A composite antibacterial material modified with vancomycin via magnetic responsive bond grafting, its preparation and application

Abstract

This invention belongs to the field of antibacterial materials, specifically relating to a composite antibacterial material modified with vancomycin through magnetically responsive bond grafting. The material comprises a magnetocaloric material, a polymer coated with the magnetocaloric material having free amino and / or hydroxyl groups, and vancomycin grafted onto the polymer via magnetron sputtering linkage residues. The magnetocaloric material is a functional material capable of generating magnetocaloricity under an alternating magnetic field. The magnetron sputtering linkage residues contain magnetically responsive bonds that can break under an alternating magnetic field, and these magnetically responsive bonds are azo bonds. This invention also provides a method for preparing the material and its applications. This invention has found that the composite material, based on the combined control of the components and modification methods, can unexpectedly achieve synergistic effects, thus effectively improving antibacterial activity and antibacterial spectrum. Furthermore, it can achieve magnetron sputtering of vancomycin, thereby achieving a magnetron sputtering attenuation effect.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical materials, specifically relating to the field of antibacterial materials. Technical Background

[0002] Infection during surgery is one of the potential risks leading to implantation failure of medical materials. Bacterial attachment and subsequent bacterial proliferation and colonization on the surface of medical materials often lead to biofilm formation. In order to reduce initial bacterial attachment and prevent subsequent biofilm formation, a great deal of effort has been devoted to developing antimicrobial surfaces. In recent decades, a variety of antimicrobial surfaces have been designed, which can be divided into three categories according to their mechanism of action: (i) bactericidal surfaces that kill attached bacteria; (ii) antimicrobial surfaces that prevent bacterial adhesion; and (iii) surfaces that reduce the adhesion between bacteria and the material surface and release loosely attached bacteria by applying external stimulation. 1 .

[0003] Among these antibacterial methods, endowing surfaces with bactericidal capabilities to kill bacteria attached to the surface or suspended in the surrounding environment is the most direct approach, and significant progress has been made in the development of such bactericidal surfaces. Traditional bactericidal surfaces are based on biocides, which are either permanently fixed to the surface or preloaded / embedded within the surface before release. These include natural antibacterial molecules (such as antimicrobial enzymes and antimicrobial peptides), antimicrobial peptide mimics, synthetic cationic polymers, nitrogen oxides, metal nanoparticles, and the most commonly used biocides such as antibiotics. 2 .

[0004] Nevertheless, biocompatibility and cytotoxicity are characteristics that need to be carefully evaluated before applying bactericidal surfaces in vivo.

[0005] Antibiotics are powerful weapons against infection, but they can also cause collateral damage if used at excessively high concentrations or for extended periods. For example, vancomycin (VAN) is a glycopeptide used to prevent and treat infections caused by Gram-positive bacteria such as Staphylococcus. It inhibits cell wall synthesis by binding to the C-terminal L-Lys-D-Ala-D-Ala motif of the cell wall precursor in certain susceptible strains. 3,4 When the concentration of vancomycin in the cell culture medium exceeds 2000 μg / mL, cell proliferation is severely inhibited. 5 .

[0006] However, for vancomycin-containing implant materials, while the bactericidal surface can prevent the formation of living biofilms, these surfaces are also cytotoxic, which may hinder the integration of the implant with surrounding tissues. Therefore, improving the early antibacterial capabilities of implants and controlling their degradation to reduce drug toxicity remains an industry-wide challenge. Summary of the Invention

[0007] To address the problems of unsatisfactory antibacterial activity, uncontrollable toxicity, and limited antibacterial spectrum in existing vancomycin antibacterial materials, the primary objective of this invention is to provide a composite antibacterial material modified by grafting vancomycin with magnetic response bonds. This aims to controllably block the antibacterial function and improve the antibacterial activity and spectrum through magnetically controlled grafting.

[0008] The second objective of this invention is to provide a method for preparing the aforementioned composite antibacterial material and its application in antibacterial materials.

[0009] The third objective of this invention is to provide an antibacterial material incorporating the aforementioned composite antibacterial material.

[0010] A composite antibacterial material modified by grafting vancomycin via magnetic responsive bonds comprises a magnetothermal material, a polymer having free amino and / or hydroxyl groups coated on the magnetothermal material, and vancomycin grafted onto the polymer via magnetronically stimulated linkage residues;

[0011] The magnetocaloric material is a functional material that can generate magnetocaloricity under the action of an alternating magnetic field;

[0012] The magnetron-stimulated linker residues contain magnetic response bonds that can break under the action of an alternating magnetic field; and the magnetic response bonds are azo bonds.

[0013] The present invention has found that the composite material, based on the combined control of the components and modification methods, can unexpectedly achieve synergistic effects, thus effectively improving antibacterial activity and antibacterial spectrum. In addition, it can also achieve magnetically controlled cleavage of vancomycin, thereby achieving a magnetically controlled detoxicity effect.

[0014] In this invention, the components and modification methods are key to synergistically improving antibacterial activity and antibacterial spectrum and achieving magnetically controlled detoxification.

[0015] In this invention, one end of the magnetically stimulated linker residue is linked to a polymer, and the other end is linked to vancomycin.

[0016] In this invention, the magnetron-stimulated linker residues link the polymer and vancomycin via chemical bonds.

[0017] Preferably, the two ends of the magnetron sputtering linker residue are connected to the polymer and vancomycin via amide-base and / or ester bonds, respectively. For example, one end of the magnetron sputtering linker residue forms an amide bond with the amino group in vancomycin and / or an ester bond with the -OH group therein. The other end of the magnetron sputtering linker residue forms an amide bond with the amino group in the polymer and / or an ester bond with the -OH group therein. The bonds connecting the two ends of the residue can be the same or different.

[0018] Preferably, the magnetron-stimulated linker residues are fragments having the structure of Formula 1:

[0019]

[0020] The n mentioned is an integer from 1 to 6;

[0021] The R is H or a C1-C6 alkyl group.

[0022] In this invention, in Formula 1, the a-terminus can be linked to an amino (-NH-) and / or hydroxyl (-O-) group in the polymer, and the b-terminus can be linked to an amino and / or hydroxyl group of vancomycin. In this invention, by chemically linking vancomycin using Formula 1, the antibacterial activity and antibacterial spectrum of the grafted product can be improved. Furthermore, controlled cleavage of vancomycin can be achieved, thereby reducing its toxicity.

[0023] Preferably, the magnetocaloric material is at least one selected from iron oxide, titanium, tantalum, platinum, gold, titanium alloys, and stainless steel. In this invention, the metal can be a pure metal.

[0024] Preferably, the polymer is at least one of chitosan, chitosan oligosaccharide, collagen, polydopamine, and gelatin.

[0025] The polymer is coated on the surface of the magnetothermal material, and the thickness of the polymer layer on the surface of the functional material is 10nm to 5mm. The weight ratio of vancomycin to polymer is 1:10000 to 1:5.

[0026] The preferred composite antibacterial material of the present invention comprises iron oxide, chitosan coated on its surface, and vancomycin modified by grafting two amide bonds based on residues of formula 2.

[0027] The present invention also provides a method for preparing the composite antibacterial material, wherein a polymer is coated on the surface of a magnetothermal material, and then a first-stage chemical linkage reaction (also known as a first modification reaction) is carried out with a precursor material containing magnetron stimulation linkage residues. After the reaction, vancomycin is added to carry out a second-stage chemical linkage reaction (also known as a second modification reaction) to obtain the composite antibacterial material.

[0028] Preferably, in this invention, the free amino group in the polymer reacts with one carboxyl group of the magnetron-stimulated linkage residue precursor to perform a first modification reaction (e.g., esterification and / or amidation); subsequently, the remaining carboxyl group in the precursor and vancomycin undergo a second modification reaction (e.g., esterification and / or amidation).

[0029] In this invention, the magnetron sputtering stimulation precursor is a substance capable of condensing and dehydrating with polymers and vancomycin to form linker residues, preferably a compound having the structure of Formula 2:

[0030]

[0031] In this invention, both the first and second chemical linking reactions can be achieved using existing methods. For example, the first and second chemical linking reactions can be carried out with the assistance of a crosslinking activator.

[0032] Preferably, the crosslinking activator is at least one selected from DMTMM, DCC, EDC, isothiocyanate, isocyanate, acyl azide, NHS ester, sulfonyl chloride, aldehyde, glyoxal, epoxide, ethylene oxide, carbonate, aryl halide, imide ester, carbodiimide, acid anhydride, and fluorophenyl ester.

[0033] In the first stage of the linkage reaction, the molar ratio of the precursor to the carboxyl activator is 0.5:1 to 1:5. In the second stage of the linkage reaction, the molar ratio of the precursor to the carboxyl activator is 0.5:1 to 1:5.

[0034] The present invention discloses a specific method for preparing a composite antibacterial material, comprising the following steps:

[0035] Step (1):

[0036] Add an iron source solution containing ferric and ferrous iron to an acetic acid solution of chitosan, add alkali and react under a protective atmosphere to obtain chitosan-coated iron oxide particles.

[0037] Step (2):

[0038] The compound of Formula 2 was mixed with chitosan-coated iron oxide particles, and the pH of the mixture was adjusted to 6.5±0.3. Then, a carboxyl activator was added, and the first modification reaction was carried out at a temperature of less than or equal to 10°C.

[0039] Step (3):

[0040] After the reaction in step (2) is completed, the product is redispersed in water by magnetic separation, vancomycin is added, the pH of the system is adjusted to 6.5±0.3, a carboxyl activator is added, and the second modification reaction is carried out at a temperature of less than or equal to 10°C. After magnetic separation washing and freeze drying, the product is obtained.

[0041] The present invention also provides an application of the aforementioned composite antibacterial material, which is used to prepare antibacterial materials.

[0042] The present invention also provides an antibacterial material comprising a pharmaceutically effective amount of the aforementioned composite antibacterial material.

[0043] The antibacterial materials mentioned above include, for example, antibacterial wound dressings and implant materials.

[0044] The composite antibacterial material is added to the surface and the skeleton of the antibacterial therapeutic material.

[0045] Beneficial effects

[0046] This invention provides a novel antibacterial material that achieves antibacterial function by grafting vancomycin onto the material surface. Through the combined control of the aforementioned components and modification methods, it can unexpectedly synergistically improve antibacterial activity and antibacterial spectrum. Moreover, it can also achieve magnetically controlled detoxicity. Attached Figure Description

[0047] Figure 1 This is a flowchart illustrating the preparation process of the composite antibacterial material in Example 1.

[0048] Figure 2 The figures show the properties of different materials prepared in Example 1, including (A) infrared spectrum; (B) X-ray diffraction pattern; (C) thermogravimetric analysis (TGA); (D) transmission electron microscopy (TEM) image; (E) TEM particle size distribution map; (F) hydrated particle size distribution map; and (G) ζ-potential map. The letters in the figures represent the following material groups: a: VH, b: Fe3O4@CS, c: Fe3O4@CS-Formula 2-1, d: Fe3O4@CS-Formula 2-1-VH.

[0049] Figure 3 The diagram shows the release characteristics of the composite antibacterial material prepared in Example 1, including (A) UV absorption spectrum, (B) standard curve, (C) temperature-controlled release curves at different temperatures, (D) final drug residue at different temperatures, (E) ζ-potential diagram, and (F) Raman spectrum. (n=3)

[0050] Figure 4 This is a magnetron release diagram of the composite antibacterial material prepared in Example 1;

[0051] Figure 5 The diagram shows the antibacterial effect of the composite antibacterial material prepared in Example 1; where A represents the minimum inhibitory concentration (MIC) test result of the release solution, B represents the inhibition zone test result of the release solution and standard vancomycin reference standard, and C represents the relative potency result calculated based on the inhibition zone test. The bacteria used in this experiment was Staphylococcus aureus.

[0052] Figure 6 The diagram shows the contact antibacterial properties of the composite antibacterial material prepared in Example 1; a: Fe3O4@CS; b: Fe3O4@CS-Formula 2-1-VH; c: VH; d: PBS.

[0053] Figure 7The results of contact antibacterial performance of the composite antibacterial material prepared in Example 1 are the bacterial live / dead staining results; af represent VH, PBS, Fe3O4@CS, Fe3O4@CS 80℃ 48h treatment, Fe3O4@CS-Formula 2-1-VH, and Fe3O4@CS-Formula 2-1-VH 80℃ 48h treatment, respectively.

[0054] Figure 8 The results of the bacterial live / dead staining ratio for the contact antibacterial performance of the composite antibacterial material prepared in Example 1 are shown; af represents VH, PBS, Fe3O4@CS, Fe3O4@CS treated at 80℃ for 48h, Fe3O4@CS-Formula 2-1-VH, and Fe3O4@CS-Formula 2-1-VH treated at 80℃ for 48h, respectively; N=3

[0055] Figure 9 The cytotoxicity results of the composite antibacterial material prepared in Example 1 are shown, including: (A) Cytotoxicity graph of Fe3O4@CS-Formula 2-1-VH release solution; (B) Cytotoxicity graph of Fe3O4@CS-Formula 2-1-VH powder; and (C) Live / dead cell staining results (where af represents VH, PBS, Fe3O4@CS, Fe3O4@CS treatment at 80℃ for 48h, Fe3O4@CS-Formula 2-1-VH, and Fe3O4@CS-Formula 2-1-VH treatment at 80℃ for 48h, respectively). (n=3)

[0056] Figure 10 The image shows the antibacterial spectrum results, where A: bacterial coating results; B: statistical analysis of coating counts. ** indicates a significant difference between groups, p<0.01; a: Fe3O4@CS; b: Fe3O4@CS-Formula 2-1-VH; c: VH control group (450 μg / mL); d: PBS control group.

[0057] Figure 11 For sustained antibacterial results: A. Contact antibacterial ability test over 4 consecutive days; B. Bacterial growth curve under co-culture conditions of material and bacteria within 24 hours; C. Biofilm formation on material surface; D. Quantitative comparison of biofilm formation on material surface. N=3, ** indicates significant difference between groups, p<0.01. a: Fe3O4@CS; b: Fe3O4@CS-Formula 2-1-VH; c: Blank control group without bacteria; d: PBS control group. Detailed Implementation

[0058] Medicine:

[0059]

[0060] Example 1: Antibacterial material achieving antibacterial function by grafting vancomycin onto the surface of chitosan.

[0061] The reaction formula is:

[0062] Take 100 ml of water, add 1 ml of acetic acid to prepare a 1% acetic acid solution, then add 1 g of chitosan to prepare a 1% chitosan-acetic acid solution (named Solution A). Next, weigh 5.406 g (0.02 mol) FeCl3·6H2O and 2.78 g (0.01 mol) FeSO4·7H2O and dissolve them in 50 ml of water (named Solution B). Measure 50 ml of Solution A and mix it with Solution B. Transfer the mixture to a three-necked flask and continuously purge with nitrogen for protection. Heat and stir in a water bath. When the temperature reaches 60 degrees Celsius, add 20 ml of ammonia water, continue stirring and heating for 1 hour, and then cool to room temperature under nitrogen. After washing three times with pure water and alcohol respectively by magnetic separation, freeze-dry under vacuum and store at 4 degrees Celsius (product labeled Fe3O4@CS).

[0063] 756.756 mg of Formula 2-1 (2.7 mmol) was dissolved in 100 ml of 1% NaOH aqueous solution. After complete dissolution, the pH was adjusted to 6-7. A certain amount of 1% Fe3O4@CS nanoparticles was weighed and uniformly dispersed in the above solution. The pH was adjusted to 6.5 ± 0.3, and finally 2.7 mmol of DMTMM was added. The mixture was stirred in an ice-water bath for 5 days. After the reaction was completed, the product was washed five times with pure water by magnetic separation. The product was labeled as Fe3O4@CS-Formula 2-1. The washed product was redispersed in 100 ml of water, and 1304.28 mg of VH (0.9 mmol) was added. The two were mixed, and the pH was adjusted to 6.5 ± 0.3 by NaOH / HCl. 249.048 mg of DMTMM (0.9 mmol) was added, and the mixture was stirred in an ice-water bath for 5 days. The final product was magnetically separated, washed, freeze-dried, and stored in a 4°C refrigerator to obtain a composite antibacterial material, labeled as (product label: Fe3O4@CS-Formula 2-1-VH).

[0064] The properties and performance of the composite antibacterial material prepared in Example 1 (hereinafter also referred to as vancomycin grafted onto the chitosan surface in the various determinations) were measured as follows:

[0065] (1) Preparation and characterization of antibacterial material (Fe3O4@CS-Formula 2-1-VH; also known as the composite material of Example 1) that achieves antibacterial function by grafting vancomycin onto the surface of chitosan.

[0066] The Fe3O4@CS-type 2-1-VH magnetic nanoparticles were characterized by infrared spectroscopy. Figure 2Figure A shows the infrared spectrum of Fe3O4@CS, where 581 cm⁻¹... -1 The absorption peak at 1626 cm⁻¹ corresponds to the Fe-O stretching vibration. -1 The absorption peak at 1392 cm⁻¹ corresponds to the NH bending vibration. CS is at 1392 cm⁻¹. -1 2922cm -1 and 1039cm -1 The characteristic peaks at 1550 cm⁻¹ correspond to CO bending vibration, CH stretching vibration, and CN vibration, respectively. -1 A new characteristic absorption peak formed at [location] corresponds to an amide bond, confirming that Formula 2-1 was successfully linked to the surface of Fe3O4@CS magnetic nanoparticles via amide bonds. Furthermore, at 2253 cm⁻¹... -1 -CN absorption appears at 1719 cm⁻¹ -1 (C=O stretching), 1451cm -1 (CH bend) and 2904cm -1 2934cm -1 and 2981cm -1 The three absorption peaks at (CH stretching) also confirm the successful grafting of Equation 2-1 onto Fe3O4@CS. Finally, in the infrared spectrum of the Fe3O4@CS-Equation 2-1-VH sample, the absorption peak at VH at 1587 cm⁻¹... -1 The absorption phenomenon corresponding to the benzene ring skeletal vibration generated at the site confirms the conjugation of VH on the Fe3O4@CS surface (Formula 2-1). Infrared data analysis shows that the antibacterial drug VH was successfully grafted onto the surface of Fe3O4@CS magnetic nanoparticles via the bridge of Formula 2-1.

[0067] The crystal structure of Fe3O4@CS-type 2-1-VH nanoparticles was characterized using X-ray diffraction. Figure 2 As shown in Figure B, for samples Fe3O4@CS(a) and Fe3O4@CS-form 2-1-VH(b), the diffraction peaks observed at 30.06°, 35.41°, 43.03°, 53.39°, 56.91°, 62.49°, and 73.93° correspond to the spinel crystal planes of Fe3O4 (JCPDS 76-1849) at (220), (311), (400), (442), (511), (440), and (622), respectively. No other impurity peaks were found. XRD pattern analysis indicates that the Fe3O4@CS-form 2-1-VH nanoparticles prepared in this experiment have good crystallinity, and the drug grafting process did not change the structure of the Fe3O4 core.

[0068] The organic and inorganic content of Fe3O4@CS-type 2-1-VH nanoparticles was characterized and analyzed using thermogravimetric analysis. Figure 2C shows the weight change curves of Fe3O4@CS, Fe3O4@CS-Formula 2-1, and Fe3O4@CS-Formula 2-1-VH in an inert gas environment as the temperature gradually increased from ambient temperature to 800℃. The TGA curves of the three groups of samples show three different weight loss steps. In the first step, as the temperature increased from room temperature to 200℃, the mass of all three samples decreased by approximately 5% due to further removal of residual moisture in the material. Between 200 and 420℃, for sample Fe3O4@CS, the mass decreased by 10.2% due to the gradual degradation of the CS network; for sample Fe3O4@CS-Formula 2-1, the mass decreased by approximately 20% due to the gradual degradation of the CS network and the decomposition of the thermally decomposed azo compound Formula 2-1; and for Fe3O4@CS-Formula 2-1-VH, the mass decreased by approximately 23.5% due to the gradual degradation of the CS network, the decomposition of the thermally decomposed azo compound Formula 2-1, and the degradation of the VH branched polycyclic compound. At 800℃, the mass of the three groups of samples decreased by 21.6%, 28.9%, and 45.4%, respectively.

[0069] Transmission electron microscopy images and particle size distribution of Fe3O4@CS-type 2-1-VH nanoparticles are attached. Figure 2 As shown in D and E. TEM image analysis reveals that the Fe3O4@CS-type 2-1-VH nanoparticles prepared by this experimental process are spherical particles with an average particle size of 10.4 nm, and the chitosan coating on the Fe3O4 layer is approximately 1–5 nm thick. Furthermore, analysis of the Fe3O4@CS-type 2-1-VH nanoparticles using a dynamic light scattering scanner (see attached image). Figure 2 F), whose hydrated particle size is 248.9±51.5 nm, is due to the aggregation of multiple Fe3O4@CS nanoparticles during the modification process after the synthesis of Fe3O4@CS, resulting in a larger hydrated particle size distribution than the Fe3O4@CS nanoparticle size obtained by transmission electron microscopy analysis. Furthermore, the hydrated particle size distribution of the prepared Fe3O4@CS-Formula 2-1-VH nanoparticles is much larger than that of the Fe3O4@CS nanoparticles.

[0070] The Zeta potential image and particle size distribution of Fe3O4@CS-form 2-1-VH nanoparticles are shown in the attached figure. Figure 2As shown in G, the zeta potential image shows that the zeta potential of Fe3O4@CS-Formula 2-1 is +6.6 mV, much lower than that of Fe3O4@CS (+25.4 mV). This significant decrease in zeta potential indicates that the carboxylated Formula 2-1 molecule at both ends has successfully attached to the surface of Fe3O4@CS, with one end extending from the surface of Fe3O4@CS-Formula 2-1, which can be used for further conjugation with VH. VH has a negative zeta potential of -13.3 mV. The zeta potential of Fe3O4@CS-Formula 2-1-VH is -5.6 mV, confirming that the free amino group on VH conjugates with the free carboxyl group on the surface of Fe3O4@CS-Formula 2-1, proving that VH has been successfully grafted onto the surface of the magnetic nanoparticle Fe3O4@CS via the azo compound Formula 2-1.

[0071] (2) Hydrothermal controlled release of antibacterial materials that achieve antibacterial function by grafting vancomycin onto the surface of chitosan

[0072] The experimental method is as follows:

[0073] (a) Weigh a certain amount of VH solid particles and prepare VH standard solutions with concentration gradients of 0, 50, 100, 200, 300, 400, and 500 μg / mL;

[0074] (b) Measure the UV absorption curve of VH in the range of 240–320 nm using a UV spectrophotometer, and measure the absorbance of VH standard solutions of different concentrations at the maximum absorption peak, and plot the functional relationship between concentration and absorbance.

[0075] (c) Weigh a certain amount of Fe3O4@CS-type 2-1-VH magnetic nanoparticles and prepare a 1 mg / mL Fe3O4@CS-type 2-1-VH magnetic nanoparticle suspension.

[0076] (d) The suspensions prepared above were placed in water baths at 37, 57 and 80°C for incubation (ambient temperature of 23°C was used as a blank control group). The release of VH in Fe3O4@CS-type 2-1-VH magnetic nanoparticles under different hydrothermal temperatures was monitored in real time by ultraviolet spectrophotometry.

[0077] (e) Plot the relationship between the cumulative release of VH and time under different temperature control using experimental data, and analyze the drug residue under each temperature after the release reaches the plateau period using experimental data.

[0078] (f) The Zeta potential of samples treated at different temperatures was characterized and analyzed by a dynamic light scattering scanner;

[0079] (g) The Fe3O4@CS-type 2-1-VH magnetic nanoparticles before and after treatment at 80℃ were characterized and analyzed by micro-laser Raman spectroscopy.

[0080] Test results are available Figure 3 .

[0081] To monitor the release of the antibacterial drug VH in real time using ultraviolet spectrophotometry during subsequent experiments, a series of VH standard solutions with varying concentrations were first measured using an ultraviolet spectrophotometer. The ultraviolet absorbance of VH at different concentrations was recorded, and a concentration-absorbance curve was plotted. Spectral analysis within the wavelength range of 240–320 nm revealed that the maximum absorption peak of VH is at 280 nm. Statistical analysis of the absorbance values ​​of different concentrations of VH at 280 nm showed the functional relationship between absorbance and concentration as follows (see appendix). Figure 3 A, B):

[0082] Y = -0.00406 + 0.00312·X(R) 2 =0.99865) (1)

[0083] To investigate the effect of temperature on the drug release rate of the experimentally prepared Fe3O4@CS-form 2-1-VH material, we planned to incubate the drug carrier material at different temperatures and monitor the release of drug molecules in real time using ultraviolet spectrophotometry, and then statistically analyze the experimental data. Similar to previous experiments, we selected 23, 37, 57, and 80℃ as four different temperatures for this experiment to investigate the effect of temperature on the drug release rate. The prepared 1 mg / mL Fe3O4@CS-form 2-1-VH suspension was incubated at 23, 37, 57, and 80℃, respectively. The VH concentration in the suspension was monitored over time using ultraviolet spectrophotometry, and the cumulative VH release and drug residue at each time point during the release process were calculated for each group of samples at different temperatures.

[0084] Release experiments at different temperatures showed that when the sample was incubated at 23 or 37°C, after 1 hour of incubation, less than 40% of the total VH was released into the solution, as detected by UV spectrophotometry. This release was mainly due to the physical adsorption of Fe3O4@CS. With increasing incubation time, the VH content released into the solution initially increased and then gradually stabilized. At 37°C, the final VH release content was 47.8% of the total load, while at 57°C, the final release content was 62.0%. When the sample was incubated at 80°C, more than half (69.4%) of the VH was released from the drug carrier after 1 hour. After 9 hours of incubation, almost all the VH on the carrier molecules was released.

[0085] By comparing the cumulative release of VH at different temperatures, it can be seen that the release of VH exhibits a non-linear increasing trend with the increase of sample incubation temperature. This is because at lower temperatures, the released VH is mainly loaded onto the Fe3O4@CS drug carrier molecules through the physical adsorption of CS. As the temperature increases, the azo bonds in the thermally decomposed azo compound formula 2-1 of the bridging function in the carrier molecule gradually begin to break. At the same time, the VH of the drug molecules covalently grafted through the bridging molecule formula 2-1 is released synchronously, resulting in a non-linear increasing trend in the release curve. At the same temperature, due to the adsorption properties of the Fe3O4@CS drug carrier molecules themselves, the release of the drug molecules and the physical adsorption will reach a dynamic equilibrium. Once the release and adsorption reach a dynamic equilibrium, the drug release from the carrier molecules will reach its corresponding plateau.

[0086] Experimental results show that VH was loaded onto the surface of Fe3O4@CS nanoparticles in two different ways. One way was through the adsorption of CS itself, which allowed VH to be loaded onto the surface of the carrier molecules via physical adsorption. The other way was through chemical covalent grafting, as proposed in this experiment, using an azo compound of formula 2-1 as a connecting bridge, allowing VH to be covalently grafted onto the surface of Fe3O4@CS. When Fe3O4@CS-formula 2-1-VH was uniformly dispersed in deionized water to form a homogeneous suspension, the VH molecules loaded on Fe3O4@CS through physical adsorption were nearly completely released after about 4 hours, reaching the first release plateau. When the incubation temperature was increased, due to the thermal instability of the azo compound of formula 2-1, the azo chemical bonds broke at high temperatures, and the chemically covalently grafted VH molecules were released. Experimental analysis showed that the drug loading of Fe3O4@CS-Formula 2-1-VH was 158.0 μg / mg, of which the VH loading rate of physical adsorption was 40.9% and the VH bridging rate of Formula 2-1 was 59.1%.

[0087] Next, we explored the residual amount of VH loaded in each group of Fe3O4@CS-form 2-1-VH drug carriers under different temperature conditions (see appendix). Figure 3 (D) Analysis of the residual VH content in the Fe3O4@CS-Formula 2-1-VH drug carrier at different incubation times (0, 12, 24, 36, and 48 hours) for each group revealed a close correlation between the residual VH content and the incubation temperature. After 12 hours of treatment at room temperature and 37℃, the residual VH content did not change significantly. This is because at room temperature and 37℃, the drug released from the drug carrier molecules is primarily VH loaded by Fe3O4@CS through physical adsorption. Formula 2-1 gradually decomposes at 57℃, with only 51.33% remaining in the carrier after 12 hours. Formula 2-1 decomposes rapidly at 80℃, with almost all VH released from the carrier after 12 hours.

[0088] The zeta potential measurement indirectly demonstrated the breaking of the thermally decomposed azo bond 2-1, which acts as a bridge in the drug carrier molecule, after different temperature treatments. After treatment at 23, 37, 60, and 80 °C for 48 h, the zeta potentials of the Fe3O4@CS-2-1-VH sample changed to -2.7, -1.5, 4.7, and 9.3 mV, respectively (see attached figure). Figure 3 E). The zeta potential values ​​of the sample after heat treatment at 37℃ were very similar to those of the untreated control sample, indicating that the azo chemical bond is approximately stable at physiological temperatures. However, as the temperature increased to 57℃, the carbon-nitrogen bond in the 2-1 molecule on the support surface broke, the electronegative VH dissociated, and the zeta potential became positive.

[0089] In addition, Raman spectroscopy analysis showed (attached) Figure 3 F), After heat treatment at 80℃ for 48 h, the CH stretching vibration corresponding to the VH benzene ring was at 1585 cm⁻¹. -1 The position and CC are at 3083cm -1 The fact that it almost disappeared indicates that vancomycin was almost completely released from the drug-loaded surface.

[0090] (3) Multiple magnetothermal controlled release of antibacterial materials that achieve antibacterial function by grafting vancomycin onto the surface of chitosan

[0091] The experimental method is as follows:

[0092] (a) Weigh a certain amount of Fe3O4@CS-type 2-1-VH magnetic nanoparticles and prepare a 1 mg / mL Fe3O4@CS-type 2-1-VH magnetic nanoparticle suspension.

[0093] (b) The prepared suspension was incubated at 37°C, and the release of VH in the suspension was monitored in real time by ultraviolet spectrophotometry.

[0094] (c) After the VH release in the suspension reached a plateau, the sample was continuously monitored for 48 hours. Then, before each sampling and testing, the sample was treated with AMF for 10 minutes (AC frequency of 831 kHz, induction power of 5.8 kW). The VH content in the sample was tested once a day. This operation lasted for 6 days.

[0095] (d) Plot the relationship between the cumulative release of VH and the duration of magnetic field action using experimental data, and perform corresponding analysis on the experimental data.

[0096] Test results are available Figure 4 .

[0097] To investigate the effects of different applied magnetic field durations and number of applications on the VH release rate of antibacterial materials that achieve antibacterial function through vancomycin grafting onto the chitosan surface, a suspension of 1 mg / ml of magnetic nanoparticles was prepared and incubated at room temperature. The release of VH from these nanoparticles was monitored in real-time using ultraviolet spectrophotometry. After the release stabilized at room temperature, alternating magnetic fields with varying durations (each application lasting 10 min) were applied, and subsequent release was monitored in real-time using ultraviolet spectrophotometry. In this experiment, the test sample was subjected to the aforementioned alternating magnetic field six times.

[0098] We investigated the regulatory effect of an external alternating magnetic field on the release of vancomycin (VH) from a magnetically controlled drug carrier of an antibacterial material that achieves antibacterial function by grafting vancomycin onto a chitosan surface. We first prepared a 1 mg / ml suspension of the antibacterial material with vancomycin grafted onto a chitosan surface and incubated it at physiological temperature. The released VH content in the solution was monitored by ultraviolet spectrophotometry. The figure shows that the release of vancomycin stabilized within two days, reaching a plateau. Subsequently, an external alternating magnetic field was applied to the sample for 10 min daily for a total of six cycles of AMF-driven monitoring (Figure 4). The analysis of the figures shows that the desired VH release can be controlled by an external alternating magnetic field within a specific time period. Furthermore, the release trend was similar across the six experimental cycles. These data indicate that the magnetically controlled drug carrier of the antibacterial material with vancomycin grafted onto a chitosan surface prepared in this experiment can achieve timed and quantitative release of VH through external AMF control.

[0099] (4) Antibacterial activity and cytotoxicity of the material release fluid

[0100] The method for evaluating the antibacterial properties of Fe3O4@CS-Formula 2-1-VH release solution is as follows:

[0101] (a) Weigh a certain amount of Fe3O4@CS-form 2-1-VH magnetic nanoparticles and prepare a 10 mg / mL Fe3O4@CS-form 2-1-VH magnetic nanoparticle suspension.

[0102] (b) The prepared samples were incubated at 37°C for 4 hours (the release solution was named a1); after incubation at 37°C for 4 hours, they were magnetically heated for 1 minute, and the cycle was repeated 5 times (the release solution was named b1); after incubation at 37°C for 4 hours, they were magnetically heated for 1 minute, and the cycle was repeated 10 times (the release solution was named c1). In addition, VH standard solutions with the same concentration as a1, b1, and c1 were prepared as experimental control groups (a2, b2, c2).

[0103] (c) Dilute the Staphylococcus aureus bacterial suspension with LB liquid medium to adjust the bacterial concentration to 10. 5 CFU / mL was added sequentially to 10 wells of a 96-well plate, with 50 μL of bacterial culture added to each well.

[0104] (d) Dilute sample solutions a1, a2, b1, b2, c1, and c2 to the same concentration.

[0105] (e) Take the above diluted sample solution and repeat the 2-fold dilution, for a total of 9 dilutions, to obtain 10 antibiotic release solutions with different dilution factors;

[0106] (f) Take 50 μL of antibiotic release solution at different dilution ratios and add it to a 96-well plate containing 50 μL of bacterial solution in order of dilution ratio from small to large. Mix well by pipetting and incubate at 37℃ for 24 h.

[0107] (g) After the culture is completed, observe the clarity of the solution in each group, calculate the antibacterial concentration of the maximum dilution factor of each group, repeat the above steps to determine the minimum inhibitory concentration (MIC) of the antibiotic in each group, and repeat each group three times in parallel.

[0108] (h) Mix the Staphylococcus aureus bacterial suspension with the still-warmed LB solid medium and adjust the bacterial concentration to 10. 6 CFU / mL, pour the plates to prepare LB solid culture medium plates containing Staphylococcus aureus;

[0109] (i) Dilute sample solutions a1, a2, b1, b2, c1, and c2 respectively, and dilute each group to two concentration gradients of 100 μg / mL and 50 μg / mL, which are recorded as high dose and low dose respectively.

[0110] (j) Place the Oxford cups on a bacterial LB plate, inject 60 μL of diluted sample solution into each Oxford cup, and place the plate in a 4°C refrigerator for 2 h.

[0111] (k) After that, the plates were placed in a constant temperature incubator at 37°C for 24 hours. After 24 hours, the size of the inhibition zone formed by each group of samples was observed, the diameter of the inhibition zone was measured by vernier calipers and the relative potency was calculated.

[0112] The cytotoxicity evaluation method for Fe3O4@CS-Formula 2-1-VH release solution is as follows:

[0113] (a) Mouse pre-osteoblasts MC3T3-E1 were seeded in 24-well plates and numbered according to samples a1, a2, b1, b2, c1, and c2. At the same time, a control group without samples and a blank group without cells were set up. Each group was repeated three times and cultured in DMEM medium and incubated at 37°C for 48 hours.

[0114] (b) Dilute the release solution of each group of samples with DMEM complete medium to 20 μg / mL, replace the original medium in the well plate according to the number, and continue to incubate for 24 h.

[0115] (c) Replace each well with medium containing 10% CCK-8, incubate at 37°C for 3 hours, and then take 50 μL of supernatant from each well and place it in a 96-well plate. Set up 3 replicates for each well, measure OD450 with a microplate reader and record the OD value. The average OD450 measured from the 3 replicates is recorded as the actual OD value of the sample.

[0116] (d) Calculate the cell viability of each group of samples after incubation according to Formula 7;

[0117] Cell viability is calculated using the following formula:

[0118]

[0119] Where As is the OD value of the experimental group, Ab is the OD value of the blank group, and Ac is the OD value of the control group.

[0120] The experimental results for this section can be found in... Figures 5-8 The results are as follows:

[0121] To further investigate the relative potency between the antibacterial material model prepared in this experiment (which achieves antibacterial function by grafting vancomycin onto the chitosan surface) and the therapeutic effect of the released drug after AMF regulation in practical applications, this study used the tube-disc method to analyze and evaluate its relative potency. It is known that when a certain amount of antibacterial drug solution is added in the experiment, a natural concentration gradient from high to low will be formed in the colony culture medium through free diffusion of molecules in the solution. During diffusion, when the concentration of antibacterial drug molecules at the diffusion edge reaches its MIC (micronizable concentration), a transparent inhibition zone is formed because the drug concentration inside is higher than the MIC and the drug concentration outside is lower than the MIC. According to the derivation of the diffusion law, the logarithm of the total amount of antibiotic is linearly related to the square of the diameter of the inhibition zone. Furthermore, this experiment used a two-dose method to evaluate its relative potency. In the two-dose method, according to the principle of parallel lines of response, when the logarithmic dose and response of the antibacterial drug concentration are linearly related, and the experimental group samples and the control group standards have the same action properties, the two dose-response curves of the experimental group and the control group are parallel to each other. Based on this principle, by measuring the size of the inhibition zone formed by the sample group after the experiment, the relative potency of VH in each experimental group (a1, b1, c1) was calculated according to the two-dose method calculation formula.

[0122]

[0123] Wherein, UH: diameter of the inhibition zone of the high-dose experimental group; UL: diameter of the inhibition zone of the low-dose experimental group; SH: diameter of the inhibition zone of the high-dose control group; SL: diameter of the inhibition zone of the low-dose control group; θ: relative drug potency; k: ratio of high to low dose, which is 2 here. Calculations using the above formulas show that the relative potencies of the Fe3O4@CS-formula 2-1-VH release solution determined by the tube-disc method are 95.0%, 79.5%, and 90.9%, respectively. Analysis of the tube-disc method results shows that the antibacterial drug carrier prepared by this experimental process, which achieves antibacterial function by grafting vancomycin onto the chitosan surface, exhibits antibacterial activity at three different time points under AMF regulation, with a relative potency of approximately 80% or higher compared to the VH standard solution of the same concentration. This indicates that the drug carrier causes minimal loss of pharmaceutical properties during drug molecule loading and transport, largely preserving drug efficacy. Furthermore, the addition of AMF to stimulate and regulate drug molecule release does not significantly affect the pharmaceutical properties of the loaded drug. These experimental results fully demonstrate that the magnetically controlled drug release carrier based on Fe3O4@CS magnetic nanoparticles proposed in this study possesses excellent drug transport capabilities and can largely preserve drug efficacy during application, providing a sound experimental theoretical basis for the subsequent practical application of this drug carrier.

[0124] In previous work, controlled drug release systems relied on physical or chemical stimuli such as temperature[1], pH[2], light[3], and ultrasound[4], but such exogenous energy would more or less damage the target organ or surrounding tissues[5]. Normal human tissues do not respond to magnetic fields and cannot interfere with the radiation of magnetic fields, so magnetically controlled drug release systems are safer than other controlled release systems regardless of the depth of the organ[5]. Unlike the magnetothermal controlled release principle of traditional alternating magnetic field controlled release systems[6], we released loaded vancomycin by destroying the antibacterial material that achieves antibacterial function by grafting vancomycin onto the surface of chitosan using Formula 2-1. The cytotoxicity, relative potency, and antibacterial activity of vancomycin in the released solution were close to those of standard VH solutions of the same concentration.

[0125] (5) Contact antibacterial properties and contact cytotoxicity of antibacterial materials that achieve antibacterial function by grafting vancomycin onto the surface of chitosan.

[0126] The contact antibacterial test method is as follows:

[0127] (a) Weigh a certain amount of Fe3O4@CS and Fe3O4@CS-form 2-1-VH magnetic nanoparticles and prepare them into 3 mg / mL Fe3O4@CS suspension and Fe3O4@CS-form 2-1-VH suspension by 0.01 mM PBS;

[0128] (b) Prepare a VH standard solution with a concentration of 474 μg / mL;

[0129] (c) Mix 200 μL of Fe3O4@CS, Fe3O4@CS-formula 2-1-VH suspension and 474 μg / mL of VH standard solution with an equal volume of 5 × 10⁻⁶. 7 A mixture of CFU / mL Staphylococcus aureus solution or Escherichia coli was prepared, and an equal amount of PBS was added to the control group as a negative control.

[0130] (d) The mixtures of each group were incubated in a bacterial incubator at 37°C for 120 min. Then, 100 μL of the mixed culture medium from each group was incubated and diluted 10-fold to 10⁻⁶. 10 The diluted culture medium was spread onto nutrient agar plates and incubated for 24 hours before colony counting.

[0131] (e) Weigh a certain amount of Fe3O4@CS and Fe3O4@CS-form 2-1-VH solid particles and prepare them into 3 mg / mL Fe3O4@CS suspension and Fe3O4@CS-form 2-1-VH suspension by 0.01 mM PBS;

[0132] (f) After treating two groups of Fe3O4@CS suspension and Fe3O4@CS-form 2-1-VH suspension at 80℃ for 48h, they were washed with 0.01mM PBS by magnetic separation and then prepared into 3 mg / mL Fe3O4@CS suspension and Fe3O4@CS-form 2-1-VH suspension by 0.01mM PBS. At the same time, a PBS control group and a vancomycin standard solution control group were set up.

[0133] (g) Add 5×10⁻⁶ mg of Fe₃O₄@CS suspension before and after treatment at 80℃, Fe₃O₄@CS-form 2-1-VH suspension, and the two control groups to the solutions of the two groups. 7 200 μL of Staphylococcus aureus or Escherichia coli at CFU / mL;

[0134] (h) After incubating each group in a 37°C bacterial incubator for 2 hours, each sample was added with bacterial live / dead staining working solution and stained in a 37°C incubator for 1 hour. Finally, the samples were observed under a fluorescence microscope.

[0135] The method for evaluating the contact cytotoxicity of Fe3O4@CS-form 2-1-VH powder is as follows:

[0136] (a) Weigh a certain amount of Fe3O4@CS and Fe3O4@CS-form 2-1-VH solid particles and prepare them into 3 mg / mL Fe3O4@CS suspension and Fe3O4@CS-form 2-1-VH suspension by 0.01 mM PBS;

[0137] (b) After treating two groups of Fe3O4@CS and Fe3O4@CS-form 2-1-VH suspensions at 80℃ for 48h, they were washed by magnetic separation and then reconstituted into 3mg / mL Fe3O4@CS suspension and Fe3O4@CS-form 2-1-VH suspension. At the same time, PBS and VH standard solution (474μg / mL) control groups were set up.

[0138] (c) Take a certain amount of Fe3O4@CS solution and Fe3O4@CS-form 2-1-VH solution before and after treatment at 80℃, as well as the solutions of the two control groups, and add them to the MC3T3-E1 cell suspension.

[0139] (d) After incubating each group in a 37°C cell culture incubator for 24 hours, each sample was stained with cell live / dead staining working solution for 30 minutes, and finally observed under a fluorescence microscope.

[0140] Test results are shown Figure 9 Among them, (A) Cytotoxicity diagram of Fe3O4@CS-Formula 2-1-VH release solution. Cytotoxicity diagram of Fe3O4@CS-Formula 2-1-VH powder: (B) Live / dead cell staining results and (C) Live / dead cell ratio (where af represents VH, PBS, Fe3O4@CS, Fe3O4@CS 80℃ 48h treatment, Fe3O4@CS-Formula 2-1-VH, Fe3O4@CS-Formula 2-1-VH 80℃ 48h treatment, respectively). (n=3)

[0141] The contact antibacterial performance of the antibacterial material achieving antibacterial function by grafting vancomycin onto the chitosan surface was verified on two levels. First, the total number of colonies in each experimental group was determined using the plate count method. The colony count and antibacterial activity results showed that both the 3 mg / ml chitosan powder and the antibacterial material powder achieving antibacterial function by grafting vancomycin onto the chitosan surface inhibited the growth of Staphylococcus aureus. However, compared to the negative control, the chitosan powder showed almost no antibacterial function, while the antibacterial material powder achieving antibacterial function by grafting vancomycin onto the chitosan surface had an inhibition rate of 99.99% against Staphylococcus aureus. Similarly, the bacterial viability / dead staining results (see attached...) Figure 7 8) This study showed that chitosan has a bactericidal effect on a small number of bacteria, and the antibacterial effect of the chitosan material did not change significantly after being treated in a water bath at 80℃ for 24 hours. The antibacterial material that achieves its antibacterial function by grafting vancomycin onto the surface of chitosan has a significant bactericidal effect due to the loading of vancomycin, but after the VH is fully released by heat, the antibacterial effect of the material also decreases significantly with the departure of VH.

[0142] Live / dead cell staining (with) Figure 9This study analyzed the contact cytotoxicity of antibacterial materials that achieve antibacterial function by grafting vancomycin onto the chitosan surface and chitosan itself. Results showed that under normal conditions, MC3T3 cells exhibited smooth edges and a small number of dead cells under a fluorescence microscope. However, after vancomycin treatment, the number of dead cells significantly increased, and numerous irregular punctate fragments appeared in the green fluorescent area. Similarly, the antibacterial material group that achieved antibacterial function by grafting vancomycin onto the chitosan surface also showed a significant increase in cell death and a large amount of cell debris. After VH release, the number of dead cells on the antibacterial material that achieved antibacterial function by grafting vancomycin onto the chitosan surface significantly decreased. Chitosan exhibits mild contact cytotoxicity, and there was no significant difference in cell activity on the material surface before and after heat treatment.

[0143] To improve the antibacterial activity of implants, different strategies have been proposed: some researchers have introduced metal ions, such as silver ions and copper ions[7]. Among them, iron ions are not only less expensive than silver ions, but also have no potential impact on the environment[8], yet they also have antibacterial ability. Some scholars believe that the main principle is that bacteria absorb iron ions and reactive oxygen species derived from iron ions, thereby producing a bactericidal effect[9]. In our study, chitosan, which is widely used in the biomedical field, was also used[10, 11]. It is well known that chitosan has positively charged amino groups that can attract negatively charged proteins on the surface of bacteria, generating electrostatic interactions. In addition, chitosan kills bacteria by hydrolyzing peptidoglycan on the cell wall and changing the permeability of the cell membrane[12-14]. However, the inhibitory effect of CS@Fe3O4 on bacterial growth and bactericidal activity is limited. Manoranjan Arakha et al.

[15] used chitosan to change the surface potential of nano-iron oxide by encapsulating nanoparticles, thereby enhancing the antibacterial properties of iron oxide. To improve the antibacterial properties of the material, we further grafted VH onto the CS@Fe3O4 surface. The antibacterial function of this material is mainly provided by vancomycin. Vancomycin is a glycopeptide antibiotic that interferes with the synthesis of the cell wall of Gram-positive bacteria. As we expected (see attached...). Figure 7 8) CS@Fe3O4-Formula 2-1-VH exhibits strong antibacterial activity, reducing bacterial count by four orders of magnitude compared to the control group. After heat treatment, Formula 2-1 of CS@Fe3O4-Formula 2-1-VH breaks down, and the antibacterial activity of the material is significantly reduced after the full release of VH. This experiment demonstrates that we have achieved on / off control of the contact antibacterial activity of CS@Fe3O4-Formula 2-1-VH material.

[0144] The live / dead ratio of MC3T3 cells on chitosan was decreased compared to the PBS control group, but the difference was not statistically significant, indicating that chitosan did not have significant cytotoxicity. Figure 9The results showed that the antibacterial material achieving antibacterial function by grafting vancomycin onto the chitosan surface exhibited significant cytotoxicity. We loaded the vancomycin control group and the antibacterial material with vancomycin grafted onto the chitosan surface with equal amounts of the material. We found that the number of MC3T3 cells decreased significantly after culturing in 450 μg / ml vancomycin and in CS@Fe3O4-formula 2-1-VH for 24 h. After VH was released from the surface of CS@Fe3O4-formula 2-1-VH, the survival rate of MC3T3 cells was comparable to that on CS@Fe3O4. Figure 3 This indicates that the cytotoxicity of the CS@Fe3O4-Formula 2-1-VH material originates from vancomycin. Most previous studies have concluded that vancomycin inhibits cell proliferation. In this patent, after performing its bactericidal function, through remote action of AMF, Formula 2-1 in CS@Fe3O4-Formula 2-1-VH is disrupted, allowing for the complete release of vancomycin from the material. This restores the material's cytocompatibility and avoids its impact on tissue repair. Therefore, we can release pre-loaded vancomycin after the peak infection period or the inflammatory phase, thus avoiding the contradiction between the material's antibacterial and cytotoxic properties.

[0145] (6) Antibacterial spectrum study

[0146] The experimental method is as follows:

[0147] (a) Weigh a certain amount of Fe3O4@CS and Fe3O4@CS-form 2-1-VH magnetic nanoparticles and prepare them into 3 mg / mL Fe3O4@CS suspension and Fe3O4@CS-form 2-1-VH suspension by 0.01 mM PBS;

[0148] (b) Prepare a VH standard solution with a concentration of 474 μg / mL;

[0149] (c) Mix 200 μL of Fe3O4@CS, Fe3O4@CS-formula 2-1-VH suspension and 474 μg / mL of VH standard solution with an equal volume of 5 × 10⁻⁶. 7 A mixture of CFU / mL Staphylococcus aureus solution or Escherichia coli was prepared, and an equal amount of PBS was added to the control group as a negative control.

[0150] (d) The mixtures of each group were incubated in a bacterial incubator at 37°C for 120 min. Then, 100 μL of the mixed culture medium from each group was incubated and diluted 10-fold to 10⁻⁶. 10 The diluted culture medium was spread onto nutrient agar plates and incubated for 24 hours before colony counting.

[0151] (e) Weigh a certain amount of Fe3O4@CS and Fe3O4@CS-form 2-1-VH solid particles and prepare them into 3 mg / mL Fe3O4@CS suspension and Fe3O4@CS-form 2-1-VH suspension by 0.01 mM PBS;

[0152] (f) After treating two groups of Fe3O4@CS suspension and Fe3O4@CS-form 2-1-VH suspension at 80℃ for 48h, they were washed with 0.01mM PBS by magnetic separation and then prepared into 3 mg / mL Fe3O4@CS suspension and Fe3O4@CS-form 2-1-VH suspension by 0.01mM PBS. At the same time, a PBS control group and a vancomycin standard solution control group were set up.

[0153] (g) Add 5×10⁻⁶ mg of Fe₃O₄@CS suspension before and after treatment at 80℃, Fe₃O₄@CS-form 2-1-VH suspension, and the two control groups to the solutions of the two groups. 7 200 μL of Staphylococcus aureus or Escherichia coli at CFU / mL;

[0154] (h) After incubating each group in a 37°C bacterial incubator for 2 hours, each sample was added with bacterial live / dead staining working solution and stained in a 37°C incubator for 1 hour. Finally, the samples were observed under a fluorescence microscope.

[0155] Vancomycin, the first glycopeptide antibiotic, possesses a triple bactericidal mechanism (inhibiting bacterial cell wall synthesis, altering bacterial cell membrane permeability, and preventing the synthesis of RNA in the bacterial cytoplasm). The most important of these mechanisms is vancomycin's direct binding to the D-alanyl-D-alanine residue at the end of the pentapeptide side chain of the peptidoglycan precursor in the cell wall. This prevents the transpeptidation of peptidoglycan polymerase, interfering with the cross-linking of the bacterial cell wall peptidoglycan precursor and thus preventing the cell wall from forming a three-dimensional structure, thereby exerting its bactericidal effect. Thanks to this triple bactericidal mechanism, vancomycin exhibits bactericidal activity against a variety of Gram-positive bacteria, making it the first-line drug for treating MRSA infections. It also plays a crucial role in delaying the emergence of drug-resistant bacteria and maintaining long-lasting sensitivity to vancomycin. Despite its renowned reputation, vancomycin is only effective against Gram-positive bacteria, while all Gram-negative bacteria are naturally resistant to it.

[0156] The contact antibacterial performance of the antibacterial material achieving antibacterial function through amino-grafted vancomycin on the chitosan surface was verified on two levels. First, the total colony count of each experimental group was determined using the plate count method. The colony count and antibacterial activity results showed that both chitosan powder (3 mg / ml) and the antibacterial material powder achieving antibacterial function through amino-grafted vancomycin on the chitosan surface could inhibit the growth of Gram-positive Staphylococcus aureus. However, compared with the negative control PBS, the chitosan powder showed almost no inhibitory effect on Gram-negative Escherichia coli. Comparing the antibacterial material powder achieving antibacterial function through amino-grafted vancomycin on the chitosan surface, the antibacterial material powder achieving antibacterial function through amino-grafted vancomycin on the chitosan surface showed significantly better inhibition of both Gram-positive and Gram-negative bacteria than the chitosan powder.

[0157] Appendix Figure 10 The antibacterial material, which achieves antibacterial function by grafting vancomycin onto the surface of chitosan, showed contact inhibition of Staphylococcus aureus and Escherichia coli. A: Bacterial coating results; B: Statistical analysis of coating results. ** indicates a significant difference between groups, p<0.01.

[0158] By comparing the antibacterial powder containing vancomycin grafted onto the chitosan surface to achieve antibacterial function with a similar vancomycin content, the antibacterial powder containing vancomycin grafted onto the chitosan surface showed significantly better inhibition of Gram-negative bacteria than the vancomycin suspension. Bacterial coating and counting results indicated that the antibacterial material containing vancomycin grafted onto the surface of this patent endows vancomycin with a new function of inhibiting Gram-negative bacteria. This may be due to the stronger ability of vancomycin, whose residues are fixed to the material surface through magnetic stimulation, to bind to and inhibit bacteria; bacteria in contact with the material surface are attracted to the surface, deformed, and die.

[0159] (7) Test for antimicrobial activity upon prolonged contact:

[0160] The contact antibacterial method was the same as above. The number of Staphylococcus aureus cultured in contact with the material was counted by bacterial coating and plate counting methods. Bacterial count was detected for 4 consecutive days, and a bacterial growth curve was plotted based on the bacterial count results within 24 hours. The biofilm formation after 24 hours of contact culture was detected by crystal violet staining method.

[0161] Appendix Figure 11The results of sustained antibacterial activity were as follows: A. Antibacterial activity assays over 4 consecutive days; B. Bacterial growth curves during 24-hour co-culture of materials and bacteria; C. Biofilm formation on the material surface after 24 hours of co-culture with Staphylococcus aureus; D. Quantitative comparison of biofilm formation on the material surface after 24 hours of co-culture with Staphylococcus aureus. N=3, ** indicates statistically significant differences between groups, p<0.01.

[0162] We compared the sustained antibacterial activity of the antibacterial material prepared by the method of this patent with that of ordinary antibacterial materials. (See attached...) Figure 11 As shown in Figure A, the antibacterial material prepared by the method of this patent can inhibit bacterial growth for 4 consecutive days, while ordinary antibacterial materials cannot maintain antibacterial activity, and the negative control group also cannot maintain antibacterial activity.

[0163] As attached Figure 11 As shown in B, bacteria cannot proliferate with the antibacterial material prepared by the method of this patent. This further proves that vancomycin, which is fixed on the material surface by magnetic stimulation of the binding residues, has a stronger ability to bind to and inhibit bacteria. Bacteria that come into contact with the material surface will be attracted to the material surface, deformed, and die.

[0164] Finally, we verified the material's ability to inhibit biofilm formation using the conventional crystal violet staining method. (See attached image) Figure 11 As shown in C and D, the antibacterial material prepared by the method of this patent can prevent the formation of bacterial biofilms.

[0165] Example 2: Antibacterial material that achieves antibacterial function by grafting vancomycin onto the collagen surface amino group.

[0166] 1. Take 2g of formula 2-1 and dissolve it in 50mL of 1% NaOH aqueous solution until it is fully dissolved;

[0167] 2. Take about 2.960g of R-type collagen, dissolve it in 50mL of 0.1% hydrochloric acid solution, and sonicate it at low temperature for 10min until it is completely dissolved. Immerse the titanium sheet in the collagen solution for 24h, then take it out and dry it to form a film, so that the surface of the titanium sheet is covered with a collagen layer.

[0168] 3. Coat the surface of the titanium sheet with a collagen layer, then adjust the pH to 6.5±0.3 with NaOH / HCl, add 1g DMTMM and 3g chitosan oligosaccharide, and stir at low temperature for 120h;

[0169] 4. Remove impurities and residual substrates using the dialysis bag method. Select a dialysis bag with a molecular weight cutoff of 4000 and dialyze in deionized water for 24 hours, changing the dialysis fluid every 2 hours.

[0170] 5. Add 2g of Formula 2-1 to the product from the previous step, then adjust the pH to 6.5±0.3 with NaOH / HCl, add 1g of DMTMM, and stir at low temperature for 120h.

[0171] 6. Remove impurities using the dialysis bag method. Select a dialysis bag with a molecular weight cutoff of 4000 and dialyze in deionized water for 24 hours, changing the dialysis fluid every 2 hours.

[0172] 7. Add 753.61 mg of vancomycin to the product from the previous step, then adjust the pH to 6.5 ± 0.3 with NaOH / HCl, add 143.90 mg of DMTMM, and stir at low temperature for 120 h;

[0173] 8. Remove impurities using the dialysis bag method. Select a dialysis bag with a molecular weight cutoff of 4000, and dialyze in deionized water for 24 hours, changing the dialysis fluid every 2 hours.

[0174] 9. Then place in a -20°C freezer overnight;

[0175] 10. Finally, the material was taken out and placed in a vacuum freeze-drying oven for freeze drying to obtain antibacterial material that achieves antibacterial function through amino grafting of vancomycin on the collagen surface;

[0176] Next, we tested the contact antibacterial properties and contact cytotoxicity. The experimental results were similar to those in Example 1. Before the action of AMF, the antibacterial material that achieved its antibacterial function by grafting vancomycin onto the collagen surface with amino groups exhibited both antibacterial and cytotoxic properties. Through the remote action of AMF, Formula 2-1 in the collagen on the titanium sheet surface was disrupted, and the vancomycin in the collagen raw material was completely released, restoring the cell compatibility of the collagen raw material and avoiding the material's impact on tissue repair.

[0177] Example 3: Antibacterial material with antibacterial function achieved by grafting vancomycin onto the surface of polydopamine.

[0178] 1. Immerse the titanium sheet in a 1M dopamine solution, adjust the pH to 8-9, and remove it after 24 hours to obtain a titanium sheet coated with polydopamine. The following uses the amino group on the surface of polydopamine to graft vancomycin.

[0179] 2. Take 420.42 mg of Formula 2-1 and dissolve it in 50 mL of 1% NaOH aqueous solution until it is fully dissolved;

[0180] 3. Coat the surface of the titanium sheet with polydopamine, then adjust the pH to 6.5±0.3 with NaOH / HCl, add 415.08mg EDC and 1g of Formula 2-1, and stir at low temperature for 120h;

[0181] 4. Remove impurities and residual substrates using the dialysis bag method. Select a dialysis bag with a molecular weight cutoff of 4000 and dialyze in deionized water for 24 hours, changing the dialysis fluid every 2 hours.

[0182] 5. Add 753.61 mg of vancomycin to the product from the previous step, then adjust the pH to 6.5 ± 0.3 with NaOH / HCl, add 143.90 mg of DMTMM, and stir at low temperature for 120 h;

[0183] 6. Remove impurities using the dialysis bag method. Select a dialysis bag with a molecular weight cutoff of 4000 and dialyze in deionized water for 24 hours, changing the dialysis fluid every 2 hours.

[0184] 7. Then place in a -20°C freezer overnight;

[0185] 8. Finally, the material was taken out and placed in a vacuum freeze-drying oven to freeze-dry and collect the antibacterial material that achieves antibacterial function by grafting vancomycin onto the surface of polydopamine.

[0186] Next, we tested the contact antibacterial properties and contact cytotoxicity. The experimental results were similar to those in Example 1. Before the action of AMF, the antibacterial material, which achieved its antibacterial function by grafting vancomycin onto the amino group of polydopamine, exhibited both antibacterial and cytotoxic properties. Through the remote action of AMF, formula 2-1 in the polydopamine on the titanium sheet surface was disrupted, and the vancomycin in the adhesive raw material was completely released, restoring the cell compatibility of the polydopamine material and avoiding any impact on tissue repair.

[0187] Example 4: Antibacterial material achieving antibacterial function by grafting vancomycin onto the gelatin surface with amino groups.

[0188] 1. Take 420.42 mg of Formula 2-1 and dissolve it in 50 mL of 1% NaOH aqueous solution until it is fully dissolved;

[0189] 2. Take about 2.960g of gelatin, dissolve it in 50mL of 0.1% hydrochloric acid solution, and sonicate it at low temperature for 10min until it is completely dissolved. Immerse the titanium sheet in the gelatin solution for 24h and then take it out so that the surface of the titanium sheet is covered with a gelatin layer.

[0190] 3. Coat the titanium sheet with a gelatin layer, then adjust the pH to 6.5±0.3 with NaOH / HCl, add 1g DMTMM and 3g chitosan, and stir at low temperature for 120h;

[0191] 4. Remove impurities and residual substrates using the dialysis bag method. Select a dialysis bag with a molecular weight cutoff of 4000 and dialyze in deionized water for 24 hours, changing the dialysis fluid every 2 hours.

[0192] 5. Add 2g of Formula 2-1 to the product from the previous step, then adjust the pH to 6.5±0.3 with NaOH / HCl, add 1g of DMTMM, and stir at low temperature for 120h.

[0193] 6. Remove impurities using the dialysis bag method. Select a dialysis bag with a molecular weight cutoff of 4000 and dialyze in deionized water for 24 hours, changing the dialysis fluid every 2 hours.

[0194] 7. Add 753.61 mg of vancomycin to the product from the previous step, then adjust the pH to 6.5 ± 0.3 with NaOH / HCl, add 143.90 mg of DMTMM, and stir at low temperature for 120 h;

[0195] 8. Remove impurities using the dialysis bag method. Select a dialysis bag with a molecular weight cutoff of 4000, and dialyze in deionized water for 24 hours, changing the dialysis fluid every 2 hours.

[0196] 9. Then place in a -20°C freezer overnight;

[0197] 10. Finally, the material was taken out and placed in a vacuum freeze-drying oven to freeze-dry and collect the antibacterial material that achieves antibacterial function by grafting vancomycin onto the surface of gelatin;

[0198] Next, we tested the contact antibacterial properties and contact cytotoxicity. The experimental results were similar to those in Example 1. Before the action of AMF, the antibacterial material that achieved its antibacterial function by grafting vancomycin onto the gelatin surface with amino groups exhibited both antibacterial and cytotoxic properties. Through the remote action of AMF, Formula 2-1 in the gelatin on the titanium sheet surface was disrupted, and the vancomycin in the gelatin material was completely released, restoring the cell compatibility of the gelatin material and avoiding any impact on tissue repair.

[0199] Example 5: Antibacterial material achieving antibacterial function by grafting vancomycin onto the surface of titanium.

[0200] 1. Immerse the titanium sheet in a 10mM dopamine aqueous solution and treat it at 80℃ for 4 hours. Then take it out and wash it thoroughly with water to obtain a titanium sheet with amino groups on the surface. The following uses the amino groups on the titanium surface to graft vancomycin.

[0201] 2. Take 420.42 mg of Formula 2-1 and dissolve it in 50 mL of 1% NaOH aqueous solution until it is fully dissolved;

[0202] 3. Coat the surface of the titanium sheet with polydopamine, then adjust the pH to 6.5±0.3 with NaOH / HCl, add 415.08mg DMTMM, and stir at low temperature for 120h;

[0203] 4. Remove impurities and residual substrates using the dialysis bag method. Select a dialysis bag with a molecular weight cutoff of 4000 and dialyze in deionized water for 24 hours, changing the dialysis fluid every 2 hours.

[0204] 5. Add 753.61 mg of vancomycin to the product from the previous step, then adjust the pH to 6.5 ± 0.3 with NaOH / HCl, add 143.90 mg of DMTMM, and stir at low temperature for 120 h; 6. Remove impurities using a dialysis bag method, selecting a dialysis bag with a molecular weight cutoff of 4000, and dialyze in deionized water for 24 h, changing the dialysis fluid every 2 h;

[0205] 7. Then place in a -20°C freezer overnight;

[0206] 8. Finally, the material was taken out and placed in a vacuum freeze-drying oven to freeze-dry and collect the antibacterial material that achieves antibacterial function by grafting vancomycin onto the surface of polydopamine.

[0207] Next, we tested the contact antibacterial properties and contact cytotoxicity. The experimental results were similar to those in Example 1. Before the action of AMF, the antibacterial material that achieved its antibacterial function by grafting vancomycin onto the titanium surface possessed both antibacterial and cytotoxic properties. Through the remote action of AMF, Formula 2-1 on the surface of the titanium sheet was destroyed, and the vancomycin on the material surface was completely released, restoring the biocompatibility of the titanium material and avoiding any impact on tissue repair.

[0208] Example 6: Antibacterial material achieving antibacterial function by amino-grafted vancomycin on titanium alloy surface.

[0209] 1. Immerse the titanium alloy sheet in a 10mM dopamine aqueous solution and treat it at 80℃ for 4 hours. Then take it out and wash it thoroughly with water to obtain a titanium alloy sheet with amino groups on the surface. The following uses the amino groups on the surface of the titanium alloy to graft vancomycin.

[0210] 2. Take 420.42 mg of Formula 2-1 and dissolve it in 50 mL of 1% NaOH aqueous solution until it is fully dissolved;

[0211] 3. Coat the surface of the titanium alloy sheet with polydopamine, then adjust the pH to 6.5±0.3 with NaOH / HCl, add 415.08mg DMTMM, and stir at low temperature for 120h;

[0212] 4. Remove impurities and residual substrates using the dialysis bag method. Select a dialysis bag with a molecular weight cutoff of 4000 and dialyze in deionized water for 24 hours, changing the dialysis fluid every 2 hours.

[0213] 5. Add 753.61 mg of vancomycin to the product from the previous step, then adjust the pH to 6.5 ± 0.3 with NaOH / HCl, add 143.90 mg of DMTMM, and stir at low temperature for 120 h; 6. Remove impurities using a dialysis bag method, selecting a dialysis bag with a molecular weight cutoff of 4000, and dialyze in deionized water for 24 h, changing the dialysis fluid every 2 h;

[0214] 7. Then place in a -20°C freezer overnight;

[0215] 8. Finally, the material was taken out and placed in a vacuum freeze-drying oven to freeze-dry and collect the antibacterial material that achieves antibacterial function by grafting vancomycin onto the surface of polydopamine.

[0216] Next, we tested the contact antibacterial properties and contact cytotoxicity. The experimental results were similar to those in Example 1. Before the action of AMF, the antibacterial material that achieved its antibacterial function by grafting vancomycin onto the titanium alloy surface possessed both antibacterial and cytotoxic properties. Through the remote action of AMF, Formula 2-1 on the surface of the titanium alloy sheet was destroyed, and the vancomycin on the material surface was completely released, restoring the cell compatibility of the titanium alloy material and avoiding any impact on tissue repair.

[0217] Example 7: Antibacterial material achieving antibacterial function by grafting vancomycin onto tantalum surface with amino groups.

[0218] 1. Tantalum sheets were immersed in a 10mM chitosan aqueous solution and treated at 80℃ for 4 hours. After that, they were taken out and thoroughly washed with water to obtain tantalum sheets with amino groups on the surface. Vancomycin was grafted onto the amino groups on the tantalum surface.

[0219] 2. Take 420.42 mg of Formula 2-1 and dissolve it in 50 mL of 1% NaOH aqueous solution until it is fully dissolved;

[0220] 3. Tantalum sheets with chitosan on the surface are coated, and the pH is adjusted to 6.5±0.3 by NaOH / HCl. 415.08mg DMTMM is added and stirred at low temperature for 120h.

[0221] 4. Remove impurities and residual substrates using the dialysis bag method. Select a dialysis bag with a molecular weight cutoff of 4000 and dialyze in deionized water for 24 hours, changing the dialysis fluid every 2 hours.

[0222] 5. Add 753.61 mg of vancomycin to the product from the previous step, then adjust the pH to 6.5 ± 0.3 with NaOH / HCl, add 143.90 mg of DMTMM, and stir at low temperature for 120 h;

[0223] 6. Remove impurities using the dialysis bag method. Select a dialysis bag with a molecular weight cutoff of 4000 and dialyze in deionized water for 24 hours, changing the dialysis fluid every 2 hours.

[0224] 7. Then place in a -20°C freezer overnight;

[0225] 8. Finally, the material was taken out and placed in a vacuum freeze-drying oven for freeze-drying and collection to obtain an antibacterial material that achieves antibacterial function by grafting vancomycin onto the surface of chitosan;

[0226] Next, we tested the contact antibacterial properties and contact cytotoxicity. The experimental results were similar to those in Example 1. Before the action of AMF, the antibacterial material that achieved its antibacterial function by grafting vancomycin onto the tantalum surface with amino groups exhibited both antibacterial and cytotoxic properties. Through the remote action of AMF, Formula 2-1 on the surface of the tantalum sheet was destroyed, and the vancomycin on the material surface was completely released, restoring the tantalum material to its cellular compatibility and avoiding any impact on tissue repair.

[0227] Example 8: Antibacterial material achieving antibacterial function by grafting vancomycin onto an amino group on a gold surface.

[0228] 1. Gold flakes were immersed in an aqueous solution of 10 mM chitosan oligosaccharide and 10 mM collagen and treated at 80°C for 4 hours. After that, they were taken out and thoroughly washed with water to obtain gold flakes with amino groups on the surface. Vancomycin was grafted onto the amino groups on the gold surface.

[0229] 2. Take 420.42 mg of Formula 2-1 and dissolve it in 50 mL of 1% NaOH aqueous solution until it is fully dissolved;

[0230] 3. Coat the surface of the gold flakes with chitosan oligosaccharide and collagen, then adjust the pH to 6.5±0.3 with NaOH / HCl, add 415.08mg EDC, and stir at low temperature for 120h;

[0231] 4. Remove impurities and residual substrates using the dialysis bag method. Select a dialysis bag with a molecular weight cutoff of 4000 and dialyze in deionized water for 24 hours, changing the dialysis fluid every 2 hours.

[0232] 5. Add 753.61 mg of vancomycin to the product from the previous step, then adjust the pH to 6.5 ± 0.3 with NaOH / HCl, add 143.90 mg of DMTMM, and stir at low temperature for 120 h;

[0233] 6. Remove impurities using the dialysis bag method. Select a dialysis bag with a molecular weight cutoff of 4000 and dialyze in deionized water for 24 hours, changing the dialysis fluid every 2 hours.

[0234] 7. Then place in a -20°C freezer overnight;

[0235] 8. Finally, the material was taken out and placed in a vacuum freeze-drying oven to freeze-dry and collect the antibacterial material that achieves antibacterial function by grafting vancomycin onto the gold surface with amino groups.

[0236] Next, we tested the contact antibacterial properties and contact cytotoxicity. The experimental results were similar to those in Example 1. Before the action of AMF, the antibacterial material that achieved its antibacterial function by grafting vancomycin onto the gold surface possessed both antibacterial and cytotoxic properties. Through the remote action of AMF, Formula 2-1 on the gold sheet surface was destroyed, and the vancomycin on the material surface was completely released, restoring the cell compatibility of the gold material and avoiding any impact on tissue repair.

[0237] Example 9: Antibacterial material achieving antibacterial function by grafting vancomycin onto a platinum surface with amino groups.

[0238] 1. Platinum sheets were immersed in a 10mM chitosan aqueous solution and treated at 80℃ for 4 hours. After that, they were taken out and thoroughly washed with water to obtain platinum sheets with amino groups on the surface. Vancomycin was then grafted onto the amino groups on the platinum surface.

[0239] 2. Take 420.42 mg of Formula 2-1 and dissolve it in 50 mL of 1% NaOH aqueous solution until it is fully dissolved;

[0240] 3. Coat a platinum sheet with chitosan on its surface, then adjust the pH to 6.5±0.3 with NaOH / HCl, add 415.08 mg DCC and 415.08 mg HOBt, and stir at low temperature for 120 h;

[0241] 4. Remove impurities and residual substrates using the dialysis bag method. Select a dialysis bag with a molecular weight cutoff of 4000 and dialyze in deionized water for 24 hours, changing the dialysis fluid every 2 hours.

[0242] 5. Add 753.61 mg of vancomycin to the product from the previous step, then adjust the pH to 6.5 ± 0.3 with NaOH / HCl, add 143.90 mg of DMTMM, and stir at low temperature for 120 h;

[0243] 6. Remove impurities using the dialysis bag method. Select a dialysis bag with a molecular weight cutoff of 4000 and dialyze in deionized water for 24 hours, changing the dialysis fluid every 2 hours.

[0244] 7. Then place in a -20°C freezer overnight;

[0245] 8. Finally, the material was taken out and placed in a vacuum freeze-drying oven for freeze-drying and collection to obtain an antibacterial material that achieves antibacterial function by grafting vancomycin onto the surface of chitosan;

[0246] Next, we tested the contact antibacterial properties and contact cytotoxicity. The experimental results were similar to those in Example 1. Before the action of AMF, the antibacterial material that achieved its antibacterial function by grafting vancomycin onto the platinum surface with amino groups exhibited both antibacterial and cytotoxic properties. Through the remote action of AMF, Formula 2-1 on the platinum sheet surface was disrupted, and the vancomycin on the material surface was completely released, restoring the platinum material to its cytocompatibility and avoiding any impact on tissue repair.

[0247] Example 10: Antibacterial material achieving antibacterial function by grafting vancomycin onto the surface of TC4 titanium.

[0248] 1. Immerse TC4 titanium sheets in a 10mM chitosan oligosaccharide aqueous solution and treat at 80℃ for 4 hours. Then remove and wash thoroughly with water to obtain TC4 titanium sheets with amino groups on the surface. Vancomycin is grafted onto the amino groups on the surface of TC4 titanium sheets.

[0249] 2. Take 420.42 mg of Formula 2-1 and dissolve it in 50 mL of 1% NaOH aqueous solution until it is fully dissolved;

[0250] 3. Coat the surface of the TC4 titanium sheet with chitosan oligosaccharide, then adjust the pH to 6.5±0.3 with NaOH / HCl, add 415.08 mg of dicyclohexylcarbodiimide (DCC) and 415.08 mg of 1-hydroxybenzotriazole (HOBT), and stir at low temperature for 120 h;

[0251] 4. Remove impurities and residual substrates using the dialysis bag method. Select a dialysis bag with a molecular weight cutoff of 4000 and dialyze in deionized water for 24 hours, changing the dialysis fluid every 2 hours.

[0252] 5. Add 753.61 mg of vancomycin to the product from the previous step, then adjust the pH to 6.5 ± 0.3 with NaOH / HCl, add 143.90 mg of DMTMM, and stir at low temperature for 120 h;

[0253] 6. Remove impurities using the dialysis bag method. Select a dialysis bag with a molecular weight cutoff of 4000 and dialyze in deionized water for 24 hours, changing the dialysis fluid every 2 hours.

[0254] 7. Then place in a -20°C freezer overnight;

[0255] 8. Finally, the material was taken out and placed in a vacuum freeze-drying oven for freeze-drying to obtain an antibacterial material that achieves antibacterial function by grafting vancomycin onto the surface of chitosan oligosaccharide;

[0256] Next, we tested the contact antibacterial properties and contact cytotoxicity. The experimental results were similar to those in Example 1. Before AMF treatment, the antibacterial material, which achieves its antibacterial function by grafting vancomycin onto the surface of TC4 titanium with oligosaccharide amino groups, exhibited both antibacterial and cytotoxic properties. Through remote AMF treatment, Formula 2-1 on the surface of the TC4 titanium sheet was disrupted, and the vancomycin on the material surface was completely released, restoring the TC4 titanium material to its cellular compatibility and preventing any impact on tissue repair.

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Claims

1. A composite antimicrobial material modified with vancomycin grafted through magnetic response linkage, characterized in that, The invention includes a magnetocaloric material, a polymer having free amino and / or hydroxyl groups coated with the magnetocaloric material, and vancomycin with residues grafted onto the polymer via magnetron stimulation. The magnetocaloric material is a functional material that can generate magnetocaloricity under the action of an alternating magnetic field, and it is at least one of iron oxide, titanium, tantalum, platinum, gold, titanium alloy and stainless steel; The magnetron-stimulated linker residue contains a magnetically responsive bond that can break under the action of an alternating magnetic field; and the magnetically responsive bond is an azo bond; one end of the magnetron-stimulated linker residue is linked to a polymer, and the other end is linked to vancomycin; the two ends of the magnetron-stimulated linker residue are respectively linked to the polymer and vancomycin via amide-base and / or ester bonds; the magnetron-stimulated linker residue is a fragment having the structural formula of Formula 1: Formula 1 n is an integer from 1 to 6; R is H or a C1 to C6 alkyl group; The polymer is at least one of chitosan, chitosan oligosaccharide, collagen, polydopamine, and gelatin.

2. The composite antibacterial material as described in claim 1, characterized in that, In composite antibacterial materials, polymers are coated on the surface of functional materials. The thickness of the polymer layer on the surface of the functional materials is 1 nm to 5 mm, and the weight ratio of vancomycin to polymers is 1:10000 to 1:

5.

3. A method for preparing the composite antibacterial material according to any one of claims 1 to 2, characterized in that, A polymer is coated on the surface of a magnetothermal material, followed by a first-stage linkage reaction with a precursor substance of magnetron-stimulated linkage residues. Vancomycin is then added to carry out a second-stage linkage reaction to obtain the composite antibacterial material.

4. The preparation method according to claim 3, characterized in that, Magnetically stimulated linkage residue precursors are compounds with the structure of Formula 2: Equation 2.

5. The preparation method according to claim 3 or 4, characterized in that, The first and second stage linkage reactions are carried out with the assistance of a crosslinking activator. The crosslinking activator is at least one selected from DMTMM, isothiocyanate, isocyanate, acyl azide, sulfonyl chloride, aldehyde, epoxide, carbonate, aryl halide, imide ester, carbodiimide, acid anhydride and fluorophenyl ester.

6. The preparation method according to claim 5, characterized in that, The crosslinking activator is at least one of DCC, EDC, glyoxal, ethylene oxide, and NHS ester.

7. The application of the composite antibacterial material according to any one of claims 1 to 2, characterized in that, It is used to prepare antibacterial materials.

8. An antibacterial material, characterized in that, The composite antibacterial material comprising a pharmaceutically effective amount as described in any one of claims 1 to 2.

9. The antibacterial material as described in claim 8, characterized in that, The antibacterial material is an antibacterial implant material.

10. The antibacterial material as described in claim 8 or 9, characterized in that, The antibacterial material has the composite antibacterial material added to its surface and within its framework.

Images