CN112315976B

CN112315976B - Injectable antibacterial composite hydrogel crosslinked by silver-loaded attapulgite, and preparation method and application thereof

Info

Publication number: CN112315976B
Application number: CN202011280301.6A
Authority: CN
Inventors: 刘志雄; 冯锋; 秦君; 郝晓宇; 张东旭
Original assignee: Shanxi Datong University
Current assignee: Shanxi Datong University
Priority date: 2020-11-16
Filing date: 2020-11-16
Publication date: 2022-10-11
Anticipated expiration: 2040-11-16
Also published as: CN112315976A

Abstract

An injectable antibacterial composite hydrogel crosslinked by silver-loaded attapulgite, a preparation method and application thereof, which belong to the field of hydrogels and can solve the problems of aggregation and agglomeration of silver nanoparticles in a base material, sudden release, complex preparation process, long period, poor antibacterial timeliness and difficult large-scale application existing in the method of organically combining nano silver and hydrogel, and the preparation method comprises the following steps: mixing a water-soluble monomer and an initiator to initiate polymerization to obtain a pre-polymerization solution; ultrasonically dispersing attapulgite in a siloxane precursor-removed aqueous solution, adding an acid catalyst, filtering, and washing to remove redundant siloxane precursors; ultrasonically dispersing the filtered precipitate in deionized water solution containing a silver source, filtering after saturation adsorption, and washing to obtain the attapulgite material loaded with silver ions; adding the hydrogel into a prepolymerization solution, uniformly dispersing by ultrasonic, and illuminating to obtain the injectable antibacterial composite hydrogel. The injectable antibacterial composite hydrogel has the properties of spreadability, injectability and the like.

Description

Injectable antibacterial composite hydrogel crosslinked by silver-loaded attapulgite, and preparation method and application thereof

Technical Field

The invention belongs to the technical field of hydrogel, and particularly relates to an injectable antibacterial composite hydrogel crosslinked by silver-loaded attapulgite, and a preparation method and application thereof.

Background

Silver nanoparticles (AgNPs) have a high specific surface area, have a very high broad-spectrum killing effect on yeast, aerobic bacteria, fungi, and viruses, and have no report that any bacteria have resistance to them so far, compared to other common antibacterial agents, and thus, silver nanoparticles have been attracting much attention as an effective antibacterial agent. The traditional synthesis method of the silver nanoparticles is a chemical reduction method, and a large amount of toxic chemical reagents such as reducing agents, stabilizing agents and the like are usually required to be added, and the chemical reagents have high cytotoxicity and relatively poor biocompatibility. In addition, the silver nanoparticles are easy to agglomerate and disperse unevenly when the concentration of the silver nanoparticles is high, so that the antibacterial performance of the nano silver is seriously influenced.

A hydrogel is a water-swollen, three-dimensional polymer network. The water-soluble polymer forms a network structure in a physical or chemical crosslinking mode and carries a large amount of moisture, so that the hydrogel is obtained. The hydrogel has similarity with animal soft tissue, has extremely high water content, controllable porous structure and mechanical strength, allows gas exchange, and has certain barrier effect on microorganisms, so that the wound is promoted to heal quickly, and the wound can be easily removed without trauma. However, the traditional hydrogel is brittle and easy to be damaged and lose efficacy by machinery, and on the other hand, the antibacterial property is poor, and the moist environment often causes bacteria at the wound to be rapidly propagated, so that the wound is inflamed and other symptoms are caused; the nano-composite hydrogel formed by organically combining the nano-silver with excellent antibacterial performance and the hydrogel keeps the hydrophilicity and softness of the hydrogel material, and simultaneously endows the hydrogel with excellent antibacterial performance, thereby providing a new idea for developing hydrogel wet dressings. At present, the most common preparation method is that the prepared hydrogel is soaked in a silver ion-containing aqueous solution, and after adsorption saturation, chemical reagents such as a reducing agent, a stabilizing agent and the like are added to reduce silver ions into nano silver; the nano-silver composite hydrogel material prepared in the way usually has the problems of aggregation and agglomeration of AgNPs in a matrix material, burst release and the like, and in addition, harmful chemical agents in a hydrogel system are removed; therefore, the preparation process is complex, the period is long, the antibacterial timeliness is poor, and the large-scale application is difficult.

Disclosure of Invention

The invention provides an injectable antibacterial composite hydrogel crosslinked by silver-loaded attapulgite, a preparation method and application thereof, aiming at the problems of aggregation, agglomeration and 'burst release' of silver nanoparticles in a matrix material, complex preparation process, long period, poor antibacterial timeliness and difficult large-scale application of a method for organically combining nano silver and hydrogel.

The invention adopts the following technical scheme:

an injectable antibacterial composite hydrogel crosslinked by silver-loaded attapulgite comprises 5-20wt% of base material and 80-95wt% of deionized water, wherein the base material comprises 1-10wt% of attapulgite material loaded with nano silver and 90-99wt% of hydrophilic polymer.

A preparation method of injectable antibacterial composite hydrogel crosslinked by silver-loaded attapulgite comprises the following steps:

firstly, mixing a water-soluble monomer and an initiator, and initiating polymerization under the stirring condition to obtain a pre-polymerization solution;

secondly, ultrasonically dispersing the attapulgite in a siloxane-containing precursor aqueous solution, adding an acid catalyst under the stirring condition, after the reaction is finished, filtering and washing to remove redundant siloxane precursors to obtain modified attapulgite, ultrasonically dispersing the filtered modified attapulgite in a deionized water solution containing a silver source, adsorbing and saturating, filtering, and washing to obtain the attapulgite material loaded with silver ions;

and thirdly, adding the attapulgite loaded with silver ions into the prepolymerization solution, and irradiating to obtain the injectable antibacterial composite hydrogel after uniform ultrasonic dispersion.

In the first step, the water-soluble monomer includes one or more of water-soluble monomers containing a nitrogen atom.

Preferably, the water-soluble monomer in the first step includes one or more of N-isopropylacrylamide, methacrylamide, N- [ tris (hydroxymethyl) methyl ] acrylamide, N-vinylcyclohexamide, N-acryloyl-N-alkylpiperazine, diethylacrylamide, isopropylacrylamide acrylate, N-hydroxymethylpropylacrylamide, vinylmethyloxazolidinone, and N-vinylcaprolactam.

In the first step, the initiator comprises any one of benzophenone, ammonium persulfate and potassium persulfate.

In the first step, the mass ratio of the water-soluble monomer to the initiator is (0.1-1): 100.

in the second step, the particle diameter of the attapulgite is 100-325 meshes, and the concentration of the attapulgite is 0.1-10wt%.

In the second step, the siloxane precursor comprises one or more than two of siloxane precursors containing sulfydryl and/or free amino, and the concentration of the siloxane precursor is 1-10wt%.

The siloxane precursor containing sulfydryl comprises propyl trimethoxy silane, mercaptopropyl triethoxy silane, bis- [3- (triethoxy silicon) propyl ] -tetrasulfide or bis- [3- (triethoxy silicon) propyl ] -disulfide.

The siloxane precursor containing free amino comprises gamma-aminopropyltriethoxysilane, N- (beta-aminoethyl) -gamma-aminopropyltrimethoxysilane, N- (beta-aminoethyl) -gamma-aminopropyltriethoxysilane, 3-ureidopropyltrimethoxysilane, 3-ureidopropyltriethoxysilane, 4-amino-3,3-dimethylbutyltrimethoxysilane or diethylenetriaminopropyltrimethoxysilane.

The acid catalyst comprises HCOOH, C ₂ H ₅ COOH、HCl、H ₂ SO ₄ 、HNO ₃ 、H ₃ PO and HBr.

The mass ratio of the acid catalyst to the siloxane precursor is 10 ^-6 ～0.1：1。

The silver source comprises any one or more than two of silver nitrate, silver trifluoromethanesulfonate and silver trifluoromethyl acetate.

The mass ratio of the silver source to the modified attapulgite is (0.1-1): 1.

the illumination includes one of ultraviolet illumination or visible light illumination.

An injectable antibacterial composite hydrogel crosslinked by silver-carrying attapulgite has injectable and spreadable properties, and can be applied to antibacterial agents and wound dressings.

The design idea of the invention is as follows: the attapulgite is an aluminosilicate mineral which has a rod-shaped crystal structure, contains water and is rich in magnesium, has a rod crystal length of about 1 to 5um and a diameter of 20-70 nanometers, and is a natural one-dimensional nano material. The surface of the attapulgite shows negative electricity because of containing a large amount of hydroxyl, which is very favorable for modifying and loading the metal nano material. Therefore, firstly, the surface of the attapulgite is modified with sulfhydryl functional groups, and a large amount of hydroxyl and sulfhydryl functional groups on the surface of the attapulgite can greatly increase the silver carrying capacity; and then dispersing the nano silver into a hydrophilic polymer with a branched chain containing nitrogen atoms, forming nano silver after ultraviolet irradiation, taking the nano silver loaded attapulgite material as a multi-site cross-linking agent, and forming a stable hydrogel material by the dynamic coordination action of the nano silver on the surface and the nitrogen atoms in the hydrophilic polymer. The introduction of the attapulgite loaded with the nano silver can improve the mechanical property of the hydrogel material on the one hand, and on the other hand, a large number of functional groups (such as hydroxyl, sulfydryl and the like) on the surface of the attapulgite and nitrogen-containing functional groups on the surface of the hydrophilic polymer have synergistic action, so that the nano silver can be effectively dispersed and stabilized, the aggregation inactivation and 'burst release' of the nano silver groups can be avoided, the antibacterial property of the hydrogel material can be fully improved and prolonged, and the injectable antibacterial composite hydrogel has wide application prospects in the fields of wound healing and the like.

The invention has the following beneficial effects:

1. the attapulgite serving as a natural one-dimensional nano material is introduced into a hydrogel system, so that the mechanical properties (such as toughness and the like) of the hydrogel can be effectively improved.

2. The synergistic effect of a large amount of functional groups such as hydroxyl, sulfydryl and the like on the surface of the attapulgite and the nitrogen-containing functional groups on the surface of the hydrophilic polymer can effectively improve the load capacity of the nano silver, simultaneously efficiently disperse and stabilize the nano silver, avoid the aggregation inactivation and 'burst release' of the nano silver groups, and fully improve and prolong the antibacterial performance of the hydrogel material.

3. The preparation of the nano silver adopts a light reduction method, avoids using a large amount of toxic reducing agents and dispersing agents, has mild reaction conditions, and is green and environment-friendly.

4. The preparation process of the injectable antibacterial composite hydrogel crosslinked by the silver-loaded attapulgite is simple, the period is short, harsh reaction conditions are not needed, and the large-scale production is facilitated.

Drawings

FIG. 1 is a photograph of a hydrogel sample in example 1 of the present invention.

Fig. 2 is a TEM image of silver nanoparticles in example 1 of the present invention.

Fig. 3 is an ultraviolet-visible absorption spectrum of silver nanoparticles in example 1 of the present invention.

FIG. 4 is a tensile stress strain curve for the hydrogels of inventive example 1 and comparative example 1.

Fig. 5 is a release curve of elemental silver in example 1 of the present invention and comparative example 1.

FIG. 6 shows Escherichia coli (E. coli) The photographs were attached to the hydrogels of comparative example 1 (a), comparative example 2 (b) and inventive example 1 (c).

Detailed Description

secondly, ultrasonically dispersing the attapulgite in a water solution containing an organosilane precursor, adding an acid catalyst under the stirring condition, filtering and washing to remove the redundant organosilane precursor after the reaction is finished, ultrasonically dispersing the filtered precipitate in a deionized water solution containing a silver source, and filtering and washing after adsorption saturation to obtain the attapulgite material loaded with silver ions;

Example 1

(1) Dissolving 10g N-isopropylacrylamide (NIPAM) and 0.1g Ammonium Persulfate (APS) in 90mL of deionized water, introducing nitrogen for 30min to remove oxygen in the system, and heating to initiate polymerization to obtain a pre-polymerization solution.

(2) Adding 5g of attapulgite and 5g of mercaptopropyltriethoxysilane into 90mL of deionized water, ultrasonically dispersing for 3h, adding one drop (about 0.05 mL) of acetic acid solution with the concentration of 0.01M, continuously stirring for 1h, filtering, washing the precipitate with water and ethanol for three times, and drying; ultrasonically dispersing the prepared modified attapulgite into 100mL of deionized water, slowly adding 0.5g of silver nitrate, continuously stirring for 2h, filtering, washing the precipitate with the deionized water for three times, and drying to obtain the silver ion-loaded attapulgite.

(3) And (2) adding 1g of attapulgite loaded with silver ions into the pre-polymerization solution obtained in the step (1), and irradiating by ultraviolet light for 30min to obtain the injectable antibacterial composite hydrogel.

Example 2

(1) 5g of methacrylamide and 0.05g Ammonium Persulfate (APS) are dissolved in 95mL of deionized water, nitrogen is introduced for 30min to remove oxygen in the system, and polymerization is initiated by heating to obtain a pre-polymerization solution.

(2) Adding 1g of attapulgite and 10g of gamma-aminopropyltriethoxysilane into 89mL of deionized water, ultrasonically dispersing for 3h, adding one drop (about 0.05 mL) of formic acid solution with the concentration of 0.01M, continuously stirring for 1h, filtering, washing the precipitate with water and ethanol for three times, and drying; ultrasonically dispersing the prepared modified attapulgite into 100mL of deionized water, slowly adding 1g of silver triflate, continuously stirring for 2h, filtering, washing the precipitate with the deionized water for three times, and drying to obtain the attapulgite loaded with silver ions.

(3) And (2) adding 0.05g of attapulgite loaded with silver ions into the pre-polymerization solution obtained in the step (1), and irradiating by ultraviolet light for 30min to obtain the injectable antibacterial composite hydrogel.

Example 3

(1) Dissolving 10 g-hydroxymethyl propyl acrylamide and 0.01 g Ammonium Persulfate (APS) in 90mL of deionized water, introducing nitrogen for 30min to remove oxygen in the system, and heating to initiate polymerization to obtain a pre-polymerization solution.

(2) Adding 1g of attapulgite and 1g of mercaptopropyltriethoxysilane into 98mL of deionized water, ultrasonically dispersing for 3h, adding one drop (about 0.05 mL) of sulfuric acid solution with the concentration of 0.01M, continuously stirring for 1h, filtering, washing the precipitate with water and ethanol for three times, and drying; ultrasonically dispersing the prepared modified attapulgite into 100mL of deionized water, slowly adding 0.1g of silver nitrate, continuously stirring for 2h, filtering, washing the precipitate with deionized water for three times, and drying to obtain the silver ion loaded attapulgite.

(3) And (2) adding 0.5g of attapulgite loaded with silver ions into the pre-polymerization solution obtained in the step (1), and irradiating by ultraviolet light for 30min to obtain the injectable antibacterial composite hydrogel.

Comparative example 1

Dissolving 10g of N-isopropylacrylamide (NIPAM), 0.1g Ammonium Persulfate (APS) and 0.1g of N, N-Methylene Bisacrylamide (MBA) in 90mL of deionized water, introducing nitrogen for 30min to remove oxygen in the system, heating to initiate polymerization to obtain hydrogel crosslinked by covalent bonds, placing the hydrogel in 0.1 g/mL of silver nitrate solution, taking out after adsorption saturation, wiping off water on the surface by using filter paper, and irradiating by ultraviolet light for 30min to obtain the covalently crosslinked nano-silver-containing hydrogel material.

Comparative example 2

(2) Adding 5g attapulgite and 5g mercaptopropyltriethoxysilane into 90mL deionized water, ultrasonically dispersing for 3h, adding one drop (about 0.05 mL) of 0.01M acetic acid solution, stirring for 1h, filtering, washing precipitate with water and ethanol for three times, and drying

(3) And (2) adding 1g of modified attapulgite into the premixed liquid in the step (1), and irradiating by ultraviolet light for 30min to obtain the injectable antibacterial composite hydrogel.

The samples prepared by the inventive examples and the comparative samples were compared for relevant properties:

(1) Hydrogel samples prepared by the plate method were cut into small Bao Changtiao (size 5.0 cm × 1.0 cm × 0.13 cm). The pull-up rate was set to 20 mm/min. The tensile strain at break and the tensile stress at break are the stress and strain, respectively, occurring at the point of break.

From FIG. 4, it can be seen that the BIS crosslinked PHEMA hydrogel (comparative example 1) fractured at a stress of 0.42 MPa with a strain of only 30%, whereas the hydrogel in example 1 had a stress of only 0.24 MPa at fracture but had a strain of about 120% which is 4 times that of the hydrogel in comparative example 1, which indicates that the nanosilver-loaded attapulgite material effectively improved the toughness of the hydrogel.

(2) The hydrogel samples were made into cylinders of 12 mm diameter and height 15 mm and placed in the center of the test station and then compressed from top to bottom with a load compression rate of 1.0 mm/min. When the strain of the compression test reaches 80%, the compression test process ends.

The compression test of the hydrogel showed that the hydrogel in comparative example 1 was damaged at a strain of 47%, the stress at failure was 1.5 MPa, and the hydrogel loaded with nanosilver (example 1) was not damaged even when the strain reached 80%. The stress of the hydrogel reaches 2.5 Mpa when the hydrogel is compressed to 80 percent, and the stress of the hydrogel still reaches 1.6 Mpa after the hydrogel is compressed for the second time. These results show that the introduction of the attapulgite loaded with nano-silver significantly improves the mechanical properties of the hydrogel.

(3) The release of silver from the hydrogel was performed in saline and dried hydrogel pieces (1.0 + -0.2 mm in diameter, 0.2 + -0.05 mm in thickness, 10 + -2 mg in mass) were placed in sample vials containing 10 mL saline. The sample vial was shaken in a water bath shaker and the temperature was controlled at 25 ℃. After 1, 3, 5, 15, 28 days, two milliliters of solution was removed from the sample and fresh 2.0 mL saline was added. 1.5 mL concentrated nitric acid is added into the taken solution, and after digestion for 4 hours at 80 ℃, the solution is cooled to be fixed to 10 mL, and then the concentration of silver in a sample is measured.

The release kinetics of silver in the hydrogel is researched, and the content of silver in the solution is measured by controlling time sampling, so that the relation between the cumulative release amount of Ag in the hydrogel and the time can be obtained. It can be seen from fig. 5 that the cumulative release amount gradually increases with time, and does not show a linear increase but a tendency to increase sharply and then decrease. The hydrogel system in comparative example 1 released silver ions at a concentration of up to 3.8mg/g after 28 days, whereas the silver ion released concentration in example 1 was only 0.7mg/g. The results show that the introduction of the attapulgite loaded with the nano silver obviously improves the slow release performance of silver ions, thereby prolonging the action aging of the silver ions.

(4) In order to further research the antibacterial capability of the hydrogel, the adhesion condition of escherichia coli on the surface of the hydrogel is researched, the escherichia coli is adhered on the surface of the hydrogel in large quantity in a comparative example 1 (figure 6 a), after nano-silver is introduced into the system, the adhesion quantity of the escherichia coli on the surface of the hydrogel in the comparative example 1 (figure 6 b) is obviously reduced due to the excellent antibacterial performance of the nano-silver, the attapulgite carrying the nano-silver is introduced into the hydrogel system, the loading quantity of the nano-silver is further improved, and meanwhile, due to the slow release effect, the hydrogel system can effectively avoid the 'burst release' of the nano-silver, and the higher concentration of the nano-silver is maintained, so that the optimal antibacterial performance is shown (figure 6 c).

Claims

1. A preparation method of injectable antibacterial composite hydrogel crosslinked by silver-loaded attapulgite is characterized by comprising the following steps: the composite hydrogel comprises 5-20wt% of base material and 80-95wt% of deionized water, wherein the base material comprises 1-10wt% of nano-silver loaded concave-convex rod material and 90-99wt% of hydrophilic polymer; the preparation method comprises the following steps:

thirdly, adding the attapulgite loaded with silver ions into the prepolymerization solution, and after uniform ultrasonic dispersion, illuminating to obtain injectable antibacterial composite hydrogel;

wherein the water-soluble monomer in the first step comprises one or more of N-isopropylacrylamide, methacrylamide, N- [ tris (hydroxymethyl) methyl ] acrylamide, N-vinylcyclohexamide, N-acryloyl-N-alkylpiperazine, diethylacrylamide, isopropylacrylamide acrylate, N-hydroxymethylpropylacrylamide, vinylmethyloxazolidinone, and N-vinylcaprolactam;

in the second step, the siloxane precursor comprises one or more than two of siloxane precursors containing sulfydryl and/or free amino;

the silver source comprises any one or more than two of silver nitrate, silver trifluoromethanesulfonate and silver trifluoromethylacetate;

the illumination is ultraviolet illumination.

2. The preparation method of the injectable antibacterial composite hydrogel crosslinked by the silver-loaded attapulgite according to claim 1, which is characterized in that: in the first step, the initiator comprises any one of benzophenone, ammonium persulfate and potassium persulfate; the mass ratio of the water-soluble monomer to the initiator is (0.1-1): 100.

3. the preparation method of the injectable antibacterial composite hydrogel crosslinked by the silver-loaded attapulgite according to claim 1, which is characterized in that: in the second step, the particle size of the attapulgite is 100-325 meshes, and the concentration of the attapulgite is 0.1-10wt%; the concentration of the siloxane precursor in the second step is 1-10wt%.

4. The preparation method of the injectable antibacterial composite hydrogel crosslinked by the silver-loaded attapulgite according to claim 1, which is characterized in that: the siloxane precursor containing sulfydryl comprises propyl trimethoxy silane, mercaptopropyl triethoxy silane, bis- [3- (triethoxy silicon) propyl ] -tetrasulfide or bis- [3- (triethoxy silicon) propyl ] -disulfide.

5. The preparation method of the injectable antibacterial composite hydrogel crosslinked by the silver-loaded attapulgite according to claim 1, which is characterized in that: the siloxane precursor containing free amino comprises gamma-aminopropyltriethoxysilane, N- (beta-aminoethyl) -gamma-aminopropyltrimethoxysilane, N- (beta-aminoethyl) -gamma-aminopropyltriethoxysilane, 3-ureidopropyltrimethoxysilane, 3-ureidopropyltriethoxysilane, 4-amino-3,3-dimethylbutyltrimethoxysilane or diethylenetriaminopropyltrimethoxysilane.

6. The preparation method of the injectable antibacterial composite hydrogel crosslinked by the silver-loaded attapulgite according to claim 1, which is characterized in that: the acid catalyst comprises HCOOH, C ₂ H ₅ COOH、HCl、H ₂ SO ₄ 、HNO ₃ 、H ₃ Any one or more of PO and HBr; the mass ratio of the acid catalyst to the siloxane precursor is 10 ^-6 -0.1: 1; the mass ratio of the silver source to the modified attapulgite is (0.1-1): 1.

7. the application of the composite hydrogel prepared by the preparation method of the injectable antibacterial composite hydrogel crosslinked by the silver-loaded attapulgite in the preparation of antibacterial agents and wound dressings is characterized in that: the composite hydrogel has injectable and spreadable properties.