Application of cis-BBP in inactivation of virus with envelope structure

Abstract

The invention discloses an application of cis-BBP in inactivation of viruses with envelope structures. Any one of the following applications of the cis-BBP comprises an application of the cis-BBP in photodynamic inactivation of the virus with the envelope structure, an application of the cis-BBP in preparation of a product for preventing and treating the virus with the envelope structure, and an application of the cis-BBP in preparation of a product for inhibiting cells infected by the virus with the envelope structure. According to the application of the cis-BBP in photodynamic inactivation of the virus with the envelope structure, the antiviral efficiency is high, and a broad-spectrum antiviral strategy for the virus with the envelope structure is developed. The broad-spectrum antiviral ability of the cis-BBP is explored by utilizing the characteristics of the cis-BBP, and the action effect of the cis-BBP on various viruses is clarified. The cis-BBP shows antiviral activity and excellent biocompatibility in vivo. When applied to transportation and packaging, it shows significant potential in controlling virus propagation.

Description

Technical Field

[0001] This application relates to the field of biomedical technology, specifically to the application of cis-BBP in the inactivation of viruses with envelope structures. Background Technology

[0002] Emerging infectious diseases caused by viruses, such as SARS-CoV-2, influenza virus, dengue virus, and monkeypox virus, pose a significant threat to mammalian health and socioeconomic stability. A considerable number of viruses widely circulating among mammals possess envelope structures. To date, vaccination has proven to be an effective strategy for preventing and controlling the spread of epidemics. However, the high variability of viruses presents a significant challenge to the development of effective vaccines, particularly for enveloped viruses such as influenza virus and porcine reproductive and respiratory syndrome virus (PRRSV), whose rapid mutation rates lead to extensive genetic variability. This variability allows these viruses to evade immune responses, increasing the complexity of developing vaccines that provide long-term protection. Furthermore, the emergence of drug-resistant strains poses a significant challenge to the effectiveness of antiviral treatments. Due to the prevalence of these mutant strains, alternative strategies are needed to effectively manage their spread. The envelope is a highly conserved structural component of enveloped viruses and is crucial for maintaining viral integrity and infectivity. Therefore, it is important to develop a broad-spectrum antiviral strategy that targets and disrupts the viral lipid envelope to prevent its spread.

[0003] Organometallic photosensitizers are widely used as photodynamic inactivators due to their ability to generate reactive oxygen species (ROS). ROS generation is a key factor in photodynamic therapy, which is applied in multiple fields, including antibacterial and anticancer treatments. Platinum compounds are fundamental drugs in cancer chemotherapy, primarily due to their mechanism of action involving the coordination of platinum centers with DNA. Cisplatin, a typical platinum-based drug, has been widely used to treat various human cancers. Furthermore, research has shown that the addition of heavy metal centers such as platinum and ruthenium to organometallic photosensitizers (PSs) plays a crucial role in promoting intersystem crossings that facilitate ROS generation, which is essential for various applications, including photodynamic therapy and photocatalysis. Several studies have shown that platinum(II)-based organometallic photosensitizers and their derivatives have considerable potential in this field, primarily for targeting and destroying cancer cells through ROS generation. Currently, the role of photodynamic inactivators in cancer cell destruction and antibacterial applications has been extensively studied. In contrast, their potential as antiviral agents remains underestimated, especially in Pt... II Regarding PSs. Summary of the Invention

[0004] The purpose of this application is to provide the application of cis-BBP in the inactivation of viruses with envelope structures, to explore the broad-spectrum antiviral ability of cis-BBP, and to provide a reference for the application of photoinactivation reagents.

[0005] The technical solution provided in this application is as follows:

[0006] Any of the following applications of cis-BBP include:

[0007] Application of cis-BBP in photodynamic inactivation of enveloped viruses.

[0008] The application of the cis-BBP in the preparation of products for preventing and treating viruses with envelope structures.

[0009] The application of the cis-BBP in the preparation of the product that inhibits the infection of cells by the virus having an envelope structure.

[0010] Furthermore, the cis-BBP can target and destroy the virus.

[0011] Furthermore, the application environment of the cis-BBP includes temperatures ranging from -70°C to 25°C.

[0012] Furthermore, the application environment of the cis-BBP includes under illumination conditions.

[0013] Furthermore, the product includes pharmaceuticals, disinfectants, or photodynamic inactivation reagents.

[0014] A method for inactivating viruses with envelope structures, comprising photodynamic inactivation of the viruses with envelope structures using the cis-BBP photodynamic method described in any of the above applications.

[0015] A product, using the cis-BBP described in any of the above applications as an active ingredient, effectively inactivates the virus having an envelope structure.

[0016] Furthermore, the products include pharmaceuticals, disinfectants, or photodynamic inactivation reagents.

[0017] This application provides the application of cis-BBP in photodynamic inactivation of viruses with envelope structures, which has high antiviral efficiency and develops a broad-spectrum antiviral strategy against viruses with envelope structures.

[0018] This application utilizes the properties of cis-BBP to explore its broad-spectrum antiviral capabilities and elucidate its effects against various viruses. Cis-BBP exhibits antiviral activity and excellent biocompatibility in vivo. When used in transportation and packaging, it shows significant potential in controlling viral transmission.

[0019] The inactivation method of this application specifically destroys viruses with envelope structures and reduces viral titers by 50% in a dose-dependent manner, but does not show the same effect on viruses without envelope structures.

[0020] The product of this application has an inactivation effect on multiple PRV subtypes and has acceptable biocompatibility in vivo and in vitro. Attached Figure Description

[0021] Figure 1 This diagram illustrates the specific inactivation of enveloped viruses by cis-BBP. Images A and H show transmission electron microscopy (TEM) images and viral titers of cis-BBP-treated and untreated viruses. A, B, C, D, E, F, G, and H correspond to PRV / XJ5, PEDV, H1N1, PRRSV, TGEV, PPV, PCV-2, and SVA, respectively. Arrows in the diagram indicate disruption of the viral lipid envelope.

[0022] Figure 2 To inactivate different PRV subtypes with cis-BBP. A–F represent the TCID values ​​of different PRV subtypes after treatment with increased concentrations of cis-BBP for different durations under light conditions. 50 Results. Among them, A is the XJ5 strain, B is the Ra strain, C is the NT strain, D is the HA strain, E is the CM strain, and F is the TX strain.

[0023] Figure 3 This demonstrates the application of cis-BBP in transportation and packaging. Figure A shows a schematic diagram of transportation and packaging sterilization at different temperatures. Figure B shows the TCID of different PRV subtypes after treatment with cis-BBP in selected areas. 50 result.

[0024] Figure 4 This study assesses the biosafety of cis-BBP in vivo. A and B represent the toxicity of cis-BBP to PK-15B6 cells under dark and light conditions, respectively. BALB / c mice were administered 45.35 μg / kg of cis-BBP via intranasal instillation, and survival rate (C) and weight change (D) were monitored over 14 days. Organs were collected to assess organ morphological changes (E) and histopathological changes (F).

[0025] Figure 5 This study investigated the effects of intranasal administration of cis-BBP (45.35 μg / kg) on ​​blood parameters. In the data, A represents complete blood count (RBC), red blood cells (RBC), white blood cells (WBC), and hemoglobin (HGB). B represents blood biochemical parameters (AST), aspartate aminotransferase (ALT), alanine aminotransferase (ALT), and blood urea nitrogen (BUN). Data are presented as mean ± standard deviation from three independent trials. Detailed Implementation

[0026] The embodiments of this application will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of this application. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer shall apply.

[0027] In this application, cis-BBP represents the Bis-BODIPY-Pt complex, which belongs to the Pt family. II A type of PSs.

[0028] Any of the following applications of cis-BBP include:

[0029] Application of cis-BBP in photodynamic inactivation of enveloped viruses.

[0030] Application of cis-BBP in the preparation of products for the prevention and treatment of viruses with envelope structures.

[0031] Application of cis-BBP in the preparation of products that inhibit viral infection of cells with enveloped structures.

[0032] Cis-BBP can target viruses that disrupt envelope structures, exhibiting broad-spectrum antiviral capabilities, such as against various PRV subtypes.

[0033] Cis-BBP is applicable to environments with temperatures ranging from -70°C to 25°C, such as 4°C, -20°C, -70°C (cold chain low temperature) and 25°C (normal temperature), covering a wide range of applicable temperatures.

[0034] The application environment of cis-BBP includes light conditions. For viruses with envelope structures, the antiviral effect of cis-BBP under light irradiation is significantly better than that under no-light conditions.

[0035] The sources of raw materials used in the following examples are:

[0036] Cells and Viruses: PRV isolates XJ5, NT, HA, CM, and TX, as well as PEDV strains HLJBY, H1N1, PRRSV, TGEV, PPV, PCV-2, and SVA, were isolated and preserved by the Infectious Disease Laboratory of Yangzhou University. Classical PRV / Ra strains were purchased from the China Institute of Veterinary Drug Control. Cell lines PK-15B6, Vero, MDCK, Marc-145, ST, and PK-15 were cultured in DuPont modified Eagle medium containing penicillin, streptomycin, nystatin, and 4%-10% fetal bovine serum at 37°C and 5% CO2. Furthermore, all PRV and PCV-2 strains were amplified in PK-15B6 cells. PEDV HLJBY, H1N1, PRRSV, and SVA strains were amplified in Vero, MDCK, Marc-145, and PK-15 cells, respectively. TGEV and PPV strains were amplified in ST cells.

[0037] Chemicals: The synthesis and structural characterization of cis-BBP were based on previous research in our laboratory, using 520-LED irradiation for 5 minutes (wavelength: 520nm, power: 5mW / cm²). 2 CCK-8 reagent (catalog number C0038) was purchased from Beyotime Biotechnology (Shanghai, China).

[0038] The following is the detection method used in the embodiments of the present invention, specifically:

[0039] Viral titer detection: Different concentrations of cis-BBP were mixed with different viral solutions, followed by light or dark treatment for 5 minutes, and then incubated at different temperatures for different times. After centrifugation, the titer was determined using a 50% tissue culture infectious dose (TCID). 50 The detection method involved collecting viral fluid and supernatant from infected cells to determine viral titers. Cells required for each virus were seeded into 96-well plates and grown into a monolayer. Virus fluid and supernatant were serially diluted 10-fold with culture medium and then seeded into cells, with each dilution repeated 8 times. The plates were incubated at 37°C for 1 hour. After adsorption, the culture medium was discarded, and unadsorbed viruses were washed away with PBS. Maintenance medium was then added, and the plates were incubated at 37°C. Cytopathic effect (CPE) was monitored daily until all virus-containing wells showed CPE. For PCV-2, since CPE was not observable, positive wells were identified by immunofluorescence. TCID was calculated using the Reed-Muench method. 50 .

[0040] Transmission electron microscopy observation of viral particles: Viruses amplified from the corresponding cells were purified using a discontinuous sucrose density gradient to remove other molecules, following the previously described method. The purified virus was mixed with different concentrations of cis-BBP, irradiated for 5 minutes, and then incubated at 37°C for 1 hour. After incubation, the virus was fixed with 2.5% glutaraldehyde at 4°C, followed by negative staining with 2% uranium acetate for 1-2 minutes. The morphology of the prepared viral samples was photographed using a Tecnai 12 transmission electron microscope. Additionally, PK-15B6 cells were infected with 10 MOI PRV / XJ5 at 4°C for 1 hour. After infection, unadsorbed virus was removed with PBS, and then medium containing 5 μM cis-BBP was added, followed by co-incubation under light for 5 minutes. After incubation, cell samples were prepared to observe the structure of viral particles adsorbed on the cell surface.

[0041] Application of cis-BBP in packaging and transportation: Select an area (10cm²) 2 The spraying droplet concentration around the spray area is 1×10⁻⁶. 6 TCID 50 Different PRV subtypes ( / mL) were sealed in plastic film and processed at different temperatures. After zone formation, 0.907 μg / cm³ of PBS was used. 2 Cis-BBP reagent was evenly sprayed onto the surface of the treatment area, followed by 5-minute light exposure at different temperatures and incubation for 1 hour. After incubation, the cis-BBP-covered area was collected in test tubes, immediately centrifuged, and then analyzed using TCID50. 50 The method measures viral titer.

[0042] The protective effect of cis-BBP against PRV was evaluated in a mouse model: Six-week-old female BALB / c mice were randomly divided into five groups: control group (Mock), PRV treatment group (PRV / XJ5), pretreatment group, co-culture group, and post-infection treatment group. In the pretreatment and post-infection treatment groups, mice were intranasally infused with 22.675 μg / kg of cis-BBP and co-cultured with 10... 3.2 TCID 50 Mice were treated with PRV / XJ5 together for 1 hour, both before and 1 hour after infection. Following this, mice were exposed to light for 5 minutes. The co-culture group consisted of 10 mice... 3.2 TCID 50 PRV / XJ5 mice were mixed with 22.675 μg / kg cis-BBP and exposed to light for 5 minutes, then incubated for 1 hour before being administered via nasal drip. Subsequently, weight changes and mortality were monitored in mice over 7 days, and organs were collected for pathological examination by hematoxylin-eosin staining, and viral replication levels in the brain and lungs were detected by qRT-PCR.

[0043] Biocompatibility assessment of cis-BBP: In vitro, PK-15B6 cells were co-incubated with different concentrations of cis-BBP for 24 hours, followed by incubation under light or no light for 5 minutes. The viability of PK-15B6 cells at the end of incubation was detected using the CCK-8 reagent. In vivo, 6-week-old female BALB / c mice (n=6 per group) were intranasally administered cis-BBP (45.35 μg / kg), while the control group received an equal volume of culture medium. The mice were fed and their body weight changes were monitored over 14 days, and percentage changes were plotted. After 14 days, blood and serum were collected from the treated mice for routine blood tests and blood biochemical parameters. Organs were collected for HE staining to observe pathological changes in each organ.

[0044] Statistical analysis: Experimental data are expressed as mean ± standard deviation (SD) and were analyzed and plotted using GraphPadPrism software. One-way ANOVA was performed using SPSS software to compare differences between groups. Statistical significance: ns, not significant; *P<0.05, **P<0.01.

[0045] Example 1:

[0046] Cis-BBP exhibited superior antiviral activity, reducing viral titers by 50% in a dose-dependent manner from the infection dose in tissue cultures. After treatment with 10 μM cis-BBP, the viral titer of PRV / XJ5 decreased from 6.47 ± 0.04 to 0.00 ± 0.00 log₂O₅. 10 TCID 50 / mL, indicating that the virus was completely inactivated. Specifically, transmission electron microscopy showed that, compared with the control group, cis-BBP disrupted the structure of the viral spikes and lipid envelope, leading to leakage of internal viral particles, thereby exerting its ability to inactivate the virus (see...). Figure 1 A). TCID of other viruses with envelope structures 50 The results showed that cis-BBP could completely inactivate porcine epidemic diarrhea virus and porcine reproductive and respiratory syndrome virus at a dose of 10 μM, and even completely inactivate transmissible gastroenteritis virus at a dose of 5 μM (see...). Figure 1 (B, D, and E). When H1N1 influenza virus was treated with 10 μM cis-BBP, the inhibition rate reached 99.9% (see B, D, and E). Figure 1 C). For these enveloped viruses, the antiviral effect of cis-BBP under light irradiation is significantly better than that under no-light conditions (see [reference]). Figure 1 AE). Meanwhile, TEM observed that the lipid envelope structure of all enveloped viruses was disrupted by cis-BBP (see AE). Figure 1(AE). In contrast, at the same concentration, cis-BBP failed to reduce viral titers of non-enveloped viruses (such as porcine parvovirus, porcine circovirus type 2, and Seneca virus A) under both dark and light conditions (see [reference needed]). Figure 1 Furthermore, the structures of PPV, PCV-2, and SVA were not affected by the cis-BBP treatment (see FH). Figure 1 These results clearly demonstrate that cis-BBP can effectively inactivate a variety of enveloped viruses, but it did not show the same effect on non-enveloped viruses.

[0047] Example 2:

[0048] PRV was used as a model virus to verify whether cis-BBP inactivated virus is broadly applicable to various subtypes of enveloped viruses. Six PRV strains were treated with different concentrations of cis-BBP, and the results were analyzed using TCID45. 50 Experimental assessment of viral titer. TCID 50 The results showed that cis-BBP could reduce the viral titers of six PRV strains in a dose- and time-dependent manner (see [link to study]). Figure 2 AF). Five minutes after treatment with cis-BBP, the virus was significantly inactivated, and the effect stabilized after one hour of co-incubation with the virus. Simultaneously, 0.5 μM cis-BBP inactivated at least 95% of these PRV subtype strains after one hour of treatment, while at a dose of 10 μM, the inhibition rate reached 99.9%, leading to HA (see AF). Figure 2 D), CM (see Figure 2 E) and TX (see E) and TX Figure 2 F) The strains were completely inactivated, which may be related to their initial viral titers. This demonstrates that approximately 99% inhibition of all PRV subtypes can be achieved within 5 minutes using only 10 μM cis-BBP.

[0049] Example 3:

[0050] To explore the possibility of using cis-BBP as a main component of a disinfectant and to evaluate its antiviral efficacy against different PRV subtypes (including XJ5, Ra, NT, HA, CM, and TX). Figure 3 Figure A illustrates the application model of cis-BBP in transportation and packaging. At a normal transportation temperature of 25°C, cis-BBP exhibited excellent virus inactivation effects, completely inactivating all PRV subtypes within 1 hour. (The text then abruptly shifts to a different topic: using 0.907 μg / cm³...) 2 Following cis-BBP spraying and 5 minutes of light exposure, the viral titers of all PRV subtypes rapidly decreased to 0.00±0.00 (P<0.01) (see [link to product description]). Figure 3B). Similarly, this effect was observed at cold chain temperatures (including 4°C, -20°C, and -70°C) (see [link]). Figure 3 B).

[0051] Example 4:

[0052] Biosafety is one of the main concerns in this application; therefore, biosafety experiments were conducted to evaluate the biocompatibility of cis-BBP. In in vitro experiments, cis-BBP did not exhibit toxicity to PK-15B6 cells under either dark or light conditions (see [link to in vitro experiments]). Figure 4 A, B). Furthermore, intranasal administration of cis-BBP (45.35 μg / kg) did not significantly affect changes in blood parameters (see [link to relevant documentation]). Figure 5 ), weight (see Figure 4 C) and survival rate (see Figure 4 D). Meanwhile, histological examination showed no lesions or abnormalities in the major organs (see...). Figure 4 E, F). These results indicate that the designed cis-BBP has acceptable biocompatibility both in vivo and in vitro.

[0053] This application presents a novel and highly efficient photodynamic inactivation (PDI) agent, termed cis-BBP, designed based on coordination chemistry theory. This agent exhibits excellent antiviral activity against enveloped viruses while demonstrating good biocompatibility. Furthermore, cis-BBP can be successfully applied as an active disinfectant ingredient in transportation and packaging, effectively reducing the infectivity of enveloped viruses under various temperature conditions. Overall, cis-BBP, as a PDI agent, provides a broad-spectrum antiviral option against enveloped viruses.

[0054] The above description of the disclosed embodiments enables those skilled in the art to implement or use this application. While specific embodiments are given in this application, it should be understood that further modifications can be made to this application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of this application.

Claims

1. Any of the following applications of cis-BBP, characterized in that, include: Application of cis-BBP in photodynamic inactivation of enveloped viruses. The application of the cis-BBP in the preparation of products for preventing and treating viruses with envelope structures. The application of the cis-BBP in the preparation of the product that inhibits the infection of cells by the virus having an envelope structure.

2. The application according to claim 1, characterized in that, The cis-BBP can target and disrupt viruses with envelope structures.

3. The application according to claim 1, characterized in that, The application environment for the cis-BBP includes temperatures ranging from -70°C to 25°C.

4. The application according to claim 1, characterized in that, The application environment for the cis-BBP includes under light conditions.

5. The application according to claim 1, characterized in that, The products include pharmaceuticals, disinfectants, or photodynamic inactivation reagents.

6. A method for inactivating viruses with envelope structures, characterized in that, The virus having an envelope structure is inactivated by photodynamic inactivation of the cis-BBP in any of the applications described in claims 1-5.

7. A product characterized in that, Using the cis-BBP as the active ingredient in any of the applications described in claims 1-5, the virus having an envelope structure is effectively inactivated.

8. The product according to claim 7, characterized in that, This includes drugs, disinfectants, or photodynamic inactivation agents.

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