Fullerene composite silver nanoparticles capable of producing nitric oxide as well as preparation method and application of fullerene composite silver nanoparticles

Abstract

The invention discloses a fullerene composite silver nanoparticle for producing nitric oxide and a preparation method and application thereof, the fullerene composite silver nanoparticle Arg-C60 coated AgNPs for producing nitric oxide takes a silver nanoparticle as a core, fullerene is adsorbed on the outer layer of the silver nanoparticle core, and the surface of the fullerene is modified with L-arginine. The preparation method comprises the following steps: slowly dropwise adding silver salt into a hydrophilic fullerene water suspension, stirring at room temperature, and dialyzing to obtain a C60-coated AgNPs compound; the preparation method comprises the following steps: activating a hydrophilic fullerene or L-arginine functional group, modifying L-arginine to the surface of C60-coated AgNPs through a chemical bond, and dialyzing and purifying a reaction solution to obtain the fullerene composite silver nanoparticles Arg-C60-coated AgNPs. Compared with other fullerene hybrid silver compounds, Arg-C60 coated AgNPs is uniform in distribution and good in water dispersibility, and the silver nanoparticles are combined with antibacterial photodynamics and gas therapy, so that the fullerene hybrid silver compound has the advantages of good broad-spectrum antibacterial effect, strong biological membrane penetrating power and no drug resistance, and can promote fibroblast and collagen proliferation and accelerate wound healing.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and specifically relates to a fullerene composite silver nanoparticle producing nitric oxide and a preparation method and application thereof. Background Art

[0002] Bacterial infection is recognized as the main cause of damage to global health and has gradually become the second leading cause of death in the world. The emergence of multidrug-resistant bacteria (such as methicillin-resistant Staphylococcus aureus MRSA) has increased the number of patients year by year, becoming the primary pathogenic microorganism that seriously threatens human health. For example, in the treatment of common subcutaneous abscesses, it is necessary not only to control the infection, but also to make the wound heal quickly, shorten the healing time, and avoid long-term care and secondary infection. Insufficient blood supply to the abscess site leads to low antibiotic concentrations, and there are few options for broad-spectrum antibiotics and anti-resistant bacteria. The bacterial biofilm formed by the extracellular polymers (EPS) secreted by bacteria wraps itself in it, which not only becomes a penetration barrier for antibiotics, but also changes the bacterial phenotype and induces drug resistance.

[0003] Antimicrobial photodynamic therapy (aPDT) is a treatment method for controlling multidrug-resistant bacteria. It consists of three major elements: light, photosensitizer, and oxygen molecules. Photosensitizers are excited by light of a specific wavelength and transfer energy to the surrounding oxygen, generating highly active singlet oxygen, which causes photooxidation of amino acids, certain pyrimidine and purine bases in DNA / RNA, and unsaturated lipids, resulting in DNA damage and / or cell membrane damage, leading to membrane lipid destruction and bacterial death. It has the advantages of being less likely to develop drug resistance, simple operation, and a broad antibacterial spectrum. However, the life span of reactive oxygen species (ROS) is very short, and its radius of action is limited. It can only oxidize surrounding microorganisms to cause killing effects, and cannot penetrate deep into the lesion or penetrate the biofilm to exert antibacterial activity. Combined with existing gas antibacterial therapy, it is expected to achieve the purpose of combined synergy.

[0004] Gas therapy has been the research focus in the field of anti-infection in the past decade. It uses endogenous signal molecules such as CO, H2S, H2, SO2, and NO to kill bacteria and disperse biofilms. It can promote wound healing caused by bacterial infection while preventing bacteria from developing drug resistance. NO is the earliest discovered gaseous signal molecule and plays a vital role in a variety of physiological and pathological conditions, such as participating in the regulation of immune function and promoting wound healing. It is also a broad-spectrum and highly effective cytotoxic antibacterial agent. NO can directly interact with bacterial DNA and block DNA repair to exert a broad-spectrum antibacterial effect. At the same time, NO can react with endogenous superoxide in the bacterial respiration process to produce highly reactive nitrogen (RNS), such as peroxynitrite (ONOO -) and nitrogen trioxide (N2O3), which in turn causes lipid peroxidation, DNA breakage and protein dysfunction in bacteria. The combination of NO and aPDT can achieve better biofilm penetration depth and make up for the problem of insufficient ROS production caused by hypoxic environment.

[0005] In view of the current problem of wound infection, in the field of wound infection treatment, traditional antibiotics have the disadvantages of multidrug resistance, large dosage, and slow iteration. Single-component antibacterial materials such as silver particles are difficult to simultaneously meet the requirements of strong bactericidal and anti-biofilm capabilities and low toxicity, especially when facing mixed infections 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 methods on the same carrier, but there are problems such as complex preparation process, diverse material varieties, difficult surface modification, and weak photosensitizer loading capacity. There is no preparation and application of particles that can simultaneously play antibacterial, anti-biofilm and promote wound healing by combining photodynamics, 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 during the synthesis process; 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 to control the release rate and short maintenance time of effective concentration. At present, some studies have used its precursor L-arginine to convert it into NO gas under oxidative conditions. However, the efficiency of physically loading L-arginine on the carrier is low. If the carrier is connected by chemical bonds, a large number of modifiable active groups are required on the surface of the carrier. Summary of the invention

[0006] The purpose of the present invention is to provide a nitric oxide-producing fullerene composite silver nanoparticle and its preparation method and application in view of the deficiencies of the prior art. The present invention innovatively combines silver nanoparticles (AgNPs), fullerene and arginine organically. The method is convenient to react, does not require heating and reducing agent, and the size distribution of the synthesized nanocomposite material can be simply controlled by the ratio of hydrophilic fullerene and AgNO3 in the reaction. Fullerene wrapped on 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. By simplifying the synthesis steps, the production cost and environmental burden are reduced. This composite material can synergistically exert the anti-wound infection effects of ROS, AgNPs and NO, inhibit bacterial growth, reduce inflammatory response and promote tissue repair in multiple dimensions, effectively overcome the poor anti-infection effect of a single component and the cumbersome traditional preparation method, and provide an efficient, safe and novel solution for the treatment of wound infection.

[0007] In the first aspect, an embodiment of the present invention provides a nitric oxide-producing fullerene composite silver nanoparticle, wherein the nitric oxide-producing fullerene composite silver nanoparticle Arg-C60@AgNPs has a silver nanoparticle as a core, the outer layer of the silver nanocore is adsorbed with fullerene, and the surface of the fullerene is modified with L-arginine.

[0008] Furthermore, the particle size of the fullerene composite silver nanoparticles is 30 to 300 nm, and the potential is -30 to +50 mV.

[0009] Furthermore, the fullerene-composite silver nanoparticles generate ROS under visible light excitation and then oxidize arginine to generate NO.

[0010] In a second aspect, an embodiment of the present invention provides a method for preparing fullerene composite silver nanoparticles that produce nitric oxide, the preparation method comprising:

[0011] Slowly add silver salt to the hydrophilic fullerene aqueous suspension, stir and dialyze at room temperature to obtain a C60@AgNPs composite;

[0012] The hydrophilic fullerene or L-arginine functional groups were activated, L-arginine was modified to the surface of C60@AgNPs through chemical bonds, and the reaction solution was dialyzed and purified to obtain fullerene composite silver nanoparticles Arg-C60@AgNPs.

[0013] Furthermore, the hydrophilic fullerene is selected from aminofullerene C60-NH2, carboxylfullerene C60-COOH or hydroxyfullerene C60-OH; the silver salt is selected from silver nitrate, silver perchlorate or silver fluoride.

[0014] Furthermore, the mass ratio of nanosilver to fullerene in the fullerene composite silver nanoparticles Arg-C60@AgNPs is 0.025-0.2:1; and the concentration of the fullerene aqueous suspension is 0.1-100 mg / mL.

[0015] Furthermore, when the hydrophilic fullerene is aminofullerene 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 to 3:1, wherein 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, wherein the mass ratio of L-arginine to the C60@AgNPs complex is 0.05 to 5:1, and stirred sufficiently. After the reaction is completed, dialysis purification is performed to obtain Arg-C60@AgNPs.

[0016] Furthermore, 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 to 3:1, wherein 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, wherein the mass ratio of L-arginine to the C60@AgNPs complex is 0.05 to 5:1. After the reaction is completed, dialysis purification is performed to obtain Arg-C60@AgNPs.

[0017] Furthermore, when the hydrophilic fullerene is hydroxyfullerene C60-OH, DCC is added to an L-arginine solution with a concentration of 0.1-10 mg / mL to activate the carboxyl group, wherein the mass ratio of DCC to L-arginine is 1.2-1.5; then, the C60@AgNPs complex and 0.1 to 0.5 times the mass equivalent of L-arginine DMAP are added to the L-arginine reaction solution, wherein the mass ratio of L-arginine to the C60@AgNPs complex is 0.05 to 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.

[0018] In a third aspect, an embodiment of the present invention provides a use of nitric oxide-producing fullerene-composite silver nanoparticles or nitric oxide-producing fullerene-composite silver nanoparticles prepared by the above preparation method in the preparation of antibacterial or anti-infective drugs.

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

[0020] The present invention provides a nitric oxide-producing fullerene composite silver nanoparticle Arg-C60@AgNPs, which is designed based on a variety of inflammatory factors and oxidative stress signals in the wound infection microenvironment, utilizes the high affinity and multifunctionality of fullerene hybrid silver nanoparticles, forms Arg-C60@AgNPs nanoparticles through self-assembly, realizes the synergistic therapeutic effect of photodynamic therapy, NO gas therapy and AgNPs, 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. The present invention adopts a simple, efficient and green chemical reaction 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, no drug resistance, and strong biofilm penetration, overcomes the problems of drug resistance, poor biofilm penetration, and large toxic and side effects of traditional antibacterial drugs, and provides a new strategy and reference for wound infection, refractory wound infection and biofilm-related anti-infection treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative labor.

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

[0023] Figure 2 The particle size potential and characteristic absorption spectrum of Arg-C60@AgNPs prepared in Example 1;

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

[0025] Figure 4 Schematic diagram of the in vitro anti-MRSA activity of Arg-C60@AgNPs prepared in Example 1 determined by the coating plate method;

[0026] Figure 5 Schematic diagram of the in vitro anti-biofilm experiment of Arg-C60@AgNPs prepared in Example 1;

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

[0028] Figure 7 This is an evaluation of the effect of Arg-C60@AgNPs prepared in Example 1 in treating MRSA infection in mouse skin wounds. DETAILED DESCRIPTION

[0029] The present invention is further described below with reference to the embodiments but is not intended to limit the present invention.

[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, ultrasonically dispersed, and 50 mL of ethylenediamine was slowly added. After stirring at room temperature for 0.5 h, the mixture was allowed to stand and the supernatant was discarded. The remaining liquid was 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 purification by dialysis (the molecular weight cutoff of the dialysis bag was 3500), the solid C60-NH2 was obtained by freeze drying.

[0033] (2) Preparation of C60@AgNPs

[0034] Take 10 mg of C60-NH2 and disperse it in 5 mL of deionized water. Add 4 mL of silver nitrate solution (2.425 mM). After stirring for 4 h, dialysis was performed to purify the complex (the molecular weight cutoff of the dialysis bag was 3500) to obtain C60@AgNPs.

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

[0036] 7.5 mg EDC and 3 mg NHS were added to 5 mL of L-Arg aqueous solution (1 mg / mL). After stirring at room temperature for 4 h, 5 mL of C60@AgNPs suspension (1 mg / mL) was added. After stirring for 18 h, Arg-C60@AgNPs (dialysis bag cut-off molecular weight was 3500) was obtained by dialysis purification.

[0037] Embodiment 1-2:

[0038] (1) Preparation of C60-NH2

[0039] 50 mg of fullerene C60 was added to 50 mL of ethylenediamine, and ultrasonically dispersed. After stirring at room temperature for 24 h under nitrogen protection, the reaction solution was filtered with a 0.22 μm filter membrane, and ethanol was added for rotary evaporation. The solid was washed with deionized water for 3 times, and the pH was adjusted to about 6.5 with dilute nitric acid. After purification by dialysis (the molecular weight cutoff of the dialysis bag is 3500), freeze-drying was performed to obtain C60-NH2 solid.

[0040] (2) Preparation of C60@AgNPs

[0041] Take 10 mg of C60-NH2 and disperse it in 5 mL of deionized water. Add 1 mL of silver nitrate solution (2.425 mM). After stirring for 4 h, dialysis was performed to purify the complex (the molecular weight cutoff of the dialysis bag was 3500) to obtain C60@AgNPs.

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

[0043] 3 mg EDC and 6 mg NHS were added to 20 mL of L-Arg aqueous solution (0.1 mg / mL), stirred at room temperature for 4 h, and then 5 mL of C60@AgNPs suspension (1 mg / mL) was added. After stirring for 18 h, the mixture was purified by dialysis (the molecular weight cutoff of the dialysis bag was 3500) to obtain Arg-C60@AgNPs.

[0044] Embodiment 1-3:

[0045] (1) Preparation of C60-NH2

[0046] 200 mg of fullerene was added to 50 mL of toluene, ultrasonically dispersed, and 50 mL of ethylenediamine was slowly added dropwise. After stirring at room temperature for 0.5 h, the mixture was allowed to stand and the supernatant was discarded. The remaining liquid was stirred for 3 days under nitrogen protection. After the reaction was completed, ethanol was added for rotary evaporation, the solid was washed with deionized water 3 times, and freeze-dried to obtain C60-NH2 solid.

[0047] (2) Preparation of C60@AgNPs

[0048] Take 10 mg of C60-NH2 and disperse it in 5 mL of deionized water. Add 8 mL of silver nitrate solution (2.425 mM). After stirring for 4 h, dialysis was performed to purify the complex (the molecular weight cutoff of the dialysis bag was 3500) to obtain C60@AgNPs.

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

[0050] 15 mg EDC and 5 mg 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. After stirring for 18 h, the mixture was purified by dialysis (the molecular weight cutoff of the dialysis bag was 3500) to obtain Arg-C60@AgNPs.

[0051] Embodiment 1-4:

[0052] (1) Preparation of C60-OH

[0053] Take 1.2g of fullerene C60 and dissolve it in 500mL toluene in a glass flask. Move 10mL TBAH into the solution and keep stirring for 3min, then add 30mL 50% (w / v) NaOH in a dropwise manner. After stirring at room temperature for 2h under N2 protection, let stand for 10min for phase separation. Place the flask in a refrigerator at -20℃ for 2h, then pour out the organic phase. Add 200mL 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.

[0054] (2) Preparation of C60@AgNPs

[0055] 10 mg of C60-OH was dispersed in 5 mL of deionized water, and 4 mL of silver nitrate solution (2.425 mM) was added. 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.

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

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

[0058] Embodiment 1-5:

[0059] (1) Preparation of C60-OH

[0060] Take 0.5g of fullerene C60 and dissolve it in 250mL toluene in a glass flask. Move 4.1mL TBAH into the solution and keep stirring for 3min, then add 12.3mL 50% (w / v) NaOH in a dropwise manner. After stirring at room temperature for 2h under N2 protection, let stand for 10min for phase separation. Place the flask in a refrigerator at -20℃ for 2 hours, then pour out the organic phase. Add 200mLH2O 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, and 4 mL of silver nitrate solution (2.425 mM) was added. 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 DCC was added to 5 mL of L-Arg aqueous solution (2 mg / mL), and after stirring at room temperature for 4 h, 20 mL of C60@AgNPs suspension (2 mg / mL) and 5 mg of DMAP were added. After stirring for 18 h, the mixture was purified by dialysis (the molecular weight cutoff of the dialysis bag was 3500) to obtain Arg-C60@AgNPs.

[0065] Embodiment 1-6:

[0066] (1) Preparation of C60-OH

[0067] Take 1g of fullerene C60 and dissolve it in 400mL toluene in a glass flask. Move 9.6mL TBAH into the solution and keep stirring for 3min, then add 25mL 50% (w / v) NaOH in a dropwise manner. After stirring at room temperature for 2h under N2 protection, let stand for 10min for phase separation. Place the flask in a refrigerator at -20℃ for 2h, then pour out the organic phase. Add 200mL 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 and purify in a desiccator to obtain C60-OH solid.

[0068] (2) Preparation of C60@AgNPs

[0069] 10 mg of C60-OH was dispersed in 5 mL of deionized water, and 4 mL of silver nitrate solution (2.425 mM) was added. 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.

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

[0071] 37.5 mg DCC was added to 5 mL of L-Arg aqueous solution (5 mg / mL), and after stirring at room temperature for 4 h, 20 mL C60@AgNPs suspension (5 mg / mL) and 7.5 mg DMAP were added. After stirring for 18 h, the mixture was purified by dialysis (the molecular weight cutoff of the dialysis bag was 3500) to obtain Arg-C60@AgNPs.

[0072] Embodiment 1-7:

[0073] (1) Preparation of C60-COOH

[0074] Weigh 200 mg of C60 and add it to 100 mL of o-dichlorobenzene, and sonicate for 30 min to dissolve it completely. Weigh 247 mg of sarcosine and 420 mg of p-acylbenzoic acid and add them to C60, and sonicate for 60 min. React at 135 ° C with nitrogen protection for 22 h. When the reaction solution is wine red, stop heating. After purification by dialysis (cutoff molecular weight is 3500), cool, filter, and dry at low temperature to obtain C60-COOH solid.

[0075] (2) Preparation of C60@AgNPs

[0076] 10 mg of C60-COOH was dispersed in 5 mL of deionized water, and 4 mL of silver nitrate solution (2.425 mM) was added. 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.

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

[0078] 1.5 mg EDC and 3 mg NHS were added to 5 mL of C60@AgNPs aqueous solution (1 mg / mL). After stirring at room temperature for 4 h, 10 mL of L-Arg aqueous solution (2.5 mg / mL) was 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.

[0079] Embodiment 1-8:

[0080] (1) Preparation of C60-COOH

[0081] Weigh 100 mg of C60 and add it to 50 mL of o-dichlorobenzene, sonicate for 30 min to dissolve it completely, weigh 120 mg of sarcosine and 210 mg of p-acylbenzoic acid and add them to C60, sonicate for 60 min, react at 135 ° C and N2 protection for 22 h, stop heating when the reaction solution is wine red, dialysis purification (cutoff molecular weight is 3500), cool filter and low-temperature drying to obtain C60-COOH solid.

[0082] (2) Preparation of C60@AgNPs

[0083] 10 mg of C60-COOH was dispersed in 5 mL of deionized water, and 2 mL of silver nitrate solution (2.425 mM) was added. 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.

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

[0085] 3 mg EDC and 1 mg NHS were added to 5 mL of C60@AgNPs aqueous solution (2 mg / mL). After stirring at room temperature for 4 h, 0.5 mL of L-Arg aqueous solution (1 mg / mL) was added. After stirring for 18 h, the mixture was purified by dialysis (the molecular weight cutoff of the dialysis bag was 3500) to obtain Arg-C60@AgNPs.

[0086] Embodiment 1-9:

[0087] (1) Preparation of C60-COOH

[0088] Weigh 150 mg of C60 and add it to 75 mL of o-dichlorobenzene, and sonicate for 30 min to dissolve it completely. Weigh 180 mg of sarcosine and 315 mg of p-acylbenzoic acid and add them to C60, and sonicate for 60 min. React at 135 ° C and N2 protection for 22 h. When the reaction solution is wine red, stop heating. After dialysis purification (cutoff molecular weight is 3500), cool, filter, and dry at low temperature to obtain C60-COOH solid.

[0089] (2) Preparation of C60@AgNPs

[0090] 10 mg of C60-COOH was dispersed in 5 mL of deionized water, and 6 mL of silver nitrate solution (2.425 mM) was added. 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.

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

[0092] 3 mg EDC and 1 mg NHS were added to 5 mL of C60@AgNPs aqueous solution (2 mg / mL), stirred at room temperature for 4 h, and then 10 mL of L-Arg aqueous solution (1 mg / mL) was added. After stirring for 18 h, the mixture was purified by dialysis (the molecular weight cutoff of the dialysis bag was 3500) to obtain Arg-C60@AgNPs.

[0093] It should be noted that the present invention utilizes the weak reducing property of the photosensitizer fullerene to reduce Ag in one step without adding other reducing agents. + , prepared hybrid nanoparticles with silver nanoparticles as the core and fullerene layers adsorbed on the surface. Then, the NO precursor L-arginine was covalently linked through the fullerene surface active group to construct photodynamic antibacterial nanoparticles Arg-C60@AgNPs based on NO enhancement. The particles are well dispersed, uniformly distributed, chemically stable, and the preparation method is simple and environmentally friendly. Under visible light excitation, ROS can be generated and L-arginine can be automatically oxidized to release NO. Combined nanosilver, photodynamic and gas therapy can play an antibacterial and anti-biofilm infection role and promote 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 using transmission electron microscopy and high-resolution transmission electron microscopy, and their chemical composition was analyzed by EDS. The results showed that the composite particles were nearly spherical, with a relatively regular shape and uniform distribution ( Figure 1A in the figure shows the transmission electron microscopy image of Arg-C60@AgNPs. A clear core-shell structure can be observed under a high-resolution microscope, with the outer layer being a fullerene adsorption layer ( Figure 1 Figure B shows a high-resolution transmission microscopy image of Arg-C60@AgNPs. Component analysis shows that the core is silver nanoparticles, and the nitrogen element proves that the surface modification of aminofullerene is successful ( Figure 1 C in FIG. 1 shows a schematic diagram of EDS analysis of Arg-C60@AgNPs). By utilizing the weak reducing property of fullerene, the present invention reduces silver nitrate to silver nanoparticles of uniform size by simple mixing and stirring. A silver nanocomposite with aminofullerene adsorbed on the surface is successfully obtained.

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

[0098] Take appropriate amounts of the prepared C60-NH2, C60@AgNPs and Arg-C60@AgNPs, dilute with deionized water, and measure the particle size and potential using a Malvern particle size analyzer. The average particle sizes of C60-NH2, C60@AgNPs and Arg-C60@AgNPs were 273.9±3.13nm, 51.2±1.59nm and 81.03±3.72nm, respectively. After the formation of the silver complex, the dispersibility of the aminofullerene was improved, the particle agglomeration was reduced, and the average particle size was reduced. After surface modification with arginine, the particle size of the complex increased slightly. The particles are positively charged, and the potential value is reduced to 29.5±1.1mV ( due to the introduction of arginine molecules. Figure 2 A).

[0099] UV-visible spectra showed that all synthesized C60@AgNPs and Arg-C60@AgNPs had almost identical plasmon resonance peaks around 420 nm ( Figure 2 B), demonstrating the production of silver nanoparticles with similar particle sizes.

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

[0101] The Arg-C60@AgNPs reaction solution was taken and arginine was separated and quantitatively determined by ultrafiltration and 8-hydroxyquinoline colorimetry, and the arginine binding rate and encapsulation rate were determined. The calculation formula is as follows:

[0102]

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

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

[0105] Using DPBF as a ROS probe, a 635 nm laser (50 mW / cm 2 ) irradiated with 10 μg / mL Arg-C60@AgNPs, and recorded the fluorescence spectra at different times ( Figure 3 A in the figure shows the relationship between illumination time and ROS generation. As the illumination time increases, ROS production gradually increases. Using a NO test kit, the ROS production at 635 nm laser (50 mW / cm 2 ) NO production after irradiation for 10 min. Figure 3 B in the figure shows the NO generation curve under illumination conditions with different complex concentrations. As the concentration of Arg-C60@AgNPs increases, the NO generation increases. It can be seen that the Arg-C60@AgNPs prepared by the present invention can not only effectively generate ROS to exert antibacterial effects, but also further oxidize arginine, effectively release NO, and play 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 the spread plate method. 250 μL of 1×10 5 CFU / mL of MRSA bacterial solution was mixed with the above antibacterial preparations at different concentrations, incubated on a shaker at 37°C for 1 h, and then treated with light (635 nm, 50 mW / cm 2 ) for 10 min or avoid light. Take 100 mL from each well and spread it on LB agar plate, continue incubation for 18 h, and take photos to record the colony formation ( Figure 4 ). Without light, C60-NH2 has almost no effect on colony formation, because Ag + In the presence of red light, 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-illuminated group. The number of colonies was significantly reduced, and the antibacterial activity was concentration-dependent. Compared with C60-NH2 producing ROS, C60@AgNPs and Arg-C60@AgNPs produced ROS through Ag, respectively. + The combined effect of MRSA and NO further improved the anti-MRSA activity. This result was consistent with the MIC and MBC results (Table 1). MRSA bacterial solution was inoculated in a 96-well plate and antibacterial particles were added. After dilution, the final concentration of the bacterial solution was 5×10 4 CFU / mL. Illumination group 635nm, 50mW / cm 2After 10 minutes of light treatment, the culture was continued for 18 hours, and the MIC value was read by observing the turbid state of the solution. After the MIC determination was completed, the mixed solution was taken out from the 96-well plate and spread on the LB agar plate. The plate was incubated for 18 hours, and the minimum preparation concentration at which no colonies grew was observed, which was the MBC. The above results prove that the Arg-C60@AgNPs prepared by the present invention can enhance the photodynamic antibacterial effect of AgNPs and fullerenes through NO gas under light conditions.

[0108] Table 1. MIC and MBC values ​​of fullerene-composite silver nanoparticles against MRSA strains

[0109]

[0110] Example 4: In vitro anti-biofilm test

[0111] 2 mL of MRSA bacterial solution (1×10 6 CFU / mL) were added to a 6-well plate and cultured at 37°C for 48 h. TSB medium was replaced every 24 h. After MRSA biofilm formed on the bottom of the plate, the medium in the wells was discarded and phosphate buffer, C60-NH2, C60@AgNPs or Arg-C60@AgNPs (40 μg / mL) were added to the wells. After incubation for 1 h, light treatment (635 nm, 50 mW / cm 2 ) for 10 min, and the non-illuminated group was kept away from light. The supernatant was discarded and washed three times. 4% paraformaldehyde was added for 15 min to fix. 0.1% crystal violet was used for staining for 15 min. After drying and taking pictures, 33% (v / v) acetic acid was added to dissolve the crystal violet. The absorbance was measured at 560 nm, and the biofilm removal rate was calculated by comparing with the blank control group.

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

[0113] Example 5: Cell scratch assay

[0114] 5×10 5 Mouse fibroblast L929 cells were seeded in a 6-well plate and scratched using a 200 μL pipette tip. The cells were then incubated with culture medium (containing 1% serum) containing C60-NH2, C60@AgNPs, and Arg-C60@AgNPs for 1 h and illuminated for 10 min (635 nm, 50 mW / cm2 The cells were cultured for 24 h, and the wound healing was recorded under a microscope. The scratch changes were analyzed using Image J software to 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 test, compared with the Arg-C60@AgNPs illumination group. ***p<0.005, ****p<0.001, fullerene has no significant effect on cell proliferation and migration. Silver nanocomposites can significantly reduce the width of scratches and increase the migration rate of fibroblasts under both illumination and non-illumination conditions due to the promotion of cell proliferation and migration by low-concentration, small-size silver nanoparticles. Arg-C60@AgNPs release NO under red light excitation, which further promotes cell migration and proliferation, and is beneficial to promote wound healing.

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

[0118] A BALB / c mouse skin wound model was established using a hole punch and 50 μL of 10 8 CFU / mL MRSA bacterial liquid caused infection. 100μL normal saline was dripped on the wound surface of the control group, and 100μL different preparation solutions (100μg / mL) were dripped on 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 (635nm, 50mW / cm 2 ), and the non-light-exposed group was protected from light. Body weight was recorded every day, and wound diameter was recorded by taking photos every two days. Image J software was used for image processing to simulate actual wound changes. Wound healing rate was calculated according to 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 illumination group was faster. Arg-C60@AgNPs also has an antibacterial effect when relying on AgNPs in non-photodynamic therapy. 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 at different times after treatment; Figure 7B in the figure shows the wound healing rate of each group at different times after treatment, **p<0.01, ****p<0.001. The in vivo data are consistent with the in vitro experimental results. Under the action of ROS, NO and AgNPs, Arg-C60@AgNPs has the strongest effect in treating mouse skin wound infection.

[0121] In summary, the Arg-C60@AgNPs constructed in the present invention can produce ROS and NO under visible light excitation, and combined with AgNPs, play a broad-spectrum antibacterial and anti-biofilm infection role, promote fibroblast proliferation and migration, and thus promote the repair and regeneration of bacterially infected wounds.

Claims

1. A fullerene composite silver nanoparticle producing nitric oxide, characterized in that: The nitric oxide-producing fullerene composite silver nanoparticles Arg-C60@AgNPs have silver nanoparticles as the core, the outer layer of the silver nanocore is adsorbed with fullerene, and the surface of the fullerene is modified with L-arginine.

2. The nitric oxide-producing fullerene composite silver nanoparticles according to claim 1, characterized in that: The particle size of the fullerene composite silver nanoparticles is 30 to 300 nm, and the potential is -30 to +50 mV.

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

4. A method for preparing fullerene composite silver nanoparticles producing nitric oxide, characterized in that: The preparation method comprises: Slowly add silver salt to the hydrophilic fullerene aqueous suspension, stir and dialyze at room temperature to obtain a C60@AgNPs composite; The hydrophilic fullerene or L-arginine functional groups were activated, L-arginine was modified to the surface of C60@AgNPs through chemical bonds, and the reaction solution was dialyzed and purified to obtain fullerene composite silver nanoparticles Arg-C60@AgNPs.

5. The method for preparing a nitric oxide-producing fullerene composite silver nanoparticle according to claim 4, characterized in that: The hydrophilic fullerene is selected from aminofullerene C60-NH2, carboxylfullerene C60-COOH or hydroxyfullerene C60-OH; the silver salt is selected from silver nitrate, silver perchlorate or silver fluoride.

6. The method for preparing a nitric oxide-producing fullerene composite silver nanoparticle according to claim 4, characterized in that: The mass ratio of nanosilver to fullerene in the fullerene composite silver nanoparticles Arg-C60@AgNPs is 0.025-0.2:1; the concentration of the fullerene aqueous suspension is 0.1-100 mg / mL.

7. The method for preparing a nitric oxide-producing fullerene composite silver nanoparticle according to claim 4, characterized in that: When the hydrophilic fullerene is aminofullerene 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 stirred at room temperature to activate the carboxyl group; then a C60@AgNPs complex is added, wherein the mass ratio of L-arginine to the C60@AgNPs complex is 0.05-5:1, and stirred sufficiently. After the reaction is completed, dialysis purification is performed to obtain Arg-C60@AgNPs.

8. The method for preparing a nitric oxide-producing fullerene composite silver nanoparticle according to claim 4, characterized in that: 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 to 3:1, wherein 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, wherein the mass ratio of L-arginine to the C60@AgNPs complex is 0.05 to 5:

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

9. The method for preparing a nitric oxide-producing fullerene composite silver nanoparticle according to claim 4, characterized in that: 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, wherein the mass ratio of DCC to L-arginine is 1.2-1.5; then, a C60@AgNPs complex and 0.1 to 0.5 times the mass equivalent of L-arginine DMAP are added to the L-arginine reaction solution, wherein the mass ratio of L-arginine to the C60@AgNPs complex is 0.05 to 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.

10. Use of the nitric oxide-producing fullerene composite silver nanoparticles according to any one of claims 1 to 3 or the nitric oxide-producing fullerene composite silver nanoparticles prepared by the preparation method according to any one of claims 4 to 9 in antibacterial or anti-infective drugs.

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