A dextran-functionalized cobalt selenide nanoparticle, its preparation method and application

Abstract

The present invention discloses a dextran-functionalized cobalt selenide nanoparticle and its preparation method and application, belonging to the technical field of biomedicine. The ultrafine cobalt selenide nanomaterial CoSe NPs is prepared by a one-pot method from CoCl2, Na2SeO3 and glutathione, and then the dextran-functionalized cobalt selenide nanoparticle is obtained by modifying with dextran. The present invention synthesizes for the first time a novel nanomaterial with good antibacterial activity, cobalt selenide nanoparticles (CoSe NPs), and modifies dextran (Dex@CoSe NPs) that can target the extracellular polymeric matrix to achieve the dispersion of biofilms. In vitro antibacterial experiments show that Dex@CoSe NPs can inhibit and destroy biofilms and eliminate bacteria. More importantly, Dex@CoSe NPs can effectively relieve chronic wound infections caused by MRSA. In summary, the present invention provides a new idea for overcoming drug-resistant bacterial biofilm-related infections.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to a dextran-functionalized cobalt selenide nanoparticle, a preparation method thereof, and an application thereof. Background Art

[0002] Bacterial infections are associated with severe morbidity and mortality, posing a major threat to global health. The overuse and misuse of antibiotics have led to the emergence of drug-resistant bacteria (such as methicillin-resistant Staphylococcus aureus (MRSA)), which may cause 10 million deaths per year by 2050. Approximately 80% of bacterial infections are related to biofilms. Bacterial biofilms have a dense physical structure and are rich in extracellular polymeric substances (EPS) containing polysaccharides and nucleic acids. Due to the presence of EPS, the microenvironment in biofilms is hypoxic, leading to anaerobic glycolysis, ion channel disorders, and being more acidic than healthy tissues. In addition, biofilms are also the culprit of many chronic diseases. Given the rather long discovery cycle of antibiotics, there is an urgent need to develop alternative antibiotic therapies that can effectively eliminate drug-resistant bacteria, especially inhibit the formation of their biofilms.

[0003] The rapid development of nanotechnology provides a promising alternative solution for combating bacteria. It is reported that typical antibacterial materials include noble metals (Au, Pt, and AgNPs), transition metals (Fe, Cu, Mn, and Ni), and quantum dots, etc. Among these nanomaterials, silver nanoparticles have become the most widely studied antibacterial agents due to their high antibacterial efficiency. However, recent studies have shown that bacteria initially sensitive to silver nanoparticles can develop resistance to their toxic effects after repeated exposure to sub-inhibitory concentrations for a long time. In addition, nanomaterials lack sufficient bacteria capture ability, resulting in unsatisfactory antibacterial effects. The physical properties of nanomaterials, including shape, size, and structure, can be easily designed and controlled. More importantly, the surface of nanomaterials can be modified and functionalized as needed to achieve various antibacterial capabilities.

[0004] Dextran (Dex) is a polysaccharide extracted from microorganisms, which has high stability and biocompatibility. It is a polymer approved by the FDA and is widely used in the biomedical field. Dextran binds to the matrix of the biofilm through extracellular enzymes (such as glucosyltransferase derived from Streptococcus mutans glucose). This enzyme uses dextran as a receptor molecule to synthesize the extracellular polymeric matrix dextran of bacteria. Therefore, dextran-modified nanoparticles have high specificity and penetrability for biofilms. Therefore, XU et al. prepared asymmetric janus structure dextran bismuth selenide (Dex-BSe) nanoparticles targeting the extracellular polymeric matrix to achieve the dissipation of drug-resistant biofilms or near-infrared (NIR) light-activated photothermal killing respectively. Dextran has targeting properties for EPS; while the unique Janus structure of Dex-BSe enhances the photothermal killing effect through self-propelled movement. The two components work synergistically to flexibly eliminate biofilm-related infections. In addition, this research group also reported an anaerobic polymer photosensitizer (HQRB-SS-Dex) constructed with quaternary ammonium salt and dextran to potentially combat various oral bacteria through APDT. The quaternary ammonium salt and dextran promote the attachment of HQRB-SS-Dex to the bacterial surface and the penetration of bacterial biofilms, thus making more effective use of the ROS generated by the photosensitizer under laser irradiation, ultimately leading to bacterial death and biofilm collapse. Although these studies can effectively target and eliminate biofilms, they all need to be achieved through light irradiation, cannot penetrate deep tissues, are only effective and applicable in the treatment of superficial infectious diseases, and it is difficult to eliminate inner layer biofilm-related infections. Therefore, further research is still needed on antibacterial nanoparticles that target and eliminate inner layer biofilms. Summary of the Invention

[0005] The purpose of the present invention is to provide a dextran-functionalized cobalt selenide nanoparticle, its preparation method and application to solve the problems existing in the above-mentioned prior art.

[0006] To achieve the above purpose, the present invention provides the following solutions:

[0007] One of the technical solutions of the present invention is a dextran-functionalized cobalt selenide nanoparticle, which is prepared by a one-pot method from CoCl2, Na2SeO3 and GSH to obtain cobalt selenide nanomaterial CoSe NPs, and then modified with dextran to obtain the dextran-functionalized cobalt selenide nanoparticle.

[0008] Another technical solution of the present invention is the preparation method of the dextran-functionalized cobalt selenide nanoparticle, which includes the following steps:

[0009] (1) Dissolve CoCl2 and GSH in dimethyl sulfoxide, adjust the pH to weakly alkaline, add the Na2SeO3 solution after heating, and then carry out a one-pot reaction. After the reaction is completed, centrifuge and dry to obtain cobalt selenide nanomaterial CoSe NPs;

[0010] (2) Mix the cobalt selenide nanomaterial CoSe NPs with dextran, stir and react, centrifuge and lyophilize to obtain the dextran-functionalized cobalt selenide nanoparticles.

[0011] In the third technical solution of the present invention, the application of the dextran-functionalized cobalt selenide nanoparticles in the preparation of a drug for preventing and treating diseases caused by bacterial infections.

[0012] In the fourth technical solution of the present invention, a drug for preventing and treating diseases caused by bacterial infections, the drug includes the dextran-functionalized cobalt selenide nanoparticles.

[0013] Based on the above technical solutions, the present invention has the following technical effects:

[0014] The present invention for the first time synthesizes a novel nanomaterial with good antibacterial activity - cobalt selenide nanoparticles (CoSe NPs), and modifies dextran (Dex@CoSe NPs) that can target the extracellular polymeric matrix to achieve the dispersion of biofilms. In vitro antibacterial experiments show that Dex@CoSe NPs can inhibit and destroy biofilms and eliminate bacteria. More importantly, Dex@CoSe NPs can effectively relieve chronic wound infections caused by MRSA. In short, the present invention provides a new idea for overcoming drug-resistant bacterial biofilm-related infections. Description of the Drawings

[0015] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0016] Figure 1Characterization of Dex@CoSe NPs. Among them, (A) TEM of CoSe NPs. (B) TEM of Dex@CoSe NPs. (C) Zeta potential of CoSe NPs and Dex@CoSe NPs. (D) UV-visible spectrum. (E) Fluorescence spectrum. (F) Standard curve of CoSe NPs. (G) XRD of CoSe NPs and Dex@CoSe NPs. (H) Survey XPS spectra of CoSe NPs and Dex@CoSe NPs. High-resolution XPS spectra of (I) Co 2p and (J) Se 3d in Dex@CoSe NPs. High-resolution XPS spectra of (K) Co 2p and (L) Se 3d in CoSe NPs.

[0017] Figure 2 In vitro antibacterial activity. Among them, (A-C) Antibacterial activities of CoSe NPs against methicillin-resistant Staphylococcus aureus (MRSA), Escherichia coli, and Pseudomonas aeruginosa. (D-F) Antibacterial activities of different treatments against MRSA. (G-H) Photographs of MRSA and relative bacterial activities after different treatments. (I) Quantitative analysis of (J). (J) Photographs of MRSA after different treatments. (K) SEM characterization of bacterial morphology of MRSA after treatment with different drugs (the blue arrow indicates the shrunken and damaged cell membrane).

[0018] Figure 3 In vitro antibiofilm activity. Among them, (A-B) Photographs and quantification of immature biofilms stained with crystal violet (showing the inhibitory effect of CoSe NPs on biofilms). (C-D) Photographs and quantification of mature biofilms stained with crystal violet (showing the disruptive effect of CoSe NPs on biofilms). (E-F) Photographs and quantification of immature biofilms stained with crystal violet (showing the inhibitory effect of different drugs on biofilms). (G) 3D confocal images of immature MRSA biofilms after different treatments stained with SYTO 9. (H) Quantification of (G).

[0019] Figure 4 In vitro antibiofilm. Among them, (A) Schematic diagram of the treatment process for chronic wounds in infected mice. (B) Representative diagram of the wound healing process in infected mice. (C) Wound closure marks in mice. (D) Quantitative analysis of the change rate of wound area in mice. (E) Change in wound temperature in mice. (F) Change rate of body weight in mice. (G) H&E staining, Masson staining, immunohistochemical staining of CD31, TNF-α, and IL-10 in damaged skin on the 9th day of treatment.

[0020] Figure 5For the biosafety evaluation of Dex@CoSe NPs. Among them, (A-C) The cytotoxicity of Dex@CoSe NPs at different concentrations against Beas-2B, HK-2, and Lx-2. (D) Quantitative analysis and photos of red blood cell hemolysis incubated with Dex@CoSe NPs at different concentrations. (E) Blood routine test of mice on the 11th day. Detailed implementation manners

[0021] The various exemplary implementation manners of the present invention will be described in detail below. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0022] It should be understood that the terms used in the present invention are only for describing specific implementation manners and are not used to limit the present invention. In addition, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0023] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0024] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation manners of the specification of the present invention, which are obvious to those skilled in the art. Other implementation manners obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of this application are only exemplary.

[0025] Regarding the terms "comprising", "including", "having", "containing", etc. used herein, they are all open-ended terms, meaning including but not limited to.

[0026] The technical solutions described in the present invention are all conventional solutions in the art unless otherwise specified. The reagents or raw materials used are all purchased from commercial channels or are already publicly available unless otherwise specified.

[0027] An embodiment of the present invention provides a dextran-functionalized cobalt selenide nanoparticle, which is prepared by a one-pot method using CoCl2, Na2SeO3, and GSH to obtain a cobalt selenide nanomaterial CoSe NPs, and then modifying the dextran to obtain the dextran-functionalized cobalt selenide nanoparticle.

[0028] Selenium (Se) is one of the essential trace elements for the human body and plays a crucial role in maintaining human health, including wound healing. Supplementing selenium is beneficial for wound healing and even reduces the risk of bacterial infection. Cobalt is an important transition metal that has attracted the interest of researchers due to its low cost, earth abundance, and strong stability. It is an essential trace element for cell metabolism and plays a very important role throughout the life processes of animals and plants. Therefore, the present invention designed and synthesized ultra-small cobalt selenide nanoparticles (CoSe NPs), and modified Dex to obtain Dex@CoSe NPs, which can specifically inhibit and destroy biofilms and play a bactericidal role by shrinking the bacterial cell wall. In addition, in vivo wound infection models also showed that Dex@CoSe NPs can promote angiogenesis, reduce the inflammatory response, and thus promote wound healing, with good biocompatibility.

[0029] An embodiment of the present invention also provides a preparation method of the dextran-functionalized cobalt selenide nanoparticle, including the following steps:

[0030] (1) Dissolve CoCl2 and GSH in dimethyl sulfoxide, adjust the pH to weakly alkaline, add the Na2SeO3 solution after heating, and then carry out a one-pot reaction. After the reaction is completed, centrifuge and dry to obtain the cobalt selenide nanomaterial CoSe NPs;

[0031] (2) Mix the cobalt selenide nanomaterial CoSe NPs with dextran, stir and react, centrifuge and lyophilize to obtain the dextran-functionalized cobalt selenide nanoparticle.

[0032] In some specific embodiments, the molar ratio of CoCl2, GSH, and Na2SeO3 is 1:4:(11 - 12); the heating temperature is 90 - 100 °C.

[0033] In some specific embodiments, the temperature of the one-pot reaction is 120 - 150 °C, and the time is 18 - 24 h.

[0034] In some specific embodiments, the mass ratio of the cobalt selenide nanomaterial CoSe NPs to dextran is (2 - 4):1.

[0035] In some specific embodiments, the mass ratio of the cobalt selenide nanomaterial CoSe NPs to dextran is 2:1.

[0036] In some specific embodiments, adjusting the pH to weakly alkaline means adjusting the solution to pH 9 with 1 M sodium hydroxide.

[0037] In some specific embodiments, the concentration of the Na2SeO3 solution is 0.59 M and the volume is 5 mL; the Na2SeO3 solution is purged with argon before addition.

[0038] In some specific embodiments, the stirring reaction is: stirring overnight at room temperature.

[0039] The embodiments of the present invention also provide the application of the dextran-functionalized cobalt selenide nanoparticles in the preparation of drugs for preventing and treating diseases caused by bacterial infections.

[0040] The embodiments of the present invention also provide a drug for preventing and treating diseases caused by bacterial infections, and the drug includes the dextran-functionalized cobalt selenide nanoparticles.

[0041] In some specific embodiments, the bacteria include methicillin-resistant Staphylococcus aureus.

[0042] Example 1

[0043] 1 Synthesis

[0044] Add CoCl2·6H2O (0.0618 g, 0.26 mmol) and glutathione (GSH, 0.32 g, 1.04 mmol) to a reaction tube (100 ml) containing 20 mL of dimethyl sulfoxide. Adjust the solution to pH 9 with sodium hydroxide (1 M), and the solution changes from a blue suspension to a dark green suspension. Heat the mixture to 95 °C. Then, inject the Na2SeO3 solution (0.59 M, 5 mL) purged with argon, and then heat to 140 °C for reaction for 20 hours. After the reaction is completed, naturally cool to 25 °C, and the solution is black. Then, centrifuge and wash 2 - 3 times. Dry the precipitate into a powder in a vacuum oven at 25 °C to obtain cobalt selenide nanoparticles (CoSeNPs).

[0045] Dissolve 20 mg of CoSe NPs particles in 4 mL of pure water, and further mix with 1 mL of 10 mg / mL dextran (Dex) solution. Stir the reaction solution at room temperature for 10 - 12 h, then centrifuge at 10000 rpm and wash 3 times with pure water. Collect the precipitate and freeze-dry to obtain Dex@CoSe NPs (1:2).

[0046] Dex@CoSe NPs (1:4) can be obtained in the same way.

[0047] 2 Characterization

[0048] The prepared nanozymes were characterized by a variety of methods. TEM measurements were carried out on a TECNAI G2 equipped with EDS at 200 kV. The atomic structure images were characterized using a TEMARM-200 (JEOL, Tokyo, Japan) transmission electron microscope operating at 200 kV. X-ray photoelectron spectroscopy (XPS) analysis was performed by a ThermoVG Scientific ESCALAB 250 spectrometer. The ultraviolet-visible (UV-vis) absorption spectra were recorded using a UV-2600 spectrophotometer (Shimadzu Corporation).

[0049] CoSe NPs were synthesized by a solvothermal method, and the size and shape of cobalt selenide quantum dots were characterized using a transmission electron microscope (TEM). Figure 1 Figure A shows that CoSe NPs are in the form of uniformly dispersed dots with a particle size of about 2 - 3 nm. CoSe NPs and dextran (Dex) were reacted at ratios of 4:1 and 2:1, respectively, to obtain Dex@CoSe NPs (1:2) and Dex@CoSe NPs (1:4). Zeta potential tests showed that CoSe NPs, Dex@CoSe NPs (1:2), and Dex@CoSe NPs (1:4) all had relatively high negative potentials, indicating that the synthesized materials had good stability ( Figure 1 Figure C). The phase structures of Dex@CoSe NPs (1:2) and CoSe NPs were analyzed using X-ray diffraction (XRD) patterns ( Figure 1 Figure G). The XRD patterns of Dex@CoSe NPs and CoSe NPs were very similar to the standard hexagonal phase of CoSe (JCPDS card number: 89 - 2004). In addition, X-ray photoelectron spectroscopy (XPS) studies were further carried out to gain an in-depth understanding of the chemical and electronic structures of Dex@CoSe NPs (1:2). As Figure 1 shown in Figure H, Co, O, N, C, S, Co, and Se signals could be clearly detected. To further analyze the changes in functional groups, high-resolution spectral analysis of Co 2p and Se 3d was performed. The peaks of Co - Se could be well matched in the high-resolution Co 2p narrow spectra Figure 1 Figures I and J), while there were two obvious peaks at 780.0 and 795.4 eV, which corresponded to the Co 2p3 / 2 and Co 2p1 / 2 orbital peaks, respectively. The peaks attributed to Se 3d3 / 2 and Se 3d5 / 2 were located at 55.6 and 55.0 eV, respectively [40, 41] ( Figure 1 Figures K and L). The ultraviolet absorption spectra ( Figure 1Figure D) shows that at wavelengths of 200 - 800 nm, Dex, CoSe NPs, Dex@CoSe NPs (1:2), and Dex@CoSe NPs (1:4) all have absorption. Fluorescence spectrum ( Figure 1 Figure E) shows that with an excitation wavelength of 365 nm, Dex@CoSe NPs (1:2) and Dex@CoSe NPs (1:4) simultaneously have emission peaks of CoSe NPs and Dex. The above results indicate the successful synthesis of CoSe NPs and the possible connection of CoSe NPs and Dex. Subsequently, the present invention uses ultraviolet - visible spectroscopy to quantitatively analyze the drug - loading amount ( Figure 1 Figure F). In Dex@CoSe NPs (1:4), the loading rate of CoSe NPs is 49.54%, and the grafting rate of Dex is 50.46%. In Dex@CoSe NPs (1:2), the loading rate of CoSe NPs is 23.09%, and the grafting rate of Dex is 76.91%.

[0050] Example 2

[0051] 1 Minimum Inhibitory Concentration (MIC)

[0052] The broth micro - dilution method of the Clinical and Laboratory Standards Institute was used to determine the minimum inhibitory concentration of different treatments against different strains. First, the bacteria were cultured overnight in LB medium at 37 °C on a shaker (180 - 200 rpm) until reaching the logarithmic growth phase. Then, the precipitate was collected by centrifugation and washed three times with 0.85% sodium chloride solution. The concentration of the suspended bacteria was determined by the optical density (OD 600 ) measured at 600 nm. The bacterial solution was diluted with the medium to 1×10 6 CFU / mL. 100 μL of these solutions was added to a 96 - well plate containing different concentrations of the drug (100 μL) to make the final bacterial concentration approximately 5×10 5 CFU / mL. The bacteria were cultured at 37 °C for 24 h, and the OD 600 value of the bacterial suspension was measured with a multi - functional microplate reader, and the minimum inhibitory concentration (MIC) was calculated. The minimum inhibitory concentration was determined as the treatment concentration at which no microbial growth was observed. Each test was repeated 3 times.

[0053] 2 Spread - plate method

[0054] The MRSA (n = 3) containing different materials and diluted to 5×10 5 CFU / mL was diluted with LB medium to a volume of 200 μL and added to a 96 - well plate. After culturing for 24 h, the suspension was diluted 10 5Multiply by [X] times, spread 50 μL of the bacterial solution on an LB agar plate, culture it at 37 °C for 18 - 24 hours, take a photo of the LB agar plate and count the colonies.

[0055] 3 Oxford cup experiment

[0056] Add Dex@CoSe NPs (1:2), Dex@CoSe NPs (1:4), Dex, and CoSe NPs to PBS to make the final concentration 500 μg / mL. Then add 1×10 6 CFU / mL of MRSA and mix well. Place the Oxford cup vertically on the petri dish poured with agar, then pipette 10 μL of the just - mixed solution into the Oxford cup, and place it in an incubator at 37 °C for incubation. Take a photo after 24 hours.

[0057] 4 Scanning electron microscopy analysis of bacterial morphology

[0058] Treat the bacterial suspension (5×10 5 CFU / mL) with 300 μg / ml of CoSe NPs, Dex@CoSe NPs (1:2), or Dex@CoSe NPs (1:4) at 37 °C for 3 hours, then wash the MRSA bacteria (3500 rpm, 5 min) twice with PBS. Then, fix each group of samples in 2.5% glutaraldehyde solution at 4 °C for 4 hours. Wash with PBS buffer 3 times, dehydrate continuously with different concentrations of ethanol (20%, 40%, 60%, 80%, and 100%) for 5 min each time, air - dry the samples to dehydrate the bacteria, and further observe with a scanning electron microscope.

[0059] The 96 - well plate method was used to evaluate the minimum inhibitory concentration (MIC) of the synthesized CoSe NPs, Dex@CoSe NPs (1:2), and Dex@CoSe NPs (1:4) against bacteria. Methicillin - resistant Staphylococcus aureus (MRSA), Escherichia coli (E. coli), and Pseudomonas aeruginosa were used as models for broad - spectrum antibacterial tests. Figure 2 A - C in [X] show that CoSe NPs have inhibitory activity against both MRSA and E. coli, and its inhibitory activity is concentration - dependent within a certain concentration range. Given that its antibacterial activity against MRSA is better than that against E. coli, MRSA was selected as the model for subsequent experiments. Figure 2Figure D-F shows that compared with Dex and CoSe NPs, Dex@CoSe NPs (1:2) and Dex@CoSe NPs (1:4) have higher antibacterial activities. However, the antibacterial activities of Dex@CoSe NPs (1:2) and Dex@CoSe NPs (1:4) are similar, and their MIC values are both 150 μg / mL. Subsequently, the antibacterial activities were observed through the dilution plating experiment ( Figure 2 Figure G in Figure 2 and the Oxford cup experiment ( Figure 2 Figure J in Figure 2 ), and the results of the above two experiments were quantified respectively ( ​ Figures H and I in ​ ). As can be seen from ​ Figure G, there are almost no colonies in Dex@CoSe NPs (1:2) and Dex@CoSe NPs (1:4). However, it can be found from ​ Figure J that the number of colonies in Dex@CoSe NPs (1:2) is less than that in Dex@CoSe NPs (1:4). Therefore, subsequent related experiments on safety and in vivo antibacterial activity were all carried out with Dex@CoSe NPs (1:2).

[0060] Based on the good in vitro antibacterial activity of Dex@CoSe NPs (1:2), the present invention further studied the morphological changes of Staphylococcus aureus after different treatments by scanning electron microscopy to better understand the antibacterial results. As ​ shown in Figure K, untreated MRSA presents a typical round structure with a smooth surface and a complete cell wall. After treatment with CoSe NPs and Dex@CoSe NPs, the surface of MRSA becomes wrinkled, and the surface of MRSA treated with Dex@CoSe NPs has more obvious folds and damages, and the cell wall surface is severely damaged. This indicates that compared with CoSe NPs, Dex@CoSe NPs (1:2) have better antibacterial activity.

[0061] Example 3

[0062] 1 Analysis of in vitro inhibition or disruption of biofilms

[0063] To detect the ability of CoSe NPs and Dex@CoSe NPs (1:2) to inhibit biofilm formation, 1 mL of a solution with a concentration of 1×10 8MRSA bacterial suspension at CFU / mL, and after culturing at 37 °C for 24 hours, the old culture medium was replaced with a culture medium containing PBS, CoSeNPs, and Dex@CoSe NPs (1:2). After different treatments, it was washed 3 times with PBS, the biofilm was fixed with 95% methanol, and stained with 0.1% crystal violet for 30 min. After washing with PBS until the supernatant was colorless, it was air-dried and photographed. The dye was dissolved with 33% acetic acid, and the absorbance of the solution was measured at 590 nm with an enzyme-labeling instrument. In addition, the biofilm formed on the confocal dish was treated as described above, stained with SYTO 9 for 30 min, and then imaged with a confocal microscope.

[0064] To evaluate the effect of destroying mature biofilms, the bacterial suspension was cultured in LB culture medium for 48 h to form mature biofilms, and then treated with PBS, CoSe NPs, and Dex@CoSe NPs (1:2) for 24 h respectively. The residual biofilms were identified by crystal violet staining.

[0065] 2 MTT assay

[0066] The cytotoxicity of Dex@CoSe NPs (1:2) on different cell lines was studied by MTT assay. Cells (Beas-2B, HK-2, and Lx-2 cell lines) were seeded into 96-well plates at a density of 4000 - 5000 cells / well with 100 μL of culture medium. After incubation for 24 hours, the culture medium was replaced with 100 μL of fresh culture medium containing different concentrations of Dex@CoSe NPs (1:2), and the cells were incubated for another 24 hours. After treatment for 24 h, MTT solution was added to each well, and then incubated at 37 °C for 4 hours. The culture medium was aspirated, 150 μL of DMSO was added to dissolve the blue formazan in living cells, and the absorbance was measured at 570 nm with a microplate reader. Cells incubated with only culture medium were considered to have 100% viability. Cell viability (%) = OD value of each treatment group / OD value of the control group × 100%.

[0067] 3 Hemolysis assay

[0068] Commercially purchased 4% fresh red blood cells (RBC, 5 mL) were centrifuged at 3500 rpm for 5 min and gently washed 3 times with sterile PBS. 100 μL of different concentrations of Dex@CoSe NPs (1:2) were added to 300 μL of red blood cells respectively and incubated at 37 °C for 4 hours. At the end of incubation, the solution was centrifuged at 3500 rpm for 5 min, and the hemolytic activity was measured at 540 nm with an enzyme-labeling instrument. Triton X-100 (0.1% in PBS) that could completely lyse red blood cells was used as a positive control, while PBS was used as a negative control. Red blood cell hemolysis (HR) was calculated according to the following formula:

[0069] Hemolysis rate (%) = (Absorbance of sample - Absorbance of negative control) / (Absorbance of positive control - Absorbance of negative control) × 100%.

[0070] Most bacteria can form biofilms on different surfaces. Bacterial biofilms have a dense physical structure and are rich in extracellular polymers containing polysaccharides and nucleic acids, which can resist the penetration of antibacterial drugs, enabling bacteria to survive in harsh environments, leading to drug resistance and recurrent infections. Based on the good antibacterial activity of Dex@CoSe NPs (1:2), the present invention evaluated the anti-biofilm activity of Dex@CoSe NPs (1:2).

[0071] First, the present invention evaluated the ability of CoSe NPs to inhibit biofilm formation. The biofilms treated with different concentrations of CoSe NPs were stained with crystal violet, and then the absorbance was measured at a wavelength of 590 nm for quantification. It was found that as the concentration of CoSe NPs increased, the color of the biofilm gradually became lighter, indicating that CoSe NPs could inhibit the growth of biofilms ( ​ in A, B). Subsequently, the present invention also evaluated its ability to disrupt biofilms ( ​ in C, D), and it was found that compared with the Control group, the color of the CoSe NPs group was lighter, indicating that CoSe NPs could disrupt biofilms.

[0072] Since the antibacterial activity of Dex@CoSe NPs (1:2) is better than that of CoSe NPs, the present invention also compared the ability of CoSe NPs and Dex@CoSe NPs (1:2) to inhibit biofilm formation by crystal violet staining. The crystal violet staining results ( ​ in E, F) showed that the ability of Dex@CoSe NPs (1:2) to inhibit biofilm formation was better than that of CoSe NPs. Since PI can label dead bacteria with damaged bacterial membranes because it can reach the cell nucleus and embed in DNA to produce red fluorescence, while SYTO 9 can enter all bacteria. Generally, when the two dyes penetrate into bacteria, the fluorescence intensity of SYTO 9 (green) will decrease. Therefore, bacteria with intact cell membranes show green fluorescence, while damaged cell membrane structures mainly show red fluorescence. So, the present invention used three-dimensional confocal scanning microscopy to analyze the live / dead staining of biofilms ( ​ in G), and used Image J to quantify the fluorescence intensity of the stained biofilms ( ​In (H). The results were consistent with those of crystal violet staining. The biofilm treated with Dex@CoSe NPs (1:2) had less green fluorescence and showed completely intact red, indicating that Dex@CoSe NPs (1:2) damaged the cell membrane, thereby inhibiting the formation of biofilm. The above results indicate that Dex@CoSe NPs (1:2) have good anti-biofilm activity, and their anti-biofilm activity is more excellent compared with CoSe NPs.

[0073] The biocompatibility of Dex@CoSe NPs (1:2) was evaluated by the MTT method. The results showed that when the concentration of Dex@CoSe NPs (1:2) was 150 μg / mL, the cell survival rate still reached 80% ( ​ In (A-C). Subsequently, the effect of Dex@CoSe NPs (1:2) on the rupture and lysis of red blood cells (RBC) was studied by hemolysis experiment ( ​ In (D). After incubation with high-concentration (300 μg / mL) Dex@CoSe NPs (1:2), the hemolysis rate was extremely low <5%. The above results indicate that there is no obvious toxicity to normal mammalian cells at high concentrations. In addition, in chronic wound infections, there were no significant differences in routine blood tests between the treatment group and the untreated group ( ​ In (E), demonstrating that Dex@CoSe NPs (1:2) have almost no side effects within a certain concentration range and have good biocompatibility.

[0074] Example 4

[0075] 1 Establish a mouse wound infection model

[0076] Remove the hair on the back of ICR mice with a depilatory agent, create a circular wound with a diameter of about 10 mm using ophthalmic scissors, and add 50 μL of 2×10 8 CFU / mL MRSA was used to establish the infection model. Then the mice were randomly divided into 5 groups (n = 5), and drugs were administered once every other day. Each group of mice was administered 50 μL of PBS, 2.5 mg / kg CoSe NPs, Dex@CoSe NPs (1:2), or vancomycin on the back. The mice were weighed, their body temperature, wound area were measured, and photos were taken. On the 11th day, fresh whole blood was collected to evaluate in vivo biosafety. Subsequently, the mice were sacrificed by cervical dislocation, and the sections of different wounds were stained with H&E, Masson, and immunohistochemistry.

[0077] In wound care, MRSA infection is a major clinical challenge. Based on the good antibacterial and anti-biofilm formation abilities of Dex@CoSe NPs (1:2) in vitro, the present invention constructs a chronic MRSA wound infection model to evaluate the ability of Dex@CoSe NPs (1:2) to promote the healing of MRSA-infected wounds. A circular incision (about 10 - 12 mm in diameter) is made on the back skin of ICR mice. In the uninfected group, sterile saline is dropped, and in the wound infection group, 50 μL of a MRSA bacterial suspension at 2×10 8 CFU / mL is dropped (recorded as the first day), and then cultured for two days to form a biofilm. Medication is given every two days, and the treatment ends on the 11th day. In the wound infection group, 3 mg / kg of PBS, Van, CoSe NPs, and Dex@CoSe NPs (1:2) are respectively dropped to treat the infected wounds. To observe the treatment effects of different groups, the temperature, body weight, and pictures of skin wound healing of the mice on the 1st, 3rd, 5th, 7th, 9th, and 11th days are recorded after treatment ( ​ as shown in A).

[0078] First, it is evaluated by clinically assessing healthy and disease symptoms, that is, visually inspecting the wound and monitoring the body weight and activity level of the mice. Visual inspection of the wound site confirms that during the treatment process, the wound scabs and the area gradually decreases, and in the mice treated with Dex@CoSe NPs (1:2), the wound is basically completely healed on the 11th day ( ​ as shown in B - D). At the same time, during the treatment process, the body temperature of the wound surface is basically stable ( ​ as shown in E), but the body weight in the PBS group decreases ( ​ as shown in F). Inflammation and tissue formation are important processes in wound healing. To study these subtle changes in infected wounds, the present invention uses H&E, Masson, CD31, TNF-α, and IL-10 ( ​G) Histological analysis of the wound was performed. Therefore, on the 11th day, the tissue around the wound was taken in this invention for histomorphological analysis. H&E staining showed that compared with the control group, there were significantly fewer inflammatory cells in the Dex@CoSe NPs (1:2) group, indicating that Dex@CoSe NPs (1:2) could promote skin regeneration. The production of collagen in dermal fibroblasts is also important for wound healing. The results of Masson staining showed that the collagen staining intensity of the wound surface in the Dex@CoSe NPs (1:2) group was significantly enhanced, indicating that there was a large amount of collagen fiber deposition in the Dex@CoSe NPs (1:2) group. In addition, immunohistochemical analysis was also carried out in this invention. Since CD31 is mainly expressed in the tight junctions between skin endothelial cells and participates in blood vessel formation, angiogenesis is crucial for tissue regeneration by providing sufficient oxygen and nutrients, as well as promoting the transport of cytokines and cell migration. Therefore, CD31 immunostaining was performed on the wound surfaces after different treatments in this invention to show the angiogenesis level. After 11 days of treatment, immunohistochemical staining of the vascular endothelial cell marker CD31 showed stronger CD31 staining in the Dex@CoSe NPs (1:2) group, suggesting further stimulation of angiogenesis during wound healing. Inflammation is a key stage of wound healing. However, a severe inflammatory response caused by bacterial infection will delay the wound repair process. Therefore, immunohistochemical staining of TNF-α and IL-10 was performed on the wound surfaces in this invention. The results showed that compared with the PBS group, the expression of the pro-inflammatory factor TNF-α in the group treated with Dex@CoSe NPs (1:2) was significantly reduced, while the expression of the anti-inflammatory factor IL-10 increased, which contributed to the repair of skin tissue. The above results indicate that Dex@CoSe NPs (1:2) has a persistent and effective antibacterial effect on bacterial skin wound infections and promotes the healing of MRSA wound wounds.

[0079] In summary, this invention successfully constructed a new type of nanomaterial, namely CoSe NPs, by a one-pot method. It has a spherical nanostructure and a relatively small particle size of 2-3 nm. In addition, it has good antibacterial and antibiofilm activities. Then, Dex was modified to obtain Dex@CoSe NPs to target the biofilm. This invention found that after being modified with Dex, its antibacterial and antibiofilm activities increased. Finally, this invention confirmed that Dex@CoSe NPs showed healing characteristics in a skin injury model infected with MRSA, such as increased wound closure rate, increased collagen, reduced inflammation, and enhanced angiogenesis. The above results indicate that Dex@CoSe NPs is an antibacterial agent with excellent antibacterial and antibiofilm application performance and has good biocompatibility. In short, this method provides opportunities for non-antibiotic drug treatment, targeted treatment, and antibacterial treatment design.

[0080] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, rather than limitations on the implementation manners of the present invention. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation manners here. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the claims of the present invention.

Claims

1. Use of dextran-functionalized cobalt selenide nanoparticles in the preparation of a drug for preventing and treating diseases caused by bacterial infections, characterized in that, The dextran-functionalized cobalt selenide nanoparticles are prepared by a one-pot method using CoCl2·6H2O, Na2SeO3, and glutathione to prepare cobalt selenide nanomaterials CoSe NPs, and then modifying with dextran to obtain the dextran-functionalized cobalt selenide nanoparticles; Its preparation method includes the following steps: (1) Dissolve CoCl2·6H2O and glutathione in dimethyl sulfoxide, adjust the pH to weakly alkaline, add the Na2SeO3 solution after heating, and then carry out a one-pot reaction. After the reaction is completed, centrifuge and dry to obtain cobalt selenide nanomaterials CoSe NPs; the molar ratio of CoCl2·6H2O, glutathione to Na2SeO3 is 1:4:(11-12); the heating temperature is 90-100 °C; the temperature of the one-pot reaction is 120-150 °C, and the time is 18-24 h; (2) Mix the cobalt selenide nanomaterials CoSe NPs with dextran, stir and react, centrifuge and freeze-dry to obtain the dextran-functionalized cobalt selenide nanoparticles; The bacterium is methicillin-resistant Staphylococcus aureus.

2. The application according to claim 1, characterized in that The mass ratio of the cobalt selenide nanomaterials CoSe NPs to dextran is (2-4):

1.

3. The application according to claim 2, wherein The mass ratio of the cobalt selenide nanomaterials CoSe NPs to dextran is 2:

1.

4. A drug for preventing and treating diseases caused by bacterial infections, characterized in that, The drug includes the dextran-functionalized cobalt selenide nanoparticles described in claim 1.

5. The drug according to claim 4, characterized in that, The bacterium includes methicillin-resistant Staphylococcus aureus.

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