Nitric oxide producing fullerene-silver nanoparticle and preparation method and application thereof

Abstract

The application discloses a kind of fullerene composite silver nanoparticles of nitric oxide production and preparation method, application thereof, the fullerene composite silver nanoparticles Arg-C60@AgNPs of nitric oxide production with silver nanoparticle as core, silver nano core has been adsorbed with fullerene outside, and the surface of fullerene is modified with L-arginine.Its preparation method includes: slowly droping silver salt to hydrophilic fullerene water suspension, stirring at room temperature, dialysis, to obtain C60@AgNPs complex;Activation hydrophilic fullerene or L-arginine functional group, L-arginine is modified to C60@AgNPs surface by chemical bond, and reaction solution is dialyzed and purified, to obtain fullerene composite silver nanoparticles Arg-C60@AgNPs.Compared with other fullerene hybrid silver composite, Arg-C60@AgNPs is evenly distributed, and water dispersibility is good, silver nanoparticle is combined with antibacterial photodynamic and gas therapy, has the advantages of good broad-spectrum antibacterial effect, strong biofilm penetration ability and drug resistance, and can promote fibroblast and collagen proliferation, accelerate wound healing.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and particularly relates to a nitric oxide-producing fullerene composite silver nanoparticle and a preparation method and application thereof. BACKGROUND

[0002] Bacterial infection is recognized as a major cause of damage to global health, and gradually becomes the second leading cause of death worldwide. The emergence of multi-drug resistant bacteria (such as methicillin-resistant Staphylococcus aureus MRSA) makes the number of patients increase year by year, and becomes the primary pathogenic microorganism that seriously threatens human health. For the treatment of common subcutaneous abscess, not only the infection should be controlled, but also the wound should be healed quickly to shorten the healing time and avoid long-term nursing and secondary infection. The insufficient blood supply at the abscess site leads to low antibiotic concentration, few choices of broad-spectrum antibiotics and anti-drug resistant bacteria drugs, and bacterial biofilm formed by extracellular polymeric substance (EPS) secreted by bacteria, which not only becomes a penetration barrier for antibiotics, but also changes the phenotype of bacteria in it, inducing drug resistance.

[0003] Antibacterial photodynamic therapy (aPDT) is a treatment method for controlling multi-drug resistant bacteria, which is composed of light, photosensitizer and oxygen molecules. The photosensitizer is excited by a specific wavelength of light to transfer energy to the surrounding oxygen to generate strong active singlet oxygen, which causes photooxidation of certain pyrimidine and purine bases in amino acids, DNA / RNA, and unsaturated lipids, resulting in DNA damage and / or cell membrane damage, leading to membrane lipid damage and bacterial death. It has the advantages of not easy to produce drug resistance, simple operation and wide antibacterial spectrum. However, the lifetime of reactive oxygen species (ROS) is very short, and its action radius is limited, which can only oxidize the surrounding microorganisms to cause killing effect, and cannot penetrate into the internal lesion or penetrate the biofilm to exert antibacterial activity. Combined with existing gas antibacterial therapy, it is expected to achieve the purpose of combined synergistic effect.

[0004] Gas therapy is the research focus in the field of anti-infection in recent ten years, which uses CO, H2S, H2, SO2, NO and other endogenous signal molecules to kill bacteria and disperse biofilm, promotes wound healing caused by bacterial infection, and also avoids bacterial drug resistance. NO is the earliest discovered gaseous signal molecule, which plays a crucial role in various physiological and pathological conditions, such as participating in the regulation of immune function and promoting wound healing. At the same time, it is also a broad-spectrum and efficient cytotoxic antibacterial agent. NO can directly interact with bacterial DNA and block DNA repair to exert broad-spectrum antibacterial effect. At the same time, NO can react with endogenous superoxide in the bacterial respiratory process to produce highly active nitrogen (RNS), such as peroxynitrite (ONOO -) and dinitrogen trioxide (N2O3), which in turn causes lipid peroxidation, DNA breakage and protein dysfunction in bacteria. The combination of NO with aPDT can achieve better penetration depth of the bacterial biofilm and compensate for the insufficient ROS production in a low-oxygen environment.

[0005] In view of the current wound infection problem, in the field of wound infection treatment, the traditional antibiotics have the disadvantages of multi-drug resistance, large dose, slow iteration, and the single-component antibacterial material such as silver particles is difficult to meet the requirements of strong bactericidal and antibiofilm ability and small toxicity at the same time, especially in the face of mixed infection of multiple drug-resistant bacteria, the anti-infection effect is not ideal, and it is difficult to effectively promote wound healing. The current nanotechnology can integrate multiple antibacterial means on the same carrier, but there are problems such as complex preparation process, multiple material varieties, difficult surface modification, and weak loading of photosensitizer. There is no preparation and application of particles that can simultaneously play the roles of antibacterial, antibiofilm and promoting wound healing through the combination of photodynamic therapy, NO antibacterial therapy and silver. Some silver composite multifunctional nanoparticles have problems such as difficulty in accurately controlling size and morphology, uneven particle size, poor dispersibility and stability, and easy agglomeration in the synthesis process; the reaction conditions such as reduction conditions and environmental factors are difficult to accurately control, resulting in unstable performance and poor repeatability. At the same time, nitric oxide (NO) is difficult to store in the carrier, and there are problems such as difficult control of release rate and short maintenance time of effective concentration. At present, some studies use its precursor L-arginine to convert into NO gas under oxidation conditions. However, the efficiency of loading L-arginine on the carrier is low, and if the carrier is connected through chemical bonds, a large number of active groups on the surface of the carrier are required. SUMMARY

[0006] The present application aims to overcome the shortcomings of the prior art and provides a fullerene composite silver nanoparticle producing nitric oxide and a preparation method and application thereof. The present application innovatively combines silver nanoparticles (AgNPs), fullerene and arginine. The method is convenient and does not require heating and reducing agents. Moreover, the size distribution of the synthesized nanocomposite material can be simply controlled by the proportion of hydrophilic fullerene and AgNO3 in the reaction. Fullerene wrapped on the silver nanoparticles can reduce the oxidation of silver nanoparticles and is also conducive to connecting other substances (such as L-Arg) through the groups on the surface of fullerene. The prepared composite material has good dispersibility, uniform particle size and good stability. The synthesis steps are simplified, and the production cost and environmental burden are reduced. The combined material can synergistically exert the effects of ROS, AgNPs and NO on wound infection, inhibit bacterial growth in multiple dimensions, reduce inflammatory response and promote tissue repair, effectively overcoming the problems of poor anti-infection effect of single-component and complicated traditional preparation method, and providing an efficient, safe and novel solution for the treatment of wound infection.

[0007] In a first aspect, the embodiments of the present application provide a fullerene-silver nanoparticle for producing nitric oxide, wherein the fullerene-silver nanoparticle for producing nitric oxide Arg-C60@AgNPs has a silver nanoparticle as a core, and a fullerene is adsorbed on an outer layer of the silver nanoparticle core, and L-arginine is modified on a surface of the fullerene.

[0008] Further, the fullerene-silver nanoparticle has a particle size of 30-300 nm and a potential of -30-+50 mV.

[0009] Further, the fullerene-silver nanoparticle generates ROS under visible light excitation and then oxidizes arginine to generate NO.

[0010] In a second aspect, the embodiments of the present application provide a preparation method of a fullerene-silver nanoparticle for producing nitric oxide, and the preparation method comprises the following steps:

[0011] The silver salt is slowly added to the hydrophilic fullerene water suspension, and stirring and dialysis are performed at room temperature to obtain a C60@AgNPs complex;

[0012] The functional groups of the hydrophilic fullerene or L-arginine are activated, the L-arginine is modified to the surface of the C60@AgNPs through a chemical bond, and the reaction solution is dialysis purified to obtain the fullerene-silver nanoparticle Arg-C60@AgNPs.

[0013] Further, the hydrophilic fullerene is selected from amino fullerene C60-NH2, carboxyl fullerene C60-COOH or hydroxyl fullerene C60-OH; and the silver salt is selected from silver nitrate, silver perchlorate or silver fluoride.

[0014] Further, the mass ratio of the silver nanoparticle and the fullerene in the fullerene-silver nanoparticle Arg-C60@AgNPs is 0.025-0.2:1; and the concentration of the fullerene water suspension is 0.1-100 mg / mL.

[0015] Further, when the hydrophilic fullerene is amino fullerene C60-NH2, EDC and NHS are added to an L-arginine aqueous solution with a concentration of 0.1-10 mg / mL in a mass ratio of 0.5-3:1, wherein the mass ratio of EDC to L-arginine is 1.5:1, and stirring is performed at room temperature to activate the carboxyl group; then the C60@AgNPs complex is added, wherein the mass ratio of L-arginine to the C60@AgNPs complex is 0.05-5:1, and sufficient stirring is performed, and after the reaction is completed, dialysis purification is performed to obtain the Arg-C60@AgNPs.

[0016] Further, when the hydrophilic fullerene is carboxyl fullerene C60-COOH, EDC and NHS are added to the C60@AgNPs complex with a concentration of 0.1-10 mg / mL in a mass ratio of 0.5-3:1, and the mass ratio of the C60@AgNPs complex to EDC is 10:3, to activate the carboxyl group; then L-arginine aqueous solution is added for continuous stirring, and the mass ratio of L-arginine to the C60@AgNPs complex is 0.05-5:1, after the reaction is completed, dialysis purification is performed to obtain Arg-C60@AgNPs.

[0017] Further, when the hydrophilic fullerene is carboxyl fullerene C60-COOH, EDC and NHS are added to the C60@AgNPs complex with a concentration of 0.1-10 mg / mL in a mass ratio of 0.5-3:1, and the mass ratio of the C60@AgNPs complex to EDC is 10:3, to activate the carboxyl group; then L-arginine aqueous solution is added for continuous stirring, and the mass ratio of L-arginine to the C60@AgNPs complex is 0.05-5:1, after the reaction is completed, dialysis purification is performed to obtain Arg-C60@AgNPs.

[0018] In a third aspect, the application provides a use of the fullerene-silver nanoparticle for producing nitric oxide or the fullerene-silver nanoparticle for producing nitric oxide prepared by the above method in the preparation of an antibacterial or anti-infective drug.

[0019] Compared with the prior art, the application has the following beneficial effects:

[0020] The application provides a fullerene-silver nanoparticle for producing nitric oxide Arg-C60@AgNPs, which is designed based on various inflammatory factors and oxidative stress signals in a wound infection microenvironment, utilizes high affinity and multifunctionality of fullerene hybrid silver nanoparticles, forms Arg-C60@AgNPs nanoparticles through self-assembly, realizes synergistic treatment effects of photodynamic therapy, NO gas therapy and AgNPs, has the advantages of good broad-spectrum antibacterial effect, strong biological membrane penetration ability and drug resistance, and can promote fibroblast and collagen proliferation and accelerate wound healing. The application adopts simple, efficient and green chemical reactions to prepare a nano drug with good water solubility, stability and biocompatibility in one step, has the advantages of simple preparation, no additional use of reducing agents, broad-spectrum antibacterial effect, no drug resistance and strong biological membrane penetration, overcomes the problems of drug resistance, poor biological membrane penetration and large toxic side effects of traditional antibacterial drugs, and provides a new strategy and reference for wound infection, refractory wound infection and biological membrane related anti-infective treatment. BRIEF DESCRIPTION OF DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed to be used in the embodiments will be briefly introduced as follows. Obviously, the drawings in the following description only constitute some embodiments of the present application, and for those skilled in the art, other drawings can also be obtained from these drawings without any creative effort.

[0022] Figure 1 Figure 1 is a morphology and composition analysis diagram of Arg-C60@AgNPs prepared in Example 1;

[0023] Figure 2 Figure 2 is a particle size, zeta potential and characteristic absorption spectrum diagram of Arg-C60@AgNPs prepared in Example 1;

[0024] Figure 3 Figure 3 is a diagram showing the in vitro ROS and NO production of Arg-C60@AgNPs prepared in Example 1;

[0025] Figure 4 Figure 4 is a diagram showing the in vitro anti-MRSA activity of Arg-C60@AgNPs prepared in Example 1 by the spread plate method;

[0026] Figure 5 Figure 5 is a diagram showing the in vitro antibiofilm experiment of Arg-C60@AgNPs prepared in Example 1;

[0027] Figure 6 Figure 6 is a diagram showing the cell scratch experiment of Arg-C60@AgNPs prepared in Example 1;

[0028] Figure 7 Figure 7 is an evaluation of the effect of Arg-C60@AgNPs prepared in Example 1 on treating skin wound infection of mice with MRSA. DETAILED DESCRIPTION

[0029] The present application will be further described below in conjunction with the embodiments but without limiting the present application.

[0030] Example 1-1: Preparation of Arg-C60@AgNPs

[0031] (1) Preparation of C60-NH2

[0032] 100 mg of fullerene C60 was added to 100 mL of toluene and ultrasonically dispersed, and then 50 mL of ethylenediamine was slowly dropped. After stirring at room temperature for 0.5 h, it was allowed to stand, and the supernatant was discarded. The remaining liquid was continuously stirred for 3 days under nitrogen protection. After the reaction was completed, ethanol was added for rotary evaporation, and the solid was washed with deionized water for 3 times. After dialysis purification (the dialysis bag was cut off at a molecular weight of 3500), it was freeze-dried to obtain C60-NH2 solid.

[0033] (2) Preparation of C60@AgNPs

[0034] Take 10 mg of C60-NH2dispersed in 5 mL of deionized water, add 4 mL of silver nitrate solution (2.425 mM), stir for 4 h, and then purify the complex by dialysis (dialysis bag cut-off molecular weight is 3500) to obtain C60@AgNPs.

[0035] (3) Preparation of Arg-C60@AgNPs

[0036] Add 7.5 mg of EDC and 3 mg of NHS to 5 mL of L-Arg aqueous solution (1 mg / mL), stir at room temperature for 4 h, then add 5 mL of C60@AgNPs suspension (1 mg / mL), continue to stir for 18 h, and then purify by dialysis (dialysis bag cut-off molecular weight is 3500) to obtain Arg-C60@AgNPs.

[0037] Example 1-2:

[0038] (1) Preparation of C60-NH2

[0039] Add 50 mg of fullerene C60to 50 mL of ethylenediamine, ultrasonically disperse, and stir continuously at room temperature for 24 h under nitrogen protection. Filter the reaction solution with a 0.22 μm filter membrane, add ethanol and rotary evaporate, wash the solid with deionized water for 3 times, adjust the PH to about 6.5 with dilute nitric acid, purify by dialysis (dialysis bag cut-off molecular weight is 3500), and freeze-dry to obtain C60-NH2solid.

[0040] (2) Preparation of C60@AgNPs

[0041] Take 10 mg of C60-NH2dispersed in 5 mL of deionized water, add 1 mL of silver nitrate solution (2.425 mM), stir for 4 h, and then purify the complex by dialysis (dialysis bag cut-off molecular weight is 3500) to obtain C60@AgNPs.

[0042] (3) Preparation of Arg-C60@AgNPs

[0043] Add 3 mg of EDC and 6 mg of NHS to 20 mL of L-Arg aqueous solution (0.1 mg / mL), stir at room temperature for 4 h, then add 5 mL of C60@AgNPs suspension (1 mg / mL), continue to stir for 18 h, and then purify by dialysis (dialysis bag cut-off molecular weight is 3500) to obtain Arg-C60@AgNPs.

[0044] Example 1-3:

[0045] (1) Preparation of C60-NH2

[0046] C60-NH2was prepared by the reaction of C60with ethylenediamine. 200 mg of C60was added to 50 mL of toluene and sonicated to disperse. 50 mL of ethylenediamine was added dropwise and stirred at room temperature for 0.5 h. The solution was left to stand and the supernatant was discarded. The remaining solution was stirred for 3 days under N2protection. After the reaction was completed, ethanol was added and evaporated. The solid was washed with deionized water for 3 times and freeze-dried to obtain C60-NH2solid.

[0047] (2) Preparation of C60@AgNPs

[0048] 10 mg of C60-NH2was dispersed in 5 mL of deionized water, 8 mL of silver nitrate solution (2.425 mM) was added, and stirred for 4 h. The complex was purified by dialysis (dialysis bag with a molecular weight cut-off of 3500) to obtain C60@AgNPs.

[0049] (3) Preparation of Arg-C60@AgNPs

[0050] 15 mg of EDC and 5 mg of NHS were added to 1 mL of L-Arg aqueous solution (10 mg / mL), stirred at room temperature for 4 h, and then 1 mL of C60@AgNPs suspension (1 mg / mL) was added. Stirring was continued for 18 h, and then the complex was purified by dialysis (dialysis bag with a molecular weight cut-off of 3500) to obtain Arg-C60@AgNPs.

[0051] Example 1-4:

[0052] (1) Preparation of C60-OH

[0053] 1.2 g of C60fullerene was dissolved in 500 mL of toluene in a glass flask. 10 mL of TBAH was added to the solution and stirred for 3 min, and then 30 mL of 50% (w / v) NaOH was added dropwise. After stirring at room temperature for 2 h under N2protection, phase separation was performed by standing for 10 min. The flask was placed in a refrigerator at -20°C for 2 h, and then the organic phase was poured out. 200 mL of H2O was added and the solution was continuously stirred at room temperature for 4 days. The collected solution was freeze-dried, and the obtained C60-OH was washed with anhydrous methanol by centrifugation for 3 times. The purified C60-OH solid was dried in a desiccator.

[0054] (2) Preparation of C60@AgNPs

[0055] 10 mg of C60-OH was dispersed in 5 mL of deionized water, 4 mL of silver nitrate solution (2.425 mM) was added, and stirred for 4 h. The complex was purified by dialysis (dialysis bag with a molecular weight cut-off of 3500) to obtain C60@AgNPs.

[0056] (3) Preparation of Arg-C60@AgNPs

[0057] 6.5 mg of DCC was added to 5 mL of L-Arg aqueous solution (1 mg / mL), stirred at room temperature for 4 h, and then 20 mL of C60@AgNPs suspension (1 mg / mL) and 0.5 mg of DMAP were added. After stirring for 18 h, the mixture was dialyzed and purified (the molecular weight cutoff of the dialysis bag was 3500) to obtain Arg-C60@AgNPs.

[0058] Example 1-5:

[0059] (1) Preparation of C60-OH

[0060] Take 0.5g of fullerene C60 and dissolve it in 250mL of toluene in a glass flask. Pipette 4.1mL of TBAH into the solution and keep stirring for 3 minutes, then add 12.3mL of 50% (w / v) NaOH dropwise. After stirring at room temperature for 2 hours under N2 protection, let it stand for 10 minutes for phase separation. Place the flask in a refrigerator at -20℃ for 2 hours, then pour out the organic phase. Add 200mL of H2O and continue stirring the solution at room temperature for 4 days. The collected solution is freeze-dried, and the obtained C60-OH is centrifuged and washed 3 times with anhydrous methanol. Dry the purified C60-OH solid in a desiccator.

[0061] (2) Preparation of C60@AgNPs

[0062] 10 mg of C60-OH was dispersed in 5 mL of deionized water, 4 mL of silver nitrate solution (2.425 mM) was added, and after stirring for 4 h, the complex was purified by dialysis (the molecular weight cutoff of the dialysis bag was 3500) to obtain C60@AgNPs.

[0063] (3) Preparation of Arg-C60@AgNPs

[0064] 12 mg of DCC was added to 5 mL of L-Arg aqueous solution (2 mg / mL), stirred at room temperature for 4 h, and then 20 mL of C60@AgNPs suspension (2 mg / mL) and 5 mg of DMAP were added. After stirring for 18 h, the mixture was dialyzed and purified (the molecular weight cutoff of the dialysis bag was 3500) to obtain Arg-C60@AgNPs.

[0065] Examples 1-6:

[0066] (1) Preparation of C60-OH

[0067] Take 1 g of fullerene C60 dissolved in 400 mL of toluene in a glass flask. 9.6 mL of TBAH is moved into the solution and kept stirring for 3 min, then 25 mL of 50% (w / v) NaOH is added dropwise. After stirring at room temperature for 2 h under N2 protection, phase separation is carried out by standing for 10 min. The flask is placed in a refrigerator at -20°C for 2 h, and then the organic phase is poured out. 200 mL of H2O is added and the solution is continuously stirred at room temperature for 4 days. The collected solution is freeze-dried, and the obtained C60-OH is washed with anhydrous methanol by centrifugation for 3 times. C60-OH solid is obtained by drying in a desiccator.

[0068] (2) Preparation of C60@AgNPs

[0069] Take 10 mg of C60-OH dispersed in 5 mL of deionized water, add 4 mL of silver nitrate solution (2.425 mM), stir for 4 h, and then purify the complex by dialysis (dialysis bag cut-off molecular weight is 3500) to obtain C60@AgNPs.

[0070] (3) Preparation of Arg-C60@AgNPs

[0071] Add 37.5 mg of DCC to 5 mL of L-Arg aqueous solution (5 mg / mL), stir at room temperature for 4 h, then add 20 mL of C60@AgNPs suspension (5 mg / mL) and 7.5 mg of DMAP, continue stirring for 18 h, and then purify by dialysis (dialysis bag cut-off molecular weight is 3500) to obtain Arg-C60@AgNPs.

[0072] Example 1-7:

[0073] (1) Preparation of C60-COOH

[0074] Take 200 mg of C60 and add it to 100 mL of o-dichlorobenzene, ultrasonic for 30 min to make it completely dissolved, take 247 mg of sarcosine and 420 mg of p-acylbenzoic acid and add them to C60, ultrasonic for 60 min, react at 135°C under nitrogen protection for 22 h, stop heating when the reaction solution is wine red, purify by dialysis (cut-off molecular weight is 3500) and then cool and filter to obtain C60-COOH solid after drying at low temperature.

[0075] (2) Preparation of C60@AgNPs

[0076] Take 10 mg of C60-COOH and disperse it in 5 mL of deionized water, add 4 mL of silver nitrate solution (2.425 mM), stir for 4 h, and then purify the complex by dialysis (dialysis bag cut-off molecular weight is 3500) to obtain C60@AgNPs.

[0077] (3) Preparation of Arg-C60@AgNPs

[0078] Add 1.5 mg EDC and 3 mg NHS to 5 mL of C60@AgNPs aqueous solution (1 mg / mL), after stirring at room temperature for 4 h, add 10 mL of L-Arg aqueous solution (2.5 mg / mL), continue stirring for 18 h, and then purify by dialysis (dialysis bag with a molecular weight cut-off of 3500) to obtain Arg-C60@AgNPs.

[0079] Example 1-8:

[0080] (1) Preparation of C60-COOH

[0081] Weigh 100 mg of C60 and add it to 50 mL of o-dichlorobenzene, ultrasonic for 30 min to make it completely dissolved, weigh 120 mg of sarcosine and 210 mg of p-acylbenzoic acid and add them to C60, ultrasonic for 60 min, and react at 135°C under N2 protection for 22 h. When the reaction solution is wine red, stop heating, purify by dialysis (cut-off molecular weight of dialysis bag is 3500), cool, filter, and dry at low temperature to obtain C60-COOH solid.

[0082] (2) Preparation of C60@AgNPs

[0083] Take 10 mg of C60-COOH and disperse it in 5 mL of deionized water, add 2 mL of silver nitrate solution (2.425 mM), stir for 4 h, and then purify the complex by dialysis (dialysis bag with a molecular weight cut-off of 3500) to obtain C60@AgNPs.

[0084] (3) Preparation of Arg-C60@AgNPs

[0085] Add 3 mg of EDC and 1 mg of NHS to 5 mL of C60@AgNPs aqueous solution (2 mg / mL), stir at room temperature for 4 h, then add 0.5 mL of L-Arg aqueous solution (1 mg / mL), continue stirring for 18 h, and then purify by dialysis (dialysis bag with a molecular weight cut-off of 3500) to obtain Arg-C60@AgNPs.

[0086] Example 1-9:

[0087] (1) Preparation of C60-COOH

[0088] Take 150 mg of C60 and add it to 75 mL of o-dichlorobenzene, ultrasonic for 30 min, so that it is completely dissolved, take 180 mg of sarcosine and 315 mg of p-acylbenzoic acid and add them to C60, ultrasonic for 60 min, react at 135℃ and under N2 protection for 22 h, stop heating when the reaction solution is wine red, after dialysis purification (cut off molecular weight is 3500), cool, filter and dry at low temperature to obtain C60-COOH solid.

[0089] (2) Preparation of C60@AgNPs

[0090] Take 10 mg of C60-COOH and disperse it in 5 mL of deionized water, add 6 mL of silver nitrate solution (2.425 mM), stir for 4 h, then purify the complex by dialysis (dialysis bag cut off molecular weight is 3500) to obtain C60@AgNPs.

[0091] (3) Preparation of Arg-C60@AgNPs

[0092] Add 3 mg of EDC and 1 mg of NHS to 5 mL of C60@AgNPs aqueous solution (2 mg / mL), stir at room temperature for 4 h, then add 10 mL of L-Arg aqueous solution (1 mg / mL), continue to stir for 18 h, then purify by dialysis (dialysis bag cut off molecular weight is 3500) to obtain Arg-C60@AgNPs.

[0093] It should be noted that the present application utilizes the weak reducing property of the photosensitizer fullerene to reduce Ag + in one step without adding other reducing agents, to prepare hybrid nanoparticles with silver nanoparticles as the core and a fullerene layer adsorbed on the surface. Then, by covalently connecting the NO precursor L-arginine to the surface active groups of fullerene, a photodynamic antibacterial nanoparticle Arg-C60@AgNPs based on the synergistic effect of NO is constructed. The particle is well dispersed, uniformly distributed, has good chemical stability, and the preparation method is simple and environmentally friendly. Under visible light excitation, it can produce ROS and automatically oxidize L-arginine to release NO. The combination of silver nanoparticles, photodynamics and gas therapy plays a role in antibacterial, anti-biofilm infection and promoting wound repair.

[0094] Example 2: Characterization of Arg-C60@AgNPs

[0095] (1) Particle morphology

[0096] The morphology and microstructure of Arg-C60@AgNPs were observed by transmission electron microscopy and high-resolution transmission electron microscopy, and the chemical composition was analyzed by EDS. The results show that the complex particles are nearly spherical, the shape is relatively regular, and the distribution is uniform Figure 1A in FIG. 1 shows the transmission electron microscopy (TEM) image of Arg-C60@AgNPs). The obvious core-shell structure can be observed under high resolution microscopy, with the outer layer being the fullerene adsorption layer Figure 1 B in FIG. 1 shows the high resolution transmission microscopy (HRTEM) image of Arg-C60@AgNPs). The core is silver nanoparticles, and the nitrogen element proves that the amino fullerene surface modification is successful. Figure 1 C in FIG. 1 shows the EDS analysis diagram of Arg-C60@AgNPs). By using the weak reducing property of fullerene, the present application only reduces silver nitrate into silver nanoparticles with uniform size through simple mixing and stirring. The silver nanoparticle composite with the surface adsorbed with amino fullerene is successfully obtained.

[0097] (2) Particle size, zeta potential and UV characteristic spectrum

[0098] The above prepared C60-NH2, C60@AgNPs and Arg-C60@AgNPs were diluted with deionized water, and the particle size and zeta potential were measured by Malvern particle size analyzer. The average particle sizes of C60-NH2, C60@AgNPs and Arg-C60@AgNPs were 273.9±3.13 nm, 51.2±1.59 nm and 81.03±3.72 nm, respectively. After forming the silver composite, the dispersibility of the amino fullerene was improved, the particle aggregation was less, and the average particle size was reduced. After the surface modification of arginine, the particle size of the composite was slightly increased. The particles were positively charged, and the zeta potential value was reduced to 29.5±1.1 mV due to the introduction of arginine molecules. Figure 2 A in FIG. 2.

[0099] The UV-visible spectrum shows that all the synthesized C60@AgNPs and Arg-C60@AgNPs have almost the same plasmon resonance peak at about 420 nm (B in FIG. 2), which proves the generation of silver nanoparticles with similar particle sizes. Figure 2

[0100] (3) Arginine binding rate and drug loading

[0101] The Arg-C60@AgNPs reaction solution was taken, and the ultrafiltration method and 8-hydroxyquinoline colorimetric method were used to separate and quantitatively determine arginine, and the arginine binding rate and encapsulation efficiency were determined. The calculation formula is as follows:

[0102]

[0103] According to the determination results, the arginine binding rate of Arg-C60@AgNPs is 71.55±3.37%, and the drug loading is 41.56±1.16%.

[0104] (4) ROS yield and NO production of Arg-C60@AgNPs in vitro​

[0105] Using DPBF as ROS probe, 10 μg / mL Arg-C60@AgNPs were irradiated by 635 nm laser (50 mW / cm 2 ) and the fluorescence spectra were recorded at different time (A in FIG. 6 shows the curve of irradiation time and ROS production). Figure 3 With the extension of irradiation time, the ROS production gradually increased. Using NO kit, the NO production after 10 min irradiation by 635 nm laser (50 mW / cm 2 ) was determined. Figure 3 B in FIG. 6 shows the curve of NO production under light irradiation at different complex concentrations, and the NO production increased with the increase of Arg-C60@AgNPs concentration. It can be seen that the prepared Arg-C60@AgNPs can not only effectively produce ROS to play an antibacterial role, but also can further oxidize arginine to effectively release NO, thus playing a combined antibacterial role.

[0106] Example 3: In vitro antibacterial test

[0107] The in vitro antibacterial activity of C60-NH2, C60@AgNPs and Arg-C60@AgNPs was determined by using coating plate method. 250 μL of 1×10 5 CFU / mL MRSA bacterial solution was added to a 24-well plate, and different concentrations of the above antibacterial preparations were mixed. After incubation at 37℃ on a shaking table for 1 h, the samples were treated by light irradiation (635 nm, 50 mW / cm 2 ) for 10 min or dark treatment. 100 mL of each sample was taken and coated on LB agar plate, and incubated for 18 h, and the colony formation was recorded by taking photos. Figure 4 Without light irradiation, C60-NH2 had little effect on colony formation. Due to the presence of Ag + , the antibacterial activity of C60@AgNPs and Arg-C60@AgNPs was concentration-dependent. After red light treatment, the antibacterial effect of each preparation group was enhanced compared with the non-irradiation group. The number of colonies was significantly reduced, and the antibacterial activity was concentration-dependent. Compared with the ROS produced by C60-NH2, C60@AgNPs and Arg-C60@AgNPs further improved the antibacterial activity against MRSA through the combined action of Ag + and NO. The results were consistent with the MIC and MBC results (Table 1). In a 96-well plate, MRSA bacterial solution was inoculated and antibacterial particles were added, and after doubling dilution, the final concentration of the bacterial solution was 5×10 4 CFU / mL. The irradiation group was irradiated by 635 nm laser (50 mW / cm 2After 10 min of light treatment, the solution was incubated for another 18 h, and the MIC value was read by observing the turbidity of the solution. After the MIC determination, the mixture was removed from the 96-well plate and spread on LB agar plates. The plates were incubated for 18 h, and the minimum concentration of the preparation without colonies was observed as the MBC. The above results prove that the Arg-C60@AgNPs prepared in the application can enhance the antibacterial effect of AgNPs and fullerenes under light conditions through NO gas.

[0108] Table 1. MIC and MBC values of fullerene composite silver nanoparticle drug against MRSA strain

[0109]

[0110] Example 4: In vitro antibiofilm test

[0111] 2 mL of MRSA bacterial solution (1 × 10 6 CFU / mL) was added to a 6-well plate, and incubated at 37°C for 48 h. The TSB medium was replaced every 24 h. After the MRSA biofilm was formed at the bottom of the plate, the culture medium in the hole was discarded and phosphate buffer, C60-NH2, C60@AgNPs or Arg-C60@AgNPs (40 μg / mL) was added to the hole, respectively. After 1 h of incubation, light treatment (635 nm, 50 mW / cm 2 ) was performed for 10 min, and the non-light treatment group was treated in the dark. The supernatant was aspirated and washed 3 times. 4% paraformaldehyde was added for 15 min for fixation. 0.1% crystal violet was used for staining for 15 min, and after drying and taking a photo, 33% (v / v) acetic acid was added to dissolve the crystal violet. The absorbance was measured at 560 nm, and the biofilm clearance rate was calculated by comparison with the blank control group.

[0112] Figure 5 A in the figure is the crystal violet staining of the biofilm after drug administration, Figure 5 B in the figure is the quantitative analysis of the biofilm clearance rate, compared with the Arg-C60@AgNPs light treatment group. *p < 0.05, ****p < 0.001. The in vitro antibiofilm results are consistent with the in vitro antibacterial experiment results. After light treatment of Arg-C60@AgNPs, it has strong biofilm killing activity and can be used for wound treatment caused by biofilm infection.

[0113] Example 5: Cell scratch test

[0114] 5 × 10 5 Mouse fibroblasts L929 were inoculated in a 6-well plate, and a 200 μL pipette tip was used for scratching, and then the cells were incubated with culture solution containing C60-NH2, C60@AgNPs and Arg-C60@AgNPs (containing 1% serum) for 1 h, and light treatment was performed for 10 min (635 nm, 50 mW / cm2 The cells were cultured for 24 h, and the wound healing process was recorded under a microscope. Image J software was used to analyze the wound changes and calculate the cell migration rate.

[0115]

[0116] like Figure 6 As shown, Figure 6 A in the figure shows the scratch healing of L929 cells, bar=200 μm, Figure 6 Figure B shows the cell migration rate in the scratch wound assay, compared to the Arg-C60@AgNPs illumination group. ***p<0.005, ****p<0.001. Fullerene had no significant effect on cell proliferation and migration. Due to the low concentration and small particle size of silver nanoparticles, the silver nanocomposite significantly reduced the scratch width and increased the migration rate of fibroblasts under both illumination and non-illumination conditions. Arg-C60@AgNPs release NO under red light excitation, further promoting cell migration and proliferation, thereby promoting wound healing.

[0117] Example 6: Evaluation of in vivo anti-infection effect

[0118] A BALB / c mouse skin wound model was established using a punch, and 50 μL of 10 8 CFU / mL MRSA bacterial liquid caused infection. 100 μL of normal saline was dripped onto the wound surface of the control group, and 100 μL of different preparation solutions (100 μg / mL) were dripped onto the wound surface of the treatment group. After 30 minutes, the wound surface of the light group was irradiated with red light for 10 minutes (635 nm, 50 mW / cm 2 ), while the non-illuminated group was kept in the dark. Body weight was recorded daily, and wound diameters were photographed every two days. Images were processed using Image J software to simulate actual wound changes. Wound healing rates were calculated using the formula.

[0119]

[0120] The results showed that after being excited by red light, C60-NH2 and C60@AgNPs could play an antibacterial and promote wound healing compared with the negative group, proving that the photosensitizer C60 has a certain antibacterial effect in vivo. Due to the addition of silver nanoparticles, the healing speed of the C60@AgNPs light-exposed group was faster. Arg-C60@AgNPs also has an antibacterial effect under non-photodynamic therapy conditions that rely on AgNPs. After being excited by red light, its wound healing speed was significantly accelerated and the antibacterial effect was significantly improved ( Figure 7 ), Figure 7 A in the figure shows the wound healing status at different times after treatment; Figure 7B in Figure 8 shows the wound healing rate of each group at different times after treatment, **p < 0.01, ****p < 0.001. The in vivo data is consistent with the in vitro experimental results, and under the action of ROS, NO and AgNPs, the Arg-C60@AgNPs treatment has the strongest effect on the skin wound infection of mice.

[0121] In summary, the Arg-C60@AgNPs constructed by the application can generate ROS and NO under visible light excitation, and combined with AgNPs, it can play a broad-spectrum antibacterial, anti-biofilm infection, promote fibroblast proliferation and migration, and further promote wound repair and regeneration of bacterial infection.

Claims

1. A fullerene-composite silver nanoparticle that produces nitric oxide, characterized in that: The nitric oxide-producing fullerene-composite silver nanoparticles Arg-C60@AgNPs have silver nanoparticles as their core, fullerenes adsorbed on the outer layer of the silver nanocore, and the surface of the fullerenes modified with L-arginine; the fullerene-composite silver nanoparticles have a particle size of 30 to 300 nm and a potential of -30 to +50 mV.

2. The nitric oxide-producing fullerene-composite silver nanoparticles according to claim 1, wherein: The fullerene-composite silver nanoparticles generate ROS under visible light excitation and then oxidize arginine to generate NO.

3. A method for preparing fullerene-composite silver nanoparticles that produce nitric oxide, characterized in that: The preparation method comprises: Slowly adding silver salt to a hydrophilic fullerene aqueous suspension, stirring and dialyzing at room temperature to obtain a C60@AgNPs complex; the hydrophilic fullerene is selected from aminofullerene C60-NH2, carboxylfullerene C60-COOH or hydroxyfullerene C60-OH; The hydrophilic fullerene or L-arginine functional groups were activated, and L-arginine was modified to the surface of C60@AgNPs through chemical bonds. The reaction solution was dialyzed and purified to obtain fullerene-composite silver nanoparticles Arg-C60@AgNPs.

4. The method for preparing fullerene-composite silver nanoparticles for producing nitric oxide according to claim 3, wherein: The silver salt is selected from silver nitrate, silver perchlorate or silver fluoride.

5. The method for preparing fullerene-composite silver nanoparticles for producing nitric oxide according to claim 3, wherein: The mass ratio of nanosilver to fullerene in fullerene composite silver nanoparticles Arg-C60@AgNPs is 0.025~0.2:1; the concentration of the hydrophilic fullerene aqueous suspension is 0.1~100 mg / mL.

6. The method for preparing fullerene-composite silver nanoparticles for producing nitric oxide according to claim 3, wherein: When the hydrophilic fullerene is aminofullerene C60-NH2, EDC and NHS are added in a mass ratio of 0.5~3:1 to an L-arginine aqueous solution with a concentration of 0.1-10 mg / mL, where the mass ratio of EDC to L-arginine is 1.5:1, and stirred at room temperature to activate the carboxyl group; then the C60@AgNPs complex is added, where the mass ratio of L-arginine to C60@AgNPs complex is 0.05~5:1, and stirred thoroughly. After the reaction is completed, dialysis purification is performed to obtain Arg-C60@AgNPs.

7. The method for preparing fullerene-composite silver nanoparticles for producing nitric oxide according to claim 3, wherein: When the hydrophilic fullerene is carboxyl fullerene C60-COOH, EDC and NHS are added to a C60@AgNPs complex with a concentration of 0.1-10 mg / mL in a mass ratio of 0.5~3:1, where the mass ratio of the C60@AgNPs complex to EDC is 10:3, to activate the carboxyl group; then, an L-arginine aqueous solution is added and continued stirring is continued, where the mass ratio of L-arginine to the C60@AgNPs complex is 0.05~5:

1. After the reaction is completed, dialysis purification is performed to obtain Arg-C60@AgNPs.

8. The method for preparing fullerene-composite silver nanoparticles for producing nitric oxide according to claim 3, wherein: When the hydrophilic fullerene is hydroxyfullerene C60-OH, DCC is added to a 0.1-10 mg / mL L-arginine solution to activate the carboxyl group, where the mass ratio of DCC to L-arginine is 1.2-1.5:1; then, the C60@AgNPs complex and 0.1-0.5 times the mass equivalent of L-arginine DMAP are added to the L-arginine reaction solution, where the mass ratio of L-arginine to C60@AgNPs complex is 0.05-5:1, and a base-catalyzed condensation reaction is carried out. After the reaction is completed, dialysis purification is performed to obtain Arg-C60@AgNPs.

9. Use of the nitric oxide-producing fullerene-complexed silver nanoparticles according to any one of claims 1 to 2 or the nitric oxide-producing fullerene-complexed silver nanoparticles prepared by the preparation method according to any one of claims 3 to 8 in anti-infective drugs.

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