Nanoparticles with photothermal-photodynamic-gas synergistic sterilization performance and application thereof

Abstract

The present application provides a multifunctional antibacterial nanoparticle, which is a crosslinked product of a raw material composition comprising the following components (A) to (C): (A) a quinoid conjugated organic molecule represented by the following formula (I): (B) an amphiphilic aldehyde group-containing block copolymer; and, (C) L-arginine. The antibacterial nanoparticle of the present application eradicates bacteria based on the synergistic effect of the "photothermal sterilization-photodynamic sterilization-gas sterilization" principle, has good antibacterial, bacterial biofilm elimination and wound recovery promotion effects, and is particularly suitable for the treatment of drug-resistant bacteria infections such as methicillin-resistant Staphylococcus aureus (MRSA) infections.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials, specifically to a nanoparticle with photothermal-photodynamic-gas synergistic bactericidal properties and its applications. Background Technology

[0002] In recent years, the overuse and abuse of antibiotics has become a major challenge in global public health, directly leading to a rapid increase in bacterial resistance. Methicillin-resistant Staphylococcus aureus (MRSA) is a typical example of drug-resistant bacteria, exhibiting extremely strong resistance to multiple antibiotics, rendering traditional antibiotic treatments increasingly ineffective. MRSA infections not only increase the difficulty of clinical treatment but also significantly increase patient mortality and healthcare costs. Therefore, developing novel antimicrobial strategies to address the threat of drug-resistant bacteria is urgently needed.

[0003] Against this backdrop, scientists are exploring new antibacterial treatment strategies, such as photothermal antibacterial therapy (PTAT), photodynamic antibacterial therapy (PDAT), and gaseous antibacterial therapy (GAT). PTAT utilizes photothermal conversion materials (such as gold nanorods and graphene) to generate localized high temperatures under near-infrared light irradiation, converting light energy into heat energy to directly cause thermal damage to bacterial cells, thereby killing bacteria. PDAT utilizes photosensitizers to generate reactive oxygen species (such as singlet oxygen) under specific wavelengths of light irradiation. These reactive oxygen species have strong oxidizing properties and can destroy bacterial cell membranes and intracellular biomolecules, leading to bacterial death. GAT utilizes the strong oxidizing and diffusive properties of gas molecules (such as nitric oxide and chlorine dioxide) to destroy bacterial cell membranes and metabolic systems, thereby achieving highly efficient sterilization. However, research shows that improving the effectiveness of a single treatment strategy faces technical bottlenecks. For example, while photothermal heating to 40°C and above can effectively kill bacteria, excessively high temperatures may cause irreversible damage to normal tissues, and bacteria will produce heat shock proteins (HSP70) to cope with photothermal damage. Furthermore, biofilms formed by drug-resistant bacteria can hinder the entry of reactive oxygen species (ROS) into the bacteria, leading to poorer treatment efficacy. Therefore, how to design material systems to further improve antibacterial properties, especially in eradicating 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 principle of "photothermal-photodynamic-gas". The system has good antibacterial, antibacterial biofilm elimination and wound healing effects, and is especially suitable for the treatment of drug-resistant bacterial infections such as MRSA.

[0005] In a first aspect, the present invention provides antibacterial nanoparticles, said antibacterial nanoparticles being a crosslinked product of a raw material composition comprising the following components (A) to (C):

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

[0007]

[0008] (B) Amphiphilic aldehyde-containing block copolymers; and,

[0009] (C)L-arginine;

[0010] in:

[0011] The 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, the antibacterial nanoparticles contain, in the raw material composition:

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

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

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

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

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

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

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

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

[0022] Preferably, in the antibacterial nanoparticles, the n value of the quinone 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 number average molecular weight of the hydrophobic block is 1000-30000, and it is obtained by polymerization of a monomer composition containing at least one aldehyde-containing hydrophobic monomer.

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

[0026]

[0027] in:

[0028] R1 is H or methyl;

[0029] R2 is selected from C 1-12 alkylene, C 3-12 Cycloalkylene, C 5-14 The aryl, C 5-8 subheterocyclic groups, -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, pyrrolithyl, imidazolyl, pyridyl, pyrimidinyl, furanyl or thiophenyl, and the heterocyclic group is selected from tetrahydrofuranyl, piperidinyl, hexahydropyridinyl or piperazineyl;

[0030] Preferably, R2 is selected from C 1-8 alkylene, C 5-10 The aryl, -C 1-8 Alkyl-C 5-10 Aryl- or -C 1-8 Alkoxy-C 5-10 aryl-;

[0031] Preferably, R2 is selected from -C 1-6 Alkoxy-C 5-6 aryl-;

[0032] Preferably, R2 is 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 A mixture.

[0036] Preferably, in the antibacterial nanoparticles, the hydrophilic block copolymer contains at least one selected from polyethylene glycol, poly(meth)acrylic acid, poly(meth)acrylamide, polyN-isopropylacrylamide, hydrophilically substituted poly(meth)acrylate, or polyvinylpyridine; and the hydrophilic group is selected from amino, carboxyl, or di(C) groups. 1-5 Alkyl)amino, hydroxyethyl, hydroxypropyl, diethylene glycol, triethylene glycol, tetraethylene glycol, or C 10-30 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] Secondly, the present invention provides a method for preparing the antibacterial nanoparticles according to any one of the foregoing claims, the method comprising the following steps:

[0040] (S1) The quinone conjugated organic molecule shown in formula (I) and the amphiphilic aldehyde-containing block copolymer are mixed in an organic solvent, and then an aqueous solvent is added to assemble nanoparticles.

[0041] (S2) Add L-arginine to the mixture in step S1 to crosslink the reaction system.

[0042] Thirdly, the present invention provides an antibacterial preparation that eliminates bacterial biofilm and promotes wound healing, comprising the antibacterial nanoparticles described in any of the preceding claims.

[0043] Preferably, the wound in the wound-healing formulation is a wound caused by bacterial infection, or a wound that has been infected by bacteria after it is formed, or a wound that has a potential risk of bacterial infection.

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

[0045] Fourthly, the present invention provides a biomedical material comprising the antibacterial nanoparticles described in any of the preceding claims.

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

[0047] Fifthly, the present invention provides a quinone-type organic molecule with the structure shown in formula (I):

[0048]

[0049] In a sixth aspect, the present invention provides the application 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, antibacterial biofilm elimination and wound healing properties.

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

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

[0052] The antibacterial nanoparticles described in this invention have the following structure and mechanism: Figure 1 As shown, quinone-type conjugated organic molecules and amphiphilic aldehyde-containing block copolymers self-assemble into nanoparticles. The quinone-type conjugated organic molecules are uniformly dispersed and encapsulated within the amphiphilic aldehyde-containing block copolymer. L-arginine bonds with the aldehyde groups in the amphiphilic aldehyde-containing block copolymer through its amino group, cross-linking the nanoparticles and further improving their stability. This results in a nanomaterial system with a synergistic bactericidal effect based on the principles of photothermal sterilization, photodynamic sterilization, and gaseous sterilization. Specifically:

[0053] Typically, the biofilm microenvironment formed by bacteria in wounds is acidic and exhibits high hydrogen peroxide expression. By contacting the antibacterial nanoparticles of this invention with the wound surface and subjecting them to near-infrared light irradiation, the imine bonds bonded to the arginine nanoparticles break in the weakly acidic environment. Arginine is released and, at the inflamed site, is catalyzed by nitric oxide enzymes and hydrogen peroxide to produce nitric oxide gas, achieving gaseous sterilization. Simultaneously, the cross-linking structure of the nanoparticles is broken. Subsequently, quinone-conjugated organic molecules have more opportunities to come into contact with the light source, generating a photothermal effect and rapidly increasing the system temperature to achieve photothermal sterilization. Furthermore, during this process, the quinone-conjugated organic molecules generate reactive oxygen species under light irradiation, achieving photodynamic sterilization. These reactive oxygen species further promote the oxidation of arginine to produce nitric oxide; the two mutually promote each other, accelerating the sterilization process. Amphiphilic aldehyde-containing block copolymers effectively encapsulate quinone-type conjugated organic molecules, controlling the system temperature to prevent excessive heat and damage to the normal system, while improving the photothermal stability of the system and extending the service life of the material. The presence of arginine and its oxidation to nitric oxide effectively reduce the expression of heat shock protein (HSP70) in the system, preventing or mitigating bacterial stress response caused by photothermal effects, and further improving bactericidal performance. Attached Figure Description

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

[0055] Figure 2 : The 1H NMR spectrum of the P2 molecule in Synthetic Example 3.

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

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

[0058] Figure 5 Cyclic voltammetry curves of the P2 molecule from Synthetic Example 3.

[0059] Figure 6 Photothermal cycling properties of P1 to P6 molecules from Synthetic Examples 1-6

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

[0061] Figure 8 The following are the DLS (a), TEM (b), and SEM (c) results of the antibacterial nanoparticles in Example 1 of this invention.

[0062] Figure 9 The results of DLS (a) and TEM (b) of the antibacterial nanoparticles of Comparative Example 1 of this invention are shown.

[0063] Figure 10 The antibacterial nanoparticles of Example 1 of this invention were stored in DLS for one month.

[0064] Figure 11 The ultraviolet absorption curve (a) and mass extinction coefficient (b) of the antibacterial nanoparticles of Example 1 of the present invention.

[0065] Figure 12 The photothermal properties of the antibacterial nanoparticles in Example 1 of this invention: (a) at different concentrations; (b) at different power levels.

[0066] Figure 13 Schematic diagram of ESR results of different types of reactive oxygen species in antibacterial nanoparticles of Example 1 of the present invention: HO·(a); 1 O2(b); ·O 2- (c)

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

[0068] Figure 15 The photothermal stability (a) and photothermal conversion efficiency (b) of the antibacterial nanoparticles in Example 1 of this invention.

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

[0070] Figure 17 A. CLSM images of live / dead planktonic MRSA in different treatment groups and under light irradiation (scale bar: 50 μm); B. Colony electroplating of MRSA in different treatment groups and under light irradiation conditions; C. Evaluation of the anti-biofilm activity of samples against MRSA biofilms using crystal violet staining; D. Confocal laser scanning microscopy (CLSM) images of live / dead MRSA biofilms stained (scale bar: 100 μm); E. Scanning electron microscopy (SEM) images of MRSA biofilms treated in different groups (scale bar: 2 μm).

[0071] Figure 18A. Schematic diagram of bacterial wound infection model and drug administration regimen; B. Representative real-time thermal images of mice in each treatment group after treatment (under NIR irradiation, 650nm, 1W cm⁻¹). -2 C. Quantitative bacterial count in mouse wounds after treatment using standard plate counting method; D, E, and F. Wound photographs of mice after treatment in each treatment group; G: Wound size of mice after treatment in each treatment group; H. Quantitative CFU in the wounds of mice after treatment in each treatment group; I. FH&E staining, Masson trichrome staining of skin tissue, and immunohistochemical staining of IL-6 and TNF-α after treatment with different formulations (scale bar: 150 μm).

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

[0073] In the following description, numerous specific details are set forth in order to provide a more thorough understanding of the invention. However, it will be apparent to those skilled in the art that the invention can be practiced without one or more of these details. In other instances, certain technical features well-known in the art have not been described in order to avoid obscuring the invention.

[0074] The invention will now be described in detail with reference to the definitions of terms:

[0075] I Antibacterial Nanoparticles

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

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

[0078]

[0079] (B) Amphiphilic aldehyde-containing block copolymers; and,

[0080] (C)L-arginine.

[0081] A quinone-type conjugated organic molecule

[0082] Quinone-type conjugated organic molecules, through the design of conjugated backbone structures, can exhibit different properties, resulting in varying temperature profiles and temperature stability during photothermal conversion. As shown in synthesis examples 2-7 below, the inventors discovered that the alkynyl group in formula (I) enables formula (I) to possess suitable energy, making it more suitable as a photothermal conversion material for photothermal gas sterilization systems than other conjugated units such as benzene, alkenes, thiophenes, and benzodithiazoles.

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

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

[0085] first step:

[0086] Step Two:

[0087] Step 3:

[0088] Step 4:

[0089]

[0090] Step 5:

[0091]

[0092] In the quinone conjugated organic molecule of the present invention, the value of n 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 0.1-20 wt%. In some specific embodiments, the above content can be 1-10 wt% or 3-7 wt%, for example, 1 wt%, 2 wt%, 3 wt%, 4 wt%, 5 wt%, 6 wt%, 7 wt%, 8 wt%, 9 wt%, 10 wt%, 12 wt%, 15 wt%, or 18 wt%.

[0094] B amphiphilic aldehyde-containing block copolymer

[0095] The amphiphilic block copolymers of this invention are known to those skilled in the art, and typically include at least one hydrophilic block and at least one hydrophobic block. Specifically, the amphiphilic block copolymers of this invention contain aldehyde groups, and the aldehyde groups are bonded to the hydrophobic blocks of the amphiphilic aldehyde-containing block copolymer. Preferably, the aldehyde groups are substantially uniformly distributed on the hydrophobic blocks of the amphiphilic aldehyde-containing block copolymer.

[0096] In some embodiments, the amphiphilic aldehyde-containing block copolymer of the present invention is a diblock, triblock, tetrablock, or multiblock copolymer, etc., and its structure can be linear, branched, or star-shaped, etc. The hydrophilic block in the copolymer can be one, two, three, or more, and the hydrophobic block can be one, two, three, 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 one hydrophilic block A and one hydrophobic block B, or a triblock copolymer ABA formed by connecting two hydrophilic blocks A and one hydrophobic block B, or a three-armed star copolymer-(BA)3 formed by connecting three hydrophilic blocks A and three hydrophobic blocks B, etc.

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

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

[0099] In some embodiments, the ratio of hydrophilic blocks to hydrophobic blocks in the amphiphilic aldehyde-containing block copolymer of the present invention is 2:8 to 8:2, or 3:7 to 7:3, or 4:6 to 6:4, etc. After coating quinone conjugated organic molecules, they 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 blocks is 1000-30000, 2000-10000, or 3000-8000, for example, 3000, 4000, 5000, or 6000; the number-average molecular weight of the hydrophobic blocks is 1000-30000, 2000-10000, or 3000-8000, for example, 3000, 4000, 5000, or 6000.

[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, it can be 35wt%, 45wt%, 50wt%, 55wt%, or 65wt%.

[0102] In some specific embodiments, the typical hydrophilic block may be polyethylene glycol (2000, 4000, 5000, 6000 or 8000, etc.), polyacrylic acid, polymethacrylic acid, polyacrylamide, polymethacrylamide, poly-N-isopropylacrylamide, poly(meth)acrylate hydroxyethyl ester, poly(meth)acrylate hydroxypropyl ester, poly-2-vinylpyridine, poly-4-vinylpyridine, polyethylene glycol methacrylate, poly(N,N-dimethylaminoethyl methacrylate), 2-(2-methoxyethoxy)ethyl methacrylate or combinations thereof.

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

[0104] The aldehyde group in the amphiphilic aldehyde-containing block copolymer of the present invention can be derived from the polymeric monomer of the hydrophobic block, or the aldehyde group can 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 polymerization of a monomer composition comprising at least one aldehyde-containing hydrophobic monomer. The degree of polymerization of the repeating unit obtained by polymerization of the aldehyde-containing hydrophobic monomer is 5-200, 10-150, or 15-100, for example, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50.

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

[0107]

[0108] The definitions of R1 and R2 are as described 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 polymeric segments obtained by polymerization of any other monomers known in the art, wherein the content of the other polymeric segments does not exceed 75%, 50%, or 20% of the total mass of the hydrophobic block.

[0112] In one specific embodiment, the monomer composition for preparing hydrophobic blocks according to the present invention is: A mixture. Among them, The mass ratio 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 according to the present invention includes the following steps:

[0117] (S1) The quinone conjugated organic molecule shown in formula (I) and the amphiphilic aldehyde-containing block copolymer are mixed in an organic solvent, and then an aqueous solvent is added to assemble nanoparticles.

[0118] (S2) Add L-arginine to the mixture in step S1 to crosslink 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 conjugated organic molecule and the amphiphilic aldehyde-containing block copolymer in step S1 is 1:3-1:50, more preferably 1:5-1:20, and even 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 quinone conjugated organic molecules is 1:1-20:1, and more preferably 5:1-10:1.

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

[0124] II. Application of Antibacterial Nanoparticles

[0125] The antibacterial nanoparticles of this invention are suitable as materials for eradicating bacteria, eliminating bacterial biofilms, and promoting wound healing. They can be used to prepare various formulations or biomedical materials for this application environment, such as wound dressings, hand sanitizers, and antibacterial coatings. During application, the light source is preferably red light, near-infrared light, or infrared light, with a preferred wavelength of 600-1200 nm, more preferably 600-900 nm, further preferably 600-700 nm, and most preferably 630-670 nm.

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

[0127] Example

[0128] raw material

[0129] All reagents and pharmaceuticals not mentioned in this invention were purchased from Aldrich and used directly.

[0130] Test methods

[0131] Nuclear magnetic resonance (NMR) spectroscopy: 1 H-NMR (400MHz) measurements were performed on a Bruker DMX-400 spectrometer using CDCl3 or DMSO-d6 as the medium.

[0132] Size exclusion chromatography (SEC) measurements: Molecular weight and molecular weight distribution were determined using a Waters 150C gel permeation chromatography (GPC) system with two Ultrastyragel columns in series and a Wyatt Optilab DSP RI detector at 25°C, using THF as the eluent at a flow rate of 0.5 mL / min. Monodisperse polystyrene standards were used for molecular weight and molecular weight distribution determination.

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

[0134] Transmission electron microscopy (TEM): TEM observations were performed on a Thermo Fisher Talos F200C TEM at an accelerating voltage of 200 kV. Sample preparation involved depositing a drop of polymer solution in methanol onto a copper grid, followed by overnight drying 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): The SEM images were obtained using a Carl Zeiss GeminiSEM 300 microscope. The samples measured by SEM were prepared by dropping a drop of methanol nanoparticle solution onto a silicon wafer and then drying it overnight at room temperature.

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

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

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

[0139] Synthetic Example 1: Preparation of the quinone-type conjugated organic monomer PAQM

[0140] The first step involved synthesizing (3Z,6Z)-3,6-bis[(5-bromothiophene-2-yl)methylmethylene]piperazine-2,5-dione 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 an inert gas (argon), the product from the first step (8 g, 17.39 mmol), anhydrous potassium carbonate (12 g, 86.95 mmol), potassium iodide (0.29 g, 1.74 mmol), tetrabutylammonium bromide (1.12 g, 3.48 mmol), DMF (100 mL), and 2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)ethyl-4-methylbenzenesulfonate (36.34 g, 104.3 mmol) were added separately to a dry three-necked flask and heated with stirring. The reaction was carried out at 120 °C for 3 h. After the reaction was completed, the mixture was cooled to room temperature, extracted with an aqueous solution of sodium chloride and dichloromethane, and dried overnight with anhydrous sodium sulfate. The reaction mixture was then filtered, the solvent was removed by vacuum distillation, and the product was separated and purified by silica gel column chromatography (eluent EA). After removing the solvent by vacuum distillation, the product solution was dried in a vacuum drying oven to obtain the red quinone conjugated organic monomer PAQM, with the following structural formula:

[0143]

[0144] Synthetic Example 2: Preparation of quinone-type conjugated organic molecule P1

[0145] The quinone-type conjugated organic monomer PAQM (243.6 mg, 0.3 mmol) was sequentially added to a 20 mL sealed tube along with 4,7-bis(4,4,5,5-tetramethyl-1,3,2-dioxapentylborane-2-yl)-2,1,3-benzothiadiazole (116.4 mg, 0.3 mmol), tris(dibenzylacetone)palladium (Pd(dba)3) (7 mg, 0.0076 mmol), tris(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). The system was deoxygenated three times using a freeze-evacuation-thawing cycle. The tube was then sealed under vacuum using a high-temperature spray gun. The reaction system was heated in a 90°C oil bath for 3 days. After the reaction was completed, the sealing 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 sequentially with methanol, acetone, and n-hexane 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 as follows:

[0147]

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

[0149] Synthetic Example 3: Preparation of the quinone-type conjugated organic molecule P2 (hereinafter referred to as AQM) (Preferred Example)

[0150] The quinone-type conjugated organic monomer PAQM (100 mg, 0.123 mmol) was sequentially added to a 10 mL sealed tube along with (trimethyltin)acetylene (43.25 mg, 0.123 mmol), tetrakis(triphenylphosphine)palladium (8.5 mg, 0.00738 mmol), and anhydrous toluene (8 mL). The system was then deoxygenated three times using a freeze-evacuation-thawing cycle to ensure an oxygen-free environment. The tube was sealed with a high-temperature spray gun and kept under vacuum. The reaction system was then placed in a 100°C oil bath for 1 hour. After the reaction was complete, 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 sequentially with methanol, acetone, and n-hexane to remove unreacted monomers and oligomers. The remaining polymer was then 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 1H NMR spectrum and molecular weight of P2 were detected, and the results are as follows: Figure 2 and Figure 3 As shown, the number-average molecular weight of P2 is 9300 g / mol, and the dispersion factor M is... n / M w It is 1.06.

[0154] The UV absorption quasi-curve for detecting P2: P2 molecules were dissolved in DMSO to prepare a 100 μg / mL solution. The solution was successively diluted to 50, 25, 12.5, 6.25, and 3.13 μg / mL, and the concentrations were measured using a UV-Vis spectrophotometer. The results are shown below. Figure 4 As shown in Figure a. The mass extinction coefficient is calculated using Beer-Lambert's 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), and the result is as follows. Figure 4 As shown in b. The results show that in THF, the shortest absorption wavelength of P2 is 560 nm, and in DMSO, the absorption wavelength of P2 is 610 nm, with a redshift of 50 nm. Furthermore, its absorbance increases with increasing concentration and exhibits good linearity (R0). 2 =0.99997). Based on this relationship, the mass extinction coefficient was calculated to be 37.2 L g. - 1 cm -1 .

[0155] Electrochemical performance of P2 was tested using cyclic voltammetry (CV) on a CHI1200C electrochemical workstation. The results are as follows: Figure 5 As shown, cyclic voltammetry (CV) curves reveal distinct redox peaks with redox potentials of 0.88 eV and -0.73 eV, respectively. The calculated HOMO and LUMO levels of PAQMA are -5.24 eV and -3.63 eV, respectively. Density functional theory (DFT) calculations were used to determine the electronic structure and optimized geometry of PAQMA. The highest occupied molecular orbital (HOMO) is mainly distributed along the conjugated backbone, indicating 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 a longer region. Based on the calculations, its band gap was determined to be 1.56 eV. The large conjugation of PAQMA results in a narrow band gap, which is beneficial for strong absorption in the near-infrared range.

[0156] Synthesis Example 4: Preparation of the quinone-type conjugated organic molecule P3

[0157] The quinone-type conjugated organic monomer PAQM (300 mg, 0.37 mmol) was sequentially added to a 50 mL sealed tube along with 1,4-bis(tributyltinyl)benzene (242.8 mg, 0.37 mmol), tetra(triphenylphosphine)palladium (25.64 mg, 0.0222 mmol), and anhydrous toluene (30 mL). The system was deoxygenated three times using a freeze-evacuation-thawing cycle, and then the tube was sealed under vacuum using a high-temperature spray gun. The reaction system was heated in a 100 °C oil bath for 12 hours. After the reaction was complete, 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 sequentially with methanol, acetone, and n-hexane 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 index M is... n / M w It is 1.15.

[0161] Synthetic Example 5: Preparation of the quinone-type conjugated organic molecule P4

[0162] The quinone conjugated organic monomer PAQM (300 mg, 0.37 mmol) was sequentially added to a 50 mL sealed tube along with 2,5-bis(trimethyltin)thiophene (245.1 mg, 0.37 mmol), tetra(triphenylphosphine)palladium (25.64 mg, 0.0222 mmol), and anhydrous toluene (30 mL). The system was deoxygenated three times using a freeze-evacuation-thawing cycle, and then the tube was sealed under vacuum using a high-temperature spray gun. The reaction system was heated in a 100 °C oil bath for 12 hours. After the reaction was complete, 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 sequentially with methanol, acetone, and n-hexane 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 7500 g / mol, and its dispersion index M is... n / M wIt is 1.18.

[0166] Synthetic Example 6: Preparation of the quinone-type conjugated organic molecule P5

[0167] The quinone-type conjugated organic monomer PAQM (300 mg, 0.37 mmol) was sequentially added to a 50 mL sealed tube along with bis(trimethyltin)ethylene (224.3 mg, 0.37 mmol), tetra(triphenylphosphine)palladium (25.64 mg, 0.0222 mmol), and anhydrous toluene (30 mL). The system was deoxygenated three times using a freeze-evacuation-thawing cycle. The tube was then sealed under vacuum using a high-temperature spray gun. The reaction system was heated in a 100 °C oil bath for 12 hours. After the reaction was complete, 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 sequentially with methanol, acetone, and n-hexane 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 index M n / M w It is 1.30.

[0171] Synthetic Example 7: Preparation of the quinone-type conjugated organic molecule P6

[0172] The quinone-type conjugated organic monomer PAQM (300 mg, 0.37 mmol) was sequentially added to a 50 mL sealed tube along with 5,5-bis(trimethylstan-2,2'-bithiophene) (275.4 mg, 0.37 mmol), tetrakis(triphenylphosphine)palladium (25.64 mg, 0.0222 mmol), and anhydrous toluene (30 mL). The system was deoxygenated three times using a freeze-evacuation-thawing cycle, and then the tube was sealed under vacuum using a high-temperature spray gun. The reaction system was heated in a 100 °C oil bath for 12 hours. After the reaction was complete, 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 sequentially with methanol, acetone, and n-hexane 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 5000 g / mol, and its dispersion index M is... n / M w It is 1.12.

[0176] At a concentration and volume of 2 mL, 50 ug / ml, and a 650 nm laser (0.8 W cm⁻¹), -2 Under the conditions described above, molecules P1 to P6 were irradiated, and their photothermal cycling curves were tested. The results are as follows: Figure 6 As shown. In photothermal therapy, it is desirable for the wound dressing to reach a temperature of 40-50°C. Considering the photothermal efficiency loss after the conjugated molecules are made into nanoparticles, a maximum heating temperature of 45-60°C is preferred, more preferably 50-60°C. Furthermore, to achieve reusability, it should possess good photothermal cycling performance. Therefore, P1, P2, P4, and P5 molecules are preferred, with P2 molecules being more preferred.

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

[0178] Add RAFT modifier PEG to 5ml sealing tubes. 5000 -TC (250 mg, 0.05 mmol), 2-((N,N-dimethylamino)ethyl)methacrylate (DMAEMA, 471 mg, 3 mmol), p(methacryloxyethoxy)benzaldehyde (MAEBA, 234 mg, 1 mmol), tetrahydrofuran (THF, 2 mL). After three rounds of deoxygenation by freezing, the system was sealed under vacuum using a high-temperature spray gun, and then reacted at 70 °C for 6 h. The sealed tube was quickly placed in liquid nitrogen for cooling to end the reaction. The copolymer was precipitated in n-hexane, filtered, and then dried overnight in a vacuum drying oven to obtain the amphiphilic aldehyde-containing block copolymer A-1. GPC analysis showed that the number-average molecular weight of A-1 was 8300 and the dispersion index was 1.01. The chemical formula of [PEG-bP(MAEBA-co-DMAEMA)] is as follows:

[0179] Example 1: Preparation of antibacterial nanoparticles ABPL@AQM

[0180] Step S1: Weigh 3 mg of the P2 molecules and dissolve them in 3 mL of DMF to prepare a solution with a concentration of 1 mg / mL.

[0181] Step S2: Take 1 mL of solution 1, then add 10 mg of amphiphilic aldehyde-containing block copolymer A-1 to the sample vial and sonicate to dissolve it, stirring for 24 h. Under vigorous stirring, slowly add 9 mL of water at a rate of 1 mL / min using a peristaltic pump, stirring for 3 h after adding water, then add 10 mg of L-arginine and stir for 6 h. Finally, dialyze with ultrapure water (molecular weight cutoff (MWCO): 35 kDa) to remove DMF for 48 h, changing the dialysis medium periodically. Collect the dialyzed solution, sonicate it, and filter it using a 0.45 μm filter membrane to remove large particles, obtaining the antibacterial nanoparticles ABPL@AQM of this invention.

[0182] Comparative Example 1: Preparation of antibacterial nanoparticles ABP@AQM

[0183] Following the method in Example 1, without adding L-arginine, water was added directly and stirred for 9 hours to obtain reference nanoparticles ABP@AQM.

[0184] Comparative Example 2: Preparation of ABPL nanoparticles

[0185] Following the method of Example 1, without adding P2 molecules, the reference nanoparticles ABPL were obtained.

[0186] Application Example 1: Physicochemical Properties of Antibacterial Nanoparticles

[0187] Figure 7 This is a schematic diagram of the ABPL@AQM form factor. Figure 8 The images show the DLS, TEM, and SEM images of ABPL@AQM. Figure 7 As shown, the nanoparticles of this invention are prepared via a co-precipitation method, in which conjugated organic molecules and an amphiphilic aldehyde-containing polymer form water-soluble nanoparticles. Under vigorous stirring, L-arginine is inserted into the hydrophobic segment, and aggregation is prevented using an ultrasonic method. 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 a uniform particle size distribution and an average size of about 100 nm.

[0188] Figure 9 The images show DLS and TEM images of ABP@AQM. It can be seen that spherical nanoparticles with uniform particle size distribution cannot be obtained in the absence of arginine.

[0189] After storing ABPL@AQM for one month, its appearance was observed and no visible changes were found compared to its initial state. Figure 10 The DLS image of ABPL@AQM after one month of storage shows that the particle size of the antibacterial nanoparticles did not change, indicating that the antibacterial nanoparticles of the present invention have good stability.

[0190] Figure 11 The UV absorption curve and mass extinction coefficient of ABPL@AQM are shown, indicating that the antibacterial nanoparticles of the present invention have good photothermal properties.

[0191] Determination of L-arginine concentration in ABPL@AQM: After freeze-drying and dialyzing, the ABPL@AQM product was dissolved in 100 ml of deionized water, and the characteristic absorbance of L-arginine was analyzed using a UV-Vis spectrophotometer. The free L-arginine content was determined to be 3.17 mg using an L-arginine standard curve, with an encapsulation efficiency (EE) of 68.3%. The calculation formula is as follows:

[0192]

[0193] Measurement of photothermal effect: 200 μL of ABPL@AQM (5 μg / mL) was added to a 96-well plate. Different concentrations (40, 20, 10, 5, 2.5 μg / mL) of ABPL@AQM were placed in 96-well plates. A 650 nm laser (0.8 W cm⁻¹) was used for measurement. -2 Irradiate for 10 minutes, measuring the temperature every 30 seconds. Adjust the power density of the 650nm laser (0.2, 0.4, 0.6, 0.8 W / cm²). -2 The study also included measuring the temperature of NPs at a fixed concentration (5 μg / mL). Temperature fluctuations were measured using a thermal imager, and the results are as follows: Figure 12 As shown.

[0194] For example, ESR was used to detect the ability of the antibacterial nanoparticles of this invention to generate reactive oxygen species (ROS): using 2,2,6,6-tetramethylpiperidine (TEMP) as... 1 O2 indicator, 5,5-dimethyl-1-pyridine-n-oxide (DMPO) is used as an indicator of hydroxyl and superoxide anion radicals. Electron spin resonance (ESR) measurements are performed to determine the types of reactive oxygen species. 100 μL of ABPL@AQM (80 μg / mL) was mixed with 100 μL of LTEMP (100 mM) to detect O2, mixed with 10 μL of DMPO (8.79 mol / L) to detect hydroxyl radicals, and mixed with 10 μL of DMPO (8.79 mol / L) and 200 μL of methanol to determine superoxide anion radicals. Samples were added via capillary tubes and irradiated in situ for 0, 1, 2, and 3 minutes, and ESR signals were recorded. The results are shown below. Figure 13 As shown.

[0195] Furthermore, the generation of hydroxyl radicals by the nanoparticles in the embodiments and comparative examples of this invention was detected using a hydroxyl radical detection kit (O27, green fluorescence): 10 μL of 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 at a concentration of 5 μg / mL, respectively. The content of hydroxyl radicals before and after irradiation was determined by fluorescence spectrophotometry. The results are as follows: Figure 14 As shown in Figure a. In addition, a NO release experiment was conducted: equal volumes of ABP@AQM, ABPL@AQM, and ABPL (1 ml each, concentration 5 μg / mL) were prepared, and ABP@AQM and ABPL@AQM light exposure groups were established. After light exposure, H2O2 solution (1 mM) was added to each group, and after reacting for 15 min, 1 μL of 4,5-diaminofluorescein (1 mM) was added. 1 mL of the sample was taken, and the fluorescence intensity at 491 nm was measured using a fluorescence spectrometer (Hitachi, F-7000, excitation wavelength 491 nm, emission wavelength 513 nm). The results are shown in Figure a. Figure 14 As shown in b. The results show that the antibacterial nanoparticles of the present invention can effectively generate ROS under 650 nm laser irradiation, and due to the synergistic effect of L-arginine and the amphiphilic aldehyde-containing block copolymer, the NO level of ABPL@AQM increases significantly under irradiation.

[0196] Furthermore, we conducted experiments on the photothermal stability of ABPL@AQM at 5 μg mL. -1 At a concentration of [value missing], 200 μL LABPL@AQM was treated with a 650 nm laser (power 0.8 W cm⁻¹). -2 After irradiation, the temperature curve of ABPL@AQM is as follows: Figure 15 As shown in Figure a, using the cooling stage data, i.e., the negative natural logarithm of the driving force temperature (θ) with respect to 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%. The results are as follows. Figure 15 As shown in b, the calculation formula is:

[0197]

[0198] Where T max T is the highest steady-state temperature of the solvent. amb Let I be the ambient temperature, I be the laser power used in the photothermal experiment, and λ be the absorbance of the nanoparticles at 650 nm. hA can be determined by measuring the rate of temperature decrease after the light source is removed, representing the heat input of the solvent and container absorbing 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.

[0199] The results showed that the antibacterial nanoparticles of the present invention have good photothermal stability and suitable photothermal conversion efficiency. Although high temperatures can damage the function of bacterial heat shock proteins, leading to bacterial death, excessively high temperatures may also damage normal cells and even cause inflammatory responses. Therefore, a more preferable temperature is between 40-55°C, and more preferably around 45-50°C, which can induce apoptosis in normal cells while minimizing damage to normal cells. Figure 15 As shown in a, the highest temperature of the antibacterial nanoparticles of the present invention after irradiation for 10 minutes is about 43-44℃, which is beneficial to the practical application environment.

[0200] Application Example 2: Antibacterial Properties of Antibacterial Nanoparticles

[0201] The antibacterial effect of ABPL@AQM at different concentrations was evaluated using the microbroth dilution method. Concentrations of 50, 25, 12.5, 10, 5, and 2.5 μg / mL were used. -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 hours to the stationary phase. Subsequently, the cultures were diluted 1:100 with fresh LB medium (4 ml) and further grown at 37°C until mid-log phase was reached (OD600 = 0.5, SpectraMax M2 microplate reader (Molecular Devices, USA)). Figure 16 As shown in Figure A, the concentration of ABPL@AQM reached 5 μg / mL. -1 It produces an antibacterial effect when the concentration reaches 25 μg / mL. -1 At that time, the bacteria completely stopped growing. Considering the inherent photothermal effect of AQM, the study was conducted with 25 μg mL... -1 Intracellular photothermal effects of AQM, ABP@AQM and ABPL@AQM at different concentrations (650 nm, 0.8 W cm⁻¹) -2 ).from Figure 16 As can be seen from B and 16C, the temperature of the bacterial suspension increases with the extension of light exposure time, and ABPL@AQM has a good photothermal effect compared with the comparative example.

[0202] ROS levels in MRSA planktonic cells were measured. MRSA planktonic bacteria were treated with control group, ABPL, AQM, AQM+NIR, ABP@AQM, ABP@AQM+NIR, ABPL@AQM, and ABPL@AQM+NIR, respectively (laser group used 1W / cm²). 2 (650nm laser treatment for 10 minutes, the same below). The concentration of each treatment group was 25μg / mL. -1The volume was 1 mL. The temperature of the bacteria after irradiation was measured using a thermal imager. Subsequently, the bacterial suspension was centrifuged, resuspended in PBS, and the OD was ensured. 600 Consistent results were obtained. 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 (Germany, Zeiss), excitation light 488 nm). The results are as follows: Figure 16 As shown in D, the quantitative results are as follows: Figure 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 ROS production. 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 effect and ROS production, demonstrates the excellent effect of ABPL@AQM in effectively eradicating MRSA. It is evident that increased temperature in MRSA may induce the production of the heat shock protein Hsp70 to counteract photothermal damage. However, in the ABPL@AQM treatment group, the relatively high concentration of H2O2 on the biofilm surface reacted with L-arginine to generate NO, inhibiting Hsp70 expression and disrupting the MRSA defense mechanism.

[0203] Western blot analysis was used to detect the expression level of Hsp70 in MRSA after different treatment groups: MRSA suspensions from each treatment group were centrifuged to remove the culture medium, lysed on ice for 30 minutes with RIPA lysis buffer, and total bacterial protein was extracted. Total protein was then quantified using BSA. Proteins were separated using an SDS-PAGE system (Bio-Rad) and transferred to a PVDF membrane (Beyotime). The PVDF membrane was blocked in protein-free rapid blocking buffer for 30 minutes, then incubated overnight with Hsp70 primary antibody. After washing with TBST buffer, the membrane was incubated with secondary antibody, and signal detection was performed using ECL substrate. Signals were collected and analyzed using a QuickChemi 5200 system (Monad). Similarly, the expression of the reference protein β-actin was detected, and the results are shown below. Figure 16As shown in F and 16G, compared with the control group, the expression levels of Hsp70 in the AQM+NIR group and the ABP@AQM+NIR group were increased, indicating that the photothermal effect induced the upregulation of Hsp70 expression. Conversely, the ABPL group showed decreased Hsp70 expression, possibly due to the reaction of L-arginine with H2O2 to produce NO. Notably, the ABPL@AQM+NIR group had the lowest Hsp70 expression level, which may be due to the enhanced release of L-arginine from ABPL@AQM under photothermal stimulation, leading to increased NO production and increased inhibition of Hsp70. The process and mechanism of this application example are as follows. Figure 16 As shown in H.

[0204] Application Example 3: In vitro antibacterial and biofilm-disrupting properties of antibacterial nanoparticles

[0205] Collect the cultured MRSA and centrifuge twice with PBS (6000 rpm, 10 min). Before seeding the MRSA into 96-well microplates, adjust the suspension to 1.5 × 10⁻⁶ ppm with PBS. 6 Colony forming units (CFUs) mL -1 MRSA suspension (50 μL) was co-incubated for 3 h with control group, ABPL, AQM, AQM+NIR, ABP@AQM, ABP@AQM+NIR, ABPL@AQM, and ABPL@AQM+NIR (laser group at 1 W / cm). 2 (The sample was treated with a 650nm laser for 10 min). The concentration of each treatment group was 25 μg / mL. -1 The treatment volume was 1 mL. The solution was diluted 10,000 times with PBS. The diluted bacterial solution (20 μL) was placed on a TSB agar plate and incubated overnight at 37°C. Colonies were observed, and the results were as follows: Figure 17 As shown in Figure 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 these groups were alive. In contrast, a small number of bacteria in the AQM+NIR and ABP@AQM+NIR groups showed cell death, indicating that the photothermal effect of AQM has antibacterial properties. Furthermore, 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 the ABPL group, while the light-treated group showed widespread red fluorescence, indicating that the high temperature after light irradiation affected the growth of MRSA planktonic bacteria. The bacterial electroplating effect is shown in Figure A. Figure 17 As shown in Figure B, the results also indicate that the antibacterial nanoparticles of the present invention have good photothermal gas antibacterial properties.

[0206] In the early stages of biofilm infection, local hydrogen peroxide (H2O2) levels are elevated, ranging from 20 to 100 μM. Biofilms are well-known to be a major obstacle to wound healing. The antibacterial activity of ABPL@AQM against MRSA biofilms was evaluated using 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 hours. The concentration for 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 carefully rinsing the biofilm with deionized water, 33% v / v acetic acid was added to the wells to dissolve the crystal violet staining agent. Finally, the absorbance of each well at 560 nm was measured to assess the biofilm biomass. Following in vitro live / dead staining of the MRSA biofilm, it was simultaneously stained with SYTO 9 and PI dyes in the dark for 30 minutes. Fluorescence imaging was performed using confocal laser scanning microscopy, and z-stacking was compiled into a three-dimensional image. Results are as follows: Figure 17 As shown in C and 17D (quantitative), ABPL, P-AQM+NIR, ABPL@AQM and ABPL@AQM+NIR all exhibited certain biofilm scavenging activities, with biofilm scavenging rates of 50.4%, 45.5%, 60.6% and 91.2%, respectively.

[0207] SYTO 9 / PI live / dead bacterial staining: Treatment with PBS, ABPL, AQM, AQM+NIR, ABP@AQM, ABP@AQM+NIR, ABPL@AQM, and ABPL@AQM+NIR (concentration of each treatment group was 25 μg / mL). -1 After processing (1 mL), the MRSA suspension was incubated with SYTO-9 (2 μM, excitation 480 nm, emission 500 nm) and propidium iodide (PI, 1 μM, excitation 490 nm, emission 635 nm) in the dark for 15 min. After centrifugation to remove free SYTO-9 and PI, the suspension was washed twice with PBS. To fix and collect the MRSA, 4% paraformaldehyde solution was added. The synthesized MRSA suspension was transferred to a glass slide, air-dried, and fixed with impregnation oil. Finally, it was observed using a laser scanning confocal microscope. All bacteria stained green with SYTO-9, and bacteria with damaged cytoplasmic membranes stained red with PI. The results are shown below. Figure 17As shown in Figure D, almost all bacteria in the ABPL@AQM+ laser-treated group exhibited red fluorescence, indicating cell death and complete disruption of the biofilm structure. In particular, compared to the slight difference in biofilm resistance between the ABP@AQM light-treated and non-light-treated groups, ABPL@AQM+NIRLaser showed stronger anti-biofilm activity than both the non-light-treated and ABP@AQM(-)(+) groups. As previously mentioned, this is because L-arginine binds to H2O2 to form NO on the biofilm surface, thereby inhibiting the expression of heat shock proteins, i.e., suppressing the organism's heat-sensitive defense mechanism. Simultaneously, light irradiation enhanced the release of L-arginine, resulting in a stronger antibacterial effect.

[0208] The effects of different treatment methods on the microstructure of MRSA biofilms were visualized and evaluated using scanning electron microscopy (SEM, FEI Talos F200s high-resolution transmission electron microscope (USA, FEI)). MRSA biofilms from the previous treatment groups, obtained by centrifugation and PBS washing, were fixed overnight at 4°C with 2.5% glutaraldehyde. After incubation in 1% osmium tetroxide for 1 hour, the samples were dehydrated for 10 minutes in a series of fractionated ethanol solutions and tert-butanol (50%, 75%, 90%, and 100%). Subsequently, the samples were dried, coated with platinum, and observed using scanning electron microscopy. Figure 17 As shown in Figure E, the MRSA morphology in the control group was normal, while the ABPL group showed slight bacterial swelling and rupture. In the AQM+NIR group, slight wrinkling of the bacterial surface occurred due to the photothermal effect. In the ABP@AQM+NIR group, the solubility was better, and the photothermal effect was enhanced, leading to significant bacterial shrinkage and rupture. As expected, the ABPL@AQM+NIR group showed complete bacterial rupture, leakage of cell contents, and complete biofilm disruption. In conclusion, ABPL@AQM exhibits good antibacterial and antibiofilm activity.

[0209] Application Example 4: In vivo antibacterial activity and wound healing properties of antibacterial nanoparticles

[0210] The in vivo antibacterial efficacy of ABPL@AQM was evaluated using a skin wound model infected with MRSA.

[0211] Figure 18A shows the establishment of the animal model used for treatment and evaluation of treatment efficacy: BALB / c mice (6-8 weeks old, male, 20-30g) were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. A mouse skin wound infection model was established by hair removal using a hair removal device. After confirming that the paw skin was intact, typrazole was injected intraperitoneally according to the mouse's weight. After complete anesthesia, a circular wound was formed in the shaved area of ​​the hind leg, avoiding the fascia layer. 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).

[0212] Evaluation of in vivo anti-biofilm activity and wound healing status: In the control group, only PBS (200 μL) was instilled into the wound. PBS, ABPL, AQM, AQM+NIR, ABP@AQM, ABP@AQM+NIR, ABPL@AQM, and ABPL@AQM+NIR (5 μg / mL, 100 μL) were instilled into the wounds of the second, third, and fourth groups, respectively. A 650 nm laser (1 W / cm²) was used for wound observation. 2 The mice were irradiated with AQM+NIR, ABP@AQM+NIR, and ABPL@AQM+NIR for 10 minutes, and the temperature changes of the wound surface were monitored using a thermal imager. Figure 18 B). Figure 18 B indicates that the wound temperature increased over time under 650nm laser irradiation. Ten minutes after irradiation, the temperature in the AQM group was lower than that in the ABP@AQM and ABPL@AQM groups, consistent with in vitro experimental results. Under anesthesia, the wound size of mice was measured on days 1, 3, 5, and 7 after modeling. Figure 18 G and Figure 18 H). Here, CFU refers to colony-forming unit. Figure 18 The results showed that the ABPL@AQM+ light group had the strongest ability to inhibit bacterial regrowth. Figure 18Results showed that the wound healing rate in the ABPL@AQM+NIRlight group was significantly faster than that in other groups. By day 5, the wound area had decreased by 81.6%. After 7 days of treatment, the wound area decreased by 59.5%, 73.22%, 67.64%, 80%, 81.53%, 91.19%, 82.14%, and 98.05% in the control group, ABPL, AQM, AQM+NIR, ABP@AQM, ABP@AQM+NIR, ABPL@AQM, and ABPL@AQM+NIR groups, 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 assessed using the plate count method. Figure 18 C). The results showed that the control group had a large amount of biofilm growth on the wound, while the coated group had a large amount of bacterial growth. Although the wound healing rate in the ABPL group was poor, the bacterial clearance rate was improved compared with the control group. In addition, the antibacterial effects of AQM, ABP@AQM, and ABPL@AQM were significantly different under light-exposed conditions. On the 7th day of treatment, some mice were euthanized, and the skin tissue around the infected wounds was dissected. The residual amount of MRSA bacteria around various infected wounds and the healing status of the surrounding tissues were further evaluated (see photos). Figure 18 D, 18E, and 18F). Figure 18 D、 Figure 18 E represents the reduction in wound area in mice after different treatment times. It can be seen that ABPL@AQM has a better wound healing speed and effect compared to the control group. Figure 18 The F results showed neutrophil infiltration in the control group, indicating persistent inflammation. The ABPL group showed some relief, while the AQM group showed no significant effect. In contrast, the ABPL@AQM+NIR group had fewer neutrophils and higher fibrinogen levels, suggesting better wound healing. Furthermore, the residual MRSA bacteria around various infected wounds and the healing of surrounding tissues 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 (results are shown in...). Figure 18 I) The results showed that the ABPL@AQM+NIR treatment group had reduced IL-6 and TNF-α levels, indicating a reduction in systemic inflammation and suggesting that the mice had entered the late stage of wound healing.

[0213] Application Example 5: Biocompatibility and Biosafety of Antibacterial Nanoparticles

[0214] To evaluate the biosafety of the antibacterial nanoparticles of this invention, complete blood counts and serum biochemical indicators of mice in the treatment groups were measured on day 9 after treatment. Additionally, major organs such as the heart, lungs, liver, spleen, and kidneys of mice in each treatment group were excised and analyzed pathologically. All tissue specimens were fixed in 4% paraformaldehyde, dehydrated, embedded in paraffin, sectioned, and stained with H&E. The results of the above two experiments are as follows: Figure 19 As shown.

[0215] Figure 19 In group A, complete blood count analysis showed that, compared with other groups, the ABPL, AQM+NIR, ABP@AQM+NIR, and ABPL@AQM+NIR groups had decreased white blood cell (WBC) levels, indicating successful wound healing in mice. Other parameters, including red blood cell (RBC), hemoglobin (HGB), hematocrit (HCT), alanine aminotransferase (ALT), and aspartate aminotransferase (AST), were all within the normal range for mice. Histopathological examination results of major organs in different treatment groups are shown below. Figure 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 showed good biocompatibility.

[0216] In summary, the antibacterial nanoparticles of the present invention have a good photo-heating-gas response mechanism, which can synergistically produce bactericidal, antibacterial and biofilm eradication effects, reduce or eliminate inflammatory response, and are especially suitable as antibacterial dressings to promote wound healing.

[0217] All technical solutions described above that fall within the scope of this invention's conceptual framework are protected by this invention. It should be noted that for those skilled in the art, any improvements and modifications made without departing from the principles of this invention should also be considered within the scope of protection of this invention.

Claims

1. An antibacterial nanoparticle, said antibacterial nanoparticle being a crosslinked product of a raw material composition comprising the following components (A) to (C): (A) A quinone-type conjugated organic molecule as shown in formula (I): Equation (I); (B) Amphiphilic aldehyde-containing block copolymers; and, (C) L-arginine; in: The 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; The amphiphilic aldehyde-containing block copolymer is [PEG-bP(MAEBA-co-DMAEMA)]; The number-average molecular weight of the amphiphilic aldehyde-containing block copolymer is 2,000 to 50,000. In the amphiphilic aldehyde-containing block copolymer, the number average molecular weight of the hydrophobic block is 1000-30000, and it is obtained by polymerization of a monomer composition containing at least one aldehyde-containing hydrophobic monomer. The monomer composition comprising at least one hydrophobic monomer containing an aldehyde group is and A mixture; The and The mass ratio is 1:3 to 1:1; The hydrophilic block is polyethylene glycol; The number-average molecular weight of the hydrophilic block is 2000-10000; The mass ratio of the hydrophilic block to the hydrophobic block in the amphiphilic aldehyde-containing block copolymer is 2:8 to 8:

2.

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

3. The antibacterial nanoparticles according to claim 2, wherein the raw material composition comprises: (A) 1-10 wt% of quinone conjugated organic molecules as shown in formula (I): (B) 30-70 wt% amphiphilic aldehyde-containing block copolymers; and, (C) L-arginine 20-50wt%.

4. The antibacterial nanoparticles according to claim 1, wherein the n value of the quinone conjugated organic molecule represented by formula (I) is 10-100.

5. The antibacterial nanoparticles according to claim 4, wherein the n value of the quinone conjugated organic molecule represented by formula (I) is 10-40.

6. The antibacterial nanoparticles according to any one of claims 1-5, wherein the structural formula of the amphiphilic aldehyde-containing block copolymer is: ； in, m, m1, and m2 represent the degree of aggregation of the corresponding repeating units.

7. A method for preparing the antibacterial nanoparticles according to any one of claims 1-6, the method comprising the following steps: (S1) The quinone conjugated organic molecule shown in formula (I) and the amphiphilic aldehyde-containing block copolymer are mixed in an organic solvent, and then an aqueous solvent is added to assemble nanoparticles. (S2) Add L-arginine to the mixture in step S1 to crosslink the reaction system.

8. An antibacterial preparation for eliminating bacterial biofilm and promoting wound healing, comprising the antibacterial nanoparticles as described in any one of claims 1-7.

9. The formulation according to claim 8, wherein the bacteria is methicillin-resistant Staphylococcus aureus (MRSA).

10. A biomedical material comprising the antibacterial nanoparticles according to any one of claims 1-6.

11. The biomedical material according to claim 10, wherein the biomedical material is a wound dressing, hand sanitizer, or antibacterial coating.

12. A quinone-type organic molecule with the structure shown in formula (I): Equation (I); The n=5-200.

13. The use of the quinone organic molecule of claim 12 in the preparation of the antibacterial nanoparticles of any one of claims 1-6, wherein the bacterium is methicillin-resistant Staphylococcus aureus (MRSA).

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