CN115735949B - Polyoxometallate crosslinked polyethylene glycol modified chitosan sponge composite material and preparation method and application thereof - Google Patents
Polyoxometallate crosslinked polyethylene glycol modified chitosan sponge composite material and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to a polyethylene glycol modified chitosan crosslinked polyacid sponge composite material, wherein the chemical formula of the polyacid is as follows: [ HL ]] 6 H 2 [Cu(H 2 O) 3 (P 2 Mo 5 O 23 )] 2 •4H 2 O, hl=2-aminopyridine; the invention utilizes electrostatic interaction and hydrogen bond interaction between polyethylene glycol modified chitosan material and anion component polyacid with good antibacterial effect to construct a sponge composite material, and then researches on antibacterial application are carried out. The system can not only keep certain stability of polyacid under physiological conditions, but also improve the recycling efficiency and biological efficiency; in addition, the sponge composite material has the advantages of easy circulation, easy operation, low toxicity and the like, and provides a valuable reference for future application in the antibacterial field.
Description
Technical Field
The polyacid-based composite material with high-efficiency antibacterial performance is developed around serious bacterial infection and environmental management problems. According to the invention, a polyoxometallate crosslinked polyethylene glycol modified chitosan sponge composite material is constructed by utilizing a polyethylene glycol modified chitosan material and an anionic component polyacid with good bacteriostasis through electrostatic interaction, and antibacterial application is studied. The system can not only keep certain stability of polyacid under physiological conditions, but also improve the recycling efficiency and biological efficiency; in addition, the sponge composite material has the advantages of easy circulation, easy operation, low toxicity and the like, and provides a valuable reference for future application in the antibacterial field.
Background
In recent years, rapid progress in industrialization and city has caused problems of deterioration of water quality, resulting in gradual increase of pathogenic bacteria, organic pollutants and the like in water. Pathogenic bacteria are one of the most common pollutants in water bodies, and meanwhile, the wide use of antibiotics not only increases the drug resistance of bacteria, but also can cause the occurrence of superbacteria, so that the problem of bacterial infection forms a great threat to the global public safety and ecological environment. Therefore, economic, environment-friendly and efficient antibacterial materials have attracted a great deal of attention. Based on the problem of water pollution caused by pathogenic microorganisms, new strategies are urgently needed to synthesize non-toxic, environmentally friendly, efficient composites to alleviate these concerns.
Polyoxometallates are unique nanoscale polyanion clusters composed of early transition metal oxides with potential application potential in biology, medicine, materials science, and water treatment. POMs and POM-based systems are considered to be promising metal drugs in the future due to their biological and biochemical effects. In particular, POMs have outstanding biological potential in the treatment of cancer, alzheimer's disease, diabetes, and infections associated with viruses and bacteria.
Among the composite materials, polyoxometallate-based organic-inorganic hybrid materials are receiving a great deal of interest and attention due to their excellent properties. However, polyacids also suffer from the disadvantages of low solubility, difficulty in recovery and instability under physiological conditions. Detailed literature studies have shown that the loading of polyacids in low cost carriers with remarkable stability, reproducibility and various functional groups, especially carriers with biodegradability and biocompatibility, is very attractive. In some biological carrier materials, chitosan (CTs) consists of beta- (1-4) -D-glucosamine and N-acetamido-di-glucose units, and has the advantages of special structure, no toxic or side effect, good biocompatibility, high biodegradation rate, strong drug loading property and the like, so that the chitosan has excellent properties of hygroscopicity, film forming property, gel property, bacteriostasis and the like, and is widely applied to the fields of foods, medical health, biomedicine, daily necessities and the like. However, due to its low solubility and colloidal stability under physiological conditions, it remains challenging in biological applications. Therefore, preparing efficient polyacid-based functional composites remains an important and challenging task. In addition, in order to construct an effective, efficient and stable antibacterial treatment agent, the antibacterial mechanism of the synthesized composite material is known, and the multifunctional nanocomposite material is synthesized by using an ultrasonic-assisted self-assembly strategy and the antibacterial performance of the multifunctional nanocomposite material is further explored.
Disclosure of Invention
In order to overcome the defects, the application considers that polyethylene glycol chitosan decrosslinking polyacid is a viable strategy, and the invention aims to provide a polyoxometallate crosslinked polyethylene glycol modified chitosan sponge composite material, a preparation method and application thereof, wherein polyethylene glycol is used for modifying chitosan, so that new physicochemical characteristics are brought to a chitosan system, such as improvement of water solubility and stability. On the basis, the application designs a sponge composite material constructed by electrostatic interaction of a polyethylene glycol chitosan material and an anionic component polyacid. The system can not only keep certain stability of polyacid under physiological conditions, but also improve the recycling efficiency and biological efficiency; in addition, the sponge composite material has the advantages of easy circulation, easy operation, low toxicity and the like, and provides a valuable reference for future application in the antibacterial field.
In order to achieve the above purpose, the invention adopts the following technical scheme:
a preparation method of a polyoxometallate crosslinked polyethylene glycol modified chitosan sponge composite material comprises the following steps:
1) Preparation of polyoxometalates POM: preparing POM by adopting a solvent evaporation method, and accurately regulating and controlling a reaction system;
2) Preparation of polyethylene glycol chitosan CS/PEG: prepared by an optimized one-step synthesis method;
3) Preparing a polyoxometallate crosslinked polyethylene glycol modified chitosan sponge composite material: and (3) adding POM suspension with mass concentration of 2.5-10mg/mL (for example, the concentration can be 2.5mg/mL, 5mg/mL, 7.5mg/mL and 10 mg/mL) into the 3-4 mg/mLCS/PEG solution prepared by the method to form suspension, then putting into a refrigerator (-18 ℃) for freezing for 24 hours, taking out, and then putting into a vacuum freeze dryer (with the pressure of 2Pa and the temperature of-77 ℃) for working for 12-36 hours (preferably 24 hours) to obtain the sponge composite material CS/PEG/POM. Samples containing different mass concentrations of polyacid were designated C/P/P2.5, C/P/P5, C/P/P7.5 and C/P/P10.
In the above preparation method, specifically, the polyoxometalate POM is prepared by the following steps:
cu (ClO) 4 ) 2 ·6H 2 Heating and stirring the mixture of O, 2-aminopyridine (HL) and distilled water at 50-70 ℃ for 0.5-1 hour, cooling to room temperature, adding ammonium molybdate solution, adjusting the pH value to 2-4, continuously heating and stirring at 50-70 ℃ for 0.5-1 hour, cooling to room temperature, filtering, and separating out blue transparent blocky monocrystal, thus obtaining the crystal.
Further, cu (ClO) 4 ) 2 ·6H 2 The dosage of O, 2-aminopyridine and ammonium molybdate is 0.05-0.07g, 0.02-0.03g and 0.15-0.17g respectively. Preferably, cu (ClO 4 ) 2 ·6H 2 The molar ratio of O, HL to ammonium molybdate was about 3:4:163.
Specifically, by dropwise adding concentrated H 3 PO 4 The pH is adjusted to 2-4, preferably to about 3.0.
In the preparation method, specifically, the polyethylene glycol chitosan CS/PEG is prepared by the following steps:
dissolving low molecular weight chitosan in 15-25mL of 1% acetic acid solution, stirring at room temperature for 12-24h, adding polyethylene glycol solution, and stirring for 5-7 h to form CS/PEG solution.
In addition, the CS/PEG solution can be poured into a disc-shaped mold, put into a refrigerator for freezing for 24 hours, taken out, put into a vacuum freeze dryer for working for 24 hours to obtain CS/PEG sponge material, and stored for use as a control group.
Further, the dosage of the low molecular weight chitosan is 90-110mg, the dosage of the polyethylene glycol solution is 8-12mL, and the concentration is 8-12mg/mL.
Further preferably, the average molar mass mw of the low molecular weight chitosan is 100-300kda, and the degree of deacetylation is more than or equal to 85%.
The invention provides a polyoxometallate crosslinked polyethylene glycol modified chitosan sponge composite material (CS/PEG/POM) prepared by the preparation method; wherein the chemical formula of the polyacid is: [ HL ]] 6 H 2 [Cu(H 2 O) 3 (P 2 Mo 5 O 23 )] 2 •4H 2 O, HL=2-aminopyridine, while CS/PEG sponge system can undergo physical electrostatic interaction and hydrogen bond interaction with polyacid component, thereby forming ternary complex sponge material (CS/PEG/POM), and then the research of antibacterial application is carried out. The system can not only keep certain stability of polyacid under physiological conditions, but also improve the recycling efficiency and biological efficiency; in addition, the sponge composite material has the advantages of easy circulation, easy operation, low toxicity and the like, and provides a valuable reference for future application in the antibacterial field.
The invention also provides application of the polyoxometallate crosslinked polyethylene glycol modified chitosan sponge composite material as a bacteriostatic or bactericidal agent.
In the preparation process of the polyoxometallate crosslinked polyethylene glycol modified chitosan sponge composite material, the first step comprises dripping polyethylene glycol solution into acetic acid solution of chitosan under the stirring action to promote the formation of modified chitosan, namely polyethylene glycol chitosan sponge (CS/PEG). The second step is to add the synthesized polyacid compound into the CS/PEG solution under the condition of stirring, and synthesize the ternary polyacid-based composite material by utilizing the hydrogen bond and the electrostatic action among molecules, wherein the hybridized polyacid compound is successfully loaded on the surface of CS/PEG. Because the sponge composite material can damage the bacterial cell membrane to a certain extent, when the integrity of the cell membrane is damaged, leakage of some components in the bacterial body can occur. Secondly, after the composite material prepared by the invention is loaded with the polyacid with specific content, the formed sponge material has good stability and cycle performance, and the practical application efficiency is improved.
In this application, we report an example of a polyethylene glycol modified chitosan cross-linked polyacid sponge composite material to improve the physiological stability of POM, reduce the toxic effects, and thus improve the bioavailability of the POM component. Considering the excellent biological effect of POM and the carrier characteristics of polyethylene glycol chitosan (CS/PEG), we reasonably assume that the obtained composite material has dual advantages, can maintain the efficient antibacterial performance and is expected to produce synergistic effect, and meanwhile, compared with pure POM, the physiological stability of the composite material can be improved. Specifically, POM molecules are loaded on the surface of polyethylene glycol modified chitosan to form a polyacid-based composite material taking CS/PEG as a carrier, and then the obtained composite material is subjected to the exploration of antibacterial application.
According to the invention, the acidified chitosan solution and the PEG solution are mixed and stirred to generate polyethylene glycol modified CS; then, a polyacid compound is loaded on the surface of the modified CS by adopting a one-step synthesis method, so that the polyacid-based composite material is prepared and obtained, and the antibacterial effectiveness of escherichia coli, staphylococcus aureus, agrobacterium tumefaciens, bacillus subtilis and the like is respectively evaluated, and the action mechanism is simultaneously explored. Compared with the prior art, the invention has the following beneficial effects:
1) According to the invention, polyethylene glycol is introduced as a stabilizer and an adhesive for the first time, and a heteropolyacid compound is loaded on the surface of chitosan to construct a novel and green synthetic strategy of a polyacid-based composite material as a bacteriostat or bactericide;
2) The chitosan selected by the invention has the characteristics of special structure, no toxic or side effect, good biocompatibility, high biodegradation rate, strong drug loading property and the like, so that the chitosan has excellent properties of hygroscopicity, film forming property, gel property, bacteriostasis and the like, and provides a practical basis for preparing polyethylene glycol modified chitosan sponge composite material loaded with polyacid;
3) The composite material prepared by the invention has higher sterilization efficiency and good biocompatibility, and can maintain high-efficiency treatment effect in a certain time;
4) The polyacid-based sponge composite material prepared by the invention also has a certain antibacterial effect on drug-resistant bacteria. In addition, the possible bacteriostasis action mechanism of the material is systematically clarified, and the obtained hybrid polyacid-based composite material has great application prospect in the biomedical field;
5) The polyacid-based sponge composite material prepared by the method has a shape similar to that of a band-aid, and has application prospect in the field of rapid wound repair.
Drawings
FIG. 1 is a schematic diagram of the synthetic route for the sponge composite (CS/PEG/POM) according to the present invention;
in FIG. 2, a-f are SEM images of CS, CS/PEG, and supported different polyacid content (C/P/P2.5, C/P/P5, C/P/P7.5, C/P/P10), respectively;
FIG. 3 is a mapping graph of CS/PEG (upper graph) and CS/PEG/POM sponge (lower graph), respectively;
in FIG. 4, (a) is an infrared spectrum of CS, PEG, POM, CS/PEG, CS/PEG/POM; (b) X-ray powder diffraction of CS/PEG/POM loaded with 25mg, 50mg, 75mg of polyacid; (c) Powder diffraction of CS, PEG, POM, CS/PEG, CS/PEG/POM X-rays; (d) is a Raman spectrum of POM, CS/PEG/POM;
FIG. 5 (a) is a general spectrum of X-ray photoelectron spectroscopy (XPS) of a CS/PEG/POM sponge sample, and (b) - (f) are X-ray photoelectron spectroscopy of O1s, N1s, cu2p, mo3d and C1s, respectively;
FIG. 6 is an optical image of a sample material co-cultured with CS/PEG, C/P/P2.5, C/P/P5, C/P/P7.5, C/P/P10 for 24h for treatment of E.coli, staphylococcus aureus, agrobacterium tumefaciens, bacillus subtilis, pseudomonas aeruginosa, bacillus cereus, respectively;
FIG. 7 is a graph showing the bacteriostatic effects of the blank, CS/PEG, C/P/P-2.5, C/P/P-5, C/P/P-7.5, C/P/P-10 on E.coli, staphylococcus aureus, bacillus subtilis, agrobacterium tumefaciens;
FIG. 8 is a schematic diagram showing the antibacterial effect of E.coli resistant to kanamycin sulfate (a) and ampicillin (b) on agar plates;
FIG. 9 is a graph showing the bacteriostatic effect after three cycles;
FIG. 10 is an SEM image of E.coli; bacterial morphology of control group (a) and treatment group (b) with CS/PEG/POM;
FIG. 11 optical density values (OD) at 260nm at different times for a control group without sponge composite and an experimental group with sponge composite 260nm )；
FIG. 12 is a graph showing the optical density values (OD) at 575nm at various times for a control group without the sponge composite and an experimental group with the sponge composite 575nm )。
Detailed Description
The following describes the technical scheme of the present invention in further detail with reference to examples, but the scope of the present invention is not limited thereto.
In the following embodiments, the raw materials are all common commercial products which can be directly purchased or can be prepared and obtained according to the conventional technology in the field, for example, chitosan is purchased from low molecular weight chitosan produced by Beijing carbofuran technology Co., ltd, the average molar mass mw is 100-300kda, and the degree of deacetylation is more than or equal to 85%. Polyethylene glycol was purchased from beijing enokio technologies limited. Room temperature refers to 25±5 ℃.
Example 1
A polyoxometallate crosslinked polyethylene glycol modified chitosan sponge composite material (CS/PEG/POM), wherein the polyacid has the chemical formula: [ HL ]] 6 H 2 [Cu(H 2 O) 3 (P 2 Mo 5 O 23 )] 2 •4H 2 O, hl=2-aminopyridine, whereas CS/PEG sponge systems can undergo physical electrostatic interactions and hydrogen bonding interactions with polyacid components, forming ternary complex sponge materials (CS/PEG/POM).
The preparation method (synthetic route is shown in figure 1) of the polyoxometallate crosslinked polyethylene glycol modified chitosan sponge composite material (CS/PEG/POM) specifically comprises the following steps:
1) Polyoxometalate POM [ HL ]] 6 H 2 [Cu(H 2 O) 3 (P 2 Mo 5 O 23 )] 2 •4H 2 Synthesis of O:
the synthesis of the precursor POM is similar to that reported previously. In the synthesis, POM is prepared by adopting a solvent evaporation method, and the reaction system is accurately regulated and controlled. Cu (ClO) 4 ) 2 ·6H 2 A mixture of O (0.06 g), HL (2-aminopyridine, 0.025 g) and distilled water (20 mL) was heated and stirred at 60℃for half an hour and cooled to room temperature. Then ammonium molybdate solution (0.016 g/mL,10 mL) was added, using concentrated H 3 PO 4 Regulating pH to about 3.0, heating and stirring at 60deg.C for 30 min, cooling to room temperature, filtering, and separating out to obtain blue transparent blocky monocrystal (HL)] 6 H 2 [Cu(H 2 O) 3 (P 2 Mo 5 O 23 )] 2 •4H 2 O。
2) Preparation of polyethylene glycol chitosan (CS/PEG):
polyethylene glycol chitosan was prepared by an optimized one-step synthesis method, first, low molecular weight chitosan (100 mg, average molar mass mw=100-300 kDa, degree of deacetylation: 85% or more) was dissolved in 20mL, 1% (mass%) acetic acid solution, and stirred at room temperature overnight (12 h). The next day, 10mg/mL of polyethylene glycol solution was added to the chitosan solution formed, followed by stirring for 6 hours, to form a CS/PEG solution.
In addition, the CS/PEG solution is poured into a disc-shaped mould, put into a refrigerator at the temperature of minus 18 ℃ for freezing for 24 hours, taken out, put into a vacuum freeze dryer at the temperature of minus 77 ℃ under the pressure of 2Pa for 24 hours, and then the CS/PEG sponge material is obtained and stored for use as a control group.
3) Preparation of polyoxometallate crosslinked polyethylene glycol modified chitosan sponge composite material (CS/PEG/POM):
and (2) adding 2.5mg/mL, 5mg/mL, 7.5mg/mL and 10mg/mL (10 mL) of POM suspension with different mass concentrations into 10mL of CS/PEG solution with the mass concentration of 3.3 mg/mL synthesized in the step (2) to form suspension, then putting the suspension into a refrigerator with the temperature of minus 18 ℃ for freezing for 24 hours, taking out the suspension, and putting the suspension into a vacuum freeze dryer with the pressure of 2Pa and the temperature of minus 77 ℃ for working for 24 hours, thus obtaining the CS/PEG/POM supermolecule sponge composite material. Samples containing different mass concentrations of polyacid were designated C/P/P2.5, C/P/P5, C/P/P7.5 and C/P/P10, respectively.
In FIG. 2 a-f are SEM images of CS, CS/PEG, and supported different polyacid contents (C/P/P2.5, C/P/P5, C/P/P7.5, C/P/P10), respectively. As can be seen from fig. 2: the FESEM image of CS sponge alone in fig. 2 a shows a smooth surface and stacked nanoplatelet morphology and a larger pore structure on the surface. The polyethylene glycol modified CS/PEG sponge prepared according to the present invention (b in FIG. 2) showed a significant degree of wrinkling. The formation of wrinkles is due to the blending of the polyacid components in the mixed system, resulting in a difference in shrinkage between positive and negative charges. Furthermore, as the POM mass ratio increases, the degree of wrinkles is seen to change more significantly when the mass ratio of CS, PEG and polyacid is changed to 1:1:1, the maximum fold degree structure can be obtained, and the larger the mixing amount, the more likely the damage of the film surface is caused.
FIG. 3 is a mapping graph of CS/PEG (upper graph) and CS/PEG/POM (lower graph) sponges, respectively, showing the distribution of individual elements in CS/PEG and CS/PEG/POM, respectively. Mapping maps in ternary sponge composites map specific distribution positions of C, O, P, mo, cu and N in CS/PEG/POM. The positions of the measured elements in the bright field of the element mapping image scan are consistent with the envisaged element distribution positions, further proving successful synthesis of CS/PEG and CS/PEG/POM. Wherein panels (b, c) represent samples of the synthesized CS/PEG sponge and CS/PEG/POM sponge composites, respectively, the color change demonstrated successful loading of the polyacid into the precursor sponge system material.
In FIG. 4, (a) is an infrared spectrum of CS, PEG, POM, CS/PEG, CS/PEG/POM, (b) is X-ray powder diffraction of CS/PEG/POM loaded with 25mg, 50mg, 75mg of polyacid, (c) isCS, PEG, POM, CS/PEG, CS/PEG/POM, and (d) Raman spectrum of POM, CS/PEG/POM. As shown in FIG. 4 a, the infrared spectrum of CS was 3429cm -1 There appears a broad peak due to hydroxyl OH stretching and amino groups on the CS structure. Free chitosan at 1555cm -1 Exhibits stronger stretching at 1651cm, which is an N-H stretching vibration -1 The absorption band at which corresponds to the tensile vibration of the carbon-based (c=o) amide group. The structure of PEG comprises OH, C-H, C-C and C-O bonds with vibration peaks of 3424, 2878, 830-1459cm respectively -1 In addition, the infrared spectrum pairs v (P-O), v (Mo=od) and v (Mo-O-Mo) of the precursor POM are 1090-1026, 908 and 685cm, respectively -1 With characteristic telescopic vibration. The CS/PEG infrared spectrum has a vibration band at 1568cm-1, which refers to the formation of amide bonds in the mixed substrate. At the same time, CS/PEG also maintained a characteristic peak at 1654, where 1463cm -1 Ascribed to the primary amide and the protonated amino group. These specific peaks mentioned in the pegylated chitosan sponge samples are considered to be a clear evidence of the pegylation reaction. Further, as can be seen from fig. 4 b: the composite membrane (CS/PEG/POM 2.5, CS/PEG/POM5, CS/PEG/POM7.5, CS/PEG/POM/POM 10) was combined with 1112-1090, 912-885 and 704-670cm -1 The characteristic stretching and vibration of (P-O and Mo-O) correspond, indicating that POM has been successfully loaded into polyethylene glycol modified chitosan sponge material. The peaks of CS/PEG in these composite polymer sponges did not have significant peak shifts compared to the starting material, indicating successful synthesis of the polymer composite.
As can be seen from fig. 4 c, the X-ray powder diffraction pattern demonstrates characteristic peaks in each material as well as in the composite material. The original CS powder showed two peaks at 12.87 and 20.73 indicating intermolecular and intramolecular hydrogen bonds between the amino and hydroxyl groups of CS. This peak is consistent with the results reported in the literature that confirm the purity of the CS sample. Solid polyethylene glycol showed a crystalline XRD pattern with two peaks at 19.64 and 23.73. For CS/PEG XRD pattern, after PEG modification, at 2θ=14.7 and 2θThe peaks at=19.32, 23.3 correspond to the original powders (CS and PEG) in the CS/PEG polymer, indicating successful synthesis of the sample. POMs crystalsThe powder had a characteristic peak at 8.68 and some weak peaks at about 10.4, 12.13 and 26.97. In addition, the PXRD pattern for CS/PEG/POM also demonstrated the presence of CS, PEG and POM, at 2θDiffraction peaks around=7.61, 19.32 and 23.52 were consistent with the starting powder materials (PEG and POM) described above, with no other impurity peaks. At the same time, the characteristic peak of CS disappears in the composite membrane and this is considered as a blending reaction between CS/PEG and POM in the polymer system, with physical electrostatic interactions and hydrogen bonding interactions occurring between the CS/PEG sponge system and the polyacid component.
As can be seen from FIG. 4 d, in the Raman spectrum of POM, the spectrum is 800-1000cm -1 Several characteristic sharp peaks can be seen. In the CS/PEG/POM sponge material, the thickness is 800-1000cm -1 A spike also appears from side to side, probably due to electrostatic and hydrogen bonding interactions between POM and CS/PEG polymer blend systems.
Further, the valence state of the chemical components and related elements is studied by using an X-ray photoelectron spectroscopy (XPS) technology. As can be seen from FIG. 5, the prepared CS/PEG/POM sponge sample consists of C, N, O, P, mo and Cu elements, and the relevant elements are uniformly dispersed on the prepared material, which shows that XPS spectrum of the CS/PEG/POM sponge material constructed into Cu2p shows two obvious peaks at the 932.6 and 952.4eV of binding energy, namely Cu 2+ 2p1/2 and Cu 2+ 2p3/2. Meanwhile, two satellite peaks exist in the Cu2pXPS spectrum, which further proves that Cu 2+ The presence of ions. In addition, FIG. 5 also shows the Mo3dXPS spectrum of the prepared CS/PEG/POM sponge. The bond energy peaks at 230.9eV and 234.1eV for Mo3d are Mo3d5/2 and Mo3d3/2, respectively, indicating that Mo is present primarily in the CS/PEG/POM sponge in the form of Mo (VI), further demonstrating that POM was successfully added to the prepared samples.
Application test 1:
antibacterial activity study-disc diffusion method: the antibacterial activity of the composite CS/PEG/POM of the present invention was evaluated using typical bacterial strains of escherichia coli, staphylococcus aureus, agrobacterium tumefaciens, bacillus subtilis, pseudomonas aeruginosa, bacillus cereus as model microorganisms. The antibacterial activity of different sponge samples was studied using an agar diffusion test (disc diffusion). CS/PEG sponge sample material (POM is not loaded) is used as a blank control group, and polyacids with different contents are loaded as an experimental group. FIG. 6 shows optical images of different species of gram-negative bacteria co-cultured with gram-positive bacteria and different sponge sample materials for 24 h. As for the antibacterial result of the CS/PEG blank group, no obvious inhibition zone is formed, and the sponge loaded with POM shows obvious antibacterial activity, which proves that the POM plays a leading role in the antibacterial process. Meanwhile, as the POM content is increased, the inhibition area is gradually increased, which proves that the antibacterial effect of the composite material is obviously enhanced.
Application test 2:
antibacterial activity study-colony counting method: the antibacterial activity of different sponge samples is studied by adopting another method, and the antibacterial rate is mainly compared: r (%) =n 0 -N 1 /N 0 X 100%, N in 0 Colony count of control group, N 1 The colony count was the number of the test group. For colony counting, sponge samples without sponge material added as a blank, CS/PEG and sponge samples loaded with different amounts of polyacid were added as experimental groups. As shown in FIG. 7, for E.coli, the colony count of the experimental group to which the CS/PEG sponge sample was added was somewhat reduced compared with that of the blank group, and it was also observed that the antibacterial activity of the sample carrying the polyacid at different levels was different, the antibacterial rate of the CS/PEG sample was 47.85%, the antibacterial rate of the C/P/P-2.5 sample was 85.86%, the antibacterial rate of the C/P/P-5 sample was 94.49%, the antibacterial rate of the C/P/P-7.5 sample was 99.32%, and the antibacterial rate of the C/P/P-10 sample was 99.86%. For Staphylococcus aureus, the antibacterial rate of the CS/PEG sample was 68.76%, the antibacterial rate of the C/P/P-2.5 sample was 97%, the antibacterial rate of the C/P/P-5 sample was 98.67%, the antibacterial rate of the C/P/P-7.5 sample was 98.98%, and the antibacterial rate of the C/P/P-10 sample was 99.24%. For Bacillus subtilis, the antibacterial rate of the CS/PEG sample was 49.83%, the antibacterial rate of the C/P/P-2.5 sample was 92.07%, the antibacterial rate of the C/P/P-5 sample was 94.77%, the antibacterial rate of the C/P/P-7.5 sample was 98.82%, and the antibacterial rate of the C/P/P-10 sample was 99.75%. For Agrobacterium tumefaciens, the CS/PEG sample has an antibacterial rate of 75.47%, the antibacterial rate of the C/P/P-2.5 sample was 83.35%, the antibacterial rate of the C/P/P-5 sample was 97.63%, the antibacterial rate of the C/P/P-7.5 sample was 98.05%, and the antibacterial rate of the C/P/P-10 sample was 99.82%.
In addition, the antibacterial activity of the sponge sample was different for the two drug-resistant bacteria. As shown in FIG. 8, the antibacterial ratio of the CS/PEG sample was 48.73%, the antibacterial ratio of the C/P/P-5 sample was 78.87% and the antibacterial ratio of the C/P/P-10 sample was 86.98% for kanamycin-resistant E.coli. For ampicillin resistant E.coli, the antibacterial rate of the CS/PEG sample was 71.73%, the antibacterial rate of the C/P/P-5 sample was 99%, and the antibacterial rate of the C/P/P-10 sample was 99.62%.
Finally, the circularity and stability of the prepared sponge samples were investigated by a cyclic experiment, and fig. 9 shows that: even after three cycles, under the same experimental conditions, the sponge still appeared to have a distinct zone of inhibition at the top of the dish. It is further shown that the synthesized polyacid-supported sponge composite has good circulation.
Application test 3:
scanning electron microscope observation of sponge composite material-colibacillus cell interaction: the morphological changes of the bacterial cells and the degree of cell damage were measured by scanning electron microscopy, and the results are shown in FIG. 10. The scanning electron microscope image result according to fig. 10 shows that: the cell structure of E.coli cells not treated with the sample (FIG. 10 a) had a smooth surface, had a complete cell wall/membrane, and exhibited a typical rod shape, but after the bacteria and composite CS/PEG/POM composite were treated for 1 hour (FIG. 10 b), the morphology of the bacteria was significantly changed, and the cell membrane surface of the bacteria was wrinkled, damaged and collapsed. It can be found that after the composite material is processed, the direct contact effect of the material and bacterial cell membrane causes larger physical damage to the bacterial cells, and the cracking degree of the cells is quickened. Most bacteria are morphologically in a state of breakage, cell walls exhibiting division, and overall dishing. The scanning electron microscope result shows that the composite material damages the integrity of bacteria, so that the bacterial cells are seriously damaged, and finally the bacterial cells die. Thus, from the bacterial cell wall/membrane structural disruption, the cytoplasm is released and analyzed in terms of SEM images, precisely because the sponge composite material directly interacts with the bacterial cell wall/membrane to form a certain degree of dishing, disrupting the cell wall/membrane integrity, leading to leakage of intracellular components and eventual death of the cell.
Application test 4:
bacteriostasis mechanism test-detection of integrity of bacterial cell membrane (E.coli as test strain): rupture of the cell membrane results in leakage of components within the bacterial cell membrane, and thus the extent of leakage of components within the cell membrane is inferred and the integrity of the bacterial cell membrane is judged by measuring absorbance at 260 nm. The method comprises the following steps: and taking the group without the sponge composite material as a control group, and adding the experimental group of the sponge composite material. 4mg of sponge composite and bacterial suspension were mixed and then mixed at 37 0 Culturing at C for 15min, filtering with 0.22mm filter membrane, and measuring optical density value with UV-vis spectrophotometer. According to the experimental results of the test, it can be found from fig. 11 a that the optical density values of the blank control group at 260nm after 0min and 60min are not greatly different, but when the optical density value of the test group is obviously higher than that of the control group, compared with the control group, the sponge composite material can damage the cell membrane of escherichia coli to a certain extent, so that the cell membrane is damaged to some extent, and when the integrity of the cell membrane is damaged, some components in the bacterial body can leak.
Bacteriostasis mechanism test-protein leakage test (E.coli as test strain): damage to bacterial cell membranes can lead to leakage of intracellular RNA, proteins, potassium ions, etc., and thus leakage of proteins can be used as an indicator for evaluating cell membrane integrity. To the EP tube, 1 mL E.coli suspension and 4mg sponge composite were added, followed by 5mL Coomassie solution. After shaking the mixture, the mixture was reacted for 2 minutes, and the absorbance at 595, 595 nm was measured. As shown in figure 11 b, the protein content in the test group treated by the sponge composite material is obviously higher than that in the control group, the penetrating capacity of bacterial cell membranes is enhanced, and more leakage of RNA and protein in cells is caused, so that the material has obvious antibacterial activity.
Bacteriostasis mechanism test-active oxygen content test (E.coli as test strain): the level of oxidative stress in bacterial cells is assessed by the amount of ROS produced by the cells, mainly using Nitro Blue Tetrazolium (NBT) reduction. E.coli and antibiotic-resistant E.coli were suspended in Phosphate Buffered Saline (PBS) at 37. Mu.g/mLCS/PEG/Cu-POM composite 。 Treatment at C was carried out for 60min, then 4 mL of NBT solution was added and the mixture was incubated for an additional 30 min. After incubation, the reaction was stopped by adding 0.4. 0.4 mL hydrochloric acid solution (0.1M) to the EP tube, recovered NBT was extracted with DMSO solution (1 mL), and absorbance was recorded with uv-vis spectra at 575 nm. As shown in FIG. 12, we can observe the optical density value (OD) at 575nm after 60min of incubation of the control group without the sponge composite added 575nm ) Not significantly, but the optical density value (OD) at 575nm of the experimental group incorporating the sponge composite 575nm ) The result shows that the CS/PEG/POM composite material can cause the generation content of Reactive Oxygen Species (ROS) to be obviously increased, the content accumulation of the reactive oxygen species can cause a series of oxidative stress reactions, the oxidative stress reactions can interfere with the normal metabolic processes of escherichia coli cells, and some important components in bacteria are oxidized, so that the physiological activities of bacteria are damaged, and the accumulation of the Reactive Oxygen Species (ROS) can be caused to finally cause the apoptosis of the bacteria cells.
In conclusion, the polyacid-based composite material with high-efficiency antibacterial performance is developed around serious bacterial infection and environmental management problems. According to the invention, the sponge composite material is constructed by utilizing the polyethylene glycol modified chitosan material and the anionic component polyacid with good antibacterial performance through electrostatic interaction, and then the research on antibacterial application is carried out. The system can not only keep certain stability of polyacid under physiological conditions, but also improve the recycling efficiency and biological efficiency; in addition, the sponge composite material has the advantages of easy circulation, easy operation, low toxicity and the like, and provides a valuable reference for future application in the antibacterial field. The synthetic route of the polyacid-based composite material in the invention is as follows: polyethylene glycol modified chitosan solution + hybridized polyacid- & gt polyacid-based composite material. Mixing chitosan solution and polyethylene glycol solution under an acidic condition, adding polyacid, stirring at room temperature to combine the chitosan solution and the polyethylene glycol solution to form suspension, then putting the suspension into a refrigerator for freezing for 24 hours, taking the suspension out, and putting the suspension into a vacuum freeze dryer for working for 24 hours to obtain the CS/PEG/POM supermolecule sponge. Due to the superior properties of the modified chitosan and the inherent antimicrobial properties of polyacids, the polyacid-based composite is endowed with a stronger antimicrobial effect, which results in significant bacteriostasis for the following reasons: (1) The polyethylene glycol modified chitosan crosslinked polyacid sponge composite material has good stability, and can cause physical damage to cell membranes of bacteria by directly contacting with bacterial suspension, so that the integrity of the cell membranes of the bacteria is damaged, and the leakage of components in cytoplasm is caused; (2) The prepared composite material has a front surface due to the existence of biopolymer chitosan, can interact with negatively charged cell walls/membranes, and promotes adhesion of cells in an initial stage; (3) The broad-spectrum biological activity of polyacid (such as cell wall/membrane rupture, intracellular substance leakage, enzyme activity reduction, collapse of an antioxidant mechanism, biological target spot interference and the like) endows the composite material with stronger bactericidal effect; (4) In summary, the interaction of bacteria with the surface charge of the sponge, the presence of bioactive components and the self-properties of chitosan are all important advantages of the preparation of the composite material, which is an indispensable characteristic of the efficient antibacterial biological material.
Claims (6)
1. The preparation method of the polyoxometallate crosslinked polyethylene glycol modified chitosan sponge composite material is characterized by comprising the following steps of:
1) Preparation of polyoxometalates POM: the chemical formula is [ HL ]] 6 H 2 [Cu(H 2 O) 3 (P 2 Mo 5 O 23 )] 2 •4H 2 O, hl=2-aminopyridine,
2) Preparation of polyethylene glycol chitosan CS/PEG:
3) Preparing a polyoxometallate crosslinked polyethylene glycol modified chitosan sponge composite material: adding POM suspension with mass concentration of 2.5-10mg/mL into the prepared 3-4 mg/mLCS/PEG solution to form suspension, then putting into a refrigerator for freezing for 24 hours, taking out, and then putting into a vacuum freeze dryer for 12-36 hours to obtain the product;
the polyethylene glycol chitosan CS/PEG is prepared by the following steps:
dissolving low molecular weight chitosan in 15-25mL of 1% acetic acid solution, stirring at room temperature for 12-24h, adding polyethylene glycol solution, and stirring for 5-7 h to form CS/PEG solution;
the dosage of the low molecular weight chitosan is 90-110mg, the dosage of the polyethylene glycol solution is 8-12mL, and the concentration is 8-12mg/mL;
the average molar mass mw of the low molecular weight chitosan is 100-300kda, and the deacetylation degree is more than or equal to 85%;
the prepared sponge composite material has good antibacterial activity on escherichia coli, staphylococcus aureus, agrobacterium tumefaciens, bacillus subtilis, pseudomonas aeruginosa and bacillus cereus.
2. The method for preparing the polyoxometallate crosslinked polyethylene glycol modified chitosan sponge composite material according to claim 1, wherein the polyoxometallate POM is prepared by the following steps:
cu (ClO) 4 ) 2 ·6H 2 Heating and stirring the mixture of O, 2-aminopyridine and distilled water at 50-70 ℃ for 0.5-1 hour, cooling to room temperature, adding ammonium molybdate solution, adjusting the pH value to 2-4, continuously heating and stirring at 50-70 ℃ for 0.5-1 hour, cooling to room temperature, filtering, and separating out blue transparent blocky monocrystal to obtain the product.
3. The method for preparing the polyoxometallate crosslinked polyethylene glycol modified chitosan sponge composite material according to claim 2, wherein Cu (ClO 4 ) 2 ·6H 2 The dosage of O, 2-aminopyridine and ammonium molybdate is 0.05-0.07g, 0.02-0.03g and 0.15-0.17g respectively.
4. Preparation method of polyoxometallate crosslinked polyethylene glycol modified chitosan sponge composite material as claimed in claim 2The method is characterized by adding concentrated H dropwise 3 PO 4 The pH value is regulated to 2-4.
5. The polyoxometallate crosslinked polyethylene glycol modified chitosan sponge composite material prepared by the preparation method of any one of claims 1 to 4.
6. The use of the polyoxometallate cross-linked polyethylene glycol modified chitosan sponge composite material of claim 5 in preparing bactericides.
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