CN115232371A - Hydrogel, preparation method and application - Google Patents

Hydrogel, preparation method and application Download PDF

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CN115232371A
CN115232371A CN202210541202.1A CN202210541202A CN115232371A CN 115232371 A CN115232371 A CN 115232371A CN 202210541202 A CN202210541202 A CN 202210541202A CN 115232371 A CN115232371 A CN 115232371A
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parts
chs
adh
deionized water
hydrogel
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范治平
张攀
程萍
王正平
韩军
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Liaocheng University
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Liaocheng University
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J3/00—Processes of treating or compounding macromolecular substances
    • C08J3/02—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques
    • C08J3/03—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques in aqueous media
    • C08J3/075—Macromolecular gels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/20—Polysaccharides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/52—Hydrogels or hydrocolloids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/58—Materials at least partially resorbable by the body
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
    • C08B37/0024—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Glucans; (beta-1,3)-D-Glucans, e.g. paramylon, coriolan, sclerotan, pachyman, callose, scleroglucan, schizophyllan, laminaran, lentinan or curdlan; (beta-1,6)-D-Glucans, e.g. pustulan; (beta-1,4)-D-Glucans; (beta-1,3)(beta-1,4)-D-Glucans, e.g. lichenan; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
    • C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
    • C08B37/0069—Chondroitin-4-sulfate, i.e. chondroitin sulfate A; Dermatan sulfate, i.e. chondroitin sulfate B or beta-heparin; Chondroitin-6-sulfate, i.e. chondroitin sulfate C; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J3/00—Processes of treating or compounding macromolecular substances
    • C08J3/24—Crosslinking, e.g. vulcanising, of macromolecules
    • C08J3/246—Intercrosslinking of at least two polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/34—Materials or treatment for tissue regeneration for soft tissue reconstruction
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J2305/00—Characterised by the use of polysaccharides or of their derivatives not provided for in groups C08J2301/00 or C08J2303/00
    • C08J2305/02—Dextran; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J2305/00—Characterised by the use of polysaccharides or of their derivatives not provided for in groups C08J2301/00 or C08J2303/00
    • C08J2305/08—Chitin; Chondroitin sulfate; Hyaluronic acid; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J2405/00—Characterised by the use of polysaccharides or of their derivatives not provided for in groups C08J2401/00 or C08J2403/00
    • C08J2405/02—Dextran; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J2405/00—Characterised by the use of polysaccharides or of their derivatives not provided for in groups C08J2401/00 or C08J2403/00
    • C08J2405/08—Chitin; Chondroitin sulfate; Hyaluronic acid; Derivatives thereof

Abstract

The invention relates to the field of A61K9/06, in particular to hydrogel, a preparation method and application thereof, the hydrogel preparation raw materials disclosed by the invention are modified chondroitin sulfate and oxycetamide, the prepared hydrogel has strong applicability in different human tissues and pathological changes in different acid-base environments, and can be applied in the fields of drug delivery, cell 3D encapsulation, cell regulation and the like.

Description

Hydrogel, preparation method and application
Technical Field
The invention relates to the field of A61K9/06, and particularly relates to a hydrogel, a preparation method and application thereof.
Background
The hydrogel is a hydrophilic polymer material obtained through physical or chemical crosslinking, has a three-dimensional space network structure, is a basic new material with an ecological intelligent controlled release function, has the characteristics of good biocompatibility, high water absorbability, high water retention property and the like, is widely applied to the industries of biomedicine, petrochemical industry, cosmetics and the like, particularly in the aspect of biological medicine materials, can be used as a polymer medicine carrier to efficiently convey medicines to a target organ, and greatly reduces the harm of toxic and side effects of the medicines to a human body.
CN113956507A discloses an injectable hydrogel and a preparation method thereof, which uses aminopolysaccharide, aldehyde group polysaccharide and inorganic compound to prepare hydrogel with a double cross-linked structure, the hydrogel has controllable gel time, can rapidly stop bleeding and is convenient to carry. But has no drug loading capacity and single use route.
CN109810265B discloses a solvent-driven volume-changing DNA-polysaccharide hybrid hydrogel and a preparation method thereof, which adopts a DNA aqueous solution and a polysaccharide derivative such as dopamine or dextran, chondroitin sulfate, hyaluronic acid and the like to be mixed with the aqueous solution to obtain the DNA-polysaccharide hybrid hydrogel. But its applicability in different human tissues and lesions of different acid-base environments is poor.
Disclosure of Invention
In order to solve the above problems, one aspect of the present invention discloses a hydrogel, which is prepared from two precursor macromolecules: modified chondroitin sulfate (Chs-ADH) and Oxycolac (OS).
The precursor macromolecules Chs-ADH at least comprise the following preparation raw materials in parts by mass: 1-3 parts of Chondroitin sulfate (ChS), 0.3-1 part of N-hydroxysuccinimide (NHS), 1-3 parts of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide (N1- ((ethylimino) methyl) -N3, N3-dimethylpropane-1,3-diamine, EDC), 3-5 parts of Adipic Dihydrazide (ADH) and 60-110 parts of deionized water.
The precursor macromolecule OS at least comprises the following preparation raw materials in parts by mass: 0.1-0.6 part of Secoic (Salecan), 1-3 parts of sodium periodate, 0.1-1 part of diethylene glycol and 300-500 parts of deionized water.
Further, the preparation method of the precursor macromolecule Chs-ADH at least comprises the following steps:
s1: dissolving 1-3 parts of ChS in 30-60 parts of deionized water by mass, adding 0.3-1 part of NHS after dissolving, and then adding 1-3 parts of EDC;
s2: activating for 1-3h, and stabilizing the pH value at 4-5;
s3:3-5 parts of ADH is dissolved in 30-50 parts of deionized water, and the activated ChS solution is dripped into the solution, so that the pH value of the system is stabilized at 5-6.5;
s4: stirring at room temperature for 8-16h. Dialyzing and purifying to obtain Chs-ADH.
Further, the step S2 specifically includes: 1mol/L sodium hydroxide and 1mol/L hydrogen chloride are adopted to adjust the pH value of the system, so that the pH value of the system is stabilized at 4-5 and activated for 1-3h.
Further, the step S4 specifically includes: after stirring at room temperature for 8-16h, the reaction system was dialyzed using a dialysis bag with a cut-off molecular weight of 3000 Da. The dialyzed purified solution was finally lyophilized to form a white sample, which was stored at 0-5 ℃ under refrigeration.
The preparation method of the precursor macromolecule OS at least comprises the following steps:
s1:0.1-0.6 parts of Secoid (Salecan) dissolved in 300-500 parts of deionized water;
s2: adding 1-3 parts of sodium periodate, reacting for 10-14h at room temperature in a dark place, and dropping 0.1-1 part of diglycol to terminate the reaction;
s3: dialyzing for 2-4 days, and purifying to obtain OS.
Further, the step S3 specifically includes: transferring the reaction solution into a dialysis bag with cut-off molecular weight of 5000Da for dialysis with deionized water for 2-4 days, finally freeze-drying the dialyzed and purified product solution to form a white flocculent sample, and refrigerating and storing at 0-5 ℃.
The invention also discloses a preparation method of the hydrogel, which at least comprises the following steps:
s1: diluting Chs-ADH by deionized water;
s2: diluting and diluting OS with deionized water;
s3: pouring the diluted Chs-ADH and OS into a mold, uniformly swirling, and standing at room temperature for 10-14h;
s4: and (5) sterilizing and cleaning.
The hydrogel is prepared by Schiff base reaction. The applicant found during the preparation that the principle of mixing two precursor macromolecular solutions to obtain a homogeneous system for crosslinking can be attributed to the new covalent bonds that appear during crosslinking: the amido bond and the OS aldehyde group of the ChS-ADH form a covalent hydrazone bond through dynamic crosslinking. The gel time of the hydrogel samples was determined by the tilt method, with all proportions of water having gel times between a few seconds and about 1 min. The hydrazone crosslinking reaction mechanism greatly reduces the gel time of the hydrogel of the system. In terms of materials, CS hydrogels use chondroitin sulfate in combination with sertraline, similar to the composition of the extracellular matrix.
Further, the mass percentage concentration of Chs-ADH diluted by deionized water in the preparation step S1 is 8-15%.
Further, the deionized water in the preparation step S2 is used for diluting the OS, and the mass percentage concentration of the OS is 2-7%.
Further, the preparation step S3 specifically includes: mixing the diluted Chs-ADH and OS according to the volume ratio of (1-5): (0.5.
Further, the step S4 specifically includes: the gel samples were sterilized in 75% ethanol and then washed three times with pure water.
The hydrogel can be applied to the biomedical fields of skin filling materials, drug delivery carriers, 3D cell culture and the like
Has the advantages that:
1. the hydrogel disclosed by the invention is strong in applicability to different human tissues and pathological changes in different acid-base environments.
2. The hydrogel disclosed by the invention has high compressive strength, can be used as a novel drug delivery system in the pharmaceutical field, and can also be applied to 3D packaging and cell regulation of cells.
3. The hydrogel disclosed by the invention has wide pore size distribution, and is beneficial to biomedical applications such as drug delivery, cell encapsulation and the like.
Drawings
FIG. 1 scanning electron microscope image of hydrogel sample. FIG. A embodiment 1, FIG. B embodiment 3, FIG. C embodiment 2.
FIG. 2 equilibrium water contents of hydrogels in three PBS buffer solutions (pH 5.0, 7.4, and 10.0) at room temperature.
FIG. 3 compression Properties of hydrogel having a compression ratio of 20mm/min at room temperature
FIG. 4 cytotoxicity profiles of hydrogels
Figure 5 degradation and swelling characteristics of hydrogels.
FIG. 6 fluorescent image of L-O2 cells cultured in hydrogel for 7 days.
FIG. 7 in vitro drug release profiles of hydrogels pH5.0 and 7.4 PBS. The graph ABCD was fitted using zeroth order, first order, higuchi and Ritger-Peppas models in sequence.
FIG. 8A is a chart of the synthesis of ChS-ADH precursors using the EDC/NHS protocol, and B is a comparison of NMR spectra of ChS before and after ADH grafting.
FIG. 9A is a diagram of a macromolecule of the OS precursor, and B is a diagram of Salecan and OS 1 Infrared spectrum of H NMR.
Detailed Description
Example 1
In one aspect, embodiment 1 of the present invention discloses a hydrogel, wherein raw materials for preparing the hydrogel include two precursor macromolecules: chs-ADH and OS.
The precursor macromolecule Chs-ADH comprises the following preparation raw materials in parts by mass: 1 part of ChS, 0.3 part of NHS, 1 part of EDC, 3 parts of ADH and 60 parts of deionized water.
The precursor macromolecule OS comprises the following preparation raw materials in parts by mass: 0.1 part of Salecan, 1 part of sodium periodate, 0.1 part of diethylene glycol and 300 parts of deionized water.
The preparation method of the precursor macromolecule Chs-ADH comprises the following steps:
s1: dissolving 1 part of ChS in 30 parts of deionized water by mass, adding 0.3 part of NHS after dissolving, and then adding 1 part of EDC;
s2: 1mol/L sodium hydroxide and 1mol/L hydrogen chloride are adopted to adjust the pH value of the system, so that the pH value of the system is stabilized at 4, and the activation is carried out for 1 hour;
s3: dissolving 3 parts of ADH in 30 parts of deionized water, dripping activated ChS solution, and adjusting the pH value of the system to 5 by adopting 1mol/L sodium hydroxide and 1mol/L hydrogen chloride;
s4: stir at room temperature for 8h. The reaction system was dialyzed using a dialysis bag with a cut-off molecular weight of 3000 Da. The dialyzed purified solution was finally lyophilized to form a white sample, which was stored at 0 ℃ under refrigeration.
The preparation method of the precursor macromolecule OS comprises the following steps:
s1:0.1 part of Salecan is dissolved in 300 parts of deionized water;
s2: adding 1 part of sodium periodate, reacting for 10 hours at room temperature in a dark place, and dropping 0.1 part of diethylene glycol to terminate the reaction;
s3: transferring the reaction solution into a dialysis bag with cut-off molecular weight of 5000Da for dialysis with deionized water for 2 days, finally freeze-drying the dialyzed and purified product solution to form a white flocculent sample, and refrigerating and storing at 0 ℃.
In another aspect, embodiment 1 of the present invention discloses a method for preparing the hydrogel, which comprises the following steps:
s1: the mass percentage concentration of Chs-ADH diluted by deionized water is 8%;
s2: diluting OS with deionized water to 2 wt%;
s3: mixing the diluted Chs-ADH and OS in a volume ratio of 1:1, pouring the mixture into a cylindrical die with the height of 5mm, uniformly swirling, and standing for 10 hours at room temperature to ensure that the crosslinking reaction is completely finished;
s4: sterilizing the gel sample in 75% ethanol, and washing with pure water for three times.
Example 2
In one aspect, the embodiment 2 of the present invention discloses a hydrogel, and the raw materials for preparing the hydrogel include two precursor macromolecules: chs-ADH and OS.
The precursor macromolecule Chs-ADH comprises the following preparation raw materials in parts by mass: 3 parts of ChS, 1 part of NHS, 3 parts of EDC, 5 parts of ADH and 110 parts of deionized water.
The precursor macromolecule OS comprises the following preparation raw materials in parts by mass: 0.6 part of Salecan, 3 parts of sodium periodate, 1 part of diethylene glycol and 500 parts of deionized water.
The preparation method of the precursor macromolecule Chs-ADH comprises the following steps:
s1: dissolving 3 parts by mass of ChS in 60 parts by mass of deionized water, adding 1 part by mass of NHS after dissolving, and then adding 3 parts by mass of EDC;
s2: adjusting the pH of the system by adopting 1mol/L sodium hydroxide and 1mol/L hydrogen chloride to stabilize the pH of the system at 5.5, and activating for 3h;
s3: dissolving 5 parts of ADH in 50 parts of deionized water, dripping the activated ChS solution, and adjusting the pH value of the system to be 6.5 by adopting 1mol/L sodium hydroxide and 1mol/L hydrogen chloride;
s4: stir at room temperature for 16h. The reaction system was dialyzed using a dialysis bag with a cut-off molecular weight of 3000 Da. The dialyzed purified solution was finally lyophilized to form a white sample, which was stored at 5 ℃ under refrigeration.
The preparation method of the precursor macromolecule OS comprises the following steps:
s1:0.6 part of Salecan is dissolved in 500 parts of deionized water;
s2: adding 3 parts of sodium periodate, reacting for 14 hours at room temperature in a dark place, and dropping 1 part of diethylene glycol to terminate the reaction;
s3: transferring the reaction solution into a dialysis bag with the cut-off molecular weight of 5000Da for dialysis with deionized water for 4 days, finally freeze-drying the dialyzed and purified product solution to form a white flocculent sample, and refrigerating and storing at 5 ℃.
In another aspect, embodiment 2 of the present invention discloses a method for preparing the hydrogel, which comprises the following steps:
s1: diluting Chs-ADH by deionized water to reach the mass percent concentration of 15%;
s2: diluting OS with deionized water to 7 wt%;
s3: mixing the diluted Chs-ADH and OS according to the volume ratio of 3:1, pouring the mixture into a cylindrical die with the height of 5mm, uniformly swirling, and standing for 14 hours at room temperature to ensure that the crosslinking reaction is completely finished;
s4: sterilizing the gel sample in 75% ethanol, and washing with pure water for three times.
Example 3
In one aspect, embodiment 3 of the present invention discloses a hydrogel, wherein the raw materials for preparing the hydrogel include two precursor macromolecules: chs-ADH and OS.
The precursor macromolecule Chs-ADH comprises the following preparation raw materials in parts by mass: 2 parts ChS, 0.72 part NHS, 1.2 parts EDC, 3.634 parts ADH, 75 parts deionized water.
The precursor macromolecule OS comprises the following preparation raw materials in parts by mass: 0.16 part of Salecan, 2.2 parts of sodium periodate, 0.5 part of diethylene glycol and 400 parts of deionized water.
The preparation method of the precursor macromolecule Chs-ADH comprises the following steps:
s1: dissolving 2 parts by mass of ChS in 40 parts by mass of deionized water, adding 0.72 part by mass of NHS after dissolving, and then adding 1.2 parts by mass of EDC;
s2: 1mol/L sodium hydroxide and 1mol/L hydrogen chloride are adopted to adjust the pH value of the system, so that the pH value of the system is stabilized at 4.8, and the activation is carried out for 2 hours;
s3:3.634 portions of ADH is dissolved in 35 portions of deionized water, the activated ChS solution is dropped in, and 1mol/L sodium hydroxide and 1mol/L hydrogen chloride are adopted to adjust the pH value of the system to be stable at 5.8;
s4: stirred at room temperature for 12h. The reaction system was dialyzed using a dialysis bag with a cut-off molecular weight of 3000 Da. The dialyzed purified solution was finally lyophilized to form a white sample, which was stored under refrigeration at 4 ℃.
The preparation method of the precursor macromolecule OS comprises the following steps:
s1:0.16 parts of Salecan was dissolved in 400 parts of deionized water;
s2: adding 2.2 parts of sodium periodate, reacting for 12 hours at room temperature in a dark place, and dropping 0.5 part of diethylene glycol to terminate the reaction;
s3: transferring the reaction solution into a dialysis bag with cut-off molecular weight of 5000Da for dialysis with deionized water for 3 days, finally freeze-drying the dialyzed and purified product solution to form a white flocculent sample, and refrigerating and storing at 4 ℃.
The embodiment 3 of the invention also discloses a preparation method of the hydrogel, which comprises the following steps:
s1: diluting Chs-ADH with deionized water to reach the mass percent concentration of 11%;
s2: diluting OS with deionized water to 5 wt%;
s3: mixing the diluted Chs-ADH and OS according to the volume ratio of 2:1, pouring the mixture into a cylindrical die with the height of 5mm, uniformly swirling, and standing at room temperature for 12 hours to ensure that the crosslinking reaction is completely finished;
s4: sterilizing the gel sample in 75% ethanol, and washing with pure water for three times.
Example 4
In one aspect, embodiment 4 of the present invention discloses a hydrogel, wherein the raw materials for preparing the hydrogel include two precursor macromolecules: chs-ADH and OS.
The precursor macromolecule Chs-ADH comprises the following preparation raw materials in parts by mass: 2 parts ChS, 0.72 part NHS, 1.2 parts EDC, 3.634 parts ADH, 75 parts deionized water.
The precursor macromolecule OS comprises the following preparation raw materials in parts by mass: 0.16 part of Salecan, 2.2 parts of sodium periodate, 0.5 part of diethylene glycol and 400 parts of deionized water.
The preparation method of the precursor macromolecule Chs-ADH comprises the following steps:
s1: dissolving 2 parts by mass of ChS in 40 parts by mass of deionized water, adding 0.72 part of NHS after dissolving, and then adding 1.2 parts of EDC;
s2: 1mol/L sodium hydroxide and 1mol/L hydrogen chloride are adopted to adjust the pH value of the system, so that the pH value of the system is stabilized at 4.8, and the activation is carried out for 2 hours;
s3:3.634 portions of ADH is dissolved in 35 portions of deionized water, the activated ChS solution is dropped in, and 1mol/L sodium hydroxide and 1mol/L hydrogen chloride are adopted to adjust the pH value of the system to be stable at 5.8;
s4: stirred at room temperature for 12h. The reaction system was dialyzed using a dialysis bag with a cut-off molecular weight of 3000 Da. The dialyzed purified solution was finally lyophilized to form a white sample, which was stored under refrigeration at 4 ℃.
The preparation method of the precursor macromolecule OS comprises the following steps:
s1:0.16 parts of Salecan was dissolved in 400 parts of deionized water;
s2: adding 2.2 parts of sodium periodate, reacting for 12 hours at room temperature in a dark place, and dripping 0.5 part of diethylene glycol to terminate the reaction;
s3: transferring the reaction solution into a dialysis bag with cut-off molecular weight of 5000Da for dialysis with deionized water for 3 days, finally freeze-drying the dialyzed and purified product solution to form a white flocculent sample, and refrigerating and storing at 4 ℃.
In another aspect, embodiment 4 of the present invention discloses a method for preparing the hydrogel, which comprises the following steps:
s1: diluting Chs-ADH with deionized water to reach the mass percent concentration of 11%;
s2: diluting OS with deionized water to 5 wt%;
s3: mixing the diluted Chs-ADH and OS according to a volume ratio of 4:1, pouring the mixture into a cylindrical die with the height of 5mm, uniformly swirling, and standing for 12 hours at room temperature to ensure that the crosslinking reaction is completely finished;
s4: sterilizing the gel sample in 75% ethanol, and washing with pure water for three times.
Example 5
In one aspect, embodiment 5 of the present invention discloses a hydrogel, wherein the raw materials for preparing the hydrogel include two precursor macromolecules: chs-ADH and OS.
The precursor macromolecule Chs-ADH comprises the following preparation raw materials in parts by mass: 2 parts ChS, 0.72 part NHS, 1.2 parts EDC, 3.634 parts ADH, 75 parts deionized water.
The precursor macromolecule OS comprises the following preparation raw materials in parts by mass: 0.16 part of Salecan, 2.2 parts of sodium periodate, 0.5 part of diethylene glycol and 400 parts of deionized water.
The preparation method of the precursor macromolecule Chs-ADH comprises the following steps:
s1: dissolving 2 parts by mass of ChS in 40 parts by mass of deionized water, adding 0.72 part of NHS after dissolving, and then adding 1.2 parts of EDC;
s2: adjusting the pH of the system by adopting 1mol/L sodium hydroxide and 1mol/L hydrogen chloride to stabilize the pH of the system at 4.8, and activating for 2h;
s3:3.634 portions of ADH is dissolved in 35 portions of deionized water, the activated ChS solution is dropped in, and 1mol/L sodium hydroxide and 1mol/L hydrogen chloride are adopted to adjust the pH value of the system to be stable at 5.8;
s4: stirred at room temperature for 12h. The reaction system was dialyzed using a dialysis bag with a cut-off molecular weight of 3000 Da. The dialyzed purified solution was finally lyophilized to form a white sample, which was stored under refrigeration at 4 ℃.
The preparation method of the precursor macromolecule OS comprises the following steps:
s1:0.16 parts of Salecan was dissolved in 400 parts of deionized water;
s2: adding 2.2 parts of sodium periodate, reacting for 12 hours at room temperature in a dark place, and dropping 0.5 part of diethylene glycol to terminate the reaction;
s3: transferring the reaction solution into a dialysis bag with the cut-off molecular weight of 5000Da for dialysis by deionized water for 3 days, finally freeze-drying the dialyzed and purified product solution to form a white flocculent sample, and refrigerating and storing at 4 ℃.
In another aspect, embodiment 5 of the present invention discloses a method for preparing the hydrogel, which comprises the following steps:
s1: the mass percentage concentration of Chs-ADH diluted by deionized water is 11 percent;
s2: diluting OS with deionized water to 5 wt%;
s3: mixing the diluted Chs-ADH and OS in a volume ratio of 1:2, pouring the mixture into a cylindrical die with the height of 5mm, uniformly swirling, and standing at room temperature for 12 hours to ensure that the crosslinking reaction is completely finished;
s4: the gel sample was sterilized in 75% ethanol and then washed three times with pure water.
Comparative example 1
Hydrogel step S3: mixing the diluted Chs-ADH and OS according to the volume ratio of 1:3 into a cylindrical mold with a height of 5mm, vortexing uniformly, standing at room temperature for 12h to ensure complete crosslinking reaction, as in example 3.
Comparative example 2
The hydrogel preparation raw materials comprise 2 parts of ChS, 3.634 parts of ADH and 75 parts of deionized water, and the rest is the same as in example 3.
Comparative example 3
Hydrogel preparation starting material was 0.16 parts Salecan, as in example 3.
The performance test method comprises the following steps:
1. pore size distribution: the hydrogels obtained in examples 1 to 3 and comparative examples 1 to 3 were brittle-broken when frozen in a solid state in a refrigerator at-80 ℃ and then lyophilized in a lyophilizer (desktop Pro 31, virtis) to obtain SEM samples. The pore size of the samples was measured on the surface of the SEM (GeminiSEM 300, zeiss) stage by gold blasting for 1 minute at 5kV acceleration voltage, and the results are reported in the following table, examples 1-3SEM pictures are shown in FIG. 1 below.
Figure RE-GDA0003745583860000091
2. Acid and alkali resistance: the hydrogels obtained in examples 1 to 5 and comparative example 1 were incubated in PBS buffer solution (pH 5.0, 7.4 and 10.0) to reach a swollen state. After removing surface free water, the swelling weight of each sample was recorded, the hydrogel was lyophilized at-20 ℃ to dry the sample, the dry weight of each sample was recorded, and the Equilibrium Water Content (EWC) value of the sample was calculated as a measure of the acid and alkali resistance of the hydrogel, and the results are shown in fig. 2 below.
In the medium of pH 7.4, the EWC value of the hydrogel is generally lower than that in the medium of pH 6.0 and 10.0, which is attributed to the sensitivity of the polymer material to different acid-base environments and good acid-base resistance, the hydrogel in comparative example 1 is formed under a high content ratio of OS, the number of cross-linking sites is small, the structure is relatively loose, the highest EWC value is 39.7, and the acid-base resistance is poor.
3. And (3) testing mechanical properties: a general purpose system (WDW-50, hengle Xingge Instrument Co., ltd., jinan) was used to align real objects with fixed dimensions (diameter 10.7mm and height 5 mm)The samples of examples 1 to 5 were subjected to the test with the compressibility set to 5mm min -1 The load-displacement curve was used to calculate the compressive strength, and the results are shown in FIG. 3.
The mechanical properties of the hydrogel (. Apprxeq.10 kPa) also show potential applications in 3D encapsulation and cell regulation of cells.
4. In vitro cytotoxicity assay: the in vitro toxicity of examples 1-4 was determined by MTT according to ISO 10993-5, and the results are shown in FIG. 4 below.
All hydrogel samples were non-toxic in vitro, and the addition of different ratios of ChS-ADH and OS promoted cell growth, which is attributable to the good biocompatibility of the hydrogel matrix and the mildness of the crosslinking reaction. The cell viability rates were all calculated to be greater than 100%. In particular, the hydrogels of example 2 and example 3 have the most significant effect on promoting cell biological growth, and prove the nature of good biocompatibility of the hydrogels, which becomes an important guarantee for good candidate materials in the biomedical field.
5. Swelling and degradation behavior study: the hydrogel prepared in example 3 was soaked in PBS solution (0.01M, pH 7.4) containing 0.05mg/mL papain at 37 deg.C, the shaking chamber was set at 100rpm, and the degradation and swelling characteristics under constant shaking were shown in FIG. 5.
The degradation and swelling properties of the hydrogels were expressed by measuring the change in weight of the hydrogels in PBS at 37 ℃, as shown in figure 5. The hydrogel mass decreased by 24.6% in 2 hours. After 24 hours of degradation, the product still retains 59.01 percent and shows a relatively regular degradation curve. In the initial stage, the rapid degradation of the sample may be attributed to the relatively loose structure inside the sample. In the sample, the free water has stronger fluidity, and the enzyme solution and the macromolecular skeleton are easier to contact with each other, so the degradation speed of the sample is relatively higher. Over the next few hours, the degradation curve tends to be more regular and controllable. The increase in swelling ratio during degradation indicates that the total water holding capacity of the hydrogel can be maintained in an ideal state for a long period of time, although the quality of the hydrogel polymer during degradation of the hydrogel is reduced. The biodegradability of naturally derived polymers is combined with the good water retention of hydrogels, and the hydrogels disclosed herein are ideal materials for drug release and tissue engineering.
6. 3D packaging of cells: to further evaluate the ability of the hydrogels to effectively act as extracellular matrix mimics, encapsulation and culture of L-O2 cells were performed on the hydrogels prepared in example 3. The precursor macromolecule solution was prepared with complete medium (90% dmem high-glucose medium, 10% fetal bovine serum, 1% diabody (streptomycin, penicillin)). Adding one precursor macromolecule solution and cell suspension into a 24-well plate, mixing well, adding the other precursor macromolecule solution, rapidly mixing well, and adding CO at 37 deg.C 2 And standing the incubator to obtain the 3D cell culture hydrogel support. The cell density was: 40000/well. After crosslinking was complete, 1mL of complete medium was added to each well and the medium was changed every 2 days. The hydrogel sheet coated with L-O2 cells was immersed in a PBS solution containing HoeRhSt 33342 (2 μm) and propidium iodide (PI, 2 μm) at a set time and immersed at 37 ℃ for 30 minutes. After washing the dye with PBS solution, images of stained cells were taken under an inverted fluorescence microscope, as shown in fig. 6.
FIG. 6 shows inverted fluorescence microscope images of L-O2 cells in the hydrogels prepared in example 3 stained with Hoechst33342/PI after 1,3, 5 and 7 days of culture. From the images, it can be observed that the density of the cell population increased significantly during the 7 day culture period, indicating that the L-O2 cells were able to proliferate in the 3D microenvironment of the hydrogel. Example 3 the high survival and proliferation rate of L-O2 cells in the hydrogel is attributed to the good biocompatibility of the polymer and the mildness of the crosslinking reaction. Compared with the traditional permanent chemical crosslinking hydrogel, the hydrogel with the reversible connection can provide a more bionic microenvironment and shows a plurality of advantages in 3D cell culture. The hydrogel can promote the exchange of nutrients and metabolites between cells and the external environment and enable the cells to play certain cell functions (proliferation, migration and the like) through the breakage and recombination of reversible bonds. The above results indicate that hydrogel is a viable 3D cell culture matrix with great potential in cell therapy and tissue regeneration.
7. Drug release experiments: vancomycin is added into the ChS-ADH solution and mixed evenly, then the OS solution is added, the other preparation steps are the same as those in the example 3, and the mass of the vancomycin in the obtained hydrogel is 10mg.
A standard curve of vancomycin in PBS (pH 7.4) solution was established for drug release quantification. In drug release experiments, the hydrogel containing vancomycin was encapsulated in a dialysis bag with a cut-off molecular weight of 1000Da soaked in 50mL of PBS (pH 7.4) and shaken continuously at 37 ℃ at 100 rpm. At predetermined time points, 5ml of supernatant was removed and an equal volume of fresh PBS solution was added to make up the solution volume. The cumulative release of vancomycin was measured at a wavelength of 280nm using a UV spectrophotometer (S-3100, SCICON). The drug release behavior of CS hydrogels was analyzed using various kinetic models (zero order, first order, higuchi and Ritger-Peppas). The best model was determined by best fit correlation, and the results are shown in FIG. 7.
As shown in fig. 7, vancomycin was released from the hydrogel (example 3) in a sustained manner. The hydrogel samples showed a significant burst at ph5.0, with a rapid trend toward a gradual release of the drug. At 240min, the hydrogel released 5.64% and 1.46% at pH5.0 and 7.4, respectively; at 720min, the hydrogel released 22.29% and 13.27% at pH5.0 and 7.4, respectively, showing pH dependence and better sustained release behavior.
8. Performance testing of precursor molecules
Nuclear magnetic spectrum comparison of the precursor macromolecule ChS-ADH synthesized in the aqueous phase by the EDC/NHS method synthesized in example 3 for ADH grafted ChS with the ChS grafted with only ADH of comparative example 2, the results are shown in FIG. 8.
ChS-ADH precursors were synthesized using the EDC/NHS scheme, as shown in FIG. 8. Comparing the 1H NMR spectra of ChS-ADH and ChS, the additional signal at 1.40-1.60 of the former can be attributed to the hydrogen on the ADH methylene, respectively, indicating successful amidation. The degree of substitution (number of ADH molecules per 100 ChS repeat units) in the ChS-ADH precursor was approximately 39.2%.
(2) The nuclear magnetic spectrum comparison of the OS synthesized in example 3 with Salecan of comparative example 3 showed that the result is shown in FIG. 9.
As shown in FIG. 9, another precursor OS was synthesized in the aqueous phase. Comparing 1H NMR spectra of Salecan and OS, the latter signals were separable at 5.40ppm and 5.60ppmOther than due to the hemiacetal proton formed by the aldehyde and the adjacent hydroxyl group. Furthermore, comparison of the infrared spectra may also verify successful oxidation reactions. 1725cm -1 The peak at (a) correlates with aldehyde groups, indicating the formation of OS. The degree of oxidation of OS (defined as the number of aldehyde groups per 100 saccharide units) is about 18% based on the hydroxylamine hydrochloride method.

Claims (10)

1. A hydrogel prepared from starting materials comprising two precursor macromolecules: chs-ADH and OS.
2. The hydrogel according to claim 1, wherein the precursor macromolecules Chs-ADH comprise at least the following raw materials: 1-3 parts of chondroitin sulfate, 0.3-1 part of N-hydroxysuccinimide, 1-3 parts of 1-3-dimethylaminopropyl-3-ethylcarbodiimide, 3-5 parts of adipic acid dihydrazide and 60-110 parts of deionized water.
3. The hydrogel according to claim 1, wherein the precursor macromolecule OS comprises at least the following preparation raw materials in parts by mass: 0.1-0.6 part of cycoladine, 1-3 parts of sodium periodate, 0.1-1 part of diglycol and 300-500 parts of deionized water.
4. The hydrogel of claim 2, wherein the precursor macromolecules Chs-ADH are prepared by a method comprising at least the following steps:
s1: dissolving 1-3 parts by mass of chondroitin sulfate in 30-60 parts by mass of deionized water, adding 0.3-1 part by mass of N-hydroxysuccinimide after dissolving, and then adding 1-3 parts by mass of 1-3-dimethylaminopropyl-3-ethylcarbodiimide;
s2: activating for 1-3h, and stabilizing the pH value at 4-5 to obtain activated chondroitin sulfate solution;
s3:3-5 parts of adipic acid dihydrazide is dissolved in 30-50 parts of deionized water, and the activated chondroitin sulfate solution is dripped in, so that the pH value of the system is stabilized at 5-6.5;
s4: stirring at room temperature for 8-16h, and dialyzing and purifying to obtain Chs-ADH.
5. The hydrogel according to claim 3, wherein the precursor macromolecule OS is prepared by a method comprising at least the steps of:
s1:0.1-0.6 parts of sodium cellulose can be dissolved in 300-500 parts of deionized water;
s2: adding 1-3 parts of sodium periodate, reacting for 10-14h at room temperature in a dark place, and dropping 0.1-1 part of diethylene glycol to terminate the reaction;
s3: dialyzing for 2-4 days, and purifying to obtain OS.
6. A method for preparing the hydrogel according to claim 1, comprising at least the steps of:
s1: diluting Chs-ADH by deionized water;
s2: diluting and diluting OS with deionized water;
s3: pouring the diluted Chs-ADH and OS into a mold, uniformly swirling, and standing at room temperature for 10-14h;
s4: and (5) sterilizing and cleaning.
7. The method for preparing the hydrogel according to claim 6, wherein the mass percentage concentration of the Chs-ADH diluted by the deionized water in the preparation step S1 is 8-15%.
8. The method for preparing a hydrogel according to claim 6, wherein the preparing step S2 comprises diluting OS with deionized water to a concentration of 2-7% by mass.
9. The method for preparing the hydrogel according to claim 6, wherein the diluted Chs-ADH and OS in the preparation step S3 are mixed according to a volume ratio of (1-5): (0.5.
10. Use of the hydrogel according to claims 1 to 5 in biomedical applications as dermal filler material, drug delivery vehicle, 3D cell culture.
CN202210541202.1A 2022-05-17 2022-05-17 Hydrogel, preparation method and application Pending CN115232371A (en)

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KR20190012589A (en) * 2017-07-28 2019-02-11 전북대학교산학협력단 Gellan-gum Hydrogels Composition containing Chondroitin Sulfate
CN109498838A (en) * 2018-12-29 2019-03-22 广州贝奥吉因生物科技有限公司 A kind of injectable cartilage repair hydrogel and preparation method thereof
WO2021162529A1 (en) * 2020-02-14 2021-08-19 연세대학교 산학협력단 Phenol derivative-functionalized chondroitin sulfate hydrogel and use of same
CN113999404A (en) * 2021-10-09 2022-02-01 昆明理工大学 Preparation method of double-cross-linked stem cell sphere hydrogel for osteoarthritis

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20190012589A (en) * 2017-07-28 2019-02-11 전북대학교산학협력단 Gellan-gum Hydrogels Composition containing Chondroitin Sulfate
CN109498838A (en) * 2018-12-29 2019-03-22 广州贝奥吉因生物科技有限公司 A kind of injectable cartilage repair hydrogel and preparation method thereof
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