Anti-biofilm composition based on synergistic effect of amino fullerene and vancomycin and application of anti-biofilm composition
By combining aminofullerene derivatives with vancomycin, the biofilm matrix is disrupted and vancomycin penetration is promoted, solving the problem of difficulty in inhibiting and removing bacterial biofilms in existing technologies. This achieves highly efficient biofilm inhibition and removal effects and reduces bacterial resistance.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-23
- Publication Date
- 2026-04-07
AI Technical Summary
Existing technologies are insufficient to effectively inhibit and eliminate bacterial biofilms, especially mature biofilms, leading to chronic and persistent infections. Furthermore, vancomycin alone has limited efficacy.
The combination of aminofullerene derivatives and vancomycin promotes the penetration of vancomycin and synergistically removes biofilm by disrupting the biofilm matrix structure and increasing bacterial membrane permeability.
It significantly inhibits biofilm formation, eliminates more than 40% of mature biofilm, reduces the risk of bacterial resistance, and provides broad prospects for clinical application.
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Figure CN121796552A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the technical field of biological medicine, and particularly relates to an antibiofilm composition based on synergistic effect of amino fullerene and vancomycin and application thereof in preparation of a medicine for preventing and / or treating bacterial biofilm related infection. BACKGROUND Bacterial biofilm is a film-like substance formed after bacteria adhere to biological or non-biological surfaces and secrete polysaccharide matrix, fibrin, lipoprotein and other polysaccharide protein complexes to make bacteria adhere to each other and aggregate bacterial colonies therein. The formation of biofilm significantly improves the drug resistance of bacteria to antibiotics, which can be up to 10-1000 times of that of planktonic bacteria, leading to chronic infection.
[0002] Vancomycin, as a glycopeptide antibiotic, is an important medicine for treating gram-positive bacterial infection, especially methicillin-resistant Staphylococcus aureus infection. However, vancomycin alone has limited effect on bacteria in biofilm and is difficult to eradicate biofilm related infection.
[0003] At present, there is an urgent need to develop a new treatment strategy that can effectively inhibit biofilm formation and remove the formed biofilm. Although some studies have explored the combination of nanomaterials and antibiotics, the synergistic effect of amino fullerene derivatives and vancomycin in antibiofilm, especially the removal of mature biofilm, has not been reported. SUMMARY
[0004] The present application aims to provide an antibiofilm composition which can effectively inhibit biofilm formation and significantly remove the formed mature biofilm, thereby being used for preparing a medicine for preventing and / or treating bacterial biofilm related infection.
[0005] To achieve the above-mentioned purpose, the present application adopts the following technical solutions: The present application provides an antibiofilm composition, the active ingredients of which include amino fullerene derivatives and vancomycin. The amino fullerene derivative is a water-soluble amino fullerene derivative, the structure of which is as follows:
[0006] The amino fullerene derivative is prepared by a method comprising the following steps: The fullerene solution is added dropwise into ethylenediamine, and the reaction is stirred until the solution becomes red-brown. The excess ethylenediamine and solvent are removed, and the product is dissolved with hydrochloric acid. Then, the pH is adjusted to 5.0-5.4 (specifically 5), and the product is purified by ultra-pure water dialysis.
[0007] The mass ratio of the fullerene to ethylenediamine can be 200-250 mg:100 mL, and specifically can be 200 mg:100 mL. The reaction is carried out in an inert atmosphere, which can be specifically a nitrogen atmosphere. The temperature of the reaction can be 75-85℃, and specifically can be 80℃, and the time can be 18-24 h, and specifically can be 24 h.
[0008] The mass ratio of the amino fullerene derivative to vancomycin can be 10:1.
[0009] The above-mentioned antibiofilm composition is also within the protection scope of the present application in the application of preparing a medicine for inhibiting the formation of bacterial biofilm and / or eliminating the formed biofilm.
[0010] Preferably, the formed biofilm is a mature biofilm with a culture time of more than 48 hours, and the composition can eliminate at least 40% of the mature biofilm.
[0011] The bacteria are gram-positive bacteria, and preferably are Staphylococcus aureus, Staphylococcus epidermidis, and further preferably are methicillin-resistant Staphylococcus aureus.
[0012] The present application also provides an antibacterial composition.
[0013] The antibacterial composition contains the above-mentioned antibiofilm composition.
[0014] The present application also provides a method for inhibiting the formation of gram-positive bacterial biofilm and / or eliminating the formed gram-positive bacterial biofilm.
[0015] The method comprises contacting the above-mentioned antibiofilm composition with the biofilm or applying the antibiofilm composition to a surface, The surface comprises a medical device surface, a chronic wound surface. The antibiofilm composition contacts the biofilm or is applied to the surface in the form of a solution. The mass ratio of the amino fullerene derivative to vancomycin in the antibiofilm composition solution can be 10:1. The concentration of the amino fullerene derivative in the antibiofilm composition solution is not less than 25 μg / mL, and specifically can be 25 μg / mL. The biofilm comprises organisms of multiple species.
[0016] The present application has the following effects: 1. Synergistic antibiofilm effect: the present application first discovers that the combination of the amino fullerene derivative and vancomycin has a significant synergistic effect in inhibiting the formation of biofilm, and the biofilm inhibition rate reaches 93%, indicating a strong synergistic effect.
[0017] 2. Breakthrough mature biofilm clearance ability: The composition can effectively clear up to more than 40% of the formed mature biofilm (24-48 hour biofilm), which is an effect that vancomycin alone cannot achieve.
[0018] 3. Multi-mechanism synergy: Amino fullerene derivatives may promote vancomycin penetration and act on deep-seated bacteria in biofilms through mechanisms such as destroying biofilm matrix structure and increasing bacterial membrane permeability, achieving synergistic clearance effect.
[0019] 4. Reduce the risk of drug resistance: By using a combination strategy, the concentration of vancomycin used can be significantly reduced, slowing the development of bacterial drug resistance.
[0020] 5. Broad clinical application prospects: The composition provides a new solution for the treatment of biofilm-related diseases such as medical device-related infections and chronic wound infections. BRIEF DESCRIPTION OF DRAWINGS
[0021] Figure 1 The infrared spectrum of the amino fullerene derivative (C 70 -EDA) prepared in Example 1 of the present application.
[0022] Figure 2 The X-ray photoelectron spectrum of the amino fullerene derivative (C 70 -EDA) prepared in Example 1 of the present application.
[0023] Figure 3 The Zeta potential of the amino fullerene derivative (C 70 -EDA) prepared in Example 1 of the present application.
[0024] Figure 4 The thermogravimetric curve of the amino fullerene derivative (C 70 -EDA) prepared in Example 1 of the present application.
[0025] Figure 5 The inhibitory effect of the amino fullerene derivative, vancomycin alone and in combination on Staphylococcus aureus biofilm formation in Example 3 of the present application.
[0026] Figure 6 The effect of each group treatment on inhibiting the ultrastructure of biofilm formation observed by scanning electron microscopy in Example 3 of the present application.
[0027] Figure 7 The clearance effect of the amino fullerene derivative, vancomycin alone and in combination on Staphylococcus aureus mature biofilm (24 hours) in Example 4 of the present application.
[0028] Figure 8Biological toxicity test of the amino-fullerene derivative and vancomycin composition in Example 5 of the present application to 3T3 cells (both concentrations were 25 μg / mL: 2.5 μg / mL, 50 μg / mL: 5 μg / mL, 100 μg / mL: 10 μg / mL, 200 μg / mL: 20 μg / mL, 500 μg / mL: 50 μg / mL, 1000 μg / mL: 100 μg / mL). DETAILED DESCRIPTION
[0029] The present application will be further described in conjunction with the specific embodiments thereof. The following examples are presented by way of illustration and therefore should not be construed to limit the scope of the present application. The examples provided below serve as a guide for further improvement by those of ordinary skill in the art and do not in any way constitute a limitation on the present application.
[0030] The experimental methods in the following examples are all conventional methods, unless otherwise specified, and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained commercially.
[0031] Example 1, Preparation and characterization of amino-fullerene derivative (C 70 -EDA) C 70 A solution of C
[0032] Figure 1 Infrared spectrum of the prepared amino-fullerene derivative (C 70 -EDA).
[0033] Figure 2 X-ray photoelectron spectrum of the prepared amino-fullerene derivative (C 70 -EDA).
[0034] Figure 3 Zeta potential of the prepared amino-fullerene derivative (C 70 -EDA).
[0035] Figure 4 Thermogravimetric curve of the prepared amino-fullerene derivative (C 70 -EDA).
[0036] From Figures 1-4 It can be seen that the N-H stretching vibration band (3400-3200 cm-1) and C-N stretching vibration peak (about 1250 cm-1) in the infrared spectrum indicate that EDA is attached to C 70 The X-ray photoelectron spectroscopy shows that the C 1s peak is at a binding energy of 284.8 eV, which is attributed to C-C, 286.26 eV and 288.66 eV are attributed to C = C and C - N, respectively, which further confirms the connection of the amino group to C 70 The zeta potential is +40.70 ± 0.34 nm. The water content and the proportions of C, H, and N elements in the C 70 -EDA solid are determined by thermogravimetric analysis and elemental analysis, and the average structural formula of C 70 -EDA is calculated as: C 70 (NHCH2CH2NH2) ~10 •24H2O.
[0037] The organic elemental analysis is performed on the prepared amino fullerene derivative (C 70 -EDA), and the results are shown in Table 1.
[0038] Table 1 Organic elemental analysis
[0039] Example 2, Preparation of a composition An appropriate amount of the amino fullerene derivative prepared in Example 1 and vancomycin are weighed, and dissolved in sterile water according to a mass ratio of 10:1 to prepare a stock solution with a fixed concentration.
[0040] Example 3, Synergistic inhibition of biofilm formation experiment Materials and methods: Strain: methicillin-resistant Staphylococcus aureus ATCC43300 Method 1: 24-well plate crystal violet staining method Grouping: Blank control group Amino fullerene derivative alone group (25 μg / mL) Vancomycin alone group (2.5 μg / mL) Combined group (amino fullerene derivative 25 μg / mL, vancomycin 2.5 μg / mL) The results are shown in Table 2, Figure 5 .
[0041] Table 2 Inhibition rate (%) of different treatment groups on methicillin-resistant Staphylococcus aureus ATCC43300 biofilm formation
[0042] Method 2: Scanning electron microscope observation Figure 6 The results showed that the combined treatment caused significant degradation of the biofilm matrix and destruction of the bacterial cell structure.
[0043] Example 4, mature biofilm removal experiment Materials and methods: Strain: methicillin-resistant Staphylococcus aureus ATCC43300 Method: First, cultivate bacteria in a 96-well plate for 48 hours to form a mature biofilm. After discarding the planktonic bacteria, add different treatments and continue for 24 hours. Then, stain with crystal violet for quantification.
[0044] The results are shown in Table 3, Figure 6 .
[0045] Table 3, removal effect of different treatment groups on mature biofilm
[0046] Example 5, cytotoxicity test Cells: mouse embryonic fibroblast cell line (3T3) Main reagents: CCK-8 kit (containing WST-8 solution, purchased from Beijing Bailei Biotechnology Co., Ltd.) Cell complete culture medium (according to cell line selection, such as DMEM + 10% FBS + 1% penicillin-streptomycin, all purchased from Thermo Fisher Scientific) Phosphate buffered saline (PBS, purchased from Thermo Fisher Scientific) Trypsin digestion solution (containing EDTA, purchased from Thermo Fisher Scientific) Grouping: Set up blank control group (only medium, no cells), control cell group (cells + complete culture medium), experimental group (cells + culture medium containing different concentrations of test substances, amino fullerene and vancomycin concentrations are 25 μg / mL: 2.5 μg / mL, 50 μg / mL: 5 μg / mL, 100 μg / mL: 10 μg / mL, 200 μg / mL: 20 μg / mL, 500 μg / mL: 50 μg / mL, 1000 μg / mL: 100 μg / mL). Each group sets 6 replicates.
[0047] Experimental steps Take the cells in logarithmic growth phase, digest with trypsin, and prepare a single cell suspension with complete culture medium. Perform cell counting and adjust the cell density to 5×10 3 -10×10 4Cells were seeded at 1 x 104cells per well. 100 μL was seeded in each well of a 96-well plate. The seeded plates were pre-cultured for 24 hours in a 37°C, 5% CO2incubator to allow the cells to adhere fully. The old culture medium was aspirated. Experimental group: 100 μL of fresh complete culture medium containing different concentrations of the drug to be tested was added. Control group: 100 μL of fresh complete culture medium without drug was added. Blank group: only 100 μL of fresh complete culture medium was added (without seeding cells).
[0048] The plates were returned to the incubator for further incubation for 24 hours. Before reaching the detection time point, the CCK-8 reagent was taken out from 4°C and restored to room temperature. 10% of the CCK-8 solution was added to the culture medium, the old culture medium was discarded, 100 μL of culture medium containing CCK-8 reagent was added to each well, the plates were returned to the incubator, and incubated for 30-60 min in the dark. After the incubation was completed, the plates were taken out of the incubator. An enzyme-labeled instrument was used to measure the absorbance (OD value) of each well at a wavelength of 450 nm.
[0049] The CCK-8 method showed that the composition was safe and controllable at an effective concentration, and the cell survival rate was greater than 95% (as shown in the figure). Figure 8
[0050] Comparative Example The inventors also explored the effect of inhibiting biofilm under the condition that the mass ratio of aminofullerene to vancomycin was 5:1, 6:1, 7:1, 8:1, 9:1. After co-culturing for three days, obvious biofilm formation was visible to the naked eye, so the mass ratio of aminofullerene to vancomycin was selected as 10:1 for the experiment.
[0051] The above has described the present application in detail. For those skilled in the art, without departing from the purpose and scope of the present application, and without unnecessary experiments, the present application can be implemented in a wider range under equivalent parameters, concentrations and conditions. Although the present application gives a special example, it should be understood that further improvements can be made to the present application. In summary, according to the principle of the present application, the present application intends to include any changes, uses or improvements of the present application, including changes made by conventional techniques known in the art, which are outside the scope disclosed in the present application.
Claims
1. An anti-biofilm composition, wherein the active ingredients include an aminofullerene derivative and vancomycin.
2. The anti-biofilm composition according to claim 1, characterized in that, The aminofullerene derivative is a water-soluble aminofullerene derivative, and its general structural formula is shown below: The aminofullerene derivative was prepared by a method comprising the following steps: The fullerene solution was added dropwise to ethylenediamine, and the reaction was stirred until the solution turned reddish-brown. Excess ethylenediamine and solvent were removed, and the product was dissolved in hydrochloric acid. The pH was adjusted to 5.0-5.4, and the product was purified by dialysis with ultrapure water.
3. The anti-biofilm composition according to claim 1, characterized in that, The mass ratio of the aminofullerene derivative to vancomycin is 10:
1.
4. Use of the antibiofilm composition according to any one of claims 1-3 in the preparation of a medicament for inhibiting bacterial biofilm formation and / or removing existing biofilms.
5. The application according to claim 4, characterized in that, The formed biofilm is a mature biofilm that has been cultured for more than 48 hours; The bacteria are Gram-positive, preferably Staphylococcus aureus or Staphylococcus epidermidis, and more preferably methicillin-resistant Staphylococcus aureus.
6. An antibacterial composition comprising the antibiofilm composition according to any one of claims 1-3.
7. A method for inhibiting the formation of Gram-positive bacterial biofilms and / or removing existing Gram-positive bacterial biofilms, comprising contacting the biofilm with the antibiofilm composition of any one of claims 1-3 or applying the antibiofilm composition to a surface.
8. The method according to claim 7, characterized in that, The surface includes the surface of a medical device and the surface of a chronic wound; the biofilm includes organisms of multiple species.
9. The method according to claim 7, characterized in that, The anti-biofilm composition is applied to a surface in solution form or in contact with a biofilm. In the anti-biofilm composition solution, the mass ratio of aminofullerene derivative to vancomycin is 10:1; In the anti-biofilm composition solution, the concentration of the aminofullerene derivative is not less than 25 μg / mL.