Antibacterial carbon dots for treating bacteremia

Abstract

The invention provides an antibacterial carbon dot for treating bacteremia, and the antibacterial carbon dot has application potential in bacteremia treatment. The antibacterial carbon dots are prepared through a precise nano synthesis technology, and have unique physicochemical properties and excellent antibacterial activity. Specifically, the average particle size of the prepared antibacterial carbon dots is 2.0 + / -0.5 nanometers (nm), and the tiny size endows the antibacterial carbon dots with good biocompatibility and efficient cell penetrating power. Meanwhile, the hydrated particle size is 2.2 + / -0.4 nm, which shows that the nano-silver powder has good dispersion stability in an aqueous solution, and is beneficial to wide distribution and action in vivo and in vitro. Besides, the Zeta potential of the antibacterial carbon dot is as high as 36.3 + / -1.5 mV (mV), and the positive potential not only enhances the charge stability of the surface, but also contributes to interaction with negatively charged bacterial cell membranes, thereby enhancing the antibacterial effect.

Description

Technical Field

[0001] This application relates to the field of materials biotechnology, and specifically relates to an antibacterial carbon dot for treating bacteremia. Background Art

[0002] Bacteremia is a serious clinical disease, which refers to bacteria invading the blood circulation, growing and multiplying in it, producing toxins and causing systemic infections. With the widespread use of antibiotics, the problem of bacterial drug resistance has become increasingly serious, and traditional antibacterial treatment methods are facing challenges. Therefore, the development of new, efficient and low-toxic antibacterial agents has become a hot topic in medical research. In recent years, antibacterial carbon dots (CDs), as a new type of nanomaterial, have shown great potential in the treatment of infectious diseases such as bacteremia due to their good biocompatibility, antibacterial properties and photodynamic effect.

[0003] Carbon dots, as a new member of the carbon material family, have attracted extensive attention since they were first reported in 2004 due to their unique fluorescence properties and diverse chemical structures. Broadly speaking, carbon dots refer to carbon particles with fluorescence properties with a size less than 20 nanometers, mainly including graphene quantum dots, carbon nanodots and carbon quantum dots, etc. These carbon dots usually have a spherical structure, a carbon structure with sp2 and sp3 orbital hybridization in their chemical structure, and can be divided into carbon dots with obvious lattices and carbon dots without lattices. The antibacterial mechanism of carbon dots is diverse, mainly including photodynamic action, peroxidase-like action, mechanical / physical damage action and inhibition of bacterial metabolism, etc. Photodynamic action means that under light illumination, carbon dots, as photosensitizers, are excited to the excited state, generating reactive oxygen species (ROS), causing non-specific damage to bacterial cells, and thus achieving the effect of killing bacteria. The peroxidase-like action is to catalyze hydrogen peroxide to produce antibacterial active hydroxyl radicals to kill bacteria. The mechanical / physical damage action refers to carbon dots directly acting on the bacterial cell wall or outer membrane, causing physical / mechanical damage and leading to bacterial death. The inhibition of bacterial metabolism action is to act on bacterial metabolism-related proteins, interfering with the normal metabolic process of bacteria, and thus achieving the antibacterial effect.

[0004] In the treatment of bacteremia, carbon dots exhibit unique advantages. First, carbon dots have good biocompatibility and water solubility, and can be safely applied in vivo without being toxic to normal cells. Second, carbon dots can kill bacteria, including drug-resistant bacteria, through multiple mechanisms, showing broad-spectrum antibacterial effects on the treatment of bacteremia. In addition, carbon dots can be combined with different materials to form various composite antibacterial materials, such as antibacterial gels and antibacterial dressings, which are used to cover wounds, prevent bacterial infections, and accelerate wound healing. In recent years, researchers have carried out a series of heteroatom doping and surface functionalization modifications on the diversity of carbon dot structures, endowing them with better antibacterial, antioxidant, and photoluminescence properties. For example, by doping antioxidant elements such as selenium atoms and cerium atoms, the antioxidant performance of carbon dots can be enhanced, reactive oxygen species in the body can be scavenged, and the inflammatory response can be alleviated. At the same time, surface functionalization modification can improve the stability and biocompatibility of carbon dots, and enhance their application effects in vivo.

[0005] Although antibacterial carbon dots have multiple potential antibacterial mechanisms, such as photodynamic action and peroxidase-like action, the specific modes of action and effects of these mechanisms in vivo still need to be further verified. In addition, different types of bacteria may have different sensitivities to antibacterial carbon dots, so targeted research and optimization need to be carried out according to specific bacterial species. The application of antibacterial carbon dots in vivo needs to consider processes such as their distribution, metabolism, and excretion. How to ensure that antibacterial carbon dots can accurately reach the infection site and exert antibacterial effects while avoiding damage to normal tissues is an urgent problem to be solved. In addition, the metabolism and excretion processes of antibacterial carbon dots in vivo also need to be studied in depth to ensure that they do not have long-term effects on the physiological functions of the human body. Summary of the Invention

[0006] In view of the above-mentioned technical limitations, the present application proposes an antibacterial carbon dot for the treatment of bacteremia. Here, we developed a hydrothermal synthesis strategy using polyethyleneimine (PEI) and disodium ethylenediaminetetraacetate (EDTA-2Na) as precursors to produce a series of CDs (PEI-CDs) with narrow size distribution, excellent fluorescence properties, and enhanced antibacterial activity at a relatively low reaction temperature. The addition of PEI improves the water solubility and dispersibility of the CDs, and at the same time brings positive charges. This improves their stability and antibacterial effects in the aqueous environment, making them very suitable for biological applications. Importantly, the antibacterial performance of PEI-CDs can be customized by adjusting the synthesis temperature, and their efficacy against Gram-positive and Gram-negative bacteria has a significant impact on the positive charge distribution, overcoming the deficiencies and defects mentioned in the background technology.

[0007] To achieve the above object, the present application adopts the following technical solutions:

[0008] The invention point of the present application is to provide an antibacterial carbon dot for the treatment of bacteremia.

[0009] The antibacterial carbon dots for treating bacteremia are quasi-spherical nanoparticles.

[0010] Optionally, the antibacterial carbon dots for treating bacteremia have an average particle size of 2.0 ± 0.5 nm, a hydrodynamic particle size of 2.2 ± 0.4 nm, and a Zeta potential of 36.3 ± 1.5 mV.

[0011] Optionally, the atomic ratios of C1s, N1s, and O1s in the antibacterial carbon dots for treating bacteremia are 66.07%, 14.06%, and 18.28% respectively.

[0012] Optionally, the antibacterial carbon dots for treating bacteremia have broad-spectrum antibacterial properties and have a certain degree of antibacterial effect on both Staphylococcus aureus and Escherichia coli.

[0013] Optionally, the antibacterial carbon dots for treating bacteremia can effectively remove bacterial biofilms.

[0014] Optionally, the antibacterial carbon dots for treating bacteremia have excellent therapeutic effects on bacteremia and can effectively remove bacteria and inflammatory factors.

[0015] Compared with the prior art, the present application has the following advantages:

[0016] The present invention has realized an antibacterial carbon dot for treating bacteremia for the first time. The antibacterial carbon dots may play a powerful antibacterial role through mechanisms such as destroying the bacterial cell membrane, inhibiting bacterial growth, or promoting bacterial death, which are not possessed by traditional antibacterial drugs. Due to the small size effect of carbon dots and the action of surface functional groups, the antibacterial carbon dots may be able to quickly bind to bacteria and play a role, thereby quickly controlling the infection. By adjusting parameters such as the surface functional groups and particle size of the antibacterial carbon dots, specific antibacterial effects on different bacteria can be achieved, providing the possibility for personalized treatment.

[0017] The antibacterial carbon dots exhibit broad-spectrum antibacterial properties and can effectively inhibit the growth of a variety of bacteria including Gram-positive bacteria and Gram-negative bacteria. The antibacterial carbon dots developed in this study, with their unique nano-size, high Zeta potential, broad-spectrum antibacterial characteristics, and significant therapeutic effects on the bacteremia mouse model, demonstrate great potential as a new generation of antibacterial treatment means. In the future, by further optimizing their preparation process, exploring more precise mechanisms of action, and conducting more extensive preclinical and clinical studies, it is expected to push the antibacterial carbon dots into clinical applications and bring new hope for the treatment of infectious diseases such as bacteremia. Description of the Drawings

[0018] Figure 1: TEM and DLS of PEI-CDs at different temperatures. Transmission electron microscopy of PEI-CDs at different temperatures. (D-F) Particle size distribution of PEI-CDs at different temperatures (n = 3).

[0019] Figure 2 : DLS of the samples in water at different temperatures (n = 3).

[0020] Figure 3 : Full-range XPS spectrum of PEI-CDs and high-resolution XPS spectra of C1s, N1s, and O1s.

[0021] Figure 4 : In vitro antibacterial performance of PEI-CDs. Growth of Escherichia coli etc. on agar plates treated with PEI-CDs (300 μg / mL)

[0022] Figure 5 : Growth inhibitory effect of different concentration combinations of PEI-CDs-180°C on Escherichia coli.

[0023] Figure 6 : Zeta potential of PEI-CDs-180°C alone and in combination with Staphylococcus aureus and Escherichia coli, and in combination with PEI-CDs.

[0024] Figure 7 : Fluorescent staining of Escherichia coli and Staphylococcus aureus treated with 300 μg / mL PEI-CDs-180°C; scale bar, 100 μm.

[0025] Figure 8 : Transmission electron microscopy of Escherichia coli and Staphylococcus aureus after treatment with 300 μg / mL PEI-CDs-180°C for 12 h; scale bar: 1 μm.

[0026] Figure 9 : Evaluation of the in vivo therapeutic effect of PEI-CD using a murine bacteremia model.

[0027] Figure 10 : PEI-CDs treat infected mice by collecting bacterial colonies from the blood, liver, kidneys, and lungs of the infected mice.

[0028] Figure 11 : Statistical analysis of the results of bacterial colony formation.

[0029] Figure 12 : Analysis of IL-6, TNA-α, and IL-1β in the blood by qPCR (n = 3).

[0030] Figure 13 : Survival curves of infected mice treated with PEI-CDs, PBS, or penicillin / streptomycin (P / S) (n = 15). Detailed implementation manners

[0031] To make the objectives, technical solutions and advantages of the present application clearer, the present application will be further described in detail below. However, it should be understood that the description herein is only used to explain the present application and is not intended to limit the scope of the present application.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which this application belongs. The terms used in the description of the present application herein are only for the purpose of describing specific embodiments and are not intended to limit the present application. The reagents and instruments used herein are all commercially available, and the characterization means involved can be referred to the relevant descriptions in the prior art and will not be elaborated herein.

[0033] The materials and methods are as follows:

[0034] Synthesis of PEI-CDs

[0035] Briefly, EDTA-2Na (0.56 g) and branched polyethyleneimine (bPEI, Mw = 1800, 0.14 g) were dispersed in 15 mL of ultrapure water. Then the solution was transferred to a hydrothermal synthesis reactor and heated at 140, 180, and 220 °C for 6 - 8 hours respectively. After cooling, the mixture was filtered through a 0.22 μm membrane and then dialyzed in ultrapure water using a dialysis membrane with MWCO = 3500 Da for 48 h. The final solid samples were obtained after freeze-drying and named PEI-CDs-140 °C, PEI-CDs-180 °C, and PEI-CDs-220 °C respectively.

[0036] Characterization of PEI-CDs

[0037] Transmission electron microscopy (TEM, HT7700, Hitachi, Japan), high-resolution transmission electron microscopy (HR-TEM, 2010F, JEOL, Japan) were used to detect the particle morphology and size. The zeta potential and particle size distribution of PEI-CD were analyzed using a dynamic light scattering (DLS) particle size analyzer (zeta Sizer 3000HSA, Malvern Instruments, UK). The X-ray diffraction (XRD) pattern was recorded on a Bruker AXS D8 (USA) diffractometer, and elemental analysis was performed using X-ray photoelectron spectroscopy (XPS, Kratos Axis Ultra, UK).

[0038] Antibacterial properties of PEI-CDs

[0039] To determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of PEI-CDs, the following procedure is generally followed. First, bacteria are inoculated on a broth agar plate and then incubated at 37 °C to obtain single colonies. Then, a single colony is picked using an inoculation loop and introduced into an appropriate amount of broth, and shaken at 37 °C (150 rpm) for 12 h until the optical density (OD600) at 600 nm is approximately 0.3 (5×108 CFU / mL). Subsequently, the bacterial suspension is diluted to 1×10 6 CFU / mL for subsequent experiments. Freshly prepared microbial suspensions (100 μL of Staphylococcus aureus and Escherichia coli) are combined with PEI-CDs solutions (100 μL, at concentrations of 0, 100, 200, 300, 400, 500, and 1000 μg / mL) and co-incubated at 37 °C in a 96-well plate for 24 h. Compared with the control, the concentration at which the solution remains clear and non-turbid represents the MIC of PEI-CDs for that specific strain.

[0040] Antibacterial mechanism of PEI-CDs

[0041] Interaction between bacteria and PEI-CDs: 1 mL of PEI-CDs (300 μg / mL) was added to 1 mL of microbial suspension (1×106 CFU / mL), incubated for 4 h, washed 3 times with physiological saline, and imaged using a confocal laser scanning microscope (CLSM, FV 3000, Olympus, Japan).

[0042] Observation of bacteria under electron microscopy: 1 mL of PEI-CDs (300 μg / mL) was mixed with 1 mL of bacterial suspension (1×106 CFU / mL), incubated for 12 h, fixed with 2.5% glutaraldehyde, washed 2 times with sterile ultrapure water, and gradually dehydrated with different concentrations of ethanol (30% and 50% ethanol for 30 min each; 75%, 85%, 95%, and 100% ethanol for 20 min each; dispersed in absolute ethanol). Finally, 3 - 5 drops of the sample were placed on a copper grid and observed using an electron microscope (ht7700, Hitachi, Japan).

[0043] Determination of extracellular protein concentration: 1 mL of PEI-CDs (300 μg / mL) was mixed with 1 mL of microbial suspension (1×106 CFU / mL) and incubated for 4 h. After centrifugation at 4500 rpm, 100 μL of the supernatant was taken, and the extracellular protein concentration was determined using a bicinchoninic acid (BCA) detection kit from Pierce, USA.

[0044] In vivo bacteremia model

[0045] With Staphylococcus aureus (1×10 7A bacteremia model of 8-week-old male C57 mice was established by intraperitoneal injection of E. coli (1×107 CFU, 100 μL). After 2 h, intravenous injection was performed via the tail vein: PEI-CDs-180 °C (300 μg / mL, 200 μL), penicillin / streptomycin (P / S 200 μL), or PBS (200 μL, n = 15). The mice were sacrificed 24 h after injection, and blood and organs were collected. The collected samples were homogenized in 1 mL of PBS. For the quantification of bacteria, the homogenized samples were incubated overnight at 37 °C and then the bacterial colony-forming units were determined by plate counting. This method was used to evaluate the effects of PEI-CDs-180 °C, penicillin / streptomycin, and PBS on the bacterial loads in the blood and organs of bacteremia model mice.

[0046] Example 1

[0047] To achieve multi-faceted synergistic therapy in the treatment of bacterial infections, in this work, we designed and synthesized a series of PEI-CDs, which were synthesized by a hydrothermal method at different temperatures. TEM images showed that PEI-CDs-140 °C, PEI-CDs-180 °C, and PEI-CDs-220 °C were quasi-spherical, well-distributed, and monodisperse structures, with average particle sizes of 3.7 ± 0.8 nm, 2.0 ± 0.5 nm, and 3.2 ± 0.7 nm ( Figure 1 ). DLS analysis verified the consistent particle size distributions of these PEI-CDs, with hydrodynamic diameters of 4.3 ± 0.3 nm, 2.2 ± 0.4 nm, and 4.7 ± 0.5 nm ( Figure 2 ). HR-TEM images showed that the crystal spacing of PEI-CD was 0.21 nm, indicating the diffraction plane (100) of graphite.

[0048] Example 2

[0049] To analyze more deeply the percentage of elements, chemical structure, and the relationship between chemical bonds and surface charge, we used XPS. In the range of PEI-CDs-180 °C, the atomic ratios of C1s, N1s, and O1s were 66.07%, 14.06%, and 18.28%, respectively. Deconvolution of the C1s XPS spectrum of PEI-CDs-180 °C revealed that the binding energy peaks were 284.8 eV (C-C / C═C), 285.7 eV (C-N), and 287.6 eV (C═O), respectively. Similarly, the N1s spectrum consisted of three fitted peaks at 398.7, 399.9, and 401.0 eV, corresponding to pyridine N, -NH2, and pyridine N [33, 34], respectively. Deconvolution of the O1s peak had two fitted peaks at 530.5 eV and 531.7 eV, corresponding to C-O / -OH and C═O ( Figure 3 ).

[0050] Example 3

[0051] To evaluate the antibacterial performance of PEI-CDs, the antibacterial activities of PEI-CDs against 4 different strains of bacteria at different temperatures were determined by colony-forming unit (CFU) counting method. After the action of 300 μg / mL PEI-CDs for 4 h, with the synthesis temperature increasing from 140 °C to 220 °C, the antibacterial activity first increased and then decreased. Among them, PEI-CDs-180 °C with the highest positive charge and the highest -NH2 content had the strongest antibacterial activity, indicating that its antibacterial activity was proportional to its surface charge. Therefore, we selected the PEI-CDs-180 °C sample for subsequent experiments, including its antibacterial activity, antibacterial mechanism, antioxidant activity, cytotoxicity and in vivo wound healing ability. The standard plate counting method was used to explore the antibacterial effect of PEI-CDs. The experimental results showed that Gram-positive bacteria decreased significantly in a concentration-dependent manner, and the antibacterial effect of Gram-negative bacteria was slightly weaker than that of Gram-positive bacteria. 300 μg / mL PEI-CDs-180 °C could achieve the effect of completely killing Staphylococcus aureus and Staphylococcus epidermidis, that is, the minimum bactericidal concentration (MBC) was about 300 μg / mL. However, the antibacterial effect of PEI-CDs-180 °C on Gram-negative bacteria, Escherichia coli and Pseudomonas aeruginosa was weak, and the MBC was 300 μg / mL. By recording the OD600 value, a time-dependent antibacterial curve of PEI-CDs-180 °C against Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli and Pseudomonas aeruginosa was established. The results showed that the minimum inhibitory concentration (MIC) of Staphylococcus aureus and Staphylococcus epidermidis was 300 μg / mL, and the MIC of Escherichia coli and Pseudomonas aeruginosa was 500 μg / mL. To sum up, the antibacterial performance of PEI-CDs is not only closely related to the synthesis temperature, but also gradually increases with the increase of concentration, and may also be related to the surface charge of bacteria ( Figure 4 and 5 ).

[0052] Example 4

[0053] Next, taking Gram-positive bacteria represented by Staphylococcus aureus and Gram-negative bacteria represented by Escherichia coli respectively, the antibacterial mechanism of PEI-CDs-180 °C was studied. Surface charge plays a crucial role in antibacterial action by attaching to bacteria. As Figure 6As shown, the zeta potentials of Staphylococcus aureus and Escherichia coli were -34.3 mV and -9.3 mV, respectively. PEI-CDs-180℃ exhibited the highest positive charge, attributed to the abundant amino groups. After adding 300 μg / mL PEI-CDs-180℃, the zeta potential of Escherichia coli only increased to -4.5 mV, indicating a very small electrostatic interaction with PEI-CDs-180℃. In contrast, after adding PEI-CDs-180℃, the zeta potential of Staphylococcus aureus increased significantly to -0.3 mV, indicating a stronger electrostatic adsorption of PEI-CDs-180℃ to it. After incubating with PEI-CDs-180℃ for 12 hours, live / dead bacterial staining was performed using SYTO 9 and PI dyes. PI stains the cell nucleus red, but can only enter cells with damaged cell membranes, while SYTO 9 can cross intact cell membranes, bind to DNA, and emit green fluorescence( Figure 7 ). Almost all untreated Staphylococcus aureus and Staphylococcus epidermidis cells survived, while most of the Staphylococcus aureus and Staphylococcus epidermidis cells treated with PEI-CDs-180℃ died. After treatment with PEI-CDs-180℃, the number of dead cells of Escherichia coli and Pseudomonas aeruginosa was significantly lower than that of Staphylococcus aureus and Staphylococcus epidermidis. These results indicate that PEI-CDs-180℃ reduced the permeability of the bacterial cell membrane, resulting in the death of a large number of bacterial cells. In addition, the integrity of the bacterial cell membrane before and after treatment with PEI-CDs-180℃ was studied by transmission electron microscopy, as shown in the figure. The cell membrane structure of untreated Gram-positive bacteria was intact and smooth; however, after treatment with PEI-CDs-180℃, the cell membranes of the bacteria were significantly ruptured and the cell contents leaked( Figure 8 ).

[0054] Example 5

[0055] To evaluate its application value, PEI-CDs was applied to the treatment of Staphylococcus aureus-infected bacteremia. 8-week-old male C57 mice were intraperitoneally injected with Staphylococcus aureus (1×10 7(CFU). After 2 h, PEI-CDs (400 μg / mL, 200 μL), penicillin / streptomycin (P / S, 100 μg / mL, 200 μL), or PBS was injected intravenously. Infected mice treated with PBS showed typical symptoms of limb weakness and died the next day. On day 2, blood and tissues of infected mice treated with PEI-CDs, P / S, or PBS were collected for bacterial colony formation assay. The results showed that the number of bacterial colonies after treatment with PEI-CDs and P / S was significantly reduced compared with the PBS group, and there were almost no bacterial colonies in the blood after treatment with PEI-CDs. This indicates that they have a significant antibacterial effect on bacterial infection in the blood. In addition, qPCR analysis of blood samples showed that both PEI-CDs and P / S could effectively relieve blood inflammation and reduce the mRNA expression levels of inflammatory markers IL-6, TNA-α, and IL-1β, while PEI-CDs showed a higher level of significance. Finally, the survival rate of mice with bacteremia treated with PEI-CDs was the highest. In contrast, the mortality rate of control group mice exceeded 80%, while the mortality rate of the PEI-CDs treatment group was no more than 25%, even slightly higher than that of the P / S treatment group. These results highlight the antibacterial effect of PEI-CDs on bacteremia, significantly reducing blood inflammation and the expression level of inflammatory factors in the blood, and ultimately improving the survival rate( Figures 9 - 13 )

[0056] The above are only the preferred embodiments of the present application and are not intended to limit the present application. Any modifications, equivalent replacements, or improvements made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. An antibacterial carbon dot for treating bacteremia, with a quasi-spherical shape, characterized in that, The atomic ratios of C1s, N1s, and O1s are 66.07%, 14.06%, and 18.28% respectively.

2. The antibacterial carbon dots for treating bacteremia according to claim 1, characterized in that, The average particle size is 2.0 ± 0.5 nm, the hydrated particle size is 2.2 ± 0.4 nm, and the Zeta potential is 36.3 ± 1.5 mV.

3. The antibacterial carbon dots for treating bacteremia according to claim 1, wherein It has broad-spectrum antibacterial properties and has a certain degree of antibacterial effect on both Staphylococcus aureus and Escherichia coli.

4. The antibacterial carbon dots for treating bacteremia according to claim 1, characterized in that, The antibacterial carbon dots for treating bacteremia are selective for pH, and the antibacterial performance is the strongest when pH = 3 - 5.

5. The antibacterial carbon dots for treating bacteremia according to claim 1, characterized in that, The antibacterial carbon dots can effectively remove bacterial biofilms.

6. The antibacterial carbon dots for treating bacteremia according to claim 1, characterized in that The preparation method includes: dispersing EDTA-2Na and branched polyethyleneimine in ultrapure water and heating at 140 - 220 °C to obtain.

7. The antibacterial carbon dots for treating bacteremia according to claim 6, characterized in that, Heat at 180 °C for 6 hours, and filter after cooling to obtain antibacterial carbon dots.

8. The antibacterial carbon dots for treating bacteremia according to claims 1-7, characterized in that, The antibacterial carbon dots for treating bacteremia are used to remove bacteria and inflammatory factors.

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