161] The rifamycin loaded in the high-capacity antibacterial sustained-release biological patch has a drug release rate of 30-39 wt% after 4 hours.

[0162] This invention also provides the application of a highly encapsulated, antibacterial, sustained-release biological patch in the preparation of materials for abdominal and pelvic cavity repair. It can be used in all scenarios involving peritoneal defects, including but not limited to perineal wound infections, bleeding, and other abdominal and pelvic wounds, reducing bleeding, preventing adhesions, shortening surgical time, and lowering the risk of internal hernias or infections.

[0163] On the other hand, the present invention also provides the application of high-capsulation antibacterial sustained-release biological patches in the preparation of anti-infective materials.

[0164] The high-capacity antibacterial sustained-release biological patch is used to prepare medical devices for surgical procedures during periods of high infection incidence.

[0165] The peak period for infection after surgery is usually within three days.

[0166] High-capacity antibacterial sustained-release biological patches are particularly useful in the preparation of treatment materials for the high incidence of open abdominal infections.

[0167] For example, the high-capacity antibacterial sustained-release biological patch is used in the preparation of temporary abdominal closure materials for open abdominal cavities.

[0168] When using high-capacity antibacterial sustained-release biological patches, a negative pressure auxiliary system can be used in conjunction with them.

[0169] Negative pressure assist systems are currently an effective measure for protecting open abdominal wounds. They are generally composed of medical foam materials, adhesive films, and negative pressure tubing. Their main purpose is to drain waste fluid from the abdominal cavity.

[0170] The antibacterial patch obtained by the process of the high-capacity antibacterial sustained-release biological patch or biological patch according to the present invention can be provided with uniform small holes, the hole diameter is 0.1-3.0 mm, and the distance between two adjacent holes is 4-20 mm.

[0171] Compared with the prior art, the present invention has the following advantages:

[0172] The biomembrane obtained by this invention for repairing abdominopelvic wounds is a bio-patch made of two layers of membranes pressed together. The thickness and weight per unit area of ​​the first and second membrane layers are controlled, and the first and second membrane layers are not completely in contact and overlapped, but only partially in contact during pressing. The resulting bio-patch has good mechanical property stability and good operability. After repeated soaking / drying or soaking for two weeks, the changes in tensile strength and peel strength are maintained within 13% compared with soaking for 4 hours. The fat ratio of the two membrane layers is controlled again, which improves the ingrowth stability and mechanical / dimensional stability of the implanted material. A balance between degradation and regeneration is achieved, and the implanted bio-patch is not prone to rapid degradation or severe shrinkage.

[0173] High-capacity antibacterial sustained-release biological patches not only have good mechanical properties in a humid environment, which is beneficial to improving feel and operability, but also have good mechanical strength and tissue compliance. Their soft texture will not cause damage to the intestinal tract and other visceral tissues they come into contact with. Moreover, they can effectively control short-term large-scale infections, have good active antibacterial ability and drug sustained-release effect, reduce the risk of infection of implanted materials, and are conducive to smooth recovery. In this technology, the high-capacity antibacterial sustained-release biological patch is based on a lamination process and features an uncoated drug-loaded sustained-release system. It synergistically combines at least two different types of antibiotics to kill bacteria in a short period of time, preventing bacterial colonization and reproduction in the wound / patch. This achieves sustained release of rifamycin and tetracycline, preventing them from being released too quickly. Instead, they are released synergistically and continuously along with the wound healing process, while simultaneously guiding tissue regeneration and degradation. This technology can be applied during the high-incidence period of surgical infections (i.e., within the first three days after surgery), especially in open abdominal surgery, reducing the probability of adhesions, strengthening the abdominal wall, promoting the growth of granulation tissue in the wound, and achieving effective antibacterial protection during the high-incidence period of postoperative infections.

[0174] By loading specific types of hydrophobic and hydrophilic antibiotics, and controlling the encapsulation dosage of the hydrophobic antibiotic to 150-600 μg / cm³, 2 The encapsulation dosage of hydrophilic antibiotics is 150-600 μg / cm³. 2This not only helps to avoid the increased toxicity caused by the aforementioned antibiotic effects, but also overcomes the technical resistance of tetracycline, especially minocycline hydrochloride, which is rapidly released from water due to its strong hydrophilicity. This slows down the release rate of tetracycline in the patch of this invention during application, keeping the release rate of tetracycline in the patch within 24 hours below 52%. When preparing the suspension of hydrophilic antibiotics, the molar mass ratio of the added hydrophilic antibiotic to the added metal salt is 1:(1.2-1.8), which improves the stability of the hydrophobic antibiotic loading in the patch, thereby improving the uniformity and stability of the product.

[0175] When high-capacity antibacterial sustained-release biological patches are used in temporary abdominal closure materials for open abdominal cavity, they can not only reduce the incidence of intestinal air fistula to below 2%, but also reduce the risk of incisional hernia and abdominal bleeding. They can also reduce fascial retraction, thereby further avoiding adhesions between the intestine and the abdominal wall, as well as between the intestine and the abdominal dressing, and effectively promoting the closure and repair of abdominal wall fascia tissue. They can be used in conjunction with negative pressure vacuum-assisted closure of the open abdomen to protect the intestine from the effects of low blood perfusion. Attached Figure Description

[0176] Figure 1 is a schematic diagram of the structure of the biological patch used to repair the abdominal and pelvic wounds described in Example 1, where 1-first membrane layer, 2-second membrane layer, X-microscopic structure magnified view of the contact part of the first membrane layer and the second membrane layer after lamination, the shaded part is the contact adhesion area, and the white area is the gap;

[0177] Figure 2 shows the biological patch obtained in Example 3, which was implanted into the peritoneum of a rat and fixed to the peritoneum. Seven days after the operation, a scanning electron microscope test was performed, and the surface mesothelial cells were shown in the electron microscope image.

[0178] Figure 3 is a pathological image of the tissue ingrowth of the biological patch obtained in Example 1 after embedding and sectioning in an animal model experiment and then staining with HE.

[0179] Figure 4 is a pathological image of the tissue ingrowth of the biological patch obtained in Example 3 after embedding and sectioning in an animal model experiment and then staining with HE.

[0180] Figure 5 is a pathological image of the tissue ingrowth of the biological patch obtained in Comparative Example 3 after embedding and sectioning in an animal model experiment and then staining with HE.

[0181] Figure 6 is a pathological image of the tissue ingrowth of the biological patch obtained in Comparative Example 4 after embedding and sectioning in an animal model experiment and then staining with HE.

[0182] Figure 7 is a schematic diagram of the structure of the high-capacity antibacterial sustained-release biological patch described in Example 5; in Figure 7, 1-base layer, 2-intersection layer 1, 3-intersection layer 2, 4-intermediate layer, 5-top layer; (the staggered layers in the illustration are only to clearly show the structure of each layer, and the edges of each quadrilateral in the actual product are aligned.)

[0183] Figure 8 is a schematic diagram of the high-capacity antibacterial sustained-release biological patch described in Example 6; in Figure 8, 8-intermediate layer, 6-intersection layer 1’ , 7-intermittent layer 2’ (The staggered layers shown in the illustration are only for clear demonstration of each layer's structure; in the actual product, the edges of each quadrilateral are aligned.)

[0184] Figure 9(a) is a schematic cross-sectional view of the high-capacity antibacterial sustained-release biological patch described in Example 7; in the figure, 9-intermediate layer''; Figure 9(b) is a schematic cross-sectional view of the high-capacity antibacterial sustained-release biological patch described in Example 8; in the figure, 11-unit module, 10-intermediate layer''';

[0185] Figure 10 is a schematic diagram of the structure of the high-capacity antibacterial sustained-release biological patch described in Comparative Example 12; (The staggered layers in the diagram are only to clearly show the structure of each layer; in the actual product, the edges of each quadrilateral are aligned.)

[0186] Figure 11 is a structural side view of the high-encapsulation antibacterial sustained-release biological patch obtained in Example 10. In Figure 11, solid lines represent continuous layers and dashed lines represent discontinuous layers.

[0187] Figure 12(a) is a front view of the structure of the high-encapsulation dosage antibacterial sustained-release biological patch obtained in Example 10, and Figure 12(b) is a schematic diagram of the structure of the high-encapsulation dosage antibacterial sustained-release biological patch obtained in Example 10 (the staggered layers in the figures are only to clearly show the structure of each layer).

[0188] Figure 13 shows the SEM images of the sample from Example 7. Figure 13(a) is an SEM image of the upper surface of the patch, Figure 13(b) is an SEM image of the lower surface of the patch, and Figures 13(c) and 13(d) are SEM images of the cross-section of the patch. Detailed Implementation

[0189] All reagents used in the examples were commercially available. PBS buffer was purchased from Thermo Fisher Scientific, phosphatidylserine from Shanxi Guotai Biotechnology Co., Ltd., animal-derived phosphatidylcholine (PC) was purchased from Xi'an Tianzheng Pharmaceutical Excipients Co., Ltd., and its product name was egg yolk lecithin, with a PC content of 50 wt%. Chondroitin sulfate was purchased from Laiyang Xinghua Biological Products Co., Ltd. The first membrane layer and the porcine bladder basement membrane were both obtained from Zhuoran Medical Technology (Suzhou) Co., Ltd. The first membrane layer includes decellularized matrix A and decellularized matrix B. Decellularized matrix A is derived from porcine bladder, and decellularized matrix B is derived from porcine small intestine. The specific models and parameters of the first membrane layer are shown in Table 1.

[0190] Table 1

[0191]

[0192] Example

[0193] Example 1

[0194] This example provides a biological patch, which includes a first membrane layer and a second membrane layer. The first membrane layer is sourced from Zoruan Medical Technology (Suzhou) Co., Ltd., and is model A.

[0195] The second membrane was obtained by modifying the porcine bladder basement membrane. The porcine bladder basement membrane was sourced from Zoran Medical Technology (Suzhou) Co., Ltd., and its model number was E.

[0196] The modification process includes the following steps: S1. Rinsing: Rinse the porcine bladder basement membrane with PBS buffer for 10 min; S2. Pretreatment: Dissolve the pretreatment agent in PBS buffer to obtain a pretreatment agent solution. Place the porcine bladder basement membrane in 500 mL of the pretreatment agent solution and shake it at 50 r / min for 2 h at 36 °C in a shaker. Then, remove the porcine bladder basement membrane and wash it with deionized water for 5 min. The pretreatment agent includes esterified amino acids and neutral liposomes, with a mass ratio of esterified amino acids to neutral liposomes of 1:0.35. The esterified amino acid is phosphatidylserine, and the neutral liposomes are animal-derived phosphatidylcholine; S3. Immersion: Immerse the substance pretreated in step S2 in 1% active substance for 130 min. The active substance is chondroitin sulfate to obtain the second membrane layer.

[0197] The interlayer bonding ratio of the first membrane layer and the second membrane layer is 51%, and the structure of the biological patch is shown in Figure 1.

[0198] The preparation method of the biological patch is as follows: freeze-dry the first and second membrane layers respectively, and vacuum laminate for 16 hours to obtain the patch.

[0199] Example 2

[0200] This example provides a biological patch, which differs from Example 1 in that the first membrane layer is sourced from Zoru Medical Technology (Suzhou) Co., Ltd., model B.

[0201] The interlayer bonding ratio between the first and second membrane layers is 48%.

[0202] The second membrane was obtained by modifying the porcine bladder basement membrane. The porcine bladder basement membrane was sourced from Zoran Medical Technology (Suzhou) Co., Ltd., and its model number was F.

[0203] The preparation method of the biological patch is the same as that in Example 1.

[0204] Example 3

[0205] This example provides a biological patch, which differs from Example 1 in that the first membrane layer is sourced from Zoruan Medical Technology (Suzhou) Co., Ltd., model C.

[0206] The interlayer bonding ratio between the first and second membrane layers is 55%.

[0207] The second membrane was obtained by modifying the porcine bladder basement membrane. The porcine bladder basement membrane was sourced from Zoran Medical Technology (Suzhou) Co., Ltd., and its model number was G.

[0208] The preparation method of the biological patch is the same as that in Example 1.

[0209] Comparative Example 1

[0210] This example provides a biological patch, which differs from Example 1 in that the first membrane layer is sourced from Zoruan Medical Technology (Suzhou) Co., Ltd., model D.

[0211] The interlayer bonding ratio between the first and second membrane layers is 42%.

[0212] The preparation method of the biological patch is the same as that in Example 1.

[0213] In other words, in this example, the thickness of the first film layer is relatively thick, at 1.2 mm, which is outside the range of 0.1-1 mm.

[0214] Comparative Example 2

[0215] This example provides a biological patch, which differs from Example 2 in that the porcine bladder basement membrane is sourced from Zhuoran Medical Technology (Suzhou) Co., Ltd., and is model number H.

[0216] The interlayer bonding ratio between the first and second membrane layers is 53%.

[0217] The preparation method of the biological patch is the same as that in Example 1.

[0218] In other words, in this example, the second film layer is thicker, at 0.5 mm, which is outside the range of 0.02-0.4 mm.

[0219] Comparative Example 3

[0220] This example provides a biological patch, which differs from Example 3 in that the second membrane layer is obtained by modifying porcine bladder basement membrane. The modification process includes the following steps: S1. Rinsing: Rinse the porcine bladder basement membrane with PBS buffer for 15 min; S2. Pretreatment: Dissolve the pretreatment agent in PBS buffer to obtain a pretreatment agent solution. Place the porcine bladder basement membrane in 500 mL of the pretreatment agent solution and shake it at 50 r / min for 3 h at 36 °C on a shaker. Then, remove the porcine bladder basement membrane and wash it with deionized water for 5 min. The pretreatment agent includes esterified amino acids and neutral liposomes, with a mass ratio of esterified amino acids to neutral liposomes of 1:0.2. The esterified amino acid is phosphatidylserine, and the neutral liposomes are animal-derived phosphatidylcholine; S3. Immersion: Immerse the substance pretreated in step S2 in 2% active substance for 150 min. The active substance is chondroitin sulfate, to obtain the second membrane layer.

[0221] The interlayer bonding ratio between the first and second membrane layers is 87%.

[0222] The preparation method of the biological patch differs from that in Example 3 in that the first and second membrane layers are freeze-dried separately and vacuum-laminated for 16 hours to obtain the patch.

[0223] In other words, in this example, the contact area between the first and second membrane layers is relatively large, and the interlayer bonding ratio is relatively high.

[0224] Comparative Example 4

[0225] This example provides a biological patch, which differs from Example 1 in that the first membrane layer is sourced from Zoru Medical Technology (Suzhou) Co., Ltd., model X.

[0226] The interlayer bonding ratio between the first and second membrane layers is 62%.

[0227] This example also provides a method for preparing a biological patch for repairing abdominal and pelvic wounds, which is the same as in Example 1.

[0228] In other words, in this example, the ratio of fat content in the first membrane layer to fat content in the second membrane layer is relatively high (>5).

[0229] Comparative Example 5

[0230] This example provides a biological patch, which differs from Example 3 in that the second membrane layer is obtained by modifying porcine small intestinal submucosal tissue. The porcine small intestinal submucosal tissue was purchased from Zoran Medical Technology (Suzhou) Co., Ltd.

[0231] The interlayer bonding ratio between the first and second membrane layers is 55%.

[0232] This example also provides a method for preparing a biological patch for repairing abdominal and pelvic wounds, which is the same as in Example 1.

[0233] Comparative Example 6

[0234] This example provides a biological patch, which differs from Example 1 in that the second membrane layer is obtained by modifying porcine bladder basement membrane. The modification process includes the following steps: S1. Rinsing: Rinse the porcine bladder basement membrane with PBS buffer for 10 min; S2. Pretreatment: Dissolve the pretreatment agent in PBS buffer to obtain a pretreatment agent solution. Place the porcine bladder basement membrane in 450 mL of the pretreatment agent solution and shake it at 70 r / min for 2 h at 35°C in a shaker. Then, remove the porcine bladder basement membrane and wash it with deionized water for 5 min. The pretreatment agent includes esterified amino acids and neutral liposomes, with a mass ratio of esterified amino acids to neutral liposomes of 1:0.6. The esterified amino acid is phosphatidylserine, and the neutral liposomes are animal-derived phosphatidylcholine; S3. Immersion: Immerse the substance pretreated in step S2 in 0.7% active substance for 120 min. The active substance is chondroitin sulfate, to obtain the second membrane layer.

[0235] The interlayer bonding ratio between the first and second membrane layers is 35%.

[0236] The preparation method of the biological patch is the same as that in Example 1.

[0237] Comparative Example 7

[0238] This example provides a biological patch. Unlike Example 3, in the modification treatment of the porcine bladder basement membrane in the second membrane layer, steps S2 and S3 are changed. First, the active substance is soaked in step S3, followed by the pretreatment agent treatment in step S2. The interlayer bonding ratio between the first and second membrane layers is 45%.

[0239] The preparation method of the biological patch is the same as that in Example 1.

[0240] Example 4

[0241] This example provides a high-capacity antibacterial sustained-release biological patch, which is obtained by encapsulating an antibiotic in a decellularized matrix material.

[0242] All decellularized matrix materials are from Zoruan Medical Technology (Suzhou) Co., Ltd., and are prepared from the submucosa of the small intestine or the basement membrane of the bladder according to the perfusion-pressure difference method described in patent CN106075583B.

[0243] The antibiotic is a hydrophobic antibiotic, specifically rifamycin, which is rifampicin. The LogP value of rifampicin is 1.09.

[0244] The preparation method of high-capacity antibacterial sustained-release biological patch is as follows:

[0245] Preparation of continuous surface layer 1: Decellularized matrix material C (derived from porcine bladder basement membrane, purchased from Zoran Medical Technology (Suzhou) Co., Ltd.) was laid with 6 ml of water.

[0246] Preparation of the discontinuous layer: A decellularized matrix material E (derived from the submucosa of porcine small intestine, purchased from Zhuoran Medical Technology (Suzhou) Co., Ltd.) was laid on 6 mL of an organic solution of a hydrophobic antibiotic (i.e., antibiotic solution Y). This process was repeated four times. After lamination, the material was cut into rectangular strips. These strips were spaced apart and arranged parallel to each other to form the discontinuous layer. The width of each rectangular strip was 3 mm. There was a 1.5 cm gap between every two rectangular strips. The angle between the length and horizontal directions of each rectangular strip in the discontinuous layer was 45°.

[0247] The method for preparing the organic solution of hydrophobic antibiotic is as follows: add the hydrophobic antibiotic to a 90wt% aqueous ethanol solution so that the concentration of the hydrophobic antibiotic in the antibiotic solution Y is 0.01g / mL.

[0248] (3) Stack one continuous surface layer 1, one discontinuous surface layer and one continuous surface layer 1 in sequence from bottom to top, and laminate at 45℃ for 12 hours to obtain the product.

[0249] Example 5

[0250] This example provides a high-capacity antibacterial sustained-release biological patch, which is obtained by encapsulating an antibiotic in a decellularized matrix material.

[0251] All decellularized matrix materials are from Zoruan Medical Technology (Suzhou) Co., Ltd., and are prepared from the submucosa of the small intestine or the basement membrane of the bladder according to the perfusion-pressure difference method described in patent CN106075583B.

[0252] The antibiotic is a hydrophobic antibiotic, specifically rifamycin, which is rifampicin. The LogP value of rifampicin is 1.09.

[0253] The preparation method of high-capacity antibacterial sustained-release biological patch is as follows:

[0254] Preparation of continuous surface layer 1: Decellularized matrix material C (derived from porcine bladder basement membrane, purchased from Zoran Medical Technology (Suzhou) Co., Ltd.) was laid with 6 ml of water.

[0255] Preparation of discontinuous layer 1: Decellularized matrix material E (derived from the submucosa of porcine small intestine, purchased from Zhuoran Medical Technology (Suzhou) Co., Ltd.) was laid on 6 mL of an organic solution of hydrophobic antibiotics (i.e., antibiotic solution Y). This process was repeated four times. After lamination, the material was cut into rectangular strips. These strips were spaced apart and arranged parallel to each other to form the discontinuous layer. The width of each rectangular strip was 3 mm. There was a 1.5 cm gap between every two rectangular strips. The angle between the length and horizontal directions of each rectangular strip in the discontinuous layer was 45°.

[0256] The method for preparing the organic solution of hydrophobic antibiotic is as follows: add the hydrophobic antibiotic to a 90wt% aqueous ethanol solution so that the concentration of the hydrophobic antibiotic in the antibiotic solution Y is 0.01g / mL.

[0257] Preparation of discontinuous layer 2: The preparation method of discontinuous layer 2 is the same as that of discontinuous layer 1.

[0258] (3) Stack 1 continuous surface layer 1, 1 discontinuous surface layer 1, 1 discontinuous surface layer 2 and 1 continuous surface layer 1 from bottom to top, as shown in Figure 7. The angle between the length direction of the bio-based strips of discontinuous surface layer 1 and the length direction of the bio-based strips of discontinuous surface layer 2 is 90°.

[0259] The product is obtained by laminating at 45℃ for 12 hours.

[0260] Example 6

[0261] This example provides a high-capacity antibacterial sustained-release biological patch. Unlike Example 5, in step (3), the angle between the length direction of the bio-based strip of the intermediate cross-section layer 1 and the length direction of the bio-based strip of the interstitial layer 2 is 0°, and the bio-based strip of the interstitial layer 2 falls exactly between the intervals of the bio-based strip of the interstitial layer 1. See Figure 8.

[0262] Example 7

[0263] This example provides a high-capacity antibacterial sustained-release biological patch, which is obtained by encapsulating two antibiotics in a decellularized matrix material.

[0264] All decellularized matrix materials are from Zoruan Medical Technology (Suzhou) Co., Ltd., and are prepared from the submucosa of the small intestine or the basement membrane of the bladder according to the perfusion-pressure difference method described in patent CN106075583B.

[0265] Antibiotics include hydrophobic antibiotics and hydrophilic antibiotics. The hydrophobic antibiotic is rifamycin, which is rifampicin, and the LogP value of rifampicin is 1.09. The hydrophilic antibiotic is minocycline hydrochloride.

[0266] The preparation method of high-capacity antibacterial sustained-release biological patch is as follows:

[0267] Preparation of continuous surface layer 1: Decellularized matrix material C (derived from porcine bladder basement membrane, purchased from Zoran Medical Technology (Suzhou) Co., Ltd.) was laid with 6 ml of water.

[0268] Preparation of continuous surface layer 2: Decellularized matrix material D (derived from porcine small intestinal submucosa, purchased from Zhuoran Medical Technology (Suzhou) Co., Ltd.) was laid with 6 mL of antibiotic solution X (i.e., a suspension of hydrophilic antibiotics).

[0269] Antibiotic solution X is a suspension of a hydrophilic antibiotic.

[0270] In the aqueous solution of the tetracycline / metal ion mixture, the molar ratio of metal ions to tetracycline is 1.7:1.

[0271] Tetracycline is minocycline hydrochloride, and the metal ion is calcium ion.

[0272] The preparation method of antibiotic solution X is as follows: add 0.1g of minocycline hydrochloride to 10mL of water, mix well, and then add 0.0045mol / mL of calcium chloride aqueous solution to make the molar ratio of calcium ions to minocycline hydrochloride in antibiotic solution X 1.7:1. Then add sodium hydroxide aqueous solution to make the pH 7.4, and the solution is obtained.

[0273] Preparation of the discontinuous layer: Acellular matrix material E (derived from the submucosa of porcine small intestine, purchased from Zoran Medical Technology (Suzhou) Co., Ltd.) was laid down using 6 mL of an organic solution containing hydrophobic antibiotics. This process was repeated four times. After lamination, the material was cut into rectangular strips. These strips were then spaced apart and arranged parallel to form the discontinuous layer. The width of each rectangular strip was 3 mm. There was a 1.5 cm gap between every two rectangular strips. The angle between the length and horizontal directions of each rectangular strip in the discontinuous layer was 45°.

[0274] The method for preparing the organic solution of hydrophobic antibiotic is as follows: add the hydrophobic antibiotic to a 90wt% aqueous ethanol solution so that the concentration of the hydrophobic antibiotic in the antibiotic solution Y is 0.01g / mL.

[0275] One continuous surface layer 1, two continuous surface layers 2, one discontinuous surface layer 1 and one continuous surface layer 1 are stacked sequentially from bottom to top. The layers are laminated at 45°C for 12 hours to obtain a high-capacity antibacterial sustained-release biological patch. The cross-sectional structure is shown in Figure 9(a).

[0276] Example 8

[0277] This example provides a high-capacity antibacterial sustained-release biological patch, which is obtained by encapsulating two antibiotics in a decellularized matrix material.

[0278] All decellularized matrix materials are from Zoruan Medical Technology (Suzhou) Co., Ltd., and are prepared from the submucosa of the small intestine or the basement membrane of the bladder according to the perfusion-pressure difference method described in patent CN106075583B.

[0279] Antibiotics include hydrophobic antibiotics and hydrophilic antibiotics. The hydrophobic antibiotic is rifamycin, which is rifampicin, and the LogP value of rifampicin is 1.09. The hydrophilic antibiotic is minocycline hydrochloride.

[0280] The preparation method of high-capacity antibacterial sustained-release biological patch is as follows:

[0281] Preparation of continuous surface layer 1: Decellularized matrix material C (derived from porcine bladder basement membrane, purchased from Zoran Medical Technology (Suzhou) Co., Ltd.) was laid with 6 ml of water.

[0282] Preparation of continuous surface layer 2: Decellularized matrix material D (derived from porcine small intestinal submucosa, purchased from Zhuoran Medical Technology (Suzhou) Co., Ltd.) was laid with 6 mL of antibiotic solution X (i.e., a suspension of hydrophilic antibiotics).

[0283] Antibiotic solution X is a suspension of a hydrophilic antibiotic.

[0284] The preparation method of antibiotic solution X is as follows: add 0.1g of minocycline hydrochloride to 10mL of water, mix well, and then add 0.0045mol / mL of calcium chloride aqueous solution to make the molar ratio of calcium ions to minocycline hydrochloride in antibiotic solution X 1.6:1. Then add sodium hydroxide aqueous solution to make the pH 7.6, and the solution is obtained.

[0285] Tetracycline is minocycline hydrochloride, and the metal ion is calcium ion.

[0286] The preparation method of antibiotic solution X is as follows: add 0.1g of minocycline hydrochloride to 10mL of water, mix well, and continue to add 0.0045mol / mL of calcium chloride aqueous solution so that the molar ratio of calcium ions to minocycline hydrochloride in antibiotic solution X is 1.7:1.

[0287] Preparation of the discontinuous layer: Acellular matrix material E (derived from the submucosa of porcine small intestine, purchased from Zhuoran Medical Technology (Suzhou) Co., Ltd.) was laid using 6 mL of an organic solution containing hydrophobic antibiotics. This process was repeated four times. After lamination, the material was cut into rectangular strips. These strips were spaced apart and arranged parallel to each other to form the discontinuous layer. The width of each rectangular strip was 5 mm. There was a 1.5 cm gap between every two rectangular strips. The angle between the length and horizontal directions of each rectangular strip in the discontinuous layer was 45°.

[0288] The method for preparing the organic solution of hydrophobic antibiotic is as follows: add the hydrophobic antibiotic to a 95wt% aqueous ethanol solution so that the concentration of the hydrophobic antibiotic in the antibiotic solution Y is 0.04g / mL.

[0289] One continuous surface layer 2, one continuous surface layer 2 and one discontinuous surface layer 1 are stacked in sequence as a unit module. Then, one continuous surface layer 1, three unit modules and one continuous surface layer 1 are stacked in sequence from bottom to top. The layers are laminated at 43°C for 12 hours to obtain a high-capacity antibacterial sustained-release biological patch. The cross-sectional structure is shown in Figure 9(b).

[0290] Comparative Example 8

[0291] This example provides a high-capacity antibacterial sustained-release biological patch, which is prepared as follows: One layer of uncrosslinked extracellular matrix (derived from porcine bladder basement membrane, purchased from Zhuoran Medical Technology (Suzhou) Co., Ltd.), seven layers of uncrosslinked extracellular matrix (derived from porcine small intestinal submucosa, purchased from Zhuoran Medical Technology (Suzhou) Co., Ltd.), and one layer of uncrosslinked extracellular matrix (derived from porcine bladder basement membrane, purchased from Zhuoran Medical Technology (Suzhou) Co., Ltd.) are stacked sequentially from bottom to top. After lamination for 24 hours, the material is removed and immersed in antibiotic solution X for 1 hour. After 1 hour, it is immersed in antibiotic solution Y for 1 hour. After 1 hour, the above immersion operation is repeated twice (equivalent to the laminated material undergoing 3 immersions in tetracycline suspension and 3 immersions in rifamycin organic solution). There is a 1-hour interval between each immersion. Finally, the material is dried at room temperature for 24 hours and then placed in a 40°C oven for another 24 hours.

[0292] In this study, antibiotic solution X (a suspension of tetracycline) and antibiotic solution Y (an organic solution of rifamycin) were prepared according to the method in Example 7.

[0293] Comparative Example 9

[0294] This example provides a high-capacity, sustained-release antibacterial biological patch, the preparation method of which is as follows:

[0295] One layer of decellularized matrix material A (derived from porcine bladder basement membrane, purchased from Zhuoran Medical Technology (Suzhou) Co., Ltd.), four layers of decellularized matrix material B (derived from porcine small intestinal submucosa, purchased from Zhuoran Medical Technology (Suzhou) Co., Ltd.), and one layer of decellularized matrix material A (derived from porcine bladder basement membrane, purchased from Zhuoran Medical Technology (Suzhou) Co., Ltd.) were immersed in antibiotic solution X for 2 hours, then removed and immersed in antibiotic solution Y for another 2 hours. After being freeze-dried separately, the six layers of treated material were stacked from bottom to top in the aforementioned order and laminated at 80°C for 18 hours using a laminator before being removed.

[0296] In this study, antibiotic solution X (a suspension of tetracycline) and antibiotic solution Y (an organic solution of rifamycin) were prepared according to the method in Example 7.

[0297] Comparative Example 10

[0298] This example provides a high-capacity antibacterial sustained-release biological patch. Its preparation method differs from that of Comparative Example 9. The six layers of treated material are stacked from bottom to top in the aforementioned order, laminated at 35°C for 24 hours, and then removed.

[0299] Comparative Example 11

[0300] This example provides a high-capacity, sustained-release antibacterial biological patch. Referring to the impregnation preparation method described in patent number CN115006592B, the specific preparation method is as follows:

[0301] Preparation of uncrosslinked extracellular matrix slurry: Uncrosslinked extracellular matrix (derived from the submucosa of the small intestine, Zhuoran Medical Technology (Suzhou) Co., Ltd.) was granulated using a tissue homogenizer with low-temperature ultrasonic homogenization technology. 25g of the granules were taken and added to 20mL of 90wt% aqueous solution of 3-[[(4-methyl-1-piperazinyl)imino]methyl]-rifamycin at 0.01g / mL and 20mL of aqueous solution of minocycline hydrochloride at 0.01g / mL.

[0302] Uncrosslinked extracellular matrix (derived from the bladder basement membrane, Zhuoran Medical Technology (Suzhou) Co., Ltd.) was immersed in uncrosslinked extracellular matrix slurry for 24 hours, and then the composite material was obtained after vacuum freeze-drying and sterilization.

[0303] Comparative Example 12

[0304] This example provides a high-capacity antibacterial sustained-release biological patch, which is obtained by encapsulating an antibiotic in a decellularized matrix material. The preparation method of the high-capacity antibacterial sustained-release biological patch differs from that in Example 5 in that:

[0305] The angle between the length direction of the bio-based strips in discontinuous layer 1 and the length direction of the bio-based strips in discontinuous layer 2 is 0°. At the same time, the bio-based strips of discontinuous layer 1 and discontinuous layer 2 are stacked in an aligned manner and laminated at 45°C for 12 hours, as shown in Figure 10.

[0306] In this example, both discontinuous layer 1 and discontinuous layer 2 have one side that is not in contact with the continuous planar layer.

[0307] Comparative Example 13

[0308] This example provides a high-capacity, sustained-release antibacterial biological patch, the preparation method of which differs from that of Example 7:

[0309] Antibiotic solution X is a suspension of a hydrophilic antibiotic.

[0310] The preparation method of antibiotic solution X is as follows: add 0.1g of minocycline hydrochloride to 10mL of water, mix well, and then add 0.0045mol / mL of calcium chloride aqueous solution to make the molar ratio of calcium ions to minocycline hydrochloride in antibiotic solution X 1.8:1. Then add sodium hydroxide aqueous solution to make the pH 7.3, and the solution is obtained.

[0311] The preparation method of the bio-based strip is as follows: 6 mL of antibiotic solution Y is used to lay decellularized matrix material C (derived from the submucosa of porcine small intestine, purchased from Zhuoran Medical Technology (Suzhou) Co., Ltd.), so that the decellularized matrix material C is flattened. This operation is repeated 4 times. The strip is then laminated using a laminator and cut into rectangular strips with a width of 10 mm.

[0312] The preparation method of antibiotic solution Y is as follows: add hydrophobic antibiotic to 94wt% ethanol aqueous solution so that the concentration of hydrophobic antibiotic in antibiotic solution Y is 0.07g / mL.

[0313] Furthermore, the bio-based strips in the discontinuous layer are uninterrupted.

[0314] Comparative Example 14

[0315] This example provides a high-capacity antibacterial sustained-release biological patch, the preparation method of which differs from that of Example 8: 8 continuous surface layers 2 and 1 discontinuous surface layer 1 are stacked sequentially as a unit module, and then 1 continuous surface layer 1, 4 unit modules and 1 continuous surface layer 1 are stacked sequentially from bottom to top, and laminated at 43°C for 12 hours to obtain the high-capacity antibacterial sustained-release biological patch.

[0316] Example 9

[0317] This example provides a high-capacity antibacterial sustained-release biological patch, which is obtained by encapsulating two antibiotics in a decellularized matrix material.

[0318] All decellularized matrix materials are from Zoruan Medical Technology (Suzhou) Co., Ltd., and are prepared from the submucosa of the small intestine or the basement membrane of the bladder according to the perfusion-pressure difference method described in patent CN106075583B.

[0319] Antibiotics include hydrophobic antibiotics and hydrophilic antibiotics. The hydrophobic antibiotic is rifamycin, which is rifampicin, and the LogP value of rifampicin is 1.09. The hydrophilic antibiotic is minocycline hydrochloride.

[0320] The preparation method of high-capacity antibacterial sustained-release biological patch is as follows:

[0321] Preparation of continuous surface layer 1: Decellularized matrix material C (derived from porcine bladder basement membrane, purchased from Zoran Medical Technology (Suzhou) Co., Ltd.) was laid with 6 ml of water.

[0322] Preparation of continuous surface layer 2: Decellularized matrix material D (derived from porcine small intestinal submucosa, purchased from Zhuoran Medical Technology (Suzhou) Co., Ltd.) was laid with 6 mL of antibiotic solution X (i.e., a suspension of hydrophilic antibiotics).

[0323] Antibiotic solution X is a suspension of a hydrophilic antibiotic.

[0324] In the aqueous solution of the tetracycline / metal ion mixture, the molar ratio of metal ions to tetracycline is 1.7:1.

[0325] Tetracycline is minocycline hydrochloride, and the metal ion is calcium ion.

[0326] The preparation method of antibiotic solution X is as follows: Dissolve 0.12g of minocycline hydrochloride in 15ml of water, then add 300μL of 0.4g / ml calcium chloride solution. This corresponds to a molar ratio of metal ions to tetracycline in antibiotic solution X of 5.3:1. Then, continue to add sodium hydroxide aqueous solution to make the pH 7.4, thus obtaining the solution.

[0327] Preparation of continuous surface layer 3: Decellularized matrix material C (derived from porcine small intestinal submucosa, purchased from Zoran Medical Technology (Suzhou) Co., Ltd.) was laid with 6 ml of water.

[0328] Preparation of the discontinuous layer: Acellular matrix material E (derived from the submucosa of porcine small intestine, purchased from Zhuoran Medical Technology (Suzhou) Co., Ltd.) was laid using 6 mL of an organic solution containing hydrophobic antibiotics. This process was repeated 5 times. After lamination, the material was cut into rectangular strips. These strips were spaced apart and arranged parallel to each other to form the discontinuous layer. The width of each rectangular strip was 10 mm. There was a 1.8 cm gap between every two rectangular strips. The angle between the length and horizontal directions of each rectangular strip in the discontinuous layer was 45°.

[0329] The method for preparing the organic solution of hydrophobic antibiotic is as follows: add the hydrophobic antibiotic to a 92wt% aqueous ethanol solution so that the concentration of the hydrophobic antibiotic in the antibiotic solution Y is 0.06g / mL.

[0330] Two continuous surface layers 1, four continuous surface layers 2, one discontinuous surface layer 1, two continuous surface layers 3, and one continuous surface layer 1 are stacked sequentially from bottom to top. The layers are then laminated at 45°C for 12 hours to obtain a high-capacity antibacterial sustained-release biological patch.

[0331] Example 10

[0332] This example provides a high-capacity antibacterial sustained-release biological patch, as shown in Figures 11 and 12. The high-capacity antibacterial sustained-release biological patch is obtained by encapsulating two antibiotics in a decellularized matrix material. The decellularized matrix material is from Zoran Medical Technology (Suzhou) Co., Ltd., and is prepared from the submucosa of the small intestine or the basement membrane of the bladder according to the perfusion-pressure differential method described in patent CN106075583B.

[0333] Antibiotics include hydrophobic antibiotics and hydrophilic antibiotics. The hydrophobic antibiotic is rifamycin; the hydrophilic antibiotic is minocycline hydrochloride.

[0334] Rifampicin is the same as rifampicin, and the LogP value of rifampicin is 1.09.

[0335] The suspension of the hydrophilic antibiotic was obtained by adding 85 μL of a 0.5 g / mL metal salt solution to 10 mL of a 0.01 g / mL hydrophilic antibiotic aqueous solution, and then adjusting the pH to 7.4 with a 0.1 mol / L sodium hydroxide aqueous solution.

[0336] The metal salt solution is an aqueous solution of calcium chloride. The solution contains metal ions, which are divalent divalent metal cations that are diamagnetic. The metal ion is Ca. 2+ In the preparation of hydrophilic antibiotic suspensions, the molar mass ratio of the added hydrophilic antibiotic to the added metal salt is 1:1.75.

[0337] The organic solution of the hydrophobic antibiotic was prepared by adding the hydrophobic antibiotic to a 91 wt% aqueous ethanol solution; the concentration of the hydrophobic antibiotic in the organic solution was 0.01 g / mL.

[0338] The preparation method of high-capacity antibacterial sustained-release biological patch is as follows:

[0339] Preparation of continuous surface layer 1: Decellularized matrix material C (derived from porcine bladder basement membrane, purchased from Zhuoran Medical Technology (Suzhou) Co., Ltd.) was laid with water.

[0340] Preparation of continuous surface layer 2: Decellularized matrix material D (derived from porcine small intestinal submucosa, purchased from Zoran Medical Technology (Suzhou) Co., Ltd.) was laid with a suspension of hydrophilic antibiotics.

[0341] Preparation of the discontinuous layer: A decellularized matrix material E (derived from the submucosa of porcine small intestine, purchased from Zhuoran Medical Technology (Suzhou) Co., Ltd.) was laid using an organic solution of hydrophobic antibiotics. This process was repeated four times. After lamination, the material was cut into rectangular strips. These strips were then spaced apart and arranged parallel to form the discontinuous layer. The width of each rectangular strip was 5 mm. There was a 1 cm gap between every two rectangular strips. The angle between the length and horizontal directions of each rectangular strip in the discontinuous layer was 45°.

[0342] The following layers are stacked sequentially from bottom to top: one continuous surface layer 1, two continuous surface layers 2, one discontinuous surface layer, one continuous surface layer 2, and one continuous surface layer 1. The layers are then laminated at 45°C for 12 hours to obtain the final product.

[0343] Comparative Example 15

[0344] This example provides a high-capacity antibacterial sustained-release biopatch, which is obtained by encapsulating two antibiotics in a decellularized matrix material. Unlike Example 9, the uncrosslinked extracellular matrix of the anti-infective biopatch has only one source, as detailed below:

[0345] Preparation of the coherent surface layer 1: An uncrosslinked extracellular matrix (derived from the submucosa of the small intestine, Zhuoran Medical Technology (Suzhou) Co., Ltd.) was laid using a suspension of a hydrophilic antibiotic (specifically minocycline hydrochloride). The minocycline hydrochloride suspension was prepared as follows: 0.12 g of minocycline hydrochloride was dissolved in 15 ml of water, and then 200 μL of a 0.5 g / ml calcium chloride solution was added to adjust the pH to 7.5. This corresponds to a molar ratio of metal ions to tetracycline in antibiotic solution X of 3.7:1.

[0346] Preparation of continuous surface layer 2: Uncrosslinked extracellular matrix (derived from the submucosa of the small intestine, Zoran Medical Technology (Suzhou) Co., Ltd.) was laid with water.

[0347] Preparation of discontinuous layers: Uncrosslinked extracellular matrix (derived from the submucosa of the small intestine, Zhuoran Medical Technology (Suzhou) Co., Ltd.) was laid with antibiotic solution Y (an organic solution of hydrophobic antibiotic). After repeated layering 4 times, the layers were laminated and cut into rectangular strips with a width of 10 mm. The rectangular strips were placed in parallel to form a discontinuous layer with a gap between each pair of rectangular strips. The gap between the rectangular strips was 1.8 cm. The rectangular strips were placed in parallel with gaps to form a discontinuous layer.

[0348] The organic solution of the hydrophobic antibiotic was prepared by adding 3-[[(4-methyl-1-piperazinyl)imino]methyl]-rifamycin to an 89 wt% aqueous ethanol solution; the concentration of the hydrophobic antibiotic in the organic solution was 0.04 g / mL.

[0349] (3) After stacking the layers in the order of 1 continuous surface layer 2, 4 continuous surface layers 1, 1 discontinuous surface layer and 3 continuous surface layers 2 from bottom to top, the layers are laminated at 45℃ for 12 hours to obtain the final product.

[0350] Comparative Example 16

[0351] This example provides a high-capacity antibacterial sustained-release biological patch, which is obtained by encapsulating two antibiotics in a decellularized matrix material. Unlike Example 9, the uncrosslinked extracellular matrix of the anti-infective biological patch is derived from the submucosa of the small intestine and the porcine pericardium, as detailed below:

[0352] Preparation of the coherent surface layer 1: An uncrosslinked extracellular matrix (derived from the submucosa of the small intestine, Zhuoran Medical Technology (Suzhou) Co., Ltd.) was laid using a suspension of a hydrophilic antibiotic (specifically minocycline hydrochloride). The minocycline hydrochloride suspension was prepared as follows: 0.1 g of minocycline hydrochloride was dissolved in 20 ml of water, and then 250 μL of a 0.3 g / ml calcium chloride solution was added to adjust the pH to 7.3. This corresponds to a molar ratio of metal ions to tetracycline in antibiotic solution X of 3.3:1.

[0353] Preparation of continuous surface layer 2: Uncrosslinked extracellular matrix (derived from porcine pericardium, Zhuoran Medical Technology (Suzhou) Co., Ltd.) was laid with water.

[0354] Preparation of continuous surface layer 3: Uncrosslinked extracellular matrix (derived from the submucosa of the small intestine, Zhuoran Medical Technology (Suzhou) Co., Ltd.) was laid with water.

[0355] Preparation of discontinuous layers: Uncrosslinked extracellular matrix (derived from the submucosa of the small intestine, Zhuoran Medical Technology (Suzhou) Co., Ltd.) was laid with antibiotic solution Y (an organic solution of hydrophobic antibiotic). After repeated layering 6 times, the matrix was laminated and cut into rectangular strips with a width of 8 mm. The rectangular strips were placed in parallel to form discontinuous layers. There was a gap between every two rectangular strips, with a gap of 1.4 cm between the rectangular strips. The discontinuous layers were formed by the rectangular strips being spaced apart and placed in parallel.

[0356] The organic solution of the hydrophobic antibiotic was prepared by adding 3-[[(4-methyl-1-piperazinyl)imino]methyl]-rifamycin (rifampin) to a 93 wt% aqueous ethanol solution; the concentration of the hydrophobic antibiotic in the organic solution was 0.04 g / mL.

[0357] (3) After lamination, the following layers are stacked in order from bottom to top: 1 continuous surface layer 2, 4 continuous surface layers 1, 1 discontinuous surface layer, 2 continuous surface layers 3 and 1 continuous surface layer 2.

[0358] Comparative Example 17

[0359] This example provides a high-capacity antibacterial sustained-release biological patch, which is obtained by encapsulating two antibiotics in a decellularized matrix material. Unlike Example 9, a solution of antibiotics is used to lay the continuous layer, while a suspension of antibiotics is used to lay the discontinuous layer. The specific method is as follows:

[0360] Preparation of continuous surface layer 1: Uncrosslinked extracellular matrix (derived from the submucosa of the small intestine, Zhuoran Medical Technology (Suzhou) Co., Ltd.) was laid with antibiotic solution Y (an organic solution of hydrophobic antibiotic); the organic solution of hydrophobic antibiotic was prepared by adding 3-[[(4-methyl-1-piperazinyl)imino]methyl]-rifamycin (rifampin) to an 85wt% aqueous ethanol solution; the concentration of hydrophobic antibiotic in the organic solution of hydrophobic antibiotic was 0.03 g / mL.

[0361] Preparation of continuous surface layer 2: Uncrosslinked extracellular matrix (derived from bladder basement membrane, Zoran Medical Technology (Suzhou) Co., Ltd.) was laid with water.

[0362] Preparation of continuous surface layer 3: Uncrosslinked extracellular matrix (derived from the submucosa of the small intestine, Zhuoran Medical Technology (Suzhou) Co., Ltd.) was laid with water.

[0363] Preparation of discontinuous layers: Uncrosslinked extracellular matrix (derived from the submucosa of the small intestine, Zhuoran Medical Technology (Suzhou) Co., Ltd.) was laid with a suspension of hydrophilic antibiotic (specifically minocycline hydrochloride). This process was repeated 6 times. After lamination, the matrix was cut into rectangular strips with a width of 5 mm. The rectangular strips were placed in parallel to form discontinuous layers. There was a gap between every two rectangular strips, with a gap of 1 cm between the rectangular strips. The discontinuous layers were formed by the rectangular strips being placed in parallel with gaps.

[0364] The preparation of a minocycline hydrochloride suspension is as follows: 0.08 g of minocycline hydrochloride is dissolved in 15 ml of water, and then 300 μL of 0.2 g / ml calcium chloride solution is added to adjust the pH to 7.4. This corresponds to a molar ratio of metal ions to tetracycline in antibiotic solution X of 3.3:1.

[0365] (3) After lamination, the following layers are stacked in order from bottom to top: 1 continuous surface layer 2, 4 continuous surface layers 1, 1 discontinuous surface layer, 2 continuous surface layers 3 and 1 continuous surface layer 2.

[0366] Comparative Example 18

[0367] This example provides a high-capacity antibacterial sustained-release biological patch, which is obtained by encapsulating two antibiotics in a decellularized matrix material. Unlike Example 10, both encapsulated antibiotics are hydrophilic antibiotics. The hydrophilic antibiotics are minocycline hydrochloride and doxycycline hydrochloride.

[0368] The suspension of minocycline hydrochloride was obtained by adding 100 μL of a 0.45 g / mL metal salt solution to 10 mL of a 0.015 g / mL minocycline hydrochloride aqueous solution, and then adjusting the pH to 7.5 with a 0.1 mol / L sodium hydroxide aqueous solution.

[0369] The metal salt solution is an aqueous solution of calcium chloride. The solution contains metal ions, which are divalent divalent metal cations that are diamagnetic. The metal ion is Ca. 2+ In the preparation of the minocycline hydrochloride suspension, the molar mass ratio of minocycline hydrochloride to the added metal salt is 1:1.33.

[0370] Doxycycline hydrochloride is obtained by adding 100 μL of 0.45 g / mL calcium chloride aqueous solution to 10 mL of 0.015 g / mL doxycycline hydrochloride aqueous solution, and then adjusting the pH to 7.5 with 0.1 mol / L sodium hydroxide aqueous solution.

[0371] The preparation method of high-capacity antibacterial sustained-release biological patch is as follows:

[0372] Preparation of continuous surface layer 1: Decellularized matrix material A (derived from bladder basement membrane) was laid with water.

[0373] Preparation of continuous surface layer 2: Decellularized matrix material B (derived from the submucosa of the small intestine) was laid with a suspension of minocycline hydrochloride.

[0374] Preparation of the discontinuous layer: Decellularized matrix material C (derived from the submucosa of the small intestine) was laid using doxycycline hydrochloride suspension. This process was repeated four times. After lamination, the material was cut into rectangular strips. These strips were then spaced apart and arranged parallel to form the discontinuous layer. The width of each rectangular strip was 7 mm. There was a 1.3 cm gap between every two rectangular strips. The angle between the length direction and the horizontal direction of each rectangular strip in the discontinuous layer was 45°.

[0375] The following layers are stacked sequentially from bottom to top: one continuous surface layer 1, two continuous surface layers 2, one discontinuous surface layer, one continuous surface layer 2, and one continuous surface layer 1. The layers are then laminated at 45°C for 12 hours to obtain the final product.

[0376] Comparative Example 19

[0377] This example provides a high-capacity, sustained-release antibacterial biological patch. Unlike Example 10, the hydrophilic antibiotic suspension is prepared by adding 100 μL of a 0.5 g / mL metal salt solution to 10 mL of a 0.01 g / mL hydrophilic antibiotic aqueous solution, followed by adjusting the pH to 7.6 with a 0.1 mol / L sodium hydroxide aqueous solution. During the preparation of the hydrophilic antibiotic suspension, the molar ratio of the added hydrophilic antibiotic to the added metal salt is 1:2.

[0378] Test Results

[0379] 1. The thickness, weight per unit area, and fat content of the second membrane layer in the biological patches obtained in Examples 1-3 and Comparative Examples 1-7 were tested. The weight per unit area ratio and fat content ratio of the first and second membrane layers were calculated, and the results are shown in Table 2. Only lipids in the biological patches are soluble in ether. The fat content was tested by taking a 0.5g sample and determining the fat content according to the method for determining soluble matter in ether in YY / T 0330-2015.

[0380] Table 2

[0381]

[0382] 2. The biological patches obtained in Examples 1-3 and Comparative Examples 1-7 were immersed in a simulated physiological environment for 1 hour. Specifically, the simulated physiological environment was 500 mL of a mixed solution of 140 mmol sodium chloride solution and 2.5 mmol calcium chloride solution. Tensile strength (refer to GB / T328.8-2007) and peel strength (YY / T 0729.2-2009 Test Methods for Adhesive Properties of Tissue Adhesives Part 2: T-Peel Tensile Bearing Strength Test) were tested. This constitutes one set of data. The biological patches obtained in Examples 1-3 and Comparative Examples 1-7 were immersed in the simulated physiological environment for two weeks, and their tensile strength and peel strength were tested. This constitutes two sets of data. The biological patches obtained in Examples 1-3 and Comparative Examples 1-7 were immersed in the simulated physiological environment for two weeks, and a peel force of 0.3 N was continuously applied to the biological patches during these two weeks. After two weeks, the biological patches were removed, and their tensile strength and peel strength were tested. These are three sets of data; the biological patches obtained in Examples 1-3 and Comparative Examples 1-7 were soaked in a simulated physiological environment for 1 hour, then pressurized to 3 mmHg for 2 hours, then depressurized and left to stand for 1 hour, then pressurized again to 3 mmHg for 2 hours, then depressurized and left to stand for 1 hour, in order to simulate the environment of skin pressure and tension, and to test tensile strength and peel strength. These are four sets of data. The changes in tensile strength and peel strength of the second, third and fourth sets of data relative to the first set of data were calculated. The results are shown in Tables 3, 4 and 5.

[0383] Table 3

[0384]

[0385] Table 4

[0386]

[0387] Table 5

[0388]

[0389] 3. The biological patch obtained in Example 3 was implanted into the peritoneum of rats and fixed to the peritoneum. Seven days after the operation, scanning electron microscopy was performed. The electron microscopic image of the surface mesothelial cells is shown in Figure 2. It can be observed that one week after the biological patch was implanted in the peritoneum, a layer of mesothelial cells grew on the surface of the patch. The microvilli on the cell surface are a characteristic of peritoneal mesothelial cells and are also the basis for the regeneration of peritoneum to prevent adhesion.

[0390] 4. Animal model experiments:

[0391] The biological patches obtained in Examples 1, 3 and Comparative Examples 3, 4 were implanted into rat pelvic muscle partial layer defect models. After 14 days of implantation, the rats were sacrificed, and the implanted materials and surrounding tissues were removed, fixed by soaking in paraformaldehyde, embedded, sectioned, and stained with hematoxylin and eosin (HE) for pathological histological examination to evaluate the tissue ingrowth of various materials. The pathological results are shown in Figures 3-6.

[0392] The pathological results show that, in Figure 3, a certain number of cells have grown into the material, a small number of new blood vessels, and a very small amount of disordered new collagen deposition; in Figure 4, the material is completely integrated with the surrounding tissue, with a large number of cells growing into the material and at various levels, obvious angiogenesis, and the beginning of secretion and deposition of new collagen, initiating tissue remodeling, and the tissue ingrowth is the most significant; in Figure 5, a very small number of cells have grown into some layers of the material, a very small number of new blood vessels, and no new collagen deposition; in Figure 6, the implanted material is intact, with no cells or blood vessels growing into the material, no new collagen deposition, and poor tissue ingrowth effect.

[0393] 5. Mechanical property testing of drug-loaded biological patches: The patch samples obtained in Examples 4-8 and Comparative Examples 8-14 were completely immersed in PBS buffer and placed at 22±2℃ for 6 hours. After that, they were removed and the tensile strength was tested according to GB / T328.8-2007 (stretched at a stable speed of 100 mm / min until fracture, and the maximum tensile force was recorded). The tensile strength was tested according to YY / T... Peel strength was tested from 0729.2-2009. Five parallel tests were performed for each test, and the average value was taken. The results are shown in Table 6. Following the same process, nine parallel experiments were conducted again for Examples 4-8 and Comparative Examples 8-14. The average deviation of tensile strength and peel strength for each of the ten experiments was calculated (average deviation is calculated as the sum of the absolute values ​​of the deviations of individual measurements from the average value, divided by the number of measurements). The smaller the average deviation, the more stable the process of the sample. The results showed that the average deviations of Examples 7 and 8 were close, indicating that for this scheme, increasing the number of layers did not negatively affect process stability. However, the average deviation of Comparative Example 14 was much greater than that of Examples 7-8, indicating that the process stability of Comparative Example 14 was poor. In other words, in this technical scheme, having too many layers of decellularized matrix material laid with antibiotic solution X will negatively affect process stability.

[0394] Table 6

[0395]

[0396] The tensile strength of the drug-loaded sample patches obtained in Examples 4-8 is all >10 N / cm, and the peel strength is >0.7 N / cm, reflecting that the samples in Examples 4-8 have good mechanical properties and a structure that is not easily loosened when exposed to water. Among them, compared with Example 7, the patch sample obtained in Example 8 has increased tensile properties due to the increase in the number of layers. At the same time, the increase in the number of layers also slightly reduces the interlayer bonding force (slightly reduces the peel strength).

[0397] In Comparative Examples 8-11, no medium was used for laying; instead, traditional methods were employed to load the drug onto the material. Comparative Example 8 involved impregnating the entire pressed material. However, theoretically, this method only loads the drug onto the material surface, essentially creating a drug coating, making it difficult to achieve a high drug loading capacity. Furthermore, it's evident that Comparative Example 8 exhibits very poor mechanical properties. Additionally, in the experiments of Comparative Examples 9-11, the inventors observed that the edge thickness of the resulting product was significantly greater than the thickness in the center. This is likely due to shrinkage or curling of the material after impregnation, particularly noticeable curling at the edges, resulting in significant unevenness in the overall thickness of the material after lamination. This significant thickness unevenness was not observed in Examples 4-8, Comparative Example 8, and Comparative Examples 12-14.

[0398] Compared with Example 5, the bio-based strips of discontinuous layer 1 and discontinuous layer 2 in Comparative Example 12, when stacked with the two layers aligned, show a significant decrease in mechanical properties. Not only does the peel strength (reaction interlayer bonding force) decrease by 44%, but the tensile strength also decreases by 29%.

[0399] Compared to Example 7, the bio-based strips in the discontinuous layer of the sample in Comparative Example 13 were uninterrupted. Test results showed that although the mechanical properties of the samples in Comparative Example 13 and Example 7 were similar, subsequent tests on drug loss, the proportion of minocycline hydrochloride epimers, and drug release rate revealed that the sample in Comparative Example 13 exhibited higher drug loss and a faster drug release rate, especially with the hydrophobic antibiotic being almost completely released within 48 hours. Therefore, the sample in Comparative Example 13 is not suitable for practical application.

[0400] Compared to Example 8, the number of layers of decellularized matrix material laid with antibiotic solution X (i.e., a suspension of hydrophilic antibiotics) in Comparative Example 14 was significantly increased. This increase in the number of layers did not significantly increase the tensile strength; the increase was only slight (7%). Simultaneously, the significant increase in the number of layers of decellularized matrix material laid with antibiotic solution X resulted in a significant decrease in peel strength (reflecting interlayer bonding force) (a decrease of 37%). Furthermore, subsequent tests showed that the ratio of transverse tensile strength to longitudinal tensile strength of Comparative Example 14 was 1:1.36. This indicates that the increased number of layers of decellularized matrix material laid with antibiotic solution X significantly reduced the mechanical uniformity of the drug-loaded patch. In conclusion, the sample of Comparative Example 14 is not suitable for practical application.

[0401] To verify the mechanical uniformity of the drug-loaded patch, further tensile strength tests were conducted in the horizontal and vertical directions, and the ratio of transverse to longitudinal tensile strength was calculated. Samples from Examples 8, 10, and Comparative Examples 18-19 were completely immersed in PBS buffer and placed in a closed space at 37±2℃ under a CO2 gas atmosphere of 12-15 kPa for 6 hours. The tensile strength in the horizontal and vertical directions was then tested according to GB / T328.8-2007 (stretched at a stable speed of 100 mm / min until fracture, and the maximum tensile force was recorded). The ratio of transverse to longitudinal tensile strength was calculated. Ten parallel experiments were performed on each sample, and the average value was taken. The results are shown in Table 7. Furthermore, the standard deviation of the ratio of transverse to longitudinal tensile strength was calculated. A smaller standard deviation indicates more stable mechanical properties of the sample. The results showed: Example 10 > Example 8 >> Comparative Example 19 > Comparative Example 18.

[0402] Table 7

[0403]

[0404] As shown in Table 7, the transverse tensile strength and longitudinal tensile strength of Examples 8 and 10 are close to 1:1, and both have a small standard deviation of the ratio of transverse tensile strength to longitudinal tensile strength. This indicates that they have good mechanical uniformity and mechanical stability, and are not easily affected by environmental factors such as pressure and swelling, which can cause the patch performance to become unstable.

[0405] The sample of Example 10 was loaded with one hydrophilic antibiotic and one hydrophobic antibiotic. Unlike Example 10, the sample of Comparative Example 18 was loaded with two hydrophilic antibiotics. Although the transverse tensile strength and longitudinal tensile strength of the sample of Comparative Example 18 were close to 1:1, the standard deviation of the ratio of its transverse tensile strength to its longitudinal tensile strength was large, indicating that the patch performance may have become unstable due to environmental factors such as pressure and swelling.

[0406] In the preparation process of Example 10, the molar mass ratio of the hydrophilic antibiotic to the added metal salt was 1:1.75. Unlike Example 10, the molar mass ratio of the hydrophilic antibiotic to the added metal salt in Comparative Example 19 was 1:2. This reflects that if the molar mass ratio of the hydrophilic antibiotic to the added metal salt is 1:2 during the preparation of the drug-loaded patch, it is very likely to be detrimental to the mechanical uniformity of the patch.

[0407] Furthermore, to determine the overall compressive strength and edge fixation reliability of the drug-loaded patch during application, ensuring its adaptability to the complex mechanical environment in vivo, the samples obtained in Example 9 and Comparative Examples 15-17 were prepared into specimens of 50mm × 50mm and 25mm width, respectively. After immersion in water for 5 minutes, the bursting strength (tested according to GB / T 19976-2005, using a 20mm diameter spherical probe) and suture strength were tested. The suture strength test method was as follows: a surgical suture was used to suture the patch 5 mm from the edge of the sample. The suture was fixed to the other end of the product on a tensile tester and stretched at a speed of 100 mm / min until the suture point was torn. The tensile force at which the suture point was torn was recorded as the suture strength. The results are shown in Table 8.

[0408] Table 8

[0409]

[0410] In Example 9, the uncrosslinked extracellular matrix in the drug-loaded patch had two sources: SIS and UBM. Unlike Example 9, the uncrosslinked extracellular matrix in Comparative Example 15 had only one source (SIS). The results showed that the puncture strength and suture strength of Comparative Example 15 were significantly reduced, indicating the necessity of having two sources of uncrosslinked extracellular matrix in the drug-loaded patch. Unlike Example 9, the uncrosslinked extracellular matrix in Comparative Example 16 consisted of SIS and pericardium. The results showed that the puncture strength of Comparative Example 16 was slightly reduced, while the suture strength was significantly reduced, indicating the advantage of including at least UBM in the drug-loaded patch.

[0411] 6. Testing of encapsulated drug content in high-capsulation antibacterial sustained-release biological patches: Samples obtained from Examples 5, 7-10, Comparative Examples 8-13, and 15-17 were cut into 5cm × 5cm pieces and extracted in methanol. Each 24-minute extraction was performed. The extract was sampled and tested using liquid chromatography until it became colorless. The drug loading was calculated based on the standard curve (rifampin and minocycline hydrochloride standards were dissolved in methanol and diluted to prepare a series of standard solutions ranging from 1-500 µg / mL, then tested using liquid chromatography to obtain the standard curve). The drug loss after the patch preparation process was calculated based on the amount of raw materials added in the process, and the results are shown in Table 9. In the liquid chromatogram, under the same conditions, minocycline hydrochloride and its diastereomer had different retention times and peak positions. Based on the peak area ratio, the proportion of the minocycline hydrochloride diastereomer in the mixture of minocycline hydrochloride and its diastereomer in Examples 7-8 and Comparative Example 13 was calculated, and the results are shown in Table 10. Each test was performed in 5 parallel experiments, and the average value was taken.

[0412] Table 9

[0413]

[0414] Table 10

[0415]

[0416] The average deviation of drug loading in the samples of Examples 7-8, Example 10 and Comparative Examples 18-19 was calculated, and the results are shown in Table 11.

[0417] Table 11

[0418]

[0419] As shown in Tables 9 and 11, the technical solutions of this invention in Examples 7-8 and Example 10 achieve the adjustment of high drug content in the biological patch. This indicates that even with an increase in the number of layers, the drug content of the resulting product remains relatively stable in this technical solution.

[0420] The average deviation of the hydrophobic antibiotic loading in the sample obtained in Comparative Example 19 was high (greater than 20), which indicates that when the molar mass ratio of hydrophilic antibiotic to added metal salt is 1:2, it will have a certain negative impact on the stability of the hydrophobic antibiotic in the prepared drug-loaded patch.

[0421] 7. Drug release rate test of high-capacity antibacterial sustained-release biological patch: After gas anesthesia, the hair on the abdomen of rats was shaved, and a 5cm long incision was made in the midline of the abdomen to fully expose the abdominal cavity. The samples obtained in Examples 7, 9-10, 8-13, and 15-18 were cut into 2cm×2cm samples and placed on both sides of the abdominal cavity of the rats. One identical sample was placed on the abdominal wall and fixed with sutures. After implantation, the wound was closed. Postoperatively, samples were collected at different time points. The patients were then euthanized by inhalation of CO2. After euthanasia, the abdominal cavity was opened, and the morphology of the implantation sites (including adhesion and granulation tissue growth) was observed in some samples. The samples were then removed and extracted with PBS buffer for 24 hours. The drug loading was calculated based on the drug standard curve and denoted as W (standards of rifampin, minocycline hydrochloride, doxycycline hydrochloride, and clarithromycin were dissolved in PBS buffer and diluted to prepare a series of standard solutions ranging from 1-500 µg / mL; the standard curve was obtained by liquid chromatography). The in vivo release rate was calculated as (drug loading of each sample in performance test 6 - W) / drug loading of each sample in performance test 6 × 100%. The results are shown in Tables 12-14.

[0422] Table 12

[0423]

[0424] Table 13

[0425]

[0426] Table 14

[0427]

[0428] In Table 12-14, "none" indicates that the drug content on the sample patch is basically undetectable, which is equivalent to the drug loading of the sample patch being 0 at this time.

[0429] 8. Antibacterial rate test: Staphylococcus aureus, Escherichia coli, Staphylococcus epidermidis, and Pseudomonas aeruginosa were prepared as test strains. After activation, single colonies were picked and mixed in sterile physiological saline to prepare a 102... 6For the bacterial suspension at CFU / mL, sample patches of Example 9 and Comparative Examples 15-17, measuring 2 cm × 3 cm, were placed in test tubes. 9.9 mL of MH medium and 0.1 mL of bacterial suspension were added. The blank control group received no patch. The test tubes were incubated at 37℃ and 180 rpm for 1, 2, 3, and 4 h. The cultures were then serially diluted 10-fold. 0.1 mL of each dilution was spread onto TSA plates and incubated at 35℃ for 24 h. Colony counts were recorded. The same dilution was tested three times. A bactericidal curve was plotted with time on the x-axis and the logarithm of colony forming units (CFU) on the y-axis. The inhibition rate at 4 h was recorded. Inhibition rate (%) = (colony count in the blank control group - colony count in the patch group) / colony count in the blank control group × 100%. The results are shown in Table 15.

[0430] Table 15

[0431]

[0432] 9. Morphological characterization: The sample from Example 7 was characterized by scanning electron microscopy (SEM). The results are shown in Figure 13. As can be seen from Figure 13, the antibacterial drug crystals are distributed in the internal network and surface of the patch structure.

[0433] 10. Animal Model Validation: Animal experiments were conducted using the drug-loaded patch sample obtained in Example 9. Forty 6-month-old sows weighing approximately 25 kg were used to construct an animal model of intra-abdominal infection caused by cecal ligation and puncture (CLP) combined with abdominal restraint device-assisted intra-abdominal hypertension (IAH). After modeling, the abdominal cavity was reopened along the original incision, decompression was performed, the cecum was removed, and the stump was buried to expose the everted intestinal mucosa of the intestinal fistula. After debridement and drainage, the abdominal cavity was opened using negative pressure assisted temporary abdominal closure. During the treatment, the drug-loaded patch sample obtained in Example 9 was used to cover the intestinal mucosa for temporary abdominal closure. It was observed that no intra-abdominal hemorrhage or adhesions occurred during the period. The definitive abdominal closure was performed three days later. The previously placed anti-infective biological patch was not removed during the definitive abdominal closure, and the fascial closure rate was 100%. After the surgery, the animals continued to be fed. Ten days later, only one case of intestinal air fistula was observed. No incisional hernias occurred during postoperative follow-up. In other words, the incidence of intestinal air fistula was 2.5% (1 / 40) when the drug-loaded patch was used as a temporary abdominal closure material.

Claims

1. A biomembrane for repairing abdominal and pelvic wounds, the biomembrane comprising a first membrane layer and a second membrane layer, characterized in that, The thickness of the first membrane layer is 0.1-1 mm, and the thickness of the second membrane layer is 0.02-0.4 mm; the weight per unit area of ​​the first membrane layer is 1.2-5 times that of the second membrane layer, and the interlayer bonding ratio of the first and second membrane layers is 35%-75%; the ratio of fat content in the first membrane layer to fat content in the second membrane layer is 1-5; the first membrane layer includes decellularized matrix A and decellularized matrix B, wherein decellularized matrix A is derived from pig bladder, and decellularized matrix B is derived from at least one of pig small intestine, pig heart, goat lung, and goat kidney; the second membrane layer is obtained by modifying the basement membrane of pig bladder.

2. The biofilm for repairing abdominal and pelvic wounds according to claim 1, characterized in that, The modification process includes the following steps: S1. rinsing; S2. pretreatment; S3. soaking to obtain a second film layer.

3. The biofilm for repairing abdominal and pelvic wounds according to claim 2, characterized in that, In step S2, a pretreatment agent is used, which includes esterified amino acids and neutral liposomes, with a mass ratio of esterified amino acids to neutral liposomes of 1:(0.1-0.5).

4. The biofilm for repairing abdominal and pelvic wounds according to claim 3, characterized in that, The neutral liposomes are selected from one or more of sphingomyelin, animal-derived phosphatidylcholine, and plant-derived phosphatidylcholine.

5. The biofilm for repairing abdominal and pelvic wounds according to claim 2, characterized in that, Step S3 specifically involves immersing the pretreated material from step S2 in 0.15-2.2% of an active substance for 130-150 minutes; the active substance is selected from one of hyaluronic acid, polycystic acid, or chondroitin sulfate.

6. A method for preparing a biofilm for repairing abdominal and pelvic wounds according to claim 1, characterized in that, The preparation method of the biofilm used to repair abdominal and pelvic wounds is as follows: freeze-dry the first membrane layer and the second membrane layer respectively, and then vacuum laminate them to obtain the biofilm.

7. A high-capacity, sustained-release antibacterial biological patch, characterized in that, The high-capacity antibacterial sustained-release biological patch is obtained by encapsulating at least two antibiotics in a decellularized matrix material; the antibiotics include hydrophobic antibiotics and hydrophilic antibiotics, the hydrophobic antibiotics being selected from one or more of beta-lactam antibiotics, rifamycin, and macrolide antibiotics; the hydrophilic antibiotics are tetracycline antibiotics. The hydrophobic antibiotic encapsulation dosage in high-capsulation sustained-release antibacterial biological patches is 150-600 μg / cm³. 2 The encapsulation dosage of hydrophilic antibiotics in high-capsulation sustained-release antibacterial biological patches is 150-600 μg / cm³. 2 .

8. The high-capsulation, sustained-release antibacterial biological patch according to claim 7, characterized in that, The decellularized matrix material includes the biomembrane described in claim 1.

9. The high-capsulation, sustained-release antibacterial biological patch according to claim 7 or 8, characterized in that, The high-capacity antibacterial sustained-release biological patch comprises a biological membrane as described in claim 1, which encapsulates at least two antibiotics.

10. The high-capsulation, sustained-release antibacterial biological patch according to claim 8, characterized in that, The high-capacity antibacterial sustained-release biological patch comprises at least two continuous surface layers and at least one discontinuous surface layer.

11. The use of a biological patch as described in claim 1 or 7 in the preparation of a material for abdominal and pelvic cavity repair.

12. The application of the high-capsulation antibacterial sustained-release biological patch as described in claim 7 in the preparation of anti-infective materials.

13. The application of a high-capsulation, antibacterial, sustained-release biological patch as described in claim 7 in the preparation of a temporary abdominal closure material for open abdominal cavity.

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