阅读说明：本技术 生物粘附性水凝胶及其应用 (Bioadhesive hydrogels and their use ) 是由 邱凌啸 于 2020-05-20 设计创作，主要内容包括：本发明的目的在于提供一种固化速度快、胶体强度好、湿面组织粘附力强的生物粘附性水凝胶,包括交联性生物大分子以及光引发剂,其中交联性生物大分子含有作为第一交联基团的邻硝基苄基光扳机以及作为第二交联基团的糖环羟甲基,第一交联基团在光照条件下产生亚硝基,该亚硝基在光引发剂存在条件下与第二交联基团发生化学键合从而固化成胶。本发明还提供了该生物粘附性水凝胶的应用。(The invention aims to provide a bioadhesive hydrogel which is high in curing speed, good in colloid strength and strong in wet tissue adhesion, and comprises a crosslinkable biomacromolecule and a photoinitiator, wherein the crosslinkable biomacromolecule contains an o-nitrobenzyl photosetting agent as a first crosslinking group and a sugar-ring hydroxymethyl group as a second crosslinking group, the first crosslinking group generates a nitroso group under the illumination condition, and the nitroso group and the second crosslinking group generate chemical bonding under the existence of the photoinitiator so as to be cured into gel. The invention also provides the application of the bioadhesive hydrogel.)

1. A bioadhesive hydrogel, comprising:

a cross-linking biological macromolecule and a photoinitiator,

wherein the crosslinkable biomacromolecule comprises a first crosslinking group and a second crosslinking group,

the first crosslinking group generates a nitroso group under the condition of illumination, and the nitroso group is chemically bonded with the second crosslinking group under the condition of existence of the photoinitiator so as to form gel,

the first crosslinking group is an o-nitrobenzyl type photosheet device,

the second crosslinking group is a sugar ring hydroxymethyl group,

the structure of the o-nitrobenzyl plate photoscreening machine is shown as the following formula I or formula II:

in formula I and formula II:

LG is a leaving group consisting of a halogen atom or a group of the form O-R ', S-R ', N-R ';

r' is selected from hydrogen, ether bond substituent, ester bond substituent, carbonate bond substituent, carbamate bond substituent, mercapto formate bond substituent or phosphate bond substituent;

r1 is selected from hydrogen, halogen atom, hydroxyl group, mercapto group, amine group, nitro group, cyano group, aldehyde group, ketone group, ester group, amide group, phosphonic acid group, sulfonic acid ester group, sulfone group, sulfoxide group, aryl group, heteroaryl group, alkyl group, alkylene group, modified alkyl group or modified alkylene group;

any one or more of R2, R3, R4, R5 is selected from a terminal amine group, hydroxyl group, mercapto group, halogen, carboxyl or carboxylate modified aryl group, heteroaryl group, alkyl group, alkylene group, modified alkyl group or modified alkylene group, optionally selected from hydrogen, halogen atom, hydroxyl group, mercapto group, amine group, nitro group, cyano group, aldehyde group, ketone group, carboxyl group, ester group, amide group, phosphonate group, sulfonic group, sulfonate group, sulfone group, sulfoxide group, aryl group, heteroaryl group, alkyl group, alkylene group, modified alkyl group or modified alkylene group.

2. The bioadhesive hydrogel according to claim 1, wherein:

wherein the cross-linkable biological macromolecule comprises a first cross-linkable biological macromolecule and a second cross-linkable biological macromolecule,

the first crosslinkable biomacromolecule is a biomacromolecule containing the first crosslinking group,

the second crosslinkable biomacromolecule is a biomacromolecule containing the second crosslinking group.

3. The bioadhesive hydrogel according to claim 1, wherein:

wherein the cross-linking biological macromolecule is a third cross-linking biological macromolecule,

wherein the third crosslinkable biomacromolecule is a biomacromolecule containing the first crosslinking group and the second crosslinking group.

4. The bioadhesive hydrogel according to claim 3, wherein:

wherein the third crosslinkable biomacromolecule is obtained by modifying the biomacromolecule containing the second crosslinking group with the first crosslinking group.

5. The bioadhesive hydrogel according to claim 2 or 4, wherein:

wherein the biomacromolecule containing sugar ring hydroxymethyl is any one or any one derivative of hyaluronic acid, carboxymethyl cellulose, methyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, dextran, agarose, heparin, chitosan, sucrose, glucose and starch.

6. The bioadhesive hydrogel according to claim 1, wherein:

wherein any two of R', R3, R4, R5 and R6 are linked to each other to form a saturated or unsaturated aliphatic or aliphatic heterocyclic ring or an aromatic or aromatic heterocyclic ring.

7. The bioadhesive hydrogel according to claim 1, wherein:

wherein the sugar ring hydroxymethyl group has a structure of any one of the following formulas IV, V, VI and VII:

in formula IV, formula V, formula VI and formula VII, R is selected from hydrogen, halogen atom, hydroxyl, sulfydryl, amine group, nitro group, cyano group, aldehyde group, ketone group, carboxyl group, carboxylate group, ester group, amide group, phosphonate group, sulfonic group, sulfonate group, sulfone group, sulfoxide group, aryl group, heteroaryl group, alkyl group, alkylene group, modified alkyl group or modified alkylene group.

8. The bioadhesive hydrogel according to claim 1, wherein:

wherein the photoinitiator is: 2-hydroxy-4' - (2-hydroxyethoxy) -2-methyl propiophenone, lithium phenyl (2,4, 6-trimethyl benzoyl) phosphate, sodium phenyl (2,4, 6-trimethyl benzoyl) phosphate and magnesium phenyl (2,4, 6-trimethyl benzoyl) phosphate.

9. Use of a bioadhesive hydrogel according to any one of claims 1-8 for the preparation of a rapid hemostatic or tissue repair article,

wherein the nitroso group generated by the first crosslinking group under the condition of illumination and the existence of a photoinitiator is chemically bonded with the second crosslinking group to form gel, and is also chemically bonded with at least one of a glycocyclo-hydroxymethyl group and a sulfhydryl group on proteoglycan and glycosaminoglycan in an extracellular matrix of the surface of the biological tissue, so that the formed colloid is adhered to the surface of the biological tissue.

10. A drug carrier comprising the bioadhesive hydrogel according to any one of claims 1 to 8.

11. A bioprinting material comprising the bioadhesive hydrogel according to any one of claims 1 to 8.

Technical Field

The invention belongs to the field of biological materials, and relates to a bioadhesive hydrogel and application thereof.

Background

The biological hydrogel is a material which is modified by various bioaffinity macromolecules and has the gelling capacity, can be used for curing gelling after being coated on a wound or a bleeding part in a liquid state so as to perform in-situ hemostasis or in-situ wound closure, can also be used for printing various biological tissues and biological tissue scaffolds by being combined with a 3D printing technology, and can also be used for coating medicines while curing so as to obtain a medicine-containing gel product, namely the biological hydrogel product can be used as a medicine carrier.

When applied to in situ hemostasis, in situ wound closure or bioprinting, the gelling properties of the cured biohydrogel are critical due to the need to cure in a short period of time. In the prior art, a plurality of biological hydrogel materials which can be formed into gel by illumination exist, for example, CN105131315A discloses a non-free radical photochemical crosslinking hydrogel material which comprises a component A containing an o-nitrobenzyl light trigger modified macromolecule derivative and a component B containing hydrazide, hydroxylamine or primary amine macromolecule derivatives, when in use, the component A and the component B are mixed and illuminated for 30 s-1 min to form gel, thus realizing wound repair or wound tissue isolation. For another example, CN108187130A discloses a reagent for repairing biological injury or stopping bleeding, which comprises a photoinitiator and natural biological macromolecules modified by photoresponsive crosslinking groups such as o-nitrobenzyl-based photo-trigger, and can be cured by several seconds of light when in use, thereby achieving rapid hemostasis.

The biogel materials all utilize the characteristic that groups modified by o-nitrobenzyl optical triggers can be used for crosslinking. According to the disclosures of these documents, ortho-nitrobenzyl photoinitiators are capable of reacting with amide, hydroxylamine, primary amines, etc., containing a variety of groups to form chemical bonds, thereby crosslinking the biomacromolecule and curing the glue solution into a gel. It is clear that without these groups, the ortho-nitrobenzyl photoinitiator is not reactive and does not cure to a gel.

Disclosure of Invention

Based on the prior art, the inventor carries out intensive research on the reaction mechanism of the o-nitrobenzyl optical trigger, and finds that the o-nitrobenzyl optical trigger can also carry out chemical bonding reaction with sugar ring hydroxymethyl, and the reaction mechanism is as follows: the o-nitrobenzyl photoinitiator generates a nitroso group under the condition of illumination, and the nitroso group can be chemically bonded with a sugar ring hydroxymethyl under the condition of existence of a photoinitiator. When the biomacromolecule contains the o-nitrobenzyl optical trigger and the sugar ring hydroxymethyl, crosslinking can be carried out based on the reaction mechanism, so that the glue is solidified.

Thus, the present invention proposes a new bioadhesive hydrogel characterized in that it comprises: the crosslinking biological macromolecule comprises a first crosslinking group and a second crosslinking group, the first crosslinking group generates a nitroso group under the condition of illumination, the nitroso group and the second crosslinking group generate chemical bonding under the condition of existence of a photoinitiator so as to form glue, the first crosslinking group is an o-nitrobenzyl type photosheet machine, the second crosslinking group is a sugar ring hydroxymethyl group, and the structure of the o-nitrobenzyl type photosheet machine is shown as the following formula I or formula II:

in formula I and formula II:

LG is a leaving group consisting of a halogen atom or a group of the form O-R ', S-R ', N-R ', wherein the halogen atom comprises F, Cl, Br, I;

r' is selected from hydrogen, ether bond substituent, ester bond substituent, carbonate bond substituent, carbamate bond substituent, mercapto formate bond substituent or phosphate bond substituent;

r1 is selected from hydrogen, halogen atom, hydroxyl group, mercapto group, amine group, nitro group, cyano group, aldehyde group, ketone group, ester group, amide group, phosphonic acid group, sulfonic acid ester group, sulfone group, sulfoxide group, aryl group, heteroaryl group, alkyl group, alkylene group, modified alkyl group or modified alkylene group;

any one or more of R2, R3, R4, R5 is selected from a terminal amine group, hydroxyl group, mercapto group, halogen, carboxyl or carboxylate modified aryl group, heteroaryl group, alkyl group, alkylene group, modified alkyl group or modified alkylene group, optionally selected from hydrogen, halogen atom, hydroxyl group, mercapto group, amine group, nitro group, cyano group, aldehyde group, ketone group, carboxyl group, ester group, amide group, phosphonate group, sulfonic group, sulfonate group, sulfone group, sulfoxide group, aryl group, heteroaryl group, alkyl group, alkylene group, modified alkyl group or modified alkylene group.

In the above groups:

the alkyl is a saturated or unsaturated aliphatic straight chain or branched chain alkyl with 1-30 carbon atoms;

the alkylene group is a saturated or unsaturated aliphatic linear or branched alkylene group having 1 to 30 carbon atoms;

modifying alkyl to be any carbon atom of alkyl by a halogen atom, -OH, -SH, -NO₂-CN, -CHO, -COOH, ester group, amide group, aryl group, arylene group, -CO-, -O-, -S-, -SO-, -₂A group obtained by replacing at least one group of primary amino, secondary amino, tertiary amino, quaternary ammonium base, saturated or unsaturated monocyclic or bicyclic cycloalkylene and bridged lipid heterocycle, wherein the modified alkyl has 1 to 30 atoms, and the carbon-carbon single bond can be replaced by carbon-carbon double bond or carbon-carbon triple bond optionally;

the alkylene group being modified to have any carbon atom of the alkylene group being substituted by a halogen atom, -OH, -SH, -NO₂-CN, -CHO, -COOH, ester group, amide group, aryl group, arylene group, -CO-, -O-, -S-, -SO-, -₂A group obtained by replacing at least one group of primary amino, secondary amino, tertiary amino, quaternary ammonium base, saturated or unsaturated monocyclic or bicyclic cycloalkylene and bridged alicyclic ring, the modified alkylene group has 1 to 30 atoms, and the carbon-carbon single bond can be optionally replaced by a carbon-carbon double bond or a carbon-carbon double bondC, replacement of a triple bond;

the ether bond substituent is selected from the following structures:

-(CH₂)_xCH₃、-(CH₂CH₂O)_xCH₃、-(CH₂)_x(CH₂CH₂O)_yCH₃or isWherein x and y are integers not less than 0;

the ester bond substituent is selected from the following structures:

-CO(CH₂)_xCH₃、-CO(CH₂CH₂O)_xCH₃、-CO(CH2)_x(CH₂CH₂O)_yCH₃wherein x and y are integers not less than 0;

the carbonate bond substituent is selected from the following structures:

-COO(CH₂)_xCH₃、-COO(CH2CH2O)xCH3、-COO(CH₂)_x(CH₂CH₂O)_yCH₃wherein x and y are integers not less than 0;

the substituent of the urethane bond is selected from the following structures:

-CONH(CH₂)_xCH₃、-CONH(CH₂CH₂O)_xCH₃、-CONH(CH₂)_x(CH₂CH₂O)_yCH₃wherein x and y are integers not less than 0;

the mercapto formic ester bond substituent is selected from the following structures:

-COS(CH₂)_xCH₃、-COS((CH₂CH₂O)_xCH₃、-COS(CH₂)_x(CH₂CH₂O)_yCH₃wherein x and y are integers not less than 0;

the phosphate ester bond substituent is selected from the following structures: -POOO (CH)₂)_xCH₃、-POOO(CH₂CH₂O)_xCH₃、-POOO(CH₂)_x(CH₂CH₂O)_yCH₃Wherein x and y are integers not less than 0;

the aryl group is a 5-10 membered aromatic monocyclic ring or aromatic condensed bicyclic ring structure;

the heteroaryl is a 5-10 membered aromatic monocyclic ring or aromatic condensed bicyclic ring structure containing at least one heteroatom selected from O, S, N or Si on the ring;

the halogen atoms are respectively and independently selected from F, Cl, Br and I;

the alicyclic ring is a saturated or unsaturated 3-to 10-membered monocyclic or polycyclic alicyclic ring;

the aliphatic heterocyclic ring is a saturated or unsaturated 3-to 10-membered monocyclic or polycyclic aliphatic heterocyclic ring containing at least one hetero atom selected from O, S, N or Si in the ring, and when the aliphatic heterocyclic ring contains an S atom, it is optionally-S-, -SO-or-SO 2-;

h on the alicyclic or alicyclic ring can be optionally substituted by a halogen atom, nitro, aryl, alkyl or modified alkyl;

the aromatic ring is a 5-10 membered aromatic monocyclic ring or an aromatic fused bicyclic ring;

the aromatic heterocyclic ring is a 5-to 10-membered aromatic monocyclic ring or an aromatic fused bicyclic ring which contains at least one heteroatom selected from O, S, N or Si on the ring;

the H on the aromatic ring or the aromatic heterocyclic ring may be optionally substituted with a halogen atom, a nitro group, an aryl group, an alkyl group or a modified alkyl group.

The bioadhesive hydrogel according to the present invention may further comprise a first crosslinking biomacromolecule and a second crosslinking biomacromolecule, wherein the first crosslinking biomacromolecule is a biomacromolecule having a first crosslinking group, and the second crosslinking biomacromolecule is a biomacromolecule having a sugar cyclohydroxymethyl group as a second crosslinking group.

Further, the first crosslinkable biomacromolecule may be obtained by modifying a biomacromolecule with the first crosslinking group, and the biomacromolecule may be any one of the following: (1) natural polysaccharide substances such as hyaluronic acid, carboxymethyl cellulose, methyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, alginic acid, dextran, agarose, heparin, chondroitin sulfate, ethylene glycol chitosan, propylene glycol chitosan, chitosan lactate, carboxymethyl chitosan or chitosan quaternary ammonium salt; (2) various hydrophilic or water-soluble animal and plant proteins, collagen, serum protein, silk fibroin and elastin, wherein the protein degradation product comprises gelatin or polypeptide; (3) hydrophilic or water-soluble synthetic polymer, including two-arm or multi-arm polyethylene glycol, polyethyleneimine, tree branch, synthetic polypeptide, polylysine, polyglutamic acid, polyacrylic acid, polymethacrylic acid, polyacrylate, polymethacrylate, polyacrylamide, polymethacrylamide, polyvinyl alcohol, and polyvinylpyrrolidone.

The bioadhesive hydrogel according to the present invention may further comprise a third crosslinkable biopolymer, wherein the third crosslinkable biopolymer is a biopolymer containing a first crosslinking group and a second crosslinking group. Further, the third crosslinkable biomacromolecule can be obtained by modifying a biomacromolecule containing a sugar ring hydroxymethyl group as the second crosslinking group with the first crosslinking group.

In the present invention, the sugar ring-hydroxymethyl-containing biopolymer may be any one or any one of a hyaluronic acid, carboxymethyl cellulose, methyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, dextran, agarose, heparin, chitosan, sucrose, glucose, and starch, or a derivative thereof.

The bioadhesive hydrogel provided by the invention can also have the technical characteristics that any two of R', R3, R4 and R5 are connected with each other to form a saturated or unsaturated alicyclic or alicyclic heterocyclic ring or an aromatic or heteroaromatic ring.

The bioadhesive hydrogel provided by the invention can also have the technical characteristics that the sugar ring hydroxymethyl group has a structure of any one of the following formulas IV, V, VI and VII:

in formula IV, formula V, formula VI and formula VII, R is selected from hydrogen, halogen atom, hydroxyl, sulfydryl, amine group, nitro group, cyano group, aldehyde group, ketone group, carboxyl group, carboxylate group, ester group, amide group, phosphonate group, sulfonic group, sulfonate group, sulfone group, sulfoxide group, aryl group, heteroaryl group, alkyl group, alkylene group, modified alkyl group or modified alkylene group.

The bioadhesive hydrogel provided by the invention can also have the technical characteristics that the photoinitiator is: 2-hydroxy-4' - (2-hydroxyethoxy) -2-methyl propiophenone, lithium phenyl (2,4, 6-trimethyl benzoyl) phosphate, sodium phenyl (2,4, 6-trimethyl benzoyl) phosphate and magnesium phenyl (2,4, 6-trimethyl benzoyl) phosphate.

In another aspect, the present invention provides a use of the bioadhesive hydrogel according to any one of the above aspects in the preparation of a rapid hemostatic or tissue repair article, wherein the nitroso group generated by the first crosslinking group in the presence of light and a photoinitiator chemically bonds with the second crosslinking group to form gel, and also chemically bonds with at least one of the sugar cyclomethylol groups and thiol groups on proteoglycan and glycosaminoglycan in the extracellular matrix of the surface of the biological tissue, so that the formed gel adheres to the surface of the biological tissue.

In another aspect, the present invention provides a drug carrier comprising the bioadhesive hydrogel according to any one of the above aspects. Further, the present invention provides a bio-printable material characterized by containing the bioadhesive hydrogel according to any one of the above.

Action and Effect of the invention

According to the bioadhesive hydrogel provided by the invention, the bioadhesive hydrogel comprises a crosslinkable biomacromolecule and a photoinitiator, wherein the crosslinkable biomacromolecule comprises a first crosslinking group and a second crosslinking group, the first crosslinking group is an o-nitrobenzyl photoinitiator, and the second crosslinking group is a sugar-ring hydroxymethyl group, so that the first crosslinking group can generate a nitroso group under the illumination condition, and further can be chemically bonded with the second crosslinking group and be cured into gel under the existence condition of the photoinitiator. In addition, when the hemostatic agent is applied to rapid hemostasis, the adhesive strength of the wet tissue is high, so that the requirement of rapid hemostasis of high blood pressure parts such as the heart and the like can be met; when the printing paste is applied to biological printing, the glued edges are clear, so that the printing paste has excellent high-precision printing performance and can be used for biological printing of complex shapes.

Drawings

FIG. 1 is a photograph of the solidification of bioadhesive hydrogel solutions of various groups of the present invention.

FIG. 2 is a bar graph of wet-side tissue adhesion test results for various groups of bioadhesive hydrogels of the present invention.

Fig. 3 is a photograph showing the experimental process of hemostatic property according to example eight of the present invention.

Fig. 4 is a photograph of a section of a healing site of an animal tissue after an experiment of hemostatic properties according to example eight of the present invention.

Fig. 5 is a photograph showing the results of the drug loading performance test of example nine of the present invention.

Fig. 6 is a schematic diagram of a microsphere printing process and a print result photo in the tenth embodiment of the present invention.

Fig. 7 is a schematic view of a process of curing a paste by using a gear-shaped mask plate and a photograph of a curing result in accordance with a tenth embodiment of the present invention.

Fig. 8 is a photograph showing the result of colloid curing using a letter mask plate according to the eleventh embodiment of the present invention.

Detailed Description

The following describes embodiments of the present invention with reference to the drawings. The reagents used in the following examples are commercially available and the experimental procedures and experimental conditions not specified are those conventional in the art.

In the following examples, lithium phenyl (2,4, 6-trimethylbenzoyl) phosphate was used as a photoinitiator, but in the present invention, the lithium phenyl (2,4, 6-trimethylbenzoyl) phosphate can be replaced by other similar photoinitiators, such as, for example, lithium 2-hydroxy-4' - (2-hydroxyethoxy) -2-methylpropiophenone, lithium phenyl (2,4, 6-trimethylbenzoyl) phosphate, sodium phenyl (2,4, 6-trimethylbenzoyl) phosphate, magnesium phenyl (2,4, 6-trimethylbenzoyl) phosphate. Furthermore, the initiator may also be a combination of any of these photoinitiators.

The cross-linking biological macromolecules involved in each example are mainly three kinds, wherein the first cross-linking biological macromolecule is a biological macromolecule containing an o-nitrobenzyl type photosheet machine (i.e., a first cross-linking group, hereinafter abbreviated as NB); the second crosslinking biological macromolecule is a biological macromolecule containing sugar ring hydroxymethyl (namely a second crosslinking group); the third crosslinkable biomacromolecule is a biomacromolecule containing both the first crosslinking group and the second crosslinking group. The first crosslinking group and the second crosslinking group may be contained in the crosslinkable biomacromolecule itself or may be modified by a chemical reaction.

In addition, in the following examples, examples in which the first crosslinking biological macromolecule, the second crosslinking biological macromolecule are used in combination, and examples in which the third crosslinking biological macromolecule is used alone (i.e., not used in combination with the other two) are described. However, in the present invention, the third crosslinkable biomacromolecule may be used in combination with any one of the first crosslinkable biomacromolecule and the second crosslinkable biomacromolecule, or may be used in combination with the first crosslinkable biomacromolecule and the second crosslinkable biomacromolecule, which can also achieve the effects described in the following examples.

< example one >

The main components of the bioadhesive hydrogel provided in this example include the third crosslinkable biomacromolecule and the photoinitiator.

The third crosslinkable biomacromolecule of the present example is hyaluronic acid modified with NB (hereinafter abbreviated as HANB). Among them, hyaluronic acid is a common biological macromolecule, which itself contains a sugar ring hydroxymethyl structure.

The preparation process of the HANB of this example is: the preparation method comprises the following steps of (1) mixing aminated NB, hyaluronic acid, EDC and NHS in a mass ratio of 1:20:1:1, dissolving in water, adjusting the pH value to 4.5-6.5, heating to above 35 ℃, stirring for reaction for 2 hours, and then dialyzing and freeze-drying to obtain the HANB with the grafting rate of 5%.

The bioadhesive hydrogel solution (designated as the HANB group) of this example was obtained by dissolving the above-mentioned HANB and LAP in deionized water at a mass ratio of 1:0.02, wherein the HANB mass concentration was 5%.

< example two >

The main components of the bioadhesive hydrogel provided in this example include a first crosslinkable biomacromolecule, a second crosslinkable biomacromolecule, and a photoinitiator.

The first crosslinkable biomacromolecule in this example was NB-modified polyglutamic acid (hereinafter abbreviated as PGANB). Wherein, the polyglutamic acid does not contain a sugar ring hydroxymethyl structure.

The preparation process of the PGANB comprises the following steps: dissolving aminated NB, polyglutamic acid, EDC and NHS into water according to the mass ratio of 1:15:1:1, adjusting the pH to 4.5-6.5, heating to over 35 ℃, stirring for reaction for 3 hours, then dialyzing, and freeze-drying to obtain PGANB with the grafting rate of 10%.

The second cross-linking biomacromolecule in this example is Hyaluronic Acid (HA). Specifically, the following solutions were prepared in this example:

(1) dissolving PGANB and LAP in water at a mass ratio of 1:0.02 to form an aqueous solution with a PGANB mass concentration of 10%, and marking as a PGANB group. This solution contained no second cross-linking biomacromolecule and was used as a control.

(2) Mixing PGANB with Chondroitin Sulfate (CS) and LAP which do not contain sugar ring hydroxymethyl structures according to a mass ratio of 1:0.02 was dissolved in water to form a 10% aqueous solution of PGANB, which was designated as PGANB + CS group. CS is a biological macromolecule that does not itself contain sugar ring hydroxymethyl structures, and the PGANB + CS group was used as a control.

(3) Mixing PGANB with Hyaluronic Acid (HA) and LAP containing sugar ring hydroxymethyl structures according to a mass ratio of 1: 0.1: 0.02 was dissolved in water to form an aqueous solution of 5% by mass of PGANB, which was designated as PGANB + HA group.

< example three >

The main components of the bioadhesive hydrogel provided in this example include a first crosslinkable biomacromolecule, a second crosslinkable biomacromolecule, and a photoinitiator.

The first cross-linking biomacromolecule of this example is NB-modified Chondroitin Sulfate (CSNB). The preparation process of CSNB is as follows: dissolving aminated NB and chondroitin sulfate into water according to the mass ratio of 1:10, adjusting the pH value to 4.5-6.5, heating to the temperature of more than 35 ℃, stirring for reaction for half an hour, and then dialyzing and freeze-drying to obtain CSNB with the grafting rate of 25%.

The second crosslinkable biomacromolecule in this example is carboxymethyl cellulose itself having a sugar ring hydroxymethyl structure.

Specifically, the following solutions were prepared in this example:

(1) and dissolving CSNB and LAP into water according to the mass ratio of 1:0.02 to form an aqueous solution with the mass concentration of CSNB of 10%, and marking as a CSNB group. This solution contained no second cross-linking biomacromolecule and was used as a control.

(2) And dissolving CSNB, CMC and LAP into water according to the mass ratio of 1:10:0.02 to form an aqueous solution with the CSNB mass concentration of 0.5%, and marking as a CSNB + CMC group.

< example four >

The main components of the bioadhesive hydrogel provided in this example include the third crosslinkable biomacromolecule and the photoinitiator.

The third crosslinkable biomacromolecule of this example is NB-modified carboxymethyl cellulose (CMCNB). That is, CMC itself contains a sugar ring hydroxymethyl group, and after NB modification, the resulting CMCNB contains both NB and the sugar ring hydroxymethyl group.

The process of obtaining the CMCNB by modifying the CMC with NB comprises the following steps: dissolving aminated NB, CMC, EDC and NHS into water according to the mass ratio of 1:20:1:1, adjusting the pH to 4.5-6.5, heating to over 35 ℃, stirring for reaction for 2 hours, and then dialyzing and freeze-drying to obtain CMCNB with NB grafting rate of 4%.

And (3) dissolving the CMCNB and the LAP into water according to the mass ratio of 1:0.02 to form an aqueous solution with the mass concentration of the CMCNB being 3%, thus obtaining the bioadhesive hydrogel solution of the embodiment, which is marked as a CMCNB group.

< example five >

The main components of the bioadhesive hydrogel provided in this example include the third crosslinkable biomacromolecule and the photoinitiator.

Wherein the first cross-linkable biomacromolecule is NB-modified Chitosan (CTSNB). Wherein, the chitosan contains a sugar ring hydroxymethyl structure, and the CTSNB obtained by modifying the chitosan with NB contains both NB and sugar ring hydroxymethyl.

The preparation process of CTSNB comprises the following steps: dissolving NHS-treated NB, chitosan, EDC and NHS into water according to the mass ratio of 1:10:1:1, adjusting the pH value to 4.5-6.5, heating to over 35 ℃, stirring for reaction for 2 hours, then dialyzing, and freeze-drying to obtain CTSNB with the grafting rate of 100%.

And dissolving CTSNB and LAP into water according to the mass ratio of 1:0.02 to form an aqueous solution with the CTSNB mass concentration of 5%, thus obtaining the bioadhesive hydrogel solution of the embodiment, which is marked as CTSNB group.

< example six >

This example is a test for testing the curing properties of each of the bioadhesive hydrogels of the first to fifth examples.

The bioadhesive hydrogel solutions of each of the first to fifth examples were applied at a wavelength of 365nm and a light intensity of 30mW/cm²The time required from the start of irradiation to complete curing was recorded, and the results are shown in table 1 below. In Table 1, the symbol "-" indicates that no gel formation occurred during the duration of light irradiation (60 s).

TABLE 1 bioadhesive hydrogel solutions photocure time

FIG. 1 is a photograph of the solidification of bioadhesive hydrogel solutions of various groups of the present invention. In fig. 1, the photographs were taken after each centrifuge tube was inverted, the solidified glue was located on the upper side (at the bottom of the centrifuge tube) and the uncured glue was located on the lower side (at the mouth of the centrifuge tube).

As shown in table 1 and fig. 1, the haib, PGANB + HA, CSNB + CMC, CMCNB, CTSNB all cured to gel in about 2s under light conditions. In contrast, PGANB + CS did not cure to a gel, indicating that HA contains groups necessary for crosslinking with PGANB; CSNB could not cure to a gel, indicating that the groups contained in CMC are a necessary condition for crosslinking with CSNB. CMC does not contain a crosslinking group described in the prior art such as amide, hydroxylamine, primary amine, etc., and therefore NB in the present invention is crosslinked by chemically bonding with other groups. Further mechanism research shows that NB can generate nitroso under the illumination condition, and the nitroso can generate chemical bonding reaction with sugar ring hydroxymethyl under the existence of photoinitiator, so that crosslinking and curing are performed to form glue.

< example seven >

This example is a test of the wet-side tissue adhesion test using the burst pressure method for each of the groups of bioadhesive hydrogels from the first to fifth examples, and the results are shown in table 2 below.

TABLE 2 Wet side tissue adhesion test results for various groups of bioadhesive hydrogels of the present invention

FIG. 2 is a bar graph of wet-side tissue adhesion test results for various groups of bioadhesive hydrogels of the present invention.

As shown in table 2 and fig. 2, in each example, the bioadhesive hydrogel solution had a certain wet tissue adhesion in addition to the control group, indicating that it can be used as a bioremediation gel for rapid hemostasis. In addition, the wet-side tissue adhesion force of the fourth to fifth examples is higher than that of the first to third examples, which shows that the adhesion force of the third cross-linking biomacromolecule cross-linking is higher than that of the combination of the first cross-linking biomacromolecule and the second cross-linking biomacromolecule, and the reason may be that when the sugar-ring hydroxymethyl group and the NB coexist in the same biomacromolecule, the adhesion force generated by the intramolecular self-cross-linking is higher than that generated when the sugar-ring hydroxymethyl group and the NB exist in different biomacromolecules respectively.

In addition, the bioadhesive hydrogel solutions of the HANB, PGANB + HA, CMCNB, CTSNB groups all had wet-side tissue adhesion higher than 160mmHg, i.e., higher than normal arterial blood pressure. That is, the bioadhesive hydrogels of these groups have sufficient adhesion to withstand the pressure of arterial blood pressure and can be applied to rapid hemostasis in areas of high blood pressure, such as the aorta or heart. The wet tissue adhesion of the CSNB + CMC group is relatively low, and the CSNB + CMC group cannot be applied to rapid hemostasis of high blood pressure parts, but can be applied to rapid hemostasis of other parts with low blood pressure.

< example eight >

This example is a hemostatic property test of a bioadhesive hydrogel.

Fig. 3 is a photograph showing the experimental process of hemostatic property according to example eight of the present invention. In fig. 3, the surgical site for blood test is shown in dotted line.

The specific process is as follows: a5 mm wound is cut on the carotid artery of the pig by a scalpel, and the blood column is rapidly ejected. The proximal end was clamped with a hemostatic clamp, the solution of PGANB + HA of example II was applied to the wound, and the wound was irradiated with light of 60mW/cm2 at 395nm for 5 seconds, after repeated 2 times, the hemostatic clamp was released, and no blood continued to flow out within 5 minutes after release, thus demonstrating the excellent rapid hemostatic properties of PGANB + HA. The animals were kept for two weeks after the operation, and the condition was good and no abnormality was observed.

Fig. 4 is a photograph of a section of a healing site of an animal tissue after an experiment of hemostatic properties according to example eight of the present invention. In the four photographs of fig. 4, from left to right: a photograph of a section of an intact carotid artery, a magnified photograph of a section of an intact carotid artery, a photograph of a section of a carotid artery surgical site, and a magnified photograph of a section of a carotid artery surgical site.

As shown in FIG. 4, after the animals are raised for two weeks, the tissue section is sampled and analyzed at the hemostasis experimental part, and the histological result shows that the wound is healed, thereby proving that PGANB + HA HAs the effect of promoting tissue repair.

In addition, the same hemostatic performance test was performed using the bioadhesive hydrogels of the other examples, and the results are similar to those shown in fig. 4, which shows that the bioadhesive hydrogels of the present invention all have the effects of rapid hemostasis and tissue repair promotion.

< example nine >

This example is a drug loading performance experiment for bioadhesive hydrogels.

The specific process of the drug loading performance test is as follows: two round holes with the diameter of 10mm are respectively arranged on the left side and the right side of the skull of a rat, the growth factor BMP2 is uniformly mixed into the PGANB + HA solution of the second embodiment, and then the mixture is injected into the defect hole on the left side of the skull, and the wave band is 395nm, and the light intensity is 60mW/cm²Light irradiation of (2) for 15 seconds. The right defective well was used as a control group without further treatment.

Fig. 5 is a photograph showing the results of the drug loading performance test of example nine of the present invention.

As shown in fig. 5, the animals are sacrificed after being continuously raised for two weeks after operation, and sampling analysis shows that the left skull defect is obviously regenerated, the boundary between new bone and the defect is not obvious, and no ectopic ossification occurs. The right defective hole control group only HAs a small amount of membranous tissues, and no obvious bone regeneration exists at the defective part, so that the PGANB + HA gel is proved to have excellent drug-loading performance and site-specific release performance. In addition, the same drug loading performance experiment was performed using the bioadhesive hydrogel of the other examples, and the results are similar to those shown in fig. 5, which indicates that the bioadhesive hydrogel of the present invention has excellent drug loading performance and site-specific release performance.

< example ten >

This example is a performance test of the bioadhesive hydrogel of the invention containing a first cross-linking biomacromolecule and a second cross-linking biomacromolecule applied to bioprinting.

Fig. 6 is a schematic diagram of a microsphere printing process and a print result photo in the tenth embodiment of the present invention. Fig. 6(a) is a schematic diagram illustrating a principle of printing using a microsphere array model, fig. 6(b) is a photograph of the microsphere array model, and fig. 6(c) is a photograph of a printed microsphere.

As shown in FIG. 6, bioadhesive hydrogel was applied to the microspheresThe specific process of printing is as follows: adding the CSNB + CMC solution obtained in the third example into the ink box of the DLP biological 3D printer, selecting a microsphere array model with the diameter of 200, and adjusting the printing light intensity to be 30mW/cm²The layer thickness was 10 μm and the exposure time was 1 second per layer. After printing, a large number of colloidal microspheres with the diameter of 200 +/-10 mu m which can be used for stem cell expansion culture can be collected. The colloidal microspheres are regular in shape and uniform in size, and the excellent printing performance of CSNB + CMC is proved.

Fig. 7 is a schematic view of a process of curing a paste by using a gear-shaped mask plate and a photograph of a curing result in accordance with a tenth embodiment of the present invention. Fig. 7(a) is a schematic view of the principle of colloid curing using a gear mask plate, fig. 7(b) is a photograph of a gear colloid obtained by CSNB + CMC curing, and fig. 7(c) is a photograph of a colloid obtained by GelMA curing.

As shown in fig. 7(a), the process of curing the paste using the gear-shaped mask plate is as follows: GelMA (i.e., methacrylic anhydrified gelatin, a common bioprinting material of the prior art) and CSNB + CMC solution of example III were each poured into a container to form a liquid film having a thickness of 2mm, and a mask plate engraved with a gear-like pattern was placed on the liquid surface, and the liquid was covered with a mask plate having a wavelength of 395nm and a light intensity of 30mW/cm²The light is irradiated for 15 seconds from above the mask plate, and then uncured solution in the container is cleaned, so that the corresponding cured colloid is obtained.

As shown in fig. 7(b) and 7(c), a clear gear-like pattern appears at the edge of the solidified colloid of CSNB + CMC, and the included angle of the sawtooth is consistent with the original image; in contrast, GelMA formed a cured glue with a circular additional cured area with significant light scattering at the edges, and almost no saw-tooth shape was observed. This result demonstrates that when the bioadhesive hydrogel of the present invention is applied to bioprinting, the edge shape of the gel formed by curing is more clear, and the hydrogel can be applied to printing of complex shapes, i.e., has excellent high-precision printing performance.

< EXAMPLE eleven >

This example is a performance test of the bioadhesive hydrogel of the invention containing a third cross-linking biomacromolecule applied to bioprinting.

The specific process of applying the bioadhesive hydrogel to the printing of the micro-cube in this example is similar to that of applying the hydrogel to the printing of the micro-sphere in the example ten, and the specific process is as follows: the CMCNB solution of example four was added to the cartridge of a DLP bio-3D printer, a 50 side microarray model was selected, and the printing intensity was adjusted to 30mW/cm²The layer thickness was 10 μm and the exposure time was 1 second per layer. After printing, a large number of colloidal micro-cubes of 50 + -5 μm can be collected. These colloidal microblocks are regular in morphology and uniform in size, demonstrating excellent printing performance of the CMCNB.

Fig. 8 is a photograph showing the result of colloid curing using a letter mask plate according to the eleventh embodiment of the present invention. Fig. 8(a) is a schematic view of the shape of a letter mask, fig. 8(b) is a photograph of a letter-like colloid obtained by curing CMCNB, and fig. 8(c) is a photograph of a colloid obtained by curing GelMA.

The process of curing the colloid by using the letter mask plate is similar to the process of curing by using the gear-shaped mask plate in the tenth embodiment, and specifically comprises the following steps: GelMA and CMCNB solutions of the four examples were poured into a container to form a liquid film having a thickness of 2mm, and a mask plate engraved with the word "ZJT" as shown in FIG. 8(a) was placed on the surface of the liquid at a wavelength of 395nm and a light intensity of 30mW/cm²The light is irradiated for 15 seconds from above the mask plate, and then uncured solution in the container is cleaned, so that the corresponding cured colloid is obtained.

As shown in fig. 8(b) and 8(c), the cured colloid of CMCNB takes the shape of a clear "ZJT" word; in contrast, GelMA forms a cured gel with almost no "ZJT" appearance, and has rounded extra cured areas due to significant light scattering at the edges, making the glyph very blurred. This result demonstrates that the bioadhesive hydrogel of the invention, which contains a third crosslinkable biomacromolecule, also has excellent high-precision printing properties.