Use of magnetoelectrically driven magnetostrictive piezoelectric nanoparticle in antiviral therapy

Abstract

Provided is use of a magnetoelectrically driven magnetostrictive piezoelectric nanoparticle in antiviral therapy. The magnetostrictive piezoelectric nanoparticle comprises a shell structure with piezoelectric properties and a core structure with magnetoelectric response properties. The magnetostrictive piezoelectric nanoparticle, by means of up-regulating multiple immunomodulatory pathways, effectively enhances the expression of type I interferon and a host's innate immune response, ultimately inhibiting the viral activity, ameliorating hepatic and pulmonary tissue damage, and improving the survival rates. The magnetostrictive piezoelectric nanoparticle has the advantages such as high controllability, deep penetration, and non-invasiveness, and offers broad application prospects in the antiviral field.

Description

Application of magnetostrictive piezoelectric nanoparticles driven by magnetoelectricity in antiviral therapy

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to patent application number CN202410331583.X filed on March 22, 2024, entitled “Application of magnetoelectrically driven magnetostrictive piezoelectric nanoparticles in antiviral therapy”, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] The present invention belongs to the field of biomedicine, and in particular relates to the application of magnetostrictive piezoelectric nanoparticles driven by magnetoelectricity in antiviral treatment. Background Art

[0004] Emerging and re-emerging viruses are a significant hazard / risk to human health and are attracting increasing attention. Viruses have the ability to infect and spread among a wide range of organisms, including humans, animals, plants, and microorganisms, causing a variety of predictable and unpredictable infectious diseases with profound impacts on society. While vaccination is the most effective method for controlling infectious viral diseases, a large number of viruses still lack preventive or therapeutic options. Furthermore, some viruses are developing resistance to current treatments. Therefore, given the severity of viral infections, the development of effective antiviral drugs or preventive strategies has become crucial and a pressing challenge that needs to be overcome.

[0005] In recent years, nanoparticle research has garnered considerable attention as a promising antiviral strategy. However, current antiviral strategies suffer from uncontrollable duration of effective treatment, unstable therapeutic effects, and low therapeutic efficiency. Therefore, developing on-site controlled antiviral treatments is necessary to address these shortcomings of existing therapies. Summary of the Invention

[0006] To solve at least some of the technical problems in the prior art, the present invention provides magnetostrictive piezoelectric nanoparticles driven by magnetoelectricity and their application in antiviral therapy. Specifically, the present invention includes the following contents.

[0007] In a first aspect, the present invention provides the use of magnetoelectrically driven magnetostrictive piezoelectric nanoparticles in the preparation of antiviral drugs, wherein the magnetostrictive piezoelectric nanoparticles include a shell structure with piezoelectric properties and a core structure with magnetoelectric response properties.

[0008] In certain embodiments, according to the use of the present invention, the shell structure having piezoelectric properties is prepared from an inorganic piezoelectric material and / or an organic piezoelectric material.

[0009] In certain embodiments, according to the use of the present invention, the inorganic piezoelectric material is selected from at least one of ferrite, niobate, titanate, silicate and aluminate materials.

[0010] In certain embodiments, according to the use of the present invention, the organic piezoelectric material is selected from at least one of polyvinylidene fluoride, polyesters, polymethyl methacrylate, nylon, polyvinyl chloride, poly-L-lactide, poly(3-hydroxybutyrate-co-3-hydroxyvalerate), vinylidene fluoride / trifluoroethylene copolymer, vinylidene fluoride / tetrafluoroethylene copolymer, polyvinylidene fluoride-hexafluoropropylene and polydimethylsiloxane materials.

[0011] In certain embodiments, according to the use of the present invention, the ferrite material has the following structure: x Fe 3-x O y , wherein M includes at least one of Bi, Ca, Ba, Mg and Ba, x is a number from 0.25 to 1.5, and y is 4.

[0012] In certain embodiments, according to the use of the present invention, the core structure having magnetoelectric response properties is prepared from a magnetoelectric material.

[0013] In certain embodiments, according to the use of the present invention, the magnetoelectric material has the following structure: N a Fe 1-a O b , N includes at least one of Co, Mn, Zn and Ni, a is a number from 0.25 to 0.75, and b is 3.

[0014] In certain embodiments, according to the use of the present invention, the magnetostrictive piezoelectric nanoparticles are cobalt ferrite-bismuth ferrite nanoparticles.

[0015] In certain embodiments, according to the use of the present invention, the particle size of the cobalt ferrite-bismuth ferrite nanoparticles is 100-500 nm.

[0016] In certain embodiments, according to the use of the present invention, the antiviral agent comprises at least one of the following:

[0017] (1) Inhibit viral replication;

[0018] (2) Reduce viral titer;

[0019] (3) Activate the host innate immune response;

[0020] (4) Reduce tissue or organ damage caused by the virus;

[0021] (5) Activate the expression of type I interferon.

[0022] In certain embodiments, the use according to the present invention, wherein the magnetoelectric driving comprises treating the magnetostrictive piezoelectric nanoparticles with a magnetic field having a magnetic field strength of 0.01-300 mT.

[0023] The second aspect of the present invention provides the use of magnetostrictive piezoelectric nanoparticles driven by magnetoelectricity in the preparation of a drug for activating the expression of type I interferon, wherein the magnetostrictive piezoelectric nanoparticles include the magnetostrictive piezoelectric nanoparticles described in the present invention.

[0024] The third aspect of the present invention provides the use of magnetostrictive piezoelectric nanoparticles driven by magnetoelectricity in the preparation of drugs that activate type I interferon expression and / or antiviral effects in combination with magnetic field therapy, wherein the magnetostrictive piezoelectric nanoparticles include the magnetostrictive piezoelectric nanoparticles described in the present invention.

[0025] In certain embodiments, according to the use of the present invention, a therapeutically effective amount of magnetostrictive piezoelectric nanoparticles and a magnetic field of 0.01-300 mT are administered to a subject in need thereof to activate type I interferon expression and / or achieve an antiviral effect.

[0026] In a fourth aspect, the present invention provides a method for treating or ameliorating viral infection, comprising administering a therapeutically effective amount of magnetoelectrically driven magnetostrictive piezoelectric nanoparticles or a pharmaceutical composition containing the same to a subject in need thereof, wherein the magnetostrictive piezoelectric nanoparticles comprise a shell structure having piezoelectric properties and a core structure having magnetoelectric response properties, wherein the shell structure is prepared from an inorganic piezoelectric material and / or an organic piezoelectric material, and the core structure is prepared from a magnetoelectric material.

[0027] In certain embodiments, according to the method for treating or ameliorating viral infection of the present invention, the inorganic piezoelectric material is selected from at least one of ferrite, niobate, titanate, silicate and aluminate materials; the organic piezoelectric material is selected from at least one of polyvinylidene fluoride, polyesters, polymethyl methacrylate, nylon, polyvinyl chloride, poly-L-lactide, poly(3-hydroxybutyrate-co-3-hydroxyvalerate), vinylidene fluoride / trifluoroethylene copolymer, vinylidene fluoride / tetrafluoroethylene copolymer, polyvinylidene fluoride-hexafluoropropylene and polydimethylsiloxane materials.

[0028] In certain embodiments, according to the method for treating or ameliorating viral infection of the present invention, the ferrite material has the following structure: x Fe 3-x O y, wherein M includes at least one of Bi, Ca, Ba, Mg and Ba, x is a number from 0.25 to 1.5, and y is 4.

[0029] In certain embodiments, according to the method for treating or ameliorating viral infection of the present invention, the magnetoelectric material has the following structure: N a Fe 1-a O b , N includes at least one of Co, Mn, Zn and Ni, a is a number from 0.25 to 0.75, and b is 3.

[0030] In certain embodiments, according to the method for treating or ameliorating viral infection of the present invention, the magnetostrictive piezoelectric nanoparticles are cobalt ferrite-bismuth ferrite nanoparticles.

[0031] In certain embodiments, according to the method for treating or ameliorating viral infection of the present invention, the particle size of the cobalt ferrite-bismuth ferrite nanoparticles is 100-500 nm.

[0032] In certain embodiments, according to the method for treating or ameliorating viral infection of the present invention, the antiviral agent comprises at least one of the following:

[0033] (1) Inhibit viral replication;

[0034] (2) Reduce viral titer;

[0035] (3) Activate the host innate immune response;

[0036] (4) Reduce tissue or organ damage caused by the virus;

[0037] (5) Activate the expression of type I interferon.

[0038] In certain embodiments, according to the method for treating or ameliorating viral infection of the present invention, the magnetoelectric driving comprises treating the magnetostrictive piezoelectric nanoparticles with a magnetic field having a magnetic field strength of 0.01-300 mT.

[0039] The present invention utilizes CoFe2O4-BiFeO3 nanoparticles (CFO-BFO NPs) to effectively perform antiviral therapy by enhancing the host's innate immune response under an external magnetic field. Based on this, CFO-BFO magnetoelectrically coupled nanoparticles with a core-shell structure were prepared using a hydrothermal method and a sol-gel method. CFO-BFO NPs have high biocompatibility. To better simulate the diseases caused by viruses to the human body, the present invention selected mouse hepatitis virus A59 (MHV-A59), which is similar to human coronaviruses. In vivo studies were conducted using an MHV-infected mouse model. The results showed that CFO-BFO NPs combined with a magnetic field can inhibit MHV viral proliferation, thereby reducing tissue damage to the liver and lungs of MHV-infected mice and improving survival rates. In addition, CFO-BFO NPs under the action of an external magnetic field can effectively activate the expression of type I IFN and the host's innate immune response, ultimately inhibiting viral replication. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 shows the characterization of CFO-BFO NPs, where (a) is the scanning electron microscopy of CFO-BFO NPs, with a scale bar of 500 nm; (b, c) are transmission electron microscopy images of CFO-BFO NPs, with scale bars of 100 nm and 10 nm, respectively; (d) is the EDX elemental mapping image of CFO-BFO NPs, with a scale bar of 50 nm; (e, f) are the XRD and XPS images of CFO-BFO NPs; (g) is the hysteresis loop pattern of CFO and CFO-BFO NPs; (h) is the pressure response amplitude curve and phase curve of CFO-BFO NPs; (i) is the O2 captured by DMPO - and ·OH EPR spectra under magnetic field.

[0041] Figure 2 shows the antiviral effects of magnetostrictive-piezoelectric CFO-BFO NPs in vivo. (a) Schematic diagram of magnetic treatment of CFO-BFO fillers; (b, c) Time-dependent survival and body weight curves of MHV-infected mice after different treatments (survival curve: Log-rank (Mantel-Cox) test, n = 12). *P < 0.05, **P < 0.01. Body weight curve: mean ± SEM, two-way AVONA Tukey's multiple comparison test, n=12, ****P<0.0001); (d, g) Spleen images and statistical analysis of MHV-infected mice after different treatments (mean±SEM, two-tailed unpaired t-test, n=4), ns, not statistically significant (P>0.05, **P<0.01, ***P<0.001); (e, f) Liver images and statistical analysis of MHV-infected mice after different treatments (mean±SEM, two-tailed unpaired t-test, n=4), ns, not significant, P>0.05, *P<0.05, **P<0.01); (hk) H&E staining of liver (h) and lung (i) of MHV-infected mice, and statistical analysis of liver necrosis area (j), respectively. Statistical analysis of inflammatory cells in the lungs (k) (mean ± SEM, two-tailed unpaired t-test, n = 3), ns, not statistically significant, P > 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, scale bars are 200 μm and 500 μm, respectively); (ln) RT-qPCR results of MHV-N mRNA expression in liver, spleen, and lung tissues of MHV-N-infected mice after different treatments (mean ± SEM, two-tailed unpaired t-test, n = 4), ns, not significant, P > 0.05, *P < 0.05, **P < 0.01); (o) Blood biochemical analysis of MHV-infected mice with different treatments (mean ± SEM, two-way AVONA Tukey multiple comparison test, n = 3), *P < 0.05, **P < 0.01, ****P < 0.0001.

[0042] Figure 3 shows the in vivo antiviral signaling pathway of magnetostrictive-piezoelectric CFO-BFO NPs, where (a) is a volcano plot analysis of differentially expressed genes in the liver tissue of MHV-infected mice treated with CFO-BFO plus magnetism compared with the control (untreated) group. The left dots indicate downregulated genes, and the right dots indicate upregulated genes; (b) is a GO database analysis of upregulated differentially expressed genes in the liver tissue of MHV-infected mice treated with CFO-BFO plus magnetism compared with the control (untreated) group; (c, d) are GSEA of differentially expressed genes in the liver tissue of MHV-infected mice treated with CFO-BFO plus magnetism compared with the control (untreated) group; (en) are IFN-β, Isg56, is20, CXCL10, and Isg15 in the liver (ei) and lung (jn) of MHV-infected mice after different treatments. RT-qPCR results of mRNA expression (mean ± SEM, two-tailed unpaired t-test, n = 3), ns, not significant, P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

[0043] Figure 4 shows the biosafety and antiviral signaling pathway of CFO-BFO NPs combined with magnetic therapy in vitro, where (a) is a schematic diagram of magnetostrictive-piezoelectric catalytic therapy in vivo; (b) CCK-8 analysis of the effects of different concentrations of CFO-BFO NPs on BMDM (mean ± SEM, one-way AVONA Tukey multiple comparison test, n = 5), ***P < 0.001, ****P < 0.0001; (c) CCK-8 analysis of the effects of magnetic field intensity and time interval on BMDM (mean ± SEM, one-way AVONA Tukey multiple comparison test, n = 4); (d, e) flow cytometry results and in vitro cellular uptake analysis of CFO-BFO NPs (mean ± SEM, two-tailed unpaired t-test, n = 3), ns, not statistically significant, P > 0.05, ***P < 0.001, ****P < 0.0001); (f) MHV-N after different treatments RT-qPCR results of mRNA expression (mean ± SEM, two-tailed unpaired t-test, n = 4), ns, not statistically significant (P>0.05, **P<0.01, ***P<0.001); (g) Volcano plot analysis, compared with the control group (untreated group), the differentially expressed genes in MHV-infected BMDM treated with CFO-BFO plus magnetism, the left dots indicate down-regulated genes, and the right dots indicate up-regulated genes; (h) According to the GO BP database analysis, the differentially expressed genes in MHV-infected BMDM treated with CFO-BFO plus magnetism were up-regulated compared with the control group (untreated group); (i) GSEA of differentially expressed genes in MHV-infected BMDM treated with CFO-BFO plus magnetism and the control group.

[0044] Figure 5 shows the in vitro antiviral signaling pathways of magnetostrictive piezoelectric CFO-BFO NPs. (ae) RT-qPCR results for IFN-β, Isg56, Isg20, CXCL10, and Isg15 mRNA expression in MHV-infected BMDM tissues after different treatments (mean ± SEM, two-tailed unpaired t-test, n = 4). ns, not statistically significant (P > 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). (f,h) Immunoblotting analysis of TBK1 phosphorylation levels in MHV-infected BMDM tissues after different treatments (mean ± SEM, two-way AVONA Tukey multiple comparison test, n = 3). ns, not statistically significant, P > 0.05, ****P < 0.0001). (g, i) Immunoblot analysis of nuclear and cytoplasmic extracts of IRF3 subcellular localization in MHV-infected BMDM after different treatments (mean ± SEM, two-way AVONA Tukey multiple comparison test, n = 3), *P < 0.05, **P < 0.01, ****P < 0.0001); (j, k) Immunofluorescence staining images and IRF3 localization analysis of MHV-infected BMDM after different treatments (mean ± SEM, two-tailed unpaired t-test, n = 3, ns, not statistically significant, P > 0.05, ***P < 0.001, scale bar 10 μm).

[0045] Figure 6 shows the in vitro antiviral (VSV) effect of magnetostrictive piezoelectric CFO-BFO NPs. (ae) are the fluorescence images of the number of VSV in each group of VSV-infected BMDM after different treatments (ad) and the flow cytometry statistical results of the number of VSV viruses in each group (mean ± SEM, two-tailed unpaired t-test, n = 3, ****P < 0.0001, scale bar is 100 μm).

[0046] FIG7 shows the degradation curves of RHB under different magnetic field frequencies, intensities and time intervals under the action of CFO-BFO nanoparticles and magnetic catalysis, where RHB is rhodamine B dye.

[0047] Figure 8 shows the in vivo biosafety of CFO-BFO NPs. (a) Blood routine and biochemical examination of C57BL / 6J mice 14 days after injection of different concentrations of CFO-BFO NPs (mean ± SEM, two-way AVONA Tukey multiple comparison test, n = 3). (b) H&E staining of the heart, liver, spleen, lung, and kidney of corresponding C57BL / 6J mice. Scale bar, 100 μm.

[0048] Figure 9 shows the distribution of CFO-BFO NPs in mouse organs. (a) Images of CFO-BFO NPs distribution in the heart, liver, spleen, lung, and kidney of mice 12, 24, and 48 hours after CFO-BFO NP injection. (b) Fluorescence intensity statistics for the liver, lung, and kidney (mean ± SEM, two-tailed unpaired t-test, n = 3). ns, not significant, P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001).

[0049] Figure 10 shows the quantitative distribution of CFO-BFO NPs in liver lymphocytes of MHV-infected mice after magnetic treatment of CFO-BFO NPs by flow cytometry analysis (mean ± SEM, two-tailed unpaired t-test, n = 3). ns, not statistically significant, P > 0.05, ***P < 0.001, ****P < 0.0001). DETAILED DESCRIPTION

[0050] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0051] It should be understood that the terms described in the present invention are only for describing particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges in the present invention, it should be understood that the upper and lower limits of the ranges and each intermediate value therebetween are specifically disclosed. Each smaller range between any stated value or intermediate value within a stated range and any other stated value or intermediate value within the stated range is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0052] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the invention belongs. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In the event of any conflict with any incorporated document, the content of this specification shall prevail.

[0053] use

[0054] In a first aspect, the present invention provides the use of magnetoelectrically driven magnetostrictive piezoelectric nanoparticles in the preparation of antiviral drugs, wherein the magnetostrictive piezoelectric nanoparticles include a shell structure with piezoelectric properties and a core structure with magnetoelectric response properties.

[0055] As used herein, "magnetoelectric drive" refers to the relative displacement of the core-shell structure of the nanoparticles of the present invention under the influence of a magnetic field, thereby activating the expression of type I IFN and / or exerting an antiviral effect. "Magnetostriction" refers to the fact that the core structure of the nanoparticles of the present invention, when magnetized in a magnetic field, will elongate or shorten along the magnetization direction. "Piezoelectric performance" refers to the ability of the shell structure of the nanoparticles of the present invention to convert the mechanical energy generated by the core structure into electrical energy.

[0056] In a preferred embodiment, the magnetoelectric drive comprises treating the magnetostrictive piezoelectric nanoparticles with a magnetic field having a magnetic field strength of 0.01-300 mT and a frequency of 0.1-100 kHz. The magnetic field strength is also preferably 0.1-200 mT, further preferably 0.5-100 mT, more preferably 0.5-50 mT, and most preferably 0.5-10 mT, such as 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, or 10 mT. The frequency is also preferably 0.5-50 kHz, further preferably 1-10 kHz, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 kHz.

[0057] In the present invention, the shell structure with piezoelectric properties is prepared from an inorganic piezoelectric material and / or an organic piezoelectric material. In a preferred embodiment, the shell structure with piezoelectric properties is prepared from an inorganic piezoelectric material. In a more preferred embodiment, the inorganic piezoelectric material is selected from at least one of ferrites, niobates, titanates, silicates and aluminates. In a most preferred embodiment, the shell structure with piezoelectric properties is prepared from a ferrite material. In the present invention, the ferrite material has the following structure: M x Fe 3-x O y , wherein M includes at least one of Bi, Ca, Ba, Mg and Ba, wherein x is a number from 0.25 to 1.5, and y is 4.

[0058] In the present invention, the core structure with magnetoelectric response performance is prepared from magnetoelectric material, wherein the magnetoelectric material has the following structure: N a Fe 1-a O b , wherein N includes at least one of Co, Mn, Zn and Ni, a is a number from 0.25 to 0.75, and b is 3.

[0059] It is understood that the magnetostrictive piezoelectric nanoparticles of the present invention may or may not contain doped elements. When doped with elements, the doped elements include, but are not limited to, at least one of carbon, nitrogen, phosphorus, sulfur, silicon, aluminum, iron, titanium, nickel, manganese, copper, silver, and zinc, thereby improving the piezoelectric properties and / or magnetoelectric response properties of the magnetostrictive piezoelectric nanoparticles.

[0060] In a specific embodiment, the magnetostrictive piezoelectric nanoparticles are cobalt ferrite-bismuth ferrite nanoparticles.

[0061] In the present invention, the average particle size of the cobalt ferrite-bismuth ferrite nanoparticles is 100-500 nm, preferably 200-400 nm, further preferably 200-300 nm, further preferably 220-260 nm, and most preferably 220-240 nm, for example 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, and 240 nm.

[0062] The present invention does not particularly limit the preparation of magnetostrictive piezoelectric nanoparticles. A shell structure with piezoelectric properties and a core structure with magnetoelectric response properties can be prepared using methods known in the art. It is understood that any doping elements or additional layer structures may be contained between the shell structure and the core structure, in the outer layer of the shell structure, and within the core structure to improve the piezoelectric properties and / or magnetoelectric response properties of the nanoparticles.

[0063] The second aspect of the present invention provides the use of magnetostrictive piezoelectric nanoparticles driven by magnetoelectricity in the preparation of a drug for activating the expression of type I IFN or increasing the amount of type I IFN. The amount of type I IFN or the expression of its gene can be determined by any method known in the art, without particular limitation. The determination method includes, but is not limited to, chemiluminescent immunoassay, enzyme-linked immunosorbent assay, real-time fluorescence quantitative PCR, gene chip technology, or immunoblotting. Therefore, the present invention also provides a method for promoting or activating the expression of type I interferon in vitro, which is used for non-disease diagnosis and treatment purposes, comprising the steps of contacting cells with magnetostrictive piezoelectric nanoparticles in vitro and treating the cells with a magnetic field having a magnetic field strength of 0.01-300 mT and a frequency of 0.1-100 kHz.

[0064] The third aspect of the present invention provides the use of magnetostrictive piezoelectric nanoparticles driven by magnetoelectricity in the preparation of drugs for activating type I interferon expression and / or antiviral effects in combination with magnetic field therapy.

[0065] In a preferred embodiment, a therapeutically effective amount of magnetostrictive piezoelectric nanoparticles and a magnetic field of 0.01-300 mT are administered to a subject in need thereof to activate type I interferon expression and / or achieve an antiviral effect.

[0066] The term "subject" as used herein refers to any animal (e.g., mammal), including but not limited to humans, non-human primates, rodents, and the like, that is about to receive a specific treatment. Generally, "subject" and "patient" are used interchangeably herein to refer to a human subject.

[0067] The terms "administer," "apply," or "administer" as used herein are used interchangeably and refer to implanting, absorbing, ingesting, injecting, inhaling, or otherwise introducing magnetostrictive piezoelectric nanoparticles or pharmaceutical compositions containing the same.

[0068] As used herein, the term "effective amount" refers to the amount of a drug or pharmaceutical agent that elicits a biological or pharmaceutical response in a tissue, system, animal, or human being, such as that sought by a researcher or clinician. In addition, the term "therapeutically effective amount" refers to an amount that causes improved treatment, cure, prevention, or alleviation of a disease, condition, or side effect, or an amount that reduces the rate of progression of a disease or condition, compared to a corresponding subject that has not received the amount. The term also includes within its scope an amount that effectively enhances normal physiological function. Typically, the effective amount herein varies according to various factors, such as a given drug or compound, pharmaceutical formulation, route of administration, type of disease or condition, subject being treated, and the like, but can still be routinely determined by those skilled in the art.

[0069] The term "antiviral" as used herein refers to improving a condition before or after the onset of a disease or disorder. The degree of relief or prevention is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95% or 100% as measured by any standard technique compared to an untreated control group under the same conditions. Beneficial or desired clinical results include, but are not limited to, the following, whether detectable or undetectable, including relief of symptoms, reduction in the extent of the disease, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement or alleviation of the disease state, and reduction (whether partial or complete). "Treatment" also refers to a prolonged survival period compared to the expected survival period when not receiving treatment. Those in need of treatment include those who already have the disease or disorder, as well as those who are susceptible to the disease or disorder, or those who need to prevent the disease or disorder.

[0070] The total dose required for each treatment can be administered in multiple doses or as a single dose. The magnetostrictive piezoelectric nanoparticles of the present invention or pharmaceutical compositions containing the same can be taken alone or in combination with other therapeutic drugs or symptomatic drugs, with the dosage adjusted. In a preferred embodiment, the magnetic therapy is performed at the aforementioned magnetic field intensity once daily for 15-30 minutes each time for three consecutive days.

[0071] There is no particular limitation on the administration method of the magnetostrictive piezoelectric nanoparticles or the pharmaceutical composition containing the same. Representative administration methods include, but are not limited to, intramuscular injection, intravenous injection, intravenous drip, or intraperitoneal injection.

[0072] In the present invention, "virus" includes but is not limited to general DNA and RNA viruses and retroviruses. Examples of DNA viruses include Parvoviridae, Papovaviridae, Adenoviridae, Herpesviridae, Poxviridae and Hepadnaviridae. Examples of RNA viruses include but are not limited to Picornaviridae, Togaviridae, Rhabdoviridae, Orthomyxoviridae, Paramyxoviridae, Coronaviridae, Reoviridae and Filoviridae. In some embodiments, it includes mouse hepatitis virus (MHV) of Coronaviridae; varicella-zoster virus (VZV) of the genus Varicellavirus of the family Herpesviridae, herpes simplex virus type I and II (HSV-I, II) of the genus Herpes simplex virus; adenovirus type 3 and 7 of the genus Adenovirus of the family Adenoviridae; measles virus (MV) of the genus Morbillivirus of the family Paramyxoviridae, mouse pneumonia virus, respiratory syncytial virus (RSV) and parainfluenza virus-I of the genus Influenzavirus of the family Orthomyxoviridae; influenza virus (A3V) of the genus Influenzavirus of the family Orthomyxoviridae, influenza virus subtype A mouse lung adapted strain FM1; poliovirus type III (PVIII), echovirus types 6 and 11 (ECH06 and 11), coxsackievirus B types 3, 4, 5, and 6 (CVB3, CVB4, CVB5, and CVB6), coxsackievirus A16 (CVA16), and novel enterovirus 71 (EV71) of the genus Enterovirus in the family Picornaviridae; SARS virus of the genus Coronavirus in the family Coronaviridae; rubella virus (RV) of the genus Rubellavirus in the family Togaviridae; and vesicular stomatitis virus (VSV) of the genus Vesiculovirus in the family Rhabdoviridae.

[0073] Methods for treating or ameliorating viral infections

[0074] A fourth aspect of the present invention provides a method for treating or ameliorating viral infection, comprising administering to a subject in need thereof a therapeutically effective amount of magnetoelectrically driven magnetostrictive piezoelectric nanoparticles or a pharmaceutical composition comprising the same. The magnetostrictive piezoelectric nanoparticles are those described above and are not further described herein.

[0075] In the present invention, the pharmaceutical composition contains magnetostrictive piezoelectric nanoparticles driven by magnetoelectricity, and optionally, a pharmaceutically acceptable excipient or carrier. The term "pharmaceutically acceptable excipient or carrier" as used herein refers to a pharmaceutically acceptable material, composition or medium, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, and such pharmaceutically acceptable material, composition or medium participates in carrying or transporting the drug from one organ or part of the body to another organ or part of the body. Each excipient or carrier is "acceptable" in the sense that it is compatible with the other ingredients of the formulation and does not harm the patient.

[0076] Example

[0077] This example demonstrates the preparation of CFO-BFO nanoparticles.

[0078] 1. Experimental Methods

[0079] 1.1 Preparation of CoFe2O4 nanoparticles

[0080] CoFe2O4 nanoparticles were prepared by a hydrothermal method. Ethylene glycol, NaOH, FeCl3·6H2O, and CoCl3·6H2O were selected as raw materials. The content of the added mineralizer NaOH was adjusted during the experiment to obtain CoFe2O4 samples with stable morphology, particle size, and magnetic properties.

[0081] 1.2 Preparation of BiFeO3 nanoparticles

[0082] BiFeO3: Bi(NO3)3·5H2O, Fe(NO3)3·9H2O, glacial acetic acid and ethylene glycol were used as raw materials to synthesize BiFeO3 nanoparticles by sol-gel process.

[0083] 1.3 Preparation of core-shell nanostructured magnetoelectric composite CoFe2O4-BiFeO3

[0084] Core-shell nanostructure: It consists of two parts: an inner core and an outer shell. CoFe2O4-BiFeO3 is a magnetoelectric composite material. The inner core is made of the ferromagnetic material CoFe2O4, and the outer shell is made of the ferroelectric material BiFeO3. That is, the outer layer of the CoFe2O4 nanoparticles is coated with a BiFeO3 shell. First, CoFe2O4 nanoparticles with excellent performance are prepared by a hydrothermal method. Then, a BiFeO3 precursor solution is prepared separately. The CoFe2O4 nanoparticles are then placed in the BiFeO3 precursor solution. Ultrasonic vibration is used to reduce the agglomeration of the powder, so that it is evenly dispersed in the solution. The powder is then dried to form a gel. Finally, heat treatment is performed to obtain a well-crystallized BiFeO3 shell on the outer layer of the CoFe2O4 nanoparticles. That is, the BiFeO3 shell is coated on the surface of the prepared CoFe2O4 nanoparticles through a sol-gel method to obtain a magnetoelectric composite material with a core-shell nanostructure, thus completing the sample preparation.

[0085] 1.4 Characterization

[0086] The morphology and size of the materials were observed by scanning electron microscopy, transmission electron microscopy, and high-resolution transmission electron microscopy. Elemental analysis of CFO-BFO NPs was performed using an energy dispersive spectrometer. X-ray diffraction (XRD) patterns of CFO NPs, BFO NPs, and CFO-BFO NPs were recorded on a Bruker-AXSD8 Advance powder diffractometer. X-ray photoelectron spectroscopy (XPS) was measured on a spectrometer. The hysteresis loop patterns of CFO NPs and CFO-BFO NPs were measured using a vibrating sample magnetometer. Piezoelectric force microscopy (PFM) measurements were recorded using an instrument. Electron paramagnetic resonance (EPR) spectra were detected using a Bruker EMX-10 / 12. Absorption spectra were measured using a UV-3600 UV-Vis-NIR spectrophotometer.

[0087] 1.5 FITC-conjugated CFO-BFO NPs

[0088] CFO-BFO NPs were added to 20 mg of FITC aqueous solution, mixed, and stirred in the dark for 24 h. To remove excess FITC, the FITC-conjugated CFO-BFO NPs were washed twice with sterile water.

[0089] Mice: 6- to 8-week-old male C57BL / 6J mice were used for animal experiments. All animals were housed under specific pathogen-free conditions. Animal experiments were conducted according to protocols approved by the Ethics Committee of Peking University Health Science Center (LA2023482).

[0090] 1.6 Flow cytometry

[0091] MHV-infected mice were injected with FITC-CFO-BFO NPs and exposed to a magnetic field (1 kHz, 1.5 mT, 30 min) to investigate the quantitative distribution of CFO-BFO NPs in the liver. Lymphocytes were then isolated from the liver and incubated with the corresponding antibodies for 30 min in the dark. Flow cytometry was used to analyze the above data. Antibodies were labeled with APC / Cy7 for CD11b, PE for F4 / 80, PE / Cy7 for CD11c, APC for MHC II, PER·CP for CD3, and BV421 for CD19.

[0092] 1.7 H&E staining

[0093] To investigate the biosafety of magnetic fields, mice were exposed to a magnetic field (1.5 mT, 1 kHz, 30 min) daily. To investigate the biosafety of CFO-BFO NPs, different concentrations of CFO-BFO NPs (6 mg / kg, 18 mg / kg, 30 mg / kg, and 150 mg / kg) were intravenously injected into normal mice. The hearts, livers, spleens, lungs, and kidneys of each group were then fixed with paraformaldehyde. The livers and lungs of four groups of MHV-infected mice were fixed with paraformaldehyde for 5 days to assess the antiviral effects of the different treatments. The major organ specimens were then dehydrated through a series of graded alcohols and embedded in paraffin. 3 μm-thick sections were stained with H&E according to standard protocols. Images were observed under an Olympus BX53 microscope.

[0094] 1.8 Routine blood test and blood biochemistry test

[0095] To observe the biosafety of the magnetic field, mice were placed in a magnetic field of 1.5 mT, 1 kHz, for 30 minutes per day. To observe the biosafety of CFO-BFO NPs, different concentrations of CFO-BFO NPs (6 mg / kg, 18 mg / kg, 30 mg / kg, and 150 mg / kg) were intravenously injected into normal mice. Blood from the canthus of each group (NC group, pure magnetic group, 6 mg / kg CFO-BFO NPs group, 18 mg / kg CFO-BFO NPs group, 30 mg / kg CFO-BFO NPs group, and 150 mg / kg CFO-BFO NPs group) was collected on day 14 for routine blood tests, and serum was collected by high-speed centrifugation for blood biochemical tests. To study the liver and kidney function of the four groups of MHV-infected mice, blood was collected from the canthus of each group, and serum was collected by high-speed centrifugation for blood biochemical tests. Routine blood tests were performed on the samples according to the manufacturer's protocol.

[0096] 1.9 Distribution of CFO-BFO NPs in mouse organs

[0097] 18 mg / kg of FITC-conjugated CFO-BFO NPs were intraperitoneally injected into normal mice. Changes in the heart, liver, spleen, lung, and kidney were observed using a living animal imaging system at 12, 24, and 48 hours. Statistical analysis was performed using Living Image 4.3.1.

[0098] 1.10 cells

[0099] All cell-based experiments used BMDM. BMDM were purchased from Shanghai ICell Biotechnology Co., Ltd. and were derived from primary bone marrow macrophages transfected with the lentivirus SV40. Throughout the experiments, BMDM were cultured in whey-containing α-MEM medium supplemented with 10% fetal bovine serum and 1% antibiotics at 37°C in 5% CO2.

[0100] 1.11 CCK-8 assay

[0101] To investigate the effects of CFO-BFO NPs at varying concentrations and magnetic field strengths on BMDM, CCK-8 assays were used to assess biosafety. BMDM were co-cultured with CFO-BFO NPs at varying concentrations in 96-well plates for 24 hours. The biosafety assay was performed. BMDM were exposed to different magnetic field strengths for 30 minutes and then tested 24 hours later. Subsequently, 20 μL of Cell Counting Kit-8 and 180 μL of culture medium were added to each well. The cells were incubated at 37°C in the dark for 1 hour, and the samples were measured using a microplate reader at a wavelength of 450 nm.

[0102] 1.12 Cellular uptake of CFO-BFO NPs in vitro

[0103] First, 30 μg / mL of FITC-conjugated CFO-BFO NPs were co-cultured with BMDM for 0, 8, 12, and 24 h. BMDM were then washed with PBS to remove extracellular CFO-BFO NPs, and the mean fluorescence intensity was measured by flow cytometry.

[0104] 1.13 RT-qPCR

[0105] MHV-infected cells were harvested in vitro using Trizol reagent. In vivo, liver and lung samples were collected from MHV-infected mice using Trizol reagent. RNA was then reverse-transcribed into cDNA according to the manufacturer's instructions. RT-qPCR was performed using the ABI 7500 detection system.

[0106] 1.14 Confocal microscopy observation

[0107] Four groups of MHV-infected cells were incubated with an IRF-3 antibody overnight at 4°C in the dark to detect changes in IRF-3 expression in BMDM. DAPI was added to each group. Cells were evaluated using laser confocal microscopy. Relative quantitative fluorescence analysis of each group was performed using Image J 1.47.

[0108] 1.15 Virus Infection

[0109] For MHV-infected cells, 2×10 6 PFU MHV-A59. For MHV infection mouse model, 2×10 6 PFU MHV-A59 was injected intraperitoneally into 6-8 week old male C57BL / 6 mice.

[0110] 2. Results

[0111] 2.1 Characterization of CFO-BFO NPs

[0112] Core-shell CFO-BFO magnetostrictive piezoelectric NPs (CFO-BFO NPs) were fabricated by coating a BFO precursor onto a pre-prepared CFO core via hydrothermal and sol-gel methods. SEM images revealed a uniform spherical structure of 180-200 nm in size. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images (Figures 1a and b) indicated that the final CFO-BFO NPs after coating were 220-240 nm in size. TEM images revealed that the CFO core was encapsulated within the CFO-BFO NPs by a layer of BFO (Figure 1c), indicating a core-shell structure. The elemental distribution was evaluated by energy dispersive spectroscopy (EDS). Bi, Co, Fe, and O were all present in the CFO-BFO NPs, with Bi being uniformly distributed around the CFO core (Figure 1d), further confirming the core-shell structure of the CFO-BFO NPs. X-ray diffraction (XRD) patterns (Figure 1e) reveal a cubic spinel structure of CoFe2O4 and a rhombohedral perovskite phase of BiFeO3. Both structures are present in CFO-BFO NPs. X-ray photoelectron spectroscopy (XPS) patterns (Figure 1f) reveal characteristic peaks of elements in CFO-BFO NPs attributed to Co2p, Fe2p, Bi4p, Bi4d, Bi4f, O1s, and C1s. The characteristic peaks of Co2p, Fe2p, O1s, and C1s appear simultaneously in CFO (CoFe2O4) and CFO-BFO NPs, while the characteristic peaks of Bi4p, Bi4d, Bi4f, Fe2p, O1s, and C1s appear simultaneously in BFO (BiFeO3) and CFO-BFO NPs. The initial magnetization and saturation magnetic intensities of CFO and CFO-BFO NPs were measured using a vibrating sample magnetometer (VSM) to investigate their magnetic properties. The typical hysteresis loop diagram verifies that CFO and CFO-BFO NPs have excellent external magnetic field responsiveness (as shown in Figure 1g). The ferroelectric and magnetoelectric properties of CFO-BFO NPs were characterized by piezoresistive force microscopy (PFM). By sweeping the applied DC bias with a ramp voltage from -10 to +10 V and synchronously recording the phase and amplitude responses, local piezoelectric response hysteresis loops can be detected at random positions of the CFO-BFO sample, illustrating the piezoelectric and magnetostrictive coupling effects of the CFO-BFO sample. The typical butterfly amplitude curve shows that the strain of the material changes continuously with the change of the applied electric field, and the BFO shell has polarization reversibility (as shown in Figure 1h). According to the basic principle of the piezoelectric effect, the piezoelectric source is non-centrosymmetric, triggering a redox reaction to produce hydroxyl radicals (·OH) and superoxide radicals (·O2 -). At the same time, it is well known that the piezoelectricity of BFO enables BFO and CFO-BFO to generate ·OH and ·O2 under external ultrasound. - , while CFO cannot. Therefore, in order to further confirm the magnetoelectric coupling effect of the core-shell CFO-BFO sample, electron paramagnetic resonance (EPR) spectroscopy was used to qualitatively analyze the products of CFO-BFO under the action of an external magnetic field. 5,5-Dimethyl-1-pyrroline n-oxide (DMPO) was used as ·OH and ·O2 - The results show that CFO-BFO can effectively trap ·OH and ·O2 under the action of an external magnetic field. - Characteristic peaks (as shown in Figure 1i), the characteristic peaks increased with time. However, under the same experimental conditions, DMPO failed to capture ·OH and ·O2 in the CFO-only group and the BFO-only group. - On this basis, in order to explore the magnetoelectric transduction potential of CFO-BFO NPs, the degradation of Rhodamine B was tested under different magnetic field frequencies, intensities, and time intervals. As shown in Figure 7, the degradation rate of RHB accelerated with the increase of magnetic field frequency, intensity, and time interval.

[0113] 2.2 Biosafety Assessment and Biodistribution Overview

[0114] To evaluate the biosafety of the treatment, one group of mice was placed in a magnetic field every day. At the same time, other groups of mice were intravenously injected with different concentrations of CFO-BFO NPs. At 2 weeks, routine blood tests and blood biochemistry tests were performed to assess the health status, liver function, and kidney function of each group of mice. Compared with the normal control group (NC), all indicators were within the normal range (as shown in Figure 8a). In addition, at 2 weeks, HE staining was performed on the heart, liver, spleen, lungs, and kidneys of each group of mice. Compared with the NC group, the vital organs of each group of mice remained healthy, even in the high-concentration group (as shown in Figure 8b). The hematology and HE staining results showed that the magnetic field and CFO-BFO NPs have good in vivo biosafety. In addition, to study the biodistribution of CFO-BFO NPs in vivo, the present invention injected FITC-conjugated CFO-BFO NPs intraperitoneally into normal mice. The in vivo small animal imaging system showed that CFO-BFO NPs were highly enriched in the liver and lungs in vivo 12h and 24h after injection (as shown in Figure 9). Furthermore, CFO-BFO NPs were also highly enriched in the kidneys and decreased over time, suggesting that CFO-BFO NPs are most likely metabolized and excreted through the kidneys. In summary, CFO-BFO NPs do not cause damage to vital organs, indicating that CFO-BFO NPs have high biosafety.

[0115] 2.3 Antiviral effect of magnetostrictive piezoelectric CFO-BFO NPs on virus-infected mice

[0116] To verify the antiviral effect of magnetoelectrically coupled CFO-BFO NPs in vivo, the MHV-infected C57BL / 6 mouse model was used to study the antiviral activity of CFO-BFO NPs under an external magnetic field. C57BL / 6 mice infected with MHV were randomly divided into 4 groups (untreated control group, magnetic field group (1 kHz, 1.5 mT), CFO-BFO NPs group (18 mg / kg) and CFO-BFO NPs group (18 mg / kg) under magnetic field group (1 kHz, 1.5 mT). CFO-BFO NPs were intravenously injected 2 hours after the mice were infected with MHV. Then, magnetic field treatment was performed every day (30 minutes each time) after the injection of CFO-BFO NPs (as shown in Figure 2a). As shown in Figure 2b, most of the MHV-infected mice in the untreated control group, pure magnetic group and CFO-BFO-only group died within 11 days, while the survival rate of MHV-infected mice in the CFO-BFO NPs plus magnetic treatment group was higher. The weight curves of MHV-infected mice in each treatment group further confirmed that CFO-BFO NPs plus magnetic energy reduced the mortality rate by maintaining the body weight of MHV-infected mice (as shown in Figure 2c). Five days after MHV infection, except for CFO-BFO With the exception of the NPs plus magnetization group, the spleens of the other three groups of mice were significantly enlarged (Figure 2d and g). The liver surface of the CFO-BFO NPs plus magnetization group remained smooth and flexible, while the livers of the other groups showed extensive macroscopic punctate necrosis and were lighter in weight (Figure 2e and f). Histological analysis of liver tissue further revealed that liver tissue damage was significantly reduced in mice treated with CFO-BFO NPs plus magnetization compared with the other groups (Figure 2h and j). Furthermore, blood biochemical tests for liver function showed the same trend (Figure 2o). H&E staining of lung tissue revealed reduced alveolar congestion, edema, exudate, and inflammatory cells in the CFO-BFO NPs plus magnetization group (Figure 2i, k, and o). RT-qPCR analysis revealed a significant decrease in the relative expression of MHV-N in the liver, spleen, and lungs in the CFO-BFO NPs plus magnetization group (Figure 2ln). Taken together, these results indicate that CFO-BFO NPs plus magnetization therapy limits viral infection in vivo.

[0117] 2.4 The antiviral activity of magnetostrictive piezoelectric CFO-BFO NPs depends mainly on the stimulation of type I IFN response in vivo

[0118] To further analyze the therapeutic effects of magnetostrictive piezoelectric CFO-BFO NPs against MHV infection, RNA-seq was used to analyze differentially expressed genes in the liver tissue of MHV-infected mice. A cluster heat map depicts the differentially expressed genes in the liver tissue of MHV-infected mice between the treatment group (CFO-BFO NPs plus magnetism) and the control group (untreated control group). Upregulated genes were identified using a volcano plot (Figure 3a). Enrichment analysis using the GO BP database and the KEGG database revealed multiple immune regulatory pathways involved in viral defense responses and antiviral processes (Figure 3b). Of particular note, the Rig-I-like receptor pathway, which plays a key role in virus recognition and upregulation of type I interferon (IFN), was activated by CFO-BFO plus magnetization (Figures 3c and 3d). Given the important role of type I IFN in inhibiting viral replication, the relative mRNA expression levels of type I IFN-related genes in the liver and lung tissues of MHV-infected mice were investigated (Figures 3e and e). RT-qPCR results showed that CFO-BFO magnetization significantly increased the expression of IFNβ, Isg56, is20, cxcl10, and Isg15 in the liver and lung tissues of MHV-infected mice. Therefore, CFO-BFO magnetization can activate the Rig-I-like receptor pathway, upregulating type I IFNs and inhibiting viral replication.

[0119] To investigate the quantitative distribution of CFO-BFO NPs in liver tissue, we analyzed liver lymphocytes from mice infected with MHV after treatment with FITC-CFO-BFO NPs and magnetic resonance imaging. Flow cytometry revealed that the number of macrophages (CD11b+F4 / 80+) and dendritic cells (CD11c+MHCⅡ+) was approximately 40%. The number of T cells (CD3+) and B cells (CD19+) was approximately 20% (Figure 10).

[0120] 2.5 Antiviral effect of magnetostrictive piezoelectric CFO-BFO NPs in virus-infected cells

[0121] As shown in Figure 4, BMDM was used for cell experiments. BMDM was co-cultured with CFO-BFO NPs for 12 hours and then exposed to a magnetic field. MHV-A59 was then added to the BMDM culture medium and placed in a magnetic field for 12 hours, and the cells were collected 36 hours later (as shown in Figure 4a). In order to verify the biosafety of CFO-BFO NPs in cells, different concentrations of CFO-BFO NPs were co-cultured with BMDM for 24 hours. When the concentration of CFO-BFO NPs was lower than 50 μg / mL, CFO-BFO NPs had almost no effect on the proliferation of BMDM (as shown in Figure 4b). Therefore, CFO-BFO NPs have high cell compatibility, and 30 μg / mL CFO-BFO (CFO-BFO NPs concentration is 30 μg / mL) was used for cell experiments. Based on the research results in Figure 7, the CCK-8 method was used to detect the effect of the magnetic field on cells. As shown in Figure 4c, the magnetic field had almost no effect on the biological activity of the cells. To determine the quantitative distribution of CFO-BFO NPs in BMDMs after co-incubation, flow cytometry was used to assess the cellular uptake of FITC-conjugated CFO-BFO NPs. After 8 hours of co-incubation, the phagocytosis rate, as indicated by the fluorescent signal, reached 98.9% (Figure 4d), and the mean fluorescence intensity stabilized after 12 hours of co-incubation (Figure 4e). Furthermore, RT-qPCR analysis demonstrated that CFO-BFO NPs combined with magnetic treatment inhibited MHV replication and reduced MHV titers in BMDMs by 60% (Figure 4f). To analyze the therapeutic effects of magnetostrictive piezoelectric CFO-BFO NPs on virus-infected cells, RNA-seq was used to deeply analyze differentially expressed genes in MHV-infected BMDMs. Upregulated genes were identified using a volcano plot (Figure 4g). Gene oncogene kinase (GOB) database enrichment analysis revealed that the upregulated genes were involved in the detection and defense of multiple viral pathways related to antiviral processes, particularly the RIG-I signaling pathway (Figure 4h). The results of gene set enrichment analysis (GSEA) shown in Figure 4i are consistent with those in Figure 4h. In summary, the data of the present invention reveal that CFO-BFO magnetization may achieve antiviral effects in vitro by upregulating the RIG-I-like receptor pathway.

[0122] 2.6 Magnetostrictive-piezoelectric CFO-BFO NPs stimulate type I IFN responses during in vitro viral infection

[0123] Upregulation of the RIG-I-like receptor pathway is considered central to type I IFN expression. Therefore, RT-qPCR was used to examine the relative mRNA expression levels of type I IFN-related genes in MHV-infected cells. Consistent with the in vivo results (Figure 2, a), in MHV-infected BMDM, the expression levels of IFNβ, Isg56, Isg20, CXCL10, and Isg15 were significantly increased in CFO-BFO plus magnetic treatment compared with other groups (Figure 5, ae). TBK1 and IRF3 play crucial roles in the transcriptional activation of type I IFNs (host innate immunity). Generally, TBK1 is activated after autophosphorylation, which in turn promotes IRF3 activation. Activated IRF3 leads to nuclear translocation, initiating type I IFN expression and inducing the transcription of various antiviral genes. As shown in Figure 5, f and h, immunoblot analysis revealed a significant increase in TBK1 phosphorylation in MHV-infected BMDM treated with CFO-BFO plus magnetic treatment. Furthermore, the present invention confirmed whether CFO-BFO NPs plus magnetism affect the nuclear translocation of IRF3 in cells. IRF3 immunoblot analysis (shown in Figures 5g and i) and immunofluorescence staining images (shown in Figures 5j and k) showed nuclear accumulation of IRF3 in MHV-infected BMDMs treated with CFO-BFO plus magnetism compared to other treatment groups. Taken together, these results confirm the stimulatory role of CFO-BFO NPs plus magnetism in regulating type I IFN and host innate immunity.

[0124] 2.7 Antiviral Effects of Magnetostrictive-Piezoelectric CFO-BFO NPs against Viruses Other Than MHV-A59

[0125] In order to explore the antiviral effect of CFO-BFO NPs against viruses other than MHV-A59, the present invention infected BMDM with VSV virus, and performed fluorescence photography (as shown in ad of Figure 6) and corresponding flow cytometry statistics (as shown in e of Figure 6) on VSV in different treatment groups. The results showed that only when CFO-BFO NPs were treated with a magnetic field could the viral replication be effectively controlled; there was no difference between the other three groups. Therefore, the magnetostrictive piezoelectric CFO-BFO NPs of the present invention also have the effect of resisting VSV viral replication and infection.

[0126] 3. Discussion and Conclusion

[0127] The development of magnetostrictive piezoelectric nanoparticles for antiviral therapy is an issue worthy of attention. In the present invention, magnetostrictive piezoelectric CFO-BFO NPs are designed for antiviral therapy. The BFO precursor surface is coated on the prepared CFO core by a sol-gel method to prepare CFO-BFO magnetostrictive piezoelectric NPs with a core-shell structure. The present invention successfully prepared uniform spherical CFO-BFO NPs with a size of 220-240 nm, whose core is a uniform CFO with a size of 180-200 nm. TEM and EDS images show that the spherical CFO is relatively uniformly wrapped by a layer of BFO, indicating that the CFO-BFO NPs have a core-shell structure. There are only two phases in the XRD spectrum of CFO-BFO NPs (as shown in Figure 1e), namely spinel-structured CFO and perovskite-structured BFO, indicating that CFO and BFO are successfully synthesized in the form of two-phase composite CFO-BFO NPs. The positions of the characteristic peaks of the elements in the XPS spectrum (as shown in Figure 1f) are consistent with previous reports. The hysteresis loop diagram of VSM (as shown in Figure 1g) and the pressure response amplitude curve and phase curve diagram of PFM (as shown in Figure 1h) show that CFO-BFO NPs have piezoelectric and magnetoelectric coupling. In addition, the EPR spectra of CFO-only, BFO-only, and CFO-BFO NPs further prove that CFO-BFO NPs are piezoelectric, come from the shell BFO, and have magnetoelectric coupling. It can be seen from the degradation curve of RhB that increasing the magnetic field frequency, intensity and time interval can accumulate the magnetoelectric transduction of CFO-BFO (as shown in Figure 7). The above results show that the magnetoelectrically coupled CFO-BFO NPs are formed through the synergistic effect of the core CFO and shell BFO. In addition, hematological tests and HE staining show that CFO-BFO NPs have good biosafety in vivo (as shown in Figure 8). At the same time, the CFO-BFO NPs of the present invention are mainly distributed in the liver and lung tissues and are metabolized through the kidneys (as shown in Figure 9).

[0128] Based on the excellent biosafety of CFO-BFO NPs, the antiviral activity of magnetostrictive-piezocatalytic CFO-BFO NPs was evaluated in vivo using a C57BL / 6 mouse model of MHV infection. Generally, MHV-A59 causes abnormal liver parameters and weight loss, which can lead to death in severe cases. Time-dependent survival curves and corresponding weight curves showed that CFO-BFO plus magnetization treatment resulted in higher survival rates and less weight loss in MHV-infected mice compared with other treatments (as shown in Figure 2b and c). Furthermore, the liver and spleen morphology of MHV-infected mice treated with CFO-BFO plus magnetization resembled that of healthy controls. Splenomegaly may be related to the immune response of various inflammatory cells in the other treatment groups. Therefore, magnetostrictive-piezocatalytic CFO-BFO NPs can better protect the liver from viral attack and improve survival. Significant reductions in ALT, AST, and ALP levels in MHV-infected mice after CFO-BFO NPs plus magnetization correlated with liver function, confirming these findings. MHV infection often causes tissue damage. However, H&E staining revealed that CFO-BFO NPs plus magnetization treatment in MHV-infected mice showed reduced liver tissue necrosis and fewer inflammatory cells in lung tissue compared to other treatment groups. Furthermore, magnetization of CFO-BFO NPs significantly reduced the titer of MHV-A59 in liver, spleen, and lung tissue. Therefore, magnetostrictive-piezocatalytic CFO-BFO NPs limit MHV-A59 replication in the liver, lungs, and spleen, ultimately producing an antiviral effect and reducing mortality. Furthermore, the present invention found that magnetization of CFO-BFO NPs induced upregulation of antiviral-related pathways in the liver of MHV-infected mice compared to the control group. Notably, upregulation of the RIG-I-like receptor pathway is highly correlated with antiviral activity. According to recent studies, when RIG-I is activated by viral RNA, it regulates the expression of type I interferon, thereby stimulating the host's innate immune response and ultimately inhibiting viral replication. Based on RNA-seq results, the present invention detected a significant increase in the mRNA expression levels of type I IFN-related genes in the liver and lung tissues of MHV-infected mice after magnetic treatment of CFO-BFO NPs. In summary, the addition of magnetism to CFO-BFO NPs plays a central role in the antiviral activity, limiting the spread of the virus in vivo by activating the RIG-I-like receptor pathway and enhancing the host innate immune response.

[0129] Because CFO-BFO NPs are primarily phagocytosed by macrophages in liver tissue, the present invention selected BMDM for in vitro experiments. Consistent with the in vivo experimental results, CFO-BFO NPs have high cytocompatibility with magnetic fields. Subsequently, the present invention confirmed that the majority of CFO-BFO NPs were phagocytosed by BMDM after 8 hours of co-culture. Similar to the animal experimental results, treatment of MHV-infected BMDM with CFO-BFO NPs and magnetization reduced viral titers, which is associated with upregulation of the RIG-I signaling pathway. Recent studies have shown that upregulation of the RIG-I-like receptor pathway plays a crucial role in enhancing host innate immunity. As envisioned by the present invention, although type I IFN-related genes were expressed in all treatment groups, expression levels were significantly increased in the CFO-BFO + magnetic field treatment group compared to the other treatment groups. In addition, the present invention detected the expression of TBK1 and IRF3 in the RIG-I-like receptor pathway, which are involved in the transcriptional activation of type I IFN. Immunoblotting and immunofluorescence analysis revealed that magnetostrictive piezoelectric CFO-BFO NPs increased TBK1 phosphorylation compared to other treatments, thereby promoting IRF3 nuclear translocation. IRF3 nuclear translocation provides further evidence for type I IFN activation. Taken together, these data strongly confirm that magnetostrictive piezoelectric CFO-BFO NPs exert their antiviral effects / functions by enhancing host immune responses.

[0130] In addition, in order to explore the antiviral effect of CFO-BFO NPs against viruses other than MHV-A59, the present invention also used VSV virus to infect BMDM. The results showed that only when the CFO-BFO NPs were treated with a magnetic field could the viral replication be effectively controlled. There was no difference between the other three groups. Therefore, the magnetostrictive piezoelectric CFO-BFO NPs of the present invention also have the effect of resisting VSV virus replication and infection.

[0131] In summary, the present invention designs magnetostrictive piezoelectric CFO-BFO NPs with high biosafety for antiviral treatment. Magnetostrictive piezoelectric CFO-BFO NPs effectively enhance type I IFN expression and host innate immune response by upregulating multiple immunomodulatory pathways, especially the RIG-I-like receptor pathway, ultimately inhibiting viral activity. At the same time, the magnetostrictive piezoelectric CFO-BFO NPs of the present invention significantly improve liver and lung tissue damage and improve survival rate. As a highly controllable, deep-penetrating and non-invasive treatment method, the magnetostrictive piezoelectric nanoparticle therapy of the present invention enhances the host antiviral immune response and provides a sufficient strategic guarantee for future antiviral drug treatments.

[0132] Finally, it should be noted that the above embodiments are intended only to illustrate the technical solutions of the present invention, and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they may modify the technical solutions described in the above embodiments or replace some of the technical features therein with equivalents. However, such modifications or replacements do not deviate from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. Use of magnetostrictive piezoelectric nanoparticles driven by magnetoelectricity in the preparation of antiviral drugs, characterized in that: The magnetostrictive piezoelectric nanoparticles include a shell structure with piezoelectric properties and a core structure with magnetoelectric response properties. The shell structure is prepared from inorganic piezoelectric materials and / or organic piezoelectric materials, and the core structure is prepared from magnetoelectric materials.

2. The use according to claim 1, characterized in that The inorganic piezoelectric material is selected from at least one of ferrite, niobic acid, titanic acid, silicate and aluminate materials; the organic piezoelectric material is selected from at least one of polyvinylidene fluoride, polyester, polymethyl methacrylate, nylon, polyvinyl chloride, poly-L-lactide, poly(3-hydroxybutyrate-co-3-hydroxyvalerate), vinylidene fluoride / trifluoroethylene copolymer, vinylidene fluoride / tetrafluoroethylene copolymer, polyvinylidene fluoride-hexafluoropropylene and polydimethylsiloxane materials.

3. The use according to claim 2, characterized in that The ferrite material has the following structure: x Fe 3-x O y , wherein M includes at least one of Bi, Ca, Ba, Mg and Ba, x is a number from 0.25 to 1.5, and y is 4.

4. The use according to claim 1, characterized in that The magnetoelectric material has the following structure: a Fe 1-a O b , N includes at least one of Co, Mn, Zn and Ni, a is a number from 0.25 to 0.75, and b is 3.

5. The use according to claim 1, characterized in that The magnetostrictive piezoelectric nanoparticles are cobalt ferrite-bismuth ferrite nanoparticles; Wherein, the particle size of the cobalt ferrite-bismuth ferrite nanoparticles is 100-500 nm.

6. The use according to claim 1, characterized in that The antiviral agent includes at least one of the following: (1) Inhibit viral replication; (2) Reduce viral titer; (3) Activate the host innate immune response; (4) Reduce tissue or organ damage caused by the virus; (5) Activate the expression of type I interferon.

7. The use according to any one of claims 1 to 6, characterized in that The magnetoelectric driving includes treating the magnetostrictive piezoelectric nanoparticles with a magnetic field having a magnetic field strength of 0.01-300 mT.

8. Use of magnetostrictive piezoelectric nanoparticles driven by magnetoelectricity in the preparation of a drug for activating the expression of type I interferon, characterized in that: The magnetostrictive piezoelectric nanoparticles include the magnetostrictive piezoelectric nanoparticles according to any one of the above claims.

9. Use of magnetostrictive piezoelectric nanoparticles driven by magnetoelectricity in the preparation of a drug for activating type I interferon expression and / or antiviral effects in combination with magnetic field therapy, characterized in that: The magnetostrictive piezoelectric nanoparticles include the magnetostrictive piezoelectric nanoparticles according to any one of the above claims.

10. The use according to claim 9, characterized in that A therapeutically effective amount of magnetostrictive piezoelectric nanoparticles and a magnetic field of 0.01-300 mT are administered to a subject in need thereof to activate type I interferon expression and / or achieve an antiviral effect.

11. A method for treating or ameliorating viral infection, characterized in that: The method comprises administering a therapeutically effective amount of magnetoelectrically driven magnetostrictive piezoelectric nanoparticles to a subject in need thereof, wherein the magnetostrictive piezoelectric nanoparticles are the magnetostrictive piezoelectric nanoparticles according to any one of claims 1 to 10.

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