CN115735949A - 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 cross-linked polyacid sponge composite material, wherein the polyacid has a chemical formula 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 constructs the sponge composite material by utilizing the electrostatic interaction and the hydrogen bond interaction between the polyethylene glycol modified chitosan material and the anion component polyacid with good bacteriostatic effect, and then researches on antibacterial application are carried out. The system can not only keep a certain stability of the polyacid under physiological conditions, but also improve the recycling efficiency and biological efficacy of the polyacid; in addition, the sponge composite material has the advantages of easiness in circulation, easiness in operation, low toxicity and the like, and provides a valuable reference for the application of the sponge composite material in the field of bacteriostasis in the future.
Description
Technical Field
The invention develops the polyacid-based composite material with high-efficiency antibacterial performance around the problems of serious bacterial infection and environmental management. 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 anion component polyacid with good bacteriostasis through electrostatic interaction, and antibacterial application research is carried out. The system can not only keep a certain stability of the polyacid under physiological conditions, but also improve the recycling efficiency and biological efficacy of the polyacid; in addition, the sponge composite material has the advantages of easiness in circulation, easiness in operation, low toxicity and the like, and provides a valuable reference for the application of the sponge composite material in the field of bacteriostasis in the future.
Background
In recent years, rapid progress in industrialization and urbanization has caused a problem of deterioration of water quality, resulting in a gradual increase in pathogenic bacteria, organic pollutants, and the like in water. Pathogenic bacteria are one of the most common pollutants in water, meanwhile, the wide use of antibiotics not only increases the drug resistance of the bacteria, but also can cause the appearance of super bacteria, and the problem of bacterial infection poses a great threat to global public safety and ecological environment. Therefore, economic, environment-friendly and efficient antibacterial materials attract people to pay attention. Based on the problem of water pollution caused by pathogenic microorganisms, new strategies to synthesize non-toxic, environmentally friendly, and highly efficient composite materials are urgently needed to alleviate these concerns.
Polyoxometallate is a unique nanoscale polyanion cluster composed of early transition metal oxides, and has potential application potential in the aspects of biology, medicine, material science and water treatment. Due to their biological and biochemical effects, POMs and POM matrix systems are considered promising metallopharmaceuticals for the future. 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, the polyoxometallate-based organic-inorganic hybrid materials have received a wide interest and attention due to their excellent characteristics. However, polyacids also have the disadvantages of low solubility, difficulty in recovery and instability under physiological conditions. Detailed literature investigations have shown that loading polyacids onto low cost supports with significant stability, renewability and variety of functional groups, especially biodegradable and biocompatible supports, is highly attractive. In some biological carrier materials, chitosan (CTs) is composed of beta- (1-4) -D-glucosamine and N-acetamido-diglucose units, and has the characteristics of special structure, no toxic or side effect, good biocompatibility, high biodegradation rate, strong drug-carrying property and the like, so the Chitosan (CTs) has excellent properties of hygroscopicity, film forming property, gel property, bacteriostatic property and the like, and is widely applied to the fields of food, medical sanitation, biomedicine, daily necessities and the like. However, due to their low solubility and colloidal stability under physiological conditions, they remain challenging in biological applications. Therefore, the preparation of highly efficient polyacid-based functional composites remains an important and challenging task. In addition, in order to construct an effective, efficient and stable antibacterial treating agent and understand the antibacterial mechanism of the synthesized composite material, the ultrasonic-assisted self-assembly strategy is specially applied to synthesize the multifunctional nano composite material and further explore the antibacterial performance of the multifunctional nano composite material.
Disclosure of Invention
In order to overcome the defects, the pegylated chitosan decrosslinked polyacid is a feasible strategy, the invention aims to provide a polyoxometallate crosslinked polyethylene glycol modified chitosan sponge composite material, a preparation method and application thereof, and the polyethylene glycol is used for modifying chitosan to bring new physical and chemical properties to a chitosan system, such as improvement of water solubility and stability. On the basis, the application designs that the polyethylene glycol chitosan material and the anionic component polyacid construct the sponge composite material through electrostatic interaction. The system can not only keep a certain stability of the polyacid under physiological conditions, but also improve the recycling efficiency and biological efficacy of the polyacid; in addition, the sponge composite material has the advantages of easiness in circulation, easiness in operation, low toxicity and the like, and provides valuable reference for the application of the sponge composite material in the bacteriostatic field in the future.
In order to achieve the purpose, the invention adopts the following technical scheme:
a preparation method of a polyoxometallate cross-linked polyethylene glycol modified chitosan sponge composite material comprises the following steps:
1) Preparation of polyoxometallate POM: POM is prepared by adopting a solvent evaporation method, and a reaction system is accurately regulated and controlled;
2) Preparing PEGylated chitosan CS/PEG: the preparation method comprises the following steps of (1) preparing by an optimized one-step synthesis method;
3) Preparing a polyoxometallate crosslinked polyethylene glycol modified chitosan sponge composite material: and adding POM suspension with mass concentration of 2.5-10mg/mL (such as concentration of 2.5mg/mL, 5mg/mL, 7.5mg/mL and 10 mg/mL) into the 3-4 mg/mLCS/PEG solution prepared by the same volume to form suspension, freezing the suspension in a refrigerator (-18 ℃) for 24 hours, taking out the suspension, and putting the suspension in a vacuum freeze dryer (pressure of 2Pa and temperature of-77 ℃) to work 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 polyoxometallate POM is prepared by the following steps:
mixing Cu (ClO) 4 ) 2 ·6H 2 Heating and stirring mixture of O, 2-aminopyridine (HL) and distilled water at 50-70 deg.C for 0.5-1 hr, cooling to room temperature, adding ammonium molybdate solution, adjusting pH to 2-4, heating and stirring at 50-70 deg.C for 0.5-1 hr, cooling to room temperature, filteringAnd separating out blue transparent blocky single crystals to obtain the crystal.
Further, cu (ClO) 4 ) 2 ·6H 2 The dosages of O, 2-aminopyridine and ammonium molybdate are 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.
In particular, H is concentrated by dropwise addition 3 PO 4 The pH is adjusted to 2-4, preferably to about 3.0.
In the above preparation method, specifically, the pegylated 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 and put into a vacuum freeze dryer for working for 24 hours to obtain a CS/PEG sponge material, and the CS/PEG sponge material is stored for use as a control group.
Furthermore, 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 low molecular weight chitosan has an average molar mass mw of 100-300kda and a degree of deacetylation of 85% or more.
The invention provides a polyoxometallate cross-linked polyethylene glycol modified chitosan sponge composite material (CS/PEG/POM) prepared by the preparation method; 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, while the CS/PEG sponge system can undergo physical electrostatic interaction and hydrogen bond interaction with the polyacid component to form a ternary complex sponge material (CS/PEG/POM), which was then studied for antibacterial applications. The system not only can keep a certain stability of the polyacid under physiological conditions, but also can improve the circulation of the polyacidEfficiency of utilization and bioefficacy; in addition, the sponge composite material has the advantages of easiness in circulation, easiness in operation, low toxicity and the like, and provides valuable reference for the application of the sponge composite material in the bacteriostatic field in the future.
The invention also provides application of the polyoxometallate cross-linked polyethylene glycol modified chitosan sponge composite material as a bacteriostatic agent or a bactericide.
In the preparation process of the polyoxometallate cross-linked polyethylene glycol modified chitosan sponge composite material, the first step comprises the step of dripping a polyethylene glycol solution into an 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 obtained above under the condition of stirring, and synthesize the ternary polyacid-based composite material by utilizing hydrogen bonds and electrostatic interaction between molecules, wherein the hybridized polyacid compound is successfully loaded on the surface of CS/PEG. Since the sponge composite can destroy the bacterial cell membrane to a certain extent, when the integrity of the cell membrane is destroyed, some components in the bacterial body can leak. Secondly, after the composite material prepared by the invention is loaded with 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 to improve the physiological stability of POM and reduce the toxic effects, thereby improving the bioavailability of POM components. In consideration of the excellent biological effect of POM and the carrier characteristic of polyethylene glycol chitosan (CS/PEG), the composite material obtained is reasonably supposed to have double advantages, so that the high-efficiency antibacterial property can be kept, the synergistic effect is expected to be generated, and the physiological stability of the composite material can be improved compared with that of pure POM. Specifically, POM molecules are loaded on the surface of polyethylene glycol modified chitosan to form a polyacid-based composite material with CS/PEG as a carrier, and then the antibacterial application of the obtained composite material is researched.
According to the invention, the acidified chitosan solution and the PEG solution are mixed and stirred to generate the CS modified by polyethylene glycol; and then, loading the polyacid compound on the modified CS surface by adopting a one-step synthesis method to prepare and obtain the polyacid-based composite material, evaluating the bacteriostatic efficacy of escherichia coli, staphylococcus aureus, agrobacterium tumefaciens, bacillus subtilis and the like respectively, and exploring the action mechanism. Compared with the prior art, the invention has the following beneficial effects:
1) The invention firstly develops a synthetic strategy for introducing polyethylene glycol as a stabilizer and an adhesive and loading a hybrid polyacid compound on the surface of chitosan to construct a novel and green polyacid-based composite material as a bacteriostatic or bactericidal agent;
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, antibacterial property and the like, and provides a practical basis for preparing the polyacid-loaded polyethylene glycol modified chitosan sponge composite material;
3) The composite material prepared by the invention has higher sterilization efficiency and good biocompatibility, and can keep high-efficiency treatment effect within a certain time;
4) The polyacid-based sponge composite material prepared by the invention also has certain antibacterial effect on drug-resistant bacteria. In addition, a possible bacteriostasis mechanism of the material is systematically clarified, and the obtained hybrid polyacid-based composite material has a great application prospect in the field of biomedicine;
5) The shape of the polyacid-based sponge composite material prepared by the invention is similar to that of a band-aid, and the polyacid-based sponge composite material has an application prospect in the field of rapid wound repair.
Drawings
FIG. 1 is a schematic diagram of the synthetic route of the sponge composite (CS/PEG/POM) of the present invention;
in FIG. 2, a-f are SEM images of CS, CS/PEG, and different loaded polyacid contents (C/P/P2.5, C/P/P5, C/P/P7.5, C/P/P10), respectively;
FIG. 3 is a mapping chart of CS/PEG (upper panel) and CS/PEG/POM sponge (lower panel), respectively;
in FIG. 4, (a) is an infrared spectrum of CS, PEG, POM, CS/PEG/POM; (b) X-ray powder diffraction of CS/PEG/POM loaded with 25mg, 50mg, 75mg of polyacid; (c) X-ray powder diffraction for CS, PEG, POM, CS/PEG/POM; (d) Raman spectra of POM, CS/PEG/POM;
FIG. 5 (a) is a summary of X-ray photoelectron spectroscopy (XPS) for CS/PEG/POM sponge samples, (b) - (f) are X-ray photoelectron spectroscopy for O1s, N1s, cu2p, mo3d and C1s, respectively;
FIG. 6 is an optical image of a sample material of Escherichia coli, staphylococcus aureus, agrobacterium tumefaciens, bacillus subtilis, pseudomonas aeruginosa, bacillus cereus and sponge treated with CS/PEG, C/P/P2.5, C/P/P5, C/P/P7.5 and C/P/P10 respectively after co-culture for 24 h;
FIG. 7 is a blank, CS/PEG, C/P/P-2.5, C/P/P-5, C/P/P-7.5, C/P/P-10 bacteriostatic effect diagram for Escherichia coli, staphylococcus aureus, bacillus subtilis, and Agrobacterium tumefaciens;
FIG. 8 is a graph showing the antibacterial effect of kanamycin (a) sulfate-resistant and ampicillin (b) resistant E.coli on agar plates;
FIG. 9 is a graph of the bacteriostatic effect after three cycles;
FIG. 10 is an SEM image of E.coli; the bacterial morphology of the control group (a) and the group (b) treated with CS/PEG/POM;
FIG. 11 shows the optical density values (OD) at 260nm at different times for the control group without sponge composite and the experimental group with sponge composite 260nm )；
FIG. 12 is a graph showing the optical density values (OD) at 575nm at different times for a control group to which no sponge composite was added and an experimental group to which a sponge composite was added 575nm )。
Detailed Description
The technical solution of the present invention is further described in detail with reference to the following examples, but the scope of the present invention is not limited thereto.
In the following examples, the raw materials are all available as ordinary commercial products or can be prepared according to conventional techniques in the art, for example, chitosan is purchased from low molecular weight chitosan manufactured by Beijing Bailingwei science and 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 Yinoka science, inc. Room temperature refers to 25 ± 5 ℃.
Example 1
A polyoxometallate cross-linked polyethylene glycol modified chitosan sponge composite material (CS/PEG/POM), wherein the chemical formula of 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, whereas the CS/PEG sponge system can undergo physical electrostatic and hydrogen bonding interactions with the polyacid component to form a ternary complex sponge material (CS/PEG/POM).
The preparation method of the polyoxometallate crosslinked polyethylene glycol modified chitosan sponge composite material (CS/PEG/POM) (the synthetic route is shown in figure 1) specifically comprises the following steps:
1) Polyoxometallate POM [ HL)] 6 H 2 [Cu(H 2 O) 3 (P 2 Mo 5 O 23 )] 2 •4H 2 And (3) synthesis of O:
the synthesis mode of the precursor POM is similar to that of the previous reported literature. In the synthesis, POM is prepared by a solvent evaporation method, and a reaction system is accurately regulated and controlled. Mixing 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 at 60 ℃ for half an hour with stirring and cooled to room temperature. Then ammonium molybdate solution (0.016 g/mL,10 mL) was added with concentrated H 3 PO 4 Adjusting pH to about 3.0, heating and stirring at 60 deg.C for 30 min, cooling to room temperature, filtering, precipitating to obtain blue transparent block-shaped monocrystal [ HL ] after one week] 6 H 2 [Cu(H 2 O) 3 (P 2 Mo 5 O 23 )] 2 •4H 2 O。
2) Preparation of pegylated Chitosan (CS/PEG):
the pegylated chitosan was prepared by an optimized one-step synthesis, first, low molecular weight chitosan (100 mg, average molar mass mw =100-300kDa, degree of deacetylation ≥ 85%) was dissolved in 20mL, 1% (mass%) acetic acid solution and stirred at room temperature overnight (12 h). The next day, 10mL of 10mg/mL polyethylene glycol solution was added to the resulting chitosan solution, followed by stirring for 6 hours to form a CS/PEG solution.
In addition, the CS/PEG solution is poured into a disc-shaped mold, put into a refrigerator at minus 18 ℃ for freezing for 24 hours, taken out and put into a vacuum freeze dryer with the pressure of 2Pa and the temperature of minus 77 ℃ for working for 24 hours to obtain the CS/PEG sponge material, and the CS/PEG sponge material is stored for use as a control group.
3) Preparation of polyoxometallate crosslinked polyethylene glycol modified chitosan sponge composite material (CS/PEG/POM):
and (3) adding 2.5mg/mL, 5mg/mL, 7.5mg/mL and 10mg/mL (10 mL) POM suspensions with different mass concentrations into 10mL CS/PEG solution with the mass concentration of 3.3 mg/mL synthesized in the step 2) to form a suspension, then putting the suspension into a refrigerator with the temperature of 18 ℃ below zero for freezing for 24 hours, taking out the suspension, putting the suspension into a vacuum freeze dryer with the pressure of 2Pa and the temperature of 77 ℃ below zero for working for 24 hours, and 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 different loaded polyacid contents (C/P/P2.5, C/P/P5, C/P/P7.5, C/P/P10), respectively. As can be seen from fig. 2: FESEM image of CS sponge alone in fig. 2 shows smooth surface and stacked nanosheet morphology as well as having a larger pore structure on the surface. The polyethylene glycol modified CS/PEG sponge prepared according to the invention (b in fig. 2) showed a significant degree of wrinkling. The formation of wrinkles is attributed to the blending of polyacid components in the mixed system, resulting in a difference in shrinkage between positive and negative charges. In addition, as the mass ratio of POM increases, the more significant the change in degree of wrinkles is seen, when the mass ratio of CS, PEG and polyacid is changed to 1:1:1, the maximum folding degree structure can be obtained, and the more the amount is mixed, the more the damage of the film surface may be caused.
FIG. 3 is a mapping chart of CS/PEG (top panel) and CS/PEG/POM (bottom panel) sponges, respectively, showing the distribution of individual elements in CS/PEG and CS/PEG/POM, respectively. Mapping in the ternary sponge composite material maps the specific distribution positions of C, O, P, mo, cu and N in the CS/PEG/POM. The positions of the elements measured in the bright field of the element mapping image scan are consistent with the assumed element distribution positions, and further proves the successful synthesis of CS/PEG and CS/PEG/POM. Where the graphs (b, c) represent samples of the synthetic CS/PEG sponge and CS/PEG/POM sponge composites, respectively, the color change demonstrates the successful loading of the polyacid into the precursor sponge system material.
In FIG. 4, (a) shows an infrared spectrum of CS, PEG, POM, CS/PEG, and CS/PEG/POM, (b) shows an X-ray powder diffraction of CS/PEG/POM loaded with 25mg, 50mg, and 75mg of a polyacid, (c) shows an X-ray powder diffraction of CS, PEG, POM, CS/PEG, and CS/PEG/POM, and (d) shows a Raman spectrum of POM and CS/PEG/POM. As shown in a in FIG. 4, the IR spectrum of CS was 3429cm -1 A broad peak appears due to stretching of hydroxyl OH and amino groups on the CS structure. Free chitosan is 1555cm -1 It shows a strong stretching, belonging to N-H stretching vibration, and at 1651cm -1 The absorption band at (a) 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, and the vibration peaks are 3424, 2878 and 830-1459cm respectively -1 Furthermore, the infrared spectrum of the precursor POM was adjusted to v (P-O), v (Mo = Od) and v (Mo-O-Mo) at 1090-1026, 908 and 685cm, respectively -1 There is a characteristic stretching vibration. The IR spectrum of CS/PEG has a vibrational band at 1568cm-1, which indicates the formation of amide bond in the mixed substrate. Meanwhile, CS/PEG also maintains a characteristic peak at 1654, wherein 1463cm -1 Belonging to the primary amides and protonated amino groups. These specific peaks, which were mentioned in the pegylated chitosan sponge samples, are considered to be clear evidence for the pegylation reaction. In addition, as can be seen from b in fig. 4: mixing the composite membrane (CS/PEG/POM 2.5, CS/PEG/POM5, CS/PEG/POM7.5, CS/PEG/POM/POM 10) with 1112-1090, 912-885 and 704-670cm -1 The characteristic stretching and vibration of (P-O and Mo-O) correspond to each other, indicating that POM has been successfully loaded into the polyethylene glycol modified chitosan sponge material. The CS/PEG peak in these composite polymer sponges had no significant peak shift compared to the starting material, indicating successful synthesis of the polymer composite.
As can be seen from c in fig. 4, 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 sharp peaks at 12.87 and 20.73, indicating that intermolecular and intramolecular hydrogen bonds were formed between the amino and hydroxyl groups of CS. This peak is consistent with literature reports of results confirming the purity of CS samples. The solid polyethylene glycol showed a crystalline XRD pattern with two peaks at 19.64 and 23.73. For the XRD pattern of CS/PEG, after PEG modification, at 2θ=14.7 and 2θThe spikes at 19.32, 23.3 correspond to the original powders in the CS/PEG polymer (CS and PEG), indicating successful synthesis of this sample. POMs crystal powder has a characteristic peak at 8.68 and weak peaks at about 10.4, 12.13 and 26.97. In addition, the PXRD pattern for CS/PEG/POM also demonstrates the presence of CS, PEG and POM at 2θDiffraction peaks around =7.61, 19.32 and 23.52 were consistent with the above starting powder materials (PEG and POM), with no other impurity peaks. Meanwhile, the characteristic peak of CS disappears in the composite membrane, and the characteristic peak is regarded as the blending reaction between CS/PEG and POM in a polymer system, and physical electrostatic interaction and hydrogen bond interaction occur between a CS/PEG sponge system and a polyacid component.
As can be seen from d in FIG. 4, the Raman spectrum of POM is in the range of 800 to 1000cm -1 Several characteristic sharp peaks are visible. In the CS/PEG/POM sponge material, the thickness is 800 to 1000cm -1 A spike in the side was also shown, probably due to the electrostatic and hydrogen bonding interaction between POM and CS/PEG blend polymer systems.
Further using X-ray photoelectron spectroscopy (XPS) technology to research the valence state of chemical components and related elements. 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 these related elements are uniformly dispersed on the prepared material, indicating that the CS/PEG/POM sponge sample is prepared by using the methodThe XPS spectrum of Cu2p constructed by the material shows that two obvious peaks are at the binding energy 932.6 and 952.4eV, and are respectively Cu 2+ 2p1/2 and Cu 2+ 2p3/2. Meanwhile, two satellite peaks exist in the Cu2pXPS spectrum, and further proves that Cu 2+ The presence of ions. In addition, FIG. 5 also shows the Mo3dXPS spectra of the CS/PEG/POM sponges prepared. The binding energy peaks of Mo3d at 230.9eV and 234.1eV are Mo3d5/2 and Mo3d3/2, respectively, indicating that Mo exists in CS/PEG/POM sponge mainly in the form of Mo (VI), further proving that POM is successfully added to prepare samples.
Application test 1:
study of antibacterial Activity-disc diffusion method: typical bacterial strains of escherichia coli, staphylococcus aureus, agrobacterium tumefaciens, bacillus subtilis, pseudomonas aeruginosa and bacillus cereus are taken as model microorganisms, and the antibacterial activity of the composite material CS/PEG/POM is evaluated. The antibacterial activity of different sponge samples was studied using the agar diffusion test (disc diffusion). CS/PEG sponge sample material (not loaded with POM) is used as a blank control group, and the sample material loaded with different contents of polyacid is used as an experimental group. FIG. 6 shows optical images of different kinds of gram-negative and gram-positive bacteria and different sponge sample materials after 24h co-incubation. For the antibacterial results of the CS/PEG blank group, it can be found that no obvious inhibition zone is formed, and the POM loaded sponge shows obvious antibacterial activity, which proves that POM plays a dominant role in the antibacterial process. Meanwhile, the inhibition area is gradually increased along with the increase of the content of POM, which shows that the antibacterial effect of the composite material is obviously enhanced.
Application test 2:
study of antibacterial Activity-colony counting: the antibacterial activity of different sponge samples is researched by adopting another method, and the antibacterial rate is mainly compared as follows: r (%) = N 0 -N 1 /N 0 X 100%, wherein N 0 Number of colonies as control group, N 1 The number of colonies in the test group was used. For colony counting, a blank was prepared without sponge material, and samples of sponges loaded with CS/PEG and different amounts of polyacid were prepared as experimental groups. As shown in FIG. 7, for E.coliCompared with a blank control group, the colony number of the experimental group added with the CS/PEG sponge sample is reduced to a certain extent, and meanwhile, the antibacterial activity of the polyacid loaded with different contents is different, the antibacterial rate of the CS/PEG sample is 47.85%, the antibacterial rate of the C/P/P-2.5 sample is 85.86%, the antibacterial rate of the C/P/P-5 sample is 94.49%, the antibacterial rate of the C/P/P-7.5 sample is 99.32%, and the antibacterial rate of the C/P/P-10 sample is 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 ratio of the CS/PEG sample is 49.83%, the antibacterial ratio of the C/P/P-2.5 sample is 92.07%, the antibacterial ratio of the C/P/P-5 sample is 94.77%, the antibacterial ratio of the C/P/P-7.5 sample is 98.82%, and the antibacterial ratio of the C/P/P-10 sample is 99.75%. For Agrobacterium tumefaciens, the percent bacteria for the CS/PEG sample was 75.47%, the percent bacteria for the C/P/P-2.5 sample was 83.35%, the percent bacteria for the C/P/P-5 sample was 97.63%, the percent bacteria for the C/P/P-7.5 sample was 98.05%, and the percent bacteria for the C/P/P-10 sample was 99.82%.
In addition, the antibacterial activity of the sponge samples was different for the two drug-resistant bacteria. As shown in FIG. 8, in the case of kanamycin-resistant E.coli, the antibacterial ratio of the CS/PEG sample was 48.73%, that of the C/P/P-5 sample was 78.87%, and that of the C/P/P-10 sample was 86.98%. For ampicillin-resistant E.coli, the antibacterial ratio of the CS/PEG sample was 71.73%, the antibacterial ratio of the C/P/P-5 sample was 99%, and the antibacterial ratio of the C/P/P-10 sample was 99.62%.
Finally, the cyclicity and stability of the sponge samples prepared were explored by cycling experiments, and figure 9 shows: even after three cycles, the sponge still showed a distinct zone of inhibition on the top of the dish under the same experimental conditions. It is further shown that the synthetic polyacid-supported sponge composite has good recycling.
Application test 3:
observing the interaction of the sponge composite material and the Escherichia coli cells by a scanning electron microscope: 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 results according to fig. 10 show that: the cell structure of the E.coli cells (a in FIG. 10) without sample treatment has smooth surface, has intact cell wall/membrane, and takes on a typical rod shape, but when the bacteria and the composite CS/PEG/POM composite are treated for 1 hour (b in FIG. 10), the morphology of the bacteria is obviously changed, and wrinkling, damage and collapse of the cell membrane surface of the bacteria occur. It can be found that after the composite material is treated, the direct contact effect of the material and the bacterial cell membrane causes great physical damage to the bacterial cells, and the cell lysis degree is accelerated. Most bacteria are morphologically in a state of breakage, cell wall showing division, and overall depression. Scanning electron microscope results show that the composite material damages the integrity of bacteria, so that bacterial cells are seriously damaged, and the bacterial cells are finally killed. Thus, analysis of the destruction of the bacterial cell wall/membrane structure, the release of cytoplasm and SEM images is due to the direct interaction of the sponge composite with the bacterial cell wall/membrane, with a degree of dishing that destroys the cell wall/membrane integrity, resulting in leakage of intracellular components and eventual cell death.
Application test 4:
bacteriostatic mechanism test-detection of bacterial cell membrane integrity (escherichia coli as test strain): rupture of the cell membrane leads to leakage of components within the bacterial cell membrane, and thus the degree of leakage of components within the cell membrane is estimated and the integrity of the bacterial cell membrane is judged by measuring the absorbance at 260 nm. The method comprises the following specific steps: the group without the sponge composite material was used as a control group, and the experimental group with the sponge composite material was added. 4mg of sponge composite and bacterial suspension were mixed at 37 0 Culturing for 15min at C, filtering with 0.22mm filter membrane, and measuring optical density with UV-vis spectrophotometer. According to the experimental results of the test, it can be found from a in FIG. 11 that the blank control group has a small difference in optical density values at 260nm after 0min and 60min, but when compared with the control group, the optical density value of the experimental group is significantly higher than that of the control group, indicating that the sponge composite material can be obtainedSo as to destroy the cell membrane of the escherichia coli bacteria, so that the cell membrane is damaged to a certain extent, and when the integrity of the cell membrane is damaged, some components in the bacteria body can leak.
Bacteriostatic mechanism test-protein leakage test (escherichia coli as test strain): damage to the bacterial cell membrane can result in leakage of intracellular RNA, proteins, potassium ions, etc., and thus leakage of proteins can be used as an index for evaluating the integrity of the cell membrane. 1 mL of E.coli suspension and 4mg of sponge composite were added to the EP tube, followed by 5mL of Coomassie blue solution. After shaking the mixture, it was reacted for 2 min, and the absorbance at 595 nm was measured. As shown in b of fig. 11, the content of protein in the test group treated with the sponge composite material was significantly higher than that in the control group, which enhanced the penetration ability of the bacterial cell membrane, resulting in more leakage of intracellular RNA and protein, indicating that the material had significant antibacterial activity.
Bacteriostatic mechanism test-content test of active oxygen (escherichia coli as test strain): the level of oxidative stress of bacterial cells is assessed by the amount of cellular ROS produced, primarily by nitroblue tetrazolium (NBT) reduction. Suspending common Escherichia coli and antibiotic-resistant Escherichia coli in Phosphate Buffered Saline (PBS), and adding 100 μ g/mLCS/PEG/Cu-POM composite material at 37 。 Treatment at C for 60min, then 4 mL NBT solution was added and the mixture was incubated for an additional 30 min. After incubation, the reaction was stopped by adding 0.4 mL of hydrochloric acid solution (0.1M) to the EP tube, recovered NBT was extracted with DMSO solution (1 mL), and absorbance was recorded by UV-visible spectroscopy at 575 nm. As shown in FIG. 12, we can observe the optical density value (OD) at 575nm after culturing for 60min in the control group without adding the sponge composite material 575nm ) Is not greatly changed, and the experimental group added with the sponge composite material has an optical density value (OD) at 575nm 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 accumulation of the content of the ROS can cause a series of oxidative stress reactions, the oxidative stress reactions can interfere the normal metabolic process of Escherichia coli cells, a plurality of important components in the body of bacteria are oxidized, and the physiological activity of the bacteria is further damaged and influencedThis, in turn, leads to the accumulation of Reactive Oxygen Species (ROS) that ultimately cause bacterial cell apoptosis.
In conclusion, the invention develops the polyacid-based composite material with high-efficiency antibacterial performance around the serious problems of bacterial infection and environmental management. According to the invention, the sponge composite material is constructed by utilizing the polyethylene glycol modified chitosan material and the anion component polyacid with good antibacterial performance through electrostatic interaction, and then the research on antibacterial application is carried out. The system not only can keep a certain stability of the polyacid under physiological conditions, but also can improve the recycling efficiency and biological efficacy of the polyacid; in addition, the sponge composite material has the advantages of easiness in circulation, easiness in operation, low toxicity and the like, and provides a valuable reference for the application of the sponge composite material in the field of bacteriostasis in the future. The synthesis route of the polyacid-based composite material is as follows: polyethylene glycol modified chitosan solution + hybridized polyacid → polyacid-based composite material. Mixing a chitosan solution and a polyethylene glycol solution under an acidic condition, adding a polyacid, stirring at room temperature to combine the chitosan solution and the polyethylene glycol solution to form a suspension, then putting the suspension into a refrigerator for freezing for 24 hours, taking out the suspension, putting the suspension into a vacuum freeze dryer for working for 24 hours, and obtaining the CS/PEG/POM supramolecular sponge. Due to the superior characteristics of the modified chitosan and the inherent antibacterial property of the polyacid, the polyacid-based composite material is endowed with a stronger antibacterial effect, and the reasons for generating obvious bacteriostasis are as follows: (1) The polyethylene glycol modified chitosan cross-linked polyacid sponge composite material has good stability, and can cause physical damage to the cell membrane of the chitosan cross-linked polyacid sponge composite material by directly contacting with a bacterial suspension, so that the integrity of the bacterial cell membrane is damaged, and the leakage of components in cytoplasm is caused; (2) The prepared composite material has a positive surface due to the existence of the biopolymer chitosan, can interact with a negatively charged cell wall/membrane, and promotes the adhesion of cells in the initial stage; (3) Broad spectrum biological activity of the polyacid (cell wall/membrane rupture, intracellular substance leakage, enzyme activity reduction, destruction of an antioxidant mechanism, biological target interference and the like) endows the composite material with stronger bactericidal action; (4) In conclusion, the interaction between bacteria and the surface charge of the sponge, the existence of bioactive components and the characteristics of chitosan are all important advantages of the composite material prepared by the method, and the characteristics are indispensable to the efficient antibacterial biological material.
Claims (9)
1. The preparation method of the polyoxometallate cross-linked polyethylene glycol modified chitosan sponge composite material is characterized by comprising the following steps of:
1) Preparation of polyoxometallate POM:
2) Preparing PEGylated chitosan CS/PEG:
3) Preparing a polyoxometallate crosslinked polyethylene glycol modified chitosan sponge composite material: and adding the POM suspension with the mass concentration of 2.5-10mg/mL into the prepared 3-4 mg/mLCS/PEG solution to form a suspension, then putting the suspension into a refrigerator for freezing for 24 hours, taking out the suspension, and putting the suspension into a vacuum freeze dryer for working for 12-36 hours to obtain the POM suspension.
2. The method for preparing the polyoxometallate cross-linked polyethylene glycol modified chitosan sponge composite material as claimed in claim 1, wherein the polyoxometallate POM is prepared by the following steps:
mixing Cu (ClO) 4 ) 2 ·6H 2 Heating and stirring a mixture of O, 2-aminopyridine and distilled water at 50-70 ℃ for 0.5-1 h, cooling to room temperature, then adding an ammonium molybdate solution, adjusting the pH value to 2-4, continuing heating and stirring at 50-70 ℃ for 0.5-1 h, cooling to room temperature, filtering, and precipitating a blue transparent blocky monocrystal to obtain the crystal.
3. The method for preparing polyoxometallate cross-linked polyethylene glycol modified chitosan sponge composite material as claimed in 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. The method for preparing polyoxometallate cross-linked polyethylene glycol modified chitosan sponge composite material as claimed in claim 2, wherein the H is dropwise added to the composite material 3 PO 4 Adjusting the pH value to 2-4。
5. The method for preparing polyoxometallate cross-linked polyethylene glycol modified chitosan sponge composite material as claimed in claim 1, wherein the pegylated 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.
6. The method for preparing the polyoxometallate crosslinked polyethylene glycol modified chitosan sponge composite material as claimed in claim 5, wherein 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.
7. The method for preparing the polyoxometallate cross-linked polyethylene glycol modified chitosan sponge composite material as claimed in claim 6, wherein 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%.
8. The polyoxometallate cross-linked polyethylene glycol modified chitosan sponge composite material prepared by the preparation method of any one of claims 1 to 7.
9. The use of the polyoxometallate cross-linked polyethylene glycol modified chitosan sponge composite material of claim 8 as a bacteriostatic or bacteriocidal agent.
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