CN114702606A

CN114702606A - Zwitterion modified polysaccharide polymer and preparation method and application thereof

Info

Publication number: CN114702606A
Application number: CN202210153397.2A
Authority: CN
Inventors: 吴海伟; 庄泽鹏; 雷德勇; 丁蒙; 张磊; 郑立新; 周金生; 李珺
Original assignee: Shenzhen Huayuan Regeneration Medical Science Co ltd
Current assignee: Shenzhen Huayuan Regeneration Medical Science Co ltd
Priority date: 2022-02-18
Filing date: 2022-02-18
Publication date: 2022-07-05

Abstract

The invention discloses a zwitterionic modified polysaccharide polymer and a preparation method and application thereof. The polysaccharide polymer modified by the zwitterion contains a nitrogen-containing heterocyclic cationic group, and is prepared by grafting nitrogen-containing heterocyclic zwitterions onto the polysaccharide polymer through amidation reaction. The polysaccharide polymer modified by zwitterions has good biocompatibility and low foreign body reactivity, can be used as an implant material and stably exists in a human body for a long time, and has good application prospect.

Description

Zwitterion-modified polysaccharide polymer and preparation method and application thereof

Technical Field

The invention belongs to the technical field of high molecular materials, and particularly relates to a zwitterionic modified polysaccharide polymer, and a preparation method and application thereof.

Background

In the related art, the traditional implant materials such as alginate are widely used as implant materials for immune isolation tools due to their low immunogenicity, good biocompatibility, and controllable degradation. However, Foreign Body Reaction (FBR) is a complex process mainly resulting from Cell Overgrowth (CO) and fibrosis, involving protein adsorption, monocyte/granulocyte/macrophage adhesion, giant cell formation and crosstalk between macrophages/giant cells and other immune/fibroblast cells, and it is difficult to suppress the overgrowth (CO) and fibrosis of cells on an implanted device with only a single polymer material, such as alginate material, etc., and there occurs a phenomenon of reducing or even cutting off diffusion of nutrients and oxygen to encapsulated cells, thereby causing apoptosis/necrosis. Therefore, the conventional implant materials such as alginate have weak foreign body reaction inhibition performance, and there is a clinical demand for better implant materials.

Disclosure of Invention

The present invention is directed to solving at least one of the problems of the prior art described above. Therefore, the invention provides a zwitterion modified polysaccharide polymer which has the characteristic of stronger foreign body reaction inhibition performance.

The present invention also provides a method for producing the zwitterionic-modified polysaccharide polymer.

The invention also provides a hydrogel.

The invention also provides a biological material.

The invention also provides a medical product.

The invention also provides application of the zwitterion modified polysaccharide polymer.

In a first aspect of the present invention, there is provided a zwitterionic-modified polysaccharide polymer having a nitrogen-containing heterocyclic cationic group, the zwitterionic-modified polysaccharide polymer being produced by grafting a nitrogen-containing heterocyclic zwitterion onto a polysaccharide polymer through amidation.

Zwitterionic modified polysaccharide polymers according to embodiments of the invention have at least the following beneficial effects: the zwitterion-modified polysaccharide polymer can be strongly interacted with water molecules through ion solvation of nitrogen-containing heterocyclic cationic groups, can reduce nonspecific adsorption on protein and cells to the maximum extent, and can inhibit foreign body reaction and inflammation, and the effect is obviously superior to that of conventional implant materials, such as poly (2-hydroxyethyl methacrylate) (PHEMA), PEG and derivatives thereof, alginate and the like, and the foreign body reaction inhibition performance is strong.

In some embodiments of the invention, the nitrogen-containing heterocyclic cationic group comprises at least one of a nitrogen-containing five-membered ring cationic group, a nitrogen-containing six-membered ring cationic group, or a nitrogen-containing fused ring cationic group, and the nitrogen-containing heterocyclic zwitterion comprises a nitrogen-containing five-membered ring zwitterion, a nitrogen-containing six-membered ring zwitterion, or a nitrogen-containing fused ring zwitterion.

In some preferred embodiments of the invention, the nitrogen-containing heterocyclic cationic group comprises at least one of an imidazolium cationic group, a pyrrolidinium cationic group, a thiazolium cationic group, a pyridinium cationic group, a pyrazinium cationic group, a pyrimidinium cationic group, a pyridazinium cationic group, an indolium cationic group, a quinolinium cationic group, a pteridinium cationic group, an acridinium cationic group, a triazolium cationic group, a benzimidazolium cationic group, a benzothiazolium cationic group, a benzotriazole cationic group, a quinoxalinium cationic group, a quinazolinium cationic group, a benzopyridazinium cationic group, or a pyrrolopyrimidinium cationic group, the nitrogen-containing heterocyclic zwitterion comprises an imidazolium zwitterion, a pyrrolidinium zwitterion, a thiazolium zwitterion, a pyridinium zwitterion, a, At least one of a pyrazinium zwitterion, a pyrimidinium zwitterion, a pyridazinium zwitterion, an indolium zwitterion, a quinolinium zwitterion, a pteridinium zwitterion, an acridinium zwitterion, a triazolium zwitterion, a benzimidazolium zwitterion, a benzothiazolium zwitterion, a benzotriazolium zwitterion, a quinoxalinium zwitterion, a quinazolinium zwitterion, a benzpyridazinium zwitterion, or a pyrrolopyrimidinium zwitterion.

Through the implementation mode, the polysaccharide polymer modified by zwitterions contains heterocyclic onium cationic groups, positive charges are uniformly dispersed on five-membered rings, six-membered rings or multiple condensed rings through a p-pi orbital conjugation effect, and the high-stability onium zwitterions are formed through an effective energy dissipation mechanism, so that the fibrosis prevention and rejection prevention functions of the zwitterions cannot be lost due to the falling of the positive charges, and the zwitterions can be used as an implantation material to keep stability in a complex environment in vivo.

In some preferred embodiments of the present invention, the nitrogen-containing heterocyclic cationic group is an imidazolium cationic group and the nitrogen-containing heterocyclic zwitterion is an imidazolium zwitterion.

Through the above embodiment, the zwitterionic modified polysaccharide polymer in the invention can generate strong interaction with water molecules through ionic solvation of imidazolium cationic groups, can reduce nonspecific adsorption to proteins and cells to the maximum extent, and can inhibit foreign body reaction and inflammation, and the effect is obviously better than that of conventional implant materials, such as poly (2-hydroxyethyl methacrylate) (PHEMA), PEG and derivatives thereof, alginate and the like, and the foreign body reaction inhibition performance is strong.

Meanwhile, the polysaccharide polymer modified by the zwitterion contains an imidazolium cationic group (containing imidazole five-membered heterocycle), the positive charge is uniformly dispersed on the five-membered heterocycle through a p-pi orbital conjugation effect, and the high-stability imidazolium zwitterion is formed through an effective energy dissipation mechanism, so that the fibrosis prevention and rejection prevention functions of the zwitterion cannot be lost due to the falling of the positive charge, and the zwitterion can be used as an implant material to keep the stability in a complex environment in vivo. The imidazolium zwitterion is grafted to the polysaccharide polymer through amidation reaction to obtain a new material with the characteristics of good biocompatibility and low foreign body reactivity, and the new material can be used as an implant material and stably exist in a body for a long time and has a good application prospect.

In some preferred embodiments of the present invention, the imidazolium zwitterion contains an amino group, and the polysaccharide polymer contains a carboxyl group or a carboxylate group, and the carboxyl group or the carboxylate group undergoes an amidation reaction with the amino group.

In some more preferred embodiments of the present invention, the imidazolium zwitterion has an amino group, and the polysaccharide polymer has a carboxyl group or a carboxylate group in a structural unit thereof, and the carboxyl group or the carboxylate group undergoes an amidation reaction with the amino group.

In some preferred embodiments of the invention, the polysaccharide polymer comprises at least one of alginate, hyaluronic acid, carboxymethyl cellulose, carboxymethyl modified hemicellulose, carboxymethyl chitosan, carboxymethyl dextran, carboxymethyl carrageenan, carboxymethyl pachyman, carboxymethyl starch, pectin, or heparin.

In some more preferred embodiments of the invention, the alginate comprises sodium alginate.

Wherein, sodium alginate, abbreviated as: NaAlg.

In some preferred embodiments of the invention, the imidazolium zwitterion comprises at least one of a sulfonate betaine, a carboxylate betaine, or a phosphorylcholine.

In some more preferred embodiments of the invention, the imidazolium zwitterion comprises at least one of 1- (3-aminopropyl) imidazolium sulfobetaine, 1- (3-aminopropyl) imidazolium acetate betaine, or 1- (3-aminopropyl) imidazolium butyrate betaine.

In some preferred embodiments of the invention, the zwitterionic modified polysaccharide polymer comprises at least one of a sulfobetaine, a carboxybetaine, or a phosphorylcholine.

In some preferred embodiments of the invention, the zwitterionic modified polysaccharide polymer is a zwitterionic modified alginate.

In some preferred embodiments of the invention, the zwitterionic modified polysaccharide polymer comprises at least one of a polymer of formula (1), a polymer of formula (2) or a polymer of formula (3):

wherein, the polymer in the formula (1) is 1- (3-aminopropyl) imidazole sulfonic acid betaine modified sodium alginate, which is referred to as: SB-Alg; the polymer in the formula (2) is 1- (3-aminopropyl) imidazole acetic acid betaine modified sodium alginate, which is referred to as: CB (CB)₁-Alg; the polymer in the formula (3) is 1- (3-aminopropyl) imidazole butyric acid betaine modified sodium alginate, which is abbreviated as: CB (CB)₂-Alg。

In some embodiments of the invention, the zwitterionic modified polysaccharide polymer comprises a photosensitive group.

Through the embodiment, the polysaccharide polymer modified by the zwitterion comprises the photosensitive group, so that the polysaccharide polymer modified by the zwitterion can be subjected to photopolymerization or photocrosslinking reaction to obtain a corresponding biological material, and the polysaccharide polymer modified by the zwitterion can be better applied to the technical field of biological materials or medicines.

In some preferred embodiments of the invention, the zwitterionic modified polysaccharide polymer comprises at least one of a polymer of formula (4), a polymer of formula (5) or a polymer of formula (6):

the polymer in the formula (4) is methacrylated 1- (3-aminopropyl) imidazole sulfonic acid betaine modified sodium alginate, which is called SB-Alg-MA for short; the polymer in the formula (5) is methacrylated 1- (3-aminopropyl) imidazole acetic acid betaine modified sodium alginate, namely CB for short₁-Alg-MA; the polymer in the formula (6) is methacrylated 1- (3-aminopropyl) imidazole butyric acid betaine modified sodium alginate, namely CB for short₂-Alg-MA。

In a second aspect of the present invention, there is provided a method for producing a zwitterionic-modified polysaccharide polymer, comprising the steps of: the polysaccharide polymer modified by the zwitterion is prepared by grafting zwitterion containing nitrogen heterocycle on the polysaccharide polymer through amidation reaction, and the zwitterion modified polysaccharide polymer contains a nitrogen heterocycle cationic group.

In some embodiments of the invention, the nitrogen-containing heterocyclic cationic group is an imidazolium cationic group and the nitrogen-containing heterocyclic zwitterion is an imidazolium zwitterion, and the method of preparing the zwitterion-modified polysaccharide polymer comprises the steps of:

sa-1, mixing a sodium alginate solution I and an imidazolium zwitterion solution I, heating for reaction, and removing the solvent to obtain a mixture I;

sa-2, mixing the mixture I with water, adjusting the pH value to be neutral, dialyzing, precipitating and separating.

In some preferred embodiments of the present invention, in step Sa-1, 2-chloro-4, 6-dimethoxy-1, 3, 5-triazine and N-methylmorpholine are added to a mixed solution of sodium alginate, water and acetonitrile to obtain solution i of sodium alginate.

In some more preferred embodiments of the present invention, in step Sa-1, the molar ratio of sodium alginate, 2-chloro-4, 6-dimethoxy-1, 3, 5-triazine and N-methylmorpholine is (4.9-5.1): (2.4-2.6): (4.9-5.1).

In some more preferred embodiments of the present invention, in step Sa-1, the molar ratio of sodium alginate, 2-chloro-4, 6-dimethoxy-1, 3, 5-triazine and N-methylmorpholine is about 5.04: 2.56: 5.04.

in some preferred embodiments of the present invention, in step Sa-1, the molar ratio of sodium alginate to imidazolium zwitterion is 1: (1-2).

In some preferred embodiments of the present invention, in step Sa-1, the heating reaction is performed at 55-60 ℃ for 24 h.

In some preferred embodiments of the present invention, in step Sa-1, the solvent is removed by concentration under reduced pressure.

In some preferred embodiments of the present invention, in step Sa-2, the pH is adjusted to neutral using 1mol/L NaOH aqueous solution.

In some preferred embodiments of the present invention, in step Sa-2, the mixture i is mixed with water, the pH is adjusted to neutral, dialyzed against water, precipitated with methanol, and centrifuged to obtain the zwitterionic modified polysaccharide polymer.

In some more preferred embodiments of the present invention, in step Sa-2, after mixing mixture i with water, adjusting pH to neutral, dialyzing with water for 3d, precipitating with anhydrous methanol, and centrifuging, wherein the precipitating and centrifuging steps are repeated 3 times and vacuum drying is performed at about 40 ℃ for 48 h.

In some preferred embodiments of the present invention, in step Sa-2, the mixture i is mixed with water, pH is adjusted to neutral, dialyzed, separated by precipitation, subjected to nucleophilic substitution reaction with methacrylic anhydride, and dialyzed and purified to obtain the zwitterionic modified polysaccharide polymer;

in some more preferred embodiments of the present invention, in step Sa-2, the mixture i is mixed with water, pH is adjusted to neutral, dialyzed, separated by settling, subjected to nucleophilic substitution reaction of hydroxyl and anhydride with methacrylic anhydride, and dialyzed and purified to obtain the zwitterionic modified polysaccharide polymer.

In some more preferred embodiments of the present invention, in step Sa-2, the mixture i is mixed with water, pH is adjusted to neutral, dialyzed, separated by precipitation, subjected to nucleophilic substitution reaction of hydroxyl and anhydride with methacrylic anhydride in an alkaline environment, reacted at room temperature for 24 hours, and dialyzed and purified to obtain the zwitterionic modified polysaccharide polymer.

In some more preferred embodiments of the present invention, in step Sa-2, the mixture i is mixed with water, pH is adjusted to neutral, dialysis is performed, precipitation separation is performed to obtain a product i, hydroxyl groups of the product i react with methacrylic anhydride to perform nucleophilic substitution reaction, and then dialysis purification is performed to obtain the zwitterionic modified polysaccharide polymer.

In some embodiments of the invention, the preparation method comprises the following steps:

sb-1, mixing a sodium alginate solution II and an imidazolium zwitterion solution II, reacting, and performing coarse purification to obtain a mixture II;

and Sb-2, mixing the mixture II with water, adjusting the pH value to be neutral, dialyzing, precipitating and separating.

In some preferred embodiments of the present invention, in step Sb-1, a mixed solution of sodium alginate and MES buffer is activated to obtain a solution ii of sodium alginate.

In some preferred embodiments of the present invention, in step Sb-1, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide are added to a mixed solution of sodium alginate and MES buffer and activated to obtain a solution ii of said sodium alginate.

Among them, 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride, abbreviated as: EDC & HCl; n-hydroxysuccinimide, abbreviated as: NHS.

In some more preferred embodiments of the invention, the MES buffer has a solute concentration of 0.1mol/L and a pH of 6.

In some more preferred embodiments of the present invention, in step Sb-1, the molar ratio of sodium alginate, 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride and N-hydroxysuccinimide is 1: (0.9-1.1): (0.9-1.1).

In some preferred embodiments of the present invention, in step Sb-1, the molar ratio of sodium alginate to imidazolium zwitterion is 1: (1-2).

In some preferred embodiments of the present invention, in step Sb-1, the reaction condition is room temperature reaction for 24 hours.

In some preferred embodiments of the present invention, in step Sb-1, the crude purification is performed by using anhydrous methanol.

In some preferred embodiments of the present invention, in step Sb-2, the pH is adjusted to neutral using 1mol/L aqueous NaOH solution.

In some preferred embodiments of the present invention, in step Sb-2, the mixture ii is mixed with water, the pH is adjusted to neutral, dialyzed against water, and then precipitated with methanol and centrifuged to obtain the zwitterionic modified polysaccharide polymer.

In some more preferred embodiments of the present invention, in step Sb-2, dialyzing with water for 3d, precipitating with anhydrous methanol, and centrifuging, wherein the precipitating and centrifuging steps are repeated 3 times and vacuum drying is performed at about 40 ℃ for 48 hours.

In some preferred embodiments of the present invention, in step Sb-2, after mixing mixture ii with water, adjusting pH to neutral, dialyzing, precipitating, separating, performing nucleophilic substitution reaction with methacrylic anhydride, and further dialyzing and purifying to obtain the zwitterionic modified polysaccharide polymer.

In some more preferred embodiments of the present invention, in step Sb-2, the mixture ii is mixed with water, the pH is adjusted to neutral, dialyzed, separated by settling, subjected to nucleophilic substitution reaction of hydroxyl and anhydride with methacrylic anhydride, and dialyzed and purified to obtain the zwitterionic modified polysaccharide polymer.

In some more preferred embodiments of the present invention, in step Sb-2, after mixing mixture ii with water, adjusting pH to neutral, dialyzing, separating by precipitation, reacting with methacrylic anhydride in an alkaline environment for nucleophilic substitution of hydroxyl and anhydride, reacting at room temperature for 24 hours, and dialyzing for purification to obtain the zwitterionic modified polysaccharide polymer.

In some more preferred embodiments of the present invention, in step Sb-2, after mixing the mixture ii with water, adjusting pH to neutral, dialyzing, separating by precipitation to obtain a product ii, reacting hydroxyl groups of the product ii with methacrylic anhydride to perform nucleophilic substitution reaction, and dialyzing and purifying to obtain the zwitterionic modified polysaccharide polymer.

In some embodiments of the invention, the imidazolium zwitterion comprises 1- (3-aminopropyl) imidazolium sulfobetaine, and the method for preparing the 1- (3-aminopropyl) imidazolium sulfobetaine comprises the steps of:

s-1-1, reacting 1- [3- (N-Boc-amino) propyl ] imidazole with 1, 3-propane sultone to prepare 1- [3- (N-Boc-amino) propyl ] imidazole sulfonic acid betaine;

hydrolyzing the S-1-2, 1- [3- (N-Boc-amino) propyl ] imidazole sulfonic acid betaine to obtain the 1- (3-aminopropyl) imidazole sulfonic acid betaine.

1- [3- (N-Boc-amino) propyl ] imidazole, abbreviated: N-Boc-propylaminoimidazole.

1- [3- (N-Boc-amino) propyl ] imidazolium sulfonate betaine, abbreviated as: N-Boc-propylaminoimidazolidinesulfonic acid betaine.

Wherein the preparation of the 1- (3-aminopropyl) imidazole sulfonic acid betaine is shown as the following reaction formula:

in some preferred embodiments of the present invention, in step S-1-1, a tetrahydrofuran mixture of 1- [3- (N-Boc-amino) propyl ] imidazole and 3-propane sultone is heated to react to obtain a viscous liquid, which is washed with tetrahydrofuran and then filtered to obtain the 1- [3- (N-Boc-amino) propyl ] imidazole sulfonic acid betaine.

In some preferred embodiments of the present invention, in step S-1-1, the molar ratio of 1- [3- (N-Boc-amino) propyl ] imidazole to 3-propanesultone is (0.9-1.1): (0.9-1.1).

In some more preferred embodiments of the present invention, in step S-1-1, a tetrahydrofuran mixture of 1- [3- (N-Boc-amino) propyl ] imidazole and 3-propane sultone is stirred at about room temperature for 48 hours to obtain a white viscous liquid, which is washed with tetrahydrofuran, filtered, and vacuum-dried at about 40 ℃ for 48 hours to obtain the 1- [3- (N-Boc-amino) propyl ] imidazole sulfonic acid betaine.

In some preferred embodiments of the present invention, in step S-1-2, 1- [3- (N-Boc-amino) propyl ] imidazolium sulfonate betaine in methanol in hydrogen chloride is reacted at room temperature, neutralized with an ion exchange resin in acid and base, filtered, the solvent is removed, and washed.

In some more preferred embodiments of the present invention, in step S-1-2, 1- [3- (N-Boc-amino) propyl ] imidazolium sulfonate betaine in methanol in hydrogen chloride is reacted at room temperature, concentrated in vacuo to remove the solvent, redissolved in methanol and neutralized with an acid or base using an ion exchange resin, then filtered, concentrated in vacuo to remove the solvent, and washed with diethyl ether. Then concentrating and drying.

In some embodiments of the invention, the imidazolium zwitterion comprises 1- (3-aminopropyl) imidazolium acetate betaine, and the method for preparing the 1- (3-aminopropyl) imidazolium acetate betaine comprises the steps of:

s-2-1-1, reacting 1- [3- (N-Boc-amino) propyl ] imidazole with tert-butyl bromoacetate to prepare 1- [3- (N-Boc-amino) propyl ] imidazole acetic acid betaine I;

hydrolyzing S-2-1-2, 1- [3- (N-Boc-amino) propyl ] imidazole acetic acid betaine I to obtain 1- (3-aminopropyl) imidazole acetic acid betaine.

1- [3- (N-Boc-amino) propyl ] imidazoleacetic acid betaine I, abbreviated as: N-Boc-propylaminoimidazolidacetic acid betaine I.

Wherein the preparation of the 1- (3-aminopropyl) imidazole acetic acid betaine is shown as the following reaction formula:

in some preferred embodiments of the present invention, in step S-2-1-1, the 1- [3- (N-Boc-amino) propyl ] imidazole acetic acid betaine i is obtained by heating a mixture of 1- [3- (N-Boc-amino) propyl ] imidazole and tert-butyl bromoacetate in acetonitrile to react, concentrating the reaction solution, precipitating, washing and filtering.

In some preferred embodiments of the present invention, in step S-2-1-1, the molar ratio of 1- [3- (N-Boc-amino) propyl ] imidazole to tert-butyl bromoacetate is (0.9-1.1): (0.9-1.1).

In some more preferred embodiments of the present invention, in step S-2-1-1, a mixture of 1- [3- (N-Boc-amino) propyl ] imidazole and t-butyl bromoacetate in acetonitrile is reacted at about 40 ℃ with stirring for 2d, the reaction solution is concentrated, precipitated by adding anhydrous diethyl ether, washed, filtered, and dried under vacuum to obtain said betaine 1- [3- (N-Boc-amino) propyl ] imidazolium acetate.

In some preferred embodiments of the present invention, in step S-2-1-2, 1- [3- (N-Boc-amino) propyl ] imidazoleacetic acid betaine I in methanol in hydrogen chloride is reacted at room temperature, neutralized with an ion exchange resin in acid and base, filtered, the solvent is removed, and washed.

In some more preferred embodiments of the present invention, in step S-2-1-2, 1- [3- (N-Boc-amino) propyl ] imidazoleacetic acid betaine I in methanolic hydrogen chloride is reacted at room temperature, the solvent is removed by vacuum concentration, redissolved with methanol and neutralized with an acid or a base with an ion exchange resin, then filtered, the solvent is removed by vacuum concentration and washed with diethyl ether. Finally, the filtrate was concentrated and dried.

s-2-2-1, reacting 1- [3- (N-Boc-amino) propyl ] imidazole with ethyl bromoacetate to prepare 1- [3- (N-Boc-amino) propyl ] imidazole acetic acid betaine II;

hydrolyzing S-2-2-2, 1- [3- (N-Boc-amino) propyl ] imidazole acetic acid betaine II to obtain 1- (3-aminopropyl) imidazole acetic acid betaine.

1- [3- (N-Boc-amino) propyl ] imidazoleacetic acid betaine ii, abbreviated: N-Boc-propylaminoimidazoacetic acid betaine II.

in some preferred embodiments of the present invention, in step S-2-2-1, a mixture of 1- [3- (N-Boc-amino) propyl ] imidazole and ethyl bromoacetate in acetonitrile is heated for reaction, and the reaction solution is concentrated and washed to obtain betaine ii of 1- [3- (N-Boc-amino) propyl ] imidazole acetic acid.

In some preferred embodiments of the present invention, in step S-2-2-1, the molar ratio of 1- [3- (N-Boc-amino) propyl ] imidazole to ethyl bromoacetate is (1.5-1.7): (1.8-2.0).

In some preferred embodiments of the present invention, in step S-2-2-2, 1- [3- (N-Boc-amino) propyl ] imidazoleacetic acid betaine II in methanol in hydrogen chloride is reacted at room temperature, neutralized with an ion exchange resin in an acid base, filtered, the solvent is removed, and washed.

In some embodiments of the invention, the imidazolium zwitterion comprises 1- (3-aminopropyl) imidazolium butyrobetaine, and the method for preparing the 1- (3-aminopropyl) imidazolium butyrobetaine comprises the steps of:

s-3-1, reacting 1- [3- (N-Boc-amino) propyl ] imidazole with ethyl 4-bromobutyrate to prepare 1- [3- (N-Boc-amino) propyl ] imidazole butyric acid betaine;

hydrolyzing the S-3-2, 1- [3- (N-Boc-amino) propyl ] imidazole butyric acid betaine to obtain the 1- (3-aminopropyl) imidazole butyric acid betaine.

1- [3- (N-Boc-amino) propyl ] imidazole butyric acid betaine, abbreviated as: N-Boc-propylaminoimidazolam butanoic acid betaine.

Wherein the preparation of the 1- (3-aminopropyl) imidazole butyric acid betaine is shown as the following reaction formula:

in some preferred embodiments of the present invention, in step S-3-1, the 1- [3- (N-Boc-amino) propyl ] imidazole betaine is obtained by heating a mixture of 1- [3- (N-Boc-amino) propyl ] imidazole and ethyl 4-bromobutyrate in acetonitrile to react, concentrating the reaction solution, washing and concentrating.

In some preferred embodiments of the present invention, in step S-3-1, the molar ratio of 1- [3- (N-Boc-amino) propyl ] imidazole to ethyl 4-bromobutyrate is (0.9-1.1): (1-1.4).

In some more preferred embodiments of the present invention, in step S-3-1, a mixture of 1- [3- (N-Boc-amino) propyl ] imidazole and ethyl 4-bromobutyrate in acetonitrile is reacted at about 45 ℃ for 5d with stirring, the reaction solution is concentrated, washed with anhydrous diethyl ether and concentrated in vacuo to give said betaine of 1- [3- (N-Boc-amino) propyl ] imidazole butanoate.

In some preferred embodiments of the present invention, in step S-3-2, 1- [3- (N-Boc-amino) propyl ] imidazolium butanoate betaine in hydrogen chloride in methanol is reacted at room temperature, neutralized with an ion exchange resin in acid and base, filtered, the solvent is removed, and washed.

In some more preferred embodiments of the present invention, in step S-3-2, 1- [3- (N-Boc-amino) propyl ] imidazolium butanoate betaine in methanolic hydrogen chloride is reacted at room temperature, concentrated in vacuo to remove the solvent, redissolved with methanol and neutralized with an acid or base using an ion exchange resin, then filtered, concentrated in vacuo to remove the solvent, and washed with diethyl ether. Finally, the filtrate was concentrated and dried.

In a third aspect of the present invention, there is provided a hydrogel, wherein a raw material of the hydrogel comprises the above zwitterionic-modified polysaccharide polymer having a photosensitive group, and the hydrogel is obtained by a photocrosslinking reaction of the zwitterionic-modified polysaccharide polymer.

In some embodiments of the present invention, the raw material of the hydrogel further includes PEGDA, and the zwitterionic modified polysaccharide polymer and the PEGDA are subjected to a photocrosslinking reaction to obtain the hydrogel.

In a fourth aspect of the present invention, a biomaterial is provided, wherein the raw material of the biomaterial comprises the above zwitterionic modified polysaccharide polymer.

In some embodiments of the invention, the method of preparing the biomaterial comprises the steps of: the polysaccharide polymer modified by the zwitterion containing double bonds is prepared by self-photocrosslinking or photocrosslinking with PEGDA.

In some embodiments of the invention, the biomaterial is an implant material, the starting material of which comprises the above-described zwitterionic modified polysaccharide polymer.

In a fifth aspect of the present invention, there is provided a medical product comprising at least one of the above-mentioned zwitterionic modified polysaccharide polymer or the above-mentioned hydrogel.

In some embodiments of the invention, the medical product comprises at least one of a medical device or a drug.

In a sixth aspect of the present invention, there is provided use of the above zwitterionic modified polysaccharide polymer or hydrogel in the production of a biomaterial or a medical product.

In some embodiments of the present invention, the use of the above zwitterionic modified polysaccharide polymer or hydrogel in coatings for medical devices, drug encapsulation or bio-inks is proposed.

The zwitterion-modified polysaccharide polymer is prepared by grafting nitrogen-containing heterocyclic zwitterions onto the polysaccharide polymer through amidation reaction, can generate strong interaction with water molecules through ion solvation of nitrogen-containing heterocyclic cationic groups, can reduce non-specific adsorption on proteins and cells to the maximum extent, and inhibit foreign body reaction and inflammation, has obviously better effect than that of a conventional implant material, and has strong foreign body reaction inhibition performance.

Further, the polysaccharide polymer modified by zwitterions contains heterocyclic onium cationic groups, such as nitrogen-containing five-membered ring cationic groups, nitrogen-containing six-membered ring cationic groups or nitrogen-containing fused ring cationic groups (specifically, imidazolium cationic groups), positive charges are uniformly dispersed on five-membered rings, six-membered rings or multiple fused rings through a p-pi orbital conjugation effect, and the high-stability onium zwitterions are formed through an effective energy dissipation mechanism, so that the positive charges cannot fall off to cause the loss of the anti-fibrosis and anti-rejection functions of the zwitterions, and can keep stability in a complex environment in vivo as an implant material.

Furthermore, the polysaccharide polymer modified by zwitterions comprises photosensitive groups, so that the polysaccharide polymer modified by zwitterions can be subjected to photopolymerization or photocrosslinking reaction to obtain a corresponding biological material, and the polysaccharide polymer modified by zwitterions can be better applied to the technical field of biological materials or medicines.

The raw material of the biomaterial provided by the invention comprises the zwitterion modified polysaccharide polymer. The preparation method comprises the following steps: the polysaccharide polymer modified by the zwitterion containing double bonds is prepared by self-photocrosslinking or photocrosslinking with PEGDA. Thereby better applying the polysaccharide polymer modified by zwitterion to the technical field of biological materials.

Drawings

The invention is further described with reference to the following figures and examples, in which:

FIG. 1 is a schematic representation of N-Boc-propylaminoimidazole in example 1 of this invention¹HNMR spectrogram;

FIG. 2 is a graphic representation of betaine N-Boc-propylaminoimidazolidesulfonate of example 1 of the invention¹HNMR spectrogram;

FIG. 3 is a schematic representation of 1- (3-aminopropyl) imidazolium sulfonate betaine in example 1 of the present invention¹HNMR spectrogram;

FIG. 4 is a MS spectrum of betaine 1- (3-aminopropyl) imidazolium sulfonate in example 1 of the present invention;

FIG. 5 shows an embodiment of the present inventionOf SB-Alg in 1¹HNMR spectrogram;

FIG. 6 is a graphic representation of betaine I N-Boc-propylaminoimidazoacetic acid of example 2 of the present invention¹HNMR spectrogram;

FIG. 7 is a drawing showing the preparation of 1- (3-aminopropyl) imidazoleacetic acid betaine in example 2 of the present invention¹HNMR spectrogram;

FIG. 8 is a MS spectrum of 1- (3-aminopropyl) imidazoleacetic acid betaine in example 2 of the present invention;

FIG. 9 shows CB in embodiment 2 of the present invention₁of-Alg¹HNMR spectrogram;

FIG. 10 is a graph of N-Boc-propylaminoimidazoacetic acid betaine II in example 3 of the present invention¹HNMR spectrogram;

FIG. 11 is a graphic representation of N-Boc-propylamine imidazobutyric acid betaine in example 4 of the present invention¹HNMR spectrogram;

FIG. 12 is a graph showing betaine 1- (3-aminopropyl) imidazole butyrate in example 4 of the present invention¹HNMR spectrogram;

FIG. 13 is a MS spectrum of 1- (3-aminopropyl) imidazolium butanoate betaine in example 4 of this invention;

FIG. 14 shows CB in embodiment 4 of the invention₂of-Alg¹HNMR spectrogram;

FIG. 15 is a drawing of NaAlg used in the examples of the present invention¹HNMR spectrogram;

FIG. 16 is a graph showing a BSA standard curve measured in an experimental example of the present invention;

FIG. 17 shows SB-Alg, CB in the test example of the present invention₁-Alg,CB₂-graph of results of testing the adsorption content of Alg protein;

FIG. 18 shows SB-Alg, CB in the present invention₁-Alg,CB₂-Alg,SB-Alg-MA,CB₁-Alg-MA cytotoxicity test results plot;

FIG. 19 shows SB-Alg-MA in example 5 of the present invention¹HNMR spectrogram;

FIG. 20 shows CB in example 6 of the present invention₁of-Alg-MA¹HNMR spectrogram;

FIG. 21 is a diagram of SB-Alg-MA self-crosslinking hydrogel/CB prepared in examples 5 and 6 of the present invention₁-Alg-MA self-crosslinking hydrogel/SB-Alg-MA-PEGDA hydrogelglue/CB₁-Alg-MA-PEGDA hydrogel pattern.

Detailed Description

The concept and technical effects of the present invention will be clearly and completely described below in conjunction with the embodiments to fully understand the objects, features and effects of the present invention. It is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments, and those skilled in the art can obtain other embodiments without inventive effort based on the embodiments of the present invention, and all embodiments are within the protection scope of the present invention.

The invention synthesizes 1- [3- (N-Boc-amino) propyl ] imidazole (namely, N-Boc-propylamine imidazole), which comprises the following steps:

di-tert-butyl dicarbonate (10.9g, 50mmol) is dissolved in 50mL of tetrahydrofuran and cooled to 0 ℃. 1- (3-aminopropyl) imidazole (5.75g, 46mmol) was then dissolved in 20mL of 1mol/mL NaOH solution and slowly added dropwise to the solution at 0 ℃ and, after addition was complete, allowed to return to room temperature and the solution was stirred for 15 h. Finally, the solvent is removed by evaporation and concentration to obtain an oily crude product, the oily crude product is dissolved in ethyl acetate again, and the oily crude product is washed with deionized water for 3 to 5 times. Reuse of the organic phase with anhydrous MgSO₄Drying for 2-3h, and filtering to remove MgSO₄The hydrate crystallized and was finally concentrated to remove ethyl acetate to give a pale yellow viscous liquid with a yield of about 75%. The reaction of this step is as follows:

it is composed of¹The HNMR spectrum is shown in FIG. 1, and the structure is analyzed as follows:

¹HNMR(400MHz,CDCl₃),δ(ppm)：

7.49(s,1H),7.05(s,1H),6.94(s,1H),4.86(bs,NH),3.98-4.01(t,2H),3.11-3.16(m,2H),1.94-1.99(q,2H),1.45(s,9H)。

synthetic methods reference: weiss E, Gertopski D, Gupta M K, et al.encapsulation of ionic liquid BMIm [ PF6] with polymeric microorganisms [ J ]. Reactive & Functional Polymers,2015,96(NOV.) 32-38.

Example 1

This example discloses a zwitterionic modified polysaccharide polymer, which is a polymer of formula (1), and the preparation process includes:

preparation of N-Boc-propylamine imidazole sulfonic acid betaine:

N-Boc-propylamine imidazole (5.4g, 24mmol) and 1, 3-propanesultone (2.93g, 24mmol) were weighed out and dissolved in 35mL of tetrahydrofuran, and the reaction was stirred at room temperature for 48 h. After 48h, a white viscous liquid appeared at the bottom. Finally, washing with tetrahydrofuran was carried out 3-5 times, filtration was carried out, and finally vacuum drying at 40 ℃ for 48h gave a pure white powder product with a yield of about 97.7%. The reaction of this step is as follows:

it is composed of¹The HNMR spectrum is shown in FIG. 2, and the structure is analyzed as follows:

¹HNMR(400MHz,D₂O),δ(ppm)：

8.87(s,1H),7.55-7.60(d,2H),4.38-4.41(t,2H),4.26-4.29(t,2H),3.12-3.16(t,2H),2.93-2.97(t,2H),2.35-2.39(m,2H),2.06-2.11(m,2H),1.43(s,9H)。

(II) 1- (3-aminopropyl) imidazosulfonic acid betaine (abbreviation: SB-NH)₂) The preparation of (1):

5g N-Boc-propylamine imidazolesulfonic acid betaine was dissolved in 50mL of 1.25mol/L HCl in methanol. The solution was then stirred at room temperature overnight. Finally, the solvent is removed by vacuum concentration, redissolved with methanol and then ion-exchange resin (OH)^-) Neutralizing with acid and alkali, filtering, vacuum concentrating to remove methanol solvent, and washing with diethyl ether. Finally, the ether is poured out, and the purified viscous product is concentrated and dried. The reaction of the step is shown as follows, wherein a sulfonic group is introduced, the sulfonic group has excellent biocompatibility, and an amino group is introduced to provide a reactive group for the next amidation reaction:

it is composed of¹The HNMR spectrum is shown in FIG. 3, and the structure is analyzed as follows:

¹HNMR(400MHz,D₂O),δ(ppm)：

8.89(s,1H),7.56-7.59(d,2H),4.32-4.39(m,4H),3.04-3.08(t,2H),2.90-2.94(t,2H),2.24-2.35(m,2H)。

the MS spectrum is shown in FIG. 4, and the analysis is as follows:

[M+H]＝248，[2M+H]495, this and M_(SB-NH2)The theoretical value of 247 corresponds to.

(III) Synthesis of Polymer of formula (1), 1- (3-aminopropyl) Imidazosulfonic acid betaine-modified sodium alginate (SB-Alg):

1g (5.04mmol) of sodium alginate was dissolved in a mixed solvent of 80mL of deionized water and 20mL of acetonitrile. 450mg (2.56mmol) of 2-chloro-4, 6-dimethoxy-1, 3, 5-triazine and 560. mu.L (5.04mmol) of N-methylmorpholine are then added. 1.254g (5.04mmol) of 1- (3-aminopropyl) imidazolium sulfonate betaine was then dissolved in 20mL deionized water and added to the above mixed solution. Reacting for 24 hours at 55-60 ℃. Finally, the solvent was removed by concentration under reduced pressure and redissolved in deionized water and the pH was adjusted to neutral with 1mol/L NaOH solution. Then dialyzing with deionized water for 3d, precipitating with anhydrous methanol, centrifuging, repeating for 3 times, and vacuum drying at 40 deg.C for 48 h.

The reaction of this step is as follows:

wherein, is composed of SB-NH₂The SB-Alg can be prepared by the following steps:

1g (5.04mmol) of sodium alginate was dissolved in 100mL of a 0.1mol/L MES buffer solution at pH 6.0. Then, 0.97g (5.04mmol) of 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride (EDC. HCl) and 0.582g (5.04mmol) of N-hydroxysuccinimide (NHS) were added, and activation was performed for 15min, followed by dissolving 1.254g (5.04mmol) of 1- (3-aminopropyl) imidazolesulfonic acid betaine in 6mL of MES buffer solution having pH of 6.0, 0.1mol/L, and adding to the above mixed solution. The reaction was carried out at room temperature for 24 h. Then, the crude product was purified with anhydrous methanol. Finally, separating, dissolving again by using deionized water, adjusting to be neutral by using 1mol/L NaOH solution, dialyzing for 3d, precipitating by using anhydrous methanol, centrifuging, repeating for 3 times, and drying for 48 hours in vacuum at 40 ℃.

It is provided with¹The HNMR spectrum is shown in FIG. 5, and the structure is analyzed as follows:

¹HNMR(400MHz,D₂O),δ(ppm)：

8.91(s,1H),7.58-7.60(d,2H),4.35-4.40(t,2H),4.26(t,2H), 3.78-4.04(m, H on NaAlg), 3.35(t,2H),3.05-3.09(t,2H),2.92-2.97(t,2H),2.28-2.36(t, 2H).

This example provides a biomaterial comprising the zwitterionic modified polysaccharide polymer prepared in this example.

This example provides a medical device comprising the zwitterionic modified polysaccharide polymer prepared in this example.

This example provides a medicament comprising the zwitterionic modified polysaccharide polymer prepared in this example.

Example 2

This example discloses a zwitterionic modified polysaccharide polymer of formula (2), prepared by a process comprising:

preparation of N-Boc-propylamine imidazole acetic acid betaine I:

N-Boc-propylamine imidazole (4.5g, 20mmol) and tert-butyl bromoacetate (3.90g, 20mmol) were weighed out and dissolved in 75mL of acetonitrile, and the reaction was stirred at 40 ℃ for 2 d. Finally, after concentrating most of the solvent, adding anhydrous ether for precipitation and washing for 3-5 times, sequentially filtering and drying in vacuum to obtain a white powder product with the yield of about 82%. The reaction of this step is as follows:

it is composed of¹The HNMR spectrum is shown in FIG. 6, and the structure is analyzed as follows:

¹HNMR(400MHz,D₂O),δ(ppm)：

8.92(s,1H),7.56-7.62(d,2H),5.10(s,2H),4.31-4.35(t,2H),3.14-3.16(t,2H),2.09-2.16(m,2H),1.45-1.51(d,18H)。

(II) 1- (3-aminopropyl) imidazoleacetic acid betaine (abbreviation: CB)₁-NH₂) The preparation of (1):

5g N-Boc-propylaminoimidazolidacetic acid betaine I was dissolved in 100mL of 1.25mol/L HCl in methanol. The solution was then stirred at room temperature overnight. Then, the solvent was removed by vacuum concentration, redissolved with methanol and then ion-exchanged with ion-exchange resin (OH)^-) Neutralizing with acid and alkali, filtering, vacuum concentrating to remove solvent, and washing with diethyl ether. Finally, the filtrate was concentrated and dried. The reaction of this step is shown below:

it is provided with¹The HNMR spectrum is shown in FIG. 7, and the structure is analyzed as follows:

¹HNMR(400MHz,D₂O),δ(ppm)：

8.93(s,1H),7.56-7.63(d,2H),4.97(s,2H),4.40-4.44(t,2H),3.11-3.15(t,2H),2.32-2.39(m,2H).

the MS spectrum is shown in FIG. 8, and the analysis is as follows:

[M+H]＝184，[2M+H]＝367，[3M+H]550, this sum M_(CB1-NH2)The theoretical value of 183 corresponds.

To sum up, CB₁-NH₂The synthesis of (2) was successful.

(III) Synthesis of Polymer of formula (2), 1- (3-aminopropyl) Imidazoacetic acid betaine modified sodium alginate (CB)₁-Alg)：

1g (5.04mmol) of sodium alginate was dissolved in a mixed solvent of 80mL of deionized water and 20mL of acetonitrile. 450mg (2.56mmol) of 2-chloro-4, 6-dimethoxy-1, 3, 5-triazine and 560. mu.L (5.04mmol) of N-methylmorpholine are then added. Then 0.924g (5.04mmol) of 1- (3-aminopropyl) imidazoleacetic acid betaine was dissolved in 20mL of deionized water and added to the above mixed solution. Reacting for 24 hours at 55-60 ℃. Finally, the solvent was removed by concentration under reduced pressure and redissolved in deionized water and the pH was adjusted to neutral with 1mol/L NaOH solution. Finally dialyzing with deionized water for 3d, precipitating with anhydrous methanol, centrifuging, repeating for 3 times, and vacuum drying at 40 deg.C for 48 h.

The reaction of the step is as follows, carboxyl participates in ion crosslinking, exogenous carboxyl is introduced, and the influence of the reduction of the ion crosslinking strength after the carboxyl of the sodium alginate is modified is reduced:

wherein, from CB₁-NH₂Preparation of CB₁-Alg, also obtainable by the following steps:

1g (5.04mmol) of sodium alginate was dissolved in 100mL of a 0.1mol/L MES buffer solution at pH 6.0. Then, 0.97g (5.04mmol) of 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride (EDC. HCl) and 0.582g (5.04mmol) of N-hydroxysuccinimide (NHS) were added, and activation was performed for 15min, followed by dissolving 0.924g (5.04mmol) of 1- (3-aminopropyl) imidazoleacetic acid betaine in 6mL of MES buffer solution having a pH of 6.0 and 0.1mol/L, and adding to the above mixed solution. The reaction was carried out at room temperature for 24 h. Then, the crude product was purified with anhydrous methanol. Finally, separating, dissolving again by using deionized water, adjusting to be neutral by using 1mol/L NaOH solution, dialyzing for 3d, precipitating by using anhydrous methanol, centrifuging, repeating for 3 times, and drying for 48h in vacuum at 40 ℃.

It is composed of¹The HNMR spectrum is shown in FIG. 9, and the structure is analyzed as follows:

¹HNMR(400MHz,D₂O),δ(ppm)：

8.82(s,1H),7.48-7.56(d,2H),4.84(s,2H),4.47(t,2H), 3.76-4.29 (m, H on NaAlg), 3.33(m,2H),2.17(t, 2H).

Example 3

This example discloses a zwitterionic modified polysaccharide polymer of formula (2), which is prepared according to the process described in example 2, wherein:

preparation of N-Boc-propylamine imidazole acetic acid betaine II:

N-Boc-propylamine imidazole (3.6g, 16mmol) and ethyl bromoacetate (3.206g, 19.2mmol) were weighed out and dissolved in 12mL of acetonitrile, and the reaction was stirred at 45 ℃ for 24 h. After 24h most of the solvent was removed by rotary evaporator, the crude product was washed 4 times with anhydrous ether and finally concentrated to remove all solvent to give a yellow oily liquid with a yield of about 85%. The reaction of this step is shown below:

it is composed of¹The HNMR spectrum is shown in FIG. 10, and the structure is analyzed as follows:

¹HNMR(400MHz,D₂O),δ(ppm)：

8.95(s,1H),7.59-7.64(d,2H),5.21(s,2H),4.31-4.36(m,4H),3.15-3.18(t,2H),2.10-2.17(m,2H),1.46(s,9H),1.31-1.34(t,3H)。

5g N-Boc-propylaminoimidazolidacetic acid betaine II was dissolved in 100mL of 1.25mol/L HCl in methanol. The solution was then stirred at room temperature overnight. Vacuum concentrating to remove solvent, dissolving with methanol, and purifying with ion exchange resin (OH)^-) Neutralizing with acid and alkali, filtering, vacuum concentrating to remove solvent, and washing with diethyl ether. Finally, the filtrate was concentrated and dried. The reaction of this step is shown below:

it is composed of¹Of HNMR and MS spectraThe test results are substantially the same as those in example 2, indicating that this example also successfully produces 1- (3-aminopropyl) imidazoleacetic acid betaine (CB)₁-NH₂)。

The results of the various experimental tests on the zwitterionic modified polysaccharide polymers of examples 3 and 2 are comparable.

Example 4

This example discloses a zwitterionic modified polysaccharide polymer, which is a polymer of formula (3), and the preparation process includes:

preparation of N-Boc-propylamine imidazole butyric acid betaine:

N-Boc-propylamine imidazole (4.5g, 20mmol) and ethyl 4-bromobutyrate (3.74g, 24mmol) were weighed out and dissolved in 20mL of acetonitrile, and the reaction was stirred at 45 ℃ for 5 d. Then, the reaction solution was concentrated to remove the solvent, and then, anhydrous ether was added thereto to wash for 3 to 5 times, and finally, vacuum concentration was performed to obtain a yellow viscous liquid with a yield of about 85%. The reaction of this step is shown below:

it is composed of¹The HNMR spectrum is shown in FIG. 11, and the structure is analyzed as follows:

¹HNMR(400MHz,D₂O),δ(ppm)：

8.88(s,1H),7.58-7.60(d,2H),4.28-4.33(m,4H),4.14-4.20(q,2H),3.14-3.17(t,2H),2.48-2.51(t,2H),2.21-2.56(m,2H),2.09-2.15(m,2H),1.47(s,9H),1.26-1.30(t,3H)。

(II) 1- (3-aminopropyl) imidazole butyric acid betaine (abbreviation: CB)₂-NH₂) The preparation of (1):

5g N-Boc-propylamine imidazole butyric acid betaine was dissolved in 100mL of 1.25mol/L HCl in methanol, and the solution was stirred at room temperature overnight. Then, the solvent was removed by vacuum concentration, redissolved with methanol and then ion-exchanged with ion-exchange resin (OH)^-) Acid and base neutralization, filtering, vacuum concentration to remove solvent, and washing with ether. Finally, the filtrate was concentrated and dried. The reaction of this step is shown below：

It is composed of¹The HNMR spectrum is shown in FIG. 12, and the structure is analyzed as follows:

¹HNMR(400MHz,D₂O)，δ(ppm)：

8.88(s,1H),7.55-7.56(d,2H),4.32-4.37(t,2H),4.21-4.25(t,2H),3.05-3.09(t,2H),2.23-2.35(m,2H),2.20-2.22(t,2H),2.10-2.15(m,2H).

the MS spectrum is shown in FIG. 13, and is analyzed as follows:

[M+H]＝212，[2M+H]413, this sum M_(CB2-NH2)The theoretical value of 211 corresponds.

To sum up, CB₂-NH₂The synthesis of (2) was successful.

(III) Synthesis of Polymer of formula (3), 1- (3-aminopropyl) Imidazobutyric acid betaine modified sodium alginate (CB)₂-Alg)：

1g (5.04mmol) of sodium alginate was dissolved in a mixed solvent of 80mL of deionized water and 20mL of acetonitrile. 450mg (2.56mmol) of 2-chloro-4, 6-dimethoxy-1, 3, 5-triazine and 560. mu.L (5.04mmol) of N-methylmorpholine are then added. 1.066g (5.04mmol) of 1- (3-aminopropyl) imidazole butyric acid betaine was dissolved in 20mL of deionized water and added to the above mixed solution, and reacted at 55-60 ℃ for 24 hours. Then, the solvent was removed by concentration under reduced pressure and redissolved in deionized water, and the pH was adjusted to neutral with 1mol/L NaOH solution. Finally dialyzing with deionized water for 3d, precipitating with anhydrous methanol, centrifuging, repeating for 3 times, and vacuum drying at 40 deg.C for 48 h.

wherein, from CB₂-NH₂Preparation of CB₂-Alg, also obtainable by the following steps:

1g (5.04mmol) of sodium alginate was dissolved in 100mL of a 0.1mol/L MES buffer solution at pH 6.0. Then, 0.97g (5.04mmol) of 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride (EDC. HCl) and 0.582g (5.04mmol) of N-hydroxysuccinimide (NHS) were added thereto, and activation was carried out for 15min, and then 1.066g (5.04mmol) of 1- (3-aminopropyl) imidazolium butanoate betaine was dissolved in 6mL of a MES buffer solution having a pH of 6.0 and 0.1mol/L and added to the above mixed solution, and reacted at room temperature for 24h, followed by crude purification with anhydrous methanol. Then, the separation is carried out, the deionized water is used for dissolving again, 1mol/L NaOH solution is used for adjusting to be neutral, the dialysis is carried out for 3d, finally, absolute methanol is used for settling out, the centrifugation is carried out, the 3 times of the three steps are carried out, and the vacuum drying is carried out for 48h at the temperature of 40 ℃.

CB₂of-Alg¹The HNMR spectrum is shown in FIG. 14, and the structure is analyzed as follows:

¹HNMR(400MHz,D₂O),δ(ppm)：

8.90(s,1H),7.58(d,2H),4.32-4.38(t,4H), 3.74-4.01 (m, H on NaAlg), 3.04-3.08(t,2H),2.91-2.95(m,2H),2.26-2.36(m, 4H).

Example 5

This example discloses a zwitterionic modified polysaccharide polymer of formula (4): methacryloylated 1- (3-aminopropyl) imidazolium sulfonate betaine modified sodium alginate (SB-Alg-MA), which was prepared from the starting material comprising SB-Alg prepared in example 1, the procedure for preparation of SB-Alg-MA comprising:

2.5g (12.6mmol) of SB-Alg are dissolved in 125ml of deionized water, 11.28ml (75.6mmol) of methacrylic anhydride are subsequently added dropwise to the solution and the pH is raised to 8 by adding a small amount of 5mol/L sodium hydroxide (NaOH) solution. The mixture was reacted at room temperature for 24 hours. Thereafter, dialysis was performed for 3d with deionized water to remove unreacted methacrylic anhydride and formed methacrylic acid, and finally freeze-drying was performed.

Of SB-Alg-MA¹The HNMR spectrum is shown in FIG. 19, and the structure is analyzed as follows:

¹HNMR(400MHz,D₂O),δ(ppm)：

8.87(s,1H),7.57(d,2H),6.22(s,1H),5.79(s,1H),4.36-4.30(t,2H),4.26(t,2H),3.93-4.05(m, H on NaAlg), 3.31(t,2H),2.93-2.97(m,2H),2.32-2.35(t,2H),2.16(t,2H),1.95(t, 2H).

This example provides a biomaterial, wherein the raw material for preparation comprises SB-Alg-MA prepared in this example, and the process for preparation comprises:

preparing 0.2% blue light initiator LAP solution, then weighing 25mg of SB-Alg-MA to dissolve to 1.25mL blue light initiator LAP solution, and finally crosslinking and curing for 1-2min under the action of blue light to obtain SB-Alg-MA self-crosslinking hydrogel as shown in figure 21 (a);

preparing 0.2% blue light initiator LAP solution, then respectively weighing 25mg of SB-Alg-MA and 12.5mg of polyethylene glycol diacrylate PEGDA, dissolving into 1.25mL of blue light initiator LAP solution, and finally crosslinking and curing for 1-2min under the action of blue light to obtain SB-Alg-MA-PEGDA hydrogel, as shown in FIG. 21 (c).

Example 6

This example discloses a zwitterionic modified polysaccharide polymer of formula (5): methacryloylated 1- (3-aminopropyl) imidazoleacetic acid betaine modified sodium alginate (CB)₁-Alg-MA) prepared from the raw material comprising CB prepared in example 2₁-Alg，CB₁-the preparation process of Alg-MA comprises:

2.5g (12.6mmol) of betaine acetate-sodium alginate are dissolved in 125ml of deionized water, 11.28ml (75.6mmol) of methacrylic anhydride are added dropwise to the solution and the pH is raised to 8 by adding a small amount of 5mol/L sodium hydroxide (NaOH) solution. The mixture was reacted at room temperature for 24 hours. Thereafter, dialysis was performed for 3d with deionized water to remove unreacted methacrylic anhydride and formed methacrylic acid, and finally freeze-drying was performed.

CB₁of-Alg-MA¹The HNMR spectrum is shown in fig. 20, and the structure is resolved as follows:

¹HNMR(400MHz,D₂O),δ(ppm)：

8.82(s,1H),7.48-7.56(d,2H),6.23(s,1H),5.78(s,1H),4.84(s,2H),4.29(t,2H),3.78-4.04(m, H on NaAlg), 3.33-3.34(t,2H),2.15-2.18(t,3H),1.92-1.95(m, 2H).

This example provides a biomaterial prepared from the raw materials including the CB prepared in this example₁-Alg-MA, the preparation process of the biomaterial comprising:

preparing 0.2% blue light initiator LAP solution, and weighing 25mg CB₁dissolving-Alg-MA to 1.25mL of blue light initiator LAP solution, and finally crosslinking and curing for 1-2min under the action of blue light to obtain CB₁-Alg-MA self-crosslinking hydrogel, as shown in FIG. 21 (b).

Preparing 0.2% blue light initiator LAP solution, and respectively weighing 25mg CB₁dissolving-Alg-MA and 12.5mg of polyethylene glycol diacrylate PEGDA into 1.25mL of blue light initiator LAP solution, and finally performing crosslinking curing for 1-2min under the action of blue light to obtain CB₁-Alg-MA-PEGDA hydrogel, as shown in FIG. 21 (d).

Test examples

In this test example, the zwitterionic modified polysaccharide polymer and sodium alginate obtained in examples 1, 2 and 4 were tested for their performance, specifically:

in vitro anti-protein adhesion test of betaine modified alginate

1. Determination of BSA Standard Curve

(1) Preparation of protein standards

a. Adding 0.8mL of protein standard preparation solution into a tube of protein standard (20mg BSA), and fully dissolving to prepare 25mg/mL of protein standard solution;

b. the protein standard was diluted to 0.5mg/mL with 0.9% NaCl.

(2) Preparation of BCA working solution

BCA working solution was prepared by adding 50 volumes of BCA reagent A to 1 volume of BCA reagent B (50:1), and mixed well.

(3) BSA standard curve

a. Adding the standard substance into standard substance wells of a 96-well plate according to the proportion of 0. mu.L, 1. mu.L, 2. mu.L, 4. mu.L, 8. mu.L, 12. mu.L, 16. mu.L and 20. mu.L, and adding 0.9% NaCl to make up to 20. mu.L, wherein the concentrations of the standard substance are respectively 0mg/mL, 0.025mg/mL, 0.05mg/mL, 0.1mg/mL, 0.2mg/mL, 0.3mg/mL, 0.4mg/mL and 0.5 mg/mL;

b. adding 200 mu L of BCA working solution into each hole, and standing at 37 ℃ for 20-30 minutes;

c. the absorbance at 562nm was measured with a microplate reader.

The results of the BSA absorbance test at different concentrations are shown in Table 1, and the obtained BSA standard curve is shown in FIG. 16.

TABLE 1 BSA Absorbance test results at different concentrations

2、SB-Alg，CB₁-Alg，CB₂Detection of protein adsorption content of Alg

(1) Respectively preparing NaAlg, SB-Alg and CB with the concentration of 2% by using 0.9% NaCl₁-Alg、CB₂-an Alg solution;

(2) prepare 20mmol/L BaCl₂A solution;

(3) according to V_{Betaine modified sodium alginate}：V_{Sodium alginate}6: 4 preparing SB-Alg + NaAlg and CB respectively₁-Alg+NaAlg、CB₂-Alg + NaAlg solution;

(4) add 400. mu.L of SB-Alg + NaAlg, CB to 96-well plates, respectively₁-Alg+NaAlg、CB₂Alg + NaAlg solutions, 400. mu.L of BaCl each being then added₂A solution;

(5) curing for 1h to form a gel having a size of about 10mm by 4 mm;

(6) mixing SB-Alg + NaAlg, CB₁-Alg+NaAlg、CB₂-Alg + NaAlg gels were transferred to small petri dishes and washed 5 times with PBS;

(7) adding 800 mu L of 1.5mg/mL BSA respectively, and incubating for 4h at 37 ℃;

(8) taking out SB-Alg + NaAlg, CB₁-Alg+NaAlg、CB₂-Alg + NaAlg gel;

(9) mu.L of each of the remaining 20. mu.L of BSA was added to 200. mu.L of BCA working solution, and the protein adsorption amount was measured at 562 nm. The test results are shown in fig. 17 and table 2:

table 2 table of protein adsorption amount test results

As shown in Table 2, 60% of SB-Alg and CB were found to be present in comparison with sodium alginate₁-Alg and CB₂The Alg can improve the protein adhesion resistance effect of the sodium alginate, so that foreign body reaction is reduced, and the immunological rejection phenomenon is reduced.

(II), SB-Alg, CB₁-Alg、CB₂-Alg、SB-Alg-MA、CB₁Cytotoxicity assay of-Alg-MA

(1) Respectively preparing 2% of SB-Alg and CB₁-Alg、CB₂-Alg、SB-Alg-MA、CB₁-sterile DMEM culture solution of Alg-MA

Respectively weighing 500mg of SB-Alg and CB₁-Alg、CB₂-Alg、SB-Alg-MA、CB₁-Alg-MA was dissolved in 25mL of 10% FBS-containing DMEM medium, and then filtered through a 0.22 μm filter to remove bacteria, thereby obtaining 2% of a solution containing SB-Alg and CB₁-Alg、CB₂-Alg、SB-Alg-MA、CB₁-DMEM culture of Alg-MA.

(2) Preparing a cell suspension: fibroblast cells L929 (viable cell number) from mice with vigorous growth in logarithmic growth phase>99%), digested with 0.25% pancreatin, formulated to a concentration of 1 x 10⁵The cell suspension was kept for use.

(3) MTT assay

Adding the prepared cell suspension into a 96-well plate, adding 100 mu L of the cell suspension into each well, and placing the well in 5% CO₂Culturing in an incubator for 18-24 h. After the adherence is complete, the culture solution is removed and washed with PBS for three times, then 100 mu L of DMEM culture solution containing the material to be detected is added into each hole, and 3 holes are arranged in parallelAdding equal amount of DMEM containing 10% FBS into blank control and negative control wells, placing at 37 deg.C and 5% CO₂Culturing in an incubator. After 24 hours, 10. mu.L/well of 5mg/mL MTT solution was added and the culture was continued for 4 hours. Finally, the culture solution was removed, 100 μ L of DMSO solution was added, absorbance (λ. about.570 nm) was measured with a microplate reader, and relative proliferation rate (RGR%) was calculated by the following formula for toxicity classification.

RGR% ((assay absorbance-blank absorbance)/(negative control absorbance-blank absorbance) — 100%).

The cytotoxicity test results are shown in fig. 18: 2% of SB-Alg, CB₁-Alg、CB₂-Alg、SB-Alg-MA、CB₁The relative proliferation rate of Alg-MA is more than 85 percent and far exceeds 70 percent, and the Alg-MA is non-cytotoxic and has good biocompatibility.

(III) biomaterials prepared in examples 5 and 6 (SB-Alg-MA self-crosslinking hydrogel, CB)₁-Alg-MA self-crosslinking hydrogel, SB-Alg-MA-PEGDA hydrogel, CB₁-Alg-MA-PEGDA hydrogel) as shown in fig. 21:

SB-Alg-MA and CB₁The Alg-MA can be used as biological ink, a modified substrate of a membrane or the like together with the PEGDA, and the sodium alginate has the advantages that besides inherent ionic crosslinking, the introduction of double bonds also contributes to the formation of a stable three-dimensional network structure by chemical crosslinking, or a new crosslinking mode is added to the sodium alginate, so that the sodium alginate is applied to the field range limited by ionic crosslinking (in the related art, divalent metal ions are generally adopted by ionic crosslinking, and the metal ions are heavy metal ions and cause a poisoning phenomenon when the sodium alginate is used as an in-vivo implantation material or enters a body in the form of a carrier).

Some embodiments disclosed herein synthesize a type of zwitterion with imidazole five-membered heterocycle, utilize p-pi orbital conjugation effect to uniformly disperse the carried positive charges on the five-membered heterocycle, and form a high-stability imidazolium zwitterion (such as SB-Alg, CB) through an effective energy dissipation mechanism₁-Alg、CB₂Alg), and finally grafting such zwitterions by amidationTo sodium alginate, a material with good biocompatibility and low foreign body reactivity is obtained, and the implant material can stably exist in a body for a long time.

It should be noted that, unless otherwise specified, "normal temperature" or "room temperature" herein is about 25 ℃; reference herein to "about" or "about" a numerical value is to the error ± 2%.

The embodiments of the present invention have been described in detail with reference to the accompanying drawings, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the gist of the present invention. Furthermore, the embodiments of the present invention and the features of the embodiments may be combined with each other without conflict.

Claims

1. A zwitterionic-modified polysaccharide polymer comprising a nitrogen-containing heterocyclic cationic group, wherein the zwitterionic-modified polysaccharide polymer is prepared by grafting nitrogen-containing heterocyclic zwitterions onto a polysaccharide polymer through amidation.

2. The zwitterionic-modified polysaccharide polymer of claim 1, wherein the nitrogen-containing heterocyclic cationic group includes at least one of a nitrogen-containing five-membered ring cationic group, a nitrogen-containing six-membered ring cationic group, or a nitrogen-containing fused ring cationic group, and the nitrogen-containing heterocyclic zwitterion includes a nitrogen-containing five-membered ring zwitterion, a nitrogen-containing six-membered ring zwitterion, or a nitrogen-containing fused ring zwitterion;

preferably, the nitrogen-containing heterocyclic cationic group includes at least one of an imidazolium cationic group, a pyrrolidinium cationic group, a thiazolium cationic group, a pyridinium cationic group, a pyrazinium cationic group, a pyrimidium cationic group, a pyridazinium cationic group, an indolium cationic group, a quinolinium cationic group, a pteridinium cationic group, an acridinium cationic group, a triazolium cationic group, a benzimidazolium cationic group, a benzothiazolium cationic group, a benzotriazole cationic group, a quinoxalinium cationic group, a quinazolinium cationic group, a benzopyridazinium cationic group, or a pyrrolopyrimidinium cationic group, and the nitrogen-containing heterocyclic zwitterion includes an imidazolium zwitterion, a pyrrolidinium zwitterion, a thiazolium zwitterion, a pyridinium zwitterion, a pyrazinium zwitterion, a, At least one of a pyrimidinium zwitterion, a pyridazinium zwitterion, an indolium zwitterion, a quinolinium zwitterion, a pteridinium zwitterion, an acridinium zwitterion, a triazolium zwitterion, a benzimidazolium zwitterion, a benzothiazolium zwitterion, a benzotriazolium zwitterion, a quinoxalinium zwitterion, a quinazolinium zwitterion, a benzpyridazinium zwitterion, or a pyrrolopyrimidinium zwitterion;

preferably, the nitrogen-containing heterocyclic cationic group is an imidazolium cationic group and the nitrogen-containing heterocyclic zwitterion is an imidazolium zwitterion; preferably, the imidazolium zwitterion contains an amino group, and the polysaccharide polymer contains a carboxyl group or a carboxylate group, and the carboxyl group or the carboxylate group undergoes amidation reaction with the amino group; preferably, the imidazolium zwitterion has an amino group, and the polysaccharide polymer has a carboxyl group or a carboxylate group in a structural unit thereof, and the carboxyl group or the carboxylate group undergoes an amidation reaction with the amino group;

preferably, the polysaccharide polymer comprises at least one of alginate, hyaluronic acid, carboxymethyl cellulose, carboxymethyl modified hemicellulose, carboxymethyl chitosan, carboxymethyl dextran, carboxymethyl carrageenan, carboxymethyl pachyman, carboxymethyl starch, pectin, or heparin; preferably, the alginate comprises sodium alginate;

preferably, the imidazolium zwitterion comprises at least one of a sulfonate betaine, a carboxylate betaine, or a phosphorylcholine; preferably, the imidazolium zwitterion includes at least one of 1- (3-aminopropyl) imidazolium sulfobetaine, 1- (3-aminopropyl) imidazolium acetate betaine, or 1- (3-aminopropyl) imidazolium butanoate betaine.

3. The zwitterionic modified polysaccharide polymer of claim 1, wherein the zwitterionic modified polysaccharide polymer comprises at least one of a sulfobetaine, a carboxybetaine, or a phosphorylcholine; preferably, the zwitterionic modified polysaccharide polymer is a zwitterionic modified alginate; preferably, the zwitterionic modified polysaccharide polymer comprises at least one of a polymer of formula (1), a polymer of formula (2) or a polymer of formula (3):

4. the zwitterionic modified polysaccharide polymer of claim 1, wherein the zwitterionic modified polysaccharide polymer includes photosensitive groups; preferably, the zwitterionic modified polysaccharide polymer comprises at least one of a polymer of formula (4), a polymer of formula (5) or a polymer of formula (6):

5. a method for producing a zwitterionic-modified polysaccharide polymer, comprising the steps of: the polysaccharide polymer modified by the zwitterion is prepared by grafting zwitterion containing nitrogen heterocycle on the polysaccharide polymer through amidation reaction, and the zwitterion modified polysaccharide polymer contains a nitrogen heterocycle cationic group.

6. The method according to claim 5, wherein the heterocyclic nitrogen-containing cationic group is an imidazolium cationic group and the heterocyclic nitrogen-containing zwitterion is an imidazolium zwitterion, and wherein the method for producing the zwitterionic modified polysaccharide polymer comprises the steps of:

sa-2, mixing the mixture I with water, adjusting the pH value to be neutral, dialyzing, precipitating and separating;

alternatively, the preparation method comprises the following steps:

sb-2, mixing the mixture II with water, adjusting the pH value to be neutral, dialyzing, precipitating and separating;

preferably, in step Sa-2, after the mixture i is mixed with water, the pH is adjusted to neutral, dialysis, precipitation separation, nucleophilic substitution reaction with methacrylic anhydride is performed, and dialysis purification is performed to obtain the zwitterionic modified polysaccharide polymer;

preferably, in the step Sb-2, after the mixture ii is mixed with water, the pH is adjusted to neutral, dialyzed, precipitated and separated, nucleophilic substitution reaction is performed with methacrylic anhydride, and dialysis purification is performed to obtain the zwitterionic modified polysaccharide polymer;

preferably, in the step Sa-1, 2-chloro-4, 6-dimethoxy-1, 3, 5-triazine and N-methylmorpholine are added into a mixed solution of sodium alginate, water and acetonitrile to obtain a solution I of the sodium alginate;

preferably, in step Sb-1, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide are added to a mixed solution of sodium alginate and MES buffer to obtain a solution II of the sodium alginate.

7. A hydrogel produced by photocrosslinking reaction of a starting material comprising at least one of the zwitterionic modified polysaccharide polymer according to claim 4 or the zwitterionic modified polysaccharide polymer produced by the process according to any one of claims 5 to 6.

Preferably, the raw material of the hydrogel further comprises PEGDA, and the zwitterionic modified polysaccharide polymer and the PEGDA are subjected to a photocrosslinking reaction to obtain the hydrogel.

8. A biomaterial comprising at least one of a zwitterionic modified polysaccharide polymer as claimed in any one of claims 1 to 4 or prepared by a process as claimed in any one of claims 5 to 6.

9. A medical product comprising at least one of a zwitterionic modified polysaccharide polymer as claimed in any one of claims 1 to 4 or a zwitterionic modified polysaccharide polymer produced by a process as claimed in any one of claims 5 to 6 or a hydrogel as claimed in claim 7;

preferably, the medical product comprises at least one of a medical device or a drug.

10. Use of a zwitterionic modified polysaccharide polymer as defined in any one of claims 1 to 4 or as prepared by a process as defined in any one of claims 5 to 6 or a hydrogel as defined in claim 7 in the preparation of a biomaterial or medical product;

preferably, the zwitterionic modified polysaccharide polymer or the hydrogel is used in a coating of a medical device, a coating of a drug or bio-ink.