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、湿面组织粘附力高、成本低且没有安全疑虑的生物胶材料,具体提供了一种生物粘附性水凝胶,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第一交联基团以及第二交联基团,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成胶,第一交联基团为邻二氢吡啶硝基苯衍生物或吡啶甲基硝基烯咪唑类衍生物,第二交联基团为糖环羟甲基、双键、巯基中的任意一种或任意几种的组合。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 biological adhesive material which is high in curing speed, high in wet tissue adhesion, low in cost and free of safety concerns, and particularly provides a biological adhesive hydrogel which comprises a crosslinking biological macromolecule and a photoinitiator, wherein the crosslinking biological macromolecule comprises a first crosslinking group and a second crosslinking group, the first crosslinking group generates a nitroso group under the illumination condition, the nitroso group and the second crosslinking group generate chemical bonding under the existence condition of the photoinitiator so as to form gel, the first crosslinking group is an o-dihydropyridine nitrobenzene derivative or a picolyl nitroene imidazole derivative, and the second crosslinking group is any one or combination of any more of a sugar ring hydroxymethyl group, a double bond and a sulfhydryl group. 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-dihydropyridine nitrobenzene derivative or a picolyl nitroene imidazole derivative,

the second crosslinking group is any one or combination of any more of sugar ring hydroxymethyl, double bond and sulfydryl,

the structure of the o-dihydropyridine nitrobenzene derivative is shown as the following formula I:

in formula I:

R₁，R₂，R₃，R₄，R₅，R₆，R₇，R₈any one or more of them is selected from a terminal amine group, hydroxyl group, mercapto group, halogen, carboxyl group, carboxylate group or reactive ester group-modified aryl group, heteroaryl group, alkyl group, alkylene group, modified alkyl group or modified alkylene group, and may be freely selected from hydrogen, halogen atom, hydroxyl group, mercapto group, 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 structure of the pyridylmethyl nitroene imidazole derivative is shown as the following formula II:

in the formula II, R₉，R₁₀，R₁₁，R₁₂，R₁₃，R₁₄Any one or more of which is selected from the group consisting of terminal amine, hydroxyl, sulfhydryl, halogen, carboxyl, carboxylate or reactive ester modified aryl, heteroaryl, alkyl, alkylene, modified alkyl or modified alkyleneAnd a group, which may be freely selected from hydrogen, halogen atom, hydroxyl group, mercapto group, amine group, nitro group, cyano group, aldehyde group, ketone group, carboxyl group, carboxylate group, ester group, amide group, phosphonate group, sulfonic acid 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,

wherein 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,

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 1, 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,

the biomacromolecule containing double-bond groups specifically contains any one or combination of any more of methacrylamide, methacrylic anhydride and methyl maleic anhydride,

the biological macromolecule containing sulfydryl is gelatin, collagen or silk fibroin.

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

wherein R is₁，R₂，R₃，R₄，R₅，R₆，R₇，R₈Any two of which are linked to each other to form a saturated or unsaturated alicyclic or alicyclic heterocyclic ring, or alternatively, linked to each other to form an aromatic ring or aromatic heterocyclic ring.

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

wherein the structure of the o-dihydropyridine nitrobenzene derivative is any one of the following structural formulas NF-1 to NF-6:

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

wherein R is₁₃，R₁₄Are linked to each other to form a saturated or unsaturated aliphatic or aliphatic heterocyclic ring, or are linked to each other to form an aromatic ring or aromatic heterocyclic ring.

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

wherein the structure of the pyridylmethyl nitroene imidazole derivative is any one of the following structural formulas NF-7-NF-30:

10. the bioadhesive hydrogel according to claim 1, wherein:

wherein the sugar ring hydroxymethyl group has a structure shown in any one of a formula IV, a formula V, a formula VI and a formula VII:

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

11. 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.

12. Use of a bioadhesive hydrogel according to any one of claims 1-11 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.

13. A drug carrier comprising the bioadhesive hydrogel according to any one of claims 1 to 11.

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

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. However, the o-nitrobenzyl optical trigger compound has high overall price, and correspondingly, the cost of the modified biomacromolecule is high, so that the o-nitrobenzyl optical trigger compound is difficult to popularize and apply. Moreover, most of the o-nitrobenzyl light trigger compounds used for modification in the prior art are not verified in the aspect of biological safety, and if the o-nitrobenzyl light trigger compounds are degraded in organisms to generate toxic small molecules, the o-nitrobenzyl light trigger compounds bring extremely high use risk, so that the application of the o-nitrobenzyl light trigger compounds in organisms, particularly human bodies is influenced.

Disclosure of Invention

The present invention has been made to solve the above problems, and an object of the present invention is to provide a biomaterial material which has a high curing speed, a high wet-side tissue adhesion, a low cost, and no safety concerns. The inventor of the invention researches and discovers that if o-dihydropyridine nitrobenzene derivatives or pyridylmethyl nitroene imidazole derivatives are adopted to modify some common biological macromolecules, the modification groups can generate nitroso groups in illumination, and the nitroso groups can be chemically bonded with sugar ring hydroxymethyl, double bonds and sulfydryl under the action of photoinitiator free radicals, so that the biological macromolecules are crosslinked. That is, under the conditions of illumination and photoinitiator, the modification group of the o-dihydropyridine nitrobenzene derivative or the pyridylmethyl nitroene imidazole derivative can be bonded with sugar ring hydroxymethyl, double bond or sulfydryl to crosslink biological macromolecules, thereby curing to form glue.

Based on the above findings, the present invention provides a bioadhesive hydrogel characterized by comprising: the crosslinking biological macromolecule comprises a first crosslinking group and a second crosslinking group, wherein 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 a gel, the first crosslinking group is an o-dihydropyridine nitrobenzene derivative or a picolyl nitroene imidazole derivative, the second crosslinking group is any one or combination of any several of sugar ring hydroxymethyl, double bonds and sulfydryl, and the structure of the o-dihydropyridine nitrobenzene derivative is shown as the following formula I:

in formula I:

R₁，R₂，R₃，R₄，R₅，R₆，R₇，R₈any one or more of them is selected from a terminal amine group, hydroxyl group, mercapto group, halogen, carboxyl group, carboxylate group or reactive ester group-modified aryl group, heteroaryl group, alkyl group, alkylene group, modified alkyl group or modified alkylene group, and may be freely selected from hydrogen, halogen atom, hydroxyl group, mercapto group, 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 structure of the pyridylmethyl nitroene imidazole derivative is shown as the following formula II:

in the formula II, R₉，R₁₀，R₁₁，R₁₂，R₁₃，R₁₄Any one or more of them is selected from a terminal amine group, hydroxyl group, mercapto group, halogen, carboxyl group, carboxylate group or reactive ester group-modified aryl group, heteroaryl group, alkyl group, alkylene group, modified alkyl group or modified alkylene group, and may be freely selected from hydrogen, halogen atom, hydroxyl group, mercapto group, 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.

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, wherein the modified alkylene has 1 to 30 atoms, and a carbon-carbon single bond of the modified alkylene can be replaced by a carbon-carbon double bond or a carbon-carbon 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 second crosslinking group.

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 may be obtained by modifying a biomacromolecule having a second crosslinking group with the first crosslinking group.

The bioadhesive hydrogel provided by the invention can also have the technical characteristics that the biomacromolecule containing the 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, the biomacromolecule containing the double bond group specifically contains any one or any combination of several of methacrylamide, methacrylic anhydride and methyl maleic anhydride, and the biomacromolecule containing the sulfhydryl group is gelatin, collagen or fibroin.

The bioadhesive hydrogel provided by the present invention may also have the technical feature that R is₁，R₂，R₃，R₄，R₅，R₆，R₇，R₈Any two of which are linked to each other to form a saturated or unsaturated alicyclic or alicyclic heterocyclic ring, or alternatively, linked to each other to form an aromatic ring or aromatic heterocyclic ring.

The bioadhesive hydrogel provided by the invention can also have the technical characteristics that the structure of the o-dihydropyridine nitrobenzene derivative is any one of the following structural formulas NF-1-NF-6:

the bioadhesive hydrogel provided by the present invention may also have the technical feature that R is₁₃，R₁₄Are linked to each other to form a saturated or unsaturated aliphatic or aliphatic heterocyclic ring, or are linked to each other to form an aromatic ring or aromatic heterocyclic ring.

The bioadhesive hydrogel provided by the invention can also have the technical characteristics that the structure of the picolyl nitroene imidazole derivative is any one of the following structural formulas NF-7-NF-30:

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

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

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 a gel, and at the same time chemically bonds with at least one of a sugar cyclomethylol group and a thiol group on a proteoglycan and a glycosaminoglycan in an extracellular matrix of a biological tissue surface to adhere the formed gel to the biological tissue surface.

In another aspect, the present invention provides a drug carrier comprising the bioadhesive hydrogel according to any one of the above aspects.

In another aspect, the present invention provides a bio-adhesive hydrogel according to any one of the above embodiments, wherein the bio-adhesive hydrogel is a hydrogel.

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-dihydropyridine nitrobenzene derivative or a pyridylmethyl nitroene imidazole derivative, and the second crosslinking group is any one or a combination of any more of a sugar ring hydroxymethyl group, a double bond and a sulfhydryl group, so that the first crosslinking group can generate a nitroso group under a lighting condition, and then is chemically bonded with the second crosslinking group in the presence of the photoinitiator and is cured to form gel.

Because the first crosslinking group is an o-dihydropyridine nitrobenzene derivative or a picolyl nitroene imidazole derivative which has precedent of drug application in the pharmaceutical industry, the safety risk is low, and the derivatives can be applied to organisms; in addition, since the use of these derivatives is mature and the price is relatively low, the bioadhesive hydrogel of the invention can be manufactured at a lower cost.

Drawings

FIG. 1 is a photograph of a set of bioadhesive hydrogel solutions according to various embodiments of the present invention;

FIG. 2 is a bar graph of wet side tissue adhesion test results for various embodiments of the present invention;

FIG. 3 is a photograph of a hemostatic property experimental procedure of example nine of the present invention;

FIG. 4 is a schematic diagram of the experimental process of tissue regeneration performance and a photograph of a tenth embodiment of the present invention;

FIG. 5 is a photograph of a drug loading performance test of example eleven of the present invention;

fig. 6 is a schematic view of a bio-printing process and a print result photo according to a twelfth 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.

In an embodiment, the first cross-linkable biomacromolecule is a biomacromolecule comprising a first cross-linking group, the second cross-linkable biomacromolecule is a biomacromolecule comprising a second cross-linking group, and the third cross-linkable biomacromolecule is a biomacromolecule comprising both the first cross-linking group and the second cross-linking group. In general, some common biological macromolecules contain second crosslinking groups, so that the biological macromolecules can be directly used as second crosslinking biological macromolecules without additional modification; since the common biological macromolecule does not usually contain the first crosslinking group, the first crosslinking biological macromolecule is obtained by modifying the biological macromolecule with the first crosslinking group, and the third crosslinking biological macromolecule is obtained by modifying the biological macromolecule (equivalent to the second crosslinking biological macromolecule) containing the second crosslinking group with the first crosslinking group.

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 a first crosslinkable biomacromolecule, a second crosslinkable biomacromolecule, and a photoinitiator.

The first crosslinkable biomacromolecule is chondroitin sulfate (hereinafter abbreviated as CSNF) modified with a compound NF-1. Wherein, the Chondroitin Sulfate (CS) does not contain sugar ring hydroxymethyl, sulfydryl and double bonds; the compound NF-1 is an o-dihydropyridine nitrobenzene derivative, and the structural formula is as follows:

the preparation process of CSNF in this example was: dissolving NF-1, chondroitin sulfate, EDC and NHS in 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, and then dialyzing and freeze-drying to obtain CSNF with the grafting rate of 20%.

In this embodiment, the second crosslinkable biomacromolecule capable of being conjugated with CSNF may be a macromolecular compound containing any one or more of a mercapto group, a sugar ring hydroxymethyl group, and a double bond, including: gelatin (Gelatin), which itself contains a thiol group; hyaluronic Acid (HA), itself containing a sugar ring hydroxymethyl structure; chondroitin Sulphate (CSMA) containing double bond modifications. In addition, several second cross-linkable biological macromolecules used in other embodiments may also be used in this embodiment.

The bioadhesive hydrogel solution of this example was obtained by dissolving the first cross-linking biomacromolecule, the second cross-linking biomacromolecule, and LAP in deionized water. This example prepared three bioadhesive hydrogels using CSNF as follows:

(1) dissolving CSNF and LAP into water according to the mass ratio of 1:0.02 to form a water solution (marked as CSNF group), wherein the mass concentration of CSNF is 3%, and the CSNF + LAP solution does not contain second cross-linking biological macromolecules and is used as a control;

(2) dissolving CSNF, Gelatin and LAP in water according to the mass ratio of 1:0.5:0.02 to form a water solution (marked as CSNF + Gelatin group), wherein the mass concentration of CSNF is 3%;

(3) dissolving CSNF, HA and LAP into water according to the mass ratio of 1:1:0.02 to form a water solution (marked as CSNF + HA group), wherein the mass concentration of CSNF is 3%;

(4) and dissolving the CSNF, the CSMA and the LAP into water according to the mass ratio of 1:1:0.02 to form an aqueous solution (marked as a CSNF + CSMA group), wherein the mass concentration of the CSNF is 3%.

< 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 is polyglutamic acid (hereinafter abbreviated as PGANF) modified with a compound NF-7. Wherein, the polyglutamic acid does not contain sugar ring hydroxymethyl, sulfydryl and double bond; the compound NF-7 is a pyridylmethyl nitroene imidazole derivative, and has a structural formula as follows:

the preparation process of PGANF of this example is: dissolving NF-7, polyglutamic acid, EDC and NHS in 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, and then dialyzing and freeze-drying to obtain PGANF with the grafting rate of 10%.

In this embodiment, the second crosslinkable biomacromolecule capable of being matched with PGANF may be a macromolecular compound including any one or more of a thiol group, a sugar ring hydroxymethyl group, and a double bond, where the thiol group, the sugar ring hydroxymethyl group, and the double bond may be self-contained structures in the biomacromolecule, or may be modified to the biomacromolecule without these structures by a chemical reaction.

The second cross-linkable biomacromolecule used in this example includes: fibroin (Silk), itself containing a thiol group; carboxymethyl cellulose (CMC), which itself contains sugar ring hydroxymethyl groups; polyethylene glycol (PEGDA) containing double bond modifications. In addition, several second cross-linkable biological macromolecules used in other embodiments may also be used in this embodiment.

The bioadhesive hydrogel solution of this example was obtained by dissolving the first cross-linking biomacromolecule, the second cross-linking biomacromolecule, and LAP in deionized water. This example prepared three bioadhesive hydrogels using PGANF as follows:

(1) dissolving PGANF and LAP in water at a mass ratio of 1:0.02 to form an aqueous solution (marked as PGANF group), wherein the mass concentration of PGANF is 10%. The PGANF + LAP solution contained no second cross-linking biomacromolecule and was used as a control.

(2) Dissolving PGANF, Silk and LAP in water according to the mass ratio of 1:1:0.02 to form an aqueous solution (marked as PGANF + Silk group), wherein the mass concentration of the PGANF is 10%.

(3) Dissolving PGANF, CMC and LAP in a mass ratio of 1:0.1:0.02 in water to form an aqueous solution (marked as PGANF + CMC group), wherein the mass concentration of PGANF is 5%.

(4) Mixing PGANF, PEGDA and LAP according to the mass ratio of 1:0.5:0.02 dissolved in water to form an aqueous solution (designated as PGANF + PEGDA group) with a PGANF concentration of 5% by mass.

< 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 crosslinkable biomacromolecule is a four-arm polyethylene glycol (hereinafter abbreviated as 4PNF) modified with a compound NF-2. Wherein, the four-arm polyethylene glycol does not contain sugar ring hydroxymethyl, sulfydryl and double bonds, and the structure of the four-arm polyethylene glycol has amino end capping; the compound NF-2 is an o-dihydropyridine nitrobenzene derivative, and the structural formula is as follows:

the preparation process of PGANF of this example is: dissolving NF-2, amino-terminated four-arm polyethylene glycol, EDC and NHS in water according to the mass ratio of 1:15:1:1, adjusting the pH to 4.5-6.5, heating to above 35 ℃, stirring for reaction for 3 hours, and then dialyzing and freeze-drying to obtain the 4PNF with the grafting rate of 100%.

In this embodiment, the second crosslinkable biomacromolecule capable of coordinating with PGANF may be a macromolecular compound containing any one or more of thiol, sugar ring hydroxymethyl, and double bond, for example, Polylysine (PLS) containing double bond modification, Polycysteine (PCYS) containing thiol, or the second crosslinkable biomacromolecule described in other embodiments.

The bioadhesive hydrogel solution of this example was obtained by dissolving the first cross-linking biomacromolecule, the second cross-linking biomacromolecule, and LAP in deionized water. This example prepared three bioadhesive hydrogels using PGANF as follows:

(1) dissolving 4PNF and LAP into water according to the mass ratio of 1:0.02 to form an aqueous solution (recorded as a 4PNF group), wherein the mass concentration of the 4PNF is 30%. The 4PNF + LAP contained no second cross-linking biomacromolecule and was used as a control.

(2) Dissolving 4PNF, CS and LAP into water according to the mass ratio of 1:0.02 to form an aqueous solution (recorded as 4PNF + CS group), wherein the mass concentration of 4PNF is 30%. CS does not contain thiol, sugar ring hydroxymethyl or double bond, and is used as control.

(3) Dissolving 4PNF, HA and LAP into water according to the mass ratio of 1:0.1:0.02 to form an aqueous solution (recorded as a 4PNF + HA group), wherein the mass concentration of the 4PNF is 20%.

(4) Dissolving 4PNF, PLS and LAP into an aqueous solution (recorded as a 4PNF + PLS group) according to a mass ratio of 1:1:0.02, wherein the mass concentration of the formed 4PNF is 10%.

(5) Dissolving 4PNF, PCYS and LAP into water according to the mass ratio of 1:0.1:0.02 to form an aqueous solution (recorded as a group of 4PNF and PCYS), wherein the mass concentration of 4PNF is 10%.

< 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 in this example was carboxymethyl cellulose (hereinafter abbreviated as CMCNF) modified with compound NF-13. NF-13 is a pyridylmethyl nitroene imidazole derivative, and the structure is as follows:

the preparation process of the CMCNF comprises the following steps: dissolving NF-13, 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 and reacting for 2 hours, and then dialyzing and freeze-drying to obtain CMCNF with the grafting rate of 5%.

The third cross-linking biological macromolecule and LAP are dissolved in deionized water to obtain the bioadhesive hydrogel solution of the present example. Specifically, in this example, CMCNF and LAP were dissolved in an aqueous solution (denoted as CMCNF group) in a mass ratio of 1:0.02 to form a CMCNF mass concentration of 3%.

< example five >

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

The third crosslinkable biomacromolecule in this embodiment is hyaluronic acid (hereinafter abbreviated as HANF) modified by a compound NF-19, wherein hyaluronic acid itself contains a sugar ring hydroxymethyl structure, and NF-19 is a pyridylmethyl nitroene imidazole derivative having the following structure:

the preparation process of the HANF comprises the following steps: mixing NF-19: hyaluronic acid: EDC: NHS in a mass ratio of 1: 30: dissolving 1:1 into water, adjusting the pH to 4.5-6.5, heating to above 35 ℃, stirring for reacting for 2 hours, and then dialyzing and freeze-drying to obtain the HANF with the grafting rate of 3%.

The specific preparation process of the bioadhesive hydrogel of this example was: dissolving the HANF and the LAP into water according to the mass ratio of 1:0.02 to form an aqueous solution (marked as a HANF group), wherein the mass concentration of the HANF is 5%.

< example six >

The main components of the bioadhesive hydrogel provided in this embodiment include a third crosslinkable biomacromolecule and a photoinitiator, the third crosslinkable biomacromolecule is chitosan modified by a compound NF-3 (hereinafter abbreviated as CTSNF), wherein Chitosan (CTS) itself contains a sugar ring hydroxymethyl structure, and NF-3 is an o-dihydropyridine nitrobenzene derivative, and its structure is as follows:

the preparation process of CTSNF comprises the following steps: dissolving NF-3, chitosan, EDC and NHS into water according to the mass ratio of 1:10:1:1, 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 CTSNF with the grafting rate of 100%.

The specific preparation process of the bioadhesive hydrogel of this example was: the CTSNF and LAP are dissolved into water according to the mass ratio of 1:0.02 to form an aqueous solution (marked as a CTSNF group), wherein the mass concentration of the CTSNF is 5%.

< example seven >

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

The bioadhesive hydrogel solutions of examples one to six were each 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 showing the solidification of a bioadhesive hydrogel solution according to each example of the present invention, wherein FIG. 1(a) corresponds to each of the first to second examples, and FIG. 1(b) corresponds to each of the third to sixth examples. In fig. 1, the photo is taken after each centrifuge tube is inverted, the solidified glue is located at the upper side (the bottom of the centrifuge tube), and the uncured glue is located at the lower side (the mouth of the centrifuge tube).

As shown in table 1 and fig. 1, in the first example, the CSNF group did not cure to form a gel under the conditions of the photoinitiator and the light, which indicates that the NF-modified biomacromolecule alone could not crosslink under the conditions of the photoinitiator and the light. In addition, the CSNF + Gelatin group, the CSNF + HA group and the CSNF + CSMA group are all cured into glue after being irradiated for about 2 seconds, which shows that macromolecules containing sulfydryl, sugar ring hydroxymethyl or double bond can be subjected to crosslinking reaction with CSNF under the conditions of photoinitiator and irradiation. Similarly, in example two, the PGANF group did not cure to a gel under the conditions of photoinitiator and light, but the PGANF + Silk, PGANF + CMC, and PGANF + PEGDA groups were all able to cure to a gel under the conditions of photoinitiator and light; in example three, neither 4PNF nor 4PNF + CS cured to a gel under photoinitiator and light conditions, and 4PNF + CMC cured to a gel.

Comparing the group 4PNF + CMC with the group CMCNF of example IV, it can be seen that the CMCNF is obtained by modifying CMC containing sugar ring hydroxymethyl with a pyridylmethyl nitroene imidazole derivative NF-13, which contains sugar ring hydroxymethyl and NF groups, and thus can crosslink with itself under the conditions of light and photoinitiator. In addition, similar to the fourth example, the HANF of the fifth example and the CTSNF of the sixth example can be crosslinked with themselves under the conditions of light and a photoinitiator, and these crosslinking results show that the biomacromolecule modified by NF (including an o-dihydropyridine nitrobenzene derivative and a picolyl nitroene imidazole derivative) can be crosslinked with sugar ring hydroxymethyl, double bond or sulfhydryl under the conditions of light and a photoinitiator; further mechanism research shows that the crosslinking process is as follows: the NF group generates nitroso under the illumination condition, and the nitroso is chemically bonded with sugar ring hydroxymethyl, double bond or sulfydryl under the action of photoinitiator free radicals, thereby realizing crosslinking.

< example eight >

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

Table 2 wet side tissue adhesion test results for various embodiments of the invention

FIG. 2 is a bar graph of wet side tissue adhesion test results for various embodiments 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 could be used as a bioremediation gel for rapid hemostasis. In addition, the wet side tissue adhesion was generally lower for the four to six examples than for the one to three examples, probably because the adhesion after cross-linking of the two cross-linking bio-macromolecules was higher than for the cross-linking of one cross-linking bio-macromolecule by itself.

In addition, the above groups of curable bioadhesive hydrogel solutions 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.

< example nine >

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

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

As shown in fig. 3, the specific process of the hemostatic performance test of the bioadhesive hydrogel is as follows: the wall of the ventricle of the left ventricle of the pig is punctured by a steel pipe with the diameter of 8mm, and the blood column is rapidly ejected. The HANF solution of the fifth embodiment is smeared on the defect blood-spraying position, and the wave band is 365nm, and the light intensity is 60mW/cm²After the operation is repeated for 3-5 times after the light irradiation is carried out for 5 seconds, the wound can be observed to stop bleeding (the leftmost picture in figure 3), and the blood can not continuously flow out after waiting and observing for 15 minutes, thus proving the excellent rapid hemostasis performance of the HANF.

In addition, the results of the same hemostatic performance test using CSNF + Gelatin of example one and PGANF + PEGDA of example two instead of the above HANF were similar to those shown in fig. 3, i.e., hemostasis was achieved after repeating the procedure 3 to 5 times, and no blood further flowed out within 15 minutes, indicating that the bioadhesive hydrogel of each example of the present invention was applicable to rapid hemostasis of the heart region. In addition, since the blood pressure of other parts is lower than that of the heart part, the requirement on the adhesion force of the wet-surface tissue is lower, and therefore, the bioadhesive hydrogel can be obviously used for rapid hemostasis of other parts.

< example ten >

This example is a tissue regeneration performance test of a bioadhesive hydrogel.

Fig. 4 is a schematic view and a photograph of an experimental process of tissue regeneration performance according to a tenth embodiment of the present invention. Wherein, fig. 4(a) is a schematic diagram of an experimental process, fig. 4(B) is a photograph of a corresponding experimental process, fig. 4(C) is a bar chart of a wound defect healing area result, and fig. 4(D) is a bar chart of a wound defect healing thickness result. In fig. 4(B), 4(C), and 4(D), Control is a Control group not treated with hydrogel, and FLS is an experimental group skin; the ordinate of fig. 4(C) is the defect healing area (%), the ordinate of fig. 4(D) is the defect healing thickness (μm), and Normal of fig. 4(D) is the skin thickness of the non-defective portion.

As shown in fig. 4, the specific process of the tissue regeneration performance test is as follows: is applied to the skin of pigsThe model of 4.5cm by 0.5cm massive skin lesion was cut with a scalpel, the wound was filled with the CSNF + Gelatin solution of example one, and then the band was 365nm with a light intensity of 60mW/cm²After 20 seconds of light irradiation, the wound surface was protected by gauze covering. After 30 days of operation, perfect wound regeneration was observed, and the regenerated skin pore structure was consistent with the surrounding undamaged skin with no significant scarring, as shown in FLS group in fig. 4 (B). The control group without gel treatment had significant scar tissue after 30 days and no pore structure on the newborn skin, as shown in the control group in fig. 4 (B). This result demonstrates the good tissue regeneration performance of CSNF + Gelatin.

In addition, the same tissue regeneration performance test was carried out by replacing the CSNF + Gelatin with 4PNF + PLS of example three and CTSNF of example six, and the results were similar to those shown in fig. 4, i.e., wound regeneration was promoted after 30 days of treatment, and the regenerated skin pore structure and the like were consistent with those of the peripheral non-damaged skin. Therefore, the bioadhesive hydrogel of the present invention has an effect of promoting skin tissue regeneration, and can be used for defective tissue repair.

< EXAMPLE eleven >

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

Fig. 5 is a photograph of a drug loading performance test of the eleventh embodiment of the present invention. In fig. 5, the inner part of the dotted line frame is the experimental part on the right side of the skull.

As shown in fig. 5, the specific process of the drug loading performance test is as follows: two round holes with the diameter of 10mm are respectively arranged at the left side and the right side of the skull of a rat, the growth factor BMP2 is uniformly mixed into the PGANF + Silk solution of the second embodiment, and then the mixture is injected into the defect hole at the left side of the skull, the wave band is 395nm, and the light intensity is 60mW/cm²The light irradiation was carried out for 15 seconds. The right defective well was used as a control group without further treatment. After operation, animals are sacrificed after four weeks of feeding, sampling analysis shows that the left skull defect part is perfectly regenerated, the boundary of injury regeneration can not be found, and no heterotopic ossification occurs, while the right defect hole control group only has a small amount of membranous tissues and no obvious bone regeneration is generated at the defect part, which proves that the bioadhesive hydrogel provided by the invention has the advantages of good biocompatibility, high stability, and the likeHas drug loading performance and site-specific release performance, and can be used as drug carrier.

< example twelve >

This example is a performance test of the application of bioadhesive hydrogels to bioprinting.

Fig. 6 is a schematic view of a bio-printing process and a print result photo according to a twelfth embodiment of the present invention. Fig. 6(a) is a schematic diagram of a printing process, fig. 6(B) is a photograph of a structure at a printing head, fig. 6(C) is a design diagram of a printing model, fig. 6(D) is an internal structure diagram of the printing model, and fig. 6(E) is a photograph of a printed product.

As shown in fig. 6, the specific process of applying the bioadhesive hydrogel to bioprinting is as follows: the CTSNF solution obtained in the sixth example is added into an ink box of a DLP biological 3D printer, a scaffold model with the aperture of 200 is selected, and the printing light intensity is adjusted to be 30mW/cm²The layer thickness was 10 μm and the exposure time was 1 second per layer. After the above printing process is completed, the scaffold support with full communication holes having a hole diameter of 200 μm can be obtained, as shown in fig. 6 (E). The experiment can prove that the curing time and the gel forming strength of the bioadhesive hydrogel can meet the performance requirements of bioprinting, and can be used as a bioprinting material.

The foregoing detailed description of the preferred embodiments of the invention has been presented. It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions available to those skilled in the art through logic analysis, reasoning and limited experiments based on the prior art according to the concept of the present invention should be within the scope of protection defined by the claims.