Preparation method of antibacterial dual-oxygen-supply manganese-doped carbon dot-oxygen-carrying microbubble

Abstract

The invention discloses a preparation method of antibacterial dual-oxygen-supply manganese-doped carbon dot-oxygen-carrying microbubbles, and belongs to the field of preparation of medical configuration products, and the preparation method comprises the following steps: attaching biotinylated manganese-doped carbon dots to oxygen-carrying microbubbles by using biotin-avidin connection to prepare micron-level manganese-doped carbon dot-oxygen-carrying microbubbles; the size of the micron-level manganese-doped carbon dot-oxygen-carrying microbubble is 1000-2500nm. The antibacterial dual-oxygen-supply manganese-doped carbon dot-oxygen-carrying microbubble prepared by the invention has the advantages of simple synthesis, clear structure, adjustable size and the like, and the material preparation method is simple, mild in reaction condition and suitable for large-scale production; manganese-doped carbon dot-oxygen-carrying microbubbles provide dual oxygen supply to enhance the sonodynamic therapy effect, so that efficient antibacterial activity is achieved, angiogenesis is promoted, wound healing is accelerated, and the method is a promising wound healing therapy method.

Description

Technical Field

[0001] The invention relates to a method for preparing antibacterial dual-oxygen-supply manganese-doped carbon dot-oxygen-carrying microbubbles, and belongs to the field of preparation of medical preparations. Background Art

[0002] Approximately 18.6 million people worldwide suffer from diabetic wounds each year, of which at least a quarter fail to heal, increasing the risk of amputation. Approximately 50% of diabetic wounds are further complicated by infection, causing diabetic wounds to remain in an inflammatory phase, which seriously hinders the wound healing process. Biofilms act as a solid physical and metabolic barrier that prevents the penetration of antimicrobial agents, leading to persistent infection and inflammation in diabetic wounds, which in turn reduces the effectiveness of antimicrobial treatment. In addition, biofilms exacerbate the chronic hypoxia caused by diabetic microangiopathy damage, thereby hindering angiogenesis and further delaying the healing process of diabetic wounds. This pathological hypoxia not only hinders the natural antimicrobial activity of neutrophils, but also reduces the effectiveness of other bactericidal treatments such as antibiotics and photodynamic / sonic dynamic therapy (PDT / SDT). Therefore, there is an urgent need for an oxygen delivery method that can alleviate the hypoxia caused by microangiopathy and the presence of biofilm at the diabetic wound site to improve the bactericidal effect and accelerate wound healing.

[0003] SDT has been demonstrated to be an effective antimicrobial approach that utilizes ultrasound to activate sonosensitizers to generate reactive oxygen species (ROS) to control bacterial infections without being limited by tissue penetration depth. These ROS are lethal to bacteria, disrupting the integrity of bacterial cell walls and membranes and causing severe oxidative stress while avoiding the problem of drug resistance. However, the efficacy of ROS is limited by the limited oxygen supply in chronic diabetic wounds. In addition, the delivery efficiency of sonosensitizers is limited by biofilms, which act as a barrier to access the core of the infection. Therefore, the development of advanced nanosystems that can effectively deliver sonosensitizers to the site of infection and overcome the challenges associated with hypoxia provides a promising approach to improve the effectiveness of SDT.

[0004] Carbon dots (CDs) are a new class of carbon-based nanomaterials that have attracted great interest due to their roles as nanoprobes, carriers, and agents. The synthesis of CDs is simple and economical, but they exhibit outstanding physicochemical properties, such as excellent biocompatibility, minimal toxicity, tunable hydrophilicity, tunable fluorescence emission properties, and abundant surface functional groups. These properties bode well for their wide potential applications in the antimicrobial field. Furthermore, recent studies have highlighted the enzyme-like catalytic capabilities of CDs, especially those doped with metals, which have been shown to mimic catalase activity. Considering these properties and our research foundation in CDs, this project aims to develop metal-doped CDs that can not only act as sonosensitizers but also chemically catalyze the decomposition of hydrogen peroxide to release oxygen. By generating oxygen through a chemical reaction, this approach seeks to enhance the impact of SDT by combining antimicrobial activity with an intrinsic O2 supply, thereby improving the antimicrobial efficacy.

[0005] The cavitation effect of ultrasound-contacted microbubbles (MB) has been found to be effective in removing biofilm barriers, especially in the field of dental and medical implants. By changing the filling gas inside the microbubbles, different types of gases such as oxygen (O2), nitric oxide (NO), hydrogen (H2) and xenon (Xe) can be delivered to the lesion site to achieve therapeutic purposes. Summary of the invention

[0006] In view of this, the present invention provides a method for preparing antibacterial dual oxygen supply manganese-doped carbon dots-oxygen-carrying microbubbles. MnCD is designed to exhibit customized sonodynamic activity and enzyme-like catalytic ability, while MnCDs@O2MBs are prepared using biotin-avidin linkage. This system combines the ability of microbubbles to transport and release oxygen with the in situ oxygen production ability of MnCD, solving the common hypoxia condition in chronic diabetic wounds. This dual oxygen supply strategy physically loads O2 and chemically generates O2, aiming to enhance the antibacterial effect of SDT.

[0007] In addition, the cavitation effect of MnCDs@O2MBs helps to overcome the limited diffusion of antimicrobial agents through biofilms, improve their distribution and enhance their antimicrobial effects. This integrated approach of dual oxygenation, SDT amplification and cavitation-promoted antimicrobial film therapy aims to accelerate wound healing. Proteomic analysis of treated wound tissues showed significant improvements as this strategy effectively controlled bacterial infection, reduced inflammation by inhibiting key inflammatory pathways and proinflammatory cytokines, promoted angiogenesis and keratinocyte differentiation, improved the skin barrier, and ultimately promoted the healing of chronic diabetic wounds.

[0008] In order to achieve the above object, the present invention adopts the following technical solution:

[0009] A method for preparing antibacterial dual oxygen supply manganese-doped carbon dots-oxygen-carrying microbubbles, the steps are as follows:

[0010] (1) Reduced glutathione, anhydrous manganese chloride and PEI-600 were mixed and dissolved in formamide, maintained at 160°C for 4 h in a polytetrafluoroethylene autoclave, dialyzed and purified, and then freeze-dried to obtain MnCDs powder;

[0011] (2) MnCDs and NHS-biotin were mixed in a Na2CO3 / NaHCO3 buffer solution and stirred evenly, and the resulting solution was dialyzed and purified, and then freeze-dried and collected to obtain MnCDs-biotin powder;

[0012] (3) DPPC, DSPE-PEG2000 and DSPE-PEG2000-biotin were mixed in chloroform, rotary evaporated until the chloroform was completely eliminated, and glycerol / PBS solution was added and ultrasonically shaken to obtain a lipid solution;

[0013] (4) completely filling a sealed container with a mixture of the lipid solution, octafluoropropane and O2, mixing and stirring to produce O2MBs, stirring the O2MBs and avidin together, washing with PBS, and centrifuging to obtain O2MBs-avidin;

[0014] (5) The O2MBs-avidin and MnCDs-Biotin powders were mixed evenly, washed with PBS and centrifuged to obtain manganese-doped carbon dots-oxygen-carrying microbubbles MnCDs@O2MBs.

[0015] On the basis of the above technical solution, the present invention further defines the solution as follows:

[0016] Further, in step (1), the molar ratio of reduced glutathione, anhydrous manganese chloride and PEI-600 is 1:1:0.5;

[0017] The solute solvent ratio of reduced glutathione, anhydrous manganese chloride, PEI-600 and formamide was 1 mmol:4 ml.

[0018] Furthermore, the mixed dissolution in step (1) is carried out by ultrasonic mixed dissolution, the ultrasonic temperature is 10 to 40 minutes, the time is 1 to 30 minutes, and the power is 100 to 1000W;

[0019] The molecular cutoff for dialysis purification is 3500Da;

[0020] The freeze-drying temperature is -40 to -60°C and the time is 48 hours.

[0021] Furthermore, the ultrasonic mixed dissolution is performed by using at least any one of a cell disruptor and an ultrasonic cleaning machine.

[0022] Furthermore, in order to eliminate larger impurities, step (1) also includes diluting three times with deionized water and filtering through a 0.22 μm membrane filter before dialysis purification.

[0023] Further, in step (2), the molar ratio of MnCDs to NHS-biotin is 1:80;

[0024] The pH value of Na2CO3 / NaHCO3 buffer solution is 9.

[0025] Furthermore, the uniform stirring in step (2) is performed at 37° C. for 6 h;

[0026] The molecular cutoff for dialysis purification is 3500Da;

[0027] The freeze-drying temperature is -40 to -60°C and the time is 24 hours.

[0028] Further, in step (3), the molar ratio of DPPC, DSPE-PEG2000 and DSPE-PEG2000-biotin is 82:9:9;

[0029] The concentration of glycerol / PBS solution was 0.05%;

[0030] Ultrasonic shaking was carried out at 37° C. for 10 min.

[0031] Furthermore, in step (4), the volume ratio of octafluoropropane to O2 is 1:1.

[0032] Furthermore, the size of manganese-doped carbon dots-oxygen-carrying microbubbles MnCDs@O2MBs is 1000-2500nm.

[0033] The beneficial effects of the present invention are that the preparation method of the antibacterial dual oxygen supply manganese-doped carbon dot-oxygen-carrying microbubbles of the present invention has the advantages of simple synthesis, clear structure, adjustable size, etc., and the material preparation method is simple, the reaction conditions are mild and suitable for large-scale production; the prepared manganese-doped carbon dot-oxygen-carrying microbubbles provide dual oxygen supply to amplify the sonodynamic therapy effect to achieve efficient antibacterial activity, and promote angiogenesis to accelerate wound healing, which is a promising wound healing treatment method. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is a transmission electron microscopy image of manganese-doped carbon dots;

[0035] Figure 2 This is a fluorescence microscopy image of manganese-doped carbon dots-oxygen-carrying microbubbles;

[0036] Figure 3 This is a statistical diagram of oxygen production of manganese-doped carbon dots-oxygen-carrying microbubbles under ultrasound;

[0037] Figure 4 This is a statistical diagram of the active oxygen production of manganese-doped carbon dots-oxygen-carrying microbubbles under ultrasound;

[0038] Figure 5 This is a statistical diagram of biofilm permeability of manganese-doped carbon dots-oxygen-carrying microbubbles under ultrasound;

[0039] Figure 6 This is a statistical diagram of the survival rate of biofilm bacteria in manganese-doped carbon dots-oxygen-carrying microbubbles under ultrasound.

[0040] Figure 7 This is a fluorescence statistical diagram of CD31 staining of microvessels under ultrasound in manganese-doped carbon dots-oxygen-carrying microbubbles. DETAILED DESCRIPTION

[0041] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0042] Example 1

[0043] The preparation method of antibacterial dual oxygen supply manganese-doped carbon dots-oxygen-carrying microbubbles comprises the following steps:

[0044] (1) 1 mmol reduced glutathione (GSH), 1 mmol anhydrous MnCl2 and 0.5 mmol PEI-600 were dissolved in 10 mL formamide and fully mixed using ultrasound. The temperature of the ultrasound treatment was 10-40°C, the time was 1-30 min, the ultrasound power was 100-1000 W, and the ultrasound device used included at least one of a cell crusher and an ultrasonic cleaner; the solution was transferred to a polytetrafluoroethylene autoclave and kept at 160°C for 4 hours. In order to eliminate larger impurities, the solution was diluted three times with deionized water and filtered through a 0.22 μm membrane filter. The solution was then purified by dialyzing against water for 7 days (MWCO was 3500 Da), and the MnCDs powder was collected by freeze drying.

[0045] (2) MnCDs and NHS-biotin were mixed in a Na2CO3 / NaHCO3 buffer solution (pH 9) at a molar ratio of 1:80. After being fully dissolved, the mixture was stirred at 37°C for 6 hours. The resulting solution was purified by dialyzing against water for 1 day (MWCO of 3500Da). Finally, the MnCDs-Biotin powder was collected by freeze drying.

[0046] (3) DPPC, DSPE-PEG2000 and DSPE-PEG2000-biotin were mixed in chloroform at a molar ratio of 82:9:9, and then the solution was evaporated using a rotary evaporator, and the pressure was reduced to 0 mbar for 2 hours to completely eliminate the chloroform, thereby forming a lipid film, and 5 mL of 0.05% glycerol / PBS solution was added to the lipid film, and ultrasonic oscillation was performed at 37°C for 10 minutes to obtain a lipid solution.

[0047] (4) In a sealed glass bottle, the solution was drawn out and released three times, the air in the upper part of the bottle was replaced with a mixture of octafluoropropane (C3F8) and O2 (volume ratio of 1:1), and then the mixture was stirred for 30 seconds using a mercury stirrer to produce O2MBs, which were then stirred with avidin and washed three times with PBS (300g, 3 minutes) and then centrifuged to obtain O2MBs-avidin.

[0048] (5) The O2MBs-avidin and MnCDs-Biotin powders were mixed, washed with PBS, and centrifuged three times (300 g, 3 min) to obtain MnCDs@O2MBs.

[0049] The performance of MnCDs@O2MBs prepared by the above method was characterized. The transmission electron microscopy image of manganese-doped carbon dots is shown in Figure 1 As shown in ab, MnCDs are monodispersed nanoparticles with an average particle size of 2.33±0.38nm. High-resolution transmission electron microscopy (HR-TEM) images show a lattice fringe of 0.21nm ( Figure 1 c), consistent with the (100) plane of graphene, indicating the presence of a graphite-like structure;

[0050] Fluorescence microscopy of manganese-doped carbon dots-oxygen-carrying microbubbles Figure 2 As shown, the red fluorescence represents the manganese-doped carbon dots. The biotinylated manganese-carbon dots were linked to the oxygen-carrying microbubbles via biotin-avidin, and the assembly process was confirmed by confocal laser scanning microscopy;

[0051] The statistical diagram of oxygen production of manganese-doped carbon dots-oxygen-carrying microbubbles under ultrasound is shown in Figure 3 As shown in the figure, under the action of ultrasound, the oxygen-carrying microbubbles quickly released the stored oxygen within 3-4 minutes. In contrast, the manganese-doped carbon dots showed a stable oxygen generation process under the action of their peroxidase-like activity. When the two were combined, the oxygen generation curve reflected both the characteristics of rapid release and the ability of continuous generation, confirming the effectiveness of manganese-doped carbon dots-oxygen-carrying microbubbles as a viable oxygen source in an excess H2O2 environment.

[0052] The statistical diagram of active oxygen production of manganese-doped carbon dots-oxygen-carrying microbubbles under ultrasound is shown in Figure 4As shown, the active oxygen production of manganese-doped carbon dots-oxygen-carrying microbubbles increased with the increase of manganese-doped carbon dots concentration and ultrasound exposure time;

[0053] The statistical diagram of biomembrane permeability of manganese-doped carbon dots-oxygen-carrying microbubbles under ultrasound is shown in Figure 5 As shown, the drug fluorescence intensity was most significant in the lower layer of the biofilm in the manganese-doped carbon dots-oxygen-carrying microbubbles + ultrasound group, and the penetration distance was also the farthest;

[0054] The statistical diagram of the survival rate of biofilm bacteria under ultrasound is shown in Figure 2. Figure 6 As shown, crystal violet dye was used to quantitatively analyze the biofilm, and the absorbance measurement results showed that the crystal violet absorbance of the manganese-doped carbon dots-oxygen-carrying microbubbles + ultrasound group was the lowest, indicating that this group had the most significant effect in reducing the survival rate of biofilm bacteria;

[0055] The fluorescence statistics of CD31 staining of microvessels under ultrasound are shown in the figure. Figure 7 As shown, the microvascular CD31 staining fluorescence results showed that the manganese-doped carbon dots-oxygen-carrying microbubbles + ultrasound group accelerated the progression of vascular normalization in diabetic biofilm-infected wounds.

Claims

1. A method for preparing antibacterial dual oxygen supply manganese-doped carbon dots-oxygen-carrying microbubbles, characterized in that: Here are the steps: (1) Reduced glutathione, anhydrous manganese chloride and PEI-600 were mixed and dissolved in formamide, maintained at 160°C for 4 h in a polytetrafluoroethylene autoclave, dialyzed and purified, and then freeze-dried to obtain MnCDs powder; (2) MnCDs and NHS-biotin were mixed in a Na2CO3 / NaHCO3 buffer solution and stirred evenly, and the resulting solution was dialyzed and purified, and then freeze-dried and collected to obtain MnCDs-biotin powder; (3) DPPC, DSPE-PEG2000 and DSPE-PEG2000-biotin were mixed in chloroform, rotary evaporated until the chloroform was completely eliminated, and glycerol / PBS solution was added and ultrasonically shaken to obtain a lipid solution; (4) completely filling a sealed container with a mixture of the lipid solution, octafluoropropane and O2, mixing and stirring to produce O2MBs, stirring the O2MBs and avidin together, washing with PBS, and centrifuging to obtain O2MBs-avidin; (5) The O2MBs-avidin and MnCDs-Biotin powders were mixed evenly, washed with PBS and centrifuged to obtain manganese-doped carbon dots-oxygen-carrying microbubbles MnCDs@O2MBs.

2. The preparation method according to claim 1, characterized in that: In step (1), the molar ratio of reduced glutathione, anhydrous manganese chloride and PEI-600 is 1:1:0.5; The solute solvent ratio of reduced glutathione, anhydrous manganese chloride, PEI-600 and formamide was 1 mmol:4 ml.

3. The preparation method according to claim 1, characterized in that: The mixed dissolution in step (1) is carried out by ultrasonic mixed dissolution, the ultrasonic temperature is 10 to 40 minutes, the time is 1 to 30 minutes, and the power is 100 to 1000W; The molecular cutoff for dialysis purification is 3500Da; The freeze-drying temperature is -40 to -60°C and the time is 48 hours.

4. The preparation method according to claim 3, characterized in that: The ultrasonic mixed dissolution is performed by using at least any one of a cell disruptor and an ultrasonic cleaning machine.

5. The preparation method according to any one of claims 1 to 4, characterized in that: The step (1) also includes diluting the solution three times with deionized water and filtering the solution through a 0.22 μm membrane filter before dialysis purification.

6. The preparation method according to claim 1, characterized in that: In step (2), the molar ratio of MnCDs to NHS-biotin is 1:80; The pH value of Na2CO3 / NaHCO3 buffer solution is 9.

7. The preparation method according to claim 1, characterized in that: The stirring operation in step (2) is stirring at 37° C. for 6 h; The molecular cutoff for dialysis purification is 3500Da; The freeze-drying temperature is -40℃~-60℃, and the time is 24h.

8. The preparation method according to claim 1, characterized in that: In step (3), the molar ratio of DPPC, DSPE-PEG2000 and DSPE-PEG2000-biotin is 82:9:9; The concentration of glycerol / PBS solution was 0.05%; Ultrasonic shaking was carried out at 37° C. for 10 min.

9. The preparation method according to claim 1, characterized in that: In step (4), the volume ratio of octafluoropropane to O2 is 1:

1.

10. The preparation method according to claim 1, characterized in that: The size of manganese-doped carbon dots-oxygen-carrying microbubbles MnCDs@O2MBs is 1000~2500nm.

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