Glucan-modified cerium dioxide nanoparticles as well as preparation method and application thereof

Abstract

The invention belongs to the field of nano biological materials, and particularly relates to dextran-modified cerium dioxide nanoparticles as well as a preparation method and application thereof. The preparation method comprises the following steps: dissolving glucan in water, adding cerium nitrate hexahydrate, and standing until the glucan and the cerium nitrate hexahydrate are completely dissolved to obtain a solution 1; the concentration of the glucan in the solution 1 is 48-52 mg / mL, and the concentration of the cerium nitrate hexahydrate in the solution 1 is 35-40 mg / mL; dropwise adding an alkali solution into the solution 1 under a stirring state until the color of the solution 1 is changed from colorless to yellow, and keeping the stirring condition until the solution 1 is changed into clear dark brown to obtain a solution 2; and centrifuging the solution 2 to remove large particles, and dialyzing and purifying to remove unreacted raw materials to obtain the dextran-modified cerium dioxide nanoparticles. The dextran-modified cerium dioxide nanoparticles have broad-spectrum antibacterial activity, high efficiency and low toxicity, and have a good immune activation effect and a wide application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of nanobiomaterials, and particularly relates to a dextran-modified cerium dioxide nanoparticle, a preparation method thereof and an application thereof. Background Art

[0002] The effectiveness of conventional antibiotics has decreased, but the number of clinical bacterial infection cases has increased year by year. Antibiotic resistance has become the primary problem in global health and hygiene. Therefore, it is urgent to develop a new generation of antibacterial agents with excellent antibacterial effects and not easily inducing bacterial drug resistance. Unmodified cerium dioxide nanoparticles have excellent characteristics such as a large specific surface area, a high degree of surface functionalization, and a flexible adjustable valence state. At the same time, their good biocompatibility, excellent antibacterial activity and simple synthesis method make them the best choice for new antibacterial agents. Dextran can enhance the body's resistance to pathogenic bacteria and viruses, and can regulate the function of the immune system, enhancing the body's immunity. β-Dextran has also been widely used as a protective agent in the preparation of nanoparticles, and the preparation method has the advantages of simplicity, environmental protection and low cost.

[0003] Scholars at home and abroad have made some explorations in the preparation of nano-material immunological antibacterial agents. At present, Chinese Patent (Patent Publication No.: CN115671281A) discloses a preparation method of metal polyphenol-modified gold and silver nanoparticles, which is prepared from Au seeds, an aqueous solution of HAuCl4, an AgNO3 solution, hydroquinone and polyvinylpyrrolidone at room temperature. While having the antibacterial advantage of photothermal therapy, it can also restore the immune dysfunction caused by bacteria and enhance the regenerative ability of periodontal collagen fibers. However, nano-silver is unstable and has strong toxicity to the human body. The US FDA has clearly prohibited the use of nano-silver in the medical field, and the National Medical Products Administration (NMPA) of China has also restricted nano-silver in the medical field. Chinese Patent (Patent Publication No.: CN116059381A) discloses a nanoparticle with both antibacterial and anti-inflammatory effects, a preparation method thereof and an application thereof. By controlling the excessive immune response in the host, the effective treatment of pneumonia and sepsis can be achieved. However, it is impossible to avoid the use of antibiotics, which may lead to the emergence of bacterial drug resistance and there is a hidden danger of inducing super bacteria. Chinese Patent (Patent Publication No.: CN116328824A) discloses a defective molecular sieve-anchored cerium oxide cluster nanozyme, a preparation method thereof and an application thereof. The cerium oxide clusters in the prepared nanozyme are highly dispersed and stable, and highly dispersed cerium oxide nanoclusters are anchored at the defects of the molecular sieve, having excellent catalytic activity. However, it needs to be obtained through soaking, washing, precipitation and high-temperature calcination. The product preparation process is complex, and the conditions required for the reaction are relatively harsh. Summary of the Invention

[0004] To solve the above technical problems, the present invention provides a dextran-modified cerium dioxide nanoparticle, a preparation method thereof, and an application thereof.

[0005] The first aspect of the present invention is to provide a preparation method of a dextran-modified cerium dioxide nanoparticle, comprising the following steps:

[0006] Dissolve dextran in water, then add cerium nitrate hexahydrate, and let it stand until the dextran and cerium nitrate hexahydrate are completely dissolved to obtain Solution 1; the concentration of dextran in Solution 1 is 48-52 mg / mL, and the concentration of cerium nitrate hexahydrate is 35-40 mg / mL;

[0007] While stirring, dropwise add an alkali solution to Solution 1 until the color of Solution 1 changes from colorless to yellow, and maintain this stirring condition until Solution 1 becomes a clear dark brown to obtain Solution 2;

[0008] Centrifuge Solution 2 to remove large particles, and purify by dialysis to remove unreacted raw materials to obtain the dextran-modified cerium dioxide nanoparticle.

[0009] Further, the stirring speed is 1000-2000 r / min.

[0010] Furthermore, the alkali solution is an ammonium hydroxide solution with a mass percentage concentration of 24%-26%.

[0011] The second aspect of the present invention is to provide a dextran-modified cerium dioxide nanoparticle prepared by the above preparation method.

[0012] The third aspect of the present invention is to provide the application of the dextran-modified cerium dioxide nanoparticle in the preparation of an immune activator.

[0013] The fourth aspect of the present invention is to provide the application of the dextran-modified cerium dioxide nanoparticle in the preparation of a Gram-negative bacteria inhibitor, or in the preparation of a drug for treating diseases caused by infection with the Gram-negative bacteria.

[0014] Further, the Gram-negative bacteria include: Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Multidrug-resistant Escherichia coli.

[0015] The fifth aspect of the present invention is to provide the application of the dextran-modified cerium dioxide nanoparticle in the preparation of a Gram-positive bacteria inhibitor, or in the preparation of a drug for treating diseases caused by infection with the Gram-positive bacteria.

[0016] Further, the Gram-positive bacteria include: Staphylococcus aureus, Enterococcus faecalis, Vancomycin-resistant Enterococcus, and Methicillin-resistant Staphylococcus aureus.

[0017] The present invention has the following beneficial effects:

[0018] (1) The preparation process of the dextran-modified cerium dioxide nanoparticles of the present invention is simple, the reaction conditions are mild, and the cost is low.

[0019] (2) The present invention uses a macromolecule dextran that has an activating effect on the immune system to modify cerium dioxide nanoparticles, synergistically achieving a broad-spectrum antibacterial effect with high efficiency and low toxicity. The dextran-modified cerium dioxide nanoparticles of the present invention significantly reduce the minimum inhibitory concentration of various Gram-negative bacteria and Gram-positive bacteria such as Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Multidrug-resistant Escherichia coli, Staphylococcus aureus, Enterococcus faecalis, Vancomycin-resistant Enterococcus, and Methicillin-resistant Staphylococcus aureus.

[0020] (3) The dextran-modified cerium dioxide nanoparticles of the present invention also have a good immune activation effect and have application value in the field of chronic infections with underlying diseases. Description of the Drawings

[0021] Figure 1 For the morphological characterization of Dex-CeO2 NPs and CeO2 NPs, in the figure, A is the transmission electron microscope image of Dex-CeO2 NPs, B is the transmission electron microscope image of CeO2 NPs, C is the particle size distribution diagram of Dex-CeO2 NPs, and D is the particle size distribution diagram of CeO2 NPs.

[0022] Figure 2 Dynamic light scattering diagrams of Dex-CeO2 NPs and CeO2 NPs (A) and Zeta potential diagrams of Dex, CeO2 NPs, and Dex-CeO2 NPs (B).

[0023] Figure 3 UV absorption spectra of Dex, CeO2 NPs, and Dex-CeO2 NPs.

[0024] Figure 4 Infrared spectra of Dex, CeO2 NPs, and Dex-CeO2 NPs.

[0025] Figure 5 XPS spectrum of Dex-CeO2 NPs.

[0026] Figure 6 XRD pattern of Dex-CeO2 NPs.

[0027] Figure 7 Bright-field images of macrophages after adding Dex, CeO2 NPs, and Dex-CeO2 NPs. The arrows indicate the cells that have changed.

[0028] Figure 8 Photos of the catalytic oxidation of H2O2 by Dex, CeO2 NPs, and Dex-CeO2 NPs, respectively.

[0029] Figure 9 UV-visible absorption spectra of the catalytic oxidation of H2O2 by Dex, CeO2 NPs, and Dex-CeO2 NPs, respectively.

[0030] Figure 10 Cell viability of RAW264.7 cells (A) and 3T3 cells (B) treated with Dex, CeO2 NPs, and Dex-CeO2 NPs. Specific implementation manners

[0031] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments, but it should not be construed as a limitation of the present invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0032] Example 1: Preparation of dextran-modified cerium oxide nanoparticles

[0033] (1) In a sample bottle, dissolve dextran (Dex, molecular weight 40000, Macklin) in deionized aqueous solution, and then add cerium(III) nitrate hexahydrate (molecular weight 434.22, Macklin). Mix at room temperature (20 - 25 °C) for 2 h until the molecules are completely dissolved to obtain Solution 1. The concentration of dextran in Solution 1 is 50 mg / mL, and the concentration of cerium(III) nitrate hexahydrate is 36 mg / mL.

[0034] (2) Add 100 μL of ammonium hydroxide solution (25%, AR, Macklin) dropwise to the sample bottle under vigorous stirring at 1500 r / min. The color of the solution in the bottle changes from colorless to yellow. After maintaining this stirring condition and reacting for another 24 h, the solution becomes a clear dark brown to obtain Solution 2.

[0035] (3) First, centrifuge the obtained Solution 2 (8000 r / min, 10 min) to remove large aggregated particles, then dialyze it in a dialysis bag with a molecular weight cut-off of 30000 in ultrapure water for 24 h until the final pH is 7. Finally, concentrate it using an ultrafiltration centrifugal tube with a molecular weight cut-off of 30000 (Amicon Ultra 15 mL, 30 kDa MW), adjust the concentration to 24 mg / mL, and store it in a 4 °C refrigerator to obtain the dextran-modified cerium oxide nanoparticles (Dex-CeO2 NPs).

[0036] Comparative Example 1: Preparation of cerium oxide nanoparticles

[0037] (1) In a sample bottle, dissolve cerium(III) nitrate hexahydrate (molecular weight 434.22, Macklin) in deionized aqueous solution, and mix at room temperature until the molecules are completely dissolved to obtain Solution 1. The concentration of cerium(III) nitrate hexahydrate in Solution 1 is 36 mg / mL.

[0038] (2) Add 100 μL of ammonium hydroxide solution (25%, AR, Macklin) dropwise to the sample bottle under vigorous stirring at 1500 r / min. The color of the solution in the bottle changes from colorless to white. After maintaining this stirring condition and reacting for 24 h, obtain Solution 2.

[0039] (3) Centrifuge the obtained Solution 2 (8000 r / min, 10 min), and repeat the operation 3 times to remove the supernatant. Freeze-dry the obtained precipitate and store it in a -20 °C refrigerator, denoted as CeO2 NPs.

[0040] Example 2: Characterization of CeO2 NPs and Dex-CeO2 NPs

[0041] The morphology of Dex-CeO2 NPs prepared in Example 1 and CeO2 NPs prepared in Comparative Example 1 was characterized by transmission electron microscopy (TEM, Tecnai G2 20S-TWIN, FEI company, USA), and the observation results are as follows Figure 1 shown. The dynamic light scattering (DLS) of the samples was tested by Zeta Potential Analytical Instruments (NanoBrook Omni, Brookhaven Instruments Corporation, USA), and the results are as follows Figure 2 shown. The ultraviolet absorption wavelength of the samples was tested by an ultraviolet-visible spectrophotometer (UV, T2602S, Shanghai Youke Instrument Co., Ltd.), and the results are as follows Figure 3 shown. The infrared absorption wavelength of the samples was tested by a Fourier transform infrared spectrometer (FT-IR, Bruker Tensor27), and the results are as follows Figure 4 shown. The XPS spectra of the samples were tested by X-ray photoelectron spectroscopy (XPS, Thermofisher EscaLab 250Xi), and the results are as follows Figure 5 shown. The XRD patterns of the samples were tested by X-ray diffraction (XRD, Rigaku ultima4 type polycrystalline powder diffractometer, Japan), and the results are as follows Figure 6 shown.

[0042] Example 3: Evaluation of the antibacterial properties of Dex, CeO2 NPs, and Dex-CeO2 NPs

[0043] Escherichia coli (ATCC25922), Pseudomonas aeruginosa (ATCC27853), Klebsiella pneumoniae (ATCC13883), Staphylococcus aureus (ATCC29213), Enterococcus faecalis (ATCC29212), multidrug-resistant Escherichia coli (MDR E. coli, isolated and identified by VITEK automatic bacterial separator, clinical isolate number: 903657), methicillin-resistant Staphylococcus aureus (MRSA, Gram-positive bacterium, ATCC43300), and vancomycin-resistant Enterococcus faecalis (VRE, Gram-positive bacterium, ATCC700221) were respectively inoculated into liquid LB medium, and the inoculation concentration was 1×10 6CFU / mL. The Dex, Dex-CeO2 NPs, and CeO2 NPs with an original concentration of 24 mg / mL were diluted 2 - 128 times and then added to the culture medium inoculated with bacteria respectively, and the minimum inhibitory concentration (MIC) of bacteria after culturing at 37 °C for 24 h was recorded.

[0044] Table 1 MIC values of Dex, CeO2 NPs, and Dex-CeO2 NPs against different bacteria

[0045]

[0046] The results are shown in Table 1. The results indicate that dextran modification can enhance the antibacterial effect of cerium oxide nanoparticles against various bacteria, significantly reducing the minimum inhibitory concentration.

[0047] Example 4: Evaluation of the immune activation performance of Dex, CeO2 NPs, and Dex-CeO2 NPs RAW264.7 cells (mouse monocyte-macrophage leukemia cells, Suzhou Haixing Biotechnology Co., Ltd.) were seeded into a well-treated 96-well plate at a density of 1000 cells / well and cultured overnight to allow cell attachment. 10 μL of Dex (40 mg / mL), CeO2 NPs (20 mg / mL), and Dex-CeO2 NPs (20 mg / mL) were added to the cell plates respectively, and the cells were incubated for another 24 h. The morphological changes of the cells were observed under a 10× microscope.

[0048] The results are as Figure 7 shown. Both Dex and CeO2 NPs can polarize RAW264.7 cells, but Dex-CeO2 NPs have a better effect.

[0049] Example 5: Redox reaction of Dex, CeO2 NPs, and Dex-CeO2 NPs

[0050] In 2 mL of ultrapure water, 16 mg of Dex, 8 mg of CeO2 NPs, and 8 mg of Dex-CeO2 NPs were added and mixed well. Then, 2 mL of H2O2 with a concentration of 200 μg / mL was added to the above solution for the redox reaction of cerium. After reacting for half an hour, the absorption spectrum of the color reaction was recorded using a UV-visible spectrophotometer.

[0051] The experimental results show that both CeO2 NPs and Dex-CeO2 NPs underwent redox reactions to varying degrees, and the degree of redox reaction of Dex-CeO2 NPs was greater. No obvious color change was observed in the H2O2 and Dex groups ( Figure 8 ), and CeO2 NPs and Dex-CeO2 NPs had absorption peaks at 300 nm ( Figure 9 ).

[0052] Example 6: Determination of cell viability treated with Dex, CeO2 NPs, and Dex-CeO2 NPs

[0053] (1) Expand and culture 3T3 (mouse embryonic fibroblasts) and RAW264.7 (mouse mononuclear macrophage leukemia cells) in a medium (90% DMEM + 10% FBS + 1% penicillin and streptomycin + 1% L-alanyl-glutamine + 1% sodium pyruvate). Add 100 μL of cell suspension to a sterile 96-well plate, ensuring that there are no fewer than 1000 cells per well, and incubate at 37 °C for 24 hours. Add 10 μL of Dex, CeO2 NPs, and Dex-CeO2 NPs at different concentrations to the culture plate, and incubate at 37 °C for 24 h. Aspirate the liquid, wash once with PBS buffer, dilute the cell counting reagent (CCK-8 reagent) to the working concentration with cell culture medium, add 100 μL of CCK-8 solution to each well, and incubate at 37 °C for 2 h. Measure the optical density at 450 nm using a multi-functional microplate reader (Infinite M Plex), and set three replicates for the samples.

[0054] Experimental results ( Figure 10 ) show that when the concentration of the Dex group is 5 mg / mL, the concentration of the CeO2 NPs group is 1.5 mg / mL, and the concentration of the Dex-CeO2 NPs is 1.5 - 3 mg / mL, the cell viability is above 75%, indicating good cell safety.

[0055] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that any value between the two endpoints of each numerical range and the two endpoints themselves can be selected. To avoid repetition, the preferred embodiments of the present invention are described.

[0056] Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications once they know the basic creative concept. Therefore, the appended claims are intended to be construed as including the preferred embodiments and all changes and modifications falling within the scope of the present invention.

[0057] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention is also intended to include these modifications and variations.

Claims

1. Preparation method of dextran-modified cerium dioxide nanoparticles, characterized in that, It includes the following steps: Dissolve dextran in water, then add cerium nitrate hexahydrate, and let it stand until the dextran and cerium nitrate hexahydrate are completely dissolved to obtain Solution 1; the concentration of dextran in Solution 1 is 48 - 52 mg / mL, and the concentration of cerium nitrate hexahydrate is 35 - 40 mg / mL; While stirring, add an alkali solution dropwise to Solution 1 until the color of Solution 1 changes from colorless to yellow, and maintain this stirring condition until Solution 1 becomes a clear dark brown to obtain Solution 2; Centrifuge Solution 2 to remove large particles, and purify it by dialysis to remove unreacted raw materials to obtain the dextran-modified cerium dioxide nanoparticles.

2. The preparation method according to claim 1, characterized in that, The stirring speed is 1000 - 2000 r / min.

3. The preparation method according to claim 2, characterized in that, The alkali solution is an ammonium hydroxide solution with a mass percentage concentration of 24% - 26%.

4. Dextran-modified cerium dioxide nanoparticles prepared by the preparation method according to claim 3.

5. Use of the dextran-modified cerium dioxide nanoparticles according to claim 4 in the preparation of an immune activator.

6. Use of the dextran-modified cerium dioxide nanoparticles according to claim 4 in the preparation of a Gram-negative bacterium inhibitor, or in the preparation of a drug for treating a disease caused by infection with the Gram-negative bacterium.

7. The application according to claim 6, characterized in that, The Gram-negative bacterium includes: Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Multidrug-resistant Escherichia coli.

8. Use of the dextran-modified cerium dioxide nanoparticles according to claim 4 in the preparation of a Gram-positive bacterium inhibitor, or in the preparation of a drug for treating a disease caused by infection with the Gram-positive bacterium.

9. The application according to claim 8, wherein The Gram-positive bacterium includes: Staphylococcus aureus, Enterococcus faecalis, Vancomycin-resistant Enterococcus faecalis, Methicillin-resistant Staphylococcus aureus.

Images