Antibacterial cationic hydrogel adhesive and preparation method thereof

Abstract

The invention provides an antibacterial cationic hydrogel adhesive and a preparation method thereof. The antibacterial cationic hydrogel adhesive is prepared from the following raw materials: polyphenol grafted epsilon-polylysine; the end group of the four-arm polyethylene glycol is phthalic dialdehyde; the polyphenol grafted epsilon-polylysine polymer has a structure as shown in a formula I or a formula II. The hydrogel adhesive contains epsilon-polylysine, so that the hydrogel adhesive can resist bacteria for a long time, and drug resistance is avoided; the grafted polyphenol functional group can effectively remove excessive free radicals and has an anti-oxidation function, so that wound healing is promoted, and tissue adhesiveness can be enhanced; and the tissue adhesiveness is provided by utilizing the rapid cross-linking gel formation of o-phthalaldehyde-amine.

Description

Technical Field

[0001] The invention belongs to the technical field of adhesives, and in particular relates to an antibacterial cationic hydrogel adhesive and a preparation method thereof. Background Art

[0002] Hydrogel is a type of material with a three-dimensional network structure composed of hydrophilic polymers, which can swell in water or biological fluids without dissolving. Because hydrogel retains a large amount of water, it has a structure similar to that of the extracellular matrix to a certain extent, showing good biocompatibility, controllable components and ideal mechanical properties. The moist and soft properties of hydrogel materials can greatly reduce damage to cells. In addition, the porous structure inside the hydrogel can absorb wound exudate, facilitate cell growth and activity, and promote the exchange of oxygen, carbon dioxide and nutrients. It is widely used in biomedical fields such as tissue repair, tissue engineering, three-dimensional cell culture, and drug delivery.

[0003] Traditional medical bonding methods include sutures, tissue staples, medical tapes, etc. However, sutures and staples usually cause secondary tissue damage, have the risk of leakage and bacterial infection, and are time-consuming and not suitable for emergency situations. The emergence of tissue adhesives provides a more convenient means of wound closure for surgical operations. The use of tissue adhesives can not only reduce the difficulty of operation, reduce the operation time, but also reduce the occurrence of surgical complications (such as infection). In some special application scenarios (such as microsurgery and liquid / gas sealing, etc.), the advantages of tissue adhesives are more obvious, and the application prospects are huge. They are used in various biomedical fields, such as wound closure, tissue sealing and tissue regeneration. This is also the main reason for the increasing market demand for tissue adhesives. However, it is still a great challenge to develop a soft tissue adhesive composed entirely of natural products, with ideal wet adhesion strength, biocompatibility, biodegradability and economic feasibility. Summary of the invention

[0004] In view of this, the object of the present invention is to provide an antibacterial cationic hydrogel adhesive and a preparation method thereof, wherein the hydrogel has an antioxidant function and relatively excellent tissue adhesion.

[0005] The present invention provides an antibacterial cationic hydrogel adhesive, the preparation raw materials of which include polyphenol-grafted ε-polylysine;

[0006] and four-arm polyethylene glycol with o-phthalaldehyde as terminal group;

[0007] The ε-polylysine of the polyphenol has a structure of Formula I or Formula II:

[0008]

[0009] Preferably, the mass ratio of the polyphenol-grafted ε-polylysine to the four-arm polyethylene glycol with terminal groups of o-phthalaldehyde is 1:1 to 1:4.

[0010] Preferably, the four-arm polyethylene glycol with o-phthalaldehyde as the terminal group has the structure of formula III:

[0011] 1≤p≤1000.

[0012] Preferably, 1≤m≤15, 1≤n≤15.

[0013] The present invention provides a method for preparing the antibacterial cationic hydrogel adhesive described in the above technical solution, comprising the following steps:

[0014] Dissolving a polymer of polyphenol-grafted ε-polylysine having structure Ⅰ or formula Ⅱ in a solvent to obtain a first solution;

[0015] dissolving four-arm polyethylene glycol having an end group of o-phthalaldehyde in a solvent to obtain a second solution;

[0016] The first solution and the second solution are mixed and reacted to obtain an antibacterial cationic hydrogel adhesive;

[0017] The reaction temperature of the first solution and the second solution is 0 to 60° C. and the reaction time is 0 to 20 minutes.

[0018] Preferably, the solvents in the first solution and the second solution are independently selected from water, physiological saline or buffer solution.

[0019] Preferably, the mass volume concentration of the polymer of polyphenol grafted ε-polylysine having structure I or II in the first solution is 5 to 15% (w / v);

[0020] The mass volume concentration of the four-arm polyethylene glycol with terminal groups of o-phthalaldehyde in the second solution is 5-15% (w / v).

[0021] Preferably, the polyphenol-grafted ε-polylysine polymer is prepared according to the following method:

[0022] Activating gallic acid or 3,4-dihydroxyphenylpropionic acid and reacting it with ε-polylysine, dialyzing the reaction product, and freeze-drying it to obtain a polymer of ε-polylysine grafted with polyphenol;

[0023] The reaction temperature is 10-40°C and the reaction time is 4-10 hours.

[0024] The present invention provides an antibacterial cationic hydrogel adhesive, the preparation raw materials include polyphenol grafted ε-polylysine polymer; and four-arm polyethylene glycol with terminal groups of o-phthalaldehyde; the polyphenol grafted ε-polylysine polymer has a structure of formula I or formula II. The hydrogel adhesive contains ε-polylysine, which can be long-lasting antibacterial and avoid drug resistance; the grafted polyphenol functional groups can effectively remove excess free radicals, have antioxidant function, thereby promoting wound healing, and can also enhance tissue adhesion; o-phthalaldehyde-amine cross-linking gelation is fast, providing tissue adhesion. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a hydrogen nuclear magnetic resonance spectrum of gallic acid grafted ε-polylysine (ε-PLL-GA) prepared in Example 1 of the present invention;

[0026] Figure 2 The hydrogen nuclear magnetic resonance spectrum of ε-polylysine (ε-PLL-CA) grafted with 3,4-dihydroxyphenylpropionic acid prepared in Example 1 of the present invention;

[0027] Figure 3 The hydrogen nuclear magnetic resonance spectrum of the four-arm polyethylene glycol with o-phthalaldehyde as the terminal group prepared in Example 5 of the present invention;

[0028] Figure 4 This is a rheological test graph of the hydrogel (ε-PLL-GA / 4aPEG-OPA) formed by the ε-polylysine grafted with gallic acid and the four-arm polyethylene glycol with o-phthalaldehyde as the terminal group in Example 6 of the present invention;

[0029] Figure 5 This is a rheological test graph of the hydrogel (ε-PLL-CA / 4aPEG-OPA) formed by ε-polylysine grafted with 3,4-dihydroxyphenylpropionic acid and four-arm polyethylene glycol with o-phthalaldehyde as the terminal group in Example 6 of the present invention;

[0030] Figure 6 This is a schematic diagram of the hydrogel prepared in Example 6 of the present invention;

[0031] Figure 7 is the lap shear strength of the two hydrogels in Example 7 of the present invention;

[0032] Figure 8 is the compressive strength of the two hydrogels in Example 8 of the present invention;

[0033] Fig. 9 is the load of the compression test of the two hydrogels in Example 8 of the present invention;

[0034] Fig.10 is the minimum inhibitory concentration of gallic acid-grafted ε-polylysine against Escherichia coli at different concentrations in Example 9 of the present invention;

[0035] Fig.11 This is a graph showing the minimum inhibitory concentration of ε-polylysine grafted with 3,4-dihydroxyphenylpropionic acid against Escherichia coli at different concentrations in Example 9 of the present invention;

[0036] Fig.12 This is a graph showing the minimum inhibitory concentration of ε-polylysine grafted with gallic acid against Staphylococcus aureus at different concentrations in Example 10 of the present invention;

[0037] Fig.13 This is a graph showing the minimum inhibitory concentration of ε-polylysine grafted with 3,4-dihydroxyphenylpropionic acid against Staphylococcus aureus at different concentrations in Example 10 of the present invention;

[0038] Fig.14 The antibacterial effect of the hydrogel formed by the polyphenol-grafted ε-polylysine and the four-arm polyethylene glycol with the terminal group of o-phthalaldehyde on Escherichia coli prepared in Example 11 of the present invention;

[0039] Fig.15 This is the antibacterial effect of the hydrogel formed by polyphenol-grafted ε-polylysine and four-arm polyethylene glycol with o-phthalaldehyde as the terminal group on Staphylococcus aureus prepared in Example 12 of the present invention. DETAILED DESCRIPTION

[0040] The present invention provides an antibacterial cationic hydrogel adhesive, the preparation raw materials of which include polyphenol-grafted ε-polylysine;

[0041] and four-arm polyethylene glycol with o-phthalaldehyde as terminal group;

[0042] The polyphenol-grafted ε-polylysine has a structure of Formula I or Formula II:

[0043]

[0044] In the present invention, the mass ratio of the polyphenol-grafted ε-polylysine polymer to the four-arm polyethylene glycol with o-phthalaldehyde as the terminal group is 1:1 to 1:4, preferably 1:1.

[0045] The four-arm polyethylene glycol with o-phthalaldehyde as the terminal group in the present invention has the structure of formula III:

[0046] 1≤p≤1000.

[0047] The value ranges of m and n in Formula I or Formula II of the present invention are 1≤m≤15 and 1≤n≤15 respectively.

[0048] In the four-arm polyethylene glycol having a structure of Formula III and having an end group of o-phthalaldehyde, p is preferably 1≤p≤800. In a specific embodiment, the molecular weight of the four-arm polyethylene glycol having a structure of Formula III and having an end group of o-phthalaldehyde is 10,000.

[0049] The present invention provides a method for preparing the antibacterial cationic hydrogel adhesive described in the above technical solution, comprising the following steps:

[0050] Dissolving a polymer of polyphenol grafted ε-polylysine having structure Ⅰ or formula Ⅱ in a solvent to obtain a first solution;

[0051] dissolving four-arm polyethylene glycol having an end group of o-phthalaldehyde in a solvent to obtain a second solution;

[0052] The first solution and the second solution are mixed and reacted to obtain an antibacterial cationic hydrogel adhesive;

[0053] The reaction temperature of the first solution and the second solution is 0 to 60° C. and the reaction time is 0 to 20 minutes.

[0054] The present invention dissolves the polymer of polyphenol grafted ε-polylysine having structure I or II in a solvent to obtain a first solution.

[0055] In the present invention, the polyphenol-grafted ε-polylysine polymer is prepared according to the following method:

[0056] Activate gallic acid or 3,4-dihydroxyphenylpropionic acid and react with ε-polylysine, dialyze the reaction product, and freeze-dry to obtain a polymer of polyphenol grafted ε-polylysine;

[0057] The reaction temperature is 10-40°C, preferably 20-30°C; the reaction time is 4-10h, preferably 5-7h.

[0058] When gallic acid is used as a raw material in the present invention, the obtained gallic acid grafted ε-polylysine has a structure of formula I; when 3,4-dihydroxyphenylpropionic acid is used as a raw material, the obtained 3,4-dihydroxyphenylpropionic acid grafted ε-polylysine has a structure of formula II.

[0059] The reaction scheme when gallic acid is used as the raw material is as follows:

[0060]

[0061] The reaction route when 3,4-dihydroxyphenylpropionic acid is used as the raw material is as follows:

[0062]

[0063] The process of activating gallic acid or 3,4-dihydroxyphenylpropionic acid in the present invention comprises:

[0064] Gallic acid or 3,4-dihydroxyphenylpropionic acid is activated, and the solvent used is MES buffer solution; the condensation reagent is preferably 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide sulfonic acid sodium salt.

[0065] In the present invention, the dialysis time of the reaction product is 3 days.

[0066] The invention dissolves four-arm polyethylene glycol whose terminal group is o-phthalaldehyde in a solvent to obtain a second solution.

[0067] In the present invention, the four-arm polyethylene glycol with o-phthalaldehyde as the terminal group has a structure of formula III; the four-arm polyethylene glycol with o-phthalaldehyde as the terminal group is prepared according to the following method:

[0068] 1,3-dimethoxy-1,3-dihydroisobenzofuran-5-carboxylic acid is dissolved and reacted with a condensation reagent, and then a four-arm polyethylene glycol solution with a hydroxyl terminal group is added to react. The reaction product after vacuum drying is deprotected, dialyzed and freeze-dried to obtain a four-arm polyethylene glycol with an o-phthalaldehyde terminal group.

[0069] The specific reaction route is:

[0070]

[0071] The structural formula of the four-arm polyethylene glycol with the terminal group of o-phthalaldehyde in the present invention is shown in Formula III:

[0072]

[0073] In the present invention, the condensation reagent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine; the reaction time with the condensation reagent is 18 to 23 minutes, preferably 20 minutes.

[0074] The deprotection in the present invention can adopt a technical solution well known to those skilled in the art; the deprotection in the present invention preferably adopts a mixed solvent of trifluoroacetic acid (TFA) and water; the volume of the mixed solvent is 5 to 20 times the mass of the reaction product, preferably 5 to 6 times; the deprotection time is 0.5 to 3 hours, preferably 1 to 1.5 hours; the deprotection temperature is 10 to 40°C, preferably 20 to 30°C; in a specific embodiment, the deprotection time is 1 hour and the temperature is 25°C.

[0075] The structural formula of 1,3-dimethoxy-1,3-dihydroisobenzofuran-5-carboxylic acid in the present invention is Preferably, it is prepared by the following method:

[0076] In the presence of a catalyst, 3,4-diformylbenzoic acid is reacted with methanol to obtain 1,3-dimethoxy-1,3-dihydroisobenzofuran-5-carboxylic acid.

[0077] The catalyst is scandium trifluoromethanesulfonate; the temperature of the reaction of 3,4-diformylbenzoic acid and methanol is 10-40°C, preferably 20-30°C; the time is 6-24h, preferably 10-15h; in a specific embodiment, the temperature of the reaction of 3,4-diformylbenzoic acid and methanol is 25°C, and the time is 12h. After the reaction is completed, spin dry and vacuum dry; the present invention preferably dissolves the reaction product with dichloromethane, washes with water, and then dries with anhydrous sodium sulfate; removes the desiccant by suction filtration, spin dry to obtain a crude product, purifies, and obtains 1,3-dimethoxy-1,3-dihydroisobenzofuran-5-carboxylic acid; the crude product of the present invention is purified by silica gel column chromatography; the mobile phase used for purification is n-hexane and ethyl acetate, and the volume ratio is (1:1) to (1:3). The present invention concentrates the solution containing 1,3-dimethoxy-1,3-dihydroisobenzofuran-5-carboxylic acid collected by purification, and vacuum dries to obtain a solid product.

[0078] The 3,4-diformylbenzoic acid described in the present invention is preferably prepared according to the following method:

[0079] 3,4-bis(dibromomethyl)benzoic acid is dissolved in an aqueous solution of sodium carbonate for hydrolysis to obtain 3,4-diformylbenzoic acid.

[0080] In the present invention, the mass-volume concentration of the sodium carbonate aqueous solution is 10%; the volume is 5 to 20 times the mass of 3,4-bis(dibromomethyl)benzoic acid, preferably 10 times, the reaction temperature is 60 to 80°C, preferably 70°C, and the reaction time is 3 to 5h, preferably 4h.

[0081] After the hydrolysis reaction is completed, the present invention preferably adds concentrated hydrochloric acid to the reaction solution until the pH is 1, extracts the acidic solution with ethyl acetate, and concentrates and vacuum-dries to obtain 3,4-diformylbenzoic acid.

[0082] The 3,4-bis(dibromomethyl)benzoic acid described in the present invention is preferably prepared according to the following method:

[0083] 3,4-dimethylbenzoic acid is subjected to bromination reaction to obtain 3,4-bis(dibromomethyl)benzoic acid.

[0084] In the bromination reaction, the present invention uses N-bromosuccinimide as a bromination reagent, benzoyl peroxide (BPO) as a free radical initiator, and carbon tetrachloride as a solvent. The temperature of the bromination reaction in the present invention is 81° C. and the time is 15 hours; the present invention preferably filters the bromination reaction product, washes it with benzene and ether in sequence, concentrates the obtained filtrate with a rotary evaporator, and then vacuum dries it; the dried solid product is recrystallized in acetonitrile to obtain 3,4-bis(dibromomethyl)benzoic acid.

[0085] Specifically, the reaction route of the 1,3-dimethoxy-1,3-dihydroisobenzofuran-5-carboxylic acid is as follows:

[0086]

[0087] In the present invention, the first solution and the second solution are prepared by using a temperature control system to control the solution temperature, and the temperature is preferably 0-60°C, more preferably 10-50°C, and most preferably 25-37°C.

[0088] In the present invention, the mass volume concentration of the polyphenol-grafted ε-polylysine polymer having structure Ⅰ or Ⅱ in the first solution is 5 to 15% (w / v), preferably 10% (w / v); the mass volume concentration of the four-arm polyethylene glycol with the terminal group of o-phthalaldehyde in the second solution is 5 to 15% (w / v), preferably 10% (w / v).

[0089] In the present invention, the solvents in the first solution and the second solution are independently selected from water, physiological saline or buffer solution.

[0090] After obtaining the first solution and the second solution, the present invention mixes the first solution and the second solution to react to obtain an antibacterial cationic hydrogel adhesive.

[0091] The present invention uses a vortex instrument to mix the first solution and the second solution evenly, and then puts them into a temperature control system for reaction; the reaction temperature is 0°C to 60°C, more preferably 10°C to 50°C, and most preferably 25°C to 37°C; the reaction time is 0 to 20 minutes.

[0092] In order to further illustrate the present invention, an antibacterial cationic hydrogel adhesive and a preparation method thereof provided by the present invention are described in detail below in conjunction with examples, but they should not be construed as limiting the scope of protection of the present invention.

[0093] Embodiment 1:

[0094] 1) Activate 0.66g of gallic acid, and the solvent is MES buffer solution. Add 1.5g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 1.7g of N-hydroxysuccinimide sulfonic acid sodium salt, and react for 1h. The reaction temperature is 25°C. After activation, add 0.5g of ε-polylysine (ε-PLL). React for 6h. Then transfer to a dialysis bag with a molecular weight cutoff of 1000 and dialyze for 72h, and then freeze-dry to obtain the reaction product, i.e., gallic acid grafted ε-polylysine (ε-PLL-GA). Figure 1 This is the H NMR spectrum of ε-polylysine grafted with gallic acid (ε-PLL-GA).

[0095] 2) Activate 0.142g 3,4-dihydroxyphenylpropionic acid with MES buffer solution as solvent. Add 0.3g 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 0.34g N-hydroxysuccinimide sulfonic acid sodium salt and react for 1h. The reaction temperature is 25°C. After activation, add 0.5g ε-polylysine (ε-PLL). React for 6h. Then transfer to a dialysis bag with a molecular weight cutoff of 1000 and dialyze for 72h, then freeze-dry to obtain the reaction product, i.e. 3,4-dihydroxyphenylpropionic acid grafted ε-polylysine (ε-PLL-CA). Figure 2 This is the H NMR spectrum of ε-polylysine grafted with 3,4-dihydroxyphenylpropionic acid.

[0096] Embodiment 2:

[0097] Weigh 18g of 3,4-dimethylbenzoic acid and 85g of N-bromosuccinimide and dissolve them in 300mL of carbon tetrachloride solution, then add 2.63g of benzoyl peroxide. The mixture is refluxed at 81°C for 15h. After filtering, it is concentrated by rotary evaporator to a residual liquid volume of 10-20mL, and then vacuum pumped to solid. The obtained solid is recrystallized in acetonitrile to obtain 3,4-bis(dibromomethyl)benzoic acid.

[0098] Embodiment 3:

[0099] 14.84 g of 3,4-bis(dibromomethyl)benzoic acid prepared in Example 2 was dissolved in a sodium carbonate aqueous solution having a mass-volume concentration of 10%, and reacted at 70° C. for 4 h. The pH of the reaction solution was adjusted to 1 with concentrated hydrochloric acid, and then extracted with ethyl acetate. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and vacuum-dried to obtain 3,4-diformylbenzoic acid.

[0100] Embodiment 4:

[0101] 2.65 g of 3,4-di(dibromomethyl)benzoic acid prepared in Example 3 was dissolved in anhydrous methanol, and then 0.398 g of the catalyst scandium trifluoromethanesulfonate was added, and the reaction was protected from light for 12 h. 1,3-dimethoxy-1,3-dihydroisobenzofuran-5-carboxylic acid was obtained by vacuum drying. The crude product of 1,3-dimethoxy-1,3-dihydroisobenzofuran-5-carboxylic acid was dissolved in dichloromethane, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, filtered, and pumped dry. Purification was performed by silica gel column chromatography to obtain 1,3-dimethoxy-1,3-dihydroisobenzofuran-5-carboxylic acid. The mobile phase was n-hexane and ethyl acetate in a volume ratio of 1:3.

[0102] Embodiment 5:

[0103] The 1,3-dimethoxy-1,3-dihydroisobenzofuran-5-carboxylic acid prepared in Example 4 was dissolved in dichloromethane, and 4.48 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 0.48 g of 4-dimethylaminopyridine were added to react for 20 min. Four-arm polyethylene glycol with hydroxyl end groups dissolved in dichloromethane was added after dehydration, and the reaction was continued for 24 h.

[0104] After the reaction, the dichloromethane was dried by spin drying. 2 g of the crude product was mixed with 0.5 g of a four-arm polyethylene glycol with a hydroxyl end group, and a TFA aqueous solution with a volume ratio of 1:1 was added to react for 1.5 h. Then it was transferred to a dialysis bag with a molecular weight cutoff of 3500 and dialyzed for 48 h, and then freeze-dried to obtain a four-arm polyethylene glycol with an o-phthalaldehyde end group and a molecular weight of about 10,000. Figure 3 This is the hydrogen nuclear magnetic resonance spectrum of four-arm polyethylene glycol with o-phthalaldehyde as the terminal group.

[0105] Embodiment 6:

[0106] 1) Weigh the four-arm polyethylene glycol solid with o-phthalaldehyde end group of Example 5, add it to a PBS solution of pH=7.4 and stir until completely dissolved, and prepare components of different concentrations, including 5%, 10%, and 15%. Adjust the temperature of the electric thermostatic water bath to 37°C, and put the obtained solution into a 37°C thermostatic water bath to obtain a first component mixed solution; weigh the gallic acid grafted ε-polylysine solid prepared in Example 1, add it to a PBS solution of pH=7.4 and stir until completely dissolved, and prepare components of different concentrations, including 5%, 10%, and 15%. Adjust the temperature of the electric thermostatic water bath to 37°C, and put the obtained solution into a 37°C thermostatic water bath to obtain a second component mixed solution; respectively mix the components of the same concentration in a volume ratio of 1:1, use a vortex instrument to mix them evenly, and then put them into a 37°C thermostatic water bath to prepare a hydrogel (ε-PLL-GA / 4aPEG-OPA). Measure 150 μL of the first component mixed solution and the second component mixed solution, place them in a 37°C water bath, and observe the gelation by inversion method. The gelation time is 60 s. Measure 150 μL of the first component mixed solution and the second component mixed solution, mix them thoroughly and quickly transfer them to the rheometer to measure the changes of the storage modulus and loss modulus of the mixed solution over time. Figure 4 This is a rheological test diagram of the ε-PLL-GA / 4aPEG-OPA hydrogel in this implementation case. Figure 4 It can be seen that with the increase of mass volume concentration, the storage modulus (G') and loss modulus (G") in the rheological test increase; when the mass volume concentration reaches 10%, the storage modulus (G') of the hydrogel formed by the ε-polylysine grafted with gallic acid and the four-arm polyethylene glycol with the terminal group of o-phthalaldehyde can reach 10 4 , with good mechanical strength.

[0107] 2) Weigh the four-arm polyethylene glycol solid with the end group of o-phthalaldehyde in Example 5, add it to the PBS solution of pH=7.4 and stir until it is completely dissolved, and prepare components of different concentrations, including 5%, 10%, and 15%. The temperature of the electric constant temperature water bath is adjusted to 37°C, and the obtained solution is placed in a constant temperature water bath of 37°C to obtain a first component mixed solution; weigh the ε-polylysine grafted with 3,4-dihydroxyphenylpropionic acid prepared in Example 1. The solid is added to the PBS solution of pH=7.4 and stirred until it is completely dissolved, and prepare components of different concentrations, including 5%, 10%, and 15%. The temperature of the electric constant temperature water bath is adjusted to 37°C, and the obtained solution is placed in a constant temperature water bath of 37°C to obtain a second component mixed solution; the obtained solution is placed in a constant temperature water bath of 37°C to obtain a second component mixed solution; the components of the same concentration are mixed in a volume ratio of 1:1, and a vortex instrument is used to mix them evenly, and then placed in a constant temperature water bath of 37°C to prepare a hydrogel (ε-PLL-CA / 4aPEG-OPA). 150 μL of the first component mixed solution and the second component mixed solution were measured and placed in a 37°C water bath. The gelation was observed by the inversion method. The gelation time was 110 s. Figure 5 is a rheological test diagram of ε-PLL-CA / 4aPEG-OPA hydrogel in this embodiment; Figure 5 It can be seen that with the increase of mass volume concentration, the storage modulus (G') and loss modulus (G") in the rheological test increase; when the mass volume concentration reaches 10%, the storage modulus (G') of the hydrogel formed by ε-polylysine grafted with 3,4-dihydroxyphenylpropionic acid and four-arm polyethylene glycol with o-phthalaldehyde as the terminal group can reach 10 4 , with good mechanical strength.

[0108] Figure 6 Schematic diagram of the hydrogel in this embodiment.

[0109] Embodiment 7:

[0110] First, the pig skin was fixed on a wooden board using industrial adhesive 502 and placed in a 37°C water bath to keep the tissue moist. Then, 50.0 mg of the four-arm polyethylene glycol solid with an end group of o-phthalaldehyde in Example 5 was weighed and added to 0.5 mL of a pH=7.4 PBS solution and stirred until completely dissolved, the temperature of the electric thermostatic water bath was adjusted to 37°C, and the resulting solution was placed in a 37°C thermostatic water bath to obtain a first component mixed solution; 50.0 mg of the gallic acid-grafted ε-polylysine solid prepared in Example 1 was weighed and added to 0.5 mL of a pH=7.4 PBS solution and stirred until completely dissolved, the temperature of the electric thermostatic water bath was adjusted to 37°C, and the resulting solution was placed in a 37°C thermostatic water bath to obtain a second component mixed solution; 50 μL of each of the first component mixed solution and the second component mixed solution were measured, and after fully mixed, they were quickly added to the overlapping area (25 mm×25 mm) of the two pieces of pig skin, and a force of 2 N was applied to the overlapping area. Then, the samples were incubated in a 37°C water bath for 2 h and tested using a universal testing machine.

[0111] First, the pig skin was fixed on a wooden board using industrial adhesive 502 and placed in a 37°C water bath to keep the tissue moist. Then, 50.0 mg of the four-arm polyethylene glycol solid with an end group of o-phthalaldehyde in Example 5 was weighed and added to 0.5 mL of a pH=7.4 PBS solution and stirred until completely dissolved, the temperature of the electric constant temperature water bath was adjusted to 37°C, and the obtained solution was placed in a 37°C constant temperature water bath to obtain a first component mixed solution; 50.0 mg of the ε-polylysine solid grafted with 3,4-dihydroxyphenylpropionic acid prepared in Example 1 was weighed and added to 0.5 mL of a pH=7.4 PBS solution and stirred until completely dissolved, the temperature of the electric constant temperature water bath was adjusted to 37°C, and the obtained solution was placed in a 37°C constant temperature water bath to obtain a second component mixed solution; 50 μL of each of the first component mixed solution and the second component mixed solution were measured, and after fully mixed, they were quickly added to the overlapping area (25 mm×25 mm) of the two pieces of pig skin, and a force of 2 N was applied to the overlapping area. Then, the samples were incubated in a 37°C water bath for 2 h and tested using a universal testing machine.

[0112] Figure 7 is the lap shear strength of the hydrogel; Figure 7 It can be seen that the adhesion strength of the two hydrogels with a mass volume concentration of 10% can reach about 40 kPa.

[0113] Embodiment 8:

[0114] 1) Weigh 50.0 mg of the four-arm polyethylene glycol solid with an end group of o-phthalaldehyde of Example 5, add 0.5 mL of PBS solution with a pH of 7.4, stir until completely dissolved, adjust the temperature of the electric thermostatic water bath to 37° C., and place the obtained solution in a 37° C. thermostatic water bath to obtain a first component mixed solution; weigh 50.0 mg of the gallic acid-grafted ε-polylysine solid prepared in Example 1, add 0.5 mL of PBS solution with a pH of 7.4, stir until completely dissolved, adjust the temperature of the electric thermostatic water bath to 37° C., and place the obtained solution in a 37° C. thermostatic water bath to obtain a second component mixed solution; measure 140 μL of the first component mixed solution and the second component mixed solution, mix them thoroughly, and quickly cast them into a round polytetrafluoroethylene mold, incubate them in a 37° C. water bath for 2 h, and then test them using a universal testing machine.

[0115] 2) Weigh 50.0 mg of the four-arm polyethylene glycol solid with o-phthalaldehyde as the terminal group of Example 5, add 0.5 mL of PBS solution with pH = 7.4 and stir until completely dissolved, adjust the temperature of the electric thermostatic water bath to 37° C., and put the obtained solution into a 37° C. constant temperature water bath to obtain a first component mixed solution; weigh 50.0 mg of the ε-polylysine solid grafted with 3,4-dihydroxyphenylpropionic acid prepared in Example 1, add 0.5 mL of PBS solution with pH = 7.4 and stir until completely dissolved, adjust the temperature of the electric thermostatic water bath to 37° C., and put the obtained solution into a 37° C. constant temperature water bath to obtain a second component mixed solution; measure 140 μL each of the first component mixed solution and the second component mixed solution, mix them thoroughly and quickly cast them into a round polytetrafluoroethylene mold, incubate them in a 37° C. water bath for 2 hours, and then test them using a universal testing machine.

[0116] Figure 8 is the compressive strength of the two hydrogels; Figure 8 It can be seen that the compressive strength of the hydrogel (ε-PLL-GA / 4aPEG-OPA) formed by 10% gallic acid grafted ε-polylysine and terminal phthalaldehyde can reach 175kPa, and the compressive strength of the hydrogel (ε-PLL-CA / 4aPEG-OPA) formed by 10% 3,4-dihydroxyphenylpropionic acid grafted ε-polylysine and terminal phthalaldehyde can reach 125kPa.

[0117] Fig. 9 is the load of the compression test of the two hydrogels; Fig. 9It can be seen that the compressive strength of the hydrogel (ε-PLL-GA / 4aPEG-OPA) formed by the grafting of ε-polylysine with gallic acid at a mass volume concentration of 10% and the four-arm polyethylene glycol with o-phthalaldehyde as the terminal group is higher than the compressive strength of the hydrogel (ε-PLL-CA / 4aPEG-OPA) formed by the grafting of ε-polylysine with 3,4-dihydroxyphenylpropionic acid at a mass volume concentration of 10%.

[0118] Embodiment 9:

[0119] 1) Weigh 20.0 mg of the gallic acid-grafted ε-polylysine solid prepared in Example 1, prepare a 1 mg / mL solution, and then dilute it by half to 200 μg / mL. 8 CFU / mL Escherichia coli solution to 10 6 CFU / mL. Add 0μL, 35μL, 70μL, 105μL, 175μL, 245μL, 350μL, 525μL, 700μL of materials into a 2mL centrifuge tube; the corresponding PBS volumes are 700μL, 665μL, 630μL, 595μL, 525μL, 455μL, 350μL, 175μL, 0μL; add 700μL 10 6 CFU / mL of bacterial solution was prepared into final concentrations of 0, 10μg / mL, 20μg / mL, 30μg / mL, 50μg / mL, 70μg / mL, 100μg / mL, 150μg / mL, and 200μg / mL. The liquid was mixed evenly and added to a 96-well plate, with a total of 9 concentrations, and 6 parallels were made. Incubate in a constant temperature incubator for 24 hours, measure the OD value, and obtain the minimum inhibitory concentration (MIC) of gallic acid grafted ε-polylysine (ε-PLL-GA). Fig.10 is the minimum inhibitory concentration of gallic acid grafted ε-polylysine against Escherichia coli at different concentrations; Fig.10 It can be seen that the minimum inhibitory concentration of gallic acid grafted ε-polylysine (ε-PLL-GA) is 70 μg / mL.

[0120] 2) Weigh 20.0 mg of the ε-polylysine solid grafted with 3,4-dihydroxyphenylpropionic acid prepared in Example 1, prepare a 1 mg / mL solution, and then dilute it by half to 200 μg / mL. 8 CFU / mL Escherichia coli solution to 10 6CFU / mL. Add 0, 35μL, 70μL, 105μL, 175μL, 245μL, 350μL, 525μL, 700μL of materials into a 2mL centrifuge tube; the corresponding PBS volumes are 700μL, 665μL, 630μL, 595μL, 525μL, 455μL, 350μL, 175μL, 0μL; add 700μL 10 6 CFU / mL of bacterial solution was prepared into final concentrations of 0, 10μg / mL, 20μg / mL, 30μg / mL, 50μg / mL, 70μg / mL, 100μg / mL, 150μg / mL, and 200μg / mL. The liquid was mixed evenly and added to a 96-well plate, with a total of 9 concentrations, and 6 parallels were made. Incubate in a constant temperature incubator for 24 hours, measure the OD value, and obtain the minimum inhibitory concentration (MIC) of 3,4-dihydroxyphenylpropionic acid grafted ε-polylysine (ε-PLL-CA). Fig.11 is the minimum inhibitory concentration of ε-polylysine grafted with 3,4-dihydroxyphenylpropionic acid against Escherichia coli at different concentrations; Fig.11 It can be seen that the minimum inhibitory concentration of ε-polylysine (ε-PLL-CA) grafted with 3,4-dihydroxyphenylpropionic acid is 100 μg / mL.

[0121] Embodiment 10:

[0122] 1) Weigh 20.0 mg of the gallic acid-grafted ε-polylysine solid prepared in Example 1, prepare a 1 mg / mL solution, and then dilute it by half to 200 μg / mL. 8 CFU / mL Escherichia coli solution to 10 6 CFU / mL. Add 0μL, 35μL, 70μL, 105μL, 175μL, 245μL, 350μL, 525μL, 700μL of materials into a 2mL centrifuge tube; the corresponding PBS volumes are 700μL, 665μL, 630μL, 595μL, 525μL, 455μL, 350μL, 175μL, 0μL; add 700μL 10 6 CFU / mL of bacterial solution was prepared into final concentrations of 0, 10μg / mL, 20μg / mL, 30μg / mL, 50μg / mL, 70μg / mL, 100μg / mL, 150μg / mL, and 200μg / mL. The liquid was mixed evenly and added to a 96-well plate, with a total of 9 concentrations, and 6 parallels were made. Incubate in a constant temperature incubator for 24 hours, measure the OD value, and obtain the minimum inhibitory concentration (MIC) of gallic acid grafted ε-polylysine (ε-PLL-GA). Fig.12 is the minimum inhibitory concentration of gallic acid grafted ε-polylysine against Staphylococcus aureus at different concentrations; Fig.12 It can be seen that the minimum inhibitory concentration of gallic acid grafted ε-polylysine (ε-PLL-GA) is 10 μg / mL.

[0123] 2) Weigh 20.0 mg of the ε-polylysine solid grafted with 3,4-dihydroxyphenylpropionic acid prepared in Example 1, prepare a 1 mg / mL solution, and then dilute it by half to 200 μg / mL. 8 CFU / mL Escherichia coli solution to 10 6 CFU / mL. Add 0, 35μL, 70μL, 105μL, 175μL, 245μL, 350μL, 525μL, 700μL of materials into a 2mL centrifuge tube; the corresponding PBS volumes are 700μL, 665μL, 630μL, 595μL, 525μL, 455μL, 350μL, 175μL, 0μL; add 700μL 10 6 CFU / mL of bacterial solution was prepared into final concentrations of 0, 10μg / mL, 20μg / mL, 30μg / mL, 50μg / mL, 70μg / mL, 100μg / mL, 150μg / mL, and 200μg / mL. The liquid was mixed evenly and added to a 96-well plate, with a total of 9 concentrations, and 6 parallels were made. Incubate in a constant temperature incubator for 24 hours, measure the OD value, and obtain the minimum inhibitory concentration (MIC) of 3,4-dihydroxyphenylpropionic acid grafted ε-polylysine (ε-PLL-CA). Fig.13 is the minimum inhibitory concentration of ε-polylysine grafted with 3,4-dihydroxyphenylpropionic acid against Staphylococcus aureus at different concentrations; Fig.13 It can be seen that the minimum inhibitory concentration of ε-polylysine (ε-PLL-CA) grafted with 3,4-dihydroxyphenylpropionic acid is 30 μg / mL.

[0124] Embodiment 11:

[0125] 1) Weigh 50.0 mg of the four-arm polyethylene glycol solid with o-phthalaldehyde as the terminal group in Example 5, add 0.5 mL of PBS solution with pH = 7.4 and stir until completely dissolved, adjust the temperature of the electric thermostatic water bath to 37° C., and place the obtained solution in a 37° C. thermostatic water bath to obtain a first component mixed solution; weigh 50.0 mg of the gallic acid-grafted ε-polylysine solid prepared in Example 1, add 0.5 mL of PBS solution with pH = 7.4 and stir until completely dissolved, adjust the temperature of the electric thermostatic water bath to 37° C., and place the obtained solution in a 37° C. thermostatic water bath to obtain a second component mixed solution; 150 μL of the two-component mixed solution are respectively taken and added to a 24-well culture plate, and after the two components are gelled, 300 μL of 10 6 CFU / mL Escherichia coli suspension was incubated at 37°C for 24 h.-1 , 10 -2 , 10 -3 , gradient dilution. Take 100 μL of the diluted liquid and spread it on the LB agar plate. The LB agar preparation ratio is prepared according to the amount given in the purchase instructions. Incubate the spread agar plate at 37°C for 24 hours, take it out, and observe the colonies on the agar plate.

[0126] 2) Weigh 50.0 mg of the four-arm polyethylene glycol solid with o-phthalaldehyde as the terminal group in Example 5, add 0.5 mL of PBS solution with pH = 7.4 and stir until completely dissolved, adjust the temperature of the electric thermostatic water bath to 37° C., and place the obtained solution in a 37° C. thermostatic water bath to obtain a first component mixed solution; weigh 50.0 mg of the ε-polylysine solid grafted with 3,4-dihydroxyphenylpropionic acid prepared in Example 1, add 0.5 mL of PBS solution with pH = 7.4 and stir until completely dissolved, adjust the temperature of the electric thermostatic water bath to 37° C., and place the obtained solution in a 37° C. thermostatic water bath to obtain a second component mixed solution; 150 μL of the two-component mixed solution are respectively taken and added to a 24-well culture plate, and after the two components are gelled, 300 μL of 10 6 CFU / mL Escherichia coli suspension was incubated at 37°C for 24 h. -1 , 10 -2 , 10 -3 , gradient dilution. Take 100 μL of the diluted liquid and spread it on the LB agar plate. The LB agar preparation ratio is prepared according to the amount given in the purchase instructions. Incubate the spread agar plate at 37°C for 24 hours, take it out, and observe the colonies on the agar plate.

[0127] Fig.14 The antibacterial effect of the polyphenol-grafted ε-lysine and the four-arm polyethylene glycol hydrogel with o-phthalaldehyde as the terminal group on Escherichia coli prepared in this example; Fig.14 It can be seen that compared with the control group, the number of bacterial colonies in the control group is extremely large, while the number of colonies in the gel group is very small.

[0128] Embodiment 12:

[0129] 1) Weigh 50.0 mg of the four-arm polyethylene glycol solid with o-phthalaldehyde as the terminal group in Example 5, add 0.5 mL of PBS solution with pH = 7.4 and stir until completely dissolved, adjust the temperature of the electric thermostatic water bath to 37° C., and place the obtained solution in a 37° C. thermostatic water bath to obtain a first component mixed solution; weigh 50.0 mg of the gallic acid-grafted ε-polylysine solid prepared in Example 1, add 0.5 mL of PBS solution with pH = 7.4 and stir until completely dissolved, adjust the temperature of the electric thermostatic water bath to 37° C., and place the obtained solution in a 37° C. thermostatic water bath to obtain a second component mixed solution; 150 μL of the two-component mixed solution are respectively taken and added to a 24-well culture plate, and after the two components are gelled, 300 μL of 10 6 CFU / mL Staphylococcus aureus suspension was incubated at 37°C for 24 h. -1 , 10 -2 , 10 -3 Gradient dilution. Take 100 μL of the diluted liquid and spread it on the LB agar plate. The LB agar preparation ratio is prepared according to the dosage given in the purchase instructions. Incubate the spread agar plate at 37°C for 24 hours, take it out, and count the colonies on the agar plate.

[0130] 2) Weigh 50.0 mg of the four-arm polyethylene glycol solid with o-phthalaldehyde as the terminal group in Example 5, add 0.5 mL of PBS solution with pH = 7.4 and stir until completely dissolved, adjust the temperature of the electric thermostatic water bath to 37° C., and place the obtained solution in a 37° C. thermostatic water bath to obtain a first component mixed solution; weigh 50.0 mg of the ε-polylysine solid grafted with 3,4-dihydroxyphenylpropionic acid prepared in Example 1, add 0.5 mL of PBS solution with pH = 7.4 and stir until completely dissolved, adjust the temperature of the electric thermostatic water bath to 37° C., and place the obtained solution in a 37° C. thermostatic water bath to obtain a second component mixed solution; 150 μL of the two-component mixed solution are respectively taken and added to a 24-well culture plate, and after the two components are gelled, 300 μL of 10 6 CFU / mL Staphylococcus aureus suspension was incubated at 37°C for 24 h. -1 , 10 -2 , 10 -3 , gradient dilution. Take 100 μL of the diluted liquid and spread it on the LB agar plate. The LB agar preparation ratio is prepared according to the amount given in the purchase instructions. Incubate the spread agar plate at 37°C for 24 hours, take it out, and count the colonies on the agar plate.

[0131] Fig.15 The antibacterial effect of the polyphenol-grafted ε-lysine and the four-arm polyethylene glycol hydrogel with o-phthalaldehyde as the terminal group on Staphylococcus aureus prepared in this example; Fig.15It can be seen that compared with the control group, the number of bacterial colonies in the control group is extremely large, while the number of colonies in the gel group is very small.

[0132] As can be seen from the above embodiments, the present invention provides an antibacterial cationic hydrogel adhesive, the raw materials for preparation include polyphenol-grafted ε-polylysine polymer; and four-arm polyethylene glycol with an end group of o-phthalaldehyde; the polyphenol-grafted ε-polylysine polymer has a structure of formula I or formula II. The hydrogel adhesive contains ε-polylysine, which can be long-lasting and antibacterial and avoid drug resistance; the grafted polyphenol functional groups can effectively remove excess free radicals, have antioxidant functions, thereby promoting wound healing, and can also enhance tissue adhesion; o-phthalaldehyde-amine cross-linking gelation is fast, providing tissue adhesion. The test results show that polyphenol-grafted ε-polylysine has effective antibacterial activity, and gels with the component of the four-arm polyethylene glycol with an end group of o-phthalaldehyde, preferably with a mass volume concentration of 10%, and exhibits strong adhesion.

[0133] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An antibacterial cationic hydrogel adhesive, wherein the raw materials for preparation include polyphenol-grafted ε-polylysine; and four-arm polyethylene glycol with o-phthalaldehyde as terminal group; The polyphenol-grafted ε-polylysine polymer has a structure of Formula I or Formula II:

2. The antibacterial cationic hydrogel adhesive according to claim 1, characterized in that: The mass ratio of the polyphenol-grafted ε-polylysine to the four-arm polyethylene glycol with o-phthalaldehyde as the terminal group is 1:1 to 1:

4.

3. The antibacterial cationic hydrogel adhesive according to claim 1, characterized in that: The four-arm polyethylene glycol with o-phthalaldehyde as the terminal group has the structure of formula III: 1≤p≤1000。 4. The antibacterial cationic hydrogel adhesive according to claim 1, characterized in that: 1≤m≤15 and 1≤n≤15.

5. A method for preparing the antibacterial cationic hydrogel adhesive according to claim 1, comprising the following steps: Dissolving polyphenol-grafted ε-polylysine having structure Ⅰ or formula Ⅱ in a solvent to obtain a first solution; dissolving four-arm polyethylene glycol having an end group of o-phthalaldehyde in a solvent to obtain a second solution; The first solution and the second solution are mixed and reacted to obtain an antibacterial cationic hydrogel adhesive; The reaction temperature of the first solution and the second solution is 0 to 60° C. and the reaction time is 0 to 20 minutes.

6. The preparation method according to claim 5, characterized in that: The solvents in the first solution and the second solution are independently selected from water, physiological saline or buffer solution.

7. The preparation method according to claim 5, characterized in that: The mass volume concentration of the polyphenol-grafted ε-polylysine having the structure of formula Ⅰ or formula Ⅱ in the first solution is 5 to 15% (w / v); The mass volume concentration of the four-arm polyethylene glycol with terminal groups of o-phthalaldehyde in the second solution is 5-15% (w / v).

8. The preparation method according to claim 5, characterized in that: The polyphenol-grafted ε-polylysine is prepared according to the following method: Activating gallic acid or 3,4-dihydroxyphenylpropionic acid and reacting it with ε-polylysine, dialyzing the reaction product, and freeze-drying it to obtain a polymer of ε-polylysine grafted with polyphenol; The reaction temperature is 10-40°C and the reaction time is 4-10 hours.

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