Nanoparticles with photo-thermal-photodynamic-gas synergistic sterilization performance and application thereof

Abstract

The invention provides a multifunctional antibacterial nano particle. The antibacterial nano particle is a cross-linked product of a raw material composition comprising the following components (A) to (C): (A) a quinonoid conjugated organic molecule as shown in the following formula (I): # imgabs0 # (B) an amphiphilic aldehyde group-containing block copolymer; and, (C) L-arginine. The antibacterial nano particle disclosed by the invention has a synergistic effect on the basis of a principle of photo-thermal sterilization, photodynamic sterilization and gas sterilization, has good effects of resisting bacteria, eliminating bacterial biofilms and promoting wound recovery, and is particularly suitable for treating drug-resistant bacterium infection such as methicillin-resistant staphylococcus (MRSA) infection.

Description

Technical Field

[0001] The present invention relates to the field of biomedical materials, and in particular to nanoparticles with photothermal-photodynamic-gas synergistic sterilization performance and applications thereof. Background Art

[0002] In recent years, the abuse and overuse of antibiotics have become a major challenge in the field of global public health, directly leading to the rapid increase of bacterial resistance. Among them, methicillin-resistant Staphylococcus aureus (MRSA) is a typical representative of drug-resistant bacteria, which shows extremely strong resistance to multiple antibiotics, making traditional antibiotic treatments gradually ineffective. MRSA infection not only increases the difficulty of clinical treatment, but also significantly increases the mortality rate and medical costs of patients. Therefore, it is urgent to develop new antibacterial strategies to deal with the threat of drug-resistant bacteria.

[0003] In this context, scientists are exploring new antibacterial treatment strategies, such as photothermal antibacterial therapy (PTAT), photodynamic antibacterial therapy (PDAT), and gas antibacterial therapy (GAT). Photothermal antibacterial therapy (PTAT): Use photothermal conversion materials (such as gold nanorods, graphene, etc.) to generate local high temperatures under near-infrared light irradiation, convert light energy into heat energy, directly cause thermal damage to bacterial cells, and thus kill bacteria. Photodynamic antibacterial therapy (PDAT): Use photosensitizers to produce reactive oxygen species (such as singlet oxygen) under specific wavelength light irradiation. These reactive oxygen species have strong oxidizing properties and can destroy bacterial cell membranes and intracellular biological molecules, leading to bacterial death. Gas antibacterial therapy (GAT): Use the strong oxidizing properties and diffusivity of gas molecules (such as nitric oxide, chlorine dioxide, etc.) to destroy the bacterial cell membrane and metabolic system, thereby achieving efficient sterilization. However, studies have shown that there are technical bottlenecks in improving the effect of a single treatment strategy. For example, photothermal heating to 40°C or above can effectively kill bacteria, but too high a temperature may cause irreversible damage to normal tissues, and bacteria will produce a stress response by producing heat shock protein (HSP70) to cope with photothermal damage; for example, the biofilm formed by drug-resistant bacteria will prevent ROS from entering the bacteria, resulting in poor treatment effects. Therefore, how to design a material system to further improve antibacterial properties, especially to eradicate bacterial biofilms, is an urgent problem to be solved. Summary of the invention

[0004] In view of this, the present invention proposes a synergistic antibacterial nanomaterial system based on the "photothermal-photodynamic-gas" principle. The system has good antibacterial, bacterial biofilm elimination and wound recovery effects, and is particularly suitable for the treatment of drug-resistant bacterial infections such as MRSA.

[0005] In a first aspect, the present invention provides an antibacterial nanoparticle, wherein the antibacterial nanoparticle is a cross-linked product of a raw material composition comprising the following components (A) to (C):

[0006] (A) A quinone-type conjugated organic molecule as shown in the following formula (I):

[0007]

[0008] (B) an amphiphilic aldehyde-containing block copolymer; and,

[0009] (C) L-arginine;

[0010] in:

[0011] said n=5-200; and,

[0012] The amphiphilic aldehyde-containing block copolymer comprises at least one hydrophilic block and at least one hydrophobic block, and the amphiphilic aldehyde-containing block copolymer comprises at least one aldehyde group bonded to the hydrophobic block.

[0013] Preferably, in the antibacterial nanoparticles, the raw material composition comprises:

[0014] (A) 0.1-20 wt% of quinone-type conjugated organic molecule represented by formula (I):

[0015] (B) 20-80 wt % of an amphiphilic aldehyde-containing block copolymer; and

[0016] (C) L-arginine 5-60 wt %.

[0017] Preferably, in the antibacterial nanoparticles, the raw material composition comprises:

[0018] (A) 1-10 wt% of a quinone-type conjugated organic molecule represented by formula (I):

[0019] (B) 30-70 wt % of an amphiphilic aldehyde-containing block copolymer; and,

[0020] (C) L-arginine 20-50 wt%.

[0021] Preferably, in the antibacterial nanoparticles, the n value of the quinone-type conjugated organic molecule represented by formula (I) is 10-100.

[0022] Preferably, in the antibacterial nanoparticles, the n value of the quinone-type conjugated organic molecule represented by formula (I) is 10-40.

[0023] Preferably, in the antibacterial nanoparticles, the number average molecular weight of the amphiphilic aldehyde-containing block copolymer is 3,000 to 50,000.

[0024] Preferably, in the antibacterial nanoparticles, in the amphiphilic aldehyde-containing block copolymer, the hydrophobic block has a number average molecular weight of 1000-30000, and is obtained by polymerizing a monomer composition comprising at least one aldehyde-containing hydrophobic monomer.

[0025] Preferably, in the antibacterial nanoparticles, the aldehyde-containing hydrophobic monomer has a structure of the following formula (II), and the degree of polymerization of the polymerized repeating unit of the hydrophobic monomer is 5-200:

[0026]

[0027] in:

[0028] R 1 is H or methyl;

[0029] R 2 Selected from C 1-12 Alkylene, C 3-12 Cycloalkylene, C 5-14 Arylene, C 5-8 Heterocyclylene, -C 1-12 Alkyl-C 3-8 Cycloalkyl-, -C 1-6 Alkoxy-C 1-6 Alkyl, -C 1-12 Alkoxy-C 3-8 Cycloalkyl-, -C 1-12 Alkyl-C 5-8 Heterocyclic-, -C 1-12 Alkoxy-C 5-8 Heterocyclic-, -C 1-12 Alkyl-C 5-14 Aryl- or -C 1-12 Alkoxy-C 5-14 Aryl-; the aryl group is selected from phenyl, naphthyl, pyrrolyl, imidazolyl, pyridyl, pyrimidinyl, furanyl or thienyl, and the heterocyclic group is selected from tetrahydrofuranyl, piperidinyl, hexahydropyridinyl or piperazinyl;

[0030] Preferably, the R 2 Selected from C 1-8 Alkylene, C 5-10 Arylene, -C 1-8 Alkyl-C 5-10 Aryl- or -C 1-8 Alkoxy-C 5-10 Aryl-;

[0031] Preferably, the R 2 Selected from -C 1-6 Alkoxy-C 5-6 Aryl-;

[0032] Preferably, the R 2 Selected from -ethoxy-phenyl-;

[0033] Preferably, the structural formula of formula (II) is:

[0034]

[0035] More preferably, the monomer composition comprising at least one hydrophobic monomer containing an aldehyde group is mixture.

[0036] Preferably, in the antibacterial nanoparticles, in the amphiphilic aldehyde-containing block copolymer, the hydrophilic block is selected from at least one of polyethylene glycol, poly(meth)acrylic acid, poly(meth)acrylamide, poly-N-isopropylacrylamide, poly(meth)acrylate substituted with a hydrophilic group, or polyvinylpyridine; the hydrophilic group is selected from amino, carboxyl, di(C 1-5 alkyl)amino, hydroxyethyl, hydroxypropyl, diethylene glycol, triethylene glycol, tetraethylene glycol or C 10-30 of polyoxyethylene.

[0037] Preferably, in the amphiphilic aldehyde-containing block copolymer, the hydrophilic block is polyethylene glycol.

[0038] Preferably, the mass ratio of the hydrophilic block to the hydrophobic block in the amphiphilic aldehyde-containing block copolymer is 2:8 to 8:2.

[0039] In a second aspect, the present invention provides a method for preparing the antibacterial nanoparticles according to any one of the above items, the method comprising the following steps:

[0040] (S1) mixing the quinone-type conjugated organic molecule represented by the formula (I) and the amphiphilic aldehyde-containing block copolymer in an organic solvent, and then adding an aqueous solvent to assemble nanoparticles;

[0041] (S2) L-arginine is added to the mixed solution of step S1 to cross-link the reaction system.

[0042] In a third aspect, the present invention provides a preparation having antibacterial, bacterial biofilm elimination and wound recovery effects, comprising the antibacterial nanoparticles described in any one of the above items.

[0043] Preferably, the wound in the wound-recovery preparation is a wound caused by bacterial infection, or the wound is infected by bacteria after it is caused, or the wound has a potential risk of bacterial infection.

[0044] Preferably, the bacteria targeted by the antibacterial, bacterial biofilm-eliminating and wound-recovering preparations are methicillin-resistant Staphylococcus aureus (MRSA).

[0045] In a fourth aspect, the present invention provides a biomedical material comprising the antibacterial nanoparticles described in any one of the above items.

[0046] Preferably, the biomedical material is a wound dressing, a hand sanitizer or an antibacterial coating.

[0047] In a fifth aspect, the present invention provides a quinone-type organic molecule, the structure of which is shown in the following formula (I):

[0048]

[0049] In a sixth aspect, the present invention provides the use of the quinone organic molecule of the aforementioned formula (I) in the preparation of antibacterial nanoparticles, wherein the antibacterial nanoparticles have photothermal, photodynamic and gas synergistic antibacterial, bacterial biofilm elimination and wound recovery properties.

[0050] Preferably, the raw materials for preparing the antibacterial nanoparticles contain L-arginine.

[0051] Preferably, the antibacterial, bacteriostatic or bactericidal bacteria are methicillin-resistant Staphylococcus aureus (MRSA).

[0052] The antibacterial nanoparticles of the present invention have a structure and mechanism as follows Figure 1 As shown. Among them, quinone-type conjugated organic molecules and amphiphilic aldehyde-containing block copolymers self-assemble into nanoparticles, quinone-type conjugated organic molecules are uniformly dispersed and wrapped in amphiphilic aldehyde-containing block copolymers, L-arginine is bonded through the amino group in its structure and the aldehyde group in the amphiphilic aldehyde-containing block copolymer, so that the nanoparticles are cross-linked, further improving the stability of the antibacterial nanoparticles, and obtaining a nanomaterial system with synergistic sterilization based on the principle of "photothermal sterilization-photodynamic sterilization-gas sterilization". Specifically:

[0053] Generally, the biofilm microenvironment formed by wound bacteria due to infection is acidic and hydrogen peroxide is highly expressed. By contacting the wound surface with the antibacterial nanoparticles of the present invention and irradiating them with near-infrared light, the imine bond of arginine bonded to the antibacterial nanoparticles is broken in a weak acid environment, and arginine is released and catalyzed by nitric oxide enzyme and hydrogen peroxide at the site of inflammation to produce nitric oxide gas to achieve gas sterilization, while the cross-linked structure of the nanoparticles is released; then the quinone-type conjugated organic molecules have more opportunities to contact the light source, produce a photothermal effect and rapidly increase the system temperature to achieve photothermal sterilization; in addition, in this process, the quinone-type conjugated organic molecules produce active oxygen under light to achieve photodynamic sterilization, and the above-mentioned active oxygen further promotes the oxidation of arginine to produce nitric oxide, and the two promote each other to self-accelerate the sterilization process. The amphiphilic aldehyde-containing block copolymer effectively assembles and encapsulates quinone-type conjugated organic molecules to control the system temperature from being too high to avoid damaging the normal system, while also improving the photothermal stability of the system and extending the service life of the material; the presence of arginine and the process of oxidation to nitric oxide effectively reduce the expression of heat shock protein (HSP70) in the system, preventing or alleviating the bacterial stress response caused by the photothermal effect, and further improving the bactericidal performance. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 : Schematic diagram of the inventive concept of the present invention.

[0055] Figure 2 : H-NMR spectrum of P2 molecule of Synthesis Example 3.

[0056] Figure 3 : Schematic diagram of the molecular weight results of the P2 molecule of Synthesis Example 3.

[0057] Figure 4 : UV absorption quasi-curve (a) and mass extinction coefficient (b) of P2 molecule of Synthesis Example 3.

[0058] Figure 5 : Cyclic voltammetry curve of P2 molecule of Synthesis Example 3.

[0059] Figure 6 : Photothermal cycling performance of molecules P1 to P6 of Synthesis Examples 1-6.

[0060] Figure 7 : Schematic diagram of the assembly of antibacterial nanoparticles in Example 1 of the present invention.

[0061] Figure 8 : DLS (a), TEM (b) and SEM (c) results of the antibacterial nanoparticles of Example 1 of the present invention.

[0062] Fig. 9 : DLS (a) and TEM (b) results of the antibacterial nanoparticles of Comparative Example 1 of the present invention.

[0063] Fig.10 : DLS of the antibacterial nanoparticles of Example 1 of the present invention after storage for 1 month.

[0064] Fig.11 : Ultraviolet absorption curve (a) and mass extinction coefficient (b) of the antibacterial nanoparticles of Example 1 of the present invention.

[0065] Fig.12 : Photothermal performance of the antibacterial nanoparticles of Example 1 of the present invention: (a) at different concentrations; (b) at different powers.

[0066] Fig.13 : Schematic diagram of the ESR results of different types of active oxygen species of the antibacterial nanoparticles of Example 1 of the present invention: HO·(a); 1 O 2 (b); ·O 2- (c).

[0067] Fig.14 : Schematic diagram of the generation of hydroxyl radicals (a) and NO (b) by nanoparticles of the embodiments of the present invention and the comparative example under light conditions.

[0068] Fig.15 : Photothermal stability (a) and photothermal conversion efficiency (b) of the antibacterial nanoparticles of Example 1 of the present invention.

[0069] Fig.16 : Antibacterial properties of antibacterial nanoparticles: bacterial growth at different concentrations and treatment groups (A); light-induced temperature increase after AQM, ABP@AQM and ABPL@AQM were given to planktonic MRSA (B) and quantification (C); expression fluorescence (D) and quantification (E) of reactive oxygen species (ROS) in MRSA after treatment with different groups; expression level (F) and quantification (G) of HSP70 after treatment with different groups; schematic diagram of the bactericidal mechanism of the antibacterial nanoparticles of the present invention (H).

[0070] Fig.17 :A. CLSM images of live / dead planktonic MRSA in different treatment groups and under illumination (scale bar: 50 μm); B. Colony plating of MRSA in different treatment groups and under illumination conditions; C. Crystal violet staining method was used to evaluate the antibiofilm activity of samples against MRSA biofilm; D. Confocal laser scanning microscopy (CLSM) images of live / dead staining of MRSA biofilm (scale bar: 100 μm); E. Scanning electron microscopy (SEM) images of MRSA biofilms treated in different groups (scale bar: 2 μm).

[0071] Fig.18:A. Schematic diagram of bacterial wound infection model and drug administration scheme; B. Representative real-time thermal imaging of mice in each treatment group after treatment (under NIR irradiation, 650nm, 1W cm -2 ); C. The bacterial counts in the wounds of mice were quantified by the standard plate counting method after treatment in each treatment group; D, E and F. Photos of the wounds of mice in each treatment group after treatment; G: The wound sizes of mice in each treatment group after treatment; H. CFU quantification of the wounds of mice in each treatment group after treatment; I. FH&E staining after treatment with different formulas, Masson trichrome staining of skin tissue, and IL-6 and TNF-α immunohistochemical staining (scale bar: 150 μm).

[0072] Fig.19 : A. Changes in blood routine and serum biochemical indexes of mice in each treatment group after treatment; B. H&E staining images of heart, liver, spleen, lung and kidney of mice in each treatment group after treatment (scale bar: 100 μm). DETAILED DESCRIPTION

[0073] In the following description, a large number of specific details are provided to provide a more thorough understanding of the present invention. However, it is apparent to those skilled in the art that the present invention can be implemented without one or more of these details. In other examples, in order to avoid confusion with the present invention, some technical features well known in the art are not described.

[0074] The present invention is described in detail below with reference to the definitions of terms:

[0075] I Antimicrobial Nanoparticles

[0076] The antibacterial nanoparticles of the present invention are cross-linked products of a raw material composition comprising the following components (A) to (C):

[0077] (A) A quinone-type conjugated organic molecule as shown in the following formula (I):

[0078]

[0079] (B) an amphiphilic aldehyde-containing block copolymer; and,

[0080] (C) L-arginine.

[0081] A Quinone-type conjugated organic molecule

[0082] Quinone-type conjugated organic molecules, by designing the conjugated main chain structure of quinone-type molecules, make the molecules have different properties, and thus have different temperature curves and temperature stability in photothermal conversion. As shown in Synthesis Examples 2-7 below, the inventors found that the alkynyl group in the molecule of formula (I) can make formula (I) have suitable energy, which is more suitable as a photothermal conversion material for photothermal gas sterilization system than other conjugated units such as benzene, alkene, thiophene, benzodithiazole, etc.

[0083] Unless otherwise specified, % in the present invention refers to wt %.

[0084] The compound of formula (I) can be prepared by the following reaction formula, and the specific preparation process is conventional in the art:

[0085] first step:

[0086] Step 2:

[0087] Step 3:

[0088] Step 4:

[0089]

[0090] Step 5:

[0091]

[0092] The value of n in the quinone-type conjugated organic molecule of the present invention is 5-200. In some embodiments, the value of n can be 5-100, 10-80, 10-50, etc., for example, 10, 20, 30, 40, 50, 60, 70, 80, etc.

[0093] In the raw material composition, the content of the conjugated organic molecule is 0.1-20wt%. In some specific embodiments, the above content can be 1-10wt% or 3-7wt%, for example, 1wt%, 2wt%, 3wt%, 4wt%, 5wt%, 6wt%, 7wt%, 8wt%, 9wt%, 10wt%, 12wt%, 15wt% or 18wt%.

[0094] B Amphiphilic block copolymer containing aldehyde groups

[0095] The definition of the amphiphilic block copolymers of the present invention is known to those skilled in the art, and generally such polymers include at least one hydrophilic block and at least one hydrophobic block. In particular, the amphiphilic block copolymers of the present invention contain aldehyde groups, and the aldehyde groups are bonded to the hydrophobic blocks of the amphiphilic aldehyde-containing block copolymers. Preferably, the aldehyde groups are substantially uniformly distributed on the hydrophobic blocks of the amphiphilic aldehyde-containing block copolymers.

[0096] In some embodiments, the amphiphilic aldehyde-containing block copolymer of the present invention is a diblock, triblock, tetrablock or multiblock, etc., and the structure can be a linear, branched or star-shaped structure, and the hydrophilic block in the copolymer can be 1, 2, 3 or more, and the hydrophobic block can be 1, 2, 3 or more. In some specific embodiments, for example, the amphiphilic aldehyde-containing block copolymer of the present invention is a diblock copolymer AB formed by connecting 1 hydrophilic block A and 1 hydrophobic block B, or a triblock copolymer ABA formed by connecting 2 hydrophilic blocks A and 1 hydrophobic block B, or a three-arm star copolymer (BA) formed by connecting 3 hydrophilic blocks A and 3 hydrophobic blocks B. 3 wait.

[0097] The hydrophilic block and the hydrophobic block of the present invention have conventional definitions known in the art. Generally, the hydrophilic block is water-soluble or water-dispersible, and may contain alkoxy chains or hydrophilic substituents such as hydroxyl, carboxyl, amino, dimethylamino, etc. in the structure; generally, the hydrophobic block is water-insoluble or water-indispersible, and may contain alkyl, cycloalkyl, aryl, heterocyclic, etc. in the structure, and generally does not contain hydrophilic substituents.

[0098] Those skilled in the art will appreciate that the amphiphilic aldehyde-containing block copolymer of the present invention can be prepared by conventional polymerization methods in the art, such as (living) free radical polymerization, condensation polymerization, ring-opening polymerization, cationic polymerization or anionic polymerization, depending on the type of polymerizable monomers.

[0099] In some embodiments, in the amphiphilic aldehyde-containing block copolymer of the present invention, the ratio of the hydrophilic block to the hydrophobic block is 2:8 to 8:2, or 3:7 to 7:3, or 4:6 to 6:4, etc., and after being coated with quinone-type conjugated organic molecules, it can be better assembled into nanoparticles in water.

[0100] In some embodiments, the number average molecular weight of the amphiphilic aldehyde-containing block copolymer of the present invention is 2000-50000, 3000-20000 or 5000-10000, for example, 5000, 6000, 7000, 8000, 9000 or 10000. The number average molecular weight of the hydrophilic block is 1000-30000, 2000-10000 or 3000-8000, for example, 3000, 4000, 5000, 6000, etc.; the number average molecular weight of the hydrophobic block is 1000-30000, 2000-10000 or 3000-8000, for example, 3000, 4000, 5000, 6000, etc.

[0101] In the raw material composition, the content of the amphiphilic aldehyde-containing block copolymer is 20-80wt%, 30-70wt% or 40-60wt%, for example, 35wt%, 45wt%, 50wt%, 55wt% or 65wt%.

[0102] In some specific embodiments, a typical hydrophilic block can be polyethylene glycol (2000, 4000, 5000, 6000 or 8000, etc.), polyacrylic acid, polymethacrylic acid, polyacrylamide, polymethacrylamide, poly-N-isopropylacrylamide, polyhydroxyethyl (meth)acrylate, polyhydroxypropyl (meth)acrylate, poly-2-vinylpyridine, poly-4-vinylpyridine, polyethylene glycol methacrylate, poly-N,N-dimethylaminoethyl methacrylate, 2-(2-methoxyethoxy)ethyl methacrylate, or a combination thereof.

[0103] In some embodiments, without affecting the hydrophilicity of the block, the hydrophilic block of the present invention may also contain any polymeric segment obtained by polymerization of other monomers known in the art, and the content of the other polymeric segment does not exceed 75%, or does not exceed 50%, or preferably does not exceed 20% of the total mass of the hydrophilic block.

[0104] The aldehyde groups of the amphiphilic aldehyde-containing block copolymer of the present invention may be derived from the polymerized monomers of the hydrophobic block, or the aldehyde groups may be bonded to the hydrophobic block of the copolymer through chemical modification after polymerization.

[0105] In some embodiments, the hydrophobic block of the present invention is obtained by polymerizing a monomer composition comprising at least one hydrophobic monomer containing an aldehyde group. The repeating unit obtained by polymerizing the hydrophobic monomer containing an aldehyde group has a degree of polymerization of 5-200, 10-150 or 15-100, etc., for example, 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50, etc.

[0106] In some specific embodiments, the aldehyde-containing hydrophobic monomer of the present invention has a structure of the following formula (II):

[0107]

[0108] The R 1 and R 2 The definition of is as mentioned above.

[0109] For example, a typical hydrophobic monomer conforming to formula (II) is:

[0110]

[0111] In some embodiments, without affecting the hydrophobicity of the block, the hydrophobic block of the present invention may also contain any polymeric segment obtained by polymerization of other monomers known in the art, and the content of the other polymeric segment does not exceed 75%, or does not exceed 50%, or does not exceed 20% of the total mass of the hydrophobic block.

[0112] In one embodiment, the monomer composition for preparing the hydrophobic block of the present invention is A mixture of The mass ratio of can be 1:5 to 5:1, 1:3 to 3:1, 1:2 to 2:1, 1:1, 2:1, 3:1, 4:1, 5:1, 1:5, 1:4, 1:3 or 1:2, etc.

[0113] C L-arginine

[0114] In the raw material composition for preparing the antibacterial nanoparticles of the present invention, the content of L-arginine is 5-60wt%, 10-50wt% or 20-40wt%, for example, it can be 10wt%, 15wt%, 20wt%, 25wt%, 30wt%, 35wt%, 40wt% or 45wt%.

[0115] D Preparation method

[0116] The method for preparing antibacterial nanoparticles of the present invention comprises the following steps:

[0117] (S1) mixing the quinone-type conjugated organic molecule represented by the formula (I) and the amphiphilic aldehyde-containing block copolymer in an organic solvent, and then adding an aqueous solvent to assemble nanoparticles;

[0118] (S2) L-arginine is added to the mixed solution of step S1 to cross-link the reaction system.

[0119] Preferably, the organic solvent in step S1 is selected from at least one of tetrahydrofuran, DMSO or DMF.

[0120] Preferably, the mass ratio of the quinone-type conjugated organic molecule to the amphiphilic aldehyde-containing block copolymer in step S1 is 1:3-1:50, preferably 1:5-1:20, and more preferably 1:5-1:10.

[0121] Preferably, the aqueous solvent is water, and the volume ratio of the organic solvent to the aqueous solvent in step S1 is 1:1-1:20, preferably 1:5-1:10.

[0122] Preferably, in step S2, the mass ratio of L-arginine to the quinone-type conjugated organic molecule is 1:1-20:1, preferably 5:1-10:1.

[0123] In some specific embodiments, the steps S1 and S2 are reacted at room temperature. The reaction time of step S1 is 2-10 hours, and the reaction time of step S2 is 1-10 hours.

[0124] II Application of Antimicrobial Nanoparticles

[0125] The antibacterial nanoparticles of the present invention are suitable as materials for eradicating bacteria, eliminating bacterial biofilms and wound recovery, and are used to prepare various preparations or biomedical materials for such application environments, such as wound dressings, hand sanitizers, antibacterial coatings, etc. In the application process, the light source is preferably a red light, near infrared light or infrared light source, and the wavelength is preferably 600-1200nm, preferably 600-900nm, further preferably 600-700nm, and most preferably 630-670nm.

[0126] The present invention is further described in detail below through specific embodiments:

[0127] Example

[0128] raw material

[0129] The reagents and drugs not mentioned in the present invention were purchased from Aldrich and used directly.

[0130] Test Method

[0131] Nuclear Magnetic Resonance (NMR) Spectroscopy: 1 H-NMR measurements (400 MHz) were performed on a Bruker DMX-400 spectrometer using CDCl as the medium. 3 or DMSO-d 6 .

[0132] Size Exclusion Chromatography (SEC) Measurement: Molecular weight and molecular weight distribution were measured using a Waters 150C gel permeation chromatograph (GPC) with two Ultrastyragel columns connected in series and a Wyatt Optilab DSP RI detector at 25°C, THF as the eluent, and a flow rate of 0.5 mL / min. Molecular weight and molecular weight distribution were calibrated using monodisperse polystyrene standards.

[0133] Cyclic voltammetry (CV) was performed on a CHI1200C electrochemical workstation, with glassy carbon, platinum and silver wire as working electrode, counter electrode and pseudo-reference electrode, respectively. Tetrabutylammonium hexafluorophosphate (0.1 M) was used as the electrolyte, ferrocene / ferrocene (Fc / Fc+) redox couple was used as the external standard, and the scan rate was 100 mV s- 1 The samples were prepared in MeCN solution. ox ) or reduction potential (E red ) and the oxidation potential of ferrocene (E HOMO =-(4.8-ed)eV, E LUMO The HOMO and LUMO energy levels of the compound were calculated by the difference between

[0134] Transmission electron microscopy (TEM): TEM observations were performed on a Thermo Fisher Talos F200C TEM at an accelerating voltage of 200 kV. The sample was prepared by depositing a drop of polymer solution in methanol on a copper grid and then drying it overnight at room temperature. The electron dose at different magnifications was: 1.9 e·nm -2 (1700×), 7e·nm -2 (4300×), 109e·nm -2 (17500×), 655e·nm -2 (28000×), 3550e·nm -2 (73000×).

[0135] Scanning Electron Microscopy (SEM): SEM images were acquired on a Carl Zeiss microscope GeminiSEM 300. The sample for SEM measurements was prepared by placing a drop of the methanol nanoparticle solution on a silicon wafer and then drying it overnight at room temperature.

[0136] Dynamic Light Scattering (DLS): The hydrodynamic size of the self-assemblies was characterized using a Malvern Z90 Zetasizer equipped with a 633 nm He-Ne laser and an avalanche photodiode detector. The scattered light was detected at an angle of 90° and the size and distribution of the scattered light were analyzed. The concentration of the carbohydrate-based compositions used for DLS measurements was in the range of 1 mg / mL.

[0137] UV-Vis Spectra: UV-Vis spectra were recorded on a SPECORD 250 equipped with an automatic temperature control accessory.

[0138] Fluorescence spectrum: Fluorescence spectrum was collected using a Hitachi F-4600 fluorescence spectrometer with a slit width of 5 nm, a scanning rate of 2400 nm / min, and an automatic temperature control accessory. The excitation wavelength of all samples was set to 320 nm.

[0139] Synthesis Example 1 Preparation of Quinone-type Conjugated Organic Molecular Monomer PAQM

[0140] In the first step, (3Z,6Z)-3,6-bis[(5-bromothiophene-2-yl)methylidene]piperazine-2,5-dione was synthesized according to the literature (X.Liu, et al, J.Am.Chem.Soc., 2017, 139, 8355-8363). The structural formula of the compound is shown below:

[0141]

[0142] In the second step, under the protection of inert gas (argon), the product of the first step (8g, 17.39mmoL), anhydrous potassium carbonate (12g, 86.95mmoL), potassium iodide (0.29g, 1.74mmoL), tetrabutylammonium bromide (1.12g, 3.48mmoL), DMF (100mL), 2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)ethyl-4-methylbenzenesulfonate (36.34g, 104.3mmoL) were added to a dry three-necked flask for heating and stirring. The reaction was carried out at 120°C for 3h. After the reaction was completed, it was cooled to room temperature, extracted with an aqueous solution of sodium chloride and dichloromethane, and dried with anhydrous sodium sulfate overnight, and the reaction mixture was filtered, the solvent was removed by reduced pressure distillation, and the product was separated and purified by silica gel column chromatography (eluent EA). After the product solution was distilled under reduced pressure to remove the solvent, it was placed in a vacuum drying oven for drying to obtain a red quinone-type conjugated organic molecular monomer PAQM, the structural formula of which is shown below:

[0143]

[0144] Synthesis Example 2 Preparation of Quinone-type Conjugated Organic Molecule P1

[0145] The quinone-type conjugated organic molecule monomer PAQM (243.6 mg, 0.3 mmol) was mixed with 4,7-bis(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2,1,3-benzothiadiazole (116.4 mg, 0.3 mmol), tris(dibenzylideneacetone)dipalladium (Pd(dba) 3 )(7 mg, 0.0076 mmol), tri(o-methylphenyl)phosphine (9 mg, 0.0296 mmol), 3 drops of N-methyl-N,N-dioctyl-1-ammonium chloride (Aliquat 336), 2M potassium carbonate solution (3 mL) and anhydrous toluene (10 mL) were added to a 20 mL sealed tube in sequence. The system was deoxygenated three times by freeze-pump-thaw cycles, and then the sealed tube was sealed with a high-temperature spray gun and maintained in a vacuum state. The reaction system was placed in a 90°C oil bath and heated for reaction for 3 days. After the reaction was completed, the sealed tube was quickly immersed in liquid nitrogen to terminate the reaction, the reaction mixture was precipitated in methanol, and the precipitate was collected by filtration. Soxhlet extraction was performed with methanol, acetone and n-hexane in sequence to remove unreacted monomers and oligomers. Subsequently, the remaining polymer was extracted with chloroform, and the chloroform fraction was collected and concentrated to obtain the target product P1.

[0146] The structural formula of P1 is shown below:

[0147]

[0148] The number average molecular weight is 9400 g / mol, and the dispersion coefficient M n / M w is 1.25.

[0149] Synthesis Example 3 Preparation of Quinone-type Conjugated Organic Molecule P2 (i.e., AQM described below) (Preferred Example)

[0150] The quinone-type conjugated organic molecule monomer PAQM (100 mg, 0.123 mmol), (trimethyltin) acetylene (43.25 mg, 0.123 mmol), tetrakis(triphenylphosphine) palladium (8.5 mg, 0.00738 mmol) and anhydrous toluene (8 mL) were added to a 10 mL sealed tube. Subsequently, three deoxygenation operations were performed through freeze-evacuation-thaw cycles to ensure that the system was oxygen-free. The sealed tube was sealed with a high-temperature spray gun and maintained in a vacuum state, and then the reaction system was placed in a 100°C oil bath for 1 hour. After the reaction was completed, the sealed tube was quickly immersed in liquid nitrogen to terminate the reaction, the reaction mixture was precipitated in methanol, and the precipitate was collected by filtration. Soxhlet extraction was performed with methanol, acetone and n-hexane in turn to remove unreacted monomers and oligomers. Subsequently, the remaining polymer was extracted with chloroform, and the chloroform fraction was collected and concentrated to obtain the target product P2.

[0151] The structural formula of P2 is shown below:

[0152]

[0153] The NMR hydrogen spectrum and molecular weight of P2 were tested, and the results were as follows Figure 2 and Figure 3 As shown, the number average molecular weight of P2 is 9300 g / mol, and the dispersion coefficient M n / M w is 1.06.

[0154] Detection of UV absorption curve of P2: Dissolve P2 molecule in DMSO to prepare 100μg / mL solution. Dilute the solution to 50, 25, 12.5, 6.25, 3.13μg / mL in sequence and measure with UV-visible spectrophotometer. The results are as follows: Figure 4 The mass extinction coefficient is calculated by the Beer-Lambert law: A / L = ε × c (L = 1 cm, A / L is the normalized absorbance at 650 nm divided by the characteristic optical path length of the test tube at different concentrations). The results are shown in Figure 4 The results show that in THF, the shortest absorption wavelength of P2 is 560nm, and in DMSO, the absorption wavelength of P2 is 610nm, with a red shift of 50nm, and its absorbance increases with the increase of concentration, and shows good linearity (R 2 =0.99997). Based on this relationship, the mass extinction coefficient is calculated to be 37.2 L g - 1 cm -1 .

[0155] Detection of the electrochemical performance of P2: Cyclic voltammetry (CV) test was performed on a CHI1200C electrochemical workstation. The results are as follows: Figure 5 As shown. The cyclic voltammetry (CV) curve shows obvious redox peaks with redox potentials of 0.88 eV and -0.73 eV, respectively. The HOMO and LUMO energy levels of PAQMA were calculated to be -5.24 eV and -3.63 eV, respectively. The electronic structure and optimized geometry of PAQMA were calculated using density functional theory (DFT). The highest occupied molecular orbital (HOMO) is mainly distributed along the conjugated main chain, indicating that it has a strong molecular conjugation effect. The lowest unoccupied molecular orbital (LUMO) contributes little to the donor, but is mainly located on the acceptor and π-bond units. The spatial separation of the HOMO and LUMO orbitals indicates significant donor-acceptor interactions and intramolecular charge transfer processes, driving the emission wavelength to move to a longer region. Based on the calculated results, its band gap was determined to be 1.56 eV. The large conjugation of PAQM leads to a narrow band gap, which is conducive to strong absorption in the near-infrared range.

[0156] Synthesis Example 4 Preparation of Quinone-type Conjugated Organic Molecule P3

[0157] The quinone-type conjugated organic molecule monomer PAQM (300 mg, 0.37 mmol), 1,4-bis(tributylstannyl)benzene (242.8 mg, 0.37 mmol), tetrakis(triphenylphosphine)palladium (25.64 mg, 0.0222 mmol) and anhydrous toluene (30 mL) were added to a 50 mL sealed tube in sequence. The system was deoxygenated three times by freeze-pump-thaw cycles, and then the sealed tube was sealed with a high-temperature spray gun and maintained in a vacuum state. The reaction system was placed in a 100°C oil bath and heated for 12 hours. After the reaction was completed, the sealed tube was quickly immersed in liquid nitrogen to terminate the reaction, the reaction mixture was precipitated in methanol, and the precipitate was collected by filtration. Soxhlet extraction was performed with methanol, acetone and n-hexane in sequence to remove unreacted monomers and oligomers. Subsequently, the remaining polymer was extracted with chloroform, and the chloroform fraction was collected and concentrated to obtain the target product P3.

[0158] The structural formula of P3 is shown below:

[0159]

[0160] Its number average molecular weight is 4300 g / mol, and its dispersion coefficient M n / M w is 1.15.

[0161] Synthesis Example 5 Preparation of Quinone-type Conjugated Organic Molecule P4

[0162] The quinone-type conjugated organic molecule monomer PAQM (300 mg, 0.37 mmol), 2,5-bis(trimethyltin)thiophene (245.1 mg, 0.37 mmol), tetrakis(triphenylphosphine)palladium (25.64 mg, 0.0222 mmol) and anhydrous toluene (30 mL) were added to a 50 mL sealed tube in sequence. The system was deoxygenated three times by freeze-pump-thaw cycles, and then the sealed tube was sealed with a high-temperature spray gun and maintained in a vacuum state. The reaction system was placed in a 100°C oil bath and heated for 12 hours. After the reaction was completed, the sealed tube was quickly immersed in liquid nitrogen to terminate the reaction, the reaction mixture was precipitated in methanol, and the precipitate was collected by filtration. Soxhlet extraction was performed with methanol, acetone and n-hexane in sequence to remove unreacted monomers and oligomers. Subsequently, the remaining polymer was extracted with chloroform, and the chloroform fraction was collected and concentrated to obtain the target product P4.

[0163] The structural formula of P4 is shown below:

[0164]

[0165] Its number average molecular weight is 7500g / mol, and its dispersion coefficient M n / M w is 1.18.

[0166] Synthesis Example 6 Preparation of Quinone-type Conjugated Organic Molecule P5

[0167] The quinone-type conjugated organic molecule monomer PAQM (300 mg, 0.37 mmol), bis(trimethyltin)ethylene (224.3 mg, 0.37 mmol), tetrakis(triphenylphosphine)palladium (25.64 mg, 0.0222 mmol) and anhydrous toluene (30 mL) were added to a 50 mL sealed tube in sequence. The system was deoxygenated three times by freeze-pump-thaw cycles, and then the sealed tube was sealed with a high-temperature spray gun and maintained in a vacuum state. The reaction system was placed in a 100°C oil bath and heated for 12 hours. After the reaction was completed, the sealed tube was quickly immersed in liquid nitrogen to terminate the reaction, the reaction mixture was precipitated in methanol, and the precipitate was collected by filtration. Soxhlet extraction was performed with methanol, acetone and n-hexane in sequence to remove unreacted monomers and oligomers. Subsequently, the remaining polymer was extracted with chloroform, and the chloroform fraction was collected and concentrated to obtain the target product P5.

[0168] The structural formula of P5 is shown below:

[0169]

[0170] Its number average molecular weight is 10600 g / mol, and its dispersion coefficient M n / M w is 1.30.

[0171] Synthesis Example 7 Preparation of Quinone-type Conjugated Organic Molecule P6

[0172] The quinone-type conjugated organic molecule monomer PAQM (300 mg, 0.37 mmol), 5,5-bistrimethyltin-2,2'-bithiophene (275.4 mg, 0.37 mmol), tetrakis(triphenylphosphine)palladium (25.64 mg, 0.0222 mmol) and anhydrous toluene (30 mL) were added to a 50 mL sealed tube in sequence. The system was deoxygenated three times by freeze-pump-thaw cycles, and then the sealed tube was sealed with a high-temperature spray gun and maintained in a vacuum state. The reaction system was placed in a 100°C oil bath and heated for 12 hours. After the reaction was completed, the sealed tube was quickly immersed in liquid nitrogen to terminate the reaction, the reaction mixture was precipitated in methanol, and the precipitate was collected by filtration. Soxhlet extraction was performed with methanol, acetone and n-hexane in sequence to remove unreacted monomers and oligomers. Subsequently, the remaining polymer was extracted with chloroform, and the chloroform fraction was collected and concentrated to obtain the target product P6.

[0173] The structural formula of P6 is shown below:

[0174]

[0175] Its number average molecular weight is 5000g / mol, and its dispersion coefficient M n / M w is 1.12.

[0176] At a concentration and volume of 2 mL and 50 ug / ml, a 650 nm laser (0.8 W cm -2 ) were used to irradiate molecules P1 to P6 and test their photothermal cycle curves. The results are as follows Figure 6 As shown. In photothermal therapy, it is hoped that the temperature of the wound dressing can reach a maximum of 40-50°C. Considering the loss of photothermal efficiency after the conjugated molecules are made into nanoparticles, the maximum temperature of the conjugated molecules is preferably 45-60°C, more preferably 50-60°C, and in order to achieve the purpose of recycling, it should have good photothermal cycle performance. It can be seen that P1, P2, P4 and P5 molecules are preferred, among which P2 molecules are more preferred.

[0177] Synthesis Example 8 Preparation of Amphiphilic Aldehyde-Containing Block Copolymer A-1 [PEG-bP (MAEBA-co-DMAEMA)]

[0178] Add RAFT modifier PEG to the 5 ml sealed tube 5000 -TC (250mg, 0.05mmol), 2-((N,N-dimethylamino)ethyl) methacrylate (DMAEMA, 471mg, 3mmol), p(methacryloyloxyethoxy)benzaldehyde (MAEBA, 234mg, 1mmol), tetrahydrofuran (THF, 2mL). After freezing and deoxygenating three times, the system was sealed with a high-temperature spray gun and placed in a vacuum state, and then reacted at 70°C for 6h. The sealed tube was quickly placed in liquid nitrogen and cooled to terminate the reaction. The copolymer was precipitated in n-hexane, filtered, and then dried in a vacuum oven overnight to obtain an amphiphilic aldehyde-containing block copolymer A-1. GPC measured the number average molecular weight of A-1 to be 8300 and the dispersion coefficient to be 1.01. The chemical formula of [PEG-bP(MAEBA-co-DMAEMA)] is as follows:

[0179]

[0180] Example 1 Preparation of antibacterial nanoparticles ABPL@AQM

[0181] Step S1: Weigh 3 mg of the P2 molecule and dissolve it in 3 mL of DMF to prepare a solution 1 with a concentration of 1 mg / mL.

[0182] Step S2: Take 1 mL of solution 1, then add 10 mg of amphiphilic aldehyde-containing block copolymer A-1 to the sample bottle and dissolve it ultrasonically, stirring for 24 hours. Under vigorous stirring, use a peristaltic pump to slowly add 9 mL of water at a rate of 1 mL / min, stir for 3 hours after adding water, then add 10 mg of L-arginine and stir for 6 hours, and finally dialyze with ultrapure water (molecular weight cutoff (MWCO): 35 kDa) to remove DMF for 48 hours, and replace the dialysis medium regularly. Collect the dialyzed solution for ultrasonication, use a 0.45 μm filter membrane to remove large particulate matter, and obtain the antibacterial nanoparticles ABPL@AQM of the present invention.

[0183] Comparative Example 1 Preparation of antibacterial nanoparticles ABP@AQM

[0184] According to the method of Example 1, without adding L-arginine, water was directly added and stirred for 9 hours to obtain reference nanoparticles ABP@AQM.

[0185] Comparative Example 2 Preparation of Nanoparticle ABPL

[0186] According to the method of Example 1, without adding P2 molecules, reference nanoparticles ABPL were obtained.

[0187] Application Example 1: Physical and Chemical Properties of Antibacterial Nanoparticles

[0188] Figure 7 Schematic diagram of the morphology of ABPL@AQM. Figure 8 DLS, TEM and SEM images of ABPL@AQM. Figure 7 As shown, the nanoparticles of the present invention are prepared by a coprecipitation method, wherein conjugated organic molecules and amphiphilic aldehyde-containing polymers form water-soluble nanoparticles, and L-arginine is inserted into the hydrophobic segment under vigorous stirring, and an ultrasonic method is used to avoid aggregation. Arginine has a certain cross-linking effect, and its addition significantly improves the stability of the nanoparticles. Figure 8 As shown, the antibacterial nanoparticles of Example 1 are spherical, with uniform particle size distribution and an average size of about 100 nm.

[0189] Fig. 9 DLS and TEM images of ABP@AQM. It can be seen that in the absence of arginine, spherical nanoparticles with uniform particle size distribution cannot be obtained.

[0190] After storing ABPL@AQM for one month, the appearance was observed and it was found that there was no visible change compared with the initial state. Fig.10 This is the DLS graph of ABPL@AQM after being stored for one month. The results show that the particle size of the antibacterial nanoparticles has not changed, which shows that the antibacterial nanoparticles of the present invention have good stability.

[0191] Fig.11 : The ultraviolet absorption curve and mass extinction coefficient of ABPL@AQM. The results show that the antibacterial nanoparticles of the present invention have good photothermal properties.

[0192] Determination of L-arginine concentration in ABPL@AQM: Freeze-dry the dialyzed ABPL@AQM, dissolve the product in 100 ml of deionized water, and analyze the characteristic absorbance of L-arginine using a UV-visible spectrophotometer. The free L-arginine content was determined to be 3.17 mg using the L-arginine standard curve, and the encapsulation efficiency (EE) was 68.3%. The calculation formula is as follows:

[0193]

[0194] Measurement of photothermal effect: 200 μL ABPL@AQM (5 μg / mL) was added to a 96-well plate. 200 μL ABPL@AQM with different concentrations (40, 20, 10, 5, 2.5 μg / mL) was placed in a 96-well plate and the photothermal effect was measured by 650 nm laser (0.8 W cm -2 ) for 10 minutes, and measure the temperature every 30 seconds. Adjust the power density of the 650nm laser (0.2, 0.4, 0.6, 0.8W cm -2 ), and measured the temperature of NPs at a fixed concentration (5 μg / mL). The temperature fluctuation was measured using a thermal imager, and the results are shown in Fig.12 shown.

[0195] For example, ESR is used to detect the ability of the antibacterial nanoparticles of the present invention to generate reactive oxygen species (ROS): 2,2,6,6-tetramethylpiperidine (TEMP) is used as 1 O 2 The indicator, 5,5-dimethyl-1-pyridine-n-oxide (DMPO) was used as the hydroxyl and superoxide anion radical indicator, and electron spin resonance (ESR) measurements were performed to determine the species of reactive oxygen species. 100 μL of ABPL@AQM (concentration of 80 μg / mL) was mixed with 100 μL of LTEMP (concentration of 100 mM) to detect O 2 , mixed with 10 μL DMPO (concentration of 8.79 mol / L) to detect hydroxyl radicals, and mixed with 10 μL DMPO (concentration of 8.79 mol / L) and 200 μL methanol to determine superoxide anion radicals. The sample was added through the capillary, illuminated in situ for 0, 1, 2, and 3 minutes, and the ESR signal was recorded. The results are as follows Fig.13 shown.

[0196] Furthermore, a hydroxyl radical detection kit (O27, green fluorescence) was used to detect the generation of hydroxyl radicals by the nanoparticles of the present invention and the comparative example: 10 μL of the O27 stock solution was diluted 1000 times with 0.1 mol / L PBS to prepare a working solution. 1.5 mL of the working solution was mixed with 0.5 mL of ABPL@AQM, ABP@AQM and ABPL nanoparticle solutions with a concentration of 5 μg / mL, respectively, and the content of hydroxyl radicals before and after irradiation was determined by fluorescence spectrophotometry. The results are shown in FIG. Fig.14 In addition, the NO release experiment was conducted: equal volumes of ABP@AQM, ABPL@AQM, and ABPL (1 ml each, 5 μg / mL) were prepared, and ABP@AQM and ABPL@AQM light exposure groups were established. After illumination, H 2 O 2 Solution (1mM), after 15min of reaction, add 1μL 4,5-diaminofluorescein (1mM). Take 1mL sample and measure the fluorescence intensity at 491nm using a fluorescence spectrometer (Hitachi, F-7000, excitation wavelength 491nm, emission wavelength 513nm). The results are as follows Fig.14 The results show that the antibacterial nanoparticles of the present invention can effectively generate ROS under 650nm laser irradiation, and due to the synergistic effect of L-arginine and the amphiphilic aldehyde-containing block copolymer, the NO level of ABPL@AQM is significantly increased under irradiation.

[0197] Furthermore, we conducted experiments on the photothermal stability of ABPL@AQM. -1 At a concentration of 200 μL ABPL@AQM, a 650 nm laser (power of 0.8 W cm -2 ) after irradiation, the temperature curve of ABPL@AQM is as follows Fig.15 As shown in a, using the cooling stage data, i.e., the negative natural logarithm of the driving force temperature (θ) versus time, the heat transfer time constant τs = 204.2 was calculated, and the photothermal conversion efficiency of ABPL@AQM was determined to be 28.2%. Fig.15 As shown in b, the calculation formula is:

[0198]

[0199] Where T max is the highest steady-state temperature of the solvent, T amb is the ambient temperature, I is the laser power used in the photothermal experiment, and λ is the absorbance of the nanoparticles at 650nm. hA can be determined by measuring the rate at which the temperature drops after the light source is removed, indicating the heat input of the solvent and container absorbing the light. h is the heat transfer coefficient, A is the container area, m is the mass of the nanoparticles, and C is the specific heat capacity of water.

[0200] The results show that the antibacterial nanoparticles of the present invention have good photothermal stability and suitable photothermal conversion efficiency. Although high temperature can damage the function of bacterial heat shock protein and cause bacterial death, too high temperature may also damage normal cells and even cause inflammatory response. Therefore, the more preferred temperature that can induce apoptosis of normal cells while minimizing damage to normal cells is between 40-55°C, more preferably around 45-50°C. Fig.15 As shown in a, the maximum temperature of the antibacterial nanoparticles of the present invention after irradiation for 10 minutes is about 43-44°C, which is beneficial to the actual application environment.

[0201] Application Example 2 Antibacterial Properties of Antibacterial Nanoparticles

[0202] The antibacterial effect of ABPL@AQM at different concentrations was evaluated by broth microdilution method. -1 Planktonic MRSA of AQM, ABP@AQM, ABPL and ABPL@AQM were tested. MRSA culture: Several single colonies were inoculated into LB medium and incubated at 37°C for 16-18 h to the stationary phase. Subsequently, the culture was diluted 1:100 with fresh LB medium (4 ml) and further grown at 37°C until it reached the mid-logarithmic phase (OD600 = 0.5, SpectraMax M2 microplate reader (Molecular Devices, USA)). Fig.16 As shown in A, the concentration of ABPL@AQM reached 5 μg mL -1 When the concentration reaches 25 μg mL -1 Considering the inherent photothermal effect of AQM, the 25 μg mL -1 Intracellular photothermal effects of AQM, ABP@AQM, and ABPL@AQM at different concentrations (650nm, 0.8W cm -2 ).from Fig.16 It can be seen from B and 16C that the temperature of the bacterial suspension increases with the extension of the illumination time, and ABPL@AQM has a good photothermal effect compared with the comparative example.

[0203] ROS detection was performed on MRSA planktonic cells. MRSA planktonic bacteria were treated with control group, ABPL, AQM, AQM+NIR, ABP@AQM, ABP@AQM+NIR, ABPL@AQM and ABPL@AQM+NIR (laser group was treated with 1W / cm 2 , 650 nm laser treatment for 10 min, the same below). The concentration of each treatment group was 25 μg mL -1, the volume was 1mL. The temperature of the bacteria after irradiation was measured with a thermal imager. Subsequently, the bacterial suspension was centrifuged and resuspended with PBS solution to ensure that the OD 600 DCFH-DA dye (10 μM) was added to the MRSA suspension and incubated in the dark for 20 minutes. After centrifugation to remove the fluorescent dye, the cells were fixed on a glass slide and ROS fluorescence was observed using a confocal laser scanning microscope (CLSM, LSM880 laser scanning confocal microscope (Zeiss, Germany), with an excitation light of 488 nm). The results are shown in Fig.16 D, and the quantitative results are shown in Fig.16 E. The results showed that the ROS level in the irradiated AQM group was significantly higher than that in the unirradiated group, indicating that the photothermal effect also affects the generation of ROS. The ABPL@AQM+near-irradiated group had the highest ROS level, which was 1.46 times that of the unirradiated ABPL@AQM group and 2.04 times that of the ABPL group. This synergistic antibacterial effect, combining photothermal action and ROS generation, demonstrated the excellent effect of ABPL@AQM in effectively eradicating MRSA. It can be seen that the increase in temperature in MRSA is expected to induce the production of heat shock protein Hsp70 to counteract photothermal damage, but in the ABPL@AQM-treated group, the relatively high concentration of H on the biofilm surface 2 O 2 It reacts with L-arginine to generate NO, which inhibits the expression of Hsp70 and destroys the defense mechanism of MRSA.

[0204] Western blot analysis was used to detect the expression level of Hsp70 in MRSA after treatment in different groups: the MRSA suspension after treatment in each treatment group in the previous step was centrifuged to remove the culture medium, and lysed on ice with RIPA lysis buffer for 30 minutes to extract total bacterial protein. The total protein was then quantified using the BSA method. The proteins were separated using the SDS-PAGE system (Bio-Rad) and transferred to a PVDF membrane (Beyotime). The PVDF membrane was blocked in a protein-free fast blocking buffer for 30 minutes and then incubated with the Hsp70 primary antibody overnight. After washing with TBST buffer, the membrane was incubated with a secondary antibody and the signal was detected using an ECL substrate. The signals were collected and analyzed using the QuickChemi 5200 system (Monad). Similarly, the expression of the reference protein β-actin was detected, and the results are shown in Figure 2. Fig.16 As shown in F and 16G, compared with the control group, the expression of Hsp70 in the AQM+NIR group and the ABP@AQM+NIR group increased, indicating that the photothermal effect induced the upregulation of Hsp70 expression. In contrast, the ABPL group showed a decrease in the expression of Hsp70, which may be due to the interaction between L-arginine and H 2 O 2The reaction produced NO. It is worth noting that the expression of Hsp70 in the ABPL@AQM+NIR group was the lowest, which may be due to the enhanced release of L-arginine in ABPL@AQM under photothermal stimulation, resulting in increased NO production and Hsp70 inhibition. Fig.16 As shown in H.

[0205] Application Example 3 In vitro antibacterial and biofilm-destroying properties of antibacterial nanoparticles

[0206] The cultured MRSA was collected and centrifuged twice with PBS (6000 rpm, 10 min). Before inoculating MRSA into a 96-well microplate, the suspension was adjusted to 1.5×10 6 Colony forming units (CFUs) mL -1 MRSA suspension (50 μL) was incubated with the control group, ABPL, AQM, AQM+NIR, ABP@AQM, ABP@AQM+NIR, ABPL@AQM, and ABPL@AQM+NIR for 3 h (laser group at 1 W / cm 2 The concentration of each treatment group was 25 μg mL -1 , the treatment volume is 1mL. Use PBS to dilute 10,000 times. Place the diluted bacterial solution (20μL) on TSB agar plate, incubate at 37℃ overnight, and observe the colonies. The results are as follows Fig.17 As shown in A. The results showed that no red fluorescence was observed in the control group, AQM group, and ABP@AQM group, indicating that the bacteria in the group were alive. In contrast, in the AQM+NIR and ABP@AQM+NIR groups, a small number of bacteria showed cell death, indicating that the photothermal effect of AQM has antibacterial properties. In addition, due to the cationic effect of L-arginine itself, it exhibited strong antibacterial activity, and some planktonic bacteria in the ABPL group also showed signs of death. In the ABPL@AQM group, the non-light group showed red fluorescence similar to that of the ABPL group, while the light-treated group showed extensive red fluorescence, indicating that the high temperature after illumination affected the growth of MRSA planktonic bacteria. The bacterial plating effect is shown in Fig.17 As shown in B, the results also show that the antibacterial nanoparticles of the present invention have good photothermal gas antibacterial properties.

[0207] In the early stages of biofilm infection, topical hydrogen peroxide (H 2 O 2) levels increased, with concentrations ranging from 20 to 100 μM. It is well known that biofilms are a major obstacle to wound healing. The antibacterial activity of ABPL@AQM against MRSA biofilms was evaluated by crystal violet staining and viability / death staining assays: MRSA biofilms were treated with PBS, ABPL, AQM, AQM+NIR, ABP@AQM, ABP@AQM+NIR, ABPL@AQM, and ABPL@AQM+NIRat at 37°C for 3 h. The concentration of each treatment group was 25 μg mL -1 , the treatment volume was 1 mL. Subsequently, the MRSA biofilm was stained with 0.1% crystal violet for 15 minutes: after the biofilm was carefully rinsed with deionized water, 33% v / v acetic acid was added to the wells to dissolve the crystal violet stain. Finally, the absorbance of each well at 560 nm was measured to assess the biomass of the biofilm. After in vitro live / dead staining of the MRSA biofilm, it was stained with SYTO 9 and PI dyes simultaneously in the dark for 30 minutes. Fluorescence was imaged using a confocal laser scanning microscope, and z stacks were compiled into three-dimensional images. The results are shown in Fig.17 C and 17D (quantitative), ABPL, P-AQM+NIR, ABPL@AQM and ABPL@AQM+NIR all exhibited certain biofilm removal activity, and their biofilm removal rates were 50.4%, 45.5%, 60.6% and 91.2%, respectively.

[0208] SYTO 9 / PI live / dead bacterial staining: PBS, ABPL, AQM, AQM+NIR, ABP@AQM, ABP@AQM+NIR, ABPL@AQM, and ABPL@AQM+NIR treatments (the concentration of each treatment group was 25 μg mL -1 , treatment volume of 1 mL), the MRSA suspension was incubated with SYTO-9 (2 μM, excitation light 480 nm, emission light 500 nm) and propidium iodide (PI was 1 μM, excitation light at 490 nm, emission light at 635 nm) and stained in the dark for 15 min. After centrifugation to remove free SYTO-9 and PI, wash twice with PBS. In order to fix and collect MRSA, a 4% paraformaldehyde solution was added. The synthesized MRSA suspension was transferred to a glass slide, air-dried, and fixed with immersion oil. Finally, it was observed with a laser scanning confocal microscope. All bacteria were stained green with SYTO-9, and bacteria with damaged cytoplasmic membranes were stained red with PI. The results are shown in Fig.17D. It can be seen that almost all bacteria in the ABPL@AQM+laser treatment group showed red fluorescence, indicating cell death and complete destruction of the biofilm structure. In particular, compared with the slight difference in biofilm resistance between the ABP@AQM light group and the non-light group, ABPL@AQM+NIRLaser showed stronger anti-biofilm activity than the non-light group and the ABP@AQM(-)(+) group. As mentioned above, this is due to the binding of L-arginine to H 2 O 2 NO is formed on the surface of the biofilm, thereby inhibiting the expression of heat shock proteins, that is, inhibiting the heat-sensitive self-defense mechanism of the organism. At the same time, light enhances the release of L-arginine, resulting in a stronger antibacterial effect.

[0209] Scanning electron microscopy (SEM, FEI Talos F200s high-resolution transmission electron microscope (FEI, USA)) was used to visualize the effects of different treatment methods on the microstructure of MRSA biofilms: the MRSA biofilms treated in each treatment group in the previous paragraph obtained by centrifugation and PBS washing were fixed with 2.5% glutaraldehyde at 4°C overnight. After incubation in 1% osmium tetroxide for 1 hour, the samples were dehydrated in a series of graded ethanol solutions and tert-butanol (50%, 75%, 90% and 100%) for 10 minutes respectively. Subsequently, the samples were dried, coated with platinum, and observed using a scanning electron microscope. As Fig.17 As shown in Figure E, the MRSA morphology in the control group showed normal, while the ABPL group showed slight bacterial swelling and rupture. In the AQM+NIR group, the bacterial surface showed slight wrinkling due to the photothermal effect. In the ABP@AQM+NIR group, the solubility was better and the photothermal effect was enhanced, resulting in a large amount of bacterial shrinkage and rupture. As expected, the ABPL@AQM+NIR group showed complete bacterial rupture, leakage of cell contents, and complete destruction of the biofilm. In summary, ABPL@AQM has good antibacterial and anti-biofilm activity.

[0210] Application Example 4 Antibacterial activity and wound healing performance of antibacterial nanoparticles in vivo

[0211] The in vivo antibacterial effect of ABPL@AQM was evaluated using a MRSA-infected skin wound model.

[0212] Fig.18A shows the establishment of the animal model for treatment and evaluation of therapeutic effects: BALB / c mice (6-8 weeks old, male, 20-30 g) were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. The mouse skin wound infection model was established by hair removal using a hair removal device. After confirming that the foot skin was intact, telazol was injected intraperitoneally according to the weight of the mouse. After complete anesthesia, a circular wound was formed in the shaved area of ​​the hind leg, avoiding the fascia layer. The mice were randomly divided into 8 groups (3 mice / group): PBS control group (group 1), ABPL group (group 2), AQM group (group 3), AQM+NIR group (group 4), ABP@AQM group (group 5), ABP@AQM+NIR group (group 6), ABPL@AQM group (group 7), and ABPL@AQM+NIR group (group 8).

[0213] Evaluation of in vivo antibiofilm activity and wound healing status: In the control group, only PBS (200 μL) was dripped into the wound surface. PBS, ABPL, AQM, AQM+NIR, ABP@AQM, ABP@AQM+NIR, ABPL@AQM and ABPL@AQM+NIR (5 μg / mL, 100 μL) were dripped into the second, third and fourth groups of wounds, respectively. The wounds were irradiated with 650 nm laser (1 W / cm 2 ) were irradiated with AQM+NIR, ABP@AQM+NIR and ABPL@AQM+NIR groups for 10 minutes, and the changes in the wound temperature of mice were monitored using a thermal imager ( Fig.18 B). Fig.18 B shows that under 650nm laser irradiation, the wound temperature increased over time. After 10 minutes of irradiation, the temperature of the AQM group was lower than that of the ABP@AQM group and the ABPL@AQM group, which is consistent with the results of in vitro experiments. Under anesthesia, the wound size of mice was measured on the 1st, 3rd, 5th, and 7th days after modeling ( Fig.18 G and Fig.18 H). Wherein, CFU refers to colony forming unit. Fig.18 The results showed that the ABPL@AQM+light group had the strongest ability to inhibit bacterial reproduction. Fig.18G The results showed that the wound healing rate in the ABPL@AQM+NIRlight group was significantly faster than that in the other groups. By the 5th day, the wound area had decreased by 81.6%. After 7 days of treatment, the wound areas of the control group, ABPL, AQM, AQM+NIR, ABP@AQM, ABP@AQM+NIR, ABPL@AQM, and ABPL@AQM+NIR groups decreased by 59.5%, 73.22%, 67.64%, 80%, 81.53%, 91.19%, 82.14%, and 98.05%, respectively. These results indicate that the photothermal effect significantly promotes wound healing. After 7 days of treatment, wound tissues from different infection groups were collected, homogenized in sterile PBS, and the elimination of MRSA biofilm was quantitatively evaluated by the plate count method ( Fig.18 C). The results showed that the control group had a large amount of biofilm growth on the wound, and a large amount of bacteria grew after coating. Although the wound recovery rate of the ABPL group was poor, the bacterial clearance rate was improved compared with the control group. In addition, there were significant differences in the antibacterial effects of AQM, ABP@AQM, and ABPL@AQM with and without light conditions. On the 7th day of treatment, some mice were euthanized and the skin tissue around the infected wound was dissected. The residual MRSA bacteria around various infected wounds and the healing of the tissue around the wound were further evaluated (photos in Fig.18 D, 18E and 18F). Fig.18 D. Fig.18 E is the reduction of the wound area of ​​mice after treatment for different periods of time. It can be found that the wound healing speed and effect of ABPL@AQM are better than those of the control group. Fig.18 The results showed that the control group had neutrophil infiltration, indicating that inflammation persisted, the ABPL group had some relief, and the AQM group had no significant effect. In contrast, the ABPL@AQM+NIR group had fewer neutrophils and higher fibrinogen levels, indicating better wound healing. In addition, the residual MRSA bacteria around various infected wounds and the healing of surrounding tissue wounds were further evaluated: after fixation with 4% paraformaldehyde, hematoxylin and eosin (H&E), Masson's trichrome (MT), TNF-α and EGF immunohistochemical staining were performed (the results are shown in Fig.18 I), the results showed that the ABPL@AQM+NIR treatment group showed decreased IL-6 and TNF-α levels, indicating reduced systemic inflammation, suggesting that the mice had entered the late stage of wound healing.

[0214] Application Example 5 Biocompatibility and biosafety of antibacterial nanoparticles

[0215] In order to evaluate the biosafety of the antibacterial nanoparticles of the present invention, the blood routine and serum biochemical indexes of the mice in the treatment group were tested on the 9th day after treatment; in addition, the heart, lung, liver, spleen, kidney and other major organs of the mice in each treatment group after treatment were removed for pathological analysis. All tissue specimens were fixed with 4% paraformaldehyde, dehydrated, embedded in paraffin, sectioned, and stained with H&E. The results of the above two experiments are shown in Figure 2. Fig.19 shown.

[0216] Fig.19 In a, complete blood cell count analysis showed that the white blood cell (WBC) levels in the ABPL, AQM+NIR, ABP@AQM+NIR, and ABPL@AQM+NIR groups decreased compared with the other groups, indicating successful wound healing in mice. Other parameters, including red blood cells (RBC), hemoglobin (HGB), hematocrit (HCT), alanine aminotransferase (ALT), and aspartate aminotransferase (AST), were within the normal range for mice. Histopathological examination of the main organs in the different treatment groups is shown in Fig.19 b, No obvious pathological damage was observed. Compared with the control group, the therapeutic dose of ABPL@AQM had no toxic side effects and had good biocompatibility.

[0217] In summary, the antibacterial nanoparticles of the present invention have a good light-heat-gas response joint action mechanism, can synergistically produce bactericidal, antibacterial and biofilm eradication effects, reduce or eliminate inflammatory reactions, and are particularly suitable as antibacterial dressings for promoting wound healing.

[0218] In the above embodiments, all technical solutions under the concept of the present invention belong to the protection scope of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications without departing from the principle of the present invention should also be regarded as the protection scope of the present invention.

Claims

1. An antibacterial nanoparticle, which is a cross-linked product of a raw material composition comprising the following components (A) to (C): (A) A quinone-type conjugated organic molecule as shown in the following formula (I): (B) an amphiphilic aldehyde-containing block copolymer; and, (C) L-arginine; in: said n=5-200; and, The amphiphilic aldehyde-containing block copolymer comprises at least one hydrophilic block and at least one hydrophobic block, and the amphiphilic aldehyde-containing block copolymer comprises at least one aldehyde group bonded to the hydrophobic block.

2. The antibacterial nanoparticles according to claim 1, wherein the raw material composition comprises: (A) 0.1-20wt% of the quinone-type conjugated organic molecule represented by formula (I), preferably 1-10wt%: (B) 20-80 wt %, preferably 30-70 wt % of an amphiphilic aldehyde-containing block copolymer; and (C) L-arginine 5-60 wt %, preferably 20-50 wt %.

3. The antibacterial nanoparticles according to claim 1, wherein the n value of the quinone-type conjugated organic molecule represented by formula (I) is 10-100, preferably 10-40; Preferably, the number average molecular weight of the amphiphilic aldehyde-containing block copolymer is from 2,000 to 60,000.

4. The antibacterial nanoparticles according to any one of claims 1 to 3, wherein in the amphiphilic aldehyde-containing block copolymer, the number average molecular weight of the hydrophobic block is 1000-30000, and the block is obtained by polymerizing a monomer composition comprising at least one aldehyde-containing hydrophobic monomer; Preferably, the aldehyde-containing hydrophobic monomer has a structure of the following formula (II), and the degree of polymerization of the polymerized repeating unit of the hydrophobic monomer is 5-200: in: R1 is H or methyl; R2 is selected from C 1-12 Alkylene, C 3-12 Cycloalkylene, C 5-14 Arylene, C 5-8 Heterocyclylene, -C 1-12 Alkyl-C 3-8 Cycloalkyl-, -C 1-6 Alkoxy-C 1-6 Alkyl, -C 1-12 Alkoxy-C 3-8 Cycloalkyl-, -C 1-12 Alkyl-C 5-8 Heterocyclic-, -C 1-12 Alkoxy-C 5-8 Heterocyclic-, -C 1-12 Alkyl-C 5-14 Aryl- or -C 1-12 Alkoxy-C 5-14 Aryl-; the aryl group is selected from phenyl, naphthyl, pyrrolyl, imidazolyl, pyridyl, pyrimidinyl, furanyl or thienyl, and the heterocyclic group is selected from tetrahydrofuranyl, piperidinyl, hexahydropyridinyl or piperazinyl; Preferably, said R2 is selected from C 1-8 Alkylene, C 5-10 Arylene, -C 1-8 Alkyl-C 5-10 Aryl- or -C 1-8 Alkoxy-C 5-10 Aryl-; Preferably, said R2 is selected from -C 1-6 Alkoxy-C 5-6 Aryl-; Preferably, said R2 is selected from -ethoxy-phenyl-; Preferably, the structural formula of formula (II) is: More preferably, the monomer composition comprising at least one hydrophobic monomer containing an aldehyde group is mixture.

5. The antibacterial nanoparticles according to claim 4, wherein in the amphiphilic aldehyde-containing block copolymer, the hydrophilic block is selected from at least one of polyethylene glycol, poly(meth)acrylic acid, poly(meth)acrylamide, poly-N-isopropylacrylamide, poly(meth)acrylate substituted with a hydrophilic group, or polyvinylpyridine; and the hydrophilic group is selected from amino, carboxyl, di(C 1-5 alkyl)amino, hydroxyethyl, hydroxypropyl, diethylene glycol, triethylene glycol, tetraethylene glycol or C 10-30 Polyoxyethylene; Preferably, the hydrophilic block is polyethylene glycol; Preferably, the number average molecular weight of the hydrophilic block is 1000-30000; Preferably, the mass ratio of the hydrophilic block to the hydrophobic block in the amphiphilic aldehyde-containing block copolymer is 2:8 to 8:

2.

6. A method for preparing the antibacterial nanoparticles according to any one of claims 1 to 5, comprising the following steps: (S1) mixing the quinone-type conjugated organic molecule represented by the formula (I) and the amphiphilic aldehyde-containing block copolymer in an organic solvent, and then adding an aqueous solvent to assemble nanoparticles; (S2) L-arginine is added to the mixed solution of step S1 to cross-link the reaction system.

7. A preparation having antibacterial, bacterial biofilm-eliminating and wound-recovering effects, comprising the antibacterial nanoparticles according to any one of claims 1 to 5, preferably, the bacteria is methicillin-resistant Staphylococcus aureus (MRSA).

8. A biomedical material comprising the antibacterial nanoparticles according to any one of claims 1 to 5; preferably, the biomedical material is a wound dressing, a hand sanitizer or an antibacterial coating.

9. A quinone-type organic molecule, the structure of which is shown in the following formula (I):

10. Use of the quinone-type organic molecule of claim 9 in the preparation of antibacterial nanoparticles, the antibacterial nanoparticles have photothermal, photodynamic and gas synergistic antibacterial, bacterial biofilm elimination and wound recovery properties, preferably, the raw materials for the preparation of the antibacterial nanoparticles contain L-arginine.

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