Antiviral composite particles, screening methods and uses thereof

Abstract

The application discloses an antiviral composite particle and a screening method and application thereof. The application utilizes a polymer reversible hydrogen bond network with hydrophilicity and hydrophobicity, precisely regulates a coordination mode of a metal oligomer, balances material dispersibility and virus capturing capacity by adjusting a charge distribution of a polymer main chain, and controls a material surface to form an electrostatic barrier through a surface potential, so that the material selectively adsorbs virus particles without entering host cells, and cell toxicity is avoided. In addition, through the synergistic design of controllable assembly of the metal oligomer and polymer interface engineering, the application breaks through the dilemma of the trade-off between "activity-toxicity" of traditional metal-based materials, and provides strong support for the development of broad-spectrum, safe antiviral drugs, coatings, protective equipment and the like.

Description

Technical Field

[0001] This invention belongs to the field of virus protection, and in particular relates to an antiviral composite particle, its screening method, and its application. Background Technology

[0002] The ongoing viral pandemic continues to threaten global public health security, while traditional antiviral strategies have significant limitations. Small molecule antiviral drugs (such as viral entry inhibitors fostemsavir, polymerase inhibitors favipiravir, and sofosbuvir) work by targeting specific viral enzymes, but they suffer from two major drawbacks: first, viruses readily develop drug resistance through genetic mutations; and second, their spectrum of action is narrow, making them ill-suited to address complex and ever-changing viral mutations. This situation urgently necessitates the development of novel formulations with broad-spectrum antiviral activity, rather than traditional drugs targeting single targets.

[0003] Metal-based materials (such as silver and copper compounds) exhibit unique potential in the field of antiviral research due to their inherent antibacterial properties. Existing metal antiviral systems are mainly divided into two categories: one is metal oxides (such as ZnO), which destroy virus macromolecules and cleave disulfide bonds on the surface of virus particles by generating reactive oxygen species (ROS); the other is metal ions (such as Ag...). + Cu 2+ Antiviral materials work by interacting with the viral envelope to block the virus's invasion of host cells. However, these materials face a key bottleneck: poor selectivity for viruses and a tendency to induce host cell toxicity at high concentrations, making it difficult to achieve both antiviral activity and biosafety. Therefore, there is an urgent need to develop an antiviral material that combines both antiviral activity and biosafety. Summary of the Invention

[0004] To address at least some of the technical problems in the prior art, the present invention provides an antiviral complex particle, a screening method therefor, and its applications. Specifically, the present invention includes the following:

[0005] A first aspect of the present invention provides a method for screening antiviral complex particles, comprising the following steps:

[0006] (1) Provide a library of composite particles, wherein the composite particles include polymer and oligomeric metal salt particles;

[0007] (2) Contact the test composite particles in the composite particle library with the virus, and then detect the virus activity; and

[0008] (3) When the virus activity decreases after the test composite particle comes into contact with the virus, the test composite particle is used as an antiviral composite particle.

[0009] In some embodiments, according to the method for screening antiviral complex particles according to the present invention, the complex particle library comprises a polymer library P and an oligomerized metal salt particle library M, wherein polymer P in the polymer library... x With oligomerized metal salt particles M in the oligomerized metal salt particle library y Capable of hybridizing to form composite particles P x M y .

[0010] In some embodiments, the method for screening antiviral complex particles according to the present invention, wherein the polymer comprises hydrophilic blocks and hydrophobic blocks.

[0011] In some embodiments, according to the method for screening antiviral complex particles according to the present invention, the monomers of the hydrophobic block of the polymer include metal-bound monomers and branched side-chain monomers, and the metal-bound monomers contain amino and / or hydroxyl groups; the branched side-chain monomers are selected from at least one of DMA, DEA, EPA, DPA, DBA and DAA.

[0012] In some embodiments, according to the method for screening antiviral complex particles according to the present invention, the metal in the oligomerized metal salt is selected from at least one of magnesium, calcium, chromium, cobalt, copper and zinc.

[0013] In some embodiments, according to the method for screening antiviral complex particles according to the present invention, step (2) includes adding the test complex particle and virus to a cell model and measuring the virus inhibition rate and / or cell survival rate.

[0014] In a second aspect, the present invention provides an antiviral composite particle prepared by the method described in the first aspect of the present invention.

[0015] In some embodiments, the antiviral complex particles according to the present invention comprise PAMO-DPA / Cu.

[0016] A third aspect of the present invention provides an antiviral composition comprising the antiviral complex particles described in the second aspect of the present invention and a pharmaceutically acceptable carrier.

[0017] A fourth aspect of the invention provides the use of the antiviral composite particles according to the second aspect of the invention in the preparation of antiviral drugs, coatings or protective equipment.

[0018] This invention utilizes the reversible hydrogen bond network of polymers (such as polyacrylates) that have both hydrophilic and hydrophobic properties to precisely control the coordination mode of metal oligomers. It balances the material's dispersibility and virus capture ability by adjusting the charge distribution of the polymer backbone, and forms an electrostatic barrier on the material surface by controlling the surface potential. This allows the material to selectively adsorb virus particles without entering the host cell, thus avoiding intracellular toxicity.

[0019] Furthermore, this invention breaks through the traditional dilemma of "activity-toxicity" trade-off in metal-based materials by controlling the assembly of metal oligomers and co-designing polymer interface engineering, providing strong support for the development of broad-spectrum and safe antiviral drugs, coatings, protective equipment, etc. Attached Figure Description

[0020] Figure 1 The screening process for antiviral composite particles is illustrated in the following example: A represents the zeta potential, particle size, and polydispersity index of the oligomerized metal salt; B represents the zeta potential of the polymer; C is a schematic diagram of the synthesis pathways of the oligomerized metal salt PAMO-Metal, the polymer PAMO-Polymer, and the composite particle PAMO-Polymer / Metal, where M represents different metals and R represents different alkyl side chains; D represents the zeta potential, particle size, and polydispersity index of the composite particles; E represents the zeta potential of the PAMO-Polymer / Cu composite particles; F represents the infrared spectral analysis results of PAMO-DPA / metal; and G represents the scanning electron microscope (SEM) image of PAMO-DPA / metal.

[0021] Figure 2 The antiviral screening process of the PAMO-Polymer / Metal composite particle is shown. A is a schematic diagram of the PAMO-DPA / Metal combo library screening; B shows the inhibitory effect of PAMO-DPA / Metal on the RNA virus VSV-GFP; C shows the inhibitory effect of PAMO-DPA / Metal on the DNA virus HSV; D is a schematic diagram of the PAMO-Polymer / Cu combo library screening; E shows the inhibitory effect of PAMO-Polymer / Cu on VSV-GFP virus; F and G show the inhibitory effects of PAMO-DPA / Cu on mouse hepatitis virus and Sendai virus, respectively.

[0022] Figure 3 A represents the median lethal concentration (IC50) analysis of PAMO-Polymer / Cu; B represents the cytotoxicity heatmap analysis of PAMO-Polymer / Cu; C represents the antiviral dose-effect heatmap of PAMO-Polymer / Cu; and D represents the comprehensive efficacy-toxicity assessment of the PAMO-Polymer / Cu system.

[0023] Figure 4The in vivo antiviral effects and safety assessment of PAMO-DPA / Cu are shown. A is a schematic diagram of the construction of the viral infection model and the dosing regimen in experimental mice; B is the effect of drug administration on the survival time of infected mice; CD is the result of viral load detection in target organs; E is the result of liver pathological analysis; and F is the result of multi-organ tissue pathological analysis.

[0024] Figure 5 The dose-dependent safety assessment results of the antiviral complex particles of the present invention are shown.

[0025] Figure 6 The effects of different hydrogen bond donor-acceptor ratios on the performance of PAMO-DPA / Cu are shown. A is a schematic diagram of the hydrogen bond interaction mechanism in PAMO-DPA / Cu; B shows the dispersion state of PAMO-DPA / Cu under different hydrogen bond donor-acceptor ratios; C shows the polydispersity index of PAMO-DPA / Cu under different hydrogen bond donor-acceptor ratios; D shows the particle size distribution characteristics of PAMO-DPA / Cu under different hydrogen bond donor-acceptor ratios; and E shows the time-dependent effect of different hydrogen bond donor-acceptor ratios on antiviral activity. Detailed Implementation

[0026] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0027] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that the upper and lower limits of the range and each intermediate value between them are specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0028] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0029] Methods for screening antiviral complex particles

[0030] One aspect of the present invention provides a method for screening antiviral complex particles, comprising steps (1)-(3):

[0031] (1) Provide a library of composite particles, wherein the composite particles include polymer and oligomeric metal salt particles;

[0032] (2) Contact the test composite particles in the composite particle library with the virus, and then detect the virus activity; and

[0033] (3) When the virus activity decreases after the test composite particle comes into contact with the virus, the test composite particle is used as an antiviral composite particle.

[0034] The following details each step.

[0035] Step (1) of the method of the present invention is to provide a library of composite particles, wherein the composite particles include polymer and oligomerized metal salt particles.

[0036] In a preferred embodiment, the composite particle library is provided by the following methods: (I) reacting metal ions (examples of which include, but are not limited to, copper, calcium, magnesium, cobalt, zinc, chromium, silver, rare earth metal ions, etc.) and inorganic acids (preferably acids that form slightly soluble or insoluble salts with metal ions, such as, but not limited to, phosphoric acid, sulfuric acid, carbonic acid, etc.) in an oligomerization initiator (examples of which include, but are not limited to, triethylamine, trimethylamine (TMA), tripropylamine (TPA), pyridine, N-methylmorpholine, choline, tetramethylammonium hydroxide, etc.) to obtain an oligomerization metal salt library; (II) polymerizing hydrophilic blocks (e.g., but not limited to, polyethylene glycol, PVP, polyamino acids, etc.), metal-bound monomers (monomers containing amino and / or hydroxyl groups, such as, but not limited to, AMA, lysine, etc.) and branched side-chain monomers (e.g., but not limited to, DMA, DEA, EPA, DPA, DBA, DAA, etc.) in the presence of an initiator to obtain a polymer library; (III) hybridizing the oligomerization metal salts and polymers to obtain the composite particle library.

[0037] To enhance the antiviral effect of the composite particles, the amounts of the oligomerized metal salt obtained in step (I) and the polymer obtained in step (II) can be controlled to ensure that the ratio of hydrogen bond donors in the polymer to hydrogen bond acceptors in the oligomerized metal salt is within a suitable range. In this invention, the ratio of hydrogen bond donors to hydrogen bond acceptors is 1:(1-15), preferably 1:(2-14), even more preferably 1:(3-13), and more preferably 1:(4-12), for example 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, and 1:12.

[0038] In another preferred embodiment, the composite particle library is provided by the following method: (I) Copper chloride dihydrate (0.5-5 g) is dissolved in anhydrous ethanol, triethylamine (5-30 mL) is added, and the mixture is stirred at room temperature for 20-40 minutes. Phosphoric acid ethanol solution (500-900 μL) is added dropwise, and the mixture is stirred overnight. The mixture is centrifuged (2,000-50,000 rpm, 5-20 minutes), the supernatant is discarded, and the precipitate is washed 3-10 times with anhydrous ethanol and dried to obtain the oligomerized metal salt PAMO-Cu. PAMO-Mg, PAMO-Ca, PAMO-Cr, PAMO-Co, and PAMO-Zn are prepared using the same method to obtain the oligomerized metal salt particle library M; (II) A hydrophilic polymer was reacted with 2-bromoisobutyryl bromide (BiBB) in the presence of triethylamine to generate a Br-terminated hydrophilic polymer. After dialysis and lyophilization purification, the Br-activated hydrophilic polymer, monomers DPA, AMA, cuprous bromide, azobisisobutyronitrile, and TPMA were dissolved in a mixed solvent in a certain proportion. The mixture was deoxygenated by argon bubbling for 20-50 minutes and reacted in an oil bath at 50-90℃ for 30-70 hours to obtain polymer PAMO-DPA. PAMO-DMA, PAMO-DEA, PAMO-EPA, PAMO-DBA, and PAMO-DAA were prepared using the same method to obtain polymer library P; (III) The polymer PAMO-DPA was dissolved in dichloromethane, and the oligomerized metal salt particles PAMO-Cu were dispersed in anhydrous ethanol. The two solutions were mixed in equal volumes and stirred overnight at room temperature. The solvent was removed by rotary evaporation, and after redissolving in ethanol / tetrahydrofuran, deionized water was added. The mixture was then sonicated to evaporate the organic solvent, thus obtaining PAMO-DPA / Cu composite particles. PAMO-DPA / Mg, PAMO-DPA / Ca, PAMO-DPA / Cr, PAMO-DPA / Co, PAMO-DPA / Zn, PAMO-DMA / Cu, PAMO-DEA / Cu, PAMO-EPA / Cu, PAMO-DBA / Cu, and PAMO-DAA / Cu were prepared using the same method to obtain a composite particle library.

[0039] Understandably, before testing the antiviral activity of composite particles, preliminary screening can be performed on the oligomerized metal salt library, polymer library, or composite particle library using steps such as particle size analysis, zeta potential analysis, and polydispersity index analysis. The order of preliminary screening for the oligomerized metal salt library and the polymer library is not particularly limited. For example, preliminary screening of the oligomerized metal salt library can be performed first, followed by preliminary screening of the polymer library, or vice versa, or both can be performed simultaneously.

[0040] In this invention, the composite particle library includes a polymer library P and an oligomerized metal salt particle library M, and the polymer P in the polymer library... x With oligomerized metal salt particles M in the oligomerized metal salt particle libraryy Capable of hybridizing to form composite particles P x M y Polymer P x It refers to any polymer in the polymer library, M y It refers to any one type of oligomerizing metal salt particle in the oligomerizing metal salt particle library, P x M y This refers to composite particles formed by random hybridization combinations of any polymer and any oligomerized metal salt particles from the aforementioned library. In a preferred embodiment, the polymer library P includes PAMO-DMA, PAMO-DEA, PAMO-EPA, PAMO-DPA, PAMO-DBA, and PAMO-DAA. In a preferred embodiment, the oligomerized metal salt particle library M includes PAMO-Cu, PAMO-Mg, PAMO-Ca, PAMO-Cr, PAMO-Co, and PAMO-Zn. In a preferred embodiment, the composite particle library includes PAMO-DPA / Cu, PAMO-DPA / Mg, PAMO-DPA / Ca, PAMO-DPA / Cr, PAMO-DPA / Co, PAMO-DPA / Zn, PAMO-DMA / Cu, PAMO-DEA / Cu, PAMO-EPA / Cu, PAMO-DBA / Cu, and PAMO-DAA / Cu.

[0041] In this invention, the polymer comprises hydrophilic and hydrophobic blocks, wherein the hydrophilic blocks are derived from, but are not limited to, PEG; the monomers of the hydrophobic blocks comprise metal-bound monomers and branched side-chain monomers, and the metal-bound monomers contain amino and / or hydroxyl groups, examples of which include, but are not limited to, AMA; examples of the branched side-chain monomers include, but are not limited to, DMA, DEA, EPA, DPA, DBA, DAA, etc. In a preferred embodiment, the metal-bound monomer is AMA, the branched side-chain monomer is DPA, and the monomer of the hydrophilic block is PEG.

[0042] In this invention, the equivalent of the branched side chain monomer is 50-150, preferably 60-140, even more preferably 70-130, and more preferably 80-120, for example 80, 85, 90, 95, 100, 105, 110, 115, 120.

[0043] In this invention, the metal in the oligomerized metal salt is not particularly limited, and examples include, but are not limited to, magnesium, calcium, chromium, cobalt, copper, zinc, selenium, rare earth metals, etc.

[0044] Step (2) of the method of the present invention involves contacting the test composite particles in the composite particle library with the virus and then detecting viral activity. The method for detecting viral activity is not particularly limited, and examples include, but are not limited to, flow cytometry, real-time quantitative PCR, ELISA / Western Blot, plaque assay, and TCID50 determination. It is understood that the test composite particles and the virus can be added to a cell model, and the viral inhibition rate and cell viability can be measured. The method for detecting cell viability is not particularly limited, and examples include, but are not limited to, the CCK-8 assay, MTT assay, XTT assay, ATP assay, and Calcein-AM assay.

[0045] Step (3) of the present invention is to determine whether the composite particle to be tested is an antiviral composite particle. When the activity of the virus decreases after the composite particle to be tested comes into contact with the virus, the composite particle to be tested is regarded as an antiviral composite particle.

[0046] Antiviral complex particles

[0047] In one aspect, the present invention provides an antiviral composite particle prepared by the method described herein. In a preferred embodiment, the antiviral composite particle comprises PAMO-DPA / Cu.

[0048] In this invention, the virus includes DNA viruses and / or RNA viruses. Examples of DNA viruses include, but are not limited to, herpes simplex virus (e.g., herpes simplex virus type 1, herpes simplex virus type 2), varicella-zoster virus, cytomegalovirus, smallpox virus, monkeypox virus, adenovirus, human papillomavirus, parvovirus, bocavirus, hepatitis B virus, etc.; examples of RNA viruses include, but are not limited to, vesicular stomatitis virus, mouse hepatitis virus, Sendai virus, encephalomyocytoxin virus, influenza A virus, influenza B virus, influenza C virus, dengue virus, Zika virus, Ebola virus, Marburg virus, Nipah virus, coronavirus, hepatitis A virus, hepatitis C virus, rotavirus, measles virus, human immunodeficiency virus, respiratory syncytial virus, rabies virus, etc. In one preferred embodiment, the DNA virus is herpes simplex virus. In another preferred embodiment, the RNA virus is vesicular stomatitis virus. In yet another preferred embodiment, the RNA virus is mouse hepatitis virus. In yet another preferred embodiment, the RNA virus is Sendai virus. In a preferred embodiment, the virus of the present invention is an enveloped virus.

[0049] In a preferred embodiment, the antiviral composite particles of the present invention can achieve a virus inhibition rate of not less than 90%, for example not less than 95%, or even more than 99%, while having a cell survival rate of not less than 95% in terms of cytotoxicity. Therefore, the antiviral composite particles of the present invention have both antiviral activity and biosafety.

[0050] Antiviral Composition

[0051] In one aspect, the present invention provides an antiviral composition comprising the antiviral complex particles described herein and a pharmaceutically acceptable carrier.

[0052] In this invention, pharmaceutically acceptable carriers are used to transport or deliver a drug from one organ or part of the body to another organ or part of the body. Each carrier is "acceptable," meaning it is compatible with other components of the formulation (e.g., antiviral complex particles) and does not harm the patient. The pharmaceutically acceptable carriers include at least one of diluents, fillers, absorbents, wetting agents, binders, disintegrants, lubricants, sweeteners, preservatives, and antioxidants. Examples of diluents include, but are not limited to, physiological saline, aqueous buffer solutions, solvents, and dispersion media; fillers include, but are not limited to, starch, lactose, mannitol, and microcrystalline cellulose; absorbents include, but are not limited to, calcium sulfate, dicalcium phosphate, and calcium carbonate; wetting agents include, but are not limited to, water and ethanol; binders include, but are not limited to, hydroxypropyl methylcellulose, povidone, and microcrystalline cellulose; disintegrants include, but are not limited to, croscarmellose sodium, croscarmellose, surfactants, and low-substituted hydroxypropyl cellulose; lubricants include, but are not limited to, magnesium stearate, talc, polyethylene glycol, sodium dodecyl sulfate, micronized silica gel, and talc; sweeteners include, but are not limited to, sucralose, acetylsupan, saccharin, sucrose, xylitol, mannitol, sorbitol, glucose, fructose, and aspartame; preservatives include, but are not limited to, parabens, chlorobutanol, phenol, and sorbic acid; and antioxidants include, but are not limited to, ascorbic acid and methionine.

[0053] use

[0054] This invention further provides the use of the composite particles in the preparation of antiviral drugs, coatings, or protective equipment. The coatings include, but are not limited to, coatings for medical devices, public facilities, household goods, and personal items. The protective equipment includes, but is not limited to, medical masks, protective suits, medical gloves, and respirators.

[0055] In this invention, antiviral action is achieved by administering a therapeutically effective amount of the antiviral drug to the subject. Subjects include, but are not limited to, mammals, including but not limited to, humans, mice, rabbits, cats, dogs, cattle, sheep, and pigs.

[0056] In this invention, there are no particular limitations on the method of administration of the drug. Representative methods of administration include, but are not limited to, oral, rectal, parenteral (intravenous, intramuscular, or subcutaneous) and local administration. Accordingly, the drug of this invention can be formulated into various clinically acceptable dosage forms, including oral dosage forms, injectable dosage forms, local dosage forms, or topical dosage forms.

[0057] The therapeutically effective dose described in this invention refers to a pharmaceutically recognized effective dosage, meaning that the amount of the active compound (i.e., the compound granules) is sufficient to significantly improve the condition without causing serious side effects. The daily dosage of the compound granules is typically 0.01-1,000 mg / kg, preferably 0.01-500 mg / kg, or 0.01-400 mg / kg, or 0.01-300 mg / kg, or 0.01-200 mg / kg, or 0.01-150 mg / kg, or 0.01-100 mg / kg, or 0.01-50 mg / kg, or 0.01-40 mg / kg, or 0.01-30 mg / kg, with 0.01-20 mg / kg being the most preferred. Exemplary effective dosages include, for example, 0.01 mg / Kg, 0.05 mg / Kg, 0.1 mg / Kg, 0.2 mg / Kg, 0.3 mg / Kg, 0.4 mg / Kg, 0.5 mg / Kg, 0.75 mg / Kg, 0.95 mg / Kg, 1 mg / Kg, 1.25 mg / Kg, 1.5 mg / Kg, 1.75 mg / Kg, 2 mg / Kg, 2.5 mg / Kg, 2.75 mg / Kg, 3 mg / Kg, 3.25 mg / Kg, 3.5 mg / Kg, 3.75 mg / Kg, 4 mg / Kg, 4.25 mg / Kg, 4.5 mg / Kg, 4.75 mg / Kg, 5 mg / Kg, 5.25 mg / Kg, 5.5 mg / Kg, 5.75 mg / Kg, 6 mg / Kg, and 6.25 mg / Kg. The recommended doses are: 6.5 mg / kg, 6.75 mg / kg, 7 mg / kg, 7.25 mg / kg, 7.5 mg / kg, 7.75 mg / kg, 8 mg / kg, 8.25 mg / kg, 8.5 mg / kg, 8.75 mg / kg, 9 mg / kg, 9.25 mg / kg, 9.5 mg / kg, 9.75 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg, and 20 mg / kg. These doses can be administered as a single daily dose, divided into multiple daily doses, or at intervals.

[0058] Example 1

[0059] This embodiment exemplifies a screening method for antiviral composite particles. In this embodiment, PAMO is a polyacrylate-metal oligomer, also known as a polyacrylate-metal oligomer; PAMO-Polymer is a polymer; PAMO-Metal is an oligomerized metal salt, also known as a metal oligomer; PAMO-Polymer / Metal is a composite particle; DMA is 2-(dimethylamino)ethyl methacrylate; DEA is 2-(diethylamino)ethyl methacrylate; EPA is 2-(ethylpropylamino)ethyl methacrylate; DPA is 2-(dipropylamino)ethyl methacrylate; DBA is 2-(dibutylamino)ethyl methacrylate; DAA is 2-(dipentylamino)ethyl methacrylate; and AMA is 2-aminoethyl methacrylate.

[0060] 1. Construction of composite particle library

[0061] 1.1 Materials and Laboratory Animals

[0062] The calcium chloride dihydrate (CaCl2), cobalt chloride hexahydrate (CoCl2), chromium chloride (CrCl2), copper chloride dihydrate (CuCl2), magnesium chloride hexahydrate (MgCl2), and zinc chloride (ZnCl2) used in the experiment were purchased from Aladdin or Anaiji Chemical; triethylamine (TEA), phosphoric acid (H3PO4), and other organic solvents were purchased from Beijing Tongguang Fine Chemical; the cell counting kit (CCK-8) was purchased from TargetMol; the reverse transcription and quantitative PCR kit was purchased from AbClonal; paraformaldehyde fixative and RNA / DNA extraction reagents were purchased from Beijing Solarbio; and fluorescent dyes were purchased from Beyotime.

[0063] BALB / c mice aged 6-8 weeks were used as experimental animals and housed in an environment with a temperature of 20-26℃, humidity of 40-70%, and a 12-hour light-dark cycle. The animal experimental protocol was approved by the Ethics Committee of Peking University School of Medicine (No.: DLASBD0605).

[0064] 1.2 Experimental Methods

[0065] 1.2.1 Preparation of oligomerized metal salt particle library

[0066] Copper chloride dihydrate (1.70 g, 10 mmol) was dissolved in anhydrous ethanol, and triethylamine (14 mL) was added. The mixture was stirred at room temperature for 30 minutes, and 700 μL of phosphate ethanol solution was added dropwise. The mixture was stirred overnight, centrifuged (10,000 rpm, 10 minutes), and the supernatant was discarded. The precipitate was washed five times with anhydrous ethanol and dried to obtain the oligomerized metal salt PAMO-Cu. PAMO-Mg, PAMO-Ca, PAMO-Cr, PAMO-Co, and PAMO-Zn were prepared using the same method to obtain the oligomerized metal salt particle library M. Dynamic light scattering (DLS) analysis was performed, and the results are as follows: Figure 1 As shown in Figure A, the particle size distribution of PAMO-Metal is in the range of 100-160 nm. The polydispersity index (PDI) exhibited by PAMO-Cu indicates its excellent stability. The zeta potential analysis results can be used to evaluate the cell binding ability of oligomeric metal salt particles and subsequent therapeutic effects.

[0067] 1.2.2 Preparation of Polymer Library

[0068] Polyethylene glycol (PEG) reacts with 2-bromoisobutyryl bromide (BiBB) in the presence of triethylamine to generate PEG-Br, which is purified by lyophilization via dialysis and then used for later use.

[0069] PEG-Br (1 equivalent), monomer DPA (100 equivalent), AMA, cuprous bromide, azobisisobutyronitrile (AIB), and TPMA were dissolved in a DMF / isopropanol (1:1) mixed solvent in a specific ratio. The mixture was deoxygenated by argon bubbling for 30 minutes and reacted in an oil bath at 70°C for 48 hours to obtain the polymer PAMO-DPA. PAMO-DMA, PAMO-DEA, PAMO-EPA, PAMO-DBA, and PAMO-DAA were prepared using the same method, resulting in polymer library P. By precisely controlling the hydrophobic block structure, six polymers with different side chain lengths (i.e., PAMO-DMA, PAMO-DEA, PAMO-EPA, PAMO-DBA, PAMO-DPA, and PAMO-DAA) were successfully synthesized. The monomer structures and reaction products were confirmed using ¹H NMR spectroscopy. Chemical shift changes in the range of 3.2–0.4 ppm confirmed the precise construction of the target polymer structures. Zeta potential analysis was performed on different polymers, and the results are as follows: Figure 1 As shown in B, the weak positive charge exhibited by PAMO-DPA indicates that it has better safety characteristics.

[0070] 1.2.3 Preparation of composite particles

[0071] The polymer PAMO-DPA was dissolved in dichloromethane, and the oligomerized metal salt particles PAMO-Cu were dispersed in anhydrous ethanol. Equal volumes of the two solutions were mixed and stirred overnight at room temperature. The solvent was removed by rotary evaporation, and the mixture was reconstituted in ethanol / tetrahydrofuran. Deionized water was then added, and the mixture was sonicated to evaporate the organic solvent, yielding PAMO-DPA / Cu composite particles. PAMO-DPA / Mg, PAMO-DPA / Ca, PAMO-DPA / Cr, PAMO-DPA / Co, PAMO-DPA / Zn, PAMO-DMA / Cu, PAMO-DEA / Cu, PAMO-EPA / Cu, PAMO-DBA / Cu, and PAMO-DAA / Cu were prepared using the same method, resulting in a composite particle library. A schematic diagram of the composite particle synthesis is shown below. Figure 1 As shown in C.

[0072] Dynamic light scattering analysis results are as follows Figure 1 As shown in Figure D, the average particle size of PAMO-DPA / Metal is significantly reduced to 30-60 nm, exhibiting better stability. In particular, the PDI values ​​of PAMO-DPA / Ca and PAMO-DPA / Cu are reduced, confirming that polymer coupling improves dispersibility. Notably, the PAMO-DPA / Metal system as a whole is positively charged, with PAMO-DPA / Ca and PAMO-DPA / Cu exhibiting relatively low zeta potentials, which are beneficial for improving biocompatibility. The zeta potential of PAMO-Polymer / Cu is shown in Figure D. Figure 1 As shown in Figure E, PAMO-DPA / Cu exhibits the lowest zeta potential and lower surface cytotoxicity. Infrared spectroscopy and scanning electron microscopy (SEM) analysis of PAMO-DPA / Metal yielded the following results: Figure 1 As shown in F and G.

[0073] 2. Evaluation of antiviral activity

[0074] 2.1 Evaluation of the antiviral activity of PAMO-DPA / Metal

[0075] In the A549 cell model, after treating VSV-GFP virus with 0.01 multiplicity of infection (MOI) and 250 μM PAMO-DPA / Metal for 24 hours, the viral infection status was observed and analyzed by flow cytometry. The results are as follows: Figure 2 As shown in Figure B, the evaluation of VSV-GFP (RNA virus) in A549 cells showed that PAMO-DPA / Cu exhibited the best antiviral effect, which was also verified in HeLa cells.

[0076] In the HeLa cell model, 0.1 MOI HSV virus was simultaneously treated with 500 μM PAMO-DPA / Metal for 48 hours, and the viral infection efficiency was assessed by quantitative PCR. Results are as follows: Figure 2 As shown in Figure C, metals such as calcium, chromium, cobalt, and copper exhibit significant antiviral effects. Among all PAMO-DPA / Metal composite particles, PAMO-DPA / Cu showed significant inhibitory effects against both RNA and DNA viruses.

[0077] 2.2 Evaluation of the antiviral activity of PAMO-Polymer / Cu

[0078] In the A549 cell model, after co-treatment with 0.01 MOI VSV virus and 250 μM composite particles for 24 hours, the viral infection status was observed and analyzed by flow cytometry. The results are as follows: Figure 2 As shown in Figure E, PAMO-DMA / Cu, PAMO-DEA / Cu, PAMO-EPA / Cu, and PAMO-DPA / Cu exhibited significant antiviral activity in A549 cells, and this result was consistent in HeLa cells.

[0079] 4T1 cells were co-treated with MHV-A59 virus (0.1 MOI) and co-particles for 48 hours, and viral RNA levels were detected by qPCR. HeLa cells were co-treated with different SeV strains (0.1 MOI) and co-particles for 48 hours, and viral RNA levels were detected by qPCR. Results are as follows: Figure 2 As shown in F and G, PAMO-DPA / Cu exhibits excellent inhibitory effects against RNA viruses such as mouse hepatitis virus (MHV) and Sendai virus (SeV), with antiviral efficiency consistently exceeding 90%, and in most cases approaching complete inhibition (100%).

[0080] This embodiment further investigated the subcellular localization of the polymer and composite particles. The results showed that PAMO-DPA was mainly localized intracellularly under all conditions, while PAMO-DPA / Cu was significantly enriched in the cell membrane region, showing a strong tendency to bind to the membrane. PAMO-DPA / Cu mainly exerted its direct antiviral effect through the extracellular pathway.

[0081] This embodiment also investigated the virus adsorption and nucleic acid adsorption capabilities of PAMO-DPA / Cu. Therefore, it was found that PAMO-DPA / Cu has a dual mechanism of action. On the one hand, the electrostatic interaction between the weak positive charge of the composite particles and the negative charge of the cell membrane forms an extracellular barrier, blocking the virus invasion pathway and shielding the virus binding receptors on the cell surface. It also effectively adsorbs free virus particles, promoting their clearance from the extracellular environment. On the other hand, the synergistic effect of DPA and copper oligomers enables the positively charged composite particles to adsorb negatively charged virus particles. By extracting negatively charged nucleic acids from the virus, its structural integrity is disrupted, thereby exerting potent antiviral activity.

[0082] 3. Cytotoxicity evaluation

[0083] The survival rate of A549 cells was determined by a 24-hour cytotoxicity assay, and the results are as follows: Figure 3 As shown in Figure A, among the different PAMO-Polymer / Cu composite particles, PAMO-DPA / Cu exhibited the lowest cytotoxicity, with its IC50 value being significantly better than other composite particles. PAMO-DPA / Cu also demonstrated the best safety in HeLa cells.

[0084] Cytotoxicity thermogram analysis of PAMO-Polymer / Cu composite particles was performed. Cell survival status under different concentration treatments was visualized using a color gradient: yellow (value 1) represents high cell activity, and purple (value 0) indicates decreased cell survival. The results are as follows: Figure 3 As shown in B.

[0085] Antiviral dose-response heatmap analysis was performed on PAMO-Polymer / Cu composite particles. A color gradient was constructed based on the VSV-GFP viral infection rate: yellow (value 1) corresponds to low infection rate, and purple (value 0) reflects high infection rate, visually demonstrating the concentration-dependent antiviral effect of the nanoparticles. The results are as follows: Figure 3 As shown in C.

[0086] A comprehensive efficacy-toxicity assessment was performed on PAMO-Polymer / Cu composite particles. Specifically, six candidate composite particles were evaluated in three dimensions: electrical potential intensity was graded using signs "- / + / ++" (<10 for -, 10-20 for +, >20 for ++); cytotoxicity was graded into five levels based on IC50 values ​​(<10 for +++, <100 for ++, 100-300 for +, 300-400 for -, >400 for --); antiviral efficiency was divided into three levels based on fluorescence microscopy observation and EC50 values ​​(completely no fluorescence and EC50 <200 for +++, completely no fluorescence but EC50 >200 for ++, and weakened but not disappeared fluorescence for +). The results are as follows: Figure 3As shown in D, PAMO-DPA / Cu maintains +++ level antiviral efficacy while exhibiting the lowest cytotoxicity characteristics, demonstrating the best overall performance.

[0087] In summary, DMA, DEA, and EPA polymers exhibit significant cytotoxicity due to their high positive charge; DBA and DAA polymers, on the other hand, show insufficient antiviral activity. Notably, PAMO-DPA / Cu demonstrates the lowest cytotoxicity while maintaining the highest antiviral activity.

[0088] 4. Evaluation of antiviral efficacy and safety in vivo

[0089] 4.1 In vivo antiviral activity

[0090] Six- to eight-week-old BALB / c mice were selected for the experiment. An infection model of MHV-A59 was established on day 0 via intraperitoneal injection. Two days post-infection (day 2), PAMO-DPA / Cu was administered at a dose of 5 mg / kg. At the experimental endpoint on day 4, liver and spleen tissues were collected from the mice for viral titer determination and pathological damage assessment. A schematic diagram of the viral infection model construction and drug administration regimen for the experimental mice is shown below. Figure 4 As shown in A.

[0091] The effect of drug administration on the survival time of infected mice is as follows: Figure 4 As shown in B, PAMO-DPA / Cu treatment significantly prolonged the survival time of infected mice.

[0092] Target organ viral load test results as follows Figure 4 As shown in the CD, the level of MHV viral RNA in the liver tissue of the PAMO-DPA / Cu treatment group was significantly reduced, and the viral load in the spleen tissue decreased even more significantly. The results indicate that PAMO-DPA / Cu composite particles have a significant antiviral effect.

[0093] Histopathological examination results as follows Figure 4 As shown in Figure E, the untreated group of mice exhibited typical characteristics of viral liver injury, while the liver tissue structure of the PAMO-DPA / Cu composite particle treatment group remained intact, with no obvious pathological changes observed. This indicates that the drug has a protective effect against viral liver injury and can effectively alleviate liver damage caused by MHV infection. Therefore, PAMO-DPA / Cu has a significant antiviral therapeutic effect in vivo.

[0094] Multi-organ histopathological analysis was performed. Using hematoxylin-eosin (HE) staining, the pathological morphological characteristics of the heart, liver, spleen, lungs, kidneys, and brain tissues of mice in different dosage groups were systematically observed on day 7 to comprehensively assess the in vivo safety of the drug. Results are as follows: Figure 4As shown in Figure F, the composite particles did not cause significant damage to vital organs such as the heart, liver, spleen, lungs, kidneys, and brain, fully demonstrating their excellent in vivo safety.

[0095] 4.2 In vivo safety

[0096] To assess the dose-dependent safety of the drug, normal mice were intravenously injected with saline, therapeutic dose, and 5-fold and 10-fold doses of PAMO-DPA / Cu composite granules. Seven days later, serum samples were collected to detect ALT, AST, ALP, UA, UREA, CREA, LDH, and CK. ALT (alanine aminotransferase), AST (aspartate aminotransferase), and ALP (alkaline phosphatase) reflect liver function; UA (uric acid), UREA (urea), and CREA (creatinine) characterize renal metabolic function; and LDH (lactate dehydrogenase) and CK (creatine kinase) serve as markers of myocardial injury.

[0097] The result is as follows Figure 5 As shown, after 7 days of treatment with the therapeutic dose and at 5 times and 10 times the dose, no significant abnormalities were observed in liver function (ALT, AST, ALP), kidney function (UA, UREA, CREA), and cardiac function indicators (LDH, CK) in any group of mice.

[0098] Example 2

[0099] This embodiment illustrates the effect of different ratios of hydrogen bond donors and acceptors on the properties of PAMO-DPA / Cu.

[0100] This embodiment measured the dispersion state, polydispersity index, particle size distribution characteristics, and time-dependent effects of different hydrogen bond donor-acceptor ratios on PAMO-DPA / Cu. A schematic diagram of the hydrogen bond interaction mechanism of PAMO-DPA / Cu is shown below. Figure 6 As shown in A, the amino hydrogen atom of PAMO-DPA acts as a hydrogen bond donor, and the phosphate group of PAMO-Cu acts as a hydrogen bond acceptor, achieving structural connection through a hydrogen bond network.

[0101] The dispersion states of PAMO-DPA / Cu with different hydrogen bond donor-acceptor ratios are as follows: Figure 6 As shown in B, when the hydrogen bond donor-acceptor ratio reaches 1:40 and 1:20, the solution exhibits obvious stratification, while at a ratio of 1:10, it presents a uniform dispersion system.

[0102] The effect of different hydrogen bond donor-acceptor ratios on the polydispersity index of PAMO-DPA / Cu is as follows: Figure 6 As shown in C, the 1:10 ratio system has the lowest polydispersity coefficient, and the polydispersity coefficient gradually increases with the increase of the hydrogen bond acceptor ratio.

[0103] The effect of different hydrogen bond donor-acceptor ratios on the particle size distribution characteristics of PAMO-DPA / Cu is as follows: Figure 6 As shown in D, the 1:10 ratio exhibits a single-peak narrow distribution; the 1:20 ratio shows a double-peak distribution; and the 1:40 ratio shows a multi-peak wide distribution with a significantly increased particle size, making it prone to sedimentation.

[0104] HeLa cells were co-treated for 24 hours with fresh / one-week-old PAMO-DPA / Cu (treatment concentration 500 μM) and VSV-GFP virus (0.01 MOI) at different hydrogen bond donor-acceptor ratios. Cell viability was detected by flow cytometry. Results are as follows: Figure 6 As shown in E, only the 1:10 ratio system maintained significant antiviral activity after one week of storage.

[0105] This invention effectively addresses the core issues of traditional antiviral strategies, such as limitations in their spectrum and the potential for drug resistance, by integrating metal ion oligomers with functional polymer materials. The invention achieves precise control over reaction conditions and structural characteristics by separately preparing polyacrylate precursors and metal ion oligomers, followed by a coupling reaction to construct a composite system. This effectively avoids the side reactions and uneven cross-linking problems common in direct polymerization methods. The self-assembly mechanism driven by dynamic hydrogen bonding interactions forms a nanostructure with controllable morphology and uniform size. These structural characteristics play a decisive role in enhancing the functional properties and antiviral efficacy of the composite particles.

[0106] At the molecular level, the cross-linked network of polyacrylate and metal ion oligomers achieves homogeneous fusion of organic and inorganic components, fundamentally solving the phase separation and interfacial compatibility problems commonly found in traditional organic-inorganic composites. The introduction of acrylate groups significantly enhances the water solubility, surface charge tunability, and physical stability of the oligomers, making them more suitable for biological systems. The optimized PAMO-DPA / Cu formulation exhibits broad-spectrum antiviral activity against various viruses, including VSV, MHV, HSV, and SeV.

[0107] This invention utilizes a comprehensive screening system combining an inorganic metal element oligomer library and an acrylate homologue library to identify copper-based PAMO-DPA as the optimal candidate, achieving the highest antiviral efficacy while maintaining the lowest cytotoxicity. The screening process also reveals structure-activity relationships, such as the correlation between side chain length and toxicity / efficacy, and the influence of polymer charge on viral activity, providing important theoretical basis for future antiviral drug development.

[0108] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. These modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An antiviral complex particle, characterized in that, The antiviral composite particles are formed by hybridization of polymer and oligomeric metal salt particles, and are prepared by the following method: (I) Reacting metal ions and phosphoric acid in the presence of an oligomerization initiator to obtain an oligomerized metal salt, wherein the metal ions are selected from copper, calcium, cobalt, zinc or chromium, and the oligomerization initiator is selected from triethylamine, trimethylamine, tripropylamine, pyridine, N-methylmorpholine, choline or tetramethylammonium hydroxide; (II) A polymer is obtained by polymerizing a hydrophilic block, a metal-bound monomer, and a branched side-chain monomer in the presence of an initiator, wherein the hydrophilic block is polyethylene glycol, the metal-bound monomer contains amino and / or hydroxyl groups, and the branched side-chain monomer is selected from at least one of 2-(dimethylamino)ethyl methacrylate, 2-(diethylamino)ethyl methacrylate, 2-(ethylpropylamino)ethyl methacrylate, 2-(dipropylamino)ethyl methacrylate, 2-(dibutylamino)ethyl methacrylate, 2-(dipentylamino)ethyl methacrylate, and 2-aminoethyl methacrylate; (III) The polymer is dissolved in dichloromethane, and the oligomerized metal salt particles are dispersed in anhydrous ethanol. The two solutions are mixed in equal volumes, stirred overnight at room temperature, and the solvent is removed by rotary evaporation. After redissolving in ethanol / tetrahydrofuran, deionized water is added, and the mixture is ultrasonically treated to evaporate the organic solvent, thereby obtaining composite particles.

2. The antiviral composite particles according to claim 1, characterized in that, The antiviral composite particles are prepared by the following method: (I) Dissolve copper chloride dihydrate in anhydrous ethanol, add triethylamine, stir at room temperature for 20-40 minutes, add phosphoric acid ethanol solution dropwise, continue stirring overnight, centrifuge and discard the supernatant, wash the precipitate 3-10 times with anhydrous ethanol, and dry to obtain oligomerized metal salt PAMO-Cu. (II) The hydrophilic polymer reacts with 2-bromoisobutyryl bromide in the presence of triethylamine to generate a Br-terminated hydrophilic polymer. After dialysis and lyophilization purification, the Br-activated hydrophilic polymer, monomers DPA, AMA, cuprous bromide, azobisisobutyronitrile, and TPMA are dissolved in a mixed solvent in proportion. The mixture is deoxygenated by argon bubbling for 20-50 minutes and reacted in an oil bath at 50-90℃ for 30-70 hours to obtain the polymer PAMO-DPA. (III) Dissolve the polymer PAMO-DPA in dichloromethane, and oligomerize the metal salt particles PAMO Cu was dispersed in anhydrous ethanol, and the two solutions were mixed in equal volumes. The mixture was stirred overnight at room temperature, and the solvent was removed by rotary evaporation. After redissolving in ethanol / tetrahydrofuran, deionized water was added, and the mixture was ultrasonically treated to evaporate the organic solvent, thus obtaining PAMO-DPA / Cu composite particles.

3. An antiviral composition, characterized in that, It includes the antiviral complex particles as described in claim 1 or 2 and a pharmaceutically acceptable carrier.

4. The use of the antiviral composite particles according to claim 1 or 2 in the preparation of antiviral drugs, coatings, or protective equipment, characterized in that, Copper-containing composite particles are used to resist VSV, HSV, MHV or SeV; calcium, cobalt or chromium-containing composite particles are used to resist VSV or HSV; zinc-containing composite particles are used to resist VSV.

5. A method for screening antiviral complex particles, characterized in that, Includes the following steps: (1) Provide a library of composite particles, wherein the composite particles include polymer and oligomeric metal salt particles; (2) Contact the test composite particles in the composite particle library with the virus, and then detect the virus activity; and (3) When the test composite particle comes into contact with the virus, the virus activity decreases, and the test composite particle is used as an antiviral composite particle. The composite particle library includes a polymer library P and an oligomerized metal salt particle library M, wherein polymer P in the polymer library... x With oligomerized metal salt particles M in the oligomerized metal salt particle library y Capable of hybridizing to form composite particles P x M y ; The polymer comprises hydrophilic blocks and hydrophobic blocks; The monomers of the hydrophobic block of the polymer include metal-bound monomers and branched side-chain monomers, wherein the metal-bound monomers contain amino and / or hydroxyl groups; and the branched side-chain monomers are selected from at least one of 2-(dimethylamino)ethyl methacrylate, 2-(diethylamino)ethyl methacrylate, 2-(ethylpropylamino)ethyl methacrylate, 2-(dipropylamino)ethyl methacrylate, 2-(dibutylamino)ethyl methacrylate, 2-(dipentylamino)ethyl methacrylate, and 2-aminoethyl methacrylate. The antiviral composite particles are prepared by the following method: (I) Reacting metal ions and phosphoric acid in the presence of an oligomerization initiator to obtain an oligomerized metal salt, wherein the metal ions are selected from copper, calcium, cobalt, zinc or chromium, and the oligomerization initiator is selected from triethylamine, trimethylamine, tripropylamine, pyridine, N-methylmorpholine, choline or tetramethylammonium hydroxide; (II) A polymer is obtained by polymerizing a hydrophilic block, a metal-bound monomer and a branched side-chain monomer in the presence of an initiator, wherein the hydrophilic block is polyethylene glycol; (III) The polymer is dissolved in dichloromethane, and the oligomerized metal salt particles are dispersed in anhydrous ethanol. The two solutions are mixed in equal volumes, stirred overnight at room temperature, and the solvent is removed by rotary evaporation. After redissolving in ethanol / tetrahydrofuran, deionized water is added, and the mixture is ultrasonically treated to evaporate the organic solvent, thereby obtaining composite particles.

6. The method for screening antiviral complex particles according to claim 5, characterized in that, Step (2) includes adding the test composite particle and virus to the cell model and measuring the virus inhibition rate and / or cell survival rate.

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