Composite nano particle as well as preparation method and application thereof

Abstract

The invention discloses a composite nano particle as well as a preparation method and application thereof, and relates to the technical field of biological medicines and nano materials. The invention particularly discloses a composite nano particle obtained by coordination self-assembly of ginsenoside and transition metal ions. According to the present invention, through the multiple synergistic mechanisms such as biological membrane physical structure destroying, catalytic generation of a large amount of reactive oxygen species (ROS), bacterial metabolism interference and the like, a variety of bacteria including gram-positive bacteria and gram-negative bacteria can be efficiently killed, particularly, the excellent removal ability on intractable biological membranes is provided, and the good biocompatibility and the good application prospect are provided.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and nanomaterials technology, and in particular to a composite nanoparticle, its preparation method, and its application. Background Technology

[0002] Bacterial infections are a major global public health problem threatening human health. With the widespread use of antibiotics, bacterial resistance has become increasingly serious, significantly weakening the effectiveness of traditional anti-infective treatments. Even more challenging is the resistance of many pathogens (such as Staphylococcus aureus...). S. aureus ), Escherichia coli ( E. coli ), Pseudomonas aeruginosa ( P. aeruginosa During infection, bacteria can form biofilms on wounds or medical device surfaces. Biofilms are complex three-dimensional structures formed by extracellular polymers (EPS) such as polysaccharides, proteins, and nucleic acids secreted by the bacteria themselves. They act as a "natural barrier," effectively blocking the penetration and attack of antibiotics and host immune cells, leading to a hundredfold or even thousandfold increase in the drug resistance of the bacteria inside, thus causing chronic and persistent infections that are difficult to cure.

[0003] In related technologies, transition metal ions (such as copper ions (Cu) that can generate reactive oxygen species (ROS) through Fenton or Fenton-like reactions are used. 2+ ), iron ions (Fe) 2+ / Fe 3+ ), manganese ions (Mn) 2+ ), cobalt ions (Co) 2+ Transition metal ions (TMEs), such as those found in bacteria, have attracted considerable attention due to their highly efficient and broad-spectrum bactericidal capabilities. The bactericidal mechanisms of these ions are multifaceted, primarily including: disrupting the integrity of bacterial cell membranes leading to leakage of contents; catalyzing the generation of reactive oxygen species (ROS) causing oxidative damage to bacterial DNA, proteins, and lipids; and binding to key enzymes within bacteria, inhibiting their metabolism and ultimately leading to bacterial death. However, free transition metal ions face significant obstacles in clinical translational applications. First, at effective bactericidal concentrations, these ions also exhibit significant cytotoxicity to mammalian cells, resulting in a very narrow therapeutic window. Second, in complex biological environments such as body fluids and blood, they are easily inactivated by various biomolecules. More importantly, these transition metal ions struggle to effectively penetrate the dense extracellular polymer matrix of biological membranes, limiting their bactericidal effect on bacteria within mature biofilms.

[0004] To overcome the aforementioned shortcomings, nanotechnology offers a promising solution. By loading these transition metal ions onto nanocarriers, their encapsulation, stabilization, and controlled release can be achieved, aiming to improve bactericidal efficacy while reducing toxic side effects. Currently, the carriers or ligands used in publicly available metal-based nanomaterials mainly fall into two categories: the first category consists of small organic molecules (such as amino acids, citric acid, and polyphenols). The second category comprises polymeric ligands. Synthetic polymers such as polyvinylpyrrolidone (PVP) and polyethylene glycol (PEG) primarily utilize steric hindrance to prevent nanoparticle aggregation and provide good colloidal stability, but they typically lack inherent biological functions and cannot produce synergistic antibacterial effects with metal ions. While natural polymers such as chitosan possess certain biocompatibility and antibacterial activity, their efficacy is limited, and they suffer from problems such as large batch-to-batch variations and difficulty in precisely controlling their properties. Therefore, the development of new antibacterial drugs is crucial. Summary of the Invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention proposes a composite nanoparticle.

[0006] The present invention also provides a method for preparing the above-mentioned composite nanoparticles.

[0007] The present invention also provides applications of the above-mentioned composite nanoparticles.

[0008] The present invention also provides a bactericide comprising the above-described composite nanoparticles.

[0009] A composite nanoparticle according to a first aspect of the present invention comprises coordinated self-assembled ginsenosides and transition metal ions.

[0010] The composite nanoparticles according to embodiments of the present invention have at least the following beneficial effects: Ginsenosides (GS) are the main bioactive components of ginseng, belonging to a class of triterpenoid saponins. They possess high biocompatibility, and their chemical structure is rich in hydroxyl groups (-OH), providing abundant lone pairs of electrons, making them ideal for complexing various transition metal ions (such as Cu). 2+ Fe 3+ Mn 2+ Co 2+Natural biocompatible ligands (such as ginsenosides) are used. Ginsenosides, acting as stabilizers, are combined with transition metal ions capable of catalyzing Fenton or Fenton-like reactions via a one-step self-assembly reaction to obtain composite nanoparticles. The composite nanoparticles of the examples can efficiently kill a variety of bacteria, including Gram-positive and Gram-negative bacteria, through multiple synergistic mechanisms such as disrupting the physical structure of biofilms, catalyzing the generation of large amounts of reactive oxygen species (ROS), and interfering with bacterial metabolism. They exhibit excellent scavenging ability, especially against stubborn biofilms, and have good biocompatibility, showing great application potential.

[0011] According to some embodiments of the present invention, the particle size of the composite nanoparticles is 5 nm to 500 nm. For example, it can be 5 nm, 10 nm, 15 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 150 nm, 200 nm, 250 nm, 300 nm, 350 nm, 400 nm, 450 nm or 500 nm.

[0012] According to some embodiments of the present invention, the ginsenosides include ginsenosides containing three glycosidic groups. Too few or too many glycosidic bonds will result in poor complexation. Furthermore, too few glycosidic bonds will lead to poor stability of the prepared nanoparticles.

[0013] According to some embodiments of the present invention, the ginsenosides include at least one of ginsenoside Re, ginsenoside Rd, and ginsenoside Ro.

[0014] According to some embodiments of the present invention, the transition metal ion includes Cu. 2+ Fe 2+ Fe 3+ Mn 2+ Co 2+ Cr 3+ V 3+ At least one of them.

[0015] The method for preparing composite nanoparticles as described in the first aspect embodiment of the present invention, according to a second aspect embodiment, includes the following steps: A mixture of ginsenoside solution and transition metal ion solution is prepared, and the mixture is reacted to obtain the composite nanoparticles.

[0016] The preparation method according to embodiments of the present invention has at least the following beneficial effects: The preparation method described in this embodiment is mild, easy to operate, and environmentally friendly.

[0017] According to some embodiments of the present invention, the concentration of ginsenosides in the ginsenoside solution is 0.1 mM-100 mM. For example, it can be 0.1 mM, 1 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, or 100 mM.

[0018] According to some embodiments of the present invention, the solvent of the ginsenoside solution includes an aqueous ethanol solution.

[0019] According to some embodiments of the present invention, the volume percentage of ethanol in the aqueous ethanol solution is 5%-95%. For example, it can be 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.

[0020] According to some embodiments of the present invention, the concentration of transition metal ions in the transition metal ion solution is 1 mM-100 mM. For example, it can be 1 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, or 100 mM.

[0021] According to some embodiments of the present invention, the solvent of the transition metal ion solution includes water.

[0022] According to some embodiments of the present invention, the transition metal ions in the transition metal ion solution are derived from soluble transition metal salts. These transition metal ions are capable of catalyzing the Fenton reaction or a Fenton-like reaction.

[0023] According to some embodiments of the present invention, the transition metal ions in the transition metal ion solution include Cu. 2+ Fe 2+ Fe 3+ Mn 2+ Co 2+ Cr 3+ V 3+ At least one of them.

[0024] According to some embodiments of the present invention, the Cu 2+ The sources include, but are not limited to, at least one of copper chloride, copper sulfate, copper nitrate, and copper acetate.

[0025] According to some embodiments of the present invention, the Fe 2+ The sources include, but are not limited to, at least one of ferrous sulfate, ferrous chloride, ferrous nitrate, and ferrous acetate.

[0026] According to some embodiments of the present invention, the Fe 3+ The sources include, but are not limited to, at least one of ferric sulfate, ferric nitrate, ferric chloride, and ferric acetate.

[0027] According to some embodiments of the present invention, the Mn 2+ The sources include, but are not limited to, at least one of manganese sulfate, manganese chloride, manganese acetate, and manganese nitrate.

[0028] According to some embodiments of the present invention, the Co 2+ The sources include, but are not limited to, at least one of cobalt sulfate, cobalt nitrate, and cobalt chloride.

[0029] According to some embodiments of the present invention, the Cr³ + The sources include, but are not limited to, at least one of chromium sulfate, chromium nitrate, and chromium chloride.

[0030] According to some embodiments of the present invention, the V³ + The sources include, but are not limited to, at least one of vanadium sulfate, vanadium nitrate, and vanadium chloride.

[0031] According to some embodiments of the present invention, the molar ratio of the ginsenoside to the transition metal ion is (0.1-10):1. For example, it can be 0.1:1, 0.5:1, 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, 5.5:1, 6:1, 6.5:1, 7:1, 7.5:1, 8:1, 8.5:1, 9:1, 9.5:1, or 10:1.

[0032] According to some embodiments of the present invention, the reaction time is 0.5 h to 24 h. For example, it can be 0.5 h, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, 12 h, 13 h, 14 h, 15 h, 16 h, 17 h, 18 h, 19 h, 20 h, 21 h, 22 h, 23 h, or 24 h.

[0033] According to some embodiments of the present invention, the pH of the reaction is a weakly acidic environment or an alkaline environment.

[0034] According to some embodiments of the present invention, the pH of the reaction is 6.5-9. For example, it can be 6.5, 7, 7.5, 8, 8.5 or 9.

[0035] According to some embodiments of the present invention, the reaction temperature is 30°C-70°C. For example, it can be 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, or 70°C.

[0036] According to some embodiments of the present invention, the preparation method further includes purification after the reaction.

[0037] According to some embodiments of the present invention, the purification process includes at least one of solid phase separation and washing.

[0038] According to some embodiments of the present invention, the separation of the solid phase includes, but is not limited to, centrifugation, ultrafiltration, or dialysis.

[0039] According to some embodiments of the present invention, the washing includes washing with ethanol.

[0040] Application of the composite nanoparticles as described in the first aspect embodiment of the present invention in any one of A1) to A4): A1) Prepare products that inhibit and / or kill bacteria; A2) Prepare products that disrupt and / or inhibit biofilm formation; A3) Prepare products that induce the generation of reactive oxygen species; A4) Prepare products that interfere with bacterial metabolism.

[0041] According to some embodiments of the present invention, the product includes at least one of pharmaceuticals, reagents, reagent kits, protective equipment, and daily chemical products.

[0042] According to some embodiments of the present invention, the bacteria include at least one of Gram-negative bacteria and Gram-positive bacteria.

[0043] According to some embodiments of the present invention, the Gram-negative bacteria include at least one of Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, and Salmonella.

[0044] According to some embodiments of the present invention, the resistance to Gram-positive bacteria includes at least one of Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, and Bacillus subtilis.

[0045] According to some embodiments of the present invention, the amount of the composite nanoparticles used, based on copper concentration, is 1.25 µg / mL to 80 µg / mL. For example, it can be 1.25 µg / mL, 5 µg / mL, 10 µg / mL, 15 µg / mL, 20 µg / mL, 25 µg / mL, 30 µg / mL, 35 µg / mL, 40 µg / mL, 45 µg / mL, 50 µg / mL, 55 µg / mL, 60 µg / mL, 65 µg / mL, 70 µg / mL, 75 µg / mL, or 80 µg / mL.

[0046] An antibacterial agent according to a fourth aspect of the present invention comprises composite nanoparticles as described in the first aspect of the present invention. Since the antibacterial agent employs all the technical solutions of the composite nanoparticles described in the above embodiments, it possesses at least all the beneficial effects brought about by the technical solutions of the above embodiments.

[0047] Other features and advantages of the invention will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the invention. Attached Figure Description

[0048] Figure 1 Characterization of GSR NPs and in vitro ROS generation mechanism. (A) Hydrated particle size and transmission electron microscopy image of GSR NPs (scale bar: 10 nm). (B) UV-Vis spectra of CuCl2, GS, and GSR NPs. (C) Infrared spectra of GS and GSR NPs. (D) Cumulative release curve of GS in PBS (pH 7.4) or PBS (pH 7.4) with 5 mM GSH added. (E) Cumulative release curve of copper in PBS (pH 7.4) or PBS (pH 7.4) with 5 mM GSH added. (F) Concentration-dependent •OH generation (methylene blue MB bleaching) ability of GSR NPs. (G) GSH-enhanced ROS generation: The MB probe bleaching rate increases with increasing GSH concentration. (H) GSH consumption curve over time after co-incubation with GSR nanoparticles. (I) Dose-dependent consumption of GSH after co-incubation with different concentrations of GSR nanoparticles. Data are expressed as mean ± standard deviation (n = 3).

[0049] Figure 2 The dose-dependent antibacterial activity of different formulations against Staphylococcus aureus is shown. (A) CuCl2. (B) Free GS. (C) Physical mixture of GS and CuCl2. (D) GSR ​​NPs.

[0050] Figure 3The dose-dependent antibacterial activity of different formulations against Escherichia coli is shown. (A) CuCl2. (B) Free GS. (C) Physical mixture of GS and CuCl2. (D) GSR ​​NPs.

[0051] Figure 4 This study illustrates the dose-dependent antibacterial activity of different formulations against Staphylococcus aureus and Escherichia coli. (A) Representative photographs of agar plates treated with different concentrations of CuCl2. (B) Quantitative analysis of the inhibition rate corresponding to Figure A. (C) Representative photographs of agar plates treated with different concentrations of free glucose solution (GS). (D) Quantitative analysis of the inhibition rate corresponding to Figure C. (E) Representative photographs of agar plates treated with physical mixtures of GS and CuCl2 at different concentrations. (F) Quantitative analysis of the inhibition rate corresponding to Figure E.

[0052] Figure 5 This study investigated the broad-spectrum antibacterial activity and mechanism of GSR NPs against Staphylococcus aureus and Escherichia coli in vitro. (A) Representative photographs of agar plates treated with different concentrations of GSR NPs. (B) Quantitative analysis of the inhibition rate corresponding to Figure A. (C) Representative photographs of agar plates treated with 10 µg / mL GSR NPs for different times. (D) Quantitative analysis of the inhibition rate corresponding to Figure C. (E) Flow cytometry analysis to assess bacterial membrane integrity. (F) Quantitative calculation of the percentage of dead bacteria based on the flow cytometry data in (E). Data are expressed as mean ± standard deviation (n = 3).

[0053] Figure 6 This study investigates the synergistic anti-biofilm effect of GSR NPs against Staphylococcus aureus and Escherichia coli. (A) SEM images showing morphological changes of Staphylococcus aureus and Escherichia coli after 4 h of treatment with different samples. (B) Quantitative assessment of biofilm removal effect. (C) Quantitative assessment of biofilm inhibition effect.

[0054] Figure 7 This study investigated the antibacterial mechanism and in vitro biocompatibility of GSR NPs. (A) Intracellular ROS levels in Staphylococcus aureus after treatment with different formulations were detected using DCFH fluorescence intensity. (B) Quantitative analysis of intracellular low molecular weight thiols (LMW thiols) consumption in Staphylococcus aureus after different treatments. (C) Evaluation of the effects of each treatment group on the metabolic activity of Staphylococcus aureus. (D) Intracellular ROS levels in Escherichia coli after treatment with different formulations were detected using DCFH fluorescence intensity. (E) Quantitative analysis of intracellular low molecular weight thiols (LMW thiols) consumption in Escherichia coli after different treatments. (F) Evaluation of the effects of each treatment group on the metabolic activity of Escherichia coli. (G) GSR ​​NPs hemolysis assay. (H) Survival rate of NIH3T3 cells under different treatment conditions. Data are expressed as mean ± standard deviation (n = 3). Detailed Implementation

[0055] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0056] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0057] When a numerical range is disclosed herein, the range is considered continuous and includes the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when the range refers to integers, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe a feature or characteristic, the ranges may be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are incorporated.

[0058] "And / or" is used to indicate that one or both of the described situations may occur, for example, A and / or B includes (A and B) and (A or B).

[0059] Unless otherwise specified, "about" in this invention means that the allowable error is within ±2%.

[0060] Unless otherwise specified, "room temperature" in this invention means (25±5)℃.

[0061] Example 1: This example provides a ginsenoside Re-copper complex nano-antibacterial agent (GSR NPs), prepared by the following method: (1) Accurately weigh 94.7 mg of ginsenoside Re (GS, molecular weight approximately 947 g / mol), dissolve it in 10 mL of ethanol aqueous solution (deionized water and ethanol are mixed in a volume ratio of 3:1), and sonicate for 5 minutes until completely dissolved to obtain a ginsenoside Re solution with a concentration of 10 mM.

[0062] (2) Accurately weigh 4.26 mg of copper chloride dihydrate (CuCl2·2H2O, molecular weight approximately 170.5 g / mol), dissolve it in 1 mL of deionized water to obtain a copper chloride solution with a concentration of 25 mM.

[0063] (3) Under the condition of continuous stirring on a magnetic stirrer (500 rpm) at 50℃ water bath, 1 mL of copper chloride solution prepared in step (2) was slowly added to 10 mL of ginsenoside Re solution prepared in step 1 (the molar ratio of ginsenoside Re to copper ions is 4:1). The pH of the mixed solution was adjusted to 8.0 with 0.1 M sodium hydroxide aqueous solution, and the reaction was stirred in the dark for 4 hours. It was observed that the solution gradually changed from colorless and transparent to a milky blue-green color, indicating that a suspension of ginsenoside-copper complex nano-antibacterial agent was formed.

[0064] (4) Centrifuge the suspension obtained in step (3) at 5000 rpm for 5 minutes, remove the supernatant, and resuspend the precipitate in ethanol; repeat 3 times to fully remove unreacted ginsenoside small molecules and copper ions. Finally, disperse the precipitate in ethanol to obtain a blue-green ethanol suspension, and store it in a sealed container at 4°C in the dark for later use.

[0065] After centrifugation, the precipitate was resuspended in physiological saline, resulting in a homogeneous blue-green suspension with an opalescent appearance and a pronounced Tyndall effect. Preliminary DLS characterization showed a particle size of approximately 10.6 nm and a PDI of 0.28. Analysis revealed that the mass of GS in the GSR NPs was approximately five times that of Cu.

[0066] Example 2 This example provides a ginsenoside Re-iron complex nano-antibacterial agent, the preparation method of which is basically the same as that in Example 1, except that the 25 mM copper chloride solution is replaced with a 25 mM ferric chloride solution.

[0067] The preparation method of 25 mM ferric chloride solution is as follows: Accurately weigh 6.76 mg of ferric chloride hexahydrate (FeCl3·6H2O, molecular weight approximately 270.3 g / mol), dissolve it in 1 mL of deionized water to obtain a ferric chloride solution with a concentration of 25 mM.

[0068] When ferric chloride solution is added to ginsenoside solution, the solution color changes to yellow or brownish-yellow, and a significant Tyndall effect is observed, indicating the formation of ginsenoside-iron complex. After purification in step (4), a brownish-yellow nanocomplex ethanol suspension is obtained.

[0069] After centrifugation, the brownish-yellow nanocomplex ethanol suspension was resuspended in physiological saline, resulting in a homogeneous brownish-yellow suspension with an opalescent appearance and a pronounced Tyndall effect. Preliminary characterization by DLS showed that the particle size was approximately 45.8 nm and the PDI was 0.18.

[0070] Example 3 This example provides a ginsenoside Re-copper complex nano-antibacterial agent, the preparation method of which is basically the same as that in Example 1, except that the 25 mM copper chloride solution is replaced with a 25 mM copper sulfate solution.

[0071] The preparation method of 25 mM copper sulfate solution is as follows: Accurately weigh 6.24 mg of copper sulfate pentahydrate (CuSO4·5H2O, molecular weight approximately 249.7 g / mol), dissolve it in 1 mL of deionized water to obtain a copper sulfate solution with a concentration of 25 mM.

[0072] Adding copper sulfate solution to ginsenoside solution yielded a blue-green ethanol suspension. After centrifugation, the precipitate was resuspended in physiological saline, resulting in a homogeneous blue-green suspension with an opalescent sheen and a pronounced Tyndall effect. Preliminary DLS characterization showed a particle size of approximately 11.4 nm and a PDI of 0.29.

[0073] Example 4: This example provides a ginsenoside Rd-copper complex nano-antibacterial agent, the preparation method of which is basically the same as that in Example 1, except that ginsenoside Re is replaced with an equimolar amount of ginsenoside Rd (GS-Rd, molecular weight approximately 947 g / mol).

[0074] Adding copper chloride solution to ginsenoside solution yielded a blue-green ethanol suspension. After centrifugation, the precipitate was resuspended in physiological saline, resulting in a homogeneous blue-green suspension with an opalescent sheen and a pronounced Tyndall effect. Preliminary DLS characterization showed a particle size of approximately 15.6 nm and a PDI of 0.26.

[0075] Example 5 This example provides a ginsenoside Re-copper complex nano-antibacterial agent, the preparation method of which is basically the same as that of Example 1, except that the pH in step (3) is replaced by 6.5 instead of 8.

[0076] Preliminary characterization by DLS revealed the formation of nanoparticles with an average particle size of approximately 10 nm, which showed no significant difference from the product of the reaction at pH 8.0.

[0077] Example 6 This example provides a ginsenoside Re-copper complex nano-antibacterial agent, the preparation method of which is basically the same as that of Example 1, except that the pH in step (3) is replaced by 9.0 instead of 8.

[0078] Preliminary characterization by DLS revealed the formation of nanoparticles with an average particle size of approximately 10 nm, which showed no significant difference from the product of the reaction at pH 8.0.

[0079] Example 7 This example provides a ginsenoside Re-copper complex nano-antibacterial agent, the preparation method of which is basically the same as that of Example 1, except that the reaction temperature in step (3) is replaced by 30°C instead of 50°C.

[0080] Preliminary characterization by DLS revealed the formation of nanoparticles with an average particle size of approximately 10 nm, which showed no significant difference from the products obtained from the reaction at 50 °C.

[0081] Example 8 This example provides a ginsenoside Re-copper complex nano-antibacterial agent. The preparation method is basically the same as that in Example 1, except that the reaction temperature in step (3) is replaced by 60°C (water bath temperature) instead of 50°C.

[0082] Preliminary characterization by DLS revealed the formation of nanoparticles with an average particle size of approximately 11 nm, which showed no significant difference from the products obtained from the reaction at 50 °C.

[0083] Example 9: This example provides a ginsenoside Re-copper complex nano-antibacterial agent, the preparation method of which is basically the same as that of Example 1, except that the reaction time of step (3) is replaced by 1 h instead of 4 h.

[0084] The blue-green color of the reaction solution was not obvious. Preliminary characterization by DLS revealed nanoparticles with an average particle size of approximately 10 nm, but there may be a small amount of unreacted raw materials.

[0085] Example 10: This example provides a ginsenoside Re-copper complex nano-antibacterial agent, the preparation method of which is basically the same as that of Example 1, except that the reaction time of step (3) is replaced by 12 h instead of 4 h.

[0086] Preliminary characterization by DLS revealed nanoparticles with an average particle size of approximately 11 nm, which showed no significant difference from the product after 4 hours of reaction.

[0087] Detection Example 1 The GSR NPs ethanol suspension prepared in Example 1 was centrifuged and then resuspended in physiological saline to obtain a GSR NPs suspension, which was then characterized as follows.

[0088] 1. Morphology and particle size: A small amount of GSR NPs suspension was dropped onto a copper grid and allowed to air dry naturally. The hydrated particle size and polydispersity index (PDI) were then observed by transmission electron microscopy (TEM) and measured by dynamic light scattering (DLS).

[0089] The results are as follows Figure 1As shown in Figure A, GSR NPs exhibit a uniform, near-spherical nanostructure with good dispersibility and no obvious agglomeration. DLS results show that the average hydrated particle size of GSR NPs in aqueous solution is 10.6 nm, and the PDI value is 0.28, indicating that their particle size is uniform in aqueous solution.

[0090] 2. Coordination structure characterization: CuCl2·2H2O and ginsenoside Re (GS) were dissolved in appropriate amounts of physiological saline, and the suspensions of CuCl2·2H2O, GS, and GSR NPs were scanned with a UV spectrophotometer. The precipitate of the ethanol suspension of GSR NPs was collected after centrifugation, dried, and its infrared spectrum was measured.

[0091] like Figure 1 As shown in Figure B, after the reaction, the UV absorption peak of copper ions shifted from 883 nm to 653 nm, indicating that the chemical environment of copper ions changed, most likely due to coordination.

[0092] like Figure 1 As shown in Figure C. Compared with the infrared spectrum of GS, at 500 cm⁻¹ -1 The presence of typical Cu-O coordination bonds indicates that copper ions in GSR NPs have coordinated with GS.

[0093] 3. Reduction-responsive release: Drug release was simulated using dialysis. The GSR NPs suspension was placed in a dialysis bag and immersed in PBS buffer (pH 7.4, simulating a normal physiological environment) and PBS buffer containing 5 mM glutathione (GSH), simulating a bacterial intracellular reducing environment, respectively. The mixture was shaken at 37°C, and the dialysis fluid was collected periodically. The cumulative release of copper ions was determined by atomic absorption spectrometry, and the cumulative release of ginsenoside Re was determined by ultraviolet spectrophotometry. The cumulative release rate was calculated.

[0094] like Figure 1 Figures D and E are shown. In PBS buffer, the cumulative release rate of copper and ginsenoside Re was less than 20% within 24 hours; while in GSH solution, copper ions and ginsenoside Re achieved a rapid release of over 80% within 4 hours. These results confirm that GSR NPs possess the characteristics of intelligent response and rapid release of active ingredients within the bacterial microenvironment.

[0095] 4. In vitro ROS generation and GSH consumption: The hydroxyl radicals (•OH) generated in the system were quantified using the methylene blue (MB) bleaching method. The principle is that •OH degrades MB, leading to a decrease in its characteristic absorbance at 665 nm; the decrease in absorbance is related to the amount of •OH generated. The specific steps are as follows: 0, 2.5, 5, 10, and 20 μg / mL of GSR NPs (calculated as copper) were mixed with 5 mM glutathione (GSH) in phosphate-buffered saline (PBS); separately, 10 μg / mL of GSR NPs (calculated as copper) was mixed with 0, 0.01, 0.05, 0.1, 1, and 5 mM glutathione (GSH) in phosphate-buffered saline (PBS). Then, MB solution and H₂O₂ solution were added to initiate the reaction. After the reaction system was allowed to stand for a period of time in the dark, the change in absorbance at 665 nm was measured using a UV-Vis spectrophotometer to evaluate the generation efficiency of •OH.

[0096] The consumption of GSH was directly determined using the Ellman reagent method. 5,5'-Dithiobis(2-nitrobenzoic acid) (DTNB) reacts with free GSH to generate a yellow product with characteristic absorption at 412 nm. The more GSH is consumed by the nanoparticles in the system, the less yellow product is generated and the lower the absorbance. The specific steps are as follows: 0, 2.5, 5, 10, and 20 μg / mL of GSR NPs (calculated as copper) were mixed with 5 mM GSH in phosphate-buffered saline (PBS), and DTNB was added; 5 mM GSH was mixed with 10 µg / mL of GSR NPs (calculated as copper). Samples were taken at different time points (0-60 minutes) and DTNB was added. Incubation was continued for 30 minutes, and the residual GSH was determined by measuring the absorbance at 412 nm.

[0097] like Figure 1 The F-plot (with a fixed GSH concentration of 5 mM) and G-plot (with a fixed copper ion concentration of 10 µg / mL) are shown. Control experiments showed that neither GSR NPs alone nor GSH alone induced much MB oxidation. However, MB oxidation was significantly enhanced in a concentration-dependent manner in the presence of both GSR NPs and GSH, confirming robust •OH generation. This sustained free radical generation implies a GSH-driven catalytic redox cycle.

[0098] like Figure 1 As shown in the H and I figures, the presence of GSR NPs leads to a rapid and significant reduction in GSH over time, confirming that GSH is continuously consumed to regenerate catalytically active Cu. + .

[0099] The antibacterial mechanism of GSR NPs is achieved through a synergistic dual attack. Once inside the bacteria, the nanoparticles are triggered by intracellular GSH, initiating a catalytic cycle that both generates lethal ROS and depletes the cell's own antioxidant defenses. This simultaneous generation of oxidative stress and the collapse of the defense system leads to rapid bacterial death.

[0100] Detection Example 2 The antibacterial effect of the GSR NPs prepared in Example 1: In order to verify and further characterize the antibacterial activity of the preparation, a standard colony counting experiment was performed.

[0101] 1. At 37℃, the final concentration is 2×10⁻⁶. 6 CFU / mL of Staphylococcus aureus ( S. aureus ) or E. coli ( E. coli The samples were incubated with different samples for 16 h. Group A consisted of 0, 2.5, 5, 10, 20, 40, or 80 μg / mL copper chloride (concentration expressed as copper ions). Group B consisted of 0, 12.5, 25, 50, 100, 200, or 400 μg / mL glucose sulfide (GS). Group C consisted of a mixture of 0, 2.5, 5, 10, 20, 40, or 80 μg / mL copper chloride and GS. Group D consisted of 0, 2.5, 5, 10, 20, 40, or 80 μg / mL GSR NPs (concentration expressed as Cu). After incubation, 50 µL of each sample (CuCl2, GS, GS & CuCl2, GSR NPs) was plated on agar plates and incubated at 37°C until single colonies appeared, followed by visual inspection.

[0102] like Figure 2 as well as Figure 3 As shown. Based on the preliminary cell viability screening results, concentrations of 5, 10, and 20 µg Cu / mL, which represent the key transition range from moderate to high bactericidal activity of GSR NPs, were selected for subsequent experiments.

[0103] like Figure 4 and Figure 5 Figures A and B are shown. All treatment groups showed a dose-dependent decrease in colony-forming units (CFUs). At a treatment concentration of 10 µg Cu / mL, GSR NPs reduced the number of Staphylococcus aureus and Escherichia coli colonies by more than 90%, significantly outperforming equivalent concentrations of copper chloride (CuCl2), ginsenosides (GS), and physical mixtures of both.

[0104] 2. At 37℃, the final concentration is 2×10⁻⁶. 6Staphylococcus aureus or Escherichia coli CFU / mL were incubated with 10 µg Cu / mL GSR NPs for 0, 0.5, 2, and 4 h, respectively. After incubation, 50 µL of sample was plated on agar plates and incubated at 37°C until single colonies appeared, which were then visually inspected. Samples after 4 h of incubation were analyzed by flow cytometry using SYTO 9 (live bacteria dye) and PI (dead bacteria dye) to assess bacterial membrane integrity and calculate bacterial mortality. Scanning electron microscopy (SEM) was used to observe ultrastructural changes in the treated bacteria.

[0105] like Figure 5 As shown in the CD diagram. GSR NPs have a rapid bactericidal effect; a sharp reduction in visible colonies can be observed after only 2 hours of incubation, and almost complete elimination is achieved within 4 hours.

[0106] like Figure 5 The EF plots are shown. GS alone had almost no effect. CuCl2 induced moderate membrane damage, resulting in approximately 30% of the bacterial population becoming PI-positive. A physical mixture of GS and CuCl2 led to a significantly higher proportion of dead cells (>50%), confirming a synergistic effect between the two, i.e., GS enhances the membrane-disrupting activity of copper. GSR NPs, on the other hand, caused a large-scale loss of membrane integrity, with over 90% of the bacteria becoming PI-positive after 4 hours of treatment. This is highly consistent with the plate count results.

[0107] like Figure 6 Figure A shows the results. The control group of *E. coli* and *Staphylococcus aureus* exhibited smooth, intact cell membranes. Bacteria treated with GSR NPs showed severe morphological damage, including extensive membrane shrinkage and rupture. This effect followed a clear hierarchical pattern: GS or CuCl2 alone caused almost no damage, their physical mixture induced moderate synergistic damage, but the destructive power of GSR NPs was significantly increased, confirming that nanoparticle structures can maximize this destructive capacity.

[0108] 3. (1) Membrane damage experiment: at 37℃, the final concentration was 2×10 6 After inoculating 96-well plates with CFU / mL Staphylococcus aureus or Escherichia coli and culturing for 24 h, centrifugation was performed, the supernatant was discarded, and different samples were added (Group A: 10 μg / mL copper chloride (concentration based on copper ions); Group B: 50 μg / mL glucose sulfoxide (GS); Group C: a mixture of 10 μg / mL copper chloride and 50 μg / mL GS; Group D: 10 μg / mL GSR NPs (concentration based on Cu)). After further culturing for 24 h, crystal violet staining was performed, and OD was measured using a microplate reader. 570 value.

[0109] (2) Membrane inhibition experiment: at 37℃, the final concentration was 2×10 6 Staphylococcus aureus or Escherichia coli at CFU / mL were cultured in 96-well plates with different samples (Group A: 10 μg / mL copper chloride (concentration as copper ions); Group B: 50 μg / mL glucose sulfoxide (GS); Group C: a mixture of 10 μg / mL copper chloride and 50 μg / mL GS; Group D: 10 μg / mL GSR NPs (concentration as Cu)) for 24 h. After crystal violet staining, OD was measured using a microplate reader. 570 value.

[0110] like Figure 6 As shown in the BC diagram, GSR NPs effectively disrupted established mature biofilms and inhibited the formation of new biofilms. Compared with the untreated control group, GSR NPs significantly reduced biofilm biomass (OD). 570 The biomass of the biofilm decreased by approximately 80-90%, far superior to other groups. Among them, GS or CuCl2 had minimal impact on biofilm biomass.

[0111] In summary, GSR NPs exhibit superior, broad-spectrum, and concentration- and time-dependent bactericidal activity. Their exceptional antibacterial efficacy stems primarily from their powerful and synergistic ability to disrupt bacterial membrane integrity, as well as their excellent capacity to disrupt and inhibit biofilm formation.

[0112] Detection Example 3 1. At 37℃, the final concentration is 2×10⁻⁶. 6 CFU / mL of Staphylococcus aureus or Escherichia coli were incubated with different samples (Group A: 10 μg / mL copper chloride (concentration as copper ions); Group B: 50 μg / mL glucose sulfoxide (GS); Group C: a mixture of 10 μg / mL copper chloride and 50 μg / mL GS; Group D: 10 μg / mL GSR NPs (concentration as Cu)) for 4 h. The ROS levels in Staphylococcus aureus and Escherichia coli were detected using the DCFH-DA probe. Thiol depletion was determined using Ellman's assay. Metabolic activity was determined using the Alamar Blue reduction assay.

[0113] like Figure 7Figures A and D are shown. GSR NPs induced a dramatic increase in ROS production, with fluorescence intensity increasing approximately 4-fold in Staphylococcus aureus and approximately 3-fold in Escherichia coli compared to the untreated control group. This effect was far stronger than any other treatment group. While CuCl2 alone caused a moderate increase in ROS, and GS alone had almost no effect, their physical mixture (GS & CuCl2) produced a synergistic ROS growth, confirming the cooperative interaction between the components. Nevertheless, the ROS burst triggered by GSR NPs was even more potent than this synergistic mixture, highlighting its unique "nanoparticle advantage."

[0114] like Figure 7 As shown in Figures B and E, GSR NPs led to catastrophic depletion of the cellular thiol pool, with losses approaching 40% in Staphylococcus aureus and exceeding 50% in Escherichia coli. This profound depletion indicates that the aggressive attack of ROS completely overwhelmed the bacteria's detoxification capacity. Similarly, GSR NPs were far more effective than synergistic physical mixtures, which in turn were more effective than their individual components.

[0115] like Figure 7 As shown in Figures C and F, the massive production of ROS destroys the cell's antioxidant shield, ultimately leading to a profound loss of metabolic activity. After treatment with GSR NPs, the metabolic activity of Staphylococcus aureus plummeted to less than 20%, and that of Escherichia coli decreased to approximately 30%. This represents a near-complete shutdown of cellular function, consistent with previously observed bactericidal effects.

[0116] Detection Example 4 1. Blood compatibility: Red blood cells (RBCs) were incubated with different concentrations (2.5, 10, 40 μg / mL) of GSR NPs at 37°C for 2 h. After centrifugation, the color of the supernatant was observed and its absorbance at 540 nm was measured.

[0117] like Figure 7 As shown in the G-plot, GSR NPs exhibit excellent blood compatibility. Observations revealed that the supernatant from the GSR NPs-treated group was clear and transparent, similar to the PBS control group, indicating that the red blood cells remained intact; while the deionized water-treated group appeared bright red due to the release of hemoglobin. Even at the highest concentration of 40 μg / mL, the hemolysis induced by these nanoparticles remained below 5%, a level comparable to the negative control group (PBS). The positive control group (deionized water) resulted in complete hemolysis of red blood cells.

[0118] 2. Cytotoxicity evaluation: At 37℃, different samples were tested (Group a: no treatment drug added; Group b: 1.25 µg Cu / mL copper chloride or 6.25 µg / mL glucose syrup or 1.25 µg Cu / mL copper chloride + 6.25 µg / mL glucose syrup or 1.25 µg Cu / mL GSR NPs; Group c: 2.5 µg Cu / mL copper chloride or 12.5 µg / mL glucose syrup or 2.5 µg Cu / mL copper chloride + 12.5 µg / mL glucose syrup or 2.5 µg Cu / mL GSR NPs; Group d: 5 µg Cu / mL copper chloride or 25 µg / mL glucose syrup or 5 µg Cu / mL copper chloride + 25 µg / mL glucose syrup or 5 µg Cu / mL GSR NPs; Group e: 10 µg Cu / mL copper chloride or 50 µg / mL glucose syrup or 10 µg Cu / mL copper chloride + 50 µg Cu / mL GSR NPs). Group f: 20 µg / mL copper chloride or 100 µg / mL copper chloride or 20 µg / mL copper chloride + 100 µg / mL copper chloride or 20 µg / mL copper chloride) with 1×10 4 After co-incubation with the mammalian fibroblast cell line (NIH3T3) for 24 h, cell viability was measured by the MTT assay.

[0119] like Figure 7 The H-plot is shown. GSR NPs exhibit excellent biocompatibility. Even at a concentration of 20 µg Cu / mL (twice the concentration required for potent bactericidal activity), cell viability remained close to 100%, indicating negligible cytotoxicity. The GS component also showed low cytotoxicity. However, free CuCl2 exhibited significant dose-dependent toxicity, with cell viability dropping below 40% at a concentration of 40 µg / mL; the physical mixture of GS and CuCl2 also showed toxicity comparable to CuCl2 alone. This suggests that although free copper is inherently toxic to mammalian cells, this nanoformulation effectively chelates copper, thereby protecting host cells from its cytotoxic effects.

[0120] In summary, the preparation method of this invention is applicable not only to copper ions but also to other metal ions with Fenton activity, such as iron ions, and to a variety of ginsenosides, demonstrating its universality. The preparation method of this invention is insensitive to pH, temperature, and time, and can successfully prepare the target product within a wide pH range (at least pH 6.5 to pH 9.0), a wide temperature range (at least 30°C to 60°C), and a wide time range (at least 1 h to 12 h). GSR NPs are not merely simple carriers of copper; their nanostructure facilitates a devastatingly effective synergistic attack. Upon entering bacteria, GSR NPs catalyze a lethal ROS burst through a Fenton-like reaction, simultaneously triggering a massive consumption of low-molecular-weight thiols, thereby effectively neutralizing the cell's main defense mechanisms. This vicious cycle of amplified oxidative stress and depleted antioxidant capacity leads to rapid and irreversible cell damage. Furthermore, it is very safe for mammalian cells at therapeutically relevant concentrations and does not cause hemolysis. This high degree of selective toxicity strongly supports GSR NPs as a promising and safe candidate drug with great potential for future preclinical and clinical applications in the treatment of bacterial infections.

[0121] The embodiments of the present invention have been described in detail above with reference to the examples. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention.

Claims

1. A composite nanoparticle, characterized in that, The complex nanoparticles comprise a coordination self-assembly of ginsenoside and transition metal ions.

2. The composite nanoparticle of claim 1, wherein, The ginsenoside includes at least one of ginsenoside Re, ginsenoside Rd, and ginsenoside Ro; and / or, the transition metal ion includes at least one of Cu 2+ , Fe 2+ , Fe 3+ , Mn 2+ , Co 2+ , Cr 3+ , V 3+ .

3. The method of producing the composite nanoparticle according to claim 1 or 2, characterized by, The method comprises the following steps: Preparation of a mixture of a ginsenoside solution and a transition metal ion solution, reaction, and thus the complex nanoparticles are obtained.

4. The production method according to claim 3, characterized by, The concentration of the ginsenoside in the ginsenoside solution is 0.1 mM-100 mM; and / or, the solvent of the ginsenoside solution comprises an aqueous ethanol solution.

5. The preparation method according to claim 3, characterized in that, a concentration of transition metal ions in the transition metal ion solution is 0.1 mM-100 mM; and / or, a solvent of the transition metal ion solution comprises water; and / or, the transition metal ions in the transition metal ion solution comprise at least one of Cu 2+ , Fe 2+ , Fe 3+ , Mn 2+ , Co 2+ , Cr 3+ , V 3+ .

6. The preparation method according to claim 5, characterized in that, The source of the Cu 2+ includes at least one of copper chloride, copper sulfate, copper nitrate, copper acetate; and / or, the source of the Fe 2+ includes at least one of ferrous sulfate, ferrous chloride, ferrous nitrate, ferrous acetate; and / or, the source of the Fe 3+ includes at least one of ferric sulfate, ferric nitrate, ferric chloride, ferric acetate; and / or, the source of the Mn 2+ includes at least one of manganese sulfate, manganese chloride, manganese acetate, manganese nitrate; and / or, the source of the Co 2+ includes at least one of cobalt sulfate, cobalt nitrate, cobalt chloride, cobalt nitrate; and / or, the source of the Cr 3+ includes at least one of chromium sulfate, chromium nitrate, chromium chloride; and / or, the source of the V 3+ includes at least one of vanadium sulfate, vanadium nitrate, vanadium chloride.

7. The preparation method according to claim 3, characterized in that, The molar ratio of the ginsenoside to the transition metal ion is (0.1-10): 1; and / or, the reaction time is 0.5 h-24 h; and / or, the pH of the reaction is 6.5-9; and / or, the temperature of the reaction is 30℃-70℃.

8. Use of the complex nanoparticles of claim 1 or 2 in any one of A1) to A4): A1) preparation of a product for inhibiting and / or killing bacteria; A2) preparation of a product for destroying and / or inhibiting the formation of biofilm; A3) preparation of a product for inducing the production of reactive oxygen species; A4) preparation of a product for interfering with bacterial metabolism.

9. Use according to claim 8, characterized in that, The bacteria comprise at least one of gram-negative bacteria and gram-positive bacteria.

10. An antibacterial agent, characterized in that, The method comprises the complex nanoparticles of claim 1 or 2.

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