Method for producing adhesive, adhesive thus obtained and use thereof

Abstract

The invention provides a method for preparing an adhesive, the adhesive obtained through the method and application of the adhesive. Specifically, the method comprises heating the gutta-percha to a temperature of 150-320 DEG C and maintaining the temperature for 60-180 minutes to modify the gutta-percha, the gutta-percha is extracted from Eucommia ulmoides, and the gutta-percha contains trans-1, 4-polyisoprene with a polymerization degree of 1000-3500. The adhesive obtained by the method comprises a polymer 1, 4-polyisoprene composed of a structural unit I and a structural unit II which are shown in the specification, and the weight ratio of the structural unit I to the structural unit II is (1-15): 1. The adhesive shows high adhesion strength, good dynamic adaptability, good subcutaneous tissue compatibility, cell biocompatibility and blood compatibility, has an active antibacterial function and tissue regeneration promoting capacity, and has the potential of being used as a medical tissue adhesive.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more particularly to a method for preparing an adhesive, the resulting adhesive, and its uses. Background Technology

[0002] In clinical practice, skin wound repair problems caused by trauma, surgery, burns, chronic ulcers, etc., are common and complex. If wounds are not closed promptly and effectively, they can easily lead to fluid loss, secondary infection, or tissue necrosis, thus delaying the healing process and even inducing systemic complications. Currently, medical adhesives, represented by cyanoacrylates, polyethylene glycol-based hydrogels, and fibrin glue, are widely used for wound closure. These materials reduce the risk of infection and promote healing to some extent by physically isolating the external environment. However, existing adhesives still have significant drawbacks: First, their adhesion performance drops sharply in moist or dynamic wounds (such as joint areas), easily leading to interface delamination due to mechanical stress or tissue fluid infiltration. Second, their functions are limited; for example, their antibacterial ability is insufficient, lacking the ability to cope with the infection risk of infected wounds; their ability to promote repair is also poor. Currently, most medical adhesives only have an adhesive effect and have no practical effect on promoting wound healing or shortening healing time. Third, insufficient biocompatibility, such as cyanoacrylate adhesives, may trigger inflammatory reactions or hinder cell migration, affecting tissue and appendage regeneration.

[0003] While natural polymer materials (such as chitosan and gelatin) possess certain biodegradability and low toxicity, their insufficient mechanical strength and poor adhesive durability remain unresolved. Therefore, developing a medical adhesive that combines high adhesion strength, dynamic adaptability, active antibacterial function, and tissue regeneration promotion capabilities has become an urgent need in wound management, especially since medical tissue adhesives derived from natural raw materials of traditional Chinese medicine are currently a gap in the field both domestically and internationally. Summary of the Invention

[0004] In view of the technical problems existing in medical adhesives in the prior art, the inventors of this invention modified Eucommia ulmoides gum by high-temperature heating and found that the modified Eucommia ulmoides gum has high adhesion strength and active antibacterial function, can promote skin tissue regeneration, and has the potential to become a medical adhesive, thus realizing the present invention.

[0005] In a first aspect of the invention, a method for preparing an adhesive is provided, the method comprising: heating Eucommia ulmoides gum to a temperature of 150°C-320°C and maintaining it for 60-180 minutes to modify the Eucommia ulmoides gum, wherein the Eucommia ulmoides gum is extracted from Eucommia ulmoides, a plant of the Eucommiaceae family, and contains trans-1,4-polyisoprene with a degree of polymerization of 1000-3500.

[0006] In a second aspect of the invention, an adhesive is provided, which is prepared by the method of the first aspect of the invention.

[0007] In a third aspect of the invention, the use of the adhesive of the second aspect of the invention as a medical adhesive is provided.

[0008] The adhesive of the present invention has the following beneficial effects:

[0009] 1) High adhesion strength, enabling better adhesion to skin wounds;

[0010] 2) It has better dynamic adaptability and is more suitable for dynamic skin wounds;

[0011] 3) It has an active antibacterial function, which is more conducive to the healing of skin wounds;

[0012] 4) It can promote tissue regeneration;

[0013] 5) No cytotoxicity;

[0014] 6) It has good biocompatibility and meets the hemolysis rate standards for biomedical materials;

[0015] 7) It has the potential to be used as a medical tissue adhesive. Attached Figure Description

[0016] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the accompanying drawings used in the description of the specific embodiments or the prior art will be briefly introduced below.

[0017] Figure 1 The results of the lap-shear tensile bearing strength test of adhesives 1-5 prepared according to embodiments of the present invention are shown.

[0018] Figure 2 The tensile strength test results of adhesives 1-5 prepared according to embodiments of the present invention are shown.

[0019] Figure 3 The T-peel tensile load-bearing results of adhesives 1-5 prepared according to embodiments of the present invention are shown.

[0020] Figure 4 The proton and carbon NMR spectra of the adhesive 1 prepared according to an embodiment of the present invention are shown, wherein A is the proton NMR spectrum of the adhesive 1 and B is the carbon NMR spectrum of the adhesive 1.

[0021] Figure 5 The cell survival results of L929 fibroblasts under different concentrations of the adhesive 1 extract prepared according to embodiments of the present invention are shown.

[0022] Figure 6The results of rabbit erythrocyte hemolysis rates under different concentrations of the extract of binder 1 prepared according to embodiments of the present invention are shown.

[0023] Figure 7 The figure shows test results of adhesive 1 prepared according to an embodiment of the present invention promoting the healing of linear skin wounds.

[0024] Figure 8 The figure shows test results of adhesive 1 prepared according to an embodiment of the present invention promoting the healing of exposed full-thickness skin injuries. Detailed Implementation

[0025] The present invention will be described in detail below. It should be understood that the following description is merely illustrative and is not intended to limit the scope of the invention; the scope of protection of the invention is defined by the appended claims. Furthermore, those skilled in the art will understand that modifications can be made to the technical solutions of the present invention without departing from its spirit and intent. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter pertains. Before a detailed description of the invention, the following definitions are provided to better understand it.

[0027] In cases where numerical ranges are provided, such as concentration ranges, percentage ranges, or ratio ranges, it should be understood that, unless the context explicitly specifies otherwise, all intermediate values ​​between the upper and lower limits of the range, up to one-tenth of the lower limit unit, and any other values ​​or intermediate values ​​within the range are included in the subject matter. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges, and such embodiments are also included in the subject matter, limited by any specific excluded limit values ​​within the range. Where the range includes one or two limit values, the range excluding any one or both of those included limit values ​​is also included in the subject matter.

[0028] In the context of this invention, many embodiments use the expressions "comprising," "including," or "basically / mainly composed of." The expressions "comprising," "including," or "basically / mainly composed of" are generally understood as open-ended expressions, indicating that they include not only the elements, components, parts, or method steps specifically listed after the expression, but also other elements, components, parts, or method steps. However, in this document, the expressions "comprising," "including," or "basically / mainly composed of" can also be understood as closed-ended expressions in certain situations, indicating that they only include the elements, components, parts, or method steps specifically listed after the expression, and do not include any other elements, components, parts, or method steps. In this case, the expression is equivalent to the expression "composed of."

[0029] In a first aspect of the invention, a method for preparing an adhesive is provided, the method comprising: heating Eucommia ulmoides gum to a temperature of 150°C-320°C and maintaining it for 60-180 minutes to modify the Eucommia ulmoides gum, wherein the Eucommia ulmoides gum is extracted from Eucommia ulmoides, a plant of the Eucommiaceae family, and contains trans-1,4-polyisoprene with a degree of polymerization of 1000-3500.

[0030] Eucommia gum is a natural product extracted from the leaves, bark, and seed hulls of the Eucommia ulmoides tree, primarily composed of trans-1,4-polyisoprene. Eucommia gum exhibits both rubber-plastic duality and shape memory properties, and displays pharmacological activities such as antibacterial activity and regulation of macrophage differentiation. Eucommia gum is widely present in fresh leaves, bark, and seed hulls of the Eucommia ulmoides tree, and can improve the mechanical properties of plant tissues. However, eucommia gum extracted from the leaves, bark, and seed hulls of the Eucommia ulmoides tree crystallizes at room temperature, transforming into a hard rubber and thus lacking adhesive properties. However, through modification treatment, eucommia gum can be endowed with good adhesive properties under room temperature conditions. Therefore, eucommia gum has the potential value for development into medical adhesives. The extraction method for obtaining high-purity eucommia gum can be found in Chinese Patent Application Publication No. CN108702930A, the entire contents of which are incorporated herein by reference.

[0031] In a preferred embodiment, the Eucommia gum (raw material) contains at least 90% trans-1,4-polyisoprene, preferably at least 96% trans-1,4-polyisoprene. The Eucommia gum (raw material) can be obtained by any extraction method known in the art, such as organic solvent extraction, biphasic extraction, biomimetic enzyme extraction, etc.

[0032] Furthermore, as verified in the Examples section, when modifying Eucommia ulmoides gum to obtain a modified product with adhesive properties, special consideration must be given to the degree of polymerization of the polymeric compound trans-1,4-polyisoprene in the raw Eucommia ulmoides gum. Only when the degree of polymerization of the polymeric compound trans-1,4-polyisoprene in Eucommia ulmoides gum is between 1000 and 3500 can it be effectively used as a raw material to prepare a product that can be used as an adhesive; if the degree of polymerization is too high or too low, an adhesive cannot be prepared. This finding suggests that when implementing the method of the present invention, if the degree of polymerization of trans-1,4-polyisoprene contained in the raw Eucommia ulmoides gum exceeds 3500, it is advisable to first reduce its degree of polymerization to the above range through methods such as ultraviolet degradation, thermal degradation, or electrochemical degradation.

[0033] In this invention, the heating temperature needs to be controlled between 150℃ and 320℃. Specifically, the eucommia gum can be heated to 150℃, 160℃, 170℃, 180℃, 190℃, 200℃, 210℃, 220℃, 230℃, 240℃, 245℃, 250℃, 255℃, 260℃, 265℃, 270℃, 275℃, 280℃, 285℃, 290℃, 295℃, 300℃, 310℃, or 320℃, or a range consisting of any two of these values. The inventors have discovered through experiments that modifying the eucommia gum within the specified temperature range can produce a modified product with adhesive properties, while modifying the eucommia gum outside this temperature range results in a product without adhesive properties.

[0034] In a preferred embodiment, the heating temperature can be 160°C-300°C.

[0035] In a more preferred embodiment, the heating temperature can be 200°C-285°C.

[0036] Additionally, the eucommia gum can be modified at this temperature for 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, or 180 minutes, or any range of two of these values.

[0037] The inventors have discovered that by modifying Eucommia ulmoides gum within the specified temperature range for the specified duration, the resulting modified product possesses adhesive properties, while products with adhesive properties cannot be obtained if the modification time is too long or too short.

[0038] In a preferred embodiment, the holding time can be 90-150 minutes.

[0039] In a more preferred embodiment, the holding time can be 100-130 minutes.

[0040] In one embodiment, the adhesive prepared by the method comprises a polymer 1,4-polyisoprene consisting of structural unit I and structural unit II as shown below, wherein the weight ratio of structural unit I to structural unit II is 1-15:1.

[0041]

[0042] In one specific embodiment, the weight ratio of structural unit I and structural unit II can be, for example, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, or 15:1, or a range consisting of any two of these values. The inventors unexpectedly discovered that by heating and modifying natural Eucommia gum to adjust the weight ratio of structural unit I and structural unit II to within the above range, the resulting modified Eucommia gum possesses adhesive properties, while modified Eucommia gum outside this ratio range does not possess adhesive properties.

[0043] In a preferred embodiment, the weight ratio of structural unit I to structural unit II is 4-13:1, preferably 5-9:1, and more preferably 6-8:1.

[0044] In yet another embodiment, in the adhesive of the present invention, the degree of polymerization of the polymer 1,4-polyisoprene is 110-1400, for example 110, 120, 130, 140, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300 or 1400, or a range consisting of any two of these values.

[0045] It is understood that the (average) degree of polymerization = the (average) molecular weight of the polymer / the molecular weight of the structural unit. The inventors unexpectedly discovered that by heating and modifying natural Eucommia gum to control the degree of polymerization of polymer 1,4-polyisoprene within the aforementioned range, the resulting modified Eucommia gum exhibits adhesive properties, while modified Eucommia gum outside this range does not possess adhesive characteristics.

[0046] In a preferred embodiment, in the adhesive of the present invention, the polymer 1,4-polyisoprene has a degree of polymerization of 400-800.

[0047] In one embodiment, the adhesive has an lap-shear tensile strength of at least 1 kPa, preferably at least 5 kPa, more preferably at least 10 kPa, as determined according to the YY / T 0729.1-2009 standard.

[0048] In one embodiment, the adhesive has a tensile bearing strength of at least 20 kPa, preferably at least 100 kPa, more preferably at least 150 kPa, as determined according to the YY / T 0729.3-2009 standard.

[0049] In one embodiment, the adhesive has a strength of at least 15 J / m³. 2 Preferably at least 40 J / m 2 More preferably at least 60 J / m 2 The interfacial toughness was determined according to the YY / T 0729.2-2009 standard.

[0050] In one embodiment, the method further includes filtering the modified Eucommia gum through a 40-60 mesh sieve, for example, a 50 mesh sieve, after heating has stopped.

[0051] As mentioned above, this invention modifies Eucommia ulmoides gum by high-temperature heating, and finds that the resulting modified Eucommia ulmoides gum exhibits high adhesion strength, good dynamic compatibility, good subcutaneous tissue compatibility, cell biocompatibility and blood compatibility, active antibacterial function and tissue regeneration promotion ability, and has the potential to be used as a medical adhesive.

[0052] In a second aspect of the invention, an adhesive is provided, which is prepared by the method of the first aspect of the invention.

[0053] In a third aspect of the invention, the use of the adhesive of the second aspect of the invention as a medical adhesive is provided.

[0054] In one embodiment, the adhesive is used to adhere an open wound to the skin.

[0055] In a preferred embodiment, the adhesive is used to promote the healing of linear skin wounds or to promote the healing of exposed full-thickness skin injuries.

[0056] The adhesive of the present invention has the following beneficial effects:

[0057] 1) High adhesion strength, enabling better adhesion to skin wounds;

[0058] 2) It has better dynamic adaptability and is more suitable for dynamic skin wounds;

[0059] 3) It has an active antibacterial function, which is more conducive to the healing of skin wounds;

[0060] 4) It can promote tissue regeneration and accelerate the healing process;

[0061] 5) No cytotoxicity;

[0062] 6) It has good biocompatibility and meets the hemolysis rate standards for biomedical materials.

[0063] 7) The healed skin surface is smooth and flat, without scars, and without damage to skin appendages or other tissues.

[0064] Example

[0065] The following examples illustrate the products, preparation methods, and related characterization of the present invention. Unless otherwise specified, all experimental methods used are conventional methods, and all experimental materials used in the following examples were purchased from conventional chemical reagent stores. % represents wt%, i.e., weight percentage. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0066] Preparation example: Preparation of adhesives

[0067] Preparation of Eucommia gum:

[0068] The crushed Eucommia ulmoides seed shells were enzymatically hydrolyzed and then extracted with petroleum ether at 60°C for 3 hours. The extract was then poured into a beaker, and 30% (by volume) of 95% ethanol was added to precipitate the Eucommia ulmoides gum. The gum was then air-dried at room temperature in a fume hood to obtain the Eucommia ulmoides gum product. Testing revealed that the trans-1,4-polyisoprene contained in this Eucommia ulmoides gum product had a degree of polymerization of 5048 and a molecular weight of 34.33 × 10⁻⁶. 4 g / mol.

[0069] The crushed Eucommia ulmoides seed shells were enzymatically hydrolyzed and then extracted with petroleum ether at 60°C for 3 hours. The extract was then poured into a beaker, and 30% (by volume) of 95% ethanol was added to precipitate the Eucommia ulmoides gum. The gum was then air-dried at room temperature in a fume hood and irradiated under ultraviolet light for 24 hours to obtain the Eucommia ulmoides gum product. Testing revealed that the trans-1,4-polyisoprene contained in this Eucommia ulmoides gum product had a degree of polymerization of 3870 and a molecular weight of 26.32 × 10⁻⁶. 4 g / mol.

[0070] The crushed Eucommia ulmoides seed shells were enzymatically hydrolyzed and then extracted with petroleum ether at 60°C for 3 hours. The extract was then poured into a beaker, and 30% (by volume) of 95% ethanol was added to precipitate the Eucommia ulmoides gum. The gum was then air-dried at room temperature in a fume hood to obtain the Eucommia ulmoides gum product. The dried gum was transferred to a high-temperature furnace and heated at 150°C for 96 hours to obtain the final Eucommia ulmoides gum product. Analysis revealed that the degree of polymerization of trans-1,4-polyisoprene in this Eucommia ulmoides gum product was 2544, and the molecular weight was 17.3 × 10⁻⁶. 4 g / mol.

[0071] After enzymatic hydrolysis of crushed Eucommia ulmoides seed shells, extraction was carried out with petroleum ether at 60℃ for 3 hours. The extract was then poured into a beaker, and 30% (by volume) of 95% ethanol was added to precipitate Eucommia ulmoides gum. The gum was then air-dried at room temperature in a fume hood to obtain the Eucommia ulmoides gum product. Alternatively, 10g of the Eucommia ulmoides gum product was dissolved in 1000mL of toluene, and 50mg of titanium dioxide and 0.3g of hydrogen peroxide solution were added. The mixture was stirred for 4 hours under a deuterium lamp or ultraviolet lamp at a controlled temperature of 25-40℃. After filtering to remove titanium dioxide, 30% (by volume) of 95% ethanol was added to precipitate Eucommia ulmoides gum. The gum was then air-dried at room temperature in a fume hood to obtain the final Eucommia ulmoides gum product. Analysis revealed that the degree of polymerization of trans-1,4-polyisoprene in this Eucommia ulmoides gum product was 1159, and the molecular weight was 7.88 × 10⁻⁶. 4 g / mol.

[0072] Adhesive preparation

[0073] Adhesive 1

[0074] (1) Weigh 50.0g of Eucommia ulmoides gum (degree of polymerization 2544, molecular weight 17.3×10). 4 (g / mol) in a high-temperature resistant glass flask.

[0075] (2) Place the high-temperature resistant glass flask containing Eucommia ulmoides gum into the MF-1200C high-temperature box furnace (Anhui Beiyike Equipment Technology Co., Ltd.), set the heating program: 0-20 minutes, heat up to 270℃, maintain heating at 270℃ for 120 minutes, and then stop heating.

[0076] (3) Pour the eucommia gum that has stopped heating into a 50-mesh (0.85 mm) pharmacopoeia sieve and cool it to below 40°C to obtain adhesive 1.

[0077] Adhesive 2

[0078] Adhesive 2 was prepared using the same method as adhesive 1, except that it was heated at 270°C for 180 minutes.

[0079] Adhesive 3

[0080] Adhesive 3 was prepared using the same method as adhesive 1, except that the temperature was raised to 285°C in 0-20 minutes and maintained at 285°C for 120 minutes.

[0081] Adhesive 4

[0082] (1) Weigh 50.0g of Eucommia ulmoides gum (degree of polymerization 1159, molecular weight 7.88×10). 4(g / mol, self-made) in a high-temperature resistant glass flask.

[0083] (2) Place the high-temperature resistant glass flask containing Eucommia ulmoides gum into an MF-1200C high-temperature box furnace (Anhui Beiyike Equipment Technology Co., Ltd.), set the heating program: 0-20 minutes, heat up to 200℃, and maintain the heating at 200℃ for 120 minutes to obtain adhesive 4.

[0084] Adhesive 5

[0085] (1) Weigh 50.0g of Eucommia ulmoides gum (degree of polymerization 1159, molecular weight 7.88×10). 4 (g / mol, self-made) in a high-temperature resistant glass flask.

[0086] (2) Place the high-temperature resistant glass flask containing Eucommia ulmoides adhesive into an MF-1200C high-temperature box furnace (Anhui Beiyike Equipment Technology Co., Ltd.), set the heating program: 0-20 minutes, heat up to 160℃, and keep heating at 160℃ for 120 minutes to obtain adhesive 5.

[0087] Comparative Example 1

[0088] The same method as for preparing adhesive 1 was used, except that the sample was charred after heating to 330°C for 0-20 minutes and then kept at 330°C for 60 minutes, and a semi-fluid adhesive could not be obtained.

[0089] Comparative Example 2

[0090] The same method as for preparing adhesive 1 was used, except that the temperature was raised to 120°C in 0-10 minutes and maintained at 120°C for 240 minutes. The resulting sample had no flowability and no adhesion.

[0091] Comparative Example 3

[0092] (1) Weigh 50.0g of Eucommia ulmoides gum (degree of polymerization 5048, molecular weight 34.33×10). 4 (g / mol, self-made) in a high-temperature resistant glass flask.

[0093] (2) Place the high-temperature resistant glass flask containing Eucommia ulmoides gum into the MF-1200C high-temperature box furnace (Anhui Beiyike Equipment Technology Co., Ltd.), set the heating program: 0-20 minutes, heat up to 270℃, and maintain the heating at 270℃ for 120 minutes.

[0094] However, the product obtained by this preparation method cannot be used as an adhesive because it lacks adhesive properties.

[0095] Comparative Example 4

[0096] (1) Weigh 50.0g of Eucommia ulmoides gum (degree of polymerization 3870, molecular weight 26.32×10⁻⁶). 4 (g / mol, self-made) in a high-temperature resistant glass flask.

[0097] (2) Place the high-temperature resistant glass flask containing Eucommia ulmoides gum into the MF-1200C high-temperature box furnace (Anhui Beiyike Equipment Technology Co., Ltd.), set the heating program: 0-20 minutes, heat up to 270℃, and maintain the heating at 270℃ for 120 minutes.

[0098] However, the product obtained by this preparation method cannot be used as an adhesive because it lacks adhesive properties.

[0099] Experimental Example 1: Overlap-Shear Tensile Bearing Strength Test of Adhesive

[0100] Experimental materials: Adhesives 1-5, porcine fibrin adhesive (Harbin Hanbang Medical Technology Co., Ltd.).

[0101] Experimental instrument: TA-XTplusC universal testing machine: Xiamen Chaoji Instrument Equipment Co., Ltd.

[0102] According to the People's Republic of China pharmaceutical industry standard "Test Methods for Tissue Adhesive Performance" YY / T0729.1-2009, the lap-shear tensile bearing strength of adhesives 1-5 and porcine fibrin adhesive was determined. The specific steps are as follows:

[0103] Take 0.2g of each of the adhesives 1-5 and porcine fibrin adhesive and apply them evenly to the overlap area of ​​the pigskin strips. The overlap area should be 10mm long and 25mm wide. Adhere the two pigskin strips of a pair of test fixtures together along the overlap area. Apply a force of approximately 2N to the overlap area or use a clamp to hold the adhesive area flat until the overlap is tightly bonded (approximately 5 minutes). Place the specimen in the fixture of the testing machine, ensuring the force direction is along the long axis of the specimen. Apply a load to the specimen at a speed of 5mm / min until failure, and record the overlap-shear tensile bearing strength. The test environment temperature is 30±1℃ and the relative humidity is 50±5%. The experimental results are as follows: Figure 1 As shown.

[0104] Depend on Figure 1It can be seen that the lap-shear tensile bearing strengths of adhesives 1-5 are 10.94 kPa, 5.16 kPa, 2.21 kPa, 1.32 kPa, and 2.07 kPa, respectively, while the lap-shear tensile bearing strength of the porcine fibrin adhesive is 5.89 kPa. It is evident that the adhesives of the present invention exhibit high lap-shear tensile bearing strengths, with adhesive 1 exhibiting the highest lap-shear tensile bearing strength, indicating that it has the strongest maximum load-bearing capacity under parallel shear stress.

[0105] Experimental Example 2: Tensile Bearing Strength Test of Adhesive

[0106] Experimental materials: Adhesives 1-5, porcine fibrin adhesive (Harbin Hanbang Medical Technology Co., Ltd.).

[0107] Experimental instrument: TA-XTplusC universal testing machine: Xiamen Chaoji Instrument Equipment Co., Ltd.

[0108] According to the People's Republic of China pharmaceutical industry standard "Test Methods for Tissue Adhesive Performance" YY / T0729.3-2009, the tensile bearing strength of adhesives 1-5 and porcine fibrin adhesives was determined. The specific steps are as follows:

[0109] Take 0.2g of each of the adhesives 1-5 and porcine fibrin adhesive and apply them evenly to the overlapping area of ​​the porcine skin strip. The overlapping area should be 25mm long and 25mm wide. Adhere the tissue surfaces of the two porcine skin strips of a pair of test fixtures together, ensuring the two fixtures are aligned and properly aligned. Apply a force of approximately 2N to the overlapping area or use a clamp to hold the adhesive area flat until the overlapping area is tightly bonded (approximately 5 minutes). Place the specimen in the fixture of the testing machine, and apply a load to the specimen at a speed of 2mm / min until failure. Record the maximum tensile strength. The test environment temperature is 30±1℃ and the relative humidity is 50±5%. The experimental results are as follows: Figure 2 As shown.

[0110] Depend on Figure 2 It can be seen that the tensile bearing strengths of adhesives 1-5 are 161.85 kPa, 107.43 kPa, 40.57 kPa, 30.20 kPa, and 19.47 kPa, respectively, while the tensile bearing strength of the porcine fibrin adhesive is 52.48 kPa. It is evident that the adhesives of the present invention exhibit high tensile bearing strength, with adhesive 1 exhibiting the highest tensile bearing strength, indicating that it has a stronger maximum load-bearing capacity under parallel shear stress.

[0111] Experimental Example 3: T-peel tensile strength test of adhesives

[0112] Experimental materials: Adhesives 1-5, porcine fibrin adhesive (Harbin Hanbang Medical Technology Co., Ltd.).

[0113] Experimental instrument: TA-XTplusC universal testing machine: Xiamen Chaoji Instrument Equipment Co., Ltd.

[0114] According to the People's Republic of China pharmaceutical industry standard "Test Methods for Tissue Adhesive Performance" YY / T0729.2-2009, the T-peel tensile strength of adhesives 1-5 and porcine fibrin adhesives was determined. The specific steps are as follows:

[0115] Take 0.2g of each of the adhesives 1-5 and porcine fibrin adhesive and apply them evenly to the bonding area of ​​the pigskin strip. The bonding area should be 125mm long and 25mm wide. Place another uncoated sample on top of the coated sample. Apply a force of approximately 5-10N to the overlap area or use a clamp to hold the bonding area flat until the overlap area is tightly bonded (approximately 5 minutes). Place the uncoated end of the sample in the clamp of the testing machine, and apply a load to the sample with the crosshead at a speed of 250mm / min. Record the maximum interfacial toughness. The test environment temperature is 30±1℃ and the relative humidity is 50±5%. The experimental results are as follows: Figure 3 As shown.

[0116] Depend on Figure 3 It can be seen that the interfacial toughness of adhesives 1-5 is 62.65 J / m. 2 40.16J / m 2 20.21 J / m 2 17.37 J / m 2 18.10 J / m 2 The interfacial toughness of the porcine fibrin adhesive is 23.81 J / m. 2 It is evident that the adhesives of the present invention exhibit high interfacial toughness, with adhesive 1 exhibiting the highest interfacial toughness, indicating that it has a stronger anti-delamination ability at the interface with pigskin tissue.

[0117] Experiment Example 4: Detection of adhesives by 1H and 1C NMR spectra

[0118] Experimental materials: Adhesives 1-5; deuterated chloroform (Saipu Ruisi (Beijing) Technology Co., Ltd.).

[0119] Experimental instruments: JNM-ECZ600R / S1 nuclear magnetic resonance spectrometer (Nippon Electron Ltd.); SQP 1 / 100,000 balance (Sartorius Scientific Instruments (Beijing) Co., Ltd.).

[0120] Accurately weigh 20 mg of adhesive 1 and place it in a test tube. Add 0.5 mL of deuterated chloroform to completely dissolve the adhesive 1. Transfer the solution to an NMR tube and place it in an NMR spectrometer to detect the proton and carbon spectra of adhesive 1. The results are as follows. Figure 4 As shown.

[0121] Depend on Figure 4 It is known that the main component of adhesive 1 is polymer 1,4-polyisoprene, which is composed of structural units derived from trans-1,4-isoprene and structural units derived from cis-1,4-isoprene, wherein the weight ratio of structural units derived from trans-1,4-isoprene to structural units derived from cis-1,4-isoprene is 7.73:1.

[0122] In addition, the NMR 1H and 1C spectra of adhesives 2-5 are determined by the same method as those of adhesive 1. Their main component is also polymer 1,4-polyisoprene, which is composed of structural units derived from trans-1,4-isoprene and structural units derived from cis-1,4-isoprene. The weight ratios of structural units derived from trans-1,4-isoprene and structural units derived from cis-1,4-isoprene are 6:1, 4:1, 13:1, and 11:1, respectively.

[0123] Experimental Example 5: Gel Chromatography Detection of Adhesives

[0124] Experimental materials: binders 1-5; tetrahydrofuran (TEDIA, USA); KF804L gel chromatography column (Showa Denko, Japan); narrow distribution polystyrene (TOSOH, Japan).

[0125] Experimental instruments: LC20 high performance liquid chromatograph (Shimadzu Corporation, Japan); RID-20 differential refractive index detector (Shimadzu Corporation, Japan); SQP 1 / 100,000 balance (Sartorius Scientific Instruments (Beijing) Co., Ltd.).

[0126] Accurately weigh 10 mg of adhesive 1 and place it in a 10 mL volumetric flask. Add 5 mL of tetrahydrofuran to completely dissolve the adhesive 1, and continue adding tetrahydrofuran to bring the volume to the mark. Accurately pipette 10 μL of the adhesive 1 sample solution for analysis. Using the polystyrene standard curve, the molecular weight of the polymer 1,4-polyisoprene (containing structural units derived from trans-1,4-isoprene and structural units derived from cis-1,4-isoprene) in adhesive 1 is calculated to be 4.41 × 10⁻⁶. 4 g / mol, degree of polymerization is 648.

[0127] The molecular weight and degree of polymerization of adhesives 2-5 were determined using the same method as those of adhesive 1. The molecular weight and degree of polymerization of polymer 1,4-polyisoprene (which contains structural units derived from trans-1,4-isoprene and structural units derived from cis-1,4-isoprene) contained therein are shown in Table 1.

[0128] Table 1

[0129]

[0130]

[0131] Experimental Example 5: In vitro cytotoxicity test of adhesives

[0132] Experimental cells: L929 fibroblasts, purchased from Xiamen Yimo Biotechnology Co., Ltd.

[0133] Experimental materials: Adhesive 1, DMEM high-glucose culture medium (Soluble Biotech, Beijing), fetal bovine serum (Gibco), MTT assay kit (Soluble Biotech, Beijing).

[0134] Experimental instrument: Thermo Fisher Scientific 1510 full-wavelength microplate reader, purchased from Thermo Fisher Scientific (China) Co., Ltd.

[0135] According to GB / T 16886.5-2017, "In vitro cytotoxicity testing" for biological evaluation of medical devices, the in vitro cell compatibility of adhesive 1 was evaluated. The specific steps are as follows:

[0136] According to the extraction conditions guidelines in GB / T 16886.5-2017 "In Vitro Cytotoxicity Tests" for biological evaluation of medical devices, considering the low polarity of Adhesive 1, a complete culture medium containing 10% (v / v) fetal bovine serum and 1% (v / v) penicillin-antibody was selected as the extraction medium. 200 mg of Adhesive 1 was placed in a petri dish, 10 mL of complete culture medium was added, and the mixture was incubated in a sterile incubator for 24 h. After filtering the extract through a 0.22 μm sterile filter membrane, the extract was diluted with complete culture medium to obtain adhesive extracts with concentrations of 20, 10, 5, 2, and 1 mg / mL.

[0137] L929 fibroblasts were respectively treated with 1×10 4 Cells were seeded at a density of 100 μL per well in 96-well plates and incubated for 24 h. After cell attachment, 100 μL of the above-mentioned binder 1 solution was added to each well, with 6 replicates per group. A negative control group (L929 cells + complete culture medium) and a reference group (complete culture medium containing only the extract) were also set up and incubated for 24 h. Then, 20 μL of MTT reagent was added to each well, and the cells were incubated for 3 h. After incubation, the liquid in the wells was removed, and dimethyl sulfoxide was added to dissolve the purple crystals. After 10 min, the absorbance at 570 nm was measured using a microplate reader. Cell viability was calculated using the following formula:

[0138] Cell viability = (As-Ab) / (Ac-Ab) × 100%

[0139] In the above formula, As represents the absorbance of the experimental group, Ab represents the absorbance of the reference group, and Ac represents the absorbance of the control group.

[0140] The results are shown in Figure 5 .Depend on Figure 5 It is known that the cell survival rate of adhesive 1 is higher than 85% in the range of extract concentration of 1-20 mg / mL, and different extract concentrations have almost no effect on cell survival rate. This indicates that the adhesive of the present invention has good cell compatibility, high safety, and can be used as a medical adhesive.

[0141] Experiment Example 6: Rabbit Erythrocyte Hemolysis Rate Test of Adhesive

[0142] Experimental materials: Adhesive 1; physiological saline (Sichuan Kelun Pharmaceutical Co., Ltd.)

[0143] Experimental instrument: Thermo Fisher Scientific 1510 full-wavelength microplate reader, purchased from Thermo Fisher Scientific (China) Co., Ltd.

[0144] According to GB / T 16886.5-2017, "In vitro cytotoxicity testing" for biological evaluation of medical devices, the in vitro cell compatibility of adhesive 1 was evaluated. The specific steps are as follows:

[0145] Take 2.5 mL of 2% rabbit erythrocyte suspension and place it into a 10 mL centrifuge tube. Add 2.5 mL of different concentrations of the adhesive extract prepared in Experiment 5 (20, 10, 5, 2, 1 mg / mL), and simultaneously add 2.5 mL of physiological saline and distilled water as the reference group and control group, respectively. After incubating in a 37℃ incubator for 3 h, centrifuge at 1500 rpm for 5 min, collect the supernatant, and measure the absorbance of each group at a wavelength of 540 nm. Calculate the hemolysis rate according to the following formula:

[0146] Hemolysis rate = (As-Ab) / (Ac-Ab) × 100%

[0147] In the above formula, As represents the absorbance of the experimental group, Ab represents the absorbance of the reference group, and Ac represents the absorbance of the control group.

[0148] The results are shown in Figure 6 .Depend on Figure 6 It can be seen that the hemolysis rate of adhesive 1 is less than 5% in the range of 1-20 mg / mL of extract concentration, and even less than 1% when the concentration of adhesive extract is 1-5 mg / mL. This indicates that the adhesive of the present invention has good blood compatibility, high safety, and can be used as a medical adhesive.

[0149] Experimental Example 7: In vivo biodegradability test of adhesives

[0150] Experimental materials: Adhesive 1

[0151] The biocompatibility and biodegradability of adhesive 1 were evaluated using a rat subcutaneous implantation model.

[0152] Experimental Procedure: Twelve male SD rats (180-200g) were acclimatized for 3 days, then anesthetized with an intraperitoneal injection of 1% sodium pentobarbital (5mL / kg). A 2cm linear skin wound was created on the back (3.5cm in a straight line from the ear). 0.3g of adhesive 1 was implanted subcutaneously, and the wound was sutured with surgical sutures. The control group had a skin wound created at the same location without implantation. After treatment, the rats were housed individually in cages with free access to food. The wound sites were photographed on days 0, 7, 14, and 28. On days 7, 14, and 28, three rats were anesthetized and euthanized, and the implanted adhesive 1 was removed, weighed, and the degradation rate was calculated. The results are shown in Table 2.

[0153] Skin tissue (3×3cm) from the wound was taken for pathological staining, and the biocompatibility of adhesive 1 with rat subcutaneous tissue was analyzed. The results are shown in Table 3.

[0154] Table 2

[0155] Time (days) Degradation rate (%) 7 6.09±1.63 14 12.99±1.67 28 37.85±2.92 56 59.00±8.90

[0156] Table 3

[0157]

[0158] As shown in Table 2, the degradation rate of adhesive 1 was as high as 59.00% after 56 days of application, indicating that the adhesive of the present invention has good in vivo biodegradability. Furthermore, no tissue necrosis or inflammatory lesions were found in the surrounding tissues, which also indicates that the product has good biosafety.

[0159] As shown in Table 3, the adhesive of the present invention has good biocompatibility in vivo.

[0160] Experiment Example 8: Test on the effect of adhesives on linear skin wound healing

[0161] Experimental materials: Adhesive 1; Surgical sutures (Shanghai Pudong Jinhuan Medical Supplies Co., Ltd.); Ankejing adhesive (Harbin Hanbang Medical Technology Co., Ltd.); Kangpaite adhesive (Beijing Kangpaite Medical Devices Co., Ltd.)

[0162] A rat linear skin incision model was used to evaluate the promoting effect of adhesive 1 on the healing of linear skin incisions in rats. The specific steps are as follows:

[0163] Fifteen male SD rats (180-200g) were acclimatized for 3 days. Twelve hours before modeling, the hair on their backs was removed. After anesthesia with an intraperitoneal injection of 1% sodium pentobarbital (5mL / kg), a 1.5cm linear skin wound was created on each side of the back (3.5cm in a straight line from the ear), with a depth equal to the skin thickness. The rats were randomly divided into 5 groups of 3 rats each. The wounds of each group underwent the following treatments: no treatment (blank control group), suturing (surgical suturing group), application of Ancocrystalline adhesive (positive control group), application of Kangpaite adhesive (positive control group), and application of adhesive 1 (experimental group). After treatment, the rats were housed individually with free access to food. On day 7, after anesthesia, skin tissue (3×3cm) from the wounds was collected for pathological staining analysis. The results are shown in Table 4. Figure 7 As shown.

[0164] Table 4

[0165]

[0166] From Table 4 and Figure 7 It is understood that the adhesive 1 of the present invention can promote the healing of linear skin wounds, and its healing effect on linear skin wounds is superior to that of surgical sutures, Compal adhesive and Anco Crystal adhesive currently used in clinical practice.

[0167] Experiment Example 9: Test on the effect of adhesives on promoting wound healing of exposed full-thickness skin injuries

[0168] Experimental materials: Adhesive 1; Alginate dressing (Minnesota Mining Manufacturing Company).

[0169] A rat model of exposed full-thickness skin injury was used to evaluate the promoting effect of adhesive 1 on wound healing of exposed full-thickness skin injury in rats. The specific steps are as follows:

[0170] Twenty-seven male SD rats (180-200g) had their back hair removed 12 hours before modeling. After anesthesia with 1% sodium pentobarbital (5mL / kg) via intraperitoneal injection, the back skin was disinfected with povidone-iodine. A 10mm diameter skin biopsy puncture device was used to establish one tissue defect model on each side of the rat's back (3.5cm in a straight line from the ear). The diameter of the skin defect on the rat's back was 10mm, and the depth was equal to the skin thickness. The rats were randomly divided into three groups of nine rats each. The wounds of each group were treated as follows: no treatment (blank control group), alginate dressing (positive control group), and adhesive 1 applied (experimental group). On day 12, after anesthesia, skin tissue (3×3cm) from the wounds was collected for pathological staining analysis. The results are shown in Table 5. Figure 8 As shown.

[0171] Table 5

[0172]

[0173] From Table 5 and Figure 8 It is understood that the adhesive of the present invention can promote the healing of exposed full-thickness skin injuries, and its healing effect on exposed full-thickness skin injuries is superior to that of currently used alginate dressings in clinical practice.

[0174] It should be noted that the terminology used in this specification is for the purpose of describing specific embodiments only and is not intended to limit the invention. The foregoing summary section and the following detailed description are for illustrative purposes only and are not intended to limit the invention in any way. The scope of the invention is defined by the appended claims without departing from its spirit and intent.

Claims

1. A method for preparing an adhesive, the method comprising: Eucommia gum is heated to 150℃-320℃, preferably 160℃-300℃, more preferably 200℃-285℃ and held for 60-180 minutes, preferably 90 minutes-150 minutes, more preferably 100 minutes-130 minutes to modify the eucommia gum, which is extracted from Eucommia ulmoides, a plant of the Eucommiaceae family, and contains trans-1,4-polyisoprene with a degree of polymerization of 1000-3500.

2. The method according to claim 1, wherein, The adhesive comprises a polymer 1,4-polyisoprene consisting of structural unit I and structural unit II as shown below, wherein the weight ratio of structural unit I to structural unit II is 1-15:

1.

3. The method according to claim 2, wherein, The weight ratio of structural unit I to structural unit II is 4-13:1, preferably 5-9:1, and even more preferably 6-8:

1.

4. The method according to claim 2 or 3, wherein, The degree of polymerization of the polymer 1,4-polyisoprene is 110-1400, preferably 400-800.

5. The method according to any one of claims 1 to 4, wherein, The adhesive has an lap-shear tensile strength of at least 1 kPa, preferably at least 5 kPa, more preferably at least 10 kPa, as determined according to YY / T 0729.1-2009 standard.

6. The method according to any one of claims 1 to 5, wherein, The adhesive has a tensile bearing strength of at least 20 kPa, preferably at least 100 kPa, and more preferably at least 150 kPa, as determined according to the YY / T 0729.3-2009 standard.

7. The method according to any one of claims 1-6, wherein, The adhesive has a strength of at least 15 J / m 2 Preferably at least 40 J / m 2 More preferably at least 60 J / m 2 The interfacial toughness was determined according to the YY / T0729.2-2009 standard.

8. An adhesive prepared by any one of claims 1 to 7.

9. Use of the adhesive of claim 8 as a medical adhesive; preferably, the adhesive is used to adhere open wounds of the skin.

10. The use according to claim 9, wherein the adhesive is used to promote the healing of linear skin wounds or to promote the healing of exposed full-thickness skin injuries.

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