Chitosan oligosaccharide-gallic acid octyl ester covalent conjugate, and preparation method and application thereof

Abstract

This invention provides a chitosan oligosaccharide-octyl gallate covalent conjugate, its preparation method, and its applications. The chitosan oligosaccharide-octyl gallate covalent conjugate is obtained by covalently grafting octyl gallate onto the chitosan oligosaccharide molecular chain. The preparation method includes: mixing an ethanol solution of octyl gallate and an aqueous solution of chitosan oligosaccharide, adding ascorbic acid, and stirring under an inert gas atmosphere; adding hydrogen peroxide to initiate the reaction, and immediately subjecting the mixture to ultrasonic treatment to obtain the reaction solution; transferring the solution to a dialysis bag for purification, followed by freeze-drying. The covalent conjugate of this invention exhibits high grafting efficiency and good water dispersibility. Under external light stimulation, it can generate reactive oxygen species, demonstrating a scavenging effect against methicillin-resistant Staphylococcus aureus (MRSA) and its biofilms. Simultaneously, it exhibits good biocompatibility with mammalian cells, showing broad application prospects in the field of anti-infective biomaterials.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, and particularly relates to a chitosan oligosaccharide-octyl gallate covalent conjugate, its preparation method and application. Background Technology

[0002] Bacterial contamination is widespread in many areas, including food safety, healthcare, and daily life environments, posing a serious threat to public health. With the long-term and widespread use of antibiotics, bacterial resistance has become increasingly prominent, especially with the emergence of multidrug-resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA), which has significantly reduced the effectiveness of traditional antibiotics. Even more serious is that MRSA readily aggregates on surfaces or host tissues and secretes extracellular polymers, forming dense biofilms. This complex microbial community structure acts as a natural physical barrier, effectively blocking the penetration of antimicrobial drugs. This results in bacteria within the biofilm exhibiting resistance hundreds to thousands of times higher than that of planktonic bacteria, further exacerbating the difficulty of controlling drug-resistant bacterial infections.

[0003] Against this backdrop, the development of safe and effective non-antibiotic antibacterial therapies has become a research hotspot in the field. Among these, octyl gallate, an esterified derivative of gallic acid and n-octanol, is a highly effective, broad-spectrum food-grade phenolic antibacterial agent. It not only exhibits significant inhibitory effects against various foodborne pathogens, but also demonstrates excellent bactericidal activity against Gram-positive bacteria such as MRSA. Furthermore, octyl gallate possesses unique photosensitivity properties; under 400-500 nm visible light excitation, it can induce the generation of reactive oxygen species such as singlet oxygen, thereby exerting a photodynamic synergistic bactericidal effect. Currently, octyl gallate has passed safety assessments by the World Health Organization, the US Food and Drug Administration, and food safety agencies in many countries, and has been approved for use as a food additive. It is a recognized safe substance and is now widely used as an antioxidant and preservative in oils, dairy products, and pharmaceutical products, with broad applications in the food industry and biopharmaceutical field.

[0004] Although octyl gallate possesses good antibacterial activity, photosensitizing properties, and biosafety, its long-chain alkyl structure results in extremely poor solubility in aqueous systems (only 20 mg / L at 30°C), which greatly limits its reactive oxygen quantum yield and photodynamic bactericidal efficiency in aqueous environments. At the same time, free octyl gallate lacks effective targeting ability and tends to have non-specific effects on non-target areas in complex biological or material environments, which seriously restricts its further application.

[0005] While existing drug delivery systems attempt to solve the application challenges of hydrophobic drugs, they generally suffer from problems such as complex preparation methods, reliance on physical adsorption or non-covalent loading, which leads to unstable drug release, poor targeting, and some systems also pose potential toxic risks to host cells, failing to simultaneously meet the application requirements of high efficiency, safety, and targeting.

[0006] In summary, there is currently a lack of ideal technical solutions that can effectively improve the water solubility of octyl gallate, enhance its photodynamic antibacterial efficiency, and achieve targeted action. Therefore, there is an urgent need to develop a novel antibacterial material that can improve the utilization rate of hydrophobic natural photosensitizer small molecules in aqueous systems, selectively eliminate multidrug-resistant bacteria and their biofilms under external light stimulation, and possess good biocompatibility with mammalian cells. To this end, the inventors of this application have conducted in-depth research and exploration to address the shortcomings of the existing technologies. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to overcome the shortcomings of the prior art and provide a chitosan oligosaccharide-octyl gallate covalent conjugate with good water dispersibility, biocompatibility and photoresponsive antibacterial and anti-biofilm properties, as well as its preparation method and application. This solves the problem that octyl gallate has poor dispersibility and limited utilization efficiency in water-based systems due to its strong hydrophobicity, while avoiding the defects of traditional antibacterial methods that are prone to inducing drug resistance and have potential biosafety risks.

[0008] To address the aforementioned technical problems, this invention provides a chitosan oligosaccharide-octyl gallate covalent conjugate, which is obtained by covalently grafting octyl gallate onto the chitosan oligosaccharide molecular chain.

[0009] Furthermore, the aforementioned chitosan oligosaccharide-octyl gallate covalent conjugate has the ability to generate reactive oxygen species under visible light excitation.

[0010] Based on a general technical concept, the present invention provides a method for preparing the above-mentioned chitosan oligosaccharide-octyl gallate covalent conjugate, the preparation method comprising the following steps: S1. Mix octyl gallate ethanol solution and chitosan oligosaccharide aqueous solution, add ascorbic acid, and stir under inert gas protection to obtain a mixture; S2. Add hydrogen peroxide to the mixture to start the reaction, and immediately perform ultrasonic treatment to obtain the reaction solution; S3. The reaction solution is transferred to a dialysis bag for purification, and then obtained by freeze drying as a chitosan oligosaccharide-octyl gallate covalent conjugate.

[0011] In the above preparation method, further, in step S1, the octyl gallate ethanol solution and the chitosan oligosaccharide aqueous solution are mixed in equal volumes, and the amount of octyl gallate added is 20% to 300% of the mass of chitosan oligosaccharide.

[0012] In the above preparation method, in step S1, the concentration of ascorbic acid is 5 mM to 20 mM (preferably 12.0 mM to 12.5 mM), and after adding ascorbic acid, the mixture is stirred under a slow nitrogen flow for 10 min to 60 min (preferably 30 min).

[0013] In the above preparation method, further, in step S2, the concentration of hydrogen peroxide is 1 M to 10 M (preferably 5 M).

[0014] In the above preparation method, further, in step S2, the reaction temperature is controlled at 20℃~35℃ during the ultrasonic treatment; the power of the ultrasonic treatment is 300W~900W (preferably 700W), the frequency is 20 kHz~60 kHz (preferably 53 kHz), and the treatment time is 1 min~10 min (preferably 5 min).

[0015] In the above preparation method, further, in step S3, the molecular weight cutoff value of the dialysis bag is 500 Da to 1000 Da. First, it is dialyzed in an ethanol aqueous solution (preferably 50% ethanol by mass) for 1 to 3 days (preferably 1 day) to remove unreacted octyl gallate, then dialyzed in ultrapure water for 3 to 6 days (preferably 3 days), and then freeze-dried to obtain the purified product.

[0016] Based on a general technical concept, the present invention provides the application of the chitosan oligosaccharide-octyl gallate covalent conjugate in the preparation of a drug for killing drug-resistant bacteria.

[0017] Based on a general technical concept, the present invention provides an application of the chitosan oligosaccharide-octyl gallate covalent conjugate in the preparation of anti-biofilm materials.

[0018] Compared with the prior art, the advantages of the present invention are as follows: (1) This invention provides a chitosan oligosaccharide-octyl gallate covalent conjugate. In view of the problem that octyl gallate has poor water solubility and easy aggregation, resulting in low effective utilization, this invention uses an ultrasound-assisted free radical grafting strategy to covalently graft it onto the chitosan oligosaccharide molecular chain. Relying on the excellent water solubility and biocompatibility of chitosan oligosaccharide, this invention fundamentally solves the problems of leakage, phase separation and insufficient stability that are easy to occur in physical encapsulation and non-covalent loading. While retaining the functional activity of octyl gallate, it significantly improves its dispersibility and stability in aqueous systems and enhances its availability in the field of photoresponsive antibacterial.

[0019] (2) This invention provides a chitosan oligosaccharide-octyl gallate covalent conjugate, which overcomes the disadvantages of short half-life, short migration distance and poor targeted killing efficiency of reactive oxygen species generated by photodynamic therapy of octyl gallate. By utilizing the natural positive charge of chitosan oligosaccharide to construct a covalent conjugate system, it can preferentially bind to the negatively charged bacterial surface through electrostatic interaction, allowing reactive oxygen species to be directionally enriched and killed in the bacterial and membrane regions, greatly improving the photodynamic antibacterial efficiency and targeting selectivity, while reducing the non-specific damage of reactive oxygen species to mammalian cells, thus achieving a synergistic improvement in antibacterial effect and biosafety.

[0020] (3) This invention provides a chitosan oligosaccharide-octyl gallate covalent conjugate. The preparation process of this invention is simple and the process conditions are mild. It abandons the toxic catalysts and heavy metal catalysts of traditional chemical modification. It adopts the ascorbic acid / hydrogen peroxide system combined with ultrasonic treatment to initiate the grafting reaction. The ultrasonic cavitation effect accelerates the generation and transfer of free radicals and shortens the reaction time. It achieves high grafting efficiency under the premise of non-toxicity and pollution-free. The process has good repeatability and is easy to control. It has good prospects for scale-up production and industrial application.

[0021] (4) This invention provides a chitosan oligosaccharide-octyl gallate covalent coupling compound. The grafting rate of the obtained chitosan oligosaccharide-octyl gallate covalent coupling compound is significantly better than that of traditional physical blending and conventional Fenton radical grafting methods. Ultrasonic assistance further improves the controllability of the reaction and the uniformity of the product. At the same time, the covalent bond gives the coupling compound better thermal stability and structural stability, avoiding the problems of easy precipitation, degradation and functional decay of free octyl gallate, and meeting the requirements of long-term stable use of antibacterial material systems.

[0022] (5) This invention provides a chitosan oligosaccharide-octyl gallate covalent conjugate, which has excellent antibacterial effect against drug-resistant bacteria and can effectively inhibit and remove MRSA biofilms; relying on a novel action mode of electrostatic attraction-cell membrane penetration-reactive oxygen killing, combined with the membrane-breaking ability and photosensitivity of octyl gallate and the positive-charge targeting of chitosan oligosaccharide, it can synergistically destroy bacterial cell membranes, proteins and DNA, triggering irreversible bacterial death; at the same time, it has a highly selective killing effect on bacteria, no obvious toxicity to mammalian cells, and excellent biocompatibility. Attached Figure Description

[0023] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings.

[0024] Figure 1 The above is a process flow diagram of the preparation method of chitosan oligosaccharide-octyl gallate covalent conjugates in Examples 1 to 8 of the present invention.

[0025] Figure 2 The ultraviolet spectra, grafting rate diagrams, particle size diagrams, and potential diagrams of the chitosan oligosaccharide-octyl gallate covalent conjugates prepared in Examples 1 to 5 of this invention and Comparative Examples 1 to 3 are shown.

[0026] Figure 3 The images show the water dispersibility and water contact angle of the chitosan oligosaccharide-octyl gallate covalent conjugates prepared in Examples 1 to 5 of this invention and Comparative Examples 1 to 2.

[0027] Figure 4 Scanning electron microscope (SEM) images of the chitosan oligosaccharide-octyl gallate covalent conjugates prepared in Examples 2 to 4 of this invention and Comparative Examples 1 to 2.

[0028] Figure 5 The X-ray diffraction spectra, Fourier transform infrared spectra, free amino content spectra, and proton nuclear magnetic resonance spectra of the chitosan oligosaccharide-octyl gallate covalent conjugates prepared in Examples 2 to 4 of this invention and Comparative Examples 1 to 3 are shown.

[0029] Figure 6 Thermogravimetric curves, differential thermogravimetric curves, and differential calorimetric scan curves of the chitosan oligosaccharide-octyl gallate covalent conjugates prepared in Examples 2 to 4 of the present invention and Comparative Examples 1 to 2 are shown.

[0030] Figure 7 The diagram shows the anti-MRSA activity of the chitosan oligosaccharide-octyl gallate covalent conjugates prepared in Examples 6 to 8 of this invention and the comparative examples 5 to 8.

[0031] Figure 8 The images show the chitosan oligosaccharide-octyl gallate covalent conjugate prepared in Example 8 of this invention and the MRSA capture test results of Comparative Examples 5 and 6.

[0032] Figure 9 The membrane flow test diagrams, live / dead staining diagrams, and quantitative analysis diagrams of the chitosan oligosaccharide-octyl gallate covalent conjugate prepared in Example 8 of this invention and Comparative Examples 5 to 6 are shown.

[0033] Figure 10 Scanning electron microscope images of the chitosan oligosaccharide-octyl gallate covalently coupled compound prepared in Example 8 of this invention and the MRSA treated in Comparative Examples 5 and 6.

[0034] Figure 11 The graph shows the free radical generation of the chitosan oligosaccharide-octyl gallate covalent conjugate prepared in Example 8 of the present invention and Comparative Example 6 in the presence or absence of blue light.

[0035] Figure 12 The diagram shows the types of free radicals generated in the chitosan oligosaccharide-octyl gallate covalently prepared in Example 8 of this invention and in Comparative Example 5, with or without blue light and in the presence of different types of free radical scavengers.

[0036] Figure 13 The images show the DNA and protein diagrams of the chitosan oligosaccharide-octyl gallate covalent conjugate prepared in Example 8 of this invention and the DNA and protein diagrams of MRSA treated in Comparative Examples 5 and 6.

[0037] Figure 14 The crystal violet staining results and quantitative diagrams of the chitosan oligosaccharide-octyl gallate covalent conjugate prepared in Example 8 of this invention and the crystal violet staining results of the immature MRSA biofilms treated with Comparative Examples 5 and 6, as well as the colony count diagrams in the biofilms, are shown.

[0038] Figure 15 The crystal violet staining results and quantitative diagrams of the chitosan oligosaccharide-octyl gallate covalent conjugate prepared in Example 8 of this invention and the crystal violet staining results of the mature MRSA biofilms treated with Comparative Examples 5 and 6, as well as the colony count diagrams in the biofilms, are shown.

[0039] Figure 16 The diagram shows the colony distribution of the chitosan oligosaccharide-octyl gallate covalent conjugate prepared in Example 8 of this invention and the colony distribution of immature and mature MRSA biofilms treated in Comparative Examples 5 and 6.

[0040] Figure 17 The graphs show the hemolysis rate, cell viability, and lactate dehydrogenase (LDH) release of chitosan oligosaccharide-octyl gallate covalent conjugates prepared in Examples 6 to 8 of this invention and Comparative Example 9 pairs of mouse erythrocytes.

[0041] Figure 18 The survival rate of bacterial cells co-culture of the chitosan oligosaccharide-octyl gallate covalent conjugate prepared in Example 8 of this invention is shown in the figure. Detailed Implementation

[0042] The present invention will be further described below with reference to specific preferred embodiments, but this does not limit the scope of protection of the present invention.

[0043] The materials, reagents, and instruments used in the following examples are all commercially available. Unless otherwise specified, the experimental methods used in the following examples are conventional methods in the art.

[0044] Example 1 A chitosan oligosaccharide-octyl gallate covalent conjugate is prepared by covalently grafting octyl gallate onto chitosan oligosaccharide using an ultrasound-assisted free radical grafting method. The preparation method is described in [link to preparation method]. Figure 1 Specifically, it includes the following steps: (1) Mix 0.2 g octyl gallate ethanol solution (50 mL) and 1 g chitosan oligosaccharide aqueous solution (50 mL), add ascorbic acid (0.216 g), and stir for 30 min under a slow nitrogen flow.

[0045] (2) After adding hydrogen peroxide (5 M, 2 mL), start the reaction and immediately sonicate the reaction system for 5 min (700 W, 53 kHz). The sonication process is carried out in an ice-water bath. The reaction temperature of the reaction system is controlled at 25℃~35℃ to obtain the reaction solution.

[0046] (3) The reaction solution was transferred to a 500 Da dialysis bag for purification, and then obtained by freeze drying as a chitosan oligosaccharide-octyl gallate covalent conjugate.

[0047] Example 2 A method for preparing the chitosan oligosaccharide-octyl gallate covalent conjugate of the present invention is basically the same as that in Example 1, except that the mass of octyl gallate in step (1) is 0.5 g.

[0048] Example 3 A method for preparing the chitosan oligosaccharide-octyl gallate covalent conjugate of the present invention is basically the same as that in Example 1, except that the mass of octyl gallate is 1 g in step (1).

[0049] Example 4 A method for preparing the chitosan oligosaccharide-octyl gallate covalent conjugate of the present invention is basically the same as that in Example 1, except that the mass of octyl gallate is 2 g in step (2).

[0050] Example 5 A method for preparing the chitosan oligosaccharide-octyl gallate covalent conjugate of the present invention is basically the same as that in Example 1, except that the mass of octyl gallate is 3 g in step (2).

[0051] Comparative Example 1 A chitosan oligosaccharide-octyl gallate covalent conjugate and its preparation method are described. The preparation process is basically the same as that in Example 1, except that octyl gallate is not added in step (1).

[0052] Comparative Example 2 A chitosan oligosaccharide-octyl gallate covalent conjugate and its preparation method are described. The preparation process is basically the same as that in Example 1, except that chitosan oligosaccharide is not added in step (1). Comparative Example 3 A chitosan oligosaccharide-octyl gallate covalent conjugate and its preparation method are described. The preparation process is basically the same as that in Example 1, except that ascorbic acid is not added in step (1) and hydrogen peroxide and ultrasonic treatment are not performed in step (2).

[0053] Comparative Example 4 A chitosan oligosaccharide-octyl gallate covalent conjugate and its preparation method are described. The preparation process is basically the same as that in Example 1, except that ultrasonic treatment is not added in step (2).

[0054] Experiment 1: UV spectra, grafting rates, particle sizes, and potentials of chitosan oligosaccharide-octyl gallate covalent conjugates in Examples 1 to 5 and Comparative Examples 1 to 3.

[0055] (1) Grafting rate determination: 200 μL of 1 mg / mL chitosan oligosaccharide-octyl gallate solution was mixed with 1 M Folin-Ciocalteu reagent (1 mL) and reacted at room temperature in the dark for 5 minutes. After the reaction was completed, 800 μL of 7.5% sodium carbonate solution was added and reacted at room temperature in the dark for 1 hour. Subsequently, the absorbance of the mixture was measured at 765 nm in a 96-well plate.

[0056] (2) Particle size, polydispersity index and zeta potential test: The particle size, polydispersity index (PDI) and zeta potential of the coupling were recorded using a Zetasizer Pro instrument.

[0057] Figure 2 The figures show the UV spectra (A), grafting ratio (B), particle size distribution (C), and potential distribution (D) of the chitosan oligosaccharide-octyl gallate covalent conjugates prepared in Examples 1-5 and Comparative Examples 1-3 of this invention. Figure A shows that the chitosan oligosaccharide-octyl gallate conjugates grafted with different ratios exhibit a characteristic absorption peak at 276 nm, corresponding to the typical absorption peak of octyl gallate.

[0058] Figure B shows that the grafting efficiency of octyl gallate reached its maximum (50.75 ± 2.65 mg octyl gallate / g) when the mass ratio of chitosan oligosaccharide to octyl gallate was 1:1. Beyond this ratio, the steric hindrance caused by the excess octyl gallate molecules reduced the grafting efficiency. Furthermore, the grafting efficiency of the sample obtained by the ultrasound-assisted Fenton method was significantly better than that obtained by physical blending (Comparative Example 3, 21.75 ± 2 mg octyl gallate / g, p < 0.001) and the traditional free radical grafting method (Comparative Example 4, 36.41 ± 1.26 mg octyl gallate / g, p < 0.001), with increases of 39.3% and 133.3%, respectively. Although physical blending may promote partial binding of octyl gallate to chitosan oligosaccharide via hydrogen bonding, the resulting complex exhibited poor stability and low grafting efficiency compared to our covalent complex. Therefore, this natural and green ultrasound-assisted free radical grafting method provides a better alternative for covalently grafting octyl gallate onto chitosan oligosaccharides.

[0059] Figures C and D show that the zeta potentials of all conjugates are higher than those of Comparative Example 1 (original chitosan oligosaccharide), and the conjugate with the highest grafting efficiency has the smallest particle size (117.3 ± 9.63 nm) and the highest zeta potential (33.34 ± 0.58 mV). The reduced particle size may be attributed to the introduction of more octyl gallate molecules, which effectively prevents the aggregation of chitosan oligosaccharide chains. The high zeta potential (>30 mV) ensures the stability of the grafts. The polydispersity index (PDI) of all conjugates is below 0.3, indicating excellent dispersibility in water.

[0060] Experiment 2: Examples 1 to 5, and Comparative Examples 1 to 3, investigated the water dispersibility and water contact angle of the chitosan oligosaccharide-octyl gallate covalent conjugates.

[0061] Water contact angle and solubility test procedure: Measure the water contact angle of the sample using a contact angle meter. Dissolve different amounts of the coupling agent in water. Gradually increase the concentration until precipitation occurs. Measure the absorbance of each solution at 425 nm using a UV-Vis spectrophotometer.

[0062] Figure 3 The images show the water dispersibility and water contact angle of the chitosan oligosaccharide-octyl gallate covalent conjugates prepared in Examples 1-5 and Comparative Examples 1-3 of this invention.

[0063] Figure A shows that Example 3 has better water dispersibility than Comparative Example 2.

[0064] Figure B shows that the water contact angle of Example 3 (chitosan oligosaccharide to octyl gallate mass ratio of 1:1) significantly increased from 32.06° ± 0.74° in Comparative Example 1 (original chitosan oligosaccharide) to 57.04° ± 3.3°. Consistent with the grafting rate results, this significant hydrophobic shift contrasts sharply with the water contact angle of simple physical blending (Comparative Example 3) (WCA only increased to 39.26° ± 2.36°), further confirming the superiority of ultrasound-assisted free radical grafting. This favorable hydrophilicity (the conjugate's solubility in water reaches 833 μg / mL, approximately 11 times that of octyl gallate), along with its improved water stability, indicates that the conjugate possesses suitable solubility and stability for aqueous applications.

[0065] Experiment 3: Microstructure investigation of the grafts of chitosan oligosaccharide-octyl gallate covalently coupled in Examples 2 to 4 and Comparative Examples 1 to 2.

[0066] Figure 4Scanning electron microscope (SEM) images of the chitosan oligosaccharide-octyl gallate covalent conjugates prepared in Examples 2-4 of this invention and Comparative Examples 1-2 are shown. As can be seen from the images, Comparative Example 1 exhibits a smooth, lamellar structure, while Comparative Example 2 exhibits a regular rod-like morphology. All examples show a more porous structure. The surfaces of Examples 2-4 become rougher and contain some pores, exhibiting a composite morphology of finer lamellar and rod-like structures. This morphological change directly reflects the successful grafting of octyl gallate onto the chitosan oligosaccharide.

[0067] Experiment 4: X-ray diffraction spectra, Fourier transform infrared spectra, free amino content, and proton nuclear magnetic resonance spectra of chitosan oligosaccharide-octyl gallate covalent conjugates in Examples 1 to 5 and Comparative Examples 1 to 3.

[0068] The UV absorption spectra of the couplings were characterized using a UV-Vis spectrophotometer in the full wavelength range of 200–800 nm. The microstructure of the couplings was observed using a scanning electron microscope. Thermal properties were determined using a differential calorimeter under a nitrogen atmosphere, increasing the temperature from 25 °C to 210 °C at a rate of 10 °C / min. The thermal stability of the samples was tested using a thermogravimetric analyzer in the range of 30–800 °C at a rate of 10 °C / min. The crystal structure was determined using a Bruker D8 Advance X-ray diffractometer in the 2θ range of 5°–75° at a scan rate of 6° / min. Fourier transform infrared spectroscopy was used to determine the crystal structure of the samples at 4000 cm⁻¹. -1 ~400cm -1 Fourier transform infrared spectra within the range. The 1H NMR spectra of the sample were measured on a Bruker AVANCE III 400 MHz spectrometer after dissolving the sample in deuterated DMSO.

[0069] Figure 5 The X-ray diffraction spectra, Fourier transform infrared spectra, free amino content spectra, and proton nuclear magnetic resonance spectra of the chitosan oligosaccharide-octyl gallate covalent conjugates prepared in Examples 2-4 of this invention and Comparative Examples 1-3 are shown.

[0070] Figure A shows the X-ray diffraction spectrum. As can be seen from the figure, compared with Comparative Example 1, the diffraction peaks of Examples 2 to 4 are significantly weakened and broadened, and shifted to 20.45°, 20.59° and 20.49°, indicating that their structure has become looser.

[0071] Figure B shows the Fourier transform infrared spectrum. It can be seen from the figure that Examples 2-4 have a spectrum at 3386 cm⁻¹. -1 The enhanced absorption peak at 2933 cm⁻¹ is due to the hydroxyl groups introduced by octyl gallate. The presence of the methylene group in octyl gallate contributes to the enhanced absorption peak of all grafts at 2933 cm⁻¹. -1 The peak intensity at 1733 cm⁻¹ is enhanced. Examples 2-4 show an increase in peak intensity at 1733 cm⁻¹. -1The new absorption peak corresponds to the C=O group of octyl gallate. Compared with Comparative Examples 1 and 3, the 1517 cm⁻¹ peak characteristic of the primary amino group in chitosan oligosaccharide... -1 The spectral band at that location disappeared in Examples 2-4, and the band at 1517-1600 cm⁻¹ disappeared. -1 The peaks broadened within the range. This indicates that the amino group in chitosan oligosaccharide is one of the reaction sites for covalent grafting with octyl gallate. Furthermore, experiments using the ninhydrin colorimetric method to determine the free amino group content further verified the consumption of amino groups. Ninhydrin can react with free primary amino groups to produce a purple product, the color intensity of which is directly proportional to the amino group content.

[0072] Figure C shows the free amino content. As can be seen, the free amino content in Examples 1-5 is significantly lower than that in Comparative Examples 1 and 3, indicating that the amino groups on the chitosan oligosaccharide chain are one of the grafting sites. Figure D shows the 1H NMR spectrum. As can be seen, Examples 2-4 all show a proton signal peak at 6.9 ppm for the benzene ring of octyl gallate, while the signal in the range of 0.8-1.5 ppm corresponds to hydrogen atoms on the long-chain alkyl group of octyl gallate. These results collectively demonstrate that the ultrasound-assisted free radical grafting reaction successfully achieved the covalent grafting of octyl gallate onto chitosan oligosaccharides, obtaining structurally stable nanografts, which are presumably capable of exhibiting highly efficient anti-MRSA activity.

[0073] Experiment 5: Thermal stability study of the chitosan oligosaccharide-octyl gallate covalent conjugates prepared in Examples 2 to 4 and Comparative Examples 1 to 2.

[0074] Thermal stability is a key property for evaluating the processing durability and storage stability of polysaccharide-based biomaterials.

[0075] Figure 6 Thermogravimetric curves, differential thermogravimetric curves, and differential calorimetric scan curves of the chitosan oligosaccharide-octyl gallate covalent conjugates prepared in Examples 2-4 of this invention and Comparative Examples 1-2 are shown.

[0076] Figure A shows the thermogravimetric curves, which indicate that Examples 2-4 experienced two typical weight loss stages. Compared to Comparative Example 1, Examples 2-4 exhibited different weight loss stages and rates.

[0077] Figure B shows the thermogravimetric curves of the chitosan oligosaccharide. The graph indicates that the maximum thermal decomposition rates for Comparative Example 1 and Examples 2-4 occurred at 203°C and 239°C, respectively. This suggests that the introduction of octyl gallate increased the decomposition temperature of the chitosan oligosaccharide.

[0078] Figure C shows the differential calorimetry (DC) curve. As can be seen, Comparative Example 1 exhibits a significant broad endothermic peak at 78.43℃. After grafting with octyl gallate, this peak shifted to 80.43℃, 90.84℃, and 82.84℃, respectively, and the shift was positively correlated with the grafting rate. These results demonstrate that grafting with octyl gallate improves the thermal stability of chitosan oligosaccharides.

[0079] Example 6 A method for preparing the chitosan oligosaccharide-octyl gallate covalent conjugate of the present invention is basically the same as that in Example 3, except that the concentration is 0.5 mg / mL in the antibacterial test.

[0080] Example 7 A method for preparing the chitosan oligosaccharide-octyl gallate covalent conjugate of the present invention is basically the same as that in Example 3, except that the concentration is 1 mg / mL in the antibacterial test.

[0081] Example 8 A method for preparing the chitosan oligosaccharide-octyl gallate covalent conjugate of the present invention is basically the same as that in Example 3, except that the concentration is 2 mg / mL in the antibacterial / cell test.

[0082] Comparative Example 5 Water is used only.

[0083] Comparative Example 6 2 mg / mL chitosan oligosaccharide.

[0084] Comparative Example 7 2 mg / mL ungrafted octyl gallate group.

[0085] Comparative Example 8 Vancomycin group with octyl gallate content equivalent to that in 2 mg / mL chitosan oligosaccharide-octyl gallate covalent conjugate.

[0086] Experiment 6: To investigate the antibacterial activity of the chitosan oligosaccharide-octyl gallate covalent conjugates of Examples 6 to 8 and Comparative Examples 5 to 8.

[0087] Antimicrobial performance testing: After MRSA was activated in LB medium, bacterial suspensions (prepared with physiological saline to 10⁶ CFU / mL) were treated with different concentrations of conjugates and irradiated under blue light (BL, irradiation for 20 min, distance 10 cm). A group without blue light irradiation was also included. Colony counts were then performed using the standard plate count method, and photographs were taken. The control group included untreated bacteria, ungrafted OG COS (2 mg / mL), OG dissolved in pure water (2 mg / mL), and vancomycin (calculated as the equivalent volume of OG in 2 mg / mL COS-OG, dispersed in pure water).

[0088] Figure 7 The figures show the anti-MRSA activity of the chitosan oligosaccharide-octyl gallate covalent conjugates prepared in Examples 6-8 and Comparative Examples 5-8. In the figures, A and C are photographs of bacterial colonies on plates irradiated with and without blue light, respectively, while B and D represent the statistical results. As can be seen from the figures, the examples exhibit concentration-dependent bactericidal activity. After 20 minutes of blue light irradiation, Example 8 reduced the bacterial concentration by 4.69 ± 0.14 logarithms, while Comparative Examples 7 and 8 reduced the bacterial concentration by only 2.17 ± 0.46 and 0.58 ± 0.064 logarithms, respectively. This significant difference can be partly attributed to the hydrophobicity of octyl gallate, which limits its excellent antibacterial potential. Covalent grafting it onto chitosan oligosaccharide not only significantly improves its dispersibility in the aqueous phase but also retains its excellent function as a photosensitizer, thereby enhancing its antibacterial ability.

[0089] Experiment 7: To investigate the capture of MRSA by the chitosan oligosaccharide-octyl gallate covalent conjugates of Examples 6 to 8 and Comparative Examples 5 to 8.

[0090] After co-culturing the bacterial suspension (OD600=1) with the conjugate for 30 min, photographs were taken, and the OD600 value of the supernatant was measured to assess the bacterial capture ability. The bacterial suspension (10⁸ CFU / mL) was treated with COS-OG (2 mg / mL) under blue light for 20 min, followed by centrifugation at 8000 rpm for 5 min. After washing three times with sterile PBS, the resulting precipitate was resuspended in PBS, and the changes in bacterial surface potential were measured using a Zetasizer Pro instrument to assess its capture mechanism.

[0091] Figure 8 The images show the chitosan oligosaccharide-octyl gallate covalent conjugate prepared in Example 8 of this invention and the MRSA capture test results of Comparative Examples 5-6.

[0092] Figure A and Figure B show the test tubes after 30 minutes of incubation with and without blue light, respectively. The figures clearly show significant bacterial aggregation and sedimentation at the bottom of the test tubes.

[0093] Figure C represents the statistical results of absorbance. As can be seen from the figure, the optical density of Example 8 was significantly reduced, and the effect of Example 8 was more significant than that of Comparative Example 6. This indicates that the introduction of octyl gallate further enhanced the targeting effect on bacteria. Furthermore, under blue light irradiation, the turbidity reduction in Example 8 was more pronounced than in the dark group. This may be related to the reactive oxygen species generated by blue light excitation attacking the bacterial cell membrane, thereby exposing more hydrophobic and negatively charged binding sites.

[0094] Figure D represents the zeta potential diagram. As can be seen from the figure, the zeta potential significantly increased after the bacteria were mixed with both Comparative Example 6 and Example 8, with Example 8 showing a more pronounced increase. Under blue light irradiation, Example 8 further increased the MRSA potential by approximately 5.49 mV compared to Comparative Example 6. These results indicate that the conjugate can rapidly attract bacteria through electrostatic interactions, and Example 8 exhibits a stronger bacterial capture ability than Comparative Example 6, which is further enhanced by blue light irradiation.

[0095] Experiment 8: To investigate the effects of the chitosan oligosaccharide-octyl gallate covalent conjugate of Example 8 and Comparative Examples 5 and 6 on the MRSA cell membrane.

[0096] Bacterial cells (10⁸ CFU / mL) were co-cultured with the conjugate and resuspended in 1 mL PBS. PI / SYTO₉ dyes (5 μL each, staining for 20 min) were added to assess COS-OG-induced damage to MRSA cell membranes. After washing away excess dye, 10 μL of the reaction solution was imaged under a fluorescence microscope, and 200 μL of the reaction solution was transferred to a black 96-well plate for fluorescence intensity quantification using a microplate reader (PI: excitation / emission wavelength = 536 / 617 nm; SYTO₉: excitation / emission wavelength = 484 / 525 nm).

[0097] Cell membrane fluidity was assessed using the Laurdan fluorescent probe. Bacterial suspension (OD600 = 0.4) was incubated with the Laurdan probe (final concentration 20 μM, dissolved in NN-dimethylformamide) at 37°C for 45 min in the dark, with the final organic solvent content controlled at 1% (v / v) to ensure bacterial viability. After the probe embedded in the membrane structure, the cells were incubated for another 45 min with COS-OG solution (2 mg / mL dissolved in sterile PBS, pH 7.4) (including 20 min of blue light irradiation). The fluorescence signal was then measured using a multi-functional microplate reader (excitation wavelength 350 nm, emission wavelength 460 nm or 500 nm), and the generalized polarization value (GP) was calculated using the following formula (1): (1).

[0098] Figure 9 The membrane flow test diagrams, live / dead staining diagrams, and quantitative analysis diagrams of the chitosan oligosaccharide-octyl gallate covalent conjugate prepared in Example 8 of this invention and Comparative Examples 5-6 are shown.

[0099] Figure A shows the membrane fluidity test results. As can be seen from the figure, compared with Comparative Examples 5 and 6, Example 8 significantly disrupted membrane fluidity, and blue light irradiation further exacerbated this disruption. This disrupted bacterial homeostasis and altered membrane permeability. Therefore, after the conjugate rapidly targets the bacteria, the long hydrophobic chain of octyl gallate can insert into the bacterial cell membrane, thereby causing structural damage.

[0100] Figure B shows the live / dead staining pattern, while figures C and D show the quantitative analysis of the combination of no blue light and the combination of blue light, respectively. As can be seen from the figures, Example 8 resulted in a significant increase in membrane permeability, manifested as a marked enhancement in PI uptake. Blue light irradiation exacerbated this damage, possibly because the reactive oxygen species produced by octyl gallate further damaged the cell membrane, leading to a sharp increase in red fluorescence intensity.

[0101] Experiment 9: Microstructure of the chitosan oligosaccharide-octyl gallate covalently coupled compound prepared in Example 8 of this invention and the MRSA treated in Comparative Examples 5 to 6.

[0102] Morphological evaluation of bacteria after conjugate treatment: After co-culturing bacterial cells (10⁸ CFU / mL) with the conjugate, the precipitate was fixed overnight with glutaraldehyde (2.5%, 4℃), dehydrated with a gradient of ethanol, then critically dried, and sprayed with gold for observation.

[0103] Figure 10The images show scanning electron microscope (SEM) images of the chitosan oligosaccharide-octyl gallate covalently coupled compound prepared in Example 8 of this invention and MRSA treated with comparative examples 5-6. As can be seen from the images, the bacteria treated in Example 8 only showed slight shrinkage and deformation, while blue light irradiation caused extensive and severe deformation, rupture, and shrinkage of the MRSA, leading to leakage of cell contents and bacterial death. This is because blue light can excite the photo-oxidation of octyl gallate, generating reactive oxygen species that attack the bacterial structure.

[0104] Experiment 10: Analysis of reactive oxygen species generation in the presence or absence of blue light on the chitosan oligosaccharide-octyl gallate covalent conjugate prepared in Example 8 and Comparative Example 6.

[0105] Evaluation of ROS generation and types in the conjugate: ROS generation after blue light irradiation was detected by electron spin resonance. Furthermore, bacterial suspension (10⁶ CFU / mL) was mixed with the conjugate (2 mg / mL), and three ROS scavengers were added: 10 mM histidine, 2.8 M dimethyl sulfoxide, and 16 mM TEMPOL. After 20 min of blue light irradiation, 100 μL of the reaction solution was evenly spread on agar plates and incubated at 37°C for 24 h for colony counting evaluation.

[0106] Figure 11 This graph shows the free radical generation of the chitosan oligosaccharide-octyl gallate covalent conjugate prepared in Example 8 of this invention and Comparative Example 6 under the presence and absence of blue light. As can be seen from the graph, Example 8 generates three types of free radicals under blue light excitation: hydroxyl radicals (·OH), singlet oxygen radicals (·OH), and singlet oxygen radicals (·OH). 1 O2) and superoxide anion (·O2) In contrast, no free radicals were generated in Comparative Example 6, indicating that the photosensitivity of octyl gallate was retained after grafting.

[0107] Figure 12 This figure shows the types of free radicals generated by the chitosan oligosaccharide-octyl gallate covalently prepared in Example 8 of this invention and Comparative Example 5 in the presence or absence of blue light and in the presence of different types of free radical scavengers. Figure A shows the bacterial count, and Figure B shows a photograph of a colony plate. As can be seen from the figure, in the absence of blue light, the three scavengers (L-histidine quenching singlet oxygen; DMSO quenching hydroxyl radicals; and tempol quenching superoxide anion radicals) had no effect on bacterial growth. However, under blue light irradiation, L-histidine, tempol, and DMSO restored the bacterial count from 2.26 log CFU / mL to 4.81 log CFU / mL, 3.57 log CFU / mL, and 4.44 log CFU / mL, respectively. Therefore, hydroxyl radicals and singlet oxygen dominate the photodynamic inactivation of octyl gallate.

[0108] Experiment 11: Effects of the chitosan oligosaccharide-octyl gallate covalent conjugate prepared in Example 8 of this invention and comparative examples 5 to 6 on the DNA and protein of MRSA.

[0109] Bacterial suspension (10⁸ CFU / mL) was co-incubated with COS-OG (2 mg / mL) under blue light for 2 h. Subsequently, bacterial genomic DNA was extracted using a commercial genomic DNA extraction kit and separated by electrophoresis at 180 V for 25 min on a 0.8% agarose-TAE gel. DNA bands were recorded using a gel imaging system. For total protein analysis, the bacterial pellet was collected by centrifugation at 12,000 rpm, washed three times with PBS, and lysed with 1×SDS loading buffer. The lysate was treated with a boiling water bath for 10 min, then clarified by centrifugation again at 12,000 rpm and 4 °C, and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Protein bands were stained with Coomassie brilliant blue, destained, and then imaged.

[0110] Nucleic acids and proteins are among the important targets of reactive oxygen species attacks.

[0111] Figure 13 The figures show the DNA (A) and protein (B) of the chitosan oligosaccharide-octyl gallate covalent conjugate prepared in Example 8 of this invention and the MRSA treated with comparative examples 5-6. As can be seen from the figures, under blue light irradiation, the DNA and protein bands of Example 8 become lighter or even disappear, indicating that the generated reactive oxygen species (ROS) severely damage the macromolecular active substances within the bacteria. The hydrophobic long chain of octyl gallate facilitates its interaction with macromolecules within the bacteria, and the generation of ROS further exacerbates this damage.

[0112] Experiment 12: Investigate the effects of the chitosan oligosaccharide-octyl gallate covalent conjugate prepared in Example 8 and comparative examples 5 to 6 on MRSA biofilms.

[0113] The anti-biofilm activity of COS-OG against MRSA was evaluated using biofilm formation inhibition and clearance assays. In the inhibition assay, MRSA and COS-OG (2 mg / mL) were co-incubated in BHI medium containing 3% sucrose, irradiated with blue light for 20 min, and then incubated statically for 48 h. The culture was then washed three times with PBS to remove airborne bacteria. The resulting biofilm was stained with crystal violet solution (0.1%, 200 μL, 37℃, 30 min) and washed five times with sterile PBS to remove unbound dye before photographing. The stained biofilm was dissolved in 33% glacial acetic acid (200 μL, 37℃, 30 min) and quantified using a microplate reader at 570 nm. Furthermore, the viable bacteria in the biofilm were counted using a plate count method. In the clearance assay, the pre-formed biofilm (37℃, 48 h) was treated with COS-OG (2 mg / mL) and irradiated with blue light (20 min), followed by a second 24 h incubation before staining. Morphological observation was performed using scanning electron microscopy to image the biofilm cultured on sterile cell slides. During growth, bacteria secrete extracellular polymers to form dense biofilms to resist the effects of antibiotics and antibacterial agents. Therefore, this invention evaluated the ability of nanografts to inhibit biofilm formation and remove mature biofilms.

[0114] Figure 14 The figures show the crystal violet staining results (A) and quantitative graph (C) of the chitosan oligosaccharide-octyl gallate covalent conjugate prepared in Example 8 of this invention, and the colony count graphs (B and D) of the biofilms after treatment with immature MRSA biofilms by Comparative Examples 5-6. As can be seen from the figures, Example 8 inhibited biofilm formation by 67.91% and reduced bacterial activity by 3.06 log CFU / mL. Under blue light irradiation, the inhibition rate of Example 8 on biofilm formation further increased to 92.05%, and the bacterial activity within the biofilm decreased by 5.28 log CFU / mL. Even under blue light irradiation, Comparative Example 6 only showed an inhibition rate of 28.69% on biofilm formation and reduced bacterial activity by only 1.29 log CFU / mL.

[0115] Figure 15The figures show the crystal violet staining results (A) and quantitative graph (C) of the chitosan oligosaccharide-octyl gallate covalent conjugate prepared in Example 8 of this invention, and the colony count graphs (B and D) of the biofilms after treatment with mature MRSA biofilms from Comparative Examples 5-6. As can be seen from the figures, Example 8 removed 53.34% of the biofilm from the mature biofilm, reducing bacterial activity by 2.97 log CFU / mL. Under blue light irradiation, the biofilm removal rate increased to 82.28%, and the bacterial activity within the biofilm decreased by 3.95 log CFU / mL. Even under blue light irradiation, Comparative Example 6 only achieved a biofilm removal rate of 37.86%, reducing bacterial activity by only 0.26 log CFU / mL.

[0116] Figure 16 The figures show the colony distribution of the chitosan oligosaccharide-octyl gallate covalent conjugate prepared in Example 8 of this invention and the colonies of immature and mature MRSA biofilms treated with comparative examples 5 and 6. As can be seen from the figures, compared with the dense and intact biofilm structures of comparative examples 5 and 6, the colonies of Example 8 activated by blue light are sparse, the biofilm structure is disrupted, and the bacteria exhibit collapsed and ruptured morphologies. These results demonstrate that the conjugate exhibits excellent anti-MRSA biofilm activity under blue light.

[0117] Experiment Thirteen: Biosafety Evaluation.

[0118] Excellent biocompatibility is a key prerequisite for the practical application of conjugates. L929 cells (2 × 10⁻⁶) were used. 3 Cells (cells / well) were seeded in 96-well plates and cultured overnight at 37°C and 5% CO2 in high-glucose DMEM medium containing 10% fetal bovine serum and 1% penicillin and streptomycin. The next day, the medium was replaced with fresh complete medium containing different concentrations of COS-OG (0, 0.5, 1 and 2 mg / mL), and the cells were co-cultured for 1, 3 and 5 days. At the set time points, the supernatant was discarded and the cells were washed three times with PBS. Then 100 μL of fresh medium containing 10% CCK-8 reagent was added, and the cells were incubated at 37°C for 1 h. The absorbance at 450 nm was measured using a microplate reader. Cell viability was calculated according to the following formula (2): (2).

[0119] In addition, cell status was assessed using 2 µM Calcein-AM and 4 µg / mL PI. Cells were incubated in the dark at room temperature for 30 min, followed by imaging using a laser confocal microscope. The in vitro biocompatibility of COS-OG was evaluated using a mouse erythrocyte hemolysis assay.

[0120] Figure 17The graphs show the hemolysis rate (A), cell viability (B), and lactate dehydrogenase (LDH) release of the chitosan oligosaccharide-octyl gallate covalent conjugates prepared in Examples 6-8 of this invention and Comparative Example 9 pairs of mouse erythrocytes.

[0121] As shown in Figure A, the hemolysis rates of all examples are well below the 5% safety threshold specified in ISO 10993-5, indicating excellent blood compatibility. As shown in Figure B, after co-culturing with Examples 6 to 8 for 1, 3, and 5 days, the viability of L929 cells remained above 90%, indicating that the conjugate does not inhibit cell proliferation.

[0122] Furthermore, given that COS-OG targets bacterial cell membranes, a lactate dehydrogenase (LDH) release assay was performed to assess its effects on mammalian cell membranes. As can be seen from Figure C, no significant LDH leakage was observed in the example, confirming that the conjugate does not compromise the integrity of mammalian cell membranes.

[0123] Experiment Fourteen: Targeting selectivity of the chitosan oligosaccharide-octyl gallate covalent conjugate prepared in Example 8 against bacteria.

[0124] Cells (2×10) 3 Cells (cells / well) were seeded in 96-well plates, with 100 μL of antibiotic-free complete DMEM medium added to each well, and incubated overnight at 37°C and 5% CO2. Bacterial cells were prepared by adjusting the concentration of antibiotic-free DMEM medium to 2 × 10⁶ CFU / mL. The supernatant in the cell wells was discarded, and 100 μL of bacterial suspension was added, incubating at 37°C for 20 min. Subsequently, COS-OG (2 mg / mL) was added to each well, and incubation was performed for 20 min under blue light or darkness. After overnight co-culture, the supernatant was carefully aspirated, and colony counting was performed using the dilution plating method. Furthermore, cell viability was assessed using the CCK-8 assay after gently washing the cells three times with PBS.

[0125] Figure 18 The graph shows the bacterial cell co-culture survival rate of the chitosan oligosaccharide-octyl gallate covalent conjugate prepared in Example 8 of this invention. Figure A shows the MRSA survival rate, and Figure B shows the cell survival rate. As can be seen from the graph, a large number of bacteria survived under dark conditions, accompanied by a decrease in cell viability. In contrast, under blue light irradiation in Example 8, mammalian cell viability remained above 85%, while bacteria were almost completely eliminated, demonstrating that the conjugate has a highly selective bactericidal effect and can effectively target and eliminate bacteria without damaging mammalian cells.

[0126] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention using the methods and techniques disclosed above, or modify them into equivalent embodiments with equivalent changes, without departing from the spirit and technical essence of the present invention. Therefore, any simple modifications, equivalent substitutions, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solutions of the present invention shall still fall within the protection scope of the technical solutions of the present invention.

Claims

1. A chitosan oligosaccharide-octyl gallate covalent conjugate, characterized in that, The chitosan oligosaccharide-octyl gallate covalent conjugate is obtained by covalently grafting octyl gallate onto the chitosan oligosaccharide molecular chain.

2. The chitosan oligosaccharide-octyl gallate covalent conjugate according to claim 1, characterized in that, The chitosan oligosaccharide-octyl gallate covalent conjugate is amphiphilic and has the ability to generate reactive oxygen species under visible light excitation.

3. A method for preparing the chitosan oligosaccharide-octyl gallate covalent conjugate as described in claim 1 or 2, characterized in that, The preparation method includes the following steps: S1. Mix octyl gallate ethanol solution and chitosan oligosaccharide aqueous solution, add ascorbic acid, and stir under inert gas protection to obtain a mixture; S2. Add hydrogen peroxide to the mixture to start the reaction, and immediately perform ultrasonic treatment to obtain the reaction solution; S3. The reaction solution is transferred to a dialysis bag for purification, and then obtained by freeze drying as a chitosan oligosaccharide-octyl gallate covalent conjugate.

4. The preparation method according to claim 3, characterized in that, In step S1, the octyl gallate ethanol solution and the chitosan oligosaccharide aqueous solution are mixed in equal volumes, and the amount of octyl gallate added is 20% to 300% of the mass of chitosan oligosaccharide.

5. The preparation method according to claim 3, characterized in that, In step S1, the concentration of ascorbic acid is 5 mM to 20 mM. After adding ascorbic acid, the mixture is stirred for 10 min to 60 min under a slow nitrogen flow.

6. The preparation method according to claim 3, characterized in that, In S2, the concentration of hydrogen peroxide is 1 M to 10 M.

7. The preparation method according to claim 3, characterized in that, In step S2, the reaction temperature is controlled at 25℃ to 35℃ during the ultrasonic treatment process; the power of the ultrasonic treatment is 300W to 900W, the frequency is 20 kHz to 60 kHz, and the treatment time is 1 min to 10 min.

8. The preparation method according to claim 3, characterized in that, In step S3, the molecular weight cutoff of the dialysis bag is 500 Da to 1000 Da. It is first dialyzed in 50% ethanol for 1 to 3 days to remove unreacted octyl gallate, and then dialyzed in ultrapure water for 3 to 6 days.

9. The use of the chitosan oligosaccharide-octyl gallate covalent conjugate according to claim 1 or 2 in the preparation of a drug for killing drug-resistant bacteria.

10. The application of the chitosan oligosaccharide-octyl gallate covalent conjugate according to claim 1 or 2 in the preparation of anti-biofilm materials.

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