Two-component tissue adhesion film

Abstract

The invention belongs to the technical field of medical films, and discloses a two-component tissue adhesion film which is a double-layer film formed by respectively heating and melting a component A and a component B and then coating a substrate with double layers in an adjustable sequence. Compared with a double-component hydrogel product, the double-component adhesive film provided by the invention has better clinical use convenience and low dependence on equipment on the basis of keeping certain adhesion performance.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical membranes, and particularly relates to a two-component tissue adhesion membrane. Background Art

[0002] At present, suture threads or staples are mostly selected as the means for closing tissue wounds clinically. Traditional suture surgeries can cause secondary damage to tissues, have a long operation time, and are prone to infection risks. Tissue adhesion membranes have the advantages of convenient operation, high wound closure efficiency, less material intervention, and no secondary tissue damage, and can replace traditional suture means in the closure of some surgical trauma wounds.

[0003] In the prior art, there are liquid adhesives and adhesive patches with functions similar to those of tissue adhesion membranes. The former can achieve tissue wound closure after curing, but it is still prone to being diluted by tissue blood during the curing process, resulting in unstable adhesion. At the same time, a large amount of adhesive will enter the blood circulation system. The latter can quickly bond wounds, but most of them use unreacted active groups to chemically react with the tissue surface, which will cause local high heat and inflammation. At the same time, excessive degradation products and foreign toxic components increase the burden on the liver and kidneys. Tissue adhesion membranes are a more effective means for wound closure, but most of the currently studied bioadhesive membranes can only be applied to the skin surface and are difficult to maintain the continuous adhesion and mechanical strength of the membrane in the in vivo liquid environment, which undoubtedly limits the further development and application of tissue adhesion membranes. Summary of the Invention

[0004] The purpose of the present invention is to overcome the disadvantages and deficiencies of the prior art, and provide a tissue adhesion membrane that can quickly adhere to wet tissues, has stable mechanical properties, and is biodegradable.

[0005] The present invention provides a two-component tissue adhesion membrane, which is a double-layer membrane formed by separately heating and melting component A and component B and then coating them on a substrate in a two-layer manner with an adjustable order;

[0006] Both component A and component B have melting stability. Melting refers to the process in which when the temperature rises, the kinetic energy of the thermal motion of molecules increases, resulting in the destruction of crystals and the transformation of the substance from the crystalline phase to the liquid phase; it is a first-order phase transition, with an increase in enthalpy, entropy, and volume. Therefore, the melting stability described in the present invention means that the melting temperature is higher than room temperature by 25 °C, and after component A or component B is heated and melted into the liquid phase and then cooled and solidified into the crystalline phase again, this continuous phase transition process does not affect the structural composition of component A or component B, that is, it does not affect its use performance.

[0007] Preferably, the substrate is a clean and dry biological membrane substrate such as sausage casing or a medical polymer film substrate.

[0008] Preferably, the mass ratio range of reaction component A and reaction component B is (2:1) to (1:2); and more preferably 1:1.

[0009] Preferably, component A is an aldehyde group-terminated multi-arm polyethylene glycol derivative in powder form; the number of arms of the multi-arm polyethylene glycol derivative is between 2 and 8, the number-average molecular weight is 5000 to 40000 Da, and the end group is selected from an aldehyde group or an NHS active ester; NHS active ester, the full name is N-Hydroxy succinimide active ester; component B is an amino group-terminated multi-arm polyethylene glycol derivative in powder form; the number of arms of the amino group-terminated multi-arm polyethylene glycol derivative is 8, and the number-average molecular weight is 5000 to 40000 Da;

[0010] During use, after the two-component tissue adhesion film contacts the wet tissue surface, when the tissue fluid on the wet tissue surface contacts the two-component tissue adhesion film, the film layer material of the two-component tissue adhesion film gradually dissolves, and component A and component B undergo a chemical cross-linking reaction during the dissolution process and establish chemical and physical connections with the tissue surface, ultimately achieving tissue adhesion. Among them, component B is a multi-amino component, and the multi-amino component is positively charged, while the human tissue surface such as the cell membrane is negatively charged. Component B in the adhesion film mainly produces an adhesion effect through electrostatic interaction; component A is an aldehyde group-terminated multi-arm polyethylene glycol derivative, which can undergo a chemical reaction with the amino group on the tissue surface, thereby producing an adhesion effect. Therefore, the adhesion function of the tissue adhesion film provided in this application is achieved by both physical and chemical actions.

[0011] As a preferred embodiment of the present invention, the two-component tissue adhesion film is a double-layer film formed by separately heating and melting component A and component B and then double-layer coating on a substrate in the order of first coating B and then coating A. And more preferably, component A is 8arm PEG-CHO with a molecular weight of 20 kDa, and component B is 8arm PEG-NH2 with a molecular weight of 20 kDa.

[0012] Beneficial effects:

[0013] Based on maintaining a certain adhesion performance, the two-component adhesion film provided by the present invention has better clinical use convenience and lower dependence on equipment compared with two-component hydrogel products; the two-component adhesion film prepared by some preferred embodiments even has better adhesion than the hydrogel with the same composition; the raw materials used in the two-component adhesion film provided by the present invention have good biocompatibility and will not cause a large rejection reaction when used in in-vivo adhesion scenarios; different components and preparation methods have a significant impact on the adhesion effect. When the polyethylene glycol derivative with reactive activity to amino groups is on the surface layer of the adhesion film, the adhesion effect is better. Brief Description of the Drawings

[0014] Figure 1 It is a demonstration diagram of the positional relationship between the two-component tissue adhesion film and the substrate;

[0015] Figure 2 It is a physical photo of the two-component tissue adhesion film fabricated on the substrate in Example 1;

[0016] Figure 3 It is a demonstration diagram of the test process for the adhesion performance of the tissue adhesion film in Test Example 1. Detailed Description of the Invention

[0017] The present invention will be further described in detail below, but the implementation manners of the present invention are not limited thereto.

[0018] The 4arm-PEG-NH2 10kDa powder is purchased from Xiamen Syno Biological Co., Ltd., with the model number 06020700209;

[0019] The 8arm-PEG-CHO 20kDa powder is purchased from Xiamen Syno Biological Co., Ltd., with the model number 06020901409;

[0020] The 4arm-PEG-SG 10kDa powder is purchased from Xiamen Syno Biological Co., Ltd., with the model number 06020702809;

[0021] The 8arm-PEG-NH2 10kDa and 20kDa powders are purchased from Xiamen Syno Biological Co., Ltd., with the model number 06020900209;

[0022] The base layer materials used in the following examples and comparative examples are all glass sheet carriers coated with dry sausage casings. The dry sausage casings are membranes with a size of 70mm×25mm after cutting, and the dry sausage casings are prepared by cleaning pig small intestines, removing fat and mucosal tissues, and then drying;

[0023] The ε-polylysine in the 5wt% ε-polylysine aqueous solution is purchased from Shanghai Macklin Biochemical Co., Ltd., with a number-average molecular weight of 2000-5000Da and a CAS number of 28211-04-3;

[0024] The sodium hyaluronate gel is purchased from Changzhou Brightgene Biopharmaceutical Co., Ltd.

[0025] Example 1: Layered coating of 8arm-PEG-CHO 20kDa and 8arm-PEG-NH2 10kDa to form a film

[0026] Step S101: First, weigh 0.2 g of 8-arm-PEG-NH2 powder with a molecular weight of 10 kDa as reagent B in a container. Transfer the container to a hot stage at 90 °C and heat it for melting for 3 min. After complete melting, take out the melted liquid and drop it onto the substrate layer, and use a scraper to evenly apply it onto the substrate layer to form an 8-arm-PEG-NH2 film. The size of the film is 25 mm × 25 mm. After uniform application, place it at room temperature and let it cool naturally for standby.

[0027] Step S102: Then, weigh 0.2 g of 8-arm-PEG-CHO powder with a molecular weight of 20 kDa as reagent A in a container. Transfer the container to a hot stage at 90 °C and heat it for melting for 3 min. After complete melting, take out the melted liquid and drop it onto the 8-arm-PEG-NH2 film prepared in Step S101, and use a scraper to evenly apply and cover the existing coating to form an 8-arm-PEG-CHO film. After uniform application, place it at room temperature and let it cool naturally to finally obtain a bilayer structured tissue adhesion film.

[0028] As Figure 2 shown is the physical photo of the two-component tissue adhesion film fabricated on the substrate in Example 1. As Figure 1 shown is Figure 2 the corresponding demonstration diagram of the positional relationship between the two-component tissue adhesion film and the substrate.

[0029] Example 2: Layered coating and film formation of 8-arm-PEG-CHO 20 kDa and 8-arm-PEG-NH2 10 kDa

[0030] The difference between Example 2 and Example 1 is only that the coating order is swapped, that is, reagent A is coated first and then reagent B. Specifically, when coating and forming the film, first coat 8-arm-PEG-CHO 20 kDa onto the substrate layer, and after cooling, coat 8-arm-PEG-NH2 10 kDa onto the existing coating to finally obtain a bilayer structured tissue adhesion film.

[0031] Example 3: Layered coating and film formation of 4-arm-PEG-SG 10 kDa and 8-arm-PEG-NH2 10 kDa

[0032] Example 3 includes Step S301 and Step S302, where Step S301 is the same as Step S101 of Example 1;

[0033] Step S302: Weigh 0.2 g of 4arm-PEG-SG 20k Da powder in a container, transfer the container to a hot plate at 90 °C and heat it for melting for 3 min. After complete melting, take out the melted liquid and drop it onto the 8arm-PEG-NH2 film prepared in Step S301. Use a scraper to evenly smear and cover the existing coating to form a 4arm-PEG-SG film. After smearing evenly, place it at room temperature and let it cool naturally to finally obtain a bilayer tissue adhesion film.

[0034] Example 4: Layered coating and film formation of 4arm-PEG-SG 10kDa and 8arm-PEG-NH2 10kDa

[0035] The difference between Example 4 and Example 3 is only that the coating order is reversed, that is, reagent A is coated first and then reagent B. Specifically, when coating and forming a film, first coat 4arm-PEG-SG 20kDa on the base layer, and after cooling, coat 8arm-PEG-NH2 10k Da on the existing coating to finally obtain a bilayer tissue adhesion film.

[0036] Example 5: Layered coating and film formation of 4arm-PEG-PA 10kDa and 8arm-PEG-NH2 10kDa

[0037] Example 5 includes Step S501 and Step S502, where Step S501 is the same as Step S101 in Example 1;

[0038] Step S502: Weigh 0.2 g of 4arm-PEG-PA 10k Da powder in a container, transfer the container to a hot plate at 90 °C and heat it for melting for 3 min. After complete melting, take out the melted liquid and drop it onto the 8arm-PEG-NH2 film prepared in Step S501. Use a scraper to evenly smear and cover the existing coating to form a 4arm-PEG-PA film. After smearing evenly, place it at room temperature and let it cool naturally to finally obtain a bilayer tissue adhesion film.

[0039] Example 6: Layered coating and film formation of 4arm-PEG-PA 10kDa and 8arm-PEG-NH2 10kDa

[0040] The difference between Example 6 and Example 5 is only that the coating order is reversed, that is, reagent A is coated first and then reagent B. Specifically, when coating and forming a film, first coat 4arm-PEG-PA 10k Da on the base layer, and after cooling, coat 8arm-PEG-NH2 10k Da on the existing coating to finally obtain a bilayer tissue adhesion film.

[0041] Example 7: Layered coating and film formation of 8-arm PEG-CHO 20 kDa and 8-arm PEG-NH2 20 kDa

[0042] Step S701, first weigh 0.2 g of 8-arm PEG-NH2 powder with a molecular weight of 20 kDa in a container, transfer the container to a hot stage at 90 °C for heating and melting for 3 min. After complete melting, take out the melted liquid and drop it onto the base layer, and use a scraper to evenly apply it onto the base layer to form an 8-arm PEG-NH2 film (size: 25 mm × 25 mm). After even application, place it at room temperature for natural cooling and set aside.

[0043] Step S702, weigh 0.2 g of 8-arm PEG-CHO powder in a container, transfer the container to a hot stage at 90 °C for heating and melting. After complete melting, take out the melted liquid and drop it onto the 8-arm PEG-NH2 film prepared in step S701, and use a scraper to evenly apply and cover the existing coating to form an 8-arm PEG-CHO film. After even application, place it at room temperature for natural cooling, and finally obtain a bilayer tissue adhesion film.

[0044] Comparative Example 1: Single-layer film formation of 8-arm PEG-CHO 20 kDa

[0045] Weigh 0.2 g of 8-arm PEG-CHO 20 kDa powder in a container, cover the container and transfer it to a hot stage at 90 °C for heating and melting. After complete melting, take out the melted liquid and drop it onto the base layer, and use a scraper to evenly apply it onto the base layer to form an 8-arm PEG-CHO 20 kDa film (size: 25 mm × 25 mm), place it at room temperature for natural cooling and set aside.

[0046] Comparative Example 2: Single-layer film formation of 8-arm PEG-NH2 20 kDa

[0047] Weigh 0.2 g of 8-arm PEG-NH2 powder in a container, cover the container and transfer it to a hot stage at 90 °C for heating and melting. After complete melting, take out the melted liquid and drop it onto the base layer, and use a scraper to evenly apply it onto the base layer to form an 8-arm PEG-NH2 film with a size of 25 mm × 25 mm, place it at room temperature for natural cooling and set aside.

[0048] Comparative Example 3: Bicomponent hydrogel adhesion 1

[0049] Select a 10 wt% aqueous solution of 8-arm PEG-CHO 20 kDa as Reagent A and a 5 wt% aqueous solution of ε-polylysine as Reagent B. Use a double-barreled medicine mixing package to extract 0.5 mL of each of Reagent A and Reagent B and inject them into a 25 mm × 25 mm area on the sausage casing. After injection, cover the injected gel area with another piece of sausage casing, and press a 200 g weight on the bonding area and hold for 10 min.

[0050] Comparative Example 4: Adhesion of Bicomponent Hydrogel 2

[0051] Select a 10 wt% aqueous solution of 8-arm PEG-CHO 20 kDa as Reagent A and a 10 wt% aqueous solution of 8-arm PEG-NH2 20 kDa as Reagent B. Use a double-barreled medicine mixing package to extract 0.5 mL of each of Reagent A and Reagent B and inject them into a 25 mm × 25 mm area on the sausage casing. After injection, cover the injected gel area with another piece of sausage casing, and press a 200 g weight on the bonding area and hold for 10 min.

[0052] Comparative Example 5: Adhesion of Monocomponent Hydrogel

[0053] Select sodium hyaluronate gel, extract 1 mL of the gel, and inject it into a 25 mm × 25 mm area on the sausage casing. After injection, cover the injected gel area with another piece of sausage casing, and press a 200 g weight on the bonding area and hold for 10 min.

[0054] Comparative Example 6: Multilayer Coating and Film Formation of 8-arm PEG-CHO 20 kDa and 8-arm PEG-NH2 20 kDa

[0055] Step S801, first weigh two portions of 0.2 g of 8-arm PEG-NH2 20 kDa powder in a container. Transfer one portion to a hot plate at 90 °C and heat and melt it for 3 min. After complete melting, take out the melted liquid and drop it onto the base layer, and use a spatula to evenly spread it on the base layer to form an 8-arm PEG-NH2 20 kDa film (size: 25 mm × 25 mm). After even spreading, place it at room temperature to cool naturally for standby.

[0056] Step S802, then weigh 0.2 g of 8-arm PEG-CHO powder in a container. Transfer the container to a hot plate at 90 °C and heat and melt it. After complete melting, take out the melted liquid and drop it onto the 8-arm PEG-NH2 film prepared in Step S801, and use a spatula to evenly spread it and cover the existing coating to form an 8-arm PEG-CHO film. After even spreading, place it at room temperature to cool naturally for standby.

[0057] Step S803: Transfer the remaining 8arm PEG-NH2 powder to a hot stage at 90°C again and heat it for 3 minutes until it melts completely. After complete melting, take out the melted liquid and drop it onto the double-layer film prepared in Step S802. Spread it evenly to cover the prepared film and then let it cool naturally at room temperature. Finally, a three-layer tissue adhesion film is obtained.

[0058] Comparative Example 7: Multilayer coating film formation of 8arm PEG-CHO 20kDa and 8arm PEG-NH2 20kDa

[0059] Step S901: First, weigh two portions of 0.2g of 8arm PEG-CHO 20kDa powder in a container. Transfer one portion to a hot stage at 90°C and heat it for 3 minutes until it melts completely. After complete melting, take out the melted liquid and drop it onto the base layer. Use a spatula to spread it evenly on the base layer to form an 8arm PEG-CHO 20kDa film (size: 25mm×25mm). After spreading evenly, let it cool naturally at room temperature for later use.

[0060] Step S902: Then, weigh 0.2g of 8arm PEG-NH2 powder in the container. Transfer the container to a hot stage at 90°C and heat it until it melts completely. After complete melting, take out the melted liquid and drop it onto the 8arm PEG-CHO film prepared in Step S901. Use a spatula to spread it evenly to cover the existing coating to form an 8arm PEG-NH2 film. After spreading evenly, let it cool naturally at room temperature for later use.

[0061] Step S903: Transfer the remaining 8arm PEG-CHO powder to a hot stage at 90°C again and heat it for 3 minutes until it melts completely. After complete melting, take out the melted liquid and drop it onto the double-layer film prepared in Step S902. Spread it evenly to cover the prepared film and then let it cool naturally at room temperature. Finally, a three-layer tissue adhesion film is obtained.

[0062] Comparative Example 8: Co-blending coating film formation of 8arm-PEG-CHO 20kDa and 4arm-PEG-NH2 20kDa

[0063] Weigh equal masses (about 0.2g) of 8arm-PEG-CHO 20kDa and 4arm-PEG-NH2 20kDa powders in a container and mix them preliminarily by gentle shaking. Then transfer the container to a hot stage at 90°C and heat it for 3 minutes until it melts completely. After complete melting, take out the liquid from the container and evenly coat it on the base layer, but no film can be formed. During co-blending melting, the two components will mix and crosslink and solidify directly after melting, so no film can be formed. Therefore, an adhesion film cannot be prepared by the method of co-blending melting.

[0064] Comparative Example 9: Layered coating of 8-arm-PEG-CHO 20 kDa and 4-arm-PEG-NH2 20 kDa to form a film

[0065] Step S1001: First, weigh 0.2 g of 8-arm-PEG-NH2 20 kDa powder in a container, transfer the container to a hot plate at 90 °C for heating and melting for 3 min. After complete melting, take out the melted liquid and drop it onto the base layer, and use a scraper to evenly coat it on the base layer to form a 4-arm-PEG-NH2 20 kDa film (size: 25 mm × 25 mm). After even coating, place it at room temperature for natural cooling and set aside.

[0066] Step S1002: Then, weigh 0.2 g of 8-arm-PEG-CHO 20 kDa powder in a container, transfer the container to a hot plate at 90 °C for heating and melting for 3 min. After complete melting, take out the melted liquid and drop it onto the 8-arm-PEG-NH2 20 kDa film prepared in Step 1, and use a scraper to evenly coat and cover the existing coating to form an 8-arm-PEG-CHO 20 kDa film. After even coating, place it at room temperature for natural cooling, and finally obtain a bilayer structured tissue adhesion film.

[0067] Among them, after the 4-arm-PEG-NH2 20 kDa powder melts, the cooling and solidification speed is relatively fast, the operable time is relatively short, and the uniformity of the 4-arm-PEG-NH2 20 kDa film is relatively poor.

[0068] It should be noted here that for the adhesion film products prepared in the above Examples 1-7, Comparative Examples 1-2, and Comparative Examples 6-7, they stay on the base layer, i.e., the dry casing, by intermolecular forces. The base layer only plays a supporting role, and it is not that the tissue adhesion film is completely combined with the base layer. When the product is actually used, for example, for adhesion to in-vivo tissues, the base layer needs to be removed. For in-vitro tissues, the glass of the base layer needs to be removed and only the casing in the base needs to be retained. In order to test the adhesiveness of the tissue adhesion film below, in Test Example 1, the base layer was not removed during the test, but the base layer was used as the stretching force point. During the test, the tissue adhesion film was attached to the wet tissue. In Examples 1-7, the moisture on the wet tissue triggered the crosslinking and curing reaction of the tissue adhesion film, and through electrostatic interaction and reaction with the amino groups on the tissue surface, an adhesive effect was produced, adhering two layers of casing tissues, i.e., the casing of the base layer and the wet casing.

[0069] Test Example 1: Adhesion experiment

[0070] Step S1101: Take the adhesion film products prepared in Examples 1 - 7, Comparative Examples 1 - 2, and Comparative Examples 6 - 7, and an appropriate amount of dry sausage casings as the blank base layer, and cut them into appropriate sizes (70 mm in length and 25 mm in width) respectively; then immerse the cut sausage casings in physiological saline for 30 s and take them out. Cover a part of the wet sausage casings on the adhesion films prepared in Examples 1 - 7, Comparative Examples 1 - 2, and Comparative Examples 6 - 7, and press a 200 g weight on the bonding part for 10 min.

[0071] Step S1102: Then directly install the samples processed in Step S1101 and the samples prepared in Comparative Examples 3 - 5 onto the fixture of a universal testing machine as Figure 3 shown, set the tensile speed to 5 mm / min for uniform testing until the maximum force required to pull the two base layers appears, that is, the maximum load F received at the bonding part of the sample.

[0072] The calculation formula for adhesion strength is as follows:

[0073] Adhesion strength = maximum load F / bonding area S;

[0074] Among them, calculate the bonding area S, accurate to 0.01 cm 2 .

[0075] Table 1: Adhesion strength test results

[0076]

[0077]

[0078] Result analysis: Different components and preparation methods have a significant impact on the adhesion effect. Through the comparison of the test results of Example 7 and Comparative Example 4, it can be verified that after forming a film by two-component melt stratification coating, there is better adhesion force, and it has better adhesion compared with the hydrogel prepared by double injection of the same components; by comparing the test results of Example 1 and Example 2, or comparing Example 3 and Example 4, or comparing Example 5 and Example 6, it can be found that under the same components, the adhesion film prepared by the stratification coating method of B first and then A has significantly better adhesion effect than the stratification coating method of A first and then B, that is, it verifies that component A with reactive activity to amino groups, as the contact layer with the adherend object when used as an adhesion film product, preferentially contacts the moisture on the surface of the wet tissue, thereby triggering a better adhesion effect of the cross-linking reaction.

[0079] Test Example 2 Reagent stability test

[0080] Step S1201, Weigh a certain amount of 8-arm PEG-CHO 20k, 4-arm PEG-SG 10k, and 4-arm PEG-PA 10k in a container respectively. Place the container on a hot stage at 90 °C and heat it for 3 minutes. After complete melting, place the container at room temperature to allow the melted 8-arm PEG-CHO 20k, 4-arm PEG-SG 10k, and 4-arm PEG-PA 10k to cool and solidify naturally;

[0081] Step S1202, Weigh a certain amount of 8-arm PEG-NH2 10k and prepare it into a 20 wt% aqueous solution as reagent B for later use;

[0082] Step S1203, Prepare the medicine that has cooled and solidified again in Step S1201 into a 20 wt% aqueous solution as reagent A for later use;

[0083] Step S1204, Place reagent A, reagent B, and the vial in a constant temperature water bath at 37 °C ± 0.5 °C and let it stand at a constant temperature for 5 minutes. Then use a pipette to draw 200 μL of reagent A and add it to the vial. Then draw another 200 μL of reagent B and add it to the vial containing reagent A and start timing. Shake the vial at a frequency of 3 times per second for 3 seconds. Then tilt the vial at a frequency of 1 time per second to observe the curing situation until there is no obvious flowing liquid in the vial and stop timing. Record the time as the curing time. Test three groups of samples in parallel as the test results of the curing time of the experimental group;

[0084] Step S1205, Similarly, use 8-arm PEG-CHO 20k, 4-arm PEG-SG 10k, and 4-arm PEG-PA 10k that have not undergone the heating and melting process to prepare a 20 wt% aqueous solution as reagent A. Repeat the above Step S204, test three groups of samples in parallel as the test results of the curing time of the control group. Use the curing time before and after heating and melting as a comparison. The test results are shown in Table 2 below and use this as a standard to evaluate the stability of different reagent A.

[0085] Table 2: Comparison table of curing time test results

[0086]

[0087] Result analysis: After the reagent A treated by heat melting is formulated into an aqueous solution again and crosslinked and solidified with the reagent B, there are certain differences in the curing time between different reagent A and reagent B crosslinked and solidified and the curing time of reagent A and reagent B crosslinked and solidified without heat melting treatment. However, for 8-arm PEG-CHO with a molecular weight of 20k at 20wt%, whether it is treated by heat melting or not, there is no significant difference in the curing time, indicating that 8-arm PEG-CHO 20k has stronger melting stability compared with 4-arm PEG-SG10k and 4-arm PEG-PA 10k.

[0088] Test Example 3: Cytotoxicity Test

[0089] Step S1301, Before the experiment, inoculate 100 μL of the suspension containing 1×10 4 cells into each well of a 96-well plate, and place it in an incubator at 37°C with 5% CO2 for 72 hours. After culturing, replace the growth medium with 100 μL of the extraction solutions of the test articles at four concentrations (100%, 75%, 50% and 25%), 100% negative control, positive control and blank (the second column and the eleventh column) respectively. Each group is repeated 6 times. Culture the 96-well plate in an environment of 37°C with 5% CO2 for 24 hours.

[0090] Step S1302, After cell culture, remove the culture medium. Then add 50 μL of MTT solution (replaced with the specification model) to each test well and place it in an incubator at 37°C with 5% CO2 for 2 hours. Then remove the MTT solution, add 100 μL of isopropanol to each well and shake gently for 10 minutes. Read the value on a microplate reader at 570 nm (reference wavelength 650 nm). Calculate the cell survival rate according to the following formula:

[0091] Viab.%=100×OD e / OD b

[0092] where OD e is the average value of the optical density measured by the extraction solution of the test article; OD b is the average value of the optical density measured by the blank solution.

[0093] Result judgment rule: When the following conditions are met, the test is valid;

[0094] 1) The average value of OD of the blank control ≥ 0.2;

[0095] 2) The difference between the average value of the blanks in the left and right columns and the average value of all blanks ≤ 15%;

[0096] 3) The survival rate of the 50% extraction solution of the test article ≥ the survival rate of the 100% extraction solution;

[0097] 4) The survival rate of the negative control is ≥70%, and the survival rate of the positive control is <70%;

[0098] When the cell survival rate of 100% of the test article extract is ≤70% of the blank control, it has potential cytotoxicity.

[0099]

[0100] Among them, both reagent A (8-arm PEG-CHO 20k) and reagent B (8-arm PEG-NH2 20k) have good biocompatibility.

Claims

1. A two-component tissue adhesive film, characterized in that: The two-component tissue adhesion film is a two-layer film formed by heating and melting component A and component B respectively and coating them on a substrate in an adjustable order; both component A and component B have melting stability.

2. A two-component tissue adhesive film as claimed in claim 1, characterized in that: The component A is a powdered multi-arm polyethylene glycol derivative terminated with aldehyde groups; the number of arms of the multi-arm polyethylene glycol derivative is between 2 and 8, the number average molecular weight is 5000 to 40000 Da, and the end group is selected from aldehyde groups or NHS active esters; the component B is a powdered multi-arm polyethylene glycol derivative terminated with amino groups; the number of arms of the multi-arm polyethylene glycol derivative terminated with amino groups is 8, and the number average molecular weight is 5000 to 40000 Da.

3. A two-component tissue adhesive film as claimed in claim 2, characterized in that: The mass ratio of reaction component A to reaction component B ranges from (2:1) to (1:2).

4. A two-component tissue adhesive film as claimed in claim 1, characterized in that: The mass ratio of reaction component A to reaction component B is 1:

1.

5. A two-component tissue adhesive film as claimed in claim 1, characterized in that: The substrate is a biofilm substrate or a medical polymer film substrate with a clean and dry surface.

6. A two-component tissue adhesive film as claimed in claim 1, characterized in that: The biofilm substrate is a casing.

7. A two-component tissue adhesive film as claimed in claim 1, characterized in that: The two-component tissue adhesion film is a double-layer film formed by heating and melting component A and component B respectively, and then coating them on a substrate in the order of coating B first and then coating A.

8. A two-component tissue adhesive film as claimed in claim 7, characterized in that: The component A is 8arm PEG-CHO with a molecular weight of 20kDa, and the component B is 8arm PEG-NH2 with a molecular weight of 20kDa.

Images