High-packaging-dose antibacterial slow-release biological patch

Abstract

The invention relates to the field of new biological materials, in particular to a high-packaging-dose antibacterial slow-release biological patch obtained by packaging at least two antibiotics with an acellular matrix material. The acellular matrix material is derived from any two of small intestinal submucosa, peritoneum, dermis, bladder basement membrane and pericardium; the antibiotics comprise hydrophobic antibiotics and hydrophilic antibiotics, and the hydrophobic antibiotics are selected from one or more of Beta-lactam antibiotics, rifamycin and macrolide antibiotics; the hydrophilic antibiotics are tetracycline antibiotics. The coating-free drug-loaded sustained-release system based on a lamination process kills bacteria in a short period, prevents colonization and reproduction of bacteria in a wound / patch, realizes synergistic and continuous release of antibiotics along with a wound healing process, guides tissue regeneration and synchronously realizes degradation, is applied to a high-incidence infection period of a surgical operation, reduces adhesion, and has a good application prospect. The abdominal wall strength is enhanced, and the growth of wound granulation tissues is promoted.

Description

Technical Field

[0001] The invention relates to the field of new biological materials, and in particular to an antibacterial sustained-release biological patch with a high encapsulated drug amount. Background Art

[0002] In the field of medical devices, biomaterials have unique advantages over synthetic polymer materials. Biomaterials are usually degradable and have lower immunogenicity. As biomaterials, decellularized matrix materials not only have high biocompatibility, but also retain the basic structure of organs, thus better adapting to the dynamic internal environment of cells. However, biomaterials, especially decellularized matrices, degrade faster than polymer materials, and the release rate of encapsulated drugs is usually much faster than that of polymer materials. This leads to the situation that the drug has been released before the therapeutic effect is achieved in clinical applications.

[0003] Patent number CN101810883B discloses a bio-derived material with high tissue compatibility and long-term anti-infection. The polylactic acid-polyglycolic acid copolymer microspheres loaded with the antibacterial agent triclosan are prepared by ultrasonic multiple emulsification-solvent evaporation method, and then the cross-linked decellularized matrix is ​​immersed in the polylactic acid microsphere aqueous solution, which achieves the long-term release of the antibacterial agent in a few weeks or months to a certain extent. It can be understood that in order to achieve long-term release, polymer materials are added to the system; at the same time, in order to delay the release, the system uses a cross-linked decellularized matrix. The higher the degree of cross-linking, the slower the material itself degrades. Summary of the invention

[0004] In order to obtain a drug-loaded antibacterial patch that can be used in the high-infection period of surgical operations, and the patch also has good softness and toughness, the present invention provides a high-encapsulated antibacterial sustained-release biological patch, wherein the high-encapsulated antibacterial sustained-release biological patch is obtained by encapsulating at least two antibiotics in a decellular matrix material; The decellularized matrix material includes decellularized matrix material A, decellularized matrix material B and decellularized matrix material C, and the decellularized matrix material is derived from any two of the following: small intestinal submucosa, peritoneum, dermis, bladder basement membrane and pericardium; In the present invention, the acellular matrix materials are all purchased from Zhuoruan Medical Technology (Suzhou) Co., Ltd., and are prepared from small intestinal submucosa, peritoneum, dermis, bladder basement membrane or pericardium according to the perfusion-pressure difference method disclosed in patent CN106075583B.

[0005] The antibiotics include hydrophobic antibiotics and hydrophilic antibiotics. The hydrophobic antibiotics are selected from one or more of Beta-lactam antibiotics, rifamycin, and macrolide antibiotics. The hydrophilic antibiotics are tetracycline antibiotics. The encapsulated drug amount of the hydrophobic antibiotics in the high-encapsulated drug amount antibacterial sustained-release biological patch is 150-600 μg / cm 2 The encapsulated drug amount of hydrophilic antibiotics in high encapsulated drug amount antibacterial sustained release biological patch is 150-600μg / cm 2 ; The ratio of the transverse tensile strength to the longitudinal tensile strength of the high encapsulated drug amount antibacterial sustained-release biological patch is 1: (0.8-1.25).

[0006] In the present invention, while ensuring a high drug encapsulation amount, in order to avoid the problem of overlapping efficacy of at least two encapsulated antibiotics and local increase in drug toxicity, the encapsulated drug amounts of hydrophobic antibiotics and hydrophilic antibiotics in the patch should not be too high or too low. The inventors also found that at the same time, if the hydrophilic antibiotic is a tetracycline antibiotic, the encapsulated drug amount of the hydrophilic antibiotic is 150-600 μg / cm 2 The encapsulated amount of hydrophobic antibiotics is 150-600μg / cm 2 It is not only conducive to avoiding the phenomenon of increased toxicity caused by the aforementioned antibiotics, but also overcomes the technical resistance of tetracycline being quickly dissolved in water and released due to its strong hydrophilicity, slowing down the release rate of tetracycline in the patch of the present invention when it is used, so that the release rate of tetracycline in the patch within 24 hours is controlled within 52%. This slowing effect is particularly obvious when the decellularized matrix material is derived from the submucosa of the small intestine and the basement membrane of the bladder.

[0007] Preferably, the encapsulated amount of hydrophobic antibiotics in the high encapsulated amount antibacterial sustained-release biological patch is 150-570 μg / cm 2 The encapsulated drug amount of hydrophilic antibiotics in high encapsulated drug amount antibacterial sustained-release biological patches is 150-570μg / cm 2 .

[0008] Preferably, the Beta-lactam antibiotic is selected from one or more of tazobactam, clindamycin and moxifloxacin.

[0009] More preferably, the Beta-lactam antibiotic is clindamycin.

[0010] Preferably, the rifamycin is selected from one or more of rifampicin, rifapentine and rifabutin.

[0011] More preferably, the rifamycin is rifampicin.

[0012] Preferably, the macrolide antibiotic is azithromycin and / or midecamycin.

[0013] Preferably, the LogP value of the hydrophobic antibiotic is 0.8-2.1.

[0014] The LogP value is used to indicate the distribution of a substance in the water and oil phases. The larger the LogP value, the higher the lipophilicity, that is, the higher the hydrophobicity.

[0015] Preferably, the tetracycline antibiotic is selected from one or more of minocycline hydrochloride, doxycycline hydrochloride, and tigecycline.

[0016] Preferably, the tetracycline antibiotic is minocycline hydrochloride.

[0017] As a preferred embodiment, the preparation method of the high encapsulated drug amount antibacterial sustained-release biological patch is: (1) Preparation of a continuous surface layer: laying acellular matrix material A and / or acellular matrix material B; (2) Preparation of the intermittent surface layer: Lay the decellularized matrix material C, and continue to repeat the laying for 2-10 times, and then cut it into rectangular strips through a molding process. The rectangular strips are placed in parallel to form the intermittent surface layer; (3) After the continuous surface layer and the discontinuous surface layer are superimposed and subjected to a molding process, the result is obtained.

[0018] Preferably, in step (2), the width of the rectangular strip is 5-50 mm. For example, the width of the rectangular strip is 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, 50 mm, etc.

[0019] Preferably, in step (2), there is a gap between every two rectangular strips, and the gap between the rectangular strips is 0.2-1.9 cm.

[0020] In step (2), the length of the rectangular strip is ≤ the length of the maximum diagonal of the continuous surface layer.

[0021] Preferably, in step (2), the rectangular strips are spaced apart and placed in parallel to form a discontinuous surface layer; the angle between the length direction and the horizontal direction of each rectangular strip of the discontinuous surface layer is 30-60°.

[0022] Preferably, the number of rectangular strips in each discontinuous surface layer is at least 2.

[0023] Preferably, the molding processes in step (2) and step (3) are selected from one or more of lamination, freeze-drying, bonding and suturing.

[0024] The purpose of the forming process is to make the laid or superimposed materials become a whole.

[0025] As a preferred embodiment, in the step (1), when preparing the continuous surface layer, the decellularized matrix material A and / or the decellularized matrix material B are laid using water and / or a suspension of a hydrophilic antibiotic.

[0026] Preferably, in the step (1), when preparing the continuous surface layer, the decellularized matrix material A and / or the decellularized matrix material B are laid using water or a suspension of a hydrophilic antibiotic.

[0027] As a preferred embodiment, in the step (2), when preparing the intermittent surface layer, an organic solution of a hydrophobic antibiotic is used for laying.

[0028] The specific method of laying with water and / or a suspension of hydrophilic antibiotics is as follows: placing a substrate flat, wetting the substrate with a suspension of water and / or a suspension of hydrophilic antibiotics, laying the decellularized matrix material A and / or the decellularized matrix material B on the substrate, flattening the decellularized matrix material on the substrate, and then scraping off excess water and / or the suspension of hydrophilic antibiotics on the surface of the decellularized matrix material with a scraper.

[0029] The purpose of wetting the substrate is to exclude air between the decellularized matrix material and the substrate, and to utilize the adhesion of water and / or hydrophilic antibiotics to the decellularized matrix material to make the decellularized matrix material flatter on the substrate.

[0030] The material of the substrate is not limited. The substrate is only used to provide an operating plane for the base layer. It can be a commercially available glass substrate, steel plate, copper plate, etc.

[0031] The operation of paving with an organic solution of a hydrophobic antibiotic is similar to the above-mentioned paving with water and / or a suspension of a hydrophilic antibiotic, and the principle is the same.

[0032] Furthermore, in step (1), the decellularized matrix material A can be laid with water to obtain a continuous surface layer, the decellularized matrix material A can be laid with a suspension of hydrophilic antibiotics to obtain a continuous surface layer, the decellularized matrix material B can be laid with water to obtain a continuous surface layer, the decellularized matrix material B can be laid with a suspension of hydrophilic antibiotics to obtain a continuous surface layer, etc.

[0033] As a preferred embodiment, both the upper and lower surfaces of the discontinuous surface layer have contact areas with the continuous surface layer.

[0034] Preferably, the contact area is 50%-100% of the area of ​​the intermittent surface layer.

[0035] In step (3) of the method for preparing a high-encapsulated drug-dose antibacterial sustained-release biological patch, the number of layers of the continuous surface layer and the discontinuous surface layer and the stacking order are not limited. For example, from bottom to top, the sequential combination is 1 continuous surface layer, 1 discontinuous surface layer and 1 continuous surface layer; for example, from bottom to top, the sequential combination is 8 continuous surface layers, 2 discontinuous surface layers and 6 continuous surface layers; for example, from bottom to top, the sequential combination is 1 continuous surface layer, 1 discontinuous surface layer, 4 continuous surface layers, 1 discontinuous surface layer and 1 continuous surface layer; for example, from bottom to top, the sequential combination is 2 continuous surface layers, 2 discontinuous surface layers, 4 continuous surface layers, 1 discontinuous surface layer and 1 continuous surface layer, etc. Although the stacking order of the continuous surface layer and the discontinuous surface layer is not limited, in order to ensure that the upper and lower surfaces of the discontinuous surface layer have contact areas with the continuous surface layer, the outermost layer of the high-encapsulated drug-dose antibacterial sustained-release biological patch must be a continuous surface layer.

[0036] Preferably, the total number of continuous surface layers and discontinuous surface layers is ≤30.

[0037] Further preferably, the total number of continuous surface layers and discontinuous surface layers is ≤25.

[0038] Further preferably, the total number of continuous surface layers and discontinuous surface layers is ≤21.

[0039] Further preferably, the total number of continuous surface layers and discontinuous surface layers is ≤17.

[0040] Tetracycline has strong hydrophilicity, which means that after it is used for patch loading, it will be released due to rapid dissolution in water. If the amount of tetracycline encapsulated in the patch is too low, tetracycline cannot be continuously released during the high-incidence period of surgical infection, and thus the antibacterial effect cannot be achieved. Since the antibacterial sustained-release biological patch obtained by the present invention is used in the high-incidence period of surgical infection, that is, the early stage of surgical operation, it is not necessary to encapsulate the amount of drug as high as possible. Generally, the more layers are stacked, the higher the encapsulated amount of drug. At the same time, as the number of layers of material stacking increases, the operability of the material will also decrease. Therefore, in the present invention, the inventor controls the total number of layers of the continuous surface layer and the discontinuous surface layer to within 30 layers. In the experiment, the inventor unexpectedly found that this approach is also conducive to the mechanical uniformity (transverse tensile strength and longitudinal tensile strength are always close to 1:1) and mechanical stability of the antibacterial sustained-release biological patch, and will not cause the patch to become unstable due to pressure, swelling and other environments, resulting in application risks. Especially when the total number of continuous surface layers and discontinuous surface layers is ≤25, the mechanical uniformity and stability are more obvious.

[0041] As 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.

[0042] 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.

[0043] Further 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.

[0044] Preferably, the alkaline solution is a 0.01-0.1 mol / L sodium hydroxide aqueous solution.

[0045] As a preferred embodiment, the hydrophilic antibiotic suspension is obtained by adding 50-600 μL of 0.18-0.95 g / mL metal salt solution to 3-15 mL of 0.003-0.02 g / mL hydrophilic antibiotic aqueous solution, and then adjusting the pH to 7.1-8.0 with an alkaline solution.

[0046] Preferably, the suspension of the hydrophilic antibiotic is obtained by adding 50-600 μL of 0.18-0.95 g / mL metal salt solution to 3-15 mL of 0.005-0.015 g / mL hydrophilic antibiotic aqueous solution, and then adjusting the pH to 7.1-8.0 with an alkaline solution.

[0047] Preferably, when preparing the suspension of hydrophilic antibiotic, the molar mass ratio of the added hydrophilic antibiotic to the added metal salt is 1:(1.2-1.8).

[0048] Tetracycline is unstable in water and prone to degradation due to diastereoisomerization. In the present invention, tetracycline is complexed with metal ions. This complexation effect is a reversible reaction, which can slowly release tetracycline in the patch of the present invention. Theoretically, in the complexation reaction, one molecule of tetracycline will bind to two molecules of metal ions. In order to avoid the waste of raw materials, in the experiment, when preparing the suspension of hydrophilic antibiotics, the inventors preferentially made the molar mass ratio of the added hydrophilic antibiotics and the added metal salts to be 1: (1.2-1.8), and found that the tetracycline diastereomers produced at this time were very few. Again, the inventors controlled the molar mass ratio of the added hydrophilic antibiotics and the added metal salts to be 1: 2, and unexpectedly found that compared with the molar mass ratio of 1: 2, the molar mass ratio of the added hydrophilic antibiotics and the added metal salts was 1: (1.2-1.8), and the tetracycline diastereomers produced were still very few, and the stability of the hydrophobic antibiotic loading in the patch was unexpectedly improved, thereby improving the product stability. The inventors speculate that this may be due to the strong interaction between hydrophobic antibiotic molecules, which are easy to agglomerate and form clusters. The clusters become the aggregation sites of electrons, and the metal ions and clusters in the tetracycline complex system have a certain coordination effect, which affects the distribution of the hydrophobic antibiotics and thus affects the stability of the encapsulated drug amount. At the same time, this coordination effect may also have some influence on the tetracycline complex system, that is, the equilibrium system of 1 molecule of tetracycline combined with 2 molecules of metal ions is affected.

[0049] As a preferred embodiment, the metal salt solution contains metal ions, and the metal ions are diamagnetic divalent metal cations.

[0050] Preferably, the metal ion is selected from Ca 2+ ,Mg 2+ , Ba 2+ , Sr. 2+ , Zn 2+ One of a kind.

[0051] As a preferred embodiment, the organic solution of the hydrophobic antibiotic is prepared by adding the hydrophobic antibiotic to 80wt%-95wt% ethanol aqueous solution; in the organic solution of the hydrophobic antibiotic, the concentration of the hydrophobic antibiotic is 0.01-0.1g / mL.

[0052] As a preferred embodiment, the high-encapsulated drug-dose antibacterial sustained-release biological patch is applied as a medical device during the high-incidence period of infection in surgical operations.

[0053] The high incidence period of surgical infection usually refers to the three days after the surgery.

[0054] Antibacterial sustained-release biological patches with high encapsulation drug content are particularly used as treatment materials during the high-incidence period of open abdominal infection.

[0055] Beneficial effects: (1) The high-encapsulated antibacterial sustained-release biological patch obtained by the present invention is a non-coated drug-loaded sustained-release system based on a lamination process, which cooperates with at least two different types of antibiotics to kill bacteria in a short period of time, prevent bacteria from colonizing and reproducing in the wound / patch, and achieve the sustained release of rifamycin and tetracycline, so that they will not be released too quickly, but will accompany the wound healing process to achieve synergistic and continuous release, while guiding tissue regeneration and achieving degradation simultaneously. It can be used in the high-incidence infection period of surgical operations (i.e., within the first three days after surgical operations), especially in open abdominal treatment, to reduce the probability of adhesion, enhance abdominal wall strength, promote the growth of wound granulation tissue, and achieve effective antibacterial in the high-incidence infection period after surgery. (2) By loading specific types of hydrophobic antibiotics and hydrophilic antibiotics, and controlling the encapsulated drug amount of hydrophobic antibiotics to 150-600μg / cm 2 The encapsulated amount of hydrophilic antibiotics is 150-600μg / cm 2 , which is not only conducive to avoiding the phenomenon of increased toxicity caused by the aforementioned antibiotics, but also overcomes the technical resistance of tetracycline, especially minocycline hydrochloride, which is released due to its strong hydrophilicity and rapid dissolution in water, and slows down the release rate of tetracycline in the patch of the present invention when it is used, so that the release rate of tetracycline in the patch within 24 hours is controlled within 52%. (3) When preparing a suspension of hydrophilic antibiotics, the molar mass ratio of the added hydrophilic antibiotics 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. (4) The high-encapsulation antibacterial sustained-release biological patch of the present invention has good mechanical properties in a wet environment, which is conducive to improving the feel and operability. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 This is a structural side view of the high encapsulated drug amount antibacterial sustained-release biological patch obtained in Example 1. Figure 1 In the figure, the solid lines represent continuous layers, and the dashed lines represent discontinuous layers; Figure 2 (a) is a front view of the structure of the high encapsulated drug amount antibacterial sustained-release biological patch obtained in Example 1, Figure 2 (b) is a schematic diagram of the structure of the high-encapsulated drug-dose antibacterial sustained-release biological patch obtained in Example 1 (the layers in the figure are staggered only to clearly show the structure of each layer). DETAILED DESCRIPTION

[0057] In the present invention, the raw materials used are all commercially available, and the decellularized matrix materials are all from Zhuoruan Medical Technology (Suzhou) Co., Ltd., which are prepared from the small intestinal submucosa, peritoneum, dermis, bladder basement membrane or pericardium according to the perfusion-pressure difference method described in patent CN106075583B.

[0058] Example 1

[0059] This example provides a high-encapsulation antibacterial sustained-release biological patch, such as Figure 1 and Figure 2 The high encapsulated drug amount antibacterial sustained-release biological patch is obtained by encapsulating two antibiotics in a decellularized matrix material.

[0060] The decellularized matrix materials include decellularized matrix material A derived from bladder basement membrane, decellularized matrix material B derived from small intestinal submucosa, and decellularized matrix material C derived from small intestinal submucosa; the antibiotics include hydrophobic antibiotics and hydrophilic antibiotics, the hydrophobic antibiotic is rifamycin; and the hydrophilic antibiotic is minocycline hydrochloride.

[0061] Rifamycin is rifampicin, and the LogP value of rifampicin is 1.09.

[0062] The suspension of the hydrophilic antibiotic was prepared 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.

[0063] The metal salt solution is a calcium chloride aqueous solution, and the metal salt solution contains metal ions, and the metal ions are diamagnetic divalent metal cations. The metal ions are Ca 2+ When preparing the suspension of the hydrophilic antibiotic, the molar mass ratio of the added hydrophilic antibiotic to the added metal salt is 1:1.75.

[0064] The organic solution of the hydrophobic antibiotic is prepared by adding the hydrophobic antibiotic to a 91 wt % ethanol aqueous solution; in the organic solution of the hydrophobic antibiotic, the concentration of the hydrophobic antibiotic is 0.01 g / mL.

[0065] The preparation method of the high encapsulated drug amount antibacterial sustained-release biological patch is as follows: (1) Preparation of the continuous surface layer 1: laying the decellularized matrix material A with water; (2) Preparation of the continuous surface layer 2: Laying the acellular matrix material B with a suspension of hydrophilic antibiotics; (3) Preparation of the intermittent surface layer: The decellularized matrix material C was laid with an organic solution of a hydrophobic antibiotic, and the layer was repeatedly stacked and laid for 4 times. After a molding process (specifically lamination), the material was cut into rectangular strips. The rectangular strips were spaced and placed in parallel to form an intermittent surface layer. The width of the rectangular strips was 5 mm. There was a gap between every two rectangular strips, and the gap between the rectangular strips was 1 cm. The angle between the length direction and the horizontal direction of each rectangular strip of the intermittent surface layer was 45°. The number of rectangular strips in the intermittent surface layer was 5.

[0066] (4) Stacking one layer of continuous surface layer 1, two layers of continuous surface layer 2, one layer of intermittent surface layer, one layer of continuous surface layer 2 and one layer of continuous surface layer 1 in sequence from bottom to top, and laminating to obtain a product.

[0067] The specific method of laying with water and / or a suspension of hydrophilic antibiotics is as follows: placing the substrate horizontally, wetting the substrate with the suspension of water and / or a suspension of hydrophilic antibiotics, laying the decellularized matrix material A and / or the decellularized matrix material B on the substrate, flattening the decellularized matrix material on the substrate, and then scraping off excess water and / or the suspension of hydrophilic antibiotics on the surface of the decellularized matrix material with a scraper.

[0068] The operation of paving with an organic solution of a hydrophobic antibiotic is similar to the above-mentioned paving with water and / or a suspension of a hydrophilic antibiotic, and the principle is the same.

[0069] Example 2

[0070] This example provides a high-encapsulated drug amount antibacterial sustained-release biological patch. The difference from Example 1 is that the preparation method of the high-encapsulated drug amount antibacterial sustained-release biological patch is: (1) Preparation of the continuous surface layer 1: laying the decellularized matrix material A with water; (2) Preparation of the continuous surface layer 2: Laying the acellular matrix material B with a suspension of hydrophilic antibiotics; (3) Preparation of the intermittent surface layer: The decellularized matrix material C was laid with an organic solution of a hydrophobic antibiotic, and the layer was repeatedly stacked and laid for 9 times. After a molding process (specifically lamination), the material was cut into rectangular strips. The rectangular strips were spaced and placed in parallel to form an intermittent surface layer. The width of the rectangular strips was 8 mm. There was a gap between every two rectangular strips, and the gap between the rectangular strips was 1.5 cm. The angle between the length direction and the horizontal direction of each rectangular strip of the intermittent surface layer was 45°. The number of rectangular strips of the intermittent surface layer was 10.

[0071] (4) From bottom to top, stack one layer of continuous surface layer 1, six layers of continuous surface layer 2, one layer of intermittent surface layer, one layer of continuous surface layer 2, one layer of intermittent surface layer, four layers of continuous surface layer 2, one layer of intermittent surface layer and two layers of continuous surface layer 1 in sequence, and laminate them to obtain a product.

[0072] Comparative Example 1 This example provides an antibacterial sustained-release biological patch with a high encapsulated drug amount. The difference from Example 1 is that the total number of continuous surface layers and discontinuous surface layers is greater than 30, and the total number of continuous surface layers and discontinuous surface layers is 36.

[0073] Specifically, the encapsulated antibacterial sustained-release biological patch is obtained by encapsulating two antibiotics in acellular matrix materials. The acellular matrix materials include acellular matrix material A derived from the bladder basement membrane, acellular matrix material B derived from the small intestinal submucosa, and acellular matrix material C derived from the small intestinal submucosa; the antibiotics include hydrophobic antibiotics and hydrophilic antibiotics, the hydrophobic antibiotic is rifamycin; the hydrophilic antibiotic is minocycline hydrochloride. Rifamycin is rifampicin, and the LogP value of rifampicin is 1.09.

[0074] The suspension of the hydrophilic antibiotic was prepared by adding 50 μL of a 0.53 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.

[0075] The metal salt solution is a calcium chloride aqueous solution, and the metal salt solution contains metal ions, and the metal ions are diamagnetic divalent metal cations. The metal ions are Ca 2+ When preparing the suspension of hydrophilic antibiotic, the molar mass ratio of the added hydrophilic antibiotic to the added metal salt is 1:1.2.

[0076] The organic solution of the hydrophobic antibiotic is prepared by adding the hydrophobic antibiotic to a 94 wt % ethanol aqueous solution; in the organic solution of the hydrophobic antibiotic, the concentration of the hydrophobic antibiotic is 0.07 g / mL.

[0077] The preparation method of the high encapsulated drug amount antibacterial sustained-release biological patch is as follows: (1) Preparation of the continuous surface layer 1: laying the decellularized matrix material A with water; (2) Preparation of the continuous surface layer 2: Laying the acellular matrix material B with a suspension of hydrophilic antibiotics; (3) Preparation of the intermittent surface layer: The decellularized matrix material C was laid with an organic solution of a hydrophobic antibiotic, and the layer was repeatedly stacked and laid for 6 times. After a molding process (specifically lamination), the material was cut into rectangular strips. The rectangular strips were spaced and placed in parallel to form an intermittent surface layer. The width of the rectangular strips was 8 mm. There was a gap between every two rectangular strips, and the gap between the rectangular strips was 1.5 cm. The angle between the length direction and the horizontal direction of each rectangular strip of the intermittent surface layer was 45°. The number of rectangular strips of the intermittent surface layer was 15.

[0078] (4) Stack in sequence from bottom to top 2 layers of continuous surface layer 1, 10 layers of continuous surface layer 2, 1 layer of intermittent surface layer, 2 layers of continuous surface layer 2, 1 layer of intermittent surface layer, 2 layers of continuous surface layer 2, 1 layer of intermittent surface layer, 2 layers of continuous surface layer 2, 1 layer of intermittent surface layer, 2 layers of continuous surface layer 2, 1 layer of intermittent surface layer, 2 layers of continuous surface layer 2, 1 layer of intermittent surface layer, 2 layers of continuous surface layer 2, 1 layer of intermittent surface layer, and 8 layers of continuous surface layer 1, and laminarly press to obtain.

[0079] Comparative Example 2 This example provides a high-encapsulated antibacterial sustained-release biological patch, which is different from Example 1 in that: The suspension of the hydrophilic antibiotic was prepared by adding 80 μL of a 0.4 g / mL metal salt solution to 10 mL of a 0.01 g / mL hydrophilic antibiotic aqueous solution and then adjusting the pH to 7.3 with a 0.1 mol / L sodium hydroxide aqueous solution.

[0080] The metal salt solution is an aqueous solution of ferrous sulfate, and the metal salt solution contains metal ions, and the metal ions are paramagnetic divalent metal cations. The metal ions are Fe 2+ When preparing the suspension of the hydrophilic antibiotic, the molar mass ratio of the added hydrophilic antibiotic to the added metal salt is 1:1.32.

[0081] The organic solution of the hydrophobic antibiotic is prepared by adding the hydrophobic antibiotic to a 90 wt % ethanol aqueous solution; in the organic solution of the hydrophobic antibiotic, the concentration of the hydrophobic antibiotic is 0.06 g / mL.

[0082] Comparative Example 3 This example provides a high-encapsulated antibacterial sustained-release biological patch. Different from Example 1, the high-encapsulated antibacterial sustained-release biological patch is obtained by encapsulating two antibiotics in a decellularized matrix material. Both of the encapsulated antibiotics are hydrophilic antibiotics. The hydrophilic antibiotics are minocycline hydrochloride and doxycycline hydrochloride.

[0083] The decellularized matrix materials include decellularized matrix material A derived from bladder basement membrane, decellularized matrix material B derived from small intestinal submucosa, and decellularized matrix material C derived from small intestinal submucosa.

[0084] The suspension of minocycline hydrochloride was prepared by adding 100 μL of 0.45 g / mL metal salt solution to 10 mL of 0.015 g / mL minocycline hydrochloride aqueous solution and then adjusting the pH to 7.5 with 0.1 mol / L sodium hydroxide aqueous solution.

[0085] The metal salt solution is a calcium chloride aqueous solution, and the metal salt solution contains metal ions, and the metal ions are diamagnetic divalent metal cations. The metal ions are Ca 2+ When preparing the suspension of minocycline hydrochloride, the molar mass ratio of the added minocycline hydrochloride to the added metal salt is 1:1.33.

[0086] Doxycycline hydrochloride was 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.

[0087] The preparation method of the high encapsulated drug amount antibacterial sustained-release biological patch is as follows: (1) Preparation of the continuous surface layer 1: laying the decellularized matrix material A with water; (2) Preparation of continuous surface layer 2: laying acellular matrix material B with a suspension of minocycline hydrochloride; (3) Preparation of the intermittent surface layer: The decellularized matrix material C was laid with a doxycycline hydrochloride suspension, and the layer was repeatedly stacked and laid for 4 times. After a molding process (specifically lamination), the material was cut into rectangular strips. The rectangular strips were spaced and placed in parallel to form an intermittent surface layer. The width of the rectangular strips was 7 mm. There was a gap between every two rectangular strips, and the gap between the rectangular strips was 1.3 cm. The angle between the length direction and the horizontal direction of each rectangular strip of the intermittent surface layer was 45°. The number of rectangular strips of the intermittent surface layer was 5.

[0088] (4) Stacking one layer of continuous surface layer 1, two layers of continuous surface layer 2, one layer of intermittent surface layer, one layer of continuous surface layer 2 and one layer of continuous surface layer 1 in sequence from bottom to top, and laminating to obtain a product.

[0089] Comparative Example 4 This example provides a high encapsulated drug amount antibacterial sustained-release biological patch. Different from Example 1, the hydrophilic antibiotic suspension is obtained by adding 100 μL of 0.5 g / mL metal salt solution to 10 mL of 0.01 g / mL hydrophilic antibiotic aqueous solution, and then adjusting the pH to 7.6 with 0.1 mol / L sodium hydroxide aqueous solution. When preparing the hydrophilic antibiotic suspension, the molar mass ratio of the added hydrophilic antibiotic to the added metal salt is 1:2.05.

[0090] Comparative Example 5 This example provides an antibacterial sustained-release biological patch with a high encapsulated drug dosage. The difference from Example 1 is that the decellularized matrix material includes decellularized matrix material A derived from the bladder basement membrane, decellularized matrix material B derived from the small intestinal submucosa, and decellularized matrix material C derived from the small intestinal submucosa; the antibiotics include hydrophobic antibiotics and hydrophilic antibiotics, the hydrophobic antibiotics are macrolide antibiotics, and the macrolide antibiotics are clarithromycin; the hydrophilic antibiotic is minocycline hydrochloride.

[0091] The LogP value of clarithromycin is 2.4.

[0092] The suspension of the hydrophilic antibiotic was prepared 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 7.5 with a 0.1 mol / L sodium hydroxide aqueous solution.

[0093] The metal salt solution is a calcium chloride aqueous solution, and the metal salt solution contains metal ions, and the metal ions are diamagnetic divalent metal cations. The metal ions are Ca 2+ When preparing the suspension of the hydrophilic antibiotic, the molar mass ratio of the added hydrophilic antibiotic to the added metal salt is 1:1.75.

[0094] The organic solution of the hydrophobic antibiotic is prepared by adding the hydrophobic antibiotic into acetone; in the organic solution of the hydrophobic antibiotic, the concentration of the hydrophobic antibiotic is 0.01 g / mL.

[0095] Performance Testing 1. Drug content test: Five samples of 5 cm×5 cm were randomly cut from each of the biological patches obtained in Examples 1-2 and Comparative Examples 1-5, and the samples were extracted in methanol. The extract was taken every 24 hours for liquid chromatography injection testing until the extract was colorless. Sampling and testing were stopped. The drug content of the sample was obtained by referring to the chromatogram of each drug standard (the average value of the five 5 cm×5 cm samples of the biological patch building material of each embodiment was taken). The results are shown in Table 1.

[0096] Table 1

[0097] The drug content of doxycycline hydrochloride in Comparative Example 3 is 149.6 μg / cm 2 The average deviation of drug loading is 8.0. The drug content of clarithromycin in Comparative Example 5 is 170.8 μg / cm 2 , the average deviation of drug loading was 16.2.

[0098] As can be seen from Table 1, in the solutions of Example 1 and Example 2, the technical solution of the present invention achieves the adjustment of high drug content of the biological patch. At the same time, in the solution of the present invention, even if the number of layers is increased, the average deviation of the drug content of the obtained high encapsulated drug amount antibacterial sustained-release biological patch is still within a relatively low range (within 20), indicating that in the technical solution, even if the number of layers is increased, the drug content of the obtained product still has good uniformity and stability.

[0099] The total number of continuous surface layers and discontinuous surface layers of the product of Comparative Example 1 is greater than 30, and the total number of continuous surface layers and discontinuous surface layers is 36. Although the drug content is increased, the average deviation of the drug content is very high (greater than 20), which indicates that the uniformity and stability of the drug content of the obtained product are low.

[0100] The average deviation of the rifampicin drug content of the high encapsulated drug amount antibacterial sustained-release biological patches obtained in Comparative Examples 2 and 4 is high (greater than 20), which indicates that the uniformity and stability of the rifampicin drug content of the obtained products are low.

[0101] 2. Mechanical property test: The biological patches obtained in Examples 1-2 and Comparative Examples 1-5 were completely immersed in PBS buffer, and placed in a closed space at a temperature of 37±2°C and a CO2 gas atmosphere of 12-15 kPa for 6 hours. The tensile strength in the horizontal and vertical directions was tested according to GB / T328.8-2007 (stretched at a steady speed of 100 mm / min until fracture, and the maximum tensile force was recorded), and the ratio of the transverse tensile strength to the longitudinal tensile strength was calculated. Ten parallel experiments were conducted for each example sample, and the average value was taken. The results are shown in Table 2. The standard deviation of the ratio of the transverse tensile strength to the longitudinal tensile strength was calculated. The smaller the standard deviation, the more stable the mechanical properties of the sample. The results showed that: Example 1> Example 2> Comparative Example 2> Comparative Example 4> Comparative Example 5> Comparative Example 1> Comparative Example 3.

[0102] Table 2 Example Ratio of transverse tensile strength to longitudinal tensile strength Example 1 1：1.02 Example 2 1：1.18 Comparative Example 1 1：1.43 Comparative Example 2 1：0.91 Comparative Example 3 1：1.15 Comparative Example 4 1：1.56 Comparative Example 5 1：0.74 3. Animal experiment: Twelve rats were given gas anesthesia, and their abdominal hair was shaved. A 5 cm long incision was made in the midline of the abdomen to fully expose the abdominal cavity. The antibacterial patches obtained in Example 1 and Comparative Example 2, Comparative Example 3, and Comparative Example 5 were cut into 2 cm × 2 cm samples, respectively, and placed on both sides of the abdominal cavity of the rats. One sample of the same sample was placed on the abdominal wall and fixed with sutures. After implantation, the wound was closed. After surgery, samples were collected at 2h, 1 day, and 3 days. After euthanasia, the abdominal cavity was opened to observe the morphology of the patch implantation site (including adhesion and granulation tissue growth). The samples were taken out and extracted with PBS buffer solution for 24h. The drug loading was calculated according to the drug standard curve, recorded as W (the standard samples of rifampicin, minocycline hydrochloride, doxycycline hydrochloride and clarithromycin were dissolved in PBS buffer solution, diluted into a series of standard solutions of 1-500μg / mL, and the standard curve was obtained by liquid chromatography injection test). In vivo release rate = (drug loading of each sample in performance test 1-W) / drug loading of each sample in performance test 1 × 100%; the results are shown in Table 3: Table 3

[0103] The in vivo release rate of doxycycline hydrochloride in comparative example 3 is 27.62% in 2 hours and 95.31% in 24 hours. The in vivo release rate of clarithromycin in comparative example 3 is 45.33% in 2 hours and 99.306% in 24 hours.

Claims

1. A high-encapsulated antibacterial sustained-release biological patch, characterized in that: The high encapsulated drug amount antibacterial sustained-release biological patch is obtained by encapsulating at least two antibiotics in a decellularized matrix material; The decellularized matrix material includes decellularized matrix material A, decellularized matrix material B and decellularized matrix material C, and the decellularized matrix material is derived from any two of the following: small intestinal submucosa, peritoneum, dermis, bladder basement membrane and pericardium; The antibiotics include hydrophobic antibiotics and hydrophilic antibiotics. The hydrophobic antibiotics are selected from one or more of Beta-lactam antibiotics, rifamycin, and macrolide antibiotics. The hydrophilic antibiotics are tetracycline antibiotics. The encapsulated drug amount of the hydrophobic antibiotics in the high-encapsulated drug amount antibacterial sustained-release biological patch is 150-600 μg / cm 2 The encapsulated drug amount of hydrophilic antibiotics in high encapsulated drug amount antibacterial sustained release biological patch is 150-600μg / cm 2 ; The ratio of the transverse tensile strength to the longitudinal tensile strength of the high encapsulated drug amount antibacterial sustained-release biological patch is 1: (0.8-1.25).

2. The high encapsulated drug amount antibacterial sustained-release biological patch according to claim 1, characterized in that: The antibacterial sustained-release biological patch with a high encapsulated drug amount comprises at least two continuous surface layers and at least one discontinuous surface layer.

3. The high encapsulated drug amount antibacterial sustained-release biological patch according to claim 1, characterized in that: The preparation method of the high encapsulated drug amount antibacterial sustained-release biological patch is as follows: (1) Preparation of a continuous surface layer: laying acellular matrix material A and / or acellular matrix material B; (2) Preparation of the intermittent surface layer: Lay the decellularized matrix material C, and continue to repeat the laying for 2-10 times, and then cut it into rectangular strips through a molding process. The rectangular strips are placed in parallel to form the intermittent surface layer; (3) After the continuous surface layer and the discontinuous surface layer are superimposed and subjected to a molding process, the result is obtained.

4. The high encapsulated drug amount antibacterial sustained-release biological patch according to claim 3, characterized in that: The upper and lower surfaces of the discontinuous surface layer both have contact areas with the continuous surface layer.

5. The high encapsulated drug amount antibacterial sustained-release biological patch according to claim 3, characterized in that: In the step (1), when preparing the continuous surface layer, water and / or a suspension of hydrophilic antibiotics is used for laying.

6. The high encapsulated drug amount antibacterial sustained-release biological patch according to claim 3, characterized in that: In the step (2), when preparing the intermittent surface layer, an organic solution of a hydrophobic antibiotic is used for laying.

7. The high-encapsulated drug-dose antibacterial sustained-release biological patch according to claim 5, characterized in that: The hydrophilic antibiotic suspension is obtained by adding a metal salt solution to a hydrophilic antibiotic aqueous solution and then adjusting the pH to 7.1-8.0 with an alkaline solution.

8. The high-encapsulated drug-dose antibacterial sustained-release biological patch according to claim 7, characterized in that: The metal salt solution contains metal ions, which are diamagnetic divalent metal cations.

9. The high encapsulated drug amount antibacterial sustained-release biological patch according to claim 6, characterized in that: The hydrophobic antibiotic organic solution is prepared by adding the hydrophobic antibiotic into 80wt%-95wt% ethanol aqueous solution; in the hydrophobic antibiotic organic solution, the concentration of the hydrophobic antibiotic is 0.01-0.1g / mL.

10. The high encapsulated drug amount antibacterial sustained-release biological patch according to any one of claims 1 to 3, characterized in that: The high-encapsulated drug-dose antibacterial sustained-release biological patch is applied to medical devices during the high-incidence period of infection in surgical operations.

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