WO2024001149A1

WO2024001149A1 - Physical gel on basis of zwitterionic modified polysaccharide and preparation method therefor

Info

Publication number: WO2024001149A1
Application number: PCT/CN2023/070719
Authority: WO
Inventors: 叶磊; 黄苏苏; 方跃霖
Original assignee: 山东大学
Priority date: 2022-07-01
Filing date: 2023-01-05
Publication date: 2024-01-04
Also published as: CN115073770A

Abstract

Disclosed in the present invention are a physical gel on the basis of a zwitterionic modified polysaccharide and a preparation method therefor. The method comprises the following steps: (1) preparing a macromolecular initiator (D-Br); (2) preparing a zwitterionic grafted modified polysaccharide derivative; and (3) preparing a physical gel on the basis of a zwitterionic modified polysaccharide. In the present invention, the control of the degree of polymerization n can be achieved by means of using the ATRP method of high molecular polymerization, so that the degree of polymerization of zwitterionic side chains is precisely regulated and controlled, and the magnitude of the ion interaction force is further controlled. Therefore, the phase transformation of "solution-gel-precipitation" of the polymer in a gradient salt ion solution is achieved. A prescription having sensitivity for a physiological solution is screened, and a biodegradable zwitterionic polysaccharide-based hydrogel is prepared.

Description

A physical gel based on zwitterionic modified polysaccharide and its preparation method

The present invention claims the priority of the Chinese patent application submitted to the China Patent Office on July 1, 2022, with the application number 202210773927.3 and the invention title "A physical gel based on zwitterionic modified polysaccharides and its preparation method", which The entire contents are incorporated herein by reference.

Technical field

The present invention relates to the technical field of polymer gels, and in particular to a physical gel based on zwitterionic modified polysaccharides and a preparation method thereof.

Background technique

All water-soluble or hydrophilic polymers can form hydrogels through certain chemical or physical cross-linking. These polymers can be divided into two categories: natural and synthetic according to their sources. Natural hydrophilic polymers include polysaccharides (starch, cellulose, alginic acid, hyaluronic acid, chitosan, etc.) and polypeptides (collagen , poly-L-lysine, poly-L-glutamic acid, etc.). Synthetic hydrophilic polymers include alcohol, acrylic acid and its derivatives (polyacrylic acid, polymethacrylic acid, polyacrylamide, etc.).

Zwitterionic materials refer to a type of material in which a molecular chain carries both a positive charge and a negative charge but is generally uncharged. Zwitterionic materials have attracted widespread attention in the field of drug delivery for tumors due to their good biocompatibility and resistance to protein adsorption. Hydrogel materials based on zwitterionic polymers and biological polysaccharides are widely used in the field of biomedicine, but each has certain shortcomings. For example, zwitterionic polymers are difficult to degrade, while polysaccharide materials have weak anti-protein adhesion effects. Among current gel materials based on zwitterionic polymers, most are nondegradable. Degradability is of great significance for nanogels. Therefore, a physical gel is needed that can combine zwitterionic polymers and biopolysaccharide hydrogel materials to prepare degradable gel materials.

Contents of the invention

In view of the shortcomings of the existing technology, the present invention provides a physical gel based on zwitterionic modified polysaccharide and a preparation method thereof. The present invention utilizes the ATRP method of polymer polymerization to achieve control of the degree of polymerization n, thereby accurately regulating the degree of polymerization of zwitterionic side chains and thereby controlling the size of the ionic interaction force. This enables the polymer to achieve a "solution-gel-precipitation" phase transition in a gradient salt ion solution. Formulations with physiological solution sensitivity were screened to prepare biodegradable zwitterionic polysaccharide-based hydrogels.

The present invention is achieved through the following technical solutions:

A first aspect of the present invention provides a method for preparing a physical gel based on zwitterionic modified polysaccharides, which includes the following steps:

(1) Dissolve the polysaccharide in dimethylformamide, then cool it in an ice-water bath, add 4-lutidine, and stir to activate the functional groups to obtain solution A;

(2) Dissolve bromo-isobutyryl bromide in dimethylformamide and stir evenly to obtain solution B;

(3) Add solution B dropwise to solution A, add triethylamine dropwise at the same time, carry out the reaction in an ice bath, disperse it in ice ethanol after the reaction, collect the precipitate, and obtain the macromolecular initiator D-Br;

(4) Dissolve the macroinitiator D-Br and complexing agent Bpy obtained in step (3) in DMSO to obtain solution C;

(5) Dissolve the zwitterionic monomer in DMSO to obtain solution D;

(6) Transfer solution C and solution D to a Schlenk tube and exclude oxygen from the reaction. Then add copper bromide under the condition of flowing nitrogen to seal the reaction. After the reaction is completed, disperse it in icy methanol and collect the product. , obtaining the zwitterionic graft-modified polysaccharide derivative DX-G-ZWIY;

(7) Preparation of physiological saline-sensitive physical gel: Add physiological saline to the DX-G-ZWIY obtained in step (6), and after standing, a physical gel based on zwitterion-modified polysaccharide is obtained.

Preferably, in step (1), the polysaccharide is selected from dextran or hyaluronic acid; the ratio of the added amounts of the polysaccharide, dimethylformamide, and 4-lutidine is (0.5 to 3) g: ( 20～150)mL: (0.1g-0.5)g.

Preferably, in step (2), the volume ratio of the bromine-isobutyryl bromide solution to dimethylformamide is 1 mL: (1-5) mL.

Preferably, in step (3), the volume ratio of solution A, solution B and triethylamine is 1 mL: (0.1-1) mL: (0.1-1) mL; the dripping acceleration rate of solution B is 0.2- 3mL/min; the dripping rate of the triethylamine is 0.2~3mL/min; the reaction time is 6~48h.

Preferably, in step (4), the added amounts of the macroinitiator D-Br, complexing agent Bpy and DMSO are (0.01~0.2)g: (0.1~1.5)g: (5~50)mL.

Preferably, in step (5), the zwitterionic monomer is SBMA or CBMA; the ratio of the added amount of the zwitterionic monomer to DMSO is (0.1-3) g: (5-30) mL.

Preferably, in step (6), the ratio of the added amounts of solution C, solution D and cuprous bromide is (5-50) mL: (5-30) mL: (0.05-3) g;

In DX-G-ZWIY, X is the degree of bromine substitution, and its degree of substitution ranges from 10% to 70%. Y is the degree of polymerization of the zwitterionic monomer (CBMA or SBMA), and its degree of polymerization ranges from 10 to 50.

Preferably, in step (7), the ratio of the added amounts of DX-G-ZWIY and physiological saline is (0.1-0.5) g: (1-5) mL; the standing time is 5 to 120 minutes.

A second aspect of the present invention provides a physical gel based on zwitterionic modified polysaccharides prepared by a preparation method, characterized in that the physical gel is a degradable physical gel.

Preferably, excess physiological saline is added to the physical gel, and the gel skeleton disintegrates accordingly.

The beneficial effects of the present invention are:

(1) The present invention uses atom transfer radical polymerization (ATRP) technology to polymerize zwitterionic monomers and polysaccharide main chains to form a molecular brush structure, and utilizes electrostatic interactions within the material to form a physical gel with anti-protein adhesion properties. The biodegradable property of the biopolysaccharide itself makes the gel skeleton biodegradable, thereby organically combining the two materials to achieve anti-protein adhesion in the early stage of implantation in the body, and later degradation to avoid surgical removal of the implant. secondary damage.

(2) The gel prepared by the present invention has good transparency and flexibility and can be used for drug loading and skin wound coating. During the drug loading process, you only need to simply dissolve the drug in physiological saline, and then add the physiological saline solution containing the drug to the DX-G-ZWIY polymer powder to achieve efficient loading of the drug. The drug loading method is simple, there are many types of drugs that can be loaded, and the carrier will gradually dissociate and degrade in the salt solution. It has broad application prospects in the fields of medicine and cosmetics.

Description of drawings

Figure 1 is the NMR image of dextran macroinitiator (D-Br);

Figure 2 is the (DX-G-PSBMAy) NMR pattern of zwitterionic modified dextran polysaccharide derivatives;

Figure 3 shows the starting physiological saline solution image (a), digital photo image (b) and SEM image (c) of DX-G-PSBMAy polymer hydrogel.

Figure 4 is a schematic diagram of the "gel-sol" transition of zwitterionic physical hydrogel;

Figure 5 is a schematic diagram of the use of zwitterionic physical hydrogel;

Figure 6 is a diagram showing the effect of zwitterionic physical hydrogel applied in anti-intestinal adhesions.

Figure 7 shows the H&E staining and Masson’s staining effects of zwitterionic physical hydrogel applied to resist intestinal adhesion.

Detailed ways

It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the present application. Unless otherwise defined, all technical and scientific terms used herein have the same meanings commonly understood by one of ordinary skill in the art to which this application belongs.

As mentioned in the background, most of the current gel materials based on zwitterionic polymers are non-degradable. Based on this, the present invention provides a physical gel based on zwitterionic modified polysaccharide and a preparation method thereof. The present invention uses atom transfer radical polymerization (ATRP) technology to polymerize zwitterionic monomers and polysaccharide main chains to form a molecular brush structure, and utilizes electrostatic interactions within the material to form a physical gel with anti-protein adhesion properties, and utilizes the biological polysaccharide itself The degradable properties endow the gel matrix with biodegradability.

In order to enable those skilled in the art to understand the technical solutions of the present application more clearly, the technical solutions of the present application will be described in detail below with reference to specific embodiments. If the specific experimental conditions are not specified in the examples, conventional conditions are usually followed, or conditions recommended by the reagent company; the reagents, consumables, etc. used in the following examples can all be obtained through commercial channels unless otherwise specified.

Example 1

1. Preparation of macroinitiator (D-Br)

①. Preparation of solution A: Weigh 3g of dextran and dissolve it in 150mL of dimethylformamide solution, then cool the system in an ice water bath, add 0.3g of 4-dimethylpyridine activator, stir for 3 hours, and activate the functional groups.

②. Preparation of solution B: Precisely weigh 3g of bromine-isobutyryl bromide reagent and dissolve it in 5g of dimethylformamide, stir evenly and set aside.

③. Add solution B into system A drop by drop through the dropping funnel. At the same time, add acid binding agent triethylamine in an equal molar amount to bromine-isobutyryl bromide, and place the system in an ice bath for 24 hours. After the reaction, the system was dispersed in glacial ethanol, and the washed precipitate was collected. The product was named D-Br single-molecule initiator.

2. Preparation of zwitterionic graft-modified polysaccharide derivatives

①. Preparation of solution C: Precisely weigh 0.1g of the prepared macroinitiator D-Br and 0.15g of the complexing agent Bpy and dissolve them in 10mL DMSO.

②. Preparation of solution D: Precisely weigh 0.6g of SBMA and dissolve it in 20mL of DMSO.

③. Transfer the prepared C and D solutions to the Schlenk tube, and vacuum the system through the Schlenk system - repeat 5 cycles of nitrogen gas to eliminate the oxygen in the reaction, and then add 0.3g under the condition of nitrogen gas. Cuprous bromide, repeat the vacuum-nitrogen cycle 5 times, seal the system, and react for 24 hours. After the reaction is completed, the system is dispersed in 10 times the amount of glacial methanol, and the product is collected by washing to obtain the product DX-G-PSBMAY.

3. Preparation of saline-sensitive physical gel

Weigh 0.2g of a zwitterionic graft-modified polysaccharide derivative with a degree of substitution .

Example 2

1. Preparation of macroinitiator (D-Br)

①. Preparation of solution A: Weigh 3g of hyaluronic acid and dissolve it in 150mL of dimethylformamide solution, then place the system in an ice water bath to cool, add 0.3g of 4-dimethylpyridine activator, stir for 3 hours, and activate Functional group.

②. Preparation of solution B: Precisely weigh 5g of bromine-isobutyryl bromide reagent and dissolve it in 11.2g of dimethylformamide, stir evenly and set aside.

③. Add solution B into system A drop by drop through the dropping funnel. At the same time, add a certain amount of acid-binding agent triethylamine dropwise, and place the system in an ice bath for 24 hours. After the reaction, the system was dispersed in glacial ethanol, and the washed precipitate was collected. The product was named D-Br single-molecule initiator.

2. Preparation of zwitterionic graft-modified polysaccharide derivatives

②. Preparation of solution D: Precisely weigh 0.3g of SBMA and dissolve it in 20mL of DMSO.

③. Transfer the prepared C and D solutions to the Schlenk tube, and vacuum the system through the Schlenk system - repeat 5 cycles of nitrogen gas to eliminate the oxygen in the reaction, and then add 0.3g under the condition of nitrogen gas. Cuprous bromide, repeat the vacuum-nitrogen cycle 5 times, seal the system, and react for 24 hours. After the reaction, the system was dispersed in 10 times the amount of glacial methanol, and the product was washed and collected to obtain the product DX-G-CBMAY.

3. Preparation of saline-sensitive physical gel

Weigh 0.15g of a zwitterionic graft-modified polysaccharide derivative with a degree of substitution , as shown in Figure 5.

Example 3

The physical gel prepared in Example 1 was added to 5 mL of physiological saline solution. As the net ion concentration in the system increased, the electrostatic interaction between the zwitterions was gradually shielded and dissociated, and the gel skeleton disintegrated.

Test example: Degradation experiment of DX-G-ZWIY gel

Two media, physiological saline and deionized water, were used for testing. Precisely weigh 0.1 g of the DX-G-ZWIY powder prepared in Example 1 and place it in a 15 mL centrifuge tube, then add 0.9 mL of physiological saline and let it stand for 1 hour to form a physical gel. Freeze-dry the gel for 48 hours to obtain a dry gel for testing (the initial gel weight is recorded as W0); add 9 mL of physiological saline (or deionized water) to the centrifuge tube, and then place it in a 37°C incubator. The normal saline group was replaced with fresh medium every day, and the deionized water group was replaced once a week for 4 consecutive weeks. At a preset time point, take out the gel, freeze-dry and weigh (Wt); the mass loss ΔW% is calculated according to the following formula (each group repeats 4 samples):

ΔW%=(W0-Wt)/W0

Physical gel has a faster mass loss process in physiological saline. At the third day of testing, the mass loss of the gel can reach 80%. In deionized water, since there is not a large amount of electrolyte, the mass loss of the sample in deionized water may be caused by the breakage of the polymer molecular chain. The mass loss of the gel samples was approximately 18% over a period of 4 weeks, see Figures 3 and 4.

The zwitterionic physical hydrogel prepared in Example 1 can be used in the field of anti-intestinal adhesion. It is used in the rat abdominal wall-cecum adhesion model, and the group treated with uncoated hydrogel is used as the control group (model Group). The hydrogel prepared in Example 1 was implanted into rats, and the abdominal cavity was opened for observation 7 days after the operation. It was found that compared with the control group, the hydrogel of the present invention had excellent anti-adhesion effect, as shown in Figures 6 and 7 .

The above descriptions are only preferred embodiments of the present application and are not intended to limit the present application. For those skilled in the art, the present application may have various modifications and changes. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of this application shall be included in the protection scope of this application.

Claims

A method for preparing a physical gel based on zwitterionic modified polysaccharide, which is characterized by including the following steps:

(1) Dissolve the polysaccharide in dimethylformamide, then cool it in an ice-water bath, add 4-lutidine, and stir to activate the functional groups to obtain solution A;

(2) Dissolve bromo-isobutyryl bromide in dimethylformamide and stir evenly to obtain solution B;

(3) Add solution B dropwise to solution A, add triethylamine dropwise at the same time, carry out the reaction in an ice bath, disperse it in ice ethanol after the reaction, collect the precipitate, and obtain the macromolecular initiator D-Br;

(4) Dissolve the macroinitiator D-Br and complexing agent Bpy obtained in step (3) in DMSO to obtain solution C;

(5) Dissolve the zwitterionic monomer in DMSO to obtain solution D;

(6) Transfer solution C and solution D to a Schlenk tube and exclude oxygen from the reaction. Then add copper bromide under the condition of flowing nitrogen to seal the reaction. After the reaction is completed, disperse it in icy methanol and collect the product. , obtaining the zwitterionic graft-modified polysaccharide derivative D X -G-ZWI Y ;

(7) Preparation of physiological saline-sensitive physical gel: Add physiological saline to the D
The preparation method according to claim 1, characterized in that, in step (1), the polysaccharide is selected from dextran or hyaluronic acid; the adding amounts of the polysaccharide, dimethylformamide, and 4-lutidine The ratio is (0.5～3)g: (20～150)mL: (0.1～0.5)g.
The preparation method according to claim 1, characterized in that, in step (2), the mass ratio of the bromine-isobutyryl bromide solution to dimethylformamide is 1 mL: (1-5) mL.
The preparation method according to claim 1, characterized in that, in step (3), the ratio of the addition amounts of solution A, solution B and triethylamine is 1 mL: (0.1 ~ 1) mL: (0.1 ~ 1) mL ; The dripping acceleration rate of the B solution is 0.2-3ml/min; the dripping acceleration rate of the triethylamine is 0.2-3ml/min; the reaction time is 6-48h.
The preparation method according to claim 1, characterized in that in step (4), the adding amounts of the macroinitiator D-Br, the complexing agent Bpy and DMSO are (0.01~0.2) g: (0.1~ 1.5)g: (5～50)ml.
The preparation method according to claim 1, characterized in that, in step (5), the zwitterionic monomer is SBMA or CBMA; the ratio of the added amount of the zwitterionic monomer to DMSO is (0.1 to 3) g: (5～30)mL.
The preparation method according to claim 1, characterized in that, in step (6), the ratio of the adding amounts of the C solution, D solution and cuprous bromide is (5~50) mL: (5~30) mL : (0.05~3)g; D ~50.
The preparation method according to claim 1, characterized in that, in step (7), the ratio of the addition amount of DX -G-ZWI Y and physiological saline is (0.1~0.5) g: (1~5) ml; The resting time is 5~120min.
The physical gel based on zwitterionic modified polysaccharide prepared by the preparation method of any one of claims 1 to 8, characterized in that the physical gel is a degradable physical gel.
The physical gel according to claim 9, characterized in that when an excess amount of physiological saline is added to the physical gel, the gel skeleton is disintegrated.