CN111909401B

CN111909401B - Bi-component cross-linked medical composite material, preparation method and application thereof

Info

Publication number: CN111909401B
Application number: CN202010816376.5A
Authority: CN
Inventors: 吴建华; 姚江平
Original assignee: Hangzhou Yiwen Biomedical Co ltd
Current assignee: Hangzhou Yiwen Biomedical Co ltd
Priority date: 2020-08-14
Filing date: 2020-08-14
Publication date: 2021-11-16
Anticipated expiration: 2040-08-14
Also published as: CN111909401A

Abstract

The invention provides a bi-component cross-linked medical composite material, a preparation method and application thereof, the bi-component cross-linked medical composite material comprises a first component and a second component, the first component is a recombinant macromolecular compound with a dead end of an active group, the recombinant macromolecular compound with the dead end of the active group is formed by bonding a branched compound with the dead end of the active group and a branched macromolecular compound or a straight-chain macromolecular compound through a chemical bond, the recombinant macromolecular compound with the dead end of the active group is provided with branched chains, the tail end of each branched chain is provided with only one active group, and the second component is a biocompatible polysaccharide cross-linking agent. The double-component cross-linked medical composite material is of a three-dimensional space network structure, can be instantly and quickly generated on a wound surface of mucosa or skin, can be seamlessly connected with the wound surface, seals, isolates and protects the wound surface, and has the advantages of tissue adhesion prevention, smooth surface of the material facing away from the wound surface, no adhesion force, and incapability of adhering adjacent tissues or foreign matters on the periphery to the wound surface.

Description

Bi-component cross-linked medical composite material, preparation method and application thereof

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a bi-component cross-linked medical composite material, and a preparation method and application thereof.

Background

The hydrogel is a first biomaterial developed for human body, shows good biocompatibility when contacting with blood, body fluid and human tissue, does not affect the metabolic process of a living body, and can discharge metabolic products through the hydrogel. However, the hydrogel has problems of rapid swelling, adhesion, etc. A hydrogel tissue adhesive produced by reacting an oxidized polysaccharide with a multi-arm polyether amine is disclosed in U.S. patent 2006/0078536. The elongation at break of the hydrogel is increased by decreasing the crosslink density of the hydrogel, but since crosslinking can prevent the hydrogel from swelling and can enhance the strength of the hydrogel, a lower crosslink density can cause the hydrogel to easily swell and lose mechanical properties. Polyfunctional polyalkylene oxides, hydrogels and tissue adhesives are disclosed in us patent CN101541857A, the polymer having two or three functional groups at the ends, the multiplicity of the functional groups increasing the statistical probability of reaction at a given chain end, but with longer gel formation times.

The medical hydrogel is a three-dimensional reticular polymer compound, is rich in water molecules, has good flexibility, is mostly nontoxic or low in toxicity, and the like, and is widely researched and applied in the fields of biological and medical engineering. However, the existing medical hydrogel still has the problems of insufficient adhesive force, poor adaptability, easy swelling, long gelling time and the like.

Disclosure of Invention

The invention provides a bi-component cross-linked medical composite material, a preparation method and application thereof, and solves the problems of insufficient adhesive force, poor adaptability, high rejection possibility, easy swelling and the like of a biomaterial in the prior art.

In a first aspect, an embodiment of the present application provides a two-component crosslinked medical composite material, including a first component and a second component, the first component is a reactive group terminated recombinant polymer compound, the reactive group terminated recombinant polymer compound is formed by chemically bonding a reactive group terminated branched compound with a branched polymer compound or a linear polymer compound, the reactive group terminated branched compound includes at least one of branched polyamidoamine, branched polylysine, branched poly L-glutamic acid, and branched polyaspartic acid, the reactive group terminated recombinant polymer compound has a branch, and the tail end of each branched chain has only one active group, the second component is a biocompatible polysaccharide cross-linking agent, and the biocompatible polysaccharide cross-linking agent is polysaccharide modified by the active groups.

Preferably, the branched polymer compound includes at least one of six-arm polyethylene glycol, eight-arm polyethylene glycol, pullulan, and the linear polymer compound includes at least one of polyvinyl alcohol, polyacrylic acid, polydextrose, and linear polyglutamic acid.

Preferably, the branched compound terminated with a reactive group has n branches, n is a natural number, n.gtoreq.8, and each branch has only one reactive group at the end.

Preferably, the molecular weight of the reactive group-terminated branched compound is in the range of 500-4000 daltons.

Preferably, the reactive group of the branched compound terminated with a reactive group includes one or more of amino group, amino acid residue, amino acid ester residue, aldehyde group, maleimide group, succinimide ester group, acrylate group, thiol group, vinylsulfonyl group, double bond, azide group and alkynyl group.

Preferably, the molecular weight of the branched polymer compound or the linear polymer compound is in the range of 4000-500000 daltons.

Preferably, the branched polymer compound or the linear polymer compound has m bonding sites, m is a natural number, m is not less than 6, and the bonding site is a chemical bonding group on the branched polymer compound or the linear polymer compound when the branched polymer compound whose active group is terminated and the branched polymer compound or the linear polymer compound form the recombinant polymer compound whose active group is terminated.

Preferably, the active group-terminated recombinant macromolecular compound has 5n to mxn or 5n to m (n-1) branched chains (the active group-terminated recombinant macromolecular compound has at least 5n branched chains and has at most (mxn) or [ mx (n-1) ] branched chains (n, m is a natural number, n is more than or equal to 8, m is more than or equal to 6), each branched chain end has an active group, the end of the active group terminated recombinant macromolecular compound has 5 n-mxn or 5 n-m (n-1) active groups, the active group terminated recombinant macromolecular compound is formed by chemically bonding a branched molecular compound with n active group terminated end and a branched macromolecular compound or a straight-chain macromolecular compound with m branched chains to form a recombinant macromolecular compound with 5 n-mxn or 5 n-mx (n-1) active groups at the end.

Preferably, the biocompatible polysaccharide cross-linking agent is a polysaccharide modified with a reactive group comprising one or more of amino groups, amino acid residues, amino acid ester residues, aldehyde groups, maleimide groups, succinimide ester groups, acrylate groups, thiol groups, vinylsulfonyl groups, double bonds, azide groups and alkyne groups.

Preferably, the biocompatible polysaccharide cross-linking agent comprises at least one of starch, carboxymethyl starch, hydroxypropyl starch, polydextrose, carboxymethyl cellulose, hydroxypropyl methyl cellulose, chitosan, alginate, and sodium hyaluronate.

Preferably, the molecular weight ranges of carboxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, chitosan, alginate and sodium hyaluronate are 10000-.

Preferably, when the active group of the active group-terminated recombinant macromolecular compound is one of an amino group and an amino acid residue, the active group of the biocompatible polysaccharide cross-linking agent is one of an aldehyde group, a maleimide group, a succinimide ester group and an acrylate group; when the active group of the active group-terminated recombinant macromolecular compound is one of aldehyde group, maleimide group, succinimide ester group and acrylate group, the active group of the biocompatible polysaccharide cross-linking agent is one of amino group and amino acid residue.

Preferably, the amino acid residues include glycine residues, alanine residues, cystine residues, serine residues, threonine residues, aspartic acid residues, and lysine residues.

Preferably, when the active group of the recombinant macromolecular compound with the end-capped active group is one of a sulfhydryl group, an amino acid residue and an amino acid ester residue, the active group of the biocompatible polysaccharide cross-linking agent is one of a vinylsulfonyl group, a double bond, a sulfhydryl group, an amino acid ester residue and an amino acid residue; the active group of the recombinant macromolecular compound with the end capped by the active group is one of vinylsulfonyl, double bond, sulfydryl, amino acid ester residue and amino acid residue, and the active group of the biocompatible polysaccharide cross-linking agent is one of sulfydryl, amino acid residue and amino acid ester residue.

Preferably, the amino acid residue is one of a cysteine residue and an N-acetylcysteine residue.

Preferably, the amino acid ester residue comprises one of a cysteine methyl ester residue and a cysteine ethyl ester residue.

Preferably, when the active group of the recombinant macromolecular compound with the end capped by the active group is azido, the active group of the biocompatible polysaccharide cross-linking agent is alkynyl; the active group of the recombinant macromolecular compound with the end capped by the active group is alkynyl, and the active group of the biocompatible polysaccharide cross-linking agent is azido. The active groups in the recombinant macromolecular compound with the end capped by the active groups comprise amino groups, amino acid residues, amino acid ester residues, aldehyde groups, maleimide groups, succinimide ester groups, acrylate groups, sulfydryl groups, vinylsulfonyl groups, double bonds, azide groups and alkynyl groups, and the active groups can form electrostatic action or chemical bonding action with damaged skin or damaged mucosal tissues of a human body.

In a second aspect, embodiments of the present application provide a method for preparing a two-component crosslinked medical composite, comprising the steps of:

s1: dissolving the first component in a first buffer solution to obtain a first component solution;

s2: dissolving the second component in a second buffer solution to obtain a second component solution;

s3: and uniformly mixing the first component solution and the second component solution to form the double-component cross-linked medical composite material.

Covering a layer of first component solution on the wound surface of the damaged skin or the damaged mucous membrane, and then covering a layer of second component solution, wherein the two-component cross-linked medical composite material is quickly formed on the wound surface in situ immediately at the moment of mixing and contacting the first component solution and the second component solution. The bi-component cross-linked medical composite material can quickly cover the damaged mucous membrane or the damaged skin and can be seamlessly connected with the wound surface.

Preferably, the concentration range of the first buffer solution is 0-0.1 mol/L, and the first buffer solution comprises a phosphate buffer solution, an acetate buffer solution and a carbonate buffer solution. The kind of the first buffer solution is not limited herein as long as the first buffer solution can perform a buffering function and can be applied to a human body.

Preferably, the concentration range of the second buffer solution is 0-0.1 mol/L, and the second buffer solution comprises a phosphate buffer solution, an acetate buffer solution and a carbonate buffer solution. The kind of the second buffer solution is not limited herein as long as the second buffer solution can perform a buffering function and can be applied to a human body.

Preferably, the two-component cross-linked medical composite can be instantaneously cross-linked in situ on a damaged mucous membrane or damaged skin for a time of < 1S. The rapid formation of the two-component crosslinked medical composite in situ in less than 1 second is due to: because the end of the active group-terminated recombinant macromolecular compound contains a large number of chemical reaction active groups, the biocompatible polysaccharide cross-linking agent can realize rapid chemical reaction cross-linking when contacting with the biocompatible polysaccharide cross-linking agent, and the first component solution and the second component solution which have strong rheological property are subjected to in-situ rapid phase change to form the bi-component cross-linking medical composite material with completely no flowability, good mechanical strength, good elasticity and a three-dimensional net-shaped rigid structure.

In a third aspect, embodiments of the present application provide for the use of a two-component cross-linked medical composite in the preparation of a formulation for damaged mucosa or damaged skin. The bi-component cross-linked medical composite material simultaneously has the following performance characteristics: (1) the two-component cross-linked medical composite material can be instantly and rapidly formed on the damaged mucous membrane or the damaged skin in situ in less than 1 s. (2) The first component solution and the second component solution are both flowable liquid with strong fluidity before contacting on the wound surface, and have no mechanical strength, elasticity and sealing effect, and after the first component solution and the second component solution contact on the wound surface, the first component solution and the second component solution instantaneously generate a crosslinking reaction to generate the bi-component crosslinking medical composite material with mechanical strength, no fluidity and fixed shape within 1 s. (3) The first component solution applied on the damaged skin or the wound surface of the damaged mucous membrane is a fluid solution with strong rheological property, and the first component solution can be flexibly filled, tiled and the like according to the wound surface form of the damaged mucous membrane or the damaged skin. Therefore, after the second component solution is contacted with the first component solution, the bi-component cross-linked medical composite material is instantly formed in situ within 1s, so that the bi-component cross-linked medical composite material has strong adaptability, adaptability adjustment is made according to the surface morphology characteristics of the wound surface of a patient, and the wound surface with various surface morphology is fully covered. (4) The bi-component cross-linked medical composite material has stronger adhesive force facing the wound surface layer, plays a role in tightly sealing and protecting the wound surface, but has smooth surface without any adhesive force action when being back to the wound surface layer, and can effectively prevent adjacent tissues or foreign matters around the wound surface from forming adhesive action with the wound surface. The bi-component cross-linked medical composite material is applied to damaged skin or damaged mucous membrane, has the functions of sealing protection, seepage absorption and drainage, adhesion prevention, water prevention, foreign body stimulation resistance, inflammation diminishing and pain relieving, bacterial infection prevention and the like, and can be used as a matrix due to the three-dimensional space three-dimensional network structure, thereby providing three-dimensional framework support for cell proliferation of the damaged skin or damaged mucous membrane, promoting cell proliferation, further promoting skin and mucous membrane repair, accelerating healing speed and shortening treatment period.

The invention relates to a bi-component cross-linked medical composite material, a preparation method and application thereof. The bi-component cross-linked medical composite material is an integral ultrahigh molecular polymer with a three-dimensional network structure, a large number of micropores and spaces exist in the composite material composition, and as the composite material composition is applied on damaged skin or damaged mucous membrane and then is integrated with the damaged skin or the damaged mucous membrane, skin and mucous membrane cells gradually permeate into the composite material composition with the three-dimensional network structure with the large number of micropores and spaces, and cell proliferation, cell differentiation and the like are carried out by utilizing the tissue fluid environment and nutrition of a human body. The bi-component cross-linked medical composite material has the advantages of strong adhesive force, no rejection, no sensitization, no toxic or side effect and the like, can accelerate wound healing, tissue repair and tissue regeneration, relieve pain, prevent wound deepening and scars, obstruct bacteria and prevent wound infection, is simple and convenient to operate, and is easy to store. The drug delivery site of the two-component cross-linked medical composite of the present invention includes, but is not limited to, application to the skin, wound, mucosa, internal organs, oral cavity, eye, etc. The current clinical experiment shows that the bi-component cross-linked medical composite material does not show rejection, and solves the problem that the current bi-component cross-linked medical composite material has larger rejection possibility.

Drawings

The accompanying drawings are included to provide a further understanding of the embodiments and are incorporated in and constitute a part of this specification. The drawings illustrate embodiments and together with the description serve to explain the principles of the invention. Other embodiments and many of the intended advantages of embodiments will be readily appreciated as they become better understood by reference to the following detailed description. The elements of the drawings are not necessarily to scale relative to each other. Like reference numerals designate corresponding similar parts.

FIG. 1 is a G2-8NH according to one embodiment of the invention₂A schematic molecular structure of a branched polyamidoamine;

FIG. 2 is a block diagram of G3-16NH according to one embodiment of the present invention₂A schematic molecular structure of a branched polyamidoamine;

FIG. 3 is a G2-8NH representation according to one embodiment of the present invention₂A schematic molecular structure diagram of branched polylysine;

FIG. 4 is a block diagram of G3-16NH according to one embodiment of the present invention₂A schematic molecular structure diagram of branched polylysine;

FIG. 5 is a schematic molecular structure diagram of G2-8SH branched polyamidoamine according to one embodiment of the present invention;

FIG. 6 is a schematic view of the molecular structure of G2-8SH branched polylysine according to one embodiment of the present invention;

FIG. 7 is a schematic representation of the molecular structure of a six-arm polyethylene glycol carboxylic acid according to one embodiment of the present invention;

FIG. 8 is a schematic representation of the molecular structure of an eight-arm polyethylene glycol carboxylic acid according to one embodiment of the present invention;

FIG. 9 is a schematic representation of the molecular structure of soluble amylopectin starch according to an embodiment of the present invention;

FIG. 10 is a schematic view of the molecular structure of a linear polylysine according to one embodiment of the present invention;

FIG. 11 is a schematic molecular structure diagram of a linear polyglutamic acid according to an embodiment of the present invention;

FIG. 12 is a schematic illustration of the molecular structure of polyglucose according to one embodiment of the present invention;

FIG. 13 is a schematic molecular structure diagram of a linear carboxymethyl cellulose according to an embodiment of the present invention;

FIG. 14 is a G2-8NH representation according to one embodiment of the present invention₂A schematic molecular structure diagram of branched polyamide-amine bonded hexa-armed polyethylene glycol carboxylic acid;

FIG. 15 is a G2-8NH representation according to one embodiment of the present invention₂A molecular structure schematic diagram of branched polyamide-amine bonded eight-arm polyethylene glycol carboxylic acid;

FIG. 16 is a schematic molecular structure diagram of G2-8SH branched polyamidoamine bonded polyglutamic acid according to one embodiment of the present invention;

FIG. 17 is a schematic diagram of the molecular structure of G2-8SH branched polylysine bonded to polyglutamic acid according to one embodiment of the present invention;

FIG. 18 is a G2-8NH representation according to one embodiment of the present invention₂A schematic molecular structure of branched polyamide-amine bonded soluble amylopectin;

FIG. 19 is a block diagram of G3-16NH according to one embodiment of the invention₂A molecular structure diagram of branched polylysine bonded polyglucose;

fig. 20(1) is a schematic illustration of a patient using a two-component cross-linked medical composite to promote wound repair healing after male urological surgery, according to one embodiment of the present invention;

FIG. 20(2) is a schematic illustration of wound repair healing after male urological surgery in a patient without the use of a two-component cross-linked medical composite, according to one embodiment of the present invention;

fig. 21(1) is a schematic illustration of a patient using a two-component cross-linked medical composite to promote wound repair healing after a medical orthopaedic procedure, according to one embodiment of the present invention;

fig. 21(2) is a schematic illustration of wound repair healing after a medical orthopedic surgery on a patient without using a two-component cross-linked medical composite, according to an embodiment of the present invention.

Detailed Description

Embodiments of the present application will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present application and should not be construed as limiting the scope of the present application. The examples do not show the specific conditions, and are conducted under the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

Example 1

Obtaining of amino reactive group terminated branched compound (first component raw material):

1、G2-8NH₂branched polyamidoamine having 8 branches, each branch having an amino reactive group at the end, the molecular structure is shown in figure 1, the generation is: g2 (second generation) available from wechen molecular new materials limited.

2、G3-16NH₂Branched polyamidoamine having 16 branches, each branch having an amino reactive group at the end, the molecular structure is shown in FIG. 2, generation: g3 (third generation) available from wechen molecular new materials limited.

3、G2-8NH₂Branched polylysine, which has 8 branches, wherein each branch end contains an amino active group, the molecular structure is shown in figure 3, and the generation number: g2 (second generation) available from wechen molecular new materials limited.

4、G3-16NH₂Branched polylysine, which has 16 branches, wherein each branch end contains an amino active group, the molecular structure is shown in figure 4, and the generation number: g3 (third generation) molecular novelty from Waishahi morningMaterials, Inc.

Example 2

Preparation of mercapto-reactive group-terminated branched Compound (first component starting Material)

1. Preparation of G2-8SH branched polyamidoamines:

preparing main raw materials: g2-8NH₂Branched polyamidoamines, N-acetylcysteine, cysteine, N' -Dicyclohexylcarbodiimide (DCC) (CAS number: 538-75-0), 1-Hydroxybenzotriazole (HOBT) (CAS number: 2592-95-2).

The preparation principle is as follows: n-acetylcysteine and carboxyl group on cysteine with G2-8NH₂The amino group on the branched polyamide-amine reacts under the catalytic action of DCC and HOBT catalysts to form a new amido bond (-CO-NH-), and G2-8SH branched polyamide-amine compound with the end containing a mercapto active group is generated.

The preparation process comprises the following steps:

(1) 160 parts by mass of N-acetylcysteine, 40 parts by mass of cysteine and 1500 parts by mass of solvent N, N-Dimethylformamide (DMF) are mixed, stirred and dissolved;

(2) continuously adding 50-80 parts by mass of G2-8NH₂Stirring and dissolving the branched polyamide-amine;

(3) respectively adding 10-25 parts by mass of DCC and 10-25 parts by mass of HOBT, and stirring for dissolving to form a mixed solution;

(4) continuously reacting the mixed solution at the temperature of 0-20 ℃ for 12-72 h to generate G2-8SH branched polyamide-amine;

(5) purifying G2-8SH branched polyamide-amine repeatedly by adopting a separation-redissolution-separation mode, wherein a separation solvent is ethanol or isopropanol, and a redissolution solvent is purified water;

(6) and (3) redissolving the purified G2-8SH branched polyamide-amine, and removing residual water and residual alcohol by using a vacuum freeze dryer to obtain the high-purity G2-8SH branched polyamide-amine compound.

G2-8SH branched polyamide-amine has 8 branched chains, each branched chain end contains a sulfydryl active group, the appearance of the branched polyamide-amine is white, porous and fluffy, the branched polyamide-amine is easy to dissolve in water, and the molecular structure is shown in figure 5.

2. Preparation of G2-8SH branched polylysine:

preparing main raw materials: g2-8NH₂Branched polylysine, N-acetylcysteine, cysteine, N' -Dicyclohexylcarbodiimide (DCC) (CAS number: 538-75-0), 1-Hydroxybenzotriazole (HOBT) (CAS number: 2592-95-2).

The preparation principle is as follows: the molecular structure of N-acetylcysteine and cysteine contains sulfydryl and carboxyl, and the carboxyl on the N-acetylcysteine and cysteine is reacted with G2-8NH₂The amino group on the branched polylysine reacts under the catalysis of DCC and HOBT catalysts to form a new imide bond (-CO-N-), and a G2-8SH branched polylysine high molecular compound with the end containing a mercapto active group end cap is generated.

The preparation process comprises the following steps:

(1) mixing, stirring and dissolving 120 parts by mass of N-acetylcysteine, 40 parts by mass of cysteine and 1500 parts by mass of solvent N, N-Dimethylformamide (DMF);

(2) continuously adding 50-80 parts by mass of G2-8NH₂Stirring and dissolving the branched polylysine;

(4) continuously reacting the mixed solution at the temperature of 0-20 ℃ for 12-72 h to generate G2-8SH branched polylysine;

(5) purifying G2-8SH branched polylysine repeatedly by adopting a separation-redissolution-separation mode, wherein a separation solvent is acetone or a mixed solvent of acetone and ethanol, and a redissolution solvent is purified water;

(6) and (3) redissolving the purified G2-8SH branched polylysine, and removing residual water and residual alcohol by using a vacuum freeze dryer to obtain the high-purity G2-8SH branched polylysine high molecular compound.

The G2-8SH branched polylysine has 8 branched chains, the tail end of each branched chain contains a sulfhydryl active group, the appearance of the branched chain is white, porous and fluffy, the branched chain is easy to dissolve in water, and the molecular structure is shown in figure 6.

Example 3

Obtaining or preparing a branched polymer compound (first component raw material):

1. six-arm polyethylene glycol carboxylic acid with average molecular weight of 10000 Dalton and 6 branched chains, molecular structure shown in FIG. 7, was purchased from Xiamen Sainuo Pong Biotech, Inc.

2. Eight-arm polyethylene glycol carboxylic acid with average molecular weight about 20000 daltons, 8 branched chains, and molecular structure shown in FIG. 8, was purchased from Xiamen Sainuo Pong Biotech, Inc.

3. The preparation process of the soluble amylopectin starch comprises the following steps:

(1) dissolving 50-100 parts by mass of amylopectin (CAS number: 9037-22-3) in 1000 parts by mass of purified water to form amylopectin suspension;

(2) adjusting the pH value of the amylopectin suspension to 1.5-2.5, and continuously degrading the solution at the temperature of 40-60 ℃ for 12-48 h;

(3) continuing the high-temperature high-pressure reaction of the degraded suspension at the temperature of 121 ℃ for 30-120 min;

(4) cooling the solution after the high-temperature and high-pressure reaction, and then dialyzing and purifying by using a dialysis bag with the molecular weight cutoff of about 40000 to remove residues and amylopectin with the molecular weight of less than 40000;

(5) and finally, freeze-drying by using a vacuum freezer to obtain the white porous fluffy soluble amylopectin with the appearance, wherein the average molecular weight of the soluble amylopectin is greater than or equal to 40000 daltons, the theoretical number of the branched chains is greater than or equal to 10, and the molecular structure is shown in figure 9.

Example 4

Obtaining of straight-chain type Polymer (first component raw Material)

1. The straight-chain polylysine has a molecular weight of 4000 daltons or more, is straight-chain, has a molecular structure shown in FIG. 10, and is purchased from Shandong Europe source bioengineering Co.

2. The preparation process of the straight-chain polyglutamic acid comprises the following steps:

(1) dissolving 20-50 parts by mass of polyglutamic acid (purchased from Shandong Furuida pharmaceutical group, Ltd.) in 1000 parts by mass of purified water, and dissolving at 45-60 ℃ to form a solution;

(2) carrying out high-temperature high-pressure reaction on the solution at the temperature of 121 ℃ for 30-120 min;

(3) cooling the solution after the high-temperature and high-pressure reaction, and then dialyzing and purifying by using a dialysis bag with the molecular weight cut-off of about 40000 to remove residues and straight-chain polyglutamic acid with the molecular weight less than 40000;

(4) and freeze drying with vacuum freezer to obtain white porous fluffy linear polyglutamic acid with molecular weight of 40000 daltons or more and molecular structure shown in FIG. 11.

3. Polydextrose, average molecular weight about 40000 daltons, CAS number: 9004-54-0, molecular structure as shown in FIG. 12, was purchased from Michelle chemical technology, Inc., Shanghai.

4. The preparation process of the carboxymethyl cellulose comprises the following steps:

(1) dissolving 20-50 parts by mass of carboxymethyl cellulose (purchased from Anhui mountain river pharmaceutic adjuvant, Inc.) in 1000 parts by mass of purified water, and dissolving at 45-60 ℃ to form a solution;

(3) cooling the solution after the high-temperature and high-pressure reaction, and dialyzing by using a dialysis bag with the molecular weight cut-off of about 40000 to remove residues and carboxymethyl cellulose with the molecular weight less than 40000;

(4) and finally, freeze-drying by using a vacuum freezer to obtain the white porous fluffy straight-chain carboxymethyl cellulose with the molecular weight of 40000 daltons or more, the viscosity of 100-200 mpa.s and the molecular structure shown in figure 13.

Example 5

Preparation of active group-terminated recombinant macromolecular Compound (first component):

1、G2-8NH₂branched polyamidoamine bonded hexa-armPreparation of polyethylene glycol carboxylic acids

Preparing main raw materials: g2-8NH from example 1₂Branched polyamidoamines, hexa-armed polyethylene glycol carboxylic acid from example 3, N' -Dicyclohexylcarbodiimide (DCC), 1-Hydroxybenzotriazole (HOBT).

The preparation principle is as follows: g2-8NH₂Reacting one amino group at the tail end of the branched polyamide-amine with the carboxyl group at the tail end of the branched polyethylene glycol carboxylic acid chain under the catalysis of DCC and HOBT catalysts to form a new amido bond (-CO-NH-), and reacting G2-8NH₂The branched polyamidoamine linkage is at the terminus of the hexa-armed polyethylene glycol carboxylic acid, resulting in a G2-8NH2 branched polyamidoamine-linked hexa-armed polyethylene glycol carboxylic acid.

The preparation process comprises the following steps:

(1) controlling the temperature of 1000 parts by mass of DMF (dimethyl formamide) solvent at 8-16 ℃;

(2) adding 50 parts by mass of G2-8NH into DMF solvent₂Dissolving the branched polyamidoamine;

(3) adding 15-25 parts by mass of hexa-arm polyethylene glycol carboxylic acid, and dissolving;

(4) continuously adding 10-20 parts by mass of DCC and 10-20 parts by mass of HOBT, and stirring and dissolving to form a mixed solution;

(5) reacting the mixed solution at the temperature of 8-16 ℃ for 12-48 h;

(6) dialyzing and purifying the solution after reaction by using a dialysis bag with molecular weight cutoff of about 3500 daltons;

(7) adding mannitol after purification, and freeze-drying with vacuum freezer to obtain G2-8NH with porous fluffy appearance₂Branched polyamidoamine bonded hexa-armed polyethylene glycol carboxylic acids, which are readily soluble in water, have a molecular structure as shown in FIG. 14.

2、G2-8NH₂Branched polyamidoamine bonded eight-armed polyethylene glycol carboxylic acid (first component)

Raw materials: g2-8NH from example 1₂Branched polyamidoamines, eight-arm polyethylene glycol carboxylic acid from example 3, N' -Dicyclohexylcarbodiimide (DCC), 1-Hydroxybenzotriazole (HOBT).

The preparation principle is as follows: g2-8NH₂One amino group on the branched polyamide-amine and one carboxyl group on the eight-arm polyethylene glycol carboxylic acid react under the action of DCC and HOBT catalysts to form a new amido bond (-CO-NH-), and G2-8NH is reacted₂The branched polyamidoamine is bonded to the carboxylic acid end of the eight-arm polyethylene glycol to form G2-8NH₂Branched polyamidoamine bonded eight-arm polyethylene glycol carboxylic acids.

The preparation process comprises the following steps:

(1) controlling the temperature of 1000 parts by mass of an anhydrous solvent DMF at 8-16 ℃;

(2) 80 parts by mass of G2-8NH was added to the solvent DMF₂Stirring and dissolving the branched polyamide-amine;

(3) adding 10-20 parts by mass of eight-arm polyethylene glycol carboxylic acid, and stirring for dissolving;

(5) reacting the mixed solution at the temperature of 8-16 ℃ for 12-48 h;

(7) purifying, adding mannitol, and freeze drying with vacuum freezer to obtain porous fluffy G2-8NH₂Branched polyamidoamine bonded octa-armed polyethylene glycol carboxylic acids, which are readily soluble in water, the molecular structure is shown in FIG. 15.

3. Preparation of G2-8SH branched polyamidoamine-bonded polyglutamic acid

Preparing main raw materials: g2-8SH branched polyamidoamine of example 2, polyglutamic acid of example 4, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC. HCl), N-hydroxysuccinimide (NHS, CAS number: 6066-82-6).

The preparation principle is as follows: one amino group (the amino group is derived from cysteine) on the G2-8SH branched polyamide-amine reacts with one carboxyl group on the polyglutamic acid under the catalysis of EDC.HCl and NHS catalysts to form a new amido bond (-CO-NH-), and the G2-8SH branched polyamide-amine is bonded on the polyglutamic acid to generate G2-8SH branched polyamide-amine bonded polyglutamic acid.

The preparation process comprises the following steps:

(1) dissolving 60 parts by mass of polyglutamic acid in 1000 parts by mass of purified water, and stirring and dissolving to form a polyglutamic acid solution;

(2) controlling the temperature of the polyglutamic acid solution to be 8-16 ℃;

(3) adding 10-20 parts by mass of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC.HCl) and 4-8 parts by mass of NHS, and stirring at the temperature of 8-16 ℃ for 30 min;

(4) then adding 10-20 parts by mass of G2-8SH branched polyamide-amine of example 2, and continuously reacting for 3-6 h;

(5) dialyzing and purifying the reacted solution by a dialysis bag with molecular weight cutoff of about 8000 daltons;

(6) adding lactose and mannitol after purification, and freeze drying with vacuum freezer to obtain G2-8SH branched polyamidoamine-bonded polyglutamic acid with porous fluffy appearance, which is easily soluble in water and has molecular structure shown in FIG. 16.

The G2-8SH branched polyamidoamine-bonded polyglutamic acid has a thiol reactive group at each branched end, and the thiol group is derived from cysteine and N-acetylcysteine. It should be noted that: cysteine contains an amino group which provides a sulfhydryl group when chemically bonded to the end of the branch of the G2-8SH branched polyamidoamine, while the amino group of the reactive group terminated G2-8SH branched polyamidoamine is not reactive with the second composition biocompatible polysaccharide. This is because, when the active group at the end of the branched G2-8SH polyamidoamine-linked polyglutamic acid is one of a mercapto group and an amino acid, the active group of the biocompatible polysaccharide crosslinking agent is one of a vinyl sulfone, a double bond, a mercapto group and an amino acid; when the active group at the end of the branched chain of the G2-8SH branched polyamidoamine-linked polyglutamic acid is one of vinyl sulfone, double bond, sulfhydryl and amino acid, the active group of the biocompatible polysaccharide cross-linking agent is one of sulfhydryl and amino acid. In this case, the amino acid bonded to the end of the branched G2-8SH branched polyamidoamine-linked polyglutamic acid is one of cysteine, N-acetylcysteine, methyl cysteine ester and ethyl cysteine ester. Therefore, since the amino group in this case does not have a condition for direct reaction with vinylsulfone, a double bond, a mercapto group, or an amino acid, the amino group in the G2-8SH branched polyamidoamine-bonded polyglutamic acid is inactive, that is, not an active group.

4. Preparation of G2-8SH branched polylysine bonded polyglutamic acid

Raw materials: g2-8 SH-branched polylysine of example 2, polyglutamic acid of example 4, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC. HCl), N-hydroxysuccinimide (NHS, CAS number: 6066-82-6).

The preparation principle is as follows: one amino group (amino is derived from cysteine) on the G2-8SH branched polylysine reacts with carboxyl on the polyglutamic acid under the catalysis of EDC.HCl and NHS catalysts to form a new amido bond (-CO-NH-), and the G2-8SH branched polylysine is bonded on the polyglutamic acid to generate the G2-8SH branched polylysine bonded polyglutamic acid.

The preparation process comprises the following steps:

(4) then adding 10-20 parts by mass of G2-8SH branched polylysine obtained in example 2, and continuously reacting for 6-12 h;

(6) and adding lactose and mannitol solvent after purification, and freeze-drying by using a vacuum freezer to obtain the G2-8SH branched polylysine bonded polyglutamic acid with porous and fluffy appearance, wherein the G2-8SH branched polylysine bonded polyglutamic acid is easily soluble in water, and the molecular structure is shown in figure 17.

The G2-8SH branched polylysine bonded polyglutamic acid has a sulfhydryl active group at each branch end, and the sulfhydryl group is derived from cysteine and N-acetylcysteine. It should be noted that: cysteine contains an amino group which provides a sulfhydryl group and an amino group when bonded to the end of a branch of the G2-8SH branched polylysine by a chemical bond, whereas the amino group in the reactive group-terminated G2-8SH branched polylysine is not reactive with the second composition biocompatible polysaccharide. This is because, when the active group at the end of the branch chain of the G2-8SH branched polylysine bonded to polyglutamic acid is one of a thiol group and an amino acid, the active group of the biocompatible polysaccharide cross-linking agent is one of a vinyl sulfone, a double bond, a thiol group and an amino acid; when the active group on the end of the branch chain of the G2-8SH branched polylysine bonded polyglutamic acid is one of vinyl sulfone, double bond, sulfydryl and amino acid, the active group of the biocompatible polysaccharide cross-linking agent is one of sulfydryl and amino acid. In this case, the amino acid bonded to the end of the branched polyglutamic acid chain of the G2-8SH branched polylysine is one of cysteine, N-acetylcysteine, cysteine methyl ester and cysteine ethyl ester. Therefore, since the amino group in this case does not have a condition for direct reaction with vinylsulfone, a double bond, a thiol group, or an amino acid, the amino group bonded to polyglutamic acid of the G2-8SH branched polylysine is inactive, that is, not an active group.

5、G2-8NH₂Preparation of branched polyamidoamine-bonded soluble amylopectin

Raw materials: g2-8NH from example 1₂Branched polyamidoamine, soluble amylopectin from example 3.

The preparation principle is as follows: first, soluble starch is oxidized by periodate to form oxidized starch, which contains aldehyde groups. Secondly, aldehyde group and primary amine can generate Schiff base reaction to form-C-N-bond, and then the-C-N-bond is reduced to-CH through the hydrogenation reduction of sodium borohydride₂-NH-bond, G2-8NH₂The branched polyamide-amine is bonded to soluble amylopectin,to generate G2-8NH₂Branched polyamide-amine bonded soluble amylopectin.

The preparation process comprises the following steps:

(1) dispersing 150 parts by mass of soluble amylopectin in 600 parts by mass of dilute sulfuric acid solution (the pH value of the dilute sulfuric acid is 1.5-2.5);

(2) adding 2-4 parts by mass of sodium periodate, stirring at 60 ℃ in a dark place, and continuously reacting for 24 hours;

(3) filtering the product after reaction, purifying the product for 2 times by using 70 to 80 percent of acetone and purified water at 0 to 8 ℃ alternately, purifying the product by using anhydrous acetone, and finally drying the product by using a vacuum drier at the temperature of 40 to 60 ℃ to obtain oxidized amylopectin for later use.

(4) Dissolving 60 parts by mass of oxidized amylopectin in 500 parts by mass of anhydrous dimethyl sulfoxide (DMSO), and then adding 2mL of glacial acetic acid;

(5) continuously adding 20-40 parts by mass of G2-8NH₂Stirring and dissolving the branched polyamide-amine to form a mixed solution;

(6) continuously reacting the mixed solution at the temperature of 20 ℃ for 12-24 h, wherein G2-8NH is generated in the process₂The amino at the end of the branched polyamide-amine reacts with the aldehyde group on the oxidized amylopectin through Schiff base reaction to form-CH-N-bond;

(7) slowly adding 5-10 parts by mass of sodium borohydride, continuously stirring, and carrying out reduction reaction for 60-200 min;

(8) dialyzing and purifying the reacted solution by a dialysis bag with molecular weight cutoff of about 8000 daltons;

(9) adding lactose and mannitol after purification, and freeze-drying with vacuum freezer to obtain G2-8NH with porous fluffy appearance₂Branched polyamide-amine bonded soluble amylopectin, which is easily soluble in water, has a molecular structure shown in FIG. 18.

6、G3-16NH₂Preparation of branched polylysine-bonded polyglucose

Raw materials: g3-16NH from example 1₂Branched polylysine, polydextrose of example 4.

The preparation principle is as follows: first, polyglucose is oxidized by periodate to form oxidized polyglucose, which contains aldehyde groups. Secondly, aldehyde group and primary amine can generate Schiff base reaction to form-C-N-bond, then the-C-N-bond is reduced to-C-N-bond through the hydrogenation reduction of sodium borohydride, and G3-16NH is added₂The branched polylysine is bonded to oxidized polyglucose to form G3-16NH2 branched polylysine bonded polyglucose.

The preparation process comprises the following steps:

(1) dissolving 80 parts by mass of polydextrose in 500 parts by mass of dilute sulfuric acid solution (the pH value of the dilute sulfuric acid is 1.5-2.5);

(2) then adding 2 parts by mass of sodium periodate, and stirring and reacting for 24 hours at the temperature of 60 ℃ in the dark;

(3) dialyzing and purifying the reacted solution by using a dialysis bag with the molecular weight cutoff of about 8000 daltons, and finally freeze-drying by using a vacuum freezer to obtain oxidized polyglucose for later use;

(4) dissolving 60 parts by mass of oxidized polydextrose in 600 parts by mass of anhydrous dimethyl sulfoxide (DMSO), and adding 2mL of glacial acetic acid;

(5) continuously adding 20-40 parts by mass of G3-16NH₂Stirring and dissolving the branched polylysine to form a mixed solution;

(6) continuously reacting the mixed solution at the temperature of 20 ℃ for 12-24 h, wherein G3-16NH is generated in the process₂The amino at the end of the branched polylysine and the aldehyde group on the oxidized polyglucose are subjected to Schiff base reaction to form-CH-N-bond;

(9) adding lactose and mannitol after purification, and freeze-drying with vacuum freezer to obtain G3-16NH with porous fluffy appearance₂Branched polylysine-bonded polyglucose, which is readily soluble in water, molecularlyThe structure is shown in fig. 19.

7、G2-8NH₂Preparation of branched polyamide-amine bonded carboxymethylcellulose

The main raw materials are as follows: carboxymethyl cellulose, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC. HCl), N-hydroxysuccinimide (NHS) from example 4, G2-8NH from example 2₂Branched polyamidoamines.

The preparation principle is as follows: g2-8NH₂One of the amino groups on the branched polyamide-amine reacts with the carboxyl group on the carboxymethyl cellulose under the catalysis of EDC.HCl and NHS catalysts to form a new amido bond (-CO-NH-), and G2-8NH is reacted₂The branched polyamidoamine is bonded to the carboxymethylcellulose to form G2-8NH₂Branched polyamide-amine bonded carboxymethylcellulose.

(1) Dissolving 50 parts by mass of the carboxymethyl cellulose of example 4 in 1000 parts by mass of purified water to form a carboxymethyl cellulose solution;

(2) controlling the temperature of the carboxymethyl cellulose solution to be 8-16 ℃;

(3) adding 10-20 parts by mass of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC.HCl) and 4-8 parts by mass of NHS, and stirring at the temperature of 8-16 ℃ for 20 min;

(4) then, 10 to 20 parts by mass of G2-8NH in example 2 was added₂Stirring and dissolving the branched polyamide-amine, and continuously reacting for 6-12 h;

(6) dialyzing and purifying the reacted solution by a dialysis bag with molecular weight cutoff of about 8000 daltons;

(7) adding lactose and mannitol after purification, and freeze-drying with vacuum freezer to obtain porous fluffy G2-8NH₂Branched polyamide-amine bonded carboxymethylcellulose, which is readily soluble in water.

Example 6

Preparation of a biocompatible polysaccharide Cross-linker (second component)

1. Preparation of oxidized starch polysaccharide cross-linking agent

The main raw materials are as follows: corn starch, sodium periodate

The preparation principle is as follows: the sugar units of the corn starch are oxidized by the oxidation of sodium periodate to form oxidized starch with a certain oxidation degree, and the oxidized starch contains aldehyde groups.

The preparation process comprises the following steps:

(1) dissolving 25 parts by mass of corn starch in 250 parts by mass of dilute sulfuric acid solution (the pH value of the dilute sulfuric acid is 1.5-2.5);

(2) then adding 5-10 parts by mass of periodic acid, and reacting at 60 ℃ in a dark place for 12-24 hours;

(3) filtering the reacted solution, purifying by using an acetone solvent containing 70-80% and purified water at 0-8 ℃ alternately, purifying by using anhydrous acetone once, and drying in vacuum at the temperature of 40-60 ℃ to obtain an oxidized starch polysaccharide cross-linking agent;

(4) dissolving an oxidized starch polysaccharide cross-linking agent in 1.0-2.0% sodium hydroxide solution, and adjusting the pH value to 5.0-7.0 to obtain an oxidized starch polysaccharide cross-linking agent solution;

(5) and adding lactose and mannitol, and freeze-drying by a vacuum freezer to obtain the oxidized starch polysaccharide cross-linking agent with porous and fluffy appearance, wherein the cross-linking agent is easily soluble in water.

2. Preparation of oxidized carboxymethyl cellulose polysaccharide cross-linking agent

The preparation principle is as follows: oxidizing sugar units of carboxymethyl cellulose by the oxidation action of sodium periodate to form oxidized carboxymethyl cellulose with a certain oxidation degree, wherein the oxidized carboxymethyl cellulose contains aldehyde groups.

The preparation process comprises the following steps:

(1) dissolving 50 parts by mass of low-viscosity carboxymethyl cellulose (the viscosity of the carboxymethyl cellulose is 200-400 mpa.s) in 1000 parts by mass of dilute sulfuric acid (the pH value is 2.0-3.0);

(2) then adding 5-10 parts by mass of periodic acid, and reacting at 60 ℃ in a dark place for 24-48 hours;

(3) dialyzing and purifying the reacted solution by a dialysis bag with molecular weight cutoff of about 8000 daltons;

(4) and adding lactose and mannitol after purification, and then carrying out freeze drying by using a vacuum freezer to obtain the oxidized carboxymethyl cellulose polysaccharide cross-linking agent which is porous and fluffy in appearance and is easy to dissolve in water.

3. Preparation of vinylsulfonyl modified polyglucose polysaccharide cross-linking agent

The preparation principle is as follows: first, vinylsulfonylpropionic acid is formed by reacting vinylsulfone with 3-mercaptopropionic acid. Secondly, the carboxyl on the vinylsulfonyl propionic acid and polyglucose are subjected to esterification reaction under the action of a catalyst Dicyclohexylcarbodiimide (DCC) and a dehydrating agent and a catalyst 4-dimethylaminopyridine p-methylbenzenesulfonic acid (DPTS), and then vinylsulfonyl is introduced into the polyglucose.

The preparation process comprises the following steps:

(1) dissolving 50 parts by mass of vinyl sulfone (DVS) in 900 parts by mass of DMSO under a nitrogen atmosphere;

(2) adding 10-25 parts by mass of 3-mercaptopropionic acid (3-MPA), stirring and dissolving, and reacting for 4-8 hours in a dark place to obtain a DVS/MPA mixed solution;

(3) dissolving 50 parts by mass of polyglucose (average molecular weight 40000) in 1000 parts by mass of anhydrous DMSO, adding 20-50 parts by mass of Dicyclohexylcarbodiimide (DCC) and 4 parts by mass of 4-dimethylaminopyridine p-methylbenzenesulfonic acid (DPTS) catalyst, and stirring to dissolve to form a mixed solution;

(4) adding the mixed solution obtained in the step (3) into the DVS/MPA mixed solution obtained in the step (2), and reacting for 24 hours in a dark place in a nitrogen atmosphere;

(5) after the reaction is finished, filtering to remove a byproduct Dicyclohexylurea (DCU), and dialyzing and purifying by adopting a dialysis bag with molecular weight cutoff of about 8000 daltons;

(6) and adding lactose and mannitol after purification, and then carrying out freeze drying by using a vacuum freezer to obtain the vinylsulfonyl modified polyglucose polysaccharide cross-linking agent which is porous and fluffy in appearance and is easy to dissolve in water.

Wherein, the preparation method of the 4-dimethylaminopyridine p-methylbenzene sulfonic acid (DPTS) catalyst comprises the following steps: boiling and distilling 10 parts by mass of p-toluenesulfonic acid (PTSA) and 250 parts by mass of toluene to remove moisture; 6.5 parts by mass of 4-Dimethylaminopyridine (DMAP) are dissolved in 70 parts by mass of hot toluene (60-80 ℃); adding the hot 4-dimethylaminopyridine solution into a p-toluenesulfonic acid solution, stirring and reacting for 4-8 hours at the temperature of 65 ℃, then cooling and filtering; then recrystallizing with dichloroethane solvent to obtain the 4-dimethylamino pyridine p-methyl benzene sulfonic acid (DPTS) with white needle-shaped appearance.

4. Preparation of sulfhydryl modified soluble starch polysaccharide cross-linking agent

The preparation principle is as follows: carboxyl on the sulfydryl modified acetic acid and soluble starch polysaccharide are subjected to esterification reaction under the action of a catalyst Dicyclohexylcarbodiimide (DCC) and a dehydrating agent and a catalyst 4-dimethylaminopyridine p-methylbenzenesulfonic acid (DPTS), and then sulfydryl is introduced into the soluble starch polysaccharide.

The preparation process comprises the following steps:

(1) dissolving 80 parts by mass of the soluble amylopectin starch of example 3 in 1000 parts by mass of the anhydrous DMSO solvent;

(2) then adding 20-40 parts by mass of sulfydryl modified acetic acid, 20-40 parts by mass of Dicyclohexylcarbodiimide (DCC) and 8 parts by mass of 4-dimethylaminopyridine p-methylbenzenesulfonic acid (DPTS) catalyst, stirring and dissolving in a nitrogen atmosphere, and reacting for 12 hours in a dark place;

(3) after the reaction is finished, filtering to remove a byproduct Dicyclohexylurea (DCU), and then dialyzing and purifying by adopting a dialysis bag with molecular weight cutoff of about 8000 daltons;

(4) and adding the mannitol after purification, and then freezing and drying by adopting a vacuum freezer to obtain the hydrosulfuryl modified soluble starch polysaccharide cross-linking agent which is porous and fluffy in appearance and is easy to dissolve in water.

5. Preparation of maleimide modified chitosan polysaccharide cross-linking agent

The preparation principle is as follows: carboxyl on 6-maleimide modified caproic acid and amino on chitosan sugar unit are subjected to chemical reaction under the action of N-hydroxysuccinimide (NHS) and 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC.HCl) catalysts to form a new amido bond (-CO-NH-), and then maleimide active groups are bonded on chitosan to generate the maleimide modified chitosan polysaccharide cross-linking agent.

The preparation process comprises the following steps:

(1) dissolving 30 parts by mass of chitosan and 15 parts by mass of N-hydroxysuccinimide (NHS) in 1000 parts by mass of purified water, and stirring for dissolution;

(2) then adding 15 parts by mass of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC.HCl) for carrying out an activation reaction for 30 min;

(3) continuously adding 10 parts by mass of 6-maleimide modified caproic acid, stirring and dissolving, and reacting for 3-6 hours in a dark place in a nitrogen atmosphere;

(4) dialyzing and purifying the reacted solution by a dialysis bag with molecular weight cutoff of about 8000 daltons;

(5) and adding lactose and mannitol after purification, and then performing freeze drying by using a vacuum freezer to obtain the maleimide-modified chitosan with porous and fluffy appearance, wherein the maleimide-modified chitosan is easily soluble in water.

Example 7

Preparation of bi-component cross-linked medical composite material, application of bi-component cross-linked medical composite material to damaged mucous membrane of pig and performance detection of bi-component cross-linked medical composite material

1. The preparation method of the bi-component cross-linked medical composite material and the application thereof on the damaged mucous membrane of the pig comprise the following steps:

s3: the first component solution is uniformly smeared or sprayed to form a thin layer on the damaged mucous membrane or the damaged skin of the pig, and then the second component solution is uniformly covered on the damaged mucous membrane or the damaged skin of the pig to quickly form the bi-component cross-linked medical composite material on the in-situ damaged mucous membrane or the damaged skin.

2. A performance test of the two-component cross-linked medical composite material on the damaged mucous membrane of the pig comprises the following steps:

the in-situ gelling time of the bi-component cross-linked medical composite material is as follows:

the method comprises the following steps:

s1: weighing 100g of the first component solution by using a 200mL beaker, and rapidly stirring the first component solution by using a magnetic stirrer (the rotating speed is 200-400 rpm) to form an obvious vortex;

s2: weighing 25g of the second component solution by using a 100mL beaker, quickly pouring the second component solution into the first component solution at one time, and immediately timing by using a stopwatch;

s3: when the fixed bi-component cross-linked medical composite material is formed in the beaker, the time for forming gel is timed and recorded, and the judgment standard for forming the fixed bi-component cross-linked medical composite material is as follows: the vortex of the solution in the beaker disappears, the mixed solution does not rotate along with the stirring, or the magnetic stirrer is obviously prevented from rotating or loses the stirring and mixing effect on the mixed system, namely the timing end point.

Remarking: because the two-component crosslinked medical composite is formed in a short time, the second component solution should be added to the first component solution at the same time as the first component solution is accurately timed.

The method 2 comprises the following steps:

s1: cutting the pig muscle biological tissue with the area of about 3cm x 4cm, and smearing or spraying a thin layer of first component solution on the pig muscle biological tissue;

s2: coating or spraying a thin layer of the second component solution on the biological tissue of the pig muscle, and using a stopwatch to time;

s3: and 3 seconds later, suspending and turning the pig muscle biological tissue for 180 ℃, and observing whether an obvious double-component cross-linked medical composite material layer is formed on the pig muscle biological tissue or not, wherein the double-component cross-linked medical composite material does not flow, is fixed in shape and seals the pig muscle biological tissue. And (4) judging the standard: the bi-component cross-linked medical composite material layer does not flow, the shape is fixed, the pig muscle biological tissue is sealed, and the gelling time of the first component solution and the second component solution is less than or equal to 3 s.

(2) The test method for the adhesive force test of the two-component cross-linked medical composite material comprises the following steps:

s1: cutting the pig muscle biological tissue with the area of about 3cm x 4cm, and smearing or spraying a thin layer of the first component solution on the pig muscle biological tissue;

s2: smearing or spraying a layer of second component solution on the pig muscle biological tissue to form a bi-component cross-linked medical composite material in situ;

s3: horizontally placing a weight of 100-250 g on the pig muscle biological tissue, horizontally pulling the weight by using a spring tension meter, enabling the tension meter to generate and display a tensile force larger than 150N, and observing whether the double-component cross-linked medical composite material layer on the pig muscle biological tissue shifts or translocates.

(3) The test method of the sealing performance of the bi-component cross-linked medical composite material layer on the damaged pig mucous membrane comprises the following steps:

s1: cutting the pig muscle biological tissue with the area of about 4cm x 4cm and the thickness of about 0.5cm, and pricking 8-16 needle holes on the pig muscle biological tissue by using an injection needle so that water can freely pass through the needle holes;

s2: smearing or spraying a first component solution thin layer on the pig muscle biological tissue, and smearing or spraying a second component solution on the pig muscle biological tissue to form a double-component cross-linked medical composite material layer;

s3: forming water pressure of 500 mm-600 mm on the pig muscle biological tissue;

s4: after 300s, it was observed whether there was significant water track penetrating the pig muscle tissue and dripping.

Example 7-1

S1: preparing a first component solution: g2-8NH of example 5₂Dissolving branched polyamide-amine bonded six-arm polyethylene glycol carboxylic acid in a phosphate buffer (the pH value of the phosphate buffer is 4.0-6.0) to form a first component solution with the content of 1.5%;

s2: preparing a second component solution: dissolving the oxidized starch polysaccharide cross-linking agent of example 6 in a phosphate buffer (the pH value of the phosphate buffer is 4.0-6.0) to form a second component solution with the content of 0.8%;

s3: the first component solution of S1 is evenly smeared or sprayed on a thin layer of damaged mucous membrane or damaged skin of pigs, and then the second component solution of S2 is evenly smeared or sprayed on the thin layer of damaged mucous membrane or damaged skin of pigs, so that the bi-component cross-linked medical composite material is quickly formed on the damaged mucous membrane or damaged skin of pigs in situ.

The in-situ gel forming time, the adhesive force and the sealing property of the bi-component cross-linked medical composite material are tested according to the test method.

Remarking: the concentrations of the first component solution and the second component solution of S1 and S2 marked by contents do not include lactose and mannitol which are auxiliary materials for freeze-drying.

Example 7-2

S1: preparing a first component solution: g2-8NH of example 5₂Dissolving branched polyamide-amine bonded eight-arm polyethylene glycol carboxylic acid in a phosphate buffer (the pH value of the phosphate buffer is 4.0-6.0) to form a first component solution with the content of 1.5%;

s3: the first component solution of S1 is evenly smeared or sprayed on a thin layer of damaged mucous membrane or damaged skin of a pig, and then the second component solution of S2 is evenly covered on the thin layer of damaged mucous membrane or damaged skin of the pig, so that the bi-component cross-linked medical composite material is quickly formed on the damaged mucous membrane or damaged skin of the pig in situ.

Remarking: the concentrations of the first component solution and the second component solution of S1 and S2 marked by contents do not include lactose and/or mannitol which are auxiliary materials for freeze-drying.

Examples 7 to 3

S1: preparing a first component solution: dissolving G2-8SH branched polyamidoamine-bound polyglutamic acid of example 5 in a phosphate buffer solution (pH of the phosphate buffer solution is 4.0-6.0) to form a first component solution with a content of 1.5%;

s2: preparing a second component solution: dissolving the vinylsulfonyl modified polyglucose polysaccharide cross-linking agent of example 6 in a phosphate buffer (the pH value of the phosphate buffer is 4.0-6.0) to form a second component solution with the content of 1.0%;

s3: the first component solution of S1 is evenly smeared or sprayed with a thin layer on the damaged mucous membrane or the damaged skin of the pig, and then the second component solution of S2 is evenly covered on the thin layer, so that the bi-component cross-linked medical composite material is quickly formed on the damaged mucous membrane or the damaged skin of the pig in situ.

Examples 7 to 4

S1: preparing a first component solution: dissolving G2-8SH branched polylysine bonded polyglutamic acid of example 5 in a phosphate buffer solution (pH value of the phosphate buffer solution is 4.0-6.0) to form a first component solution with the content of 1.5%;

s3: the first component solution of S1 is evenly smeared or sprayed on a thin layer of damaged mucous membrane or damaged skin of a pig, and then the second component solution of S2 is evenly covered on the thin layer of damaged mucous membrane or damaged skin of the pig, so that the double-component cross-linked medical composite material is quickly formed in situ.

Examples 7 to 5

S1: preparing a first component solution: dissolving G2-8SH branched polyamidoamine bonded soluble amylopectin of example 5 in a phosphate buffer solution (pH of the phosphate buffer solution is 7.0-8.0) to form a first component solution with a content of 1.5%;

s2: preparing a second component solution: dissolving the sulfhydryl-modified soluble starch polysaccharide cross-linking agent obtained in the embodiment 6 in a phosphate buffer (the pH value of the phosphate buffer is 7.0-8.0) to form a second component solution with the content of 2.0%;

s3: the first component solution of S1 is evenly smeared or sprayed with a thin layer on the damaged mucous membrane or the damaged skin of a pig, then the second component solution of S2 and 2.0 percent hydrogen peroxide solution (oxidant) are respectively and independently filled into a double-component injector, the mixed solution of the sulfhydryl-modified soluble starch polysaccharide cross-linking agent and the hydrogen peroxide is quickly sprayed on the damaged mucous membrane or the damaged skin of the same pig under the premixing action at the front end of the double-component injector, and the double-component cross-linking medical composite material is quickly formed in situ.

Examples 7 to 6

S1: preparing a first component solution: g3-16NH of example 5₂Dissolving branched polylysine bonded polydextrose in a phosphate buffer (the pH value of the phosphate buffer is 7.0-8.0) to form a first component solution with the content of 2.5%;

s2: preparing a second component solution: dissolving the maleimide modified chitosan polysaccharide cross-linking agent of example 6 in a phosphate buffer (pH value of the phosphate buffer is 5.0-6.0) to form a second component solution with the content of 1.2%;

The results of the in situ gel formation time, adhesion and seal test for 6 different two-component crosslinked medical composites are shown in tables 1, 2 and 3 below.

TABLE 1 data sheet for in situ formation of two-component crosslinked medical composites

Examples	Method	1 gelling time	Method	2 gelling time
					7-1	<1s	≤1s
7-2	<1s	≤1s
			7-3	≤1s	≤1s
7-4	≤1s	≤1s
			7-5	<1s	≤1s
7-6	<1s	≤1s

As shown in the data table of in situ formation time of the two-component crosslinked medical composite material in Table 1, the formation time of the 6 different two-component crosslinked medical composite materials in examples 7-1 to 7-6 applied or sprayed on the damaged mucosa or the damaged skin of swine was not more than 1s, indicating that the two-component crosslinked medical composite material of the present invention can be rapidly formed in situ.

TABLE 2 adhesion test results for two-component crosslinked medical composites

As can be seen from the adhesion test results of the two-component crosslinked medical composite material shown in table 2, the 6 different two-component crosslinked medical composite materials of examples 7-1 to 7-6 were applied or sprayed on the damaged mucosa or the damaged skin of swine, and the 6 different two-component crosslinked medical composite material layers did not shift or translocate on the damaged mucosa or the damaged skin under a tensile force greater than 150N, indicating that the two-component crosslinked medical composite material layer of the present invention has good adhesion.

TABLE 3 results of the sealing test of two-component crosslinked medical composites

Examples	Hydrostatic pressure	Time	Sealing property
				7-1	550mm	300s	Without leakage
7-2	542mm	300s	Without leakage
				7-3	560mm	300s	Without leakage
7-4	580mm	300s	Without leakage
				7-5	563mm	300s	Without leakage
7-6	568mm	300s	Without leakage

The results of the two-component cross-linked medical composite sealing test in table 3 show that 6 different two-component cross-linked medical composites in examples 7-1 to 7-6 applied or sprayed on the damaged mucosa or the damaged skin of the pig form a water pressure of 500mm to 600mm on the damaged mucosa or the damaged skin, and no water leakage is found, which indicates that the two-component cross-linked medical composite of the present invention has good sealing property, can prevent blood leakage, and can block bacteria.

Example 8

Preparation of bi-component cross-linked medical composite material and application thereof to clinically damaged mucous membrane or skin of human body

Example 8-1

Application of bi-component cross-linked medical composite material in promotion of wound repair and healing after male urinary surgery

Test groups:

s1: preparing a first component solution: the radiation sterilized G2-8NH from example 5₂Dissolving branched polyamide-amine bonded carboxymethyl cellulose in a phosphate buffer (the pH value of the phosphate buffer is 4.0-6.0) to form a first component solution with the content of 1.0% for later use;

s2: preparing a second component solution: dissolving the oxidized starch polysaccharide cross-linking agent of example 6 in a phosphate buffer (the pH value of the phosphate buffer is 4.0-6.0) to form a second component solution with the content of 1.0%, and performing moist heat sterilization at the temperature of 121 ℃ for 15min for later use;

s3: the first component solution of S1 is uniformly coated or sprayed on a thin layer of wound surface after male urinary surgery after debridement, and then the second component solution of S2 is uniformly covered on the wound surface to quickly form a bi-component cross-linked medical composite material in situ, so that the wound surface is sealed and isolated.

S4: after 7 days of the operation, the healing of the wound surface of the patient after the operation was observed, and the results are shown in fig. 20 (1).

Control group:

the patients were not treated with the two-component crosslinked medical composite material of the present invention according to the conventional post-operative treatment method, and after 7 days of the operation, the healing condition of the wound surface of the patients after the operation was observed, and the results are shown in fig. 20 (2).

Fig. 20(1) is a schematic diagram of a patient who uses the two-component cross-linked medical composite material of the present invention to promote wound healing after male urological surgery, and as shown in fig. 20(1), the patient uses the two-component cross-linked medical composite material of the present invention to exhibit an obvious effect of promoting wound healing, and the wound surface after using the two-component cross-linked medical composite material promotes wound healing due to the comprehensive effects of imbibition, sealing, bacteria blocking, adhesion prevention, etc. of the two-component cross-linked medical composite material. Fig. 20(2) is a schematic diagram of wound repair and healing after male urinary surgery without using the two-component cross-linked medical composite material of the present invention, as shown in fig. 20(2), the patient does not use the two-component cross-linked medical composite material, but adopts the traditional post-operative treatment method, because the wound has poor effects of imbibition, sealing, bacteria blocking and adhesion prevention, the wound has slow repair and healing, and obvious suppuration phenomenon also appears.

Example 8 to 2

Application of bi-component cross-linked medical composite material in promotion of wound repair and healing after medical plastic surgery

Test groups:

s1: preparing a first component solution: dissolving G2-8SH branched polyamide-amine bonded polyglutamic acid of example 5 subjected to radiation sterilization in a phosphate buffer (the pH value of the phosphate buffer is 7.0-8.0) to form a first component solution with the content of 1.5% for later use;

s2: preparing a second component solution: dissolving the vinylsulfonyl modified polyglucose polysaccharide cross-linking agent of example 6 in a phosphate buffer (the pH value of the phosphate buffer is 7.0-8.0) to form a second component solution with the content of 1.0%, and performing moist heat sterilization at the temperature of 121 ℃ for 15min for later use;

s3: in the medical eyebrow lifting operation (a medical plastic operation), before the eyebrow cutting opening is sutured, a thin layer of the first component solution of S1 is uniformly coated or sprayed on a wound surface after debridement, and then the second component solution of S2 is uniformly covered on the wound surface to quickly form a double-component cross-linked medical composite material on the wound surface in situ, so that the wound surface is sealed, isolated and protected.

S4: after 7 days of the operation, the healing of the wound surface of the patient after the operation was observed, and the results are shown in fig. 21 (1).

Control group:

the patients are not treated by the double-component cross-linked medical composite material, and are nursed according to the traditional postoperative treatment method, and the healing condition of the wound surface of the patients after the operation is observed after 7 days, and the result is shown in figure 21 (2).

Fig. 21(1) is a schematic view showing that a patient uses the two-component cross-linked medical composite material of the present invention to promote wound healing after a medical plastic surgery, as shown in fig. 21(1), the patient uses the two-component cross-linked medical composite material of the present invention to show an obvious effect of promoting wound healing, and due to the comprehensive effects of sealing protection, infiltration absorption, infiltration prevention, bacteria inhibition, etc. of the composite material of the present invention, the wound healing is promoted, and the patient does not have complications of redness, swelling, verification, etc. Fig. 21(2) is a schematic diagram of wound repair and healing after a medical plastic surgery without using the two-component cross-linked medical composite material of the present invention, as shown in fig. 21(2), the wound is repaired and healed slowly by a conventional post-operative treatment method without using the two-component cross-linked medical composite material of the present invention, and obvious complications such as redness, swelling, inflammation, etc. appear because the wound is not reasonably protected.

While the principles of the invention have been described in detail in connection with the preferred embodiments thereof, it will be understood by those skilled in the art that the foregoing embodiments are merely illustrative of exemplary implementations of the invention and are not limiting of the scope of the invention. The details of the embodiments are not to be interpreted as limiting the scope of the invention, and any obvious changes, such as equivalent alterations, simple substitutions and the like, based on the technical solution of the invention, can be interpreted without departing from the spirit and scope of the invention.

Claims

1. The double-component cross-linked medical composite material is characterized by comprising a first component and a second component, wherein the first component is a recombinant high-molecular compound terminated by an active group, and the recombinant high-molecular compound terminated by the active group is composed of active groupsThe end-capped branched compound and the branched polymer compound or the linear polymer compound are bonded by chemical bonds, and the active group-capped recombinant polymer compound comprises G2-8NH₂Branched polyamidoamine bonded hexa-armed polyethylene glycol carboxylic acid, G2-8NH₂Branched polyamidoamine bonded octa-armed polyethylene glycol carboxylic acid, G2-8SH branched polyamidoamine bonded polyglutamic acid, G2-8SH branched polylysine bonded polyglutamic acid, G2-8NH₂Branched polyamidoamine-bonded soluble amylopectin, G3-16NH₂Branched polylysine bonded polyglucose and G2-8NH₂At least one branched polyamide-amine bonded carboxymethylcellulose, the reactive group-terminated recombinant macromolecular compound has branches and has only one reactive group at the end of each branch, the second component is a biocompatible polysaccharide cross-linking agent, and the biocompatible polysaccharide cross-linking agent is a polysaccharide modified by reactive groups.

2. The two-component crosslinked medical composite material according to claim 1, wherein the branched compound terminated with a reactive group has n branches, n is a natural number, n.gtoreq.8, and each of the branches has only one reactive group at its end.

3. The two-component crosslinked medical composite according to claim 2, wherein the reactive group of the reactive group-terminated branched compound comprises one or more of an amino group and a mercapto group.

4. The two-component crosslinked medical composite material according to claim 1, wherein the branched polymer compound or the linear polymer compound has m bondable sites, m is a natural number, m is greater than or equal to 6, and the bondable sites are reactive groups bonded to the branched polymer compound or the linear polymer compound when the reactive group-terminated branched polymer compound and the branched polymer compound or the linear polymer compound form the reactive group-terminated recombinant polymer compound.

5. The two-component crosslinked medical composite according to claim 1, wherein the reactive groups in the biocompatible polysaccharide crosslinker comprise one or more of amino groups, amino acid residues, amino acid ester residues, aldehyde groups, maleimide groups, succinimide ester groups, acrylate groups, thiol groups, vinylsulfonyl groups, double bonds, azide groups, and alkyne groups.

6. The two-component crosslinked medical composite according to claim 5, wherein the biocompatible polysaccharide crosslinking agent is selected from the group consisting of at least one of starch, carboxymethyl starch, hydroxypropyl starch, polydextrose, carboxymethyl cellulose, hydroxypropyl methyl cellulose, chitosan, alginate, and sodium hyaluronate.

7. The two-component crosslinked medical composite according to claim 6, characterized in that the carboxymethyl cellulose, the hydroxypropyl methyl cellulose, the chitosan, the alginate and the sodium hyaluronate have a molecular weight in the range of 10000-.

8. The two-component crosslinked medical composite material according to claim 3 or 5, wherein when the reactive group of the reactive group-terminated recombinant polymer compound is an amino group, the reactive group of the biocompatible polysaccharide crosslinking agent is one of an aldehyde group, a maleimide group, a succinimide ester group, and an acrylate group.

9. The two-component crosslinked medical composite material according to claim 3 or 5, wherein when the reactive group of the reactive group-terminated recombinant macromolecular compound is a thiol group, the reactive group of the biocompatible polysaccharide crosslinking agent is one of a vinylsulfonyl group, a double bond, a thiol group, an amino acid ester residue, and an amino acid residue.

10. The two-component crosslinked medical composite according to claim 9, characterized in that the amino acid residue is one of a cysteine residue and an N-acetylcysteine residue.

11. The two-component crosslinked medical composite of claim 9, wherein the amino acid ester residue comprises one of a cysteine methyl ester residue and a cysteine ethyl ester residue.

12. A method for preparing a two-component cross-linked medical composite according to any of claims 1 to 11, comprising the steps of:

13. The method for preparing the bi-component cross-linked medical composite material as claimed in claim 12, wherein the concentration of the first buffer solution is in the range of 0 to 0.1mol/L, and the first buffer solution comprises a phosphate buffer solution, an acetate buffer solution and a carbonate buffer solution.

14. The method for preparing the bi-component cross-linked medical composite material as claimed in claim 12, wherein the concentration of the second buffer solution is in the range of 0 to 0.1mol/L, and the second buffer solution comprises a phosphate buffer solution, an acetate buffer solution and a carbonate buffer solution.

15. The method for preparing the two-component cross-linked medical composite according to claim 12, wherein the time for instantaneous cross-linking formation of the two-component cross-linked medical composite in situ of a damaged mucous membrane or damaged skin is < 1S.

16. Use of a two-component cross-linked medical composite according to any of claims 1-11 for the preparation of a formulation for damaged mucous membranes or damaged skin.