CN114306722A

CN114306722A - Injectable hydrogel type biological adhesive and preparation and application thereof

Info

Publication number: CN114306722A
Application number: CN202011052782.5A
Authority: CN
Inventors: 不公告发明人
Original assignee: East China University of Science and Technology
Current assignee: East China University of Science and Technology
Priority date: 2020-09-29
Filing date: 2020-09-29
Publication date: 2022-04-12
Anticipated expiration: 2040-09-29
Also published as: CN114306722B

Abstract

The invention relates to an injectable hydrogel type biological adhesive, and preparation and application thereof. Specifically, the hydrogel material is formed by mixing a gelling precursor liquid A formed by dissolving a water-soluble polymer or macromolecule with more than two amino functionalities in a normal saline solution containing strong base and weak acid salt and a gelling precursor liquid B formed by dissolving polyethylene glycol with a modified active ester at the tail end in a normal saline solution. By adjusting the pH value in the precursor solution, the hydrogel can have different gelation time from several seconds to several minutes after injection, so that the hydrogel can be used for rapid hemostasis, drug slow release, tissue adhesion, minimally invasive delivery treatment to deep wounds in vivo and other clinical fields.

Description

Injectable hydrogel type biological adhesive and preparation and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a gelation time regulating method based on amidation reaction, and different injectable hydrogels prepared by the method are applied to different medical clinical fields.

Background

A hydrogel is a polymer with a hydrophilic three-dimensional network structure, similar to the human extracellular matrix (ECM); in water, it can absorb a large amount of water to swell and maintain its form without being dissolved, so that the material has wide application in clinical fields. In recent years, injectable hydrogels have become a new round of research focus in the field of hydrogels. The injectable hydrogel means that hydrogel raw materials in a fluid form are simultaneously injected into a body through a syringe and then form gel in situ. The 'liquid injection, in-situ solidification' breaks through the limitation of the traditional hydrogel state. The injectable hydrogel based on the two-component chemical crosslinking has wide application in the clinical field due to the advantages of convenient delivery mode, high wound shape adaptability after delivery and the like. For tissue adhesive applications, injectable hydrogels based on amidation reactions (i.e., reactions between amino and active ester functionalities) are preferred over other chemically crosslinked hydrogels due to their inherent chemical bonding capability to the biological tissue interface.

The gelation time, i.e., the phase transition time of the hydrogel from the solution state to the gel state after injection, is an important parameter of the injectable hydrogel, and determines the applicable scene of the gel. The hydrogel for hemostasis is required to be rapidly gelated and transformed after injection, so as to stop bleeding at wounds as soon as possible. Furthermore, for certain deep tissue wound sealing applications, such as gastric perforation, lung rupture and liver injury, slow and controlled gelation would be clinically desirable to ensure that the hydrogel is delivered in a minimally invasive manner prior to curing. However, the relatively fixed gelation rate makes it technically challenging to use such hydrogels for minimally invasive delivery.

Therefore, under the precondition of not changing the adhesive strength, the mechanical property and other properties of the hydrogel based on the amidation reaction, the development of a method for accurately controlling the gelation time of the hydrogel is of great significance.

Disclosure of Invention

In a first aspect of the present invention, there is provided a hydrogel injection composition comprising:

(i) injecting the solution A; the injection A comprises a gel-forming precursor solution A, wherein the gel-forming precursor solution A is formed by dissolving water-soluble macromolecules or macromolecules with more than two amino functionalities in a normal saline solution containing strong base and weak acid salts;

(ii) injecting the solution B; the injection B solution comprises a gel-forming precursor solution B, and the gel-forming precursor solution B is formed by dissolving polyethylene glycol with a modified active ester at the tail end in a normal saline solution.

In another preferred embodiment, the strong base and weak acid salt has one or more of the following characteristics:

(a) after the strong base weak acid salt is dissolved in water, partial hydrolysis can be carried out to release hydroxide ions;

(b) the strong base and weak acid salt can be dissolved in water, and the pH value of the water solution is 7.0-10.5;

(c) the salt itself does not carry an amino group;

(d) the salt should be more than 99% pure.

In another preferred embodiment, the strong base and weak acid salt is selected from the group consisting of: borax (sodium tetraborate), sodium carbonate, sodium bicarbonate, sodium hydrogen phosphate, sodium metaaluminate, sodium acetate, potassium carbonate, potassium bicarbonate, potassium metaaluminate, calcium bicarbonate, or combinations thereof.

In another preferred embodiment, the strong base and weak acid salt is Borax (Borax).

In another preferred example, the content of the strong base and weak acid salt in the gel-forming precursor liquid a is 1 mg/mL to 30 mg/mL.

In another preferred embodiment, the polyethylene glycol derivative of the terminally modified active ester is modified with an active ester selected from the group consisting of: carbonates, acetates, propionates, succinates, valerates, or combinations thereof.

In another preferred embodiment, the polyethylene glycol has at least two active ester functional group modifications.

In another preferred embodiment, the polyethylene glycol derivative of the terminally modified active ester is a tetra-armed polyethylene glycol of a terminally modified succinimide active ester.

In another preferred embodiment, the active ester of succinimide is selected fromThe following groups: carboxylic acid succinimidyl (- (CH)₂)_m-COO succinimide, wherein m is an integer of 0 to 10), dicarboxylic acid monosuccinimidyl (- (C ═ O) - (CH)₂)_m-COO succinimide).

In another preferred embodiment, the four-armed polyethylene glycol of the terminally modified succinimide active ester is selected from the group consisting of: a tetraarmed polyethylene glycol succinimide carbonate, a tetraarmed polyethylene glycol succinimide acetate, a tetraarmed polyethylene glycol succinimide succinate, or a combination thereof.

In another preferred embodiment, the reactive substance with amino group is selected from the group consisting of: functional proteins (which refer to a class of proteins having some special physiological functions in addition to the nutritional effects of general proteins), aminopolysaccharides, or amino-modified polyethylene glycols.

In another preferred embodiment, the reactive substance with amino groups is a functional protein solution, and the functional protein has one or more of the following characteristics:

(a) the number of amino groups exposed on the surface of each molecule is more than or equal to 2;

(b) the solubility of the protein in water at 37 ℃ is more than 50 mg/ml;

(c) the protein does not aggregate and precipitate under alkaline conditions (pH 7.0-10.5).

In another preferred embodiment, the functional protein is selected from the group consisting of: lysozyme protein, serum albumin, egg white albumin, hemoglobin, or a combination thereof; preferably lysozyme protein.

In another preferred embodiment, the polyethylene glycol with the modified amino group at the end has one or more of the following characteristics:

(a) the weight average molecular weight is 5000-100000;

(b) the purity is more than 99 percent;

(c) the dispersion coefficient (PDI) of a single polyethylene glycol Polymer (PEG) is 1-1.1;

(d) the number of arms of the multi-arm poly (ethylene glycol) is more than or equal to 2.

In another preferred embodiment, the weight average molecular weight of the amino-modified polyethylene glycol is 10000 to 20000, and more preferably 10000.

In another preferred embodiment, the amino-terminally modified polyethylene glycol has the structure shown in the following formula:

wherein n is an integer of 2 to 300.

In another preferred embodiment, the polyethylene glycol is a multi-arm polyethylene glycol.

In another preferred embodiment, the gel-forming precursor solution a is a mixed solution of a functional protein and polyethylene glycol with a modified amino group at the end.

In another preferred embodiment, the viscosity of the gel-forming precursor liquid A is 0.1-2 Pas;

the viscosity of the gel-forming precursor liquid B is 0.5-1 Pas.

In another preferred embodiment, the concentration of the water-soluble polymer or macromolecule with more than two amino functionalities is 10-200 mg/mL; the concentration of the polyethylene glycol of the end modified active ester is 10-200 mg/mL.

In a second aspect of the present invention, there is provided a hydrogel material prepared by the method comprising:

(1) providing a gel-forming precursor solution A, wherein the gel-forming precursor solution A is formed by dissolving water-soluble macromolecules or macromolecules with more than two amino functionalities in a normal saline solution containing strong base and weak acid salts;

(2) providing a colloid-forming precursor liquid B, wherein the colloid-forming precursor liquid B is formed by dissolving polyethylene glycol with a modified active ester at the tail end in a normal saline solution;

(3) and mixing the gelling precursor solution A and the gelling precursor solution B to form the hydrogel material. .

In a third aspect of the present invention, there is provided a method for preparing the hydrogel material according to the second aspect of the present invention, comprising the steps of:

(a) providing the gel-forming precursor liquid A and the gel-forming precursor liquid B;

(b) and mixing the gelling precursor solution A and the gelling precursor solution B to obtain the hydrogel material.

In another preferred embodiment, the mixing comprises: and injecting the gel-forming precursor solution A and the gel-forming precursor solution B to a treatment site through a double-barrel injector, so as to mix and form the hydrogel material.

In a fourth aspect of the invention, there is provided a medical material comprising a hydrogel material according to the second aspect of the invention.

In another preferred embodiment, the medical material is selected from the group consisting of: the tissue shielding/adhesive, the rapid hemostatic material, the minimally invasive deliverable tissue sealant can be sprayed.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 shows a mechanism explored by Borax to regulate gelation rate. (a) A gelling reaction process; firstly, deprotonation is carried out to improve amino (-NH) in protein₂) Nucleophilicity of, -NH₂Attacks the 4-arm-PEG-SC terminal ester bond to form PEG-LZM hydrogel and releases acidic by-product N-hydroxysuccinimide. (b) Due to the water electrolysis, hydroxyl (OH-) is generated near the cathode to promote the gelling reaction. (c) After the addition of Borax, the same amount of boric acid (H) is hydrolyzed₃BO₃) And tetrahydroxyborate ion (B (OH)₄ ^-) And providing buffering capacity consuming protons (H)⁺). And (d) adding boric acid and hydrochloric acid into the gelling precursor liquid A, and then comparing the gelling time. (e) The gelation time of the two systems was compared after adjusting the gel-forming precursor solution a to the same pH with NaOH and Borax.

FIG. 2 shows the versatility of Borax to promote gel formation rates. (a) Under the condition of no Borax addition, different proteins and 4-arm-PEG-SC do not gel within 10 min. (b) After the Borax is added, the gelling rate of the protein and the 4-arm-PEG-SC is greatly improved.

FIG. 3 shows the relationship between the concentration of Borax in precursor solution A and the pH of precursor solution A as a function of the gelation time of the PEG-LZM/Borax hydrogel.

FIG. 4 shows the delivery pattern (tool) of PEG-LZM/Borax hydrogel.

FIG. 5 shows that the PEG-LZM/Borax gel-forming solution is sprayed to rapidly gel and rapidly close the skin and myocardial defect wound.

FIG. 6 shows a simulated in vitro minimally invasive delivery of PEG-LZM/Borax hydrogel.

Figure 7 shows hydrogel mechanical and tissue adhesion performance testing. (a) And (4) performing rheological test. (b) The adhesion performance of the hydrogel to the pigskin is shown and (c) and (d) the adhesive strength is tested. (e) A hydrogel anti-vascular-bursting pressure test device graph and (f) a result.

FIG. 8 shows the cell affinity and cytotoxicity of PEG-LZM/Borax hydrogels. (a) C2C12 cells and HaCaT cells were compared in terms of their morphology by spreading on the surface of a well plate and PEG-LZM/Borax hydrogel (light microscopy and fluorescent staining of cytoskeleton). (b) And calculating the average spreading area of each group of cells according to the fluorescent staining result. Relative cell viability and (d) live/dead cell fluorescent staining of cells seeded on different sample surfaces after 1, 3, 5 days.

FIG. 9 shows the in vivo biocompatibility of PEG-LZM/Borax hydrogels. (a) Photograph of the PEG-LZM/Borax hydrogel after implantation subcutaneously at the back of rat 1 w. (b) Hydrogel and surrounding tissue H & E staining.

FIG. 10 shows the PEG-LZM/Borax hydrogel antimicrobial activity evaluation. (a) Photograph of coated plate of bacteria after co-culturing Staphylococcus aureus (S. aureus), methicillin-resistant Staphylococcus aureus (MRSA), Escherichia coli (E. coli), Pseudomonas aeruginosa (P. aeruginosa) with PEG-LZM, PEG-LZM/Borax hydrogel. (b) Bacterial suspension turbidity measurements (OD value,600nm) after co-cultivation with different samples. (c) The living/dead bacteria fluorescence staining and SEM were used to observe the biofilm formation on the surface of different samples.

FIG. 11 shows PEG-LZM/Borax ultra-quick gel for left ventricular penetration closure evaluation. (a) The surgical procedure shows that (1) the left ventricle is exposed; (2) establishing a left ventricular penetrating lesion with a 1.2mm (inner diameter) needle; (3) spraying hydrogel; (4) the hydrogel plugs the bleeding opening. (b) Representative echocardiographic images of the preoperative normal heart and post-operative 2d, 3 w. (c) Ejection fraction (EF%) and fractional shortening (FS%) statistics. (d) Post-operative 3w photographs of the wound. (e) Histological observation of wounds (H & E).

Detailed Description

The inventor of the invention has studied extensively and deeply, and has developed for the first time that the gelation time of the self-nucleophilic addition reaction, namely the amidation reaction, between the amino group and the active ester functional group of the succinimide can be effectively controlled by adding strong base and weak acid salt such as borax. Adding strong base weak acid salt with pH regulating capacity into the reaction solution to regulate and control the protonation degree of amino group in reactant, such as protein, etc., i.e. N atom nucleophilicity in amino group, so that the injectable hydrogel has different gelation time. After the gelation time of the protein-polyethylene glycol hydrogel is adjusted, the hydrogel with the rapid gel forming capability can be applied to rapid hemostasis in emergency, and meanwhile, the adjustable gel forming time can provide convenience for minimally invasive delivery treatment of the hydrogel to deep wounds in vivo. The gelation time regulation and control method is simple, strong in operability, safe, efficient and suitable for clinical application.

Description of the terms

Unless defined otherwise, 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 belongs.

As used herein, the term "about" when used in reference to a specifically recited value means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

As used herein, the term "comprising" or "includes" can be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of …," or "consisting of ….

As used herein, the term "strong base and weak acid salt" is a salt formed by the reaction of a strong base and a weak acid. Because acid radical ions or non-metal ions consume a part of hydrogen ions in hydrolysis and ionize hydroxide ions, most of strong base weak acid salt solution is alkaline.

The term "gel-forming solvent" as used herein refers to a solvent used to dissolve the gel-forming components (precursors: PEG derivatives, proteins, etc.).

As used herein, the term "gel-forming precursor solution" refers to a homogeneous solution formed by dissolving gel-forming components (precursors: PEG derivatives, proteins, etc.) in a particular solution.

As used herein, the term "gel-forming solution" refers to a solution that is formed by uniformly mixing two gel-forming precursors a/B and is capable of forming a gel chemical reaction.

As used herein, the term "gelation time" refers to the time required for a gel liquid to transform from a flowable liquid state to a solid gel at a specified temperature.

As used herein, the term "Borax for medical purposes" (Borax) has the chemical formula Na₂B₄O₇·10H₂O, also known as sodium tetraborate (decahydrate), borax (pharmaceutical), borax sand, etc., is a very important boron-containing mineral and boron compound. The Chinese medicinal composition has the effects of removing toxic substances, diminishing inflammation and the like, and also has certain antibacterial activity.

Polyethylene glycol (PEG)

PEG is a water-soluble polyether obtained by gradually performing addition polymerization on ethylene oxide and water or ethylene glycol, is a polymer with neutral pH and no toxicity, has a linear or branched chain structure, ether bonds-O-on the chain can form a strong hydrogen bond with water to ensure that the water-soluble polyether has good hydrophilicity, and vinyl on the molecular chain ensures that the water-soluble polyether has certain hydrophobicity and can also be dissolved in various organic solvents; PEG has excellent biocompatibility, can be dissolved in interstitial fluid in vivo, does not stimulate the immune system of human body to generate rejection, and can be quickly discharged out of the body by a matrix without generating any toxic or side effect.

Multi-arm polyethylene glycol (n-arm-PEG)

As used herein, "multi-armed polyethylene glycol" and "multi-armed polyethylene glycol" are used interchangeably and refer to PEG that has been further chemically modified to modify various reactive groups for various crosslinking, assembly, etc. properties for assembling hydrogels, etc. in a variety of applications. The multi-arm polyethylene glycol is a high molecular polymer in which a plurality of linear chain polyethylene glycol chains with equal molecular weight are connected from a branch point in a radial manner. In the present invention, a preferred class of polyethylene glycols are multi-armed polyethylene glycols, more preferably tetra-armed polyethylene glycols.

The four-armed polyethylene glycol to which the present invention relates can be represented by the following structure:

where different molecular weights are used, the value of the repeating unit, n, is different, for example, when the weight average molecular weight is 10000, n is about 56.

Four-armed polyethylene glycol (4-arm-PEG-NHS) with terminal modified succinimide active ester

The four-armed polyethylene glycol of the present invention may be modified at the ends of the four chains with different active succinimidyl esters (-NHS), for example, with a carboxylic acid succinimidyl group (as shown below: - (CH)₂)_m-COO succinimide, wherein m is an integer of 0 to 10), dicarboxylic acid monosuccinimidyl group (shown below: (C ═ O) - (CH)₂)_m-COO succinimide, wherein m is an integer of 0 to 10), etc., to form a tetra-armed polyethylene glycol (4arm PEG-NHS) having a terminal-modified succinimide active ester, so that it has activity to react with other amino group-bearing substances.

The 4-arm-PEG-NHS has one or more of the following characteristics:

(a) the weight average molecular weight is 5000 to 40000, preferably 10000 to 20000, more preferably 10000;

(b) the purity is more than 99 percent;

(c) the dispersion coefficient (PDI) of a single polyethylene glycol Polymer (PEG) is 1-1.1; preferably 1.05.

For example, the active esters can be classified according to the type of succinimide ester:

a four-armed polyethylene glycol succinimide carbonate (4-arm-PEG-SC) having the chemical formula:

the chemical structural formula of the four-arm polyethylene glycol succinimide acetate (4-arm-PEG-SCM) is as follows:

the chemical structural formula of the four-arm polyethylene glycol succinimide succinate (4-arm-PEG-SS) is as follows:

the four-arm polyethylene glycol succinimide glutarate (4-arm-PEG-SG) has a chemical structural formula as follows:

in the above formulas, n is an integer of 2 to 300

Terminal modified amino four-arm polyethylene glycol (4-arm-PEG-NH)₂)

The tetra-armed polyethylene glycol of the invention may also be modified with amino groups at the ends of the four chains (e.g., - (CH)₂)m-NH₂Wherein m is an integer of 0 to 10) to form a terminal-modified amino group-containing tetra-armed polyethylene glycol (4-arm-PEG-NH)₂)。

The 4-arm-PEG-NH₂Has one or more of the following features:

(b) the purity is more than 99 percent;

(c) the dispersion coefficient (PDI) of a single polyethylene glycol Polymer (PEG) is 1-1.1; preferably, it is 1.05.

For example, the chemical formula may be specifically:

the four-arm polyethylene glycol with the amino modified at the tail end can be used for replacing part of protein when preparing the hydrogel, and the mechanical properties of the hydrogel, such as elastic modulus, can be adjusted; pore size of the three-dimensional network structure of the polymer, and the like.

Functional proteins

Functional proteins are proteins that carry physiological functions of the human body, and they mainly perform various metabolic activities of the human body. The functional protein includes catalytic protein, transport protein, immunological protein, regulatory protein, etc.

The functional protein of the present invention is a protein with amino groups exposed on the surface, and can be selected from the following group: lysozyme protein (which may be from various sources (e.g., birds, poultry), such as chicken lysozyme protein), serum albumin, egg white albumin, hemoglobin, or a combination thereof.

In the protein, the number of amino groups exposed on the surface of each protein molecule is more than or equal to 2.

The protein has a solubility in water at 37 ℃ of more than 50 mg/ml.

Tetraarmed polyethylene glycol 1- (C ═ O) -N-proteins

The "tetraarmed polyethylene glycol 1- (C ═ O) -N-protein" of the present invention is a product obtained by urethane exchange reaction between tetraarmed polyethylene glycol (4-arm-PEG-NHS) having a succinimide active ester modified at the end and a protein having an amino group exposed on the surface.

Tetraarmed polyethylene glycol 1- (C ═ O) -N-tetraarmed polyethylene glycol 2

The invention relates to a four-arm polyethylene glycol 1- (C ═ O) -N-four-arm polyethylene glycol 2 which is composed of four-arm polyethylene glycol (4-arm-PEG-NHS) with a terminal modified succinimide active ester and four-arm polyethylene glycol (4-arm-PEG-NH) with a terminal modified amino group₂) The product obtained by urethane exchange reaction.

PEG-LZM/Borax hydrogels

Hydrogels are a class of polymeric materials having three-dimensional crosslinked networks that are capable of absorbing and retaining large amounts of moisture. The PEG-LZM/Borax hydrogel is formed by dissolving a certain amount of Borax (Borax) into physiological saline as a gelling solvent to dissolve lysozyme protein (LZM) as gelling precursor solution A; dissolving a four-arm poly (ethylene glycol) derivative (4arm PEG-NHS) of the terminal modified active ester by using physiological saline to serve as a gelling precursor liquid B; and mixing the two gel-forming precursor solutions to prepare the hydrogel which is marked as PEG-LZM/Borax.

In situ injectable/sprayable hydrogels

In situ injectable hydrogels refer to hydrogels that are semisolid in situ after the hydrogel raw materials in fluid form are simultaneously injected into subcutaneous or muscular tissue by a syringe, and thus easily fill the entire defect site with irregular shape. The liquid injection and in-situ consolidation break through the limitation of the state of the traditional hydrogel, so that the hydrogel has better adaptability and fitness to wounds, and meanwhile, the simple injection means greatly reduces the wounds of surgical operations, accelerates the healing of the wounds, improves the healing efficiency and quality of diseases, and can be used for wound dressings, tissue adhesion, drug controlled release, tissue scaffolds and other aspects.

Minimally invasive delivery

Minimally invasive, that is, a technique of only causing a tiny wound to a patient in the surgical treatment process and leaving only a tiny wound after the operation, is a scientific and technological achievement relative to the traditional operation. The minimally invasive delivery is to insert a minimally invasive catheter through the tiny wound, inject glue solution through the minimally invasive catheter with the assistance of medical equipment such as an endoscope and the like, and convey the hydrogel material to the focus part for treatment.

Tissue adhesion/tissue sealing/physical occlusion hemostasis

The tissue glue (or called as tissue adhesive) has certain adhesive property and can contact with human tissues, and the tissue glue can react with the human tissues to increase adhesion in the process of gelatinizing the surface of the human tissues, so that hemostasis, adhesion of wounds instead of sutures, tissue sealing, wound sealing, postoperative tissue adhesion prevention and the like are realized.

Method for adjusting gelation time of injectable protein/polyethylene glycol-based hydrogel material

The invention provides a method for adjusting the gelation time of an injectable protein/polyethylene glycol-based hydrogel material by incorporating a strong base weak acid salt, the method comprising the steps of:

(a) firstly, dissolving quantitative strong base and weak acid salt into physiological saline;

(b) dissolving functional protein or multi-arm polyethylene glycol (n-arm-PEG-NH) with amino group modified at tail end in the strong alkali weak acid salt solution₂) As a gel-forming precursor solution A;

(c) dissolving a multi-arm poly (ethylene glycol) derivative (n-arm-PEG-NHS) of the terminal modified active ester in physiological saline to obtain a colloid precursor solution B;

(d) equivalently filling the gel-forming precursor solution into a double-barrel syringe, and forming the four-armed polyethylene glycol 1- (C ═ O) -N-protein after injection; or a polymer network of tetra-armed polyethylene glycol 1- (C ═ O) -N-tetra-armed polyethylene glycol 2, and forming a hydrogel.

The strong base and weak acid salt can adjust the pH value, so that the hydrogel material is cured at different speeds, in another preferable case, the strong base and weak acid salt doped in the gelling precursor liquid A can be borax, sodium carbonate, sodium hydrogen phosphate, sodium metaaluminate and the like, and the pH value in the gelling precursor liquid A can be continuously increased along with the increase of the doping amount.

In another preferred case, the alkali and weak acid salt added into the gel-forming precursor solution A can be medical Borax (Borax), and the addition amount in the gel-forming precursor solution A can be 4 mg/mL-23 mg/mL according to the required gelation time.

In another preferred case, the gel-forming precursor solution B may be a multi-arm poly (ethylene glycol) solution with terminally modified active ester groups, and the multi-arm poly (ethylene glycol) solution contained therein has one or more of the following characteristics:

(a) the weight average molecular weight is 5000-100000;

(b) the purity is more than 99 percent;

(c) the single polyethylene glycol Polymer (PEG) has a dispersion coefficient (PDI) of 1-1.1.

In another preferred example, the four-armed polyethylene glycol with the terminal modified succinimide active ester group in the gel-forming precursor liquid B is four-armed polyethylene glycol Succinimide Carbonate (SC), four-armed polyethylene glycol succinimide acetate (SCM), four-armed polyethylene glycol Succinimide Succinate (SS), four-armed polyethylene glycol Succinimide Glutarate (SG), and the like; preferably, it is a tetraarmed polyethylene glycol succinimide carbonate.

In another preferred embodiment, the weight average molecular weight of the four-armed polyethylene glycol with the terminal modified succinimide active ester group is 10000-20000, and more preferably 10000.

In another preferred embodiment, in the gel-forming preparation process, the concentrations of the polymer solutes in the gel-forming precursor liquid a and the gel-forming precursor liquid B may be the same or different, and are preferably the same.

In another preferred example, in the preparation process of gelling, gelling precursor liquid a and gelling precursor liquid B are added and mixed, the volumes of the two liquids are required to be consistent, and the ratio of the gelling liquid a to the gelling liquid B is 1: 1 are filled into sterile syringes (single barrels) respectively, and are injected and gelled to form the hydrogel material.

In another preferred embodiment, the sterile filter used has a mesh pore size of 0.22 μm.

In another preferred embodiment, the temperature required for gelling is 25 ℃ +/-15 ℃; preferably, it is 37 ℃.

In another preferred embodiment, the solution filled in the double-tube syringe is required to be injected in one time and cannot be injected repeatedly.

In another preferred example, after 100mg/mL lysozyme protein solution and 100mg/mL end-modified carbonate four-arm polyethylene glycol (4-arm-PEG-SC, Mw:10000) solution are respectively used as the gel-forming precursor solution A and B and are adjusted by using borax with different contents, the gelation time is 3 s-600 s when the borax concentration in the gel-forming precursor solution A is 4 mg/mL-22 mg/mL.

In another preferred embodiment, the two gel-forming precursors can be merged and instantly mixed uniformly at the outlet of the double-cylinder injector by an atomizing nozzle or a mixing needle at the outlet of the double-cylinder injector.

In another preferred example, the hydrogel with the gelation time within 3s can be injected and instantly cured to block the tissue defect.

In another preferred example, the gelation time can be adjusted to be consistent with the flowing time of the gelation liquid in the minimally invasive catheter, so that the gelation liquid can smoothly flow in the minimally invasive catheter after injection and can rapidly generate gelation at an outlet for curing (< 10 s).

In another preferred example, the mechanical properties and the like of the hydrogel are relatively stable after the gelation time is adjusted.

In another preferred example, when the solid content of the hydrogel precursor is 150mg/mL, the adhesion force to the pigskin is 20kPa, and the anti-vascular-burst pressure is 180 mmHg.

In another preferred embodiment, the antibacterial performance of the hydrogel is obviously enhanced by introducing Borax.

In another preferred embodiment, lysozyme is used as the gel-forming component, and the hydrogel has good cell adhesion ability and excellent biocompatibility.

Citation of protein/polyethylene glycol based hydrogel materials with different gelation times

The invention aims at different clinical application needs, and the method of the first aspect of the invention is applied to adjust the gelation time of the protein/polyethylene glycol-based hydrogel material so as to be suitable for different clinical applications. The material after the special gelation time adjustment can be used for tissue shielding, hemostasis, tissue plugging, tissue adhesion and the like.

In another preferred example, the medical material with adjustable gelation time according to needs is one or more of the following:

(1) as a sprayable tissue barrier/adhesive (after gel time adjustment, the detached tissue can be joined by rapid in situ curing after spraying);

(2) as a rapid hemostatic material (after the adjustment of the gelation time, the gel can be used as a rapid physical sealing hemostatic material and promoting the local repair of tissues in an emergency);

(3) as a minimally invasive delivery tissue blocking agent (after the gelation time is adjusted, the minimally invasive delivery tissue blocking agent can be applied to local open wounds of internal organs (intestines/stomach/heart/lung and the like) of a human body in a minimally invasive delivery mode, and is used for blocking the open wounds and promoting the repair of local combination);

the main advantages of the invention are:

(1) the gelation time of the hydrogel based on the amidation reaction can be accurately regulated and controlled by simply regulating and controlling the adding amount of the strong alkali and the weak acid salt in the gelation precursor liquid.

(2) Through reasonable mixed delivery design, the adding of the Borax can not generate obvious influence on the mechanical property, the tissue adhesion property, the anti-bursting pressure and the like of the hydrogel.

(3) After adjustment, the PEG-LZM/Borax can form gel within 3s at the fastest speed, and can be used for rapid hemostasis in emergency.

(4) The precisely regulated gelation time can provide convenience for minimally invasive delivery of the hydrogel to deep wounds in vivo.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. Unless otherwise indicated, percentages and parts are percentages and parts by weight.

The test materials and reagents used in the following examples are commercially available without specific reference.

Example 1: investigation of gelation time mechanism

1. Hydrogel preparation

Before preparing the hydrogel, the LZM was first purified by dialysis-lyophilization (3d) to remove acidic substances from the protein. The different Borax content saline solutions were prepared by dissolving Borax with saline (0.9%, w/v), using it to dissolve LZM or other proteins (15%, w/v) as the gelling precursor solution A, and using saline to dissolve 4arm PEG-SC (15%, w/v) as the gelling precursor solution B. Equal volumes of gel-forming precursors a and B are loaded into dual barrel blender syringes), and a needle or sprayer is mounted at the nozzle of the dual barrel syringe and then rapidly injected into different molds to form PEG-LZM/Borax or other PEG-Protein/Borax hydrogels.

2. Measurement of gelation time

The gelation time was measured in this experiment using the vial tilt method. Gel-forming precursor liquid a and gel-forming precursor liquid B were prepared and filled into double syringes according to the method described above at room temperature (25 ℃), the liquid in the syringes was filled into vials immediately after the start of the timekeeping and shaking was continued, the timekeeping was stopped when no flow of liquid in the vials was observed, and the recorded time was taken as the gelation time of the group.

pH value measurement

The pH of all solutions and hydrogels was monitored in this experiment using a microelectrode research system equipped with a pH electrode (tip diameter: 100 μm). Before measurement, the washed pH working electrode and reference electrode were first inserted into a calibration solution having pH values of 4.0, 7.0, and 9.2 to perform calibration, thereby determining voltages corresponding to different pH values. After the calibration is finished, a computer automatically generates a standard curve, the pH working electrode and the reference electrode are carefully cleaned and simultaneously inserted into the solution or gel, and the corresponding pH value is the measured pH value after the equal voltage is stable.

4. Electrochemical deposition experiments

In order to investigate the effect of OH-ions in the gelling solution on the gelling reaction, first, LZM and 4arm PEG-SC (15%, w/v) dissolved in normal saline (0.9%, w/v) added with a proper amount of pH indicator are used as gelling precursor solutions A and B, the two-phase solution is uniformly mixed to be used as electrolyte, a titanium sheet is used as a working electrode (cathode), a platinum wire is used as a counter electrode (anode), and a constant current (current density 16A/m2) is applied through an electrochemical workstation to perform an electrodeposition experiment. And recording the color change of the pH indicator in the electrolyte by using a camera, taking out the working electrode after electrifying for 20min, and observing the gelling condition on the surface of the electrode.

5. Effect of boron on gelation time

Different amounts of boric acid (H) were added to the physiological saline solution containing 20mg/mL Borax₃BO₃) Or hydrochloric acid (HCl), adjusting the pH value of the solution from initial 9.1 to 8.8, 8.6, dissolving LZM to obtain gel-forming precursor solution A, and dissolving 4-arm-PEG-NHS (15%, w/v) in physiological saline to obtain gel-forming precursor solution B. And gelation was measured under different solvent conditions according to the above methodTime.

6. Effect of strong base (NaOH) on gelation time

LZM (150mg/mL) was dissolved using a physiological saline solution containing 20mg/mL Borax, and the pH of the solution was measured. At the same time, the pH was adjusted to the same value by adding an appropriate amount of NaOH to an aqueous solution of 150mg/mL LZM. The two solutions were used as gelling precursor solution A, respectively, and gelling experiments were performed with the same gelling precursor solution B (150mg/mL 4arm PEG-NHS saline solution). And the gelation time was measured under various conditions according to the above-mentioned method.

The mechanism of amidation reaction between protein and PEG is shown in FIG. 1(a), in which-NH in protein (e.g., LZM)₂Deprotonation is carried out firstly, and then nucleophilic substitution is carried out on the 4-arm-PEG-SC terminal group to form a polymer network with stable amido bond as a cross-linking point. Due to-NH₂Is influenced by the pH of the initial buffer and the acidic by-product (N-hydroxysuccinimide) generated during the reaction, and therefore, it is expected that the alkaline gelling environment can promote-NH₂Deprotonation of the middle N atom, thereby catalyzing the amidation reaction. Electrochemical testing was used to verify this speculation, as shown in figure 1 (b). Before the potential was applied, the electrolyte solution showed yellow (acidic) color, and no gelation was observed, indicating that the acidic environment is not favorable for the gelation reaction. However, after the voltage was applied, hydrogel was rapidly formed on the cathode titanium plate. At the same time, the solution near the cathode changed from yellow to blue (alkaline), which demonstrates the large amount of OH generated by the electrolysis of water^-The quick gelation of the gel-forming liquid is facilitated. Based on this, it can be concluded that the alkaline environment can catalyze the amidation gel reaction.

As shown in FIG. 1(c), Borax, a typical strong base weak acid salt, ionizes to Na in aqueous solution⁺And B₄O₇ ^2-And B is₄O₇ ^2-Can be further hydrolyzed into boric acid (H) with the same amount₃BO₃) And tetrahydroxyborate ion (B (OH)₄ ^-) The weak alkaline buffer environment established between them can consume the protons (H) present in the gum solution⁺)。

To measureGelation time of PEG-LZM in the presence of Borax, gelation time was only about 3s in the case of adding Borax to the LZM solution at a final concentration of 20mg/mL, and no solid gel was produced without adding Borax. To further understand the driving mechanism of Borax, boronic acid (H) was used₃BO₃) Or hydrochloric acid (HCl) to reduce the buffering capacity of the Borax, the pH of the Borax-containing LZM solution is reduced from 8.9 to the same pH of 8.6. Whatever acidic substance (H) is added₃BO₃Or HCl), the gelation time of the PEG-LZM hydrogel was significantly increased, and no significant gelation time difference was observed under the same pH environment. It was thus demonstrated that the Borax-promoted gelation process can be attributed to its pH-regulating ability to gel-forming environment.

Next, to further verify that the Borax solution has a certain buffering capacity during gelation and can continuously consume H⁺So as to accelerate the gelling reaction process, and the pH value of the LZM solution is adjusted to 8.9 by using non-buffered NaOH to carry out the same gelling reaction. As shown in FIG. 1(e), while the pH of both LZM/Borax and LZM/NaOH gel-forming precursors A were the same, the LZM/Borax gel-forming precursors exhibited a faster gelation rate when exposed to 4-arm-PEG-NHS. This is because the buffer capacity of Borax prevents the pH value from dropping too quickly due to the acidic substance during the reaction process, thereby maintaining-NH-in the early stage of the gelation process₂High nucleophilicity.

Example 2: evaluation of mechanism versatility

Dissolving a series of proteins such as LZM, BSA, OVA and the like as a gel-forming precursor solution A (15%, w/v) by using normal saline or normal saline containing 20mg/mL Borax, and dissolving 4-arm-PEG-NHS as a gel-forming precursor solution B (15%, w/v) by using the normal saline; mixing the gel-forming precursor solutions, injecting, and observing gel formation.

As shown in FIG. 2, the LZM in the PEG-LZM hydrogel was replaced with Bovine Serum Albumin (BSA), human Hemoglobin (HGB), chicken Ovalbumin (OVA), etc., and their reaction rate with 4-arm-PEG-NHS was measured. It was found that all proteins and 4-arm-PEG-NHS were poorly reactive without adding Borax and no gelling occurred within 10 min. However, after the Borax is added, the gelation speed of all the systems is obviously accelerated, and various hydrogels can quickly form hydrogel within 30s after injection. Thus, a simple general strategy for adjusting the rate based on amidation reaction is provided by the way of adding Borax.

Example 3: determination of pH and gelation time in gel-forming precursor solution A

The pH and the gelation time in the gel-forming precursor solution A were determined by reference to the method of example 1.

The LZM content was first fixed, and the pH of the gelling precursor solution a with different Borax contents was measured, and the results are shown in fig. 3(a), where the concentration of Borax was above 8mg/mL, the pH of the solution decreased slowly with the decrease of Borax concentration, which indicates that at this time Borax can provide sufficient buffer capacity to maintain the pH of the gelling precursor solution a at a higher alkaline level, thereby greatly enhancing the nucleophilicity of the LZM in the solution. When the Borax concentration is reduced to below 8mg/mL, the pH value in the solution is rapidly reduced along with the reduction of the Borax concentration. This indicates that H is generated in the gel-forming precursor liquid A at this time⁺It may have exceeded the buffer capacity provided by Borax at this concentration.

Next, the gelation time of hydrogels with different Borax contents was determined, and as shown in FIG. 3(b), the gelation time varied from seconds to minutes by changing the Borax content in gel solution A to control the gelation rate of the hydrogels. The gelation time and the pH value in the gelation precursor liquid A measured before have obvious corresponding relation, and the gel can be gelled in 3s after injection at the fastest speed. Therefore, we hypothesize that the pH, i.e., the initial nucleophilicity of the LZM, in the gel-forming precursor a plays an important role in PEG-LZM gelation time. Meanwhile, fitting the corresponding relation between the gelation time and the Borax concentration to obtain a formula:

Gelation Time(s)＝3.117+4890.865×0.553^C_borax(mg/mL)

it can be found that the actual measurement value of the gelation time is substantially consistent with the fitting value when the Borax concentration is in the range of 1-20 mg/mL. Therefore, by means of the formula, the gelation time of PEG-LZM can be precisely controlled.

Example 4: PEG-LZM/Borax hydrogel application performance in-vitro verification

1. Spray hydrogel rapid plugging test

After washing fresh pigskin and pig hearts, a circular defect with a diameter of 2mm was created. LZM was dissolved in 20mg/mL Borax in saline as gel-forming precursor solution A (15%, w/v), and 4-arm-PEG-NHS in saline as gel-forming precursor solution B (15%, w/v). The gel-forming precursor was loaded into a double barrel blender (as shown in FIG. 4), and the PEG-LZM/Borax hydrogel gel-forming and defect-blocking were checked immediately after spraying the hydrogel in the notch (4 mL).

2. In vitro simulated hydrogel minimally invasive delivery test

To verify that adjustable gelation time allows for minimally invasive delivery of the hydrogel, a 30cm long, 27G id Myostar minimally invasive catheter was attached to a 2mL blender (double syringe) at one end and placed at the top of a 2mL centrifuge tube at the other end, and saline was injected at a flow rate of 2mL/min using a syringe pump and timing was started until the timing was completed with the appearance of a drop at the other end of the catheter. The Borax content was adjusted so that the PEG-LZM gelation time was the same as the time counted. And finally, filling the prepared gelling liquid into a mixer, starting an injection pump, and observing the gelling condition of the outlet of the conduit and the blocking condition of the centrifugal pipe orifice.

As shown in fig. 5, under the condition that the gel-forming precursor solution a contains 20mg/mL Borax, the gel-forming solution is sprayed on the surface of the defective pigskin, and the gel-forming solution can be uniformly sprayed and adhered on the surface of the pigskin, and simultaneously, the gel-forming solution is rapidly solidified to seal the defective wound. Next, as shown in fig. 5, a catheter is placed inside the heart, one end of the catheter is placed inside the myocardial defect, and water is continuously injected to the outer surface of the myocardial wound through a syringe pump at the other end of the catheter, so as to simulate blood flow at the wound. It was found that the spray-coated hydrogel could completely seal the wound immediately due to the fast gel formation rate of the Borax-adjusted hydrogel.

As shown in fig. 6, one end of the medical minimally invasive catheter is connected into a double-barrel syringe and is arranged on a syringe pump, and the other end of the medical minimally invasive catheter is arranged above a centrifugal tube. After the flowing time of the gel-forming liquid in the conduit is calculated, a proper amount of Borax is added according to the formula to regulate and control the gelling time so as to match the flowing time of the gel-forming liquid in the conduit. Experimental results show that after regulation and control, the glue forming liquid after injection can smoothly flow in the minimally invasive catheter, and meanwhile, in-situ gelation rapidly occurs at an outlet, so that the opening of the centrifugal tube is plugged and does not flow to the bottom of the centrifugal tube. Therefore, we believe that the hydrogel can meet the customization requirement of the gelation time of the minimally invasive treatment only by adjusting the content of the Borax.

Example 5: influence of Borax addition on physical Properties of hydrogels

1. Determination of hydrogel rheological Properties

In this experiment and the following experiments for measuring hydrogel adhesion strength, PEG-LZM/Borax hydrogels with different gelation times were prepared by dissolving LZM as a gel-forming precursor solution A (15%, w/v) in physiological saline containing 4, 8, 12, 20mg/mL Borax and dissolving 4-arm-PEG-NHS as a gel-forming precursor solution B (15%, w/v) in physiological saline.

The prepared PEG-LZM/Borax hydrogel was cut into disk-like samples with a diameter of 20 mm. The viscoelastic behavior of the hydrogels was determined using a rotary rheometer at 37 ℃. Before testing, in order to prevent the volatilization of water in the hydrogel during the testing process, the gel sample was subjected to edge sealing treatment using simethicone. During the test, the hydrogel was first amplitude scanned to determine its Linear Viscoelastic Region (LVR) and the modulus of the gel in LVR was considered independent of the strain amplitude. Setting the LVR middle amplitude value as a condition parameter, and carrying out frequency scanning on the hydrogel within the range of 0.1-10 Hz. The measured storage modulus G 'represents the elasticity of the PEG-LZM hydrogel at this shear frequency and the loss modulus G' represents its viscosity.

2. Measurement of adhesive Strength

Mixing the gel-forming solution, and spraying between two pieces of pigskin (1cm × 2.5cm) (200 μ L). After the reaction was completed (30 min), the pigskins adhered together were fixed to one end (5 cm. times.2.5 cm) of the roughened glass plate by cyanoacrylate glue golden image 508, respectively. And clamping the glass sheet by using a universal mechanical drawing machine to draw the pigskin bonded together at a drawing rate of 5 mm/min. The maximum value measured during stretching corresponds to the adhesive strength of the hydrogel.

3. Burst pressure determination

Selecting fresh pig artery blood vessels, cleaning, and establishing a circular gap with the diameter of 2 mm. And mixing and spraying the colloid-forming liquid precursor liquid at the notch of the blood vessel by using a double-barrel mixer. After the gel is completely solidified, one end of the blood vessel is blocked, and the other end is connected with a pressure gauge and a syringe pump. PBS was slowly injected using a syringe pump until the hydrogel ruptured at the notch and the internal liquid pressure at rupture was recorded.

As shown in FIG. 7(a), the PEG-LZM/Borax hydrogels with different Borax contents exhibited approximately the same storage modulus G 'and loss modulus G' as seen by rheological analysis.

Meanwhile, in the gelling process, the residual active groups on the 4-arm-PEG-SC can also be reacted with-NH on the tissue surface₂Amide bonds are formed, thereby enabling the hydrogel to have good tissue adhesion. As shown in FIG. 7(b), two pig skins were bonded together using a gel forming solution having different contents of Borax, and the bonded pig skins were stretched to measure the adhesive strength of the hydrogel to the skin tissue. As can be seen, the adhesive strength of hydrogels with different Borax contents was about the same, about 20 to 21 KPa.

Meanwhile, as shown in FIG. 7(c), the PEG-LZM/Borax hydrogel was tested for burst strength using a porcine pulmonary aortic vascular defect model. The results show that the change in the Borax content of the hydrogel does not significantly affect the burst pressure resistance of the hydrogel.

The results show that the mechanical properties, the adhesive strength and other physical properties of the hydrogel can be kept in a stable range by adding the strategy of adjusting the gelation time of the hydrogel by adding different contents of Borax, so that the subsequent practical application of the material is guaranteed.

Example 6: evaluation of PEG-LZM/Borax biocompatibility

In the subsequent biological performance-exploring experiment, LZM was dissolved as a gel-forming solution A using a 20mg/mL Borax physiological saline solution unless otherwise stated.

1. Cell culture and inoculation

In the experiment, mouse myoblast (C2C12) and human keratinocyte (HaCaT) are used as cell models, and the influence of the addition of Borax on the cytotoxicity and cell affinity of the hydrogel is evaluated. First, C2C12 cells and HaCaT cells were uniformly dispersed in a DMEM cell culture medium solution containing 10% fetal bovine serum, 100U/mL penicillin and 100. mu.g/mL streptomycin. 5mL of cell suspension was taken at 75cm²The cells were placed in a cell culture flask at 37 ℃ in a cell culture chamber (5% CO)₂) Culturing the strain.

2. Gel surface cell adhesion study

500 μ L of hydrogel was prepared in each well of a 48-well plate according to the method described above. According to a ratio of 3X 10 per hole⁴Density C2C12 cells and HaCaT cells were seeded onto the well plate or PEG-LZM/Borax hydrogel surface. After 12h of incubation, the cell morphology was observed with an inverted microscope.

Cells were then visualized by immunofluorescence staining. Cells were first fixed with 2.5% glutaraldehyde solution for 15 min, and then the sample was treated with Triton X-100 (0.1%, v/v) solution for 10min to increase the permeability of the cells. After washing 5 times with PBS, the samples were incubated with 5. mu.g/mL FITC-pHalloidin and 5. mu.g/mL DAPI for 20min for cytoskeleton and nuclear staining, respectively, and the cell adhesion state was observed with CLSM. 10 positions of different samples were randomly photographed and the average spread area of each group of cells was calculated using Image J software.

3. Cell proliferation Rate Studies

The proliferation of cells was determined using the CCK-8 kit in this experiment. First, C2C12 cells and HaCaT cells were plated at 3X 10 per well⁴The density is inoculated on the surface of the hydrogel. After the culture of 1 st, 3 th and 5 th days, the original culture medium is removed, fresh culture medium containing 10% CCK-8 is added for incubation for 2h, 100 mu L of the incubation solution is sucked from each well and transferred to a 96-well plate, the absorbance is measured at 450nm by using a microplate reader, and the cell proliferation rate at each time point is calculated.

4. Live/dead cell fluorescent staining

After the cells were seeded, the hydrogel samples were removed at 1, 3, 5d of culture, washed 3 times on their surface with PBS, stained for live/dead cell fluorescence with Calmodulin (CaM) and Propidium Iodide (PI) according to the kit instructions (Thermo Fisher, L3224) and observed with an inverted fluorescence microscope.

5. Evaluation of compatibility in hydrogel body

200 μ L of PEG-LZM/Borax gel forming solution (15%, w/v) was injected subcutaneously into the back of SD rats (200-300 g) to assess the fibrosis and inflammatory response of the hydrogel in vivo. After 1w, all experimental mice were euthanized, samples and surrounding tissues were removed, fixed with paraformaldehyde, embedded in paraffin, sectioned, and subjected to gradient dehydration for H & E staining observation. (n is 3)

We evaluated the effect on hydrogel biocompatibility in the presence of higher levels of Borax, i.e., when it rapidly gels. As previously introduced, the unique LZM composition imparts superior cellular affinity to the hydrogel. As can be seen from FIG. 8(a), the gel still had good cell affinity after the addition of Borax. Meanwhile, the MTT kit and the fluorescent staining of live/dead cells are used for evaluating the cell proliferation condition on the surface of the hydrogel, so that the cells can be normally proliferated and almost no cell death condition occurs. Meanwhile, after the implant is implanted into the body for 2w, inflammatory cells are still fewer. Therefore, the results show that the PEG-LZM has good cell affinity and histocompatibility after the borax is added.

As can be seen from the observation of FIG. 8(a), cells were normally spread on the hydrogel surface, and no significant morphological difference was observed in comparison with cells cultured on the surface of a normal well plate. Subsequent immunofluorescent staining of the cells revealed a distinct actin skeleton, as well as extended pseudopodia and plateaus (green) in the cells adhered to the hydrogel surface. The spread area of the cells on the surface of the different matrices was counted using image J software. As shown in FIG. 8(b), there was no significant difference in cell spreading area between the different groups. The above results all show that the PEG-LZM hydrogel containing Borax still has higher cell affinity. When the proliferation of cells on the surface of hydrogel was evaluated by using CCK-8 kit (fig. 8(c)) and fluorescence staining of live/dead cells (fig. 8(d)), it was found from the proliferation statistics that the cells seeded on the surface of gel showed some relative toxicity in the first day, but the cells were observed to be stably proliferated in the next 3 days and 5 days. In addition, in live/dead fluorescent staining, almost no cell death was observed.

And as shown in FIG. 9, no significant fiber-wrapped or intact collagen deposition was found on the PEG-LZM/Borax hydrogel surface after implantation at 1 w. Meanwhile, after the tissues around the hydrogel are sliced and observed by H & E staining, a large number of inflammatory cells (multinucleated cells, macrophages and the like) do not appear around the injected hydrogel, and only a small number of inflammatory cells are gathered along the boundary of materials and tissues, which is probably caused by short-term sensitive reaction of normal tissues to foreign matters. And meanwhile, tissue necrosis is avoided. The surrounding tissue in which the hydrogel was implanted did not show significant redness, ulceration, or other inflammatory response throughout the experimental observations. The above results demonstrate that PEG-LZM/Borax hydrogels have good histocompatibility.

Example 7: evaluation of PEG-LZM/Borax in vitro antibacterial performance

First, 2mL of PEG-LZM hydrogel with/without Borax was prepared in a 24-well plate according to the above method, and 1mL of bacterial solution (Staphylococcus aureus (S. aureus), methicillin-resistant Staphylococcus (MRSA), Escherichia coli (E. coli), Pseudomonas aeruginosa (P. aeruginosa), 1X 10, respectively, was added to each well⁷CFU/mL, 100% medium), turbidity at OD 600nm after 12h incubation in a 37 ℃ bacteria incubator with normally cultured bacteria as negative control. Gradient dilution of each group of bacterial solutions 10 using physiological saline³Coating the plate after doubling. Colonies were counted in each agar plate after 18h incubation in a 37 ℃ bacterial incubator.

Under the culture condition of high glucose content medium (1%), 2mL of the medium containing 1X 10⁷CFU/mL MRSA was incubated with 2mL PEG-LZM hydrogel with or without Borax at 37 ℃ for 48 h. After the incubation was completed, the hydrogel surface was gently washed 3 times with PBS. mu.L of live/dead bacterial fluorescent staining solution (1.67mM SYTO 9 and 20mM propidium iodide) was added to each hydrogel surface for 20min, and the hydrogel surface was observed for fluorescent expression using CLSM. Meanwhile, after the hydrogel is fixed for 2 hours by 2.5 percent glutaraldehyde solution, the hydrogel is freeze-dried after alcohol gradient dehydration,and observing the generation of the biofilm on the surfaces of different hydrogels by using SEM.

As shown in FIG. 10, the number of colonies was significantly reduced in the PEG-LZM/Borax group compared to the PEG-LZM hydrogel. In the meantime, the turbidity of each bacterial suspension was also significantly reduced by cocultivation with PEG-LZM/Borax hydrogel (FIG. 7 (b)). The results all prove that the antibacterial activity of the hydrogel can be obviously improved by adding the Borax.

To further examine that the addition of Borax can enhance the antimicrobial capacity of the hydrogel, anti-biofilm formation experiments were performed. Through fluorescence dyeing and SEM microscopic morphology observation of live/dead bacteria on different hydrogel surfaces, as shown in FIG. 7(c), the blank PEG-LZM hydrogel emits a layer of obvious green fluorescence on the hydrogel surface due to weak bacteriostatic ability, and a large amount of bacteria are observed to be gathered on the hydrogel surface, so that a layer of biological membrane is generated on the surface of the surface hydrogel. No biofilm structure was observed on the hydrogel surface after Borax addition, and no bacterial adhesion was observed on the hydrogel surface. Therefore, when the appropriate content of Borax is added, the gelling rate of the hydrogel is improved, the antibacterial activity of the hydrogel can be greatly improved, and the biofilm formation at the wound is inhibited.

Example 8: application of PEG-LZM/Borax in treatment of ventricular injury

According to the dose of 40-50 mg/kg, the experimental rabbits are anesthetized by intravenous injection of pentobarbital solution at the ear margin, preoperative echocardiography measurements (ECHOs) are carried out on the experimental rabbits, and the left heart function of each experimental rabbit is recorded. And then after the preparation work of trachea intubation, breathing assistance of a breathing machine, respiratory anesthesia, nutrient solution vein supply and the like is finished, opening the thoracic cavity to expose the left ventricle, establishing a transmural puncture wound on the left ventricle by using a 1.2X 38mm medical needle, observing and recording the bleeding condition of the left ventricle, directly spraying 200 mu L of PEG-LZM/Borax glue forming liquid to quickly seal the wound, and then immediately checking the beating of the heart and the sealing condition of the wound. After the normal state is ensured, the chest cavity of the rabbit is closed according to the clinical routine operation. Injection of 1mL of antibiotic prevented postoperative systemic bacterial infection. Left ventricular function recovery was monitored with

ECHOs

2d, 3w post-operatively. After 3w, the rabbits were euthanized, and the tissues at the wound were fixed with paraformaldehyde solution and then sectioned, stained (Masson trichrome), and histologically observed.

Fig. 11(a) shows the experimental procedure. After PEG-LZM/Borax is injected into the wound to form glue solution, the bleeding can be stopped within seconds. In subsequent observations, the hydrogel was tightly adhered to the surface of the beating heart without falling or cracking. It is worth mentioning that, in the experiment, the wound can be directly plugged by injecting the gel without any auxiliary means, and the bleeding can be stopped. The hydrogel thus appears to be more advantageous in emergency situations.

Postoperative recovery of cardiac function was monitored by Echocardiography (ECHOs). As shown in fig. 11(b), post-operative 2d due to the surgical trauma, ejection fraction (EF%) and fractional shortening (FS%) representing left ventricular function decreased slightly relative to pre-operative, but returned to normal levels after 3 w.

While 3w later was observed on the cardiac wound, the hydrogel remained firmly adhered to the surface of the cardiac wound (fig. 11 (d)). As can be seen by staining the wound tissue section (fig. 11(e)), new connective tissue was generated at the original wound site, and no significant inflammatory reaction and wound necrosis were observed around the hydrogel, demonstrating that the hydrogel has good biocompatibility and promotes wound healing to some extent.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims

1. A hydrogel injection composition, comprising:

2. The injection composition of claim 1, wherein the salt of a strong base and a weak acid has one or more of the following characteristics:

(c) the salt itself does not carry an amino group;

(d) the salt should be more than 99% pure.

3. The injection composition of claim 1, wherein the salt of a strong base and a weak acid is selected from the group consisting of: borax (sodium tetraborate), sodium carbonate, sodium bicarbonate, sodium hydrogen phosphate, sodium metaaluminate, sodium acetate, potassium carbonate, potassium bicarbonate, potassium metaaluminate, calcium bicarbonate, or combinations thereof.

4. The injectable composition of claim 1, wherein the polyethylene glycol derivative of the terminally modified active ester is modified with an active ester selected from the group consisting of: carbonates, acetates, propionates, succinates, valerates, or combinations thereof.

5. The injectable composition of claim 1, wherein said reactive species with amino groups are selected from the group consisting of: functional proteins (which refer to a class of proteins having some special physiological functions in addition to the nutritional effects of general proteins), aminopolysaccharides, or amino-modified polyethylene glycols.

6. The injection composition of claim 1, wherein the gel-forming precursor a has a viscosity of 0.1 to 2 Pas;

the viscosity of the gel-forming precursor liquid B is 0.5-1 Pas.

7. The injection composition of claim 1, wherein the concentration of the water-soluble polymer or macromolecule with more than two amino functionalities is 10-200 mg/mL; the concentration of the polyethylene glycol of the end modified active ester is 10-200 mg/mL.

8. A hydrogel material, wherein the hydrogel material is prepared by a method comprising:

(3) and mixing the gelling precursor solution A and the gelling precursor solution B to form the hydrogel material.

9. A method of preparing the hydrogel material of claim 8, comprising the steps of:

10. A medical material comprising the hydrogel material of claim 8.