Preparation and application of exosome and nano-drug thereof

Abstract

The invention discloses preparation and application of an exosome and a nano-drug thereof, and aims to solve the technical problem that a current anti-herpes virus drug is difficult to effectively break through a blood brain barrier. According to the application, the exosome is separated and purified from a culture supernatant of an A549 cell infected by BoHV-1, and the exosome is used as a carrier to prepare a nano Acyclovir drug. The exosome prepared by the invention is safe and non-toxic, can effectively inhibit the binding of BoHV-1 and a cell receptor, and can deliver a drug to trigeminal ganglion and brainstem in a targeted manner; the prepared nanometer Acyclovir medicine can be used for efficiently inhibiting the replication of BoHV-1 virus in trigeminal nerves and brainstems, so that the infection of the virus is inhibited.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and specifically to the preparation and application of exosomes and nanomedicines thereof. Background Art

[0002] Exosomes are vesicular lipid bodies with a single-layer membrane structure secreted by a variety of cells. They are approximately 40 to 200 nm in diameter and usually contain a variety of active molecules such as nucleic acids and proteins. They are important media for information transmission between cells. Exosomes are widely distributed in various body fluids such as blood, urine and saliva, and play an important role in regulating various physiological functions of the body. A large number of studies have shown that exosomes are involved in regulating the infection of a variety of viruses, such as hepatitis B virus, hepatitis C, influenza virus H1N1 and the new coronavirus, and are involved in regulating tissue damage caused by the corresponding viral infections.

[0003] Nanomedicines developed based on exosomes have shown good therapeutic effects. For example, exosomes containing super-repressor IκB (srIκB) injected into the peritoneal cavity of mice can significantly inhibit LPS-induced inflammatory responses; exosomes isolated from COVID-19 infected people contain viral receptors AEC, which can significantly inhibit the infection of different mutant strains, and the exosomes were used to treat COVID-19 infected mice via nasal administration, showing good therapeutic effects. However, studies have shown that exosomes from different cells or tissues have different tissue cell binding characteristics, and natural exosomes usually do not have tissue binding specificity and need to be modified to have targeted binding characteristics.

[0004] Bovine herpesvirus type 1 (BoHV-1) belongs to the Herpesviridae family and the Varicellavirus genus. The virus mainly infects cattle, especially beef and dairy cattle, and can cause inflammatory responses in the respiratory, reproductive and nervous systems. BoHV-1 can not only cause abortion storms, but its replication process can also cause mucosal damage and immunosuppression, making cattle more susceptible to secondary infection with pathogenic microorganisms such as mycoplasma, thereby causing bovine respiratory disease syndrome (BRDC). After BoHV-1 infects cattle, it first replicates in the submucosal layer such as the conjunctiva and nasal cavity, and then invades tissues such as the trigeminal ganglion and brainstem 3-6 days later and continues to replicate (this process is called lytic infection). After that, the expression of genes related to lytic infection is inhibited and latent infection is formed. Latently infected cattle do not show any clinical symptoms, but the latently infected virus can be activated by various stress factors such as long-distance transportation, sudden weather changes and drug use, thereby producing infectious virions and causing disease. After that, the lytic infection is gradually suppressed and enters the latent infection state again. In addition, the end of the trigeminal ganglion is connected to the brainstem. After the virus infects the nasal cavity, it reaches the trigeminal neurons along the afferent neurons and replicates in large quantities. Some viruses can continue to move forward and reach the brainstem. Latent infection mainly occurs in the trigeminal ganglion and brainstem. After being activated, infectious virus particles are also first assembled from this part. Due to the protective effect of the blood-brain barrier, it is difficult for antiviral drugs to reach this part. Therefore, it is urgent to develop anti-herpes virus drugs that can effectively break through the blood-brain barrier to achieve effective treatment of the virus.

[0005] Acyclovir is an analog of acrylic purine nucleotides. Current studies have shown that the drug can effectively inhibit a variety of herpes viruses, including human herpes virus type 1, human herpes virus type 2, and varicella-zoster virus. The drug enters virus-infected cells and is phosphorylated by the virus-encoded protein TK protein to form monophosphate acyclo, namely acyclo-GMP. Acyclo-GMP is further phosphorylated by protein kinases in the cell to form triphosphate acyclo, namely acyclo-GTP. Studies have found that the content of acyclo-GTP in virus-infected cells is 40 to 100 times that in normal cells. Acyclo-GTP can specifically bind to and inhibit virally encoded DNA polymerases, but it has no obvious effect on cell-encoded DNA polymerases, so it has less cytotoxicity. However, studies have found that due to the protective effect of the blood-brain barrier, it is difficult for this type of drug to enter the trigeminal nerve and brainstem to exert its effects.

[0006] The information disclosed in this background technology section is only used to deepen the understanding of the background technology of the present disclosure, and should not be regarded as acknowledging or suggesting in any form that the information constitutes the prior art known to those skilled in the art. Summary of the invention

[0007] The inventors have found through long-term research that the exosomes secreted by A549 cells infected with BoHV-1 contain a large number of viral structural proteins and non-structural proteins, including all viral proteins (such as viral protein gD, etc.) that mediate the binding of viruses to cells; further research has found that the exosomes can specifically bind to cell receptors and can enter the trigeminal nerve and brainstem neurons through nasal and intravenous injection routes to play a role. Based on this, the present disclosure provides a preparation and application of exosomes and nanomedicines thereof, aiming to solve the technical problem that current anti-herpes virus drugs are difficult to effectively break through the blood-brain barrier.

[0008] According to a first aspect of the present disclosure, an exosome is provided, which is a lipid body or / and an analog thereof obtained by separating and purifying A549 cell metabolites after infection with BoHV-1 virus.

[0009] According to a second aspect of the present disclosure, the exosomes are used in any one of the following (1) to (6): (1) Inhibiting the binding of BoHV-1 virus to cell receptors or preparing products that inhibit the binding of BoHV-1 virus to cell receptors; (2) Inhibiting the replication of BoHV-1 virus or preparing products that inhibit the replication of BoHV-1 virus; (3) Treating BoHV-1 virus infection or preparing products for treating BoHV-1 virus infection; (4) Drug products that cross the blood-brain barrier or are prepared to cross the blood-brain barrier; (5) Inducing the expression of interferon-alpha protein and / or mRNA or preparing products that induce the expression of interferon-alpha protein and / or mRNA; (6) Products formed in combination with Acyclovir.

[0010] According to a third aspect of the present disclosure, a method for preparing the above-mentioned exosomes is provided, comprising the following steps: (1) Cultivate A549 cells to a cell density of 70-80%, and add BoHV-1 virus solution to the A549 cells at an infection dose of 1-2 for infection treatment; (2) After the infection, the BoHV-1 virus solution was discarded and the residual virus was washed away with PBS, and then serum-free DMEM was added and cultured for 45-55 h; (3) After the culture is completed, the cell supernatant is collected and centrifuged at 3500-4500 rpm at 3-5°C to remove cell debris; then centrifuged at 15,000-25,000 rpm at 3-5°C to remove virions, and the supernatant is collected after the centrifugation is completed; (4) Mix 3×PEG8000 and 3×NaCl solution with the supernatant in a volume ratio of 1:0.8~1.2:0.8~1.2 to form a mixed solution, and then shake the mixed solution at 3~4°C for 10~12 h; (5) The mixture obtained after the shaking in the previous step was centrifuged at 16,000-18,000 g at 3-5°C, the precipitate was collected, and PBS was added thereto for resuspending to obtain a resuspension, and then the resuspension was centrifuged at 16,000-18,000 g at 3-5°C to remove PEG8000 and NaCl; (6) After removing PEG8000 and NaCl, the precipitate is resuspended in PBS or physiological saline and then stored at -70 to -90°C.

[0011] According to a fourth aspect of the present disclosure, a product containing or compounded with the above-mentioned exosomes is provided, wherein the product has at least one of the following functions (1) to (5): (1) Inhibit the binding of BoHV-1 virus to cell receptors; (2) Inhibit BoHV-1 virus replication; (3) Treatment of BoHV-1 virus infection; (4) Crossing the blood-brain barrier; (5) Induce the expression of alpha interferon protein and / or mRNA.

[0012] In some embodiments of the present disclosure, the product containing or compounded with the above-mentioned exosomes is a nano-Acyclovir drug formed by compounding the exosomes with Acyclovir.

[0013] According to a fifth aspect of the present disclosure, a method for preparing a nano-Acyclovir drug is provided, comprising the following steps: (1) mixing the exosomes, PBS solution and Acyclovir solution in a volume ratio of 1:45-50:45-50 to obtain a mixed solution; (2) subjecting the mixed solution to ultrasonic treatment; (3) Incubate the ultrasonic mixed solution at 35-40°C. After the incubation, separate it by ultracentrifugation at 13,000-17,000g at 2-4°C and discard the supernatant.

[0014] In some embodiments of the present disclosure, in step (1), the concentration of exosomes is 8-12 mg / mL; the concentration of Acyclovir solution is 48-52 μM.

[0015] In some embodiments of the present disclosure, in step (2), the ultrasonic treatment process is: run for 25 to 30 s, stop for 25 to 30 s, continue for 2.5 to 3 min, and then cool for 1 to 2 min. The above operation process is regarded as one cycle, and 5 to 6 cycles are performed.

[0016] One or more technical solutions provided in the embodiments of the present application have at least any of the following technical effects or advantages: 1. The method for extracting and purifying exosomes from the supernatant of A549 cells infected with BoHV-1 is simple and efficient, and the extraction efficiency is higher than that of commercial kits; the prepared exosomes are safe, non-toxic, and non-infectious to humans. They can effectively inhibit the binding of BoHV-1 to cell receptors by specifically binding to cell receptors, and the exosomes can deliver drugs to the trigeminal ganglion and brainstem, which is suitable for drug development across the blood-brain barrier.

[0017] 2. The prepared nano-Acyclovir drug can effectively inhibit the replication of BoHV-1 virus in the trigeminal nerve and brainstem, thereby inhibiting the infection of the virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The extraction efficiency of the exosome extraction method described in one embodiment of the present application is compared with that of the imported kit (Invitrogen, cat#4478359).

[0019] Figure 2 In one embodiment of the present application, proteomics is used to determine the BoHV-1 encoded protein contained in exosomes.

[0020] Figure 3 In one embodiment of the present application, the labeled exosomes compete with the virions for binding to the viral receptors on the surface of MDBK cells.

[0021] Figure 4 In one embodiment of the present application, exosomes are used to deliver siRNA to MDBK cells.

[0022] Figure 5 In one embodiment of the present application, exosomes are injected into the trigeminal nerve and brainstem via the nasal cavity and intravenous route.

[0023] Figure 6 The inhibitory effect of nano-Acyclovir on BoHV-1 infection is evaluated at the cellular level in one embodiment of the present application.

[0024] Figure 7 In one embodiment of the present application, New Zealand white rabbits were used as a model to evaluate the therapeutic results of nano-Acyclovir on BoHV-1 infection in vivo. DETAILED DESCRIPTION

[0025] In order to better understand the technical solution of the present application, the above technical solution will be described in detail below in conjunction with the accompanying drawings and specific implementation methods.

[0026] Unless otherwise specified, the instruments and equipment involved in the following embodiments are all conventional instruments and equipment; the reagents involved are all commercially available conventional reagents unless otherwise specified; the detection and preparation methods involved are all conventional methods unless otherwise specified.

[0027] Example 1. Efficient preparation of exosomes and detection of their effects This example discloses a method for separating and purifying exosomes from the supernatant of A549 cells infected with BoHV-1, that is, separating the virions and exosomes by ultracentrifugation, then precipitating with PEG8000 overnight, and finally obtaining the exosomes after ultracentrifugation. The specific steps are as follows: (1) A549 cells were inoculated in a 10 cm cell culture dish. When the cell density reached 70%~80%, BoHV-1 virus solution was added at an infection dose of MOI of 1~2. After 2 h of infection, the virus solution was removed, the residual virus was washed off with PBS, and serum-free DMEM was added for 48 h.

[0028] (2) After the culture was completed, the cell supernatant was collected and centrifuged at 4000 rpm at 4°C for 20 min to remove cell debris.

[0029] (3) Centrifuge at 20,000 rpm for 60 min at 4°C to remove the virions. The virions will precipitate and adhere tightly to the bottom of the centrifuge tube. Collect the supernatant.

[0030] (4) Mix the prepared 3×PEG8000, 3×NaCl and the supernatant in a ratio of 1:1:1 and shake horizontally at 4°C for 10 to 12 h.

[0031] (5) The mixture was centrifuged at 17,000 g for 40 min at 4°C, the precipitate was collected, and 1 mL of PBS was added thereto for resuspending to obtain a resuspension, and the resuspension was then centrifuged at 17,000 g for 40 min at 4°C to remove PEG8000 and NaCl.

[0032] (6) After removing PEG8000 and NaCl, resuspend the precipitate in 1 mL of PBS or physiological saline, and then store it at -70 to -90°C.

[0033] The extraction efficiency of the method described in this example was tested by extracting exosomes from the supernatant using an imported commercial exosome isolation kit as a control. The results are as follows Figure 1 As shown, the results show that the exosome extraction efficiency of the method described in this example is higher than that of the imported kit.

[0034] The isolated and purified exosomes were sent to a biotechnology company for proteomics analysis, and the obtained peptide sequences were compared with the protein library encoded by the virus to determine the BoHV-1 viral protein contained in the exosomes (the obtained reading is the relative content of the corresponding viral protein encoded by the virus in the exosomes). Figure 2 As shown, the exosomes contain 59 proteins encoded by the virus, including viral proteins gC and gD that can bind to cell receptors.

[0035] Further, the inhibitory effect of the prepared exosomes on the binding of viruses to cell receptors is detected, and the specific steps are as follows: (1) MDBK cells were seeded in 6-well plates. When the cell density reached 90%, different concentrations of exosomes (50 μg / mL, 150 μg / mL, and 300 μg / mL) were added and incubated at 4°C for 1 h.

[0036] (2) Discard the supernatant and add ice-cold virus diluted with PBS to the cells and incubate at 4°C for 1 h.

[0037] (3) Discard the viral supernatant and add ice-cold PBS to the cells and wash three times.

[0038] (4) Use a whole genome extraction kit to extract the viral and cellular genomes.

[0039] (5) Fluorescence quantitative PCR was used to detect the viral genome (target genes were gC and gD), and the cell genome was used as an internal reference to compare and analyze the effect of exosome treatment on the binding of the virus to the corresponding receptor. The exosomes secreted by A549 cells that were not infected with BoHV-1 virus were treated in the same way (control group). Among them, the exosomes isolated from virus-infected A549 were represented by EDV, and the exosomes secreted by uninfected A549 cells were represented by EDM.

[0040] The results are as follows Figure 3 As shown, when the exosome EDV concentration is 150 μg / mL, the binding of the virus to the cells can be slightly reduced, and when the exosome EDV concentration is 300 μg / mL, the binding of the virus to the cells can be significantly reduced, indicating that the exosomes prepared in this example can significantly inhibit the binding of the virus to the cell receptors.

[0041] Example 2: Feasibility analysis of exosomes as nanodrug carriers In this example, the exosomes obtained in Example 1 were labeled with the dye Dil, and in vitro and in vivo experiments were used to evaluate whether the exosomes were suitable as carriers for nanodrug delivery.

[0042] 1. Delivery of siRNA to MDBK cells using exosomes (1) At room temperature, the exosomes obtained in Example 1 were labeled with Dil.

[0043] (2) The labeled exosomes encapsulate siRNA labeled with the fluorescent dye FAM (siRNA-FAM).

[0044] (3) Add MDBK cells to the cell slide in a 24-well plate. After the cells grow into a monolayer, add the siRNA labeled with the fluorescent dye FAM prepared above.

[0045] (4) After 1.5 h and 24 h of treatment, the cell slides were removed, fixed with 4% paraformaldehyde, and the cell nuclei were stained with DAPI.

[0046] (5) Observe and take photos under a laser confocal microscope.

[0047] The results are as follows Figure 4 As shown, fluorescence signals can be detected at 1.5 h and 24 h of action, and the FAM green fluorescence signal is distributed in the cytoplasm and co-localized with the labeled Dil (red fluorescence signal), indicating that the exosomes successfully delivered RNA into the cells.

[0048] 2. Detection of exosomes entering the trigeminal nerve and brainstem The exosomes labeled with the dye Dil are delivered into the trigeminal nerve and brainstem via ocular and nasal application and intravenous injection. The specific steps are as follows: (1) At room temperature, exosomes are labeled with Dil.

[0049] (2) Animal experiments were conducted using the New Zealand white rabbit as a model animal: Dil-labeled exosomes were applied via the eye and nasal cavity every 30 min. The New Zealand white rabbits were anesthetized and killed 5 h after treatment, and the trigeminal nerve and brainstem tissues were immediately collected.

[0050] (3) Prepare the collected tissue into frozen paraffin sections.

[0051] (4) Observe under a microscope whether the neurons in the trigeminal nerve and brainstem are stained with Dil-labeled exosomes.

[0052] The results are as follows Figure 5As shown, the labeled Dil fluorescence signal can be detected in the trigeminal ganglion (TG on the left) and the brainstem (Brain stem on the right), indicating that after ocular and nasal application and intravenous injection, some exosomes have been successfully delivered to the trigeminal ganglion and brainstem within 5 hours of action, indicating that the exosomes can be used as a potential drug delivery carrier to deliver specific drugs to the trigeminal ganglion and brainstem.

[0053] Therefore, the exosomes prepared in the present application can be used to prepare nanomedicines, achieve targeted delivery to the trigeminal ganglion and brainstem, specifically inhibit the replication of BoHV-1 in the corresponding neurons, and can be used as an effective means or strategy for treating lytic infections or activated latent infections caused by the virus.

[0054] Example 3: Preparation of Nano-Acyclovir Drug and Detection of Its Effect This example provides a method for preparing a nano Acyclovir drug using the exosomes obtained in Example 1 as a carrier, and uses in vivo and in vitro experiments to detect the inhibitory effect of the nano drug on the BoHV-1 virus.

[0055] 1. Preparation of Nanoparticle Acyclovir (1) The exosomes (10 mg / mL) prepared in Example 1, PBS solution and Acyclovir solution (50 μM) were mixed in a volume ratio of 1:49:50 to obtain a mixed solution.

[0056] (2) The mixed solution was subjected to ultrasonic treatment with an amplitude of 20%. The ultrasonic treatment process was as follows: 30 s on, 30 s off, and continued for 3 min, followed by cooling for 2 min. The above operation process was regarded as one cycle, and six cycles were performed.

[0057] (3) After the ultrasound is completed, the mixed solution is taken out and incubated at 37°C for 6 min to allow the exosome membrane to fully recover. After the incubation, it is ultracentrifuged at 4°C and 15,000g for 95 min, and the supernatant is discarded.

[0058] (4) High performance liquid chromatography was used to determine that Acyclovir had been loaded into exosomes.

[0059] 2. Evaluation of the inhibitory effect of nano-Acyclovir on BoHV-1 replication using MDBK cells (1) Add MDBK cells to a 24-well plate and culture overnight until the cells form a monolayer.

[0060] (2) MDBK cells were infected with BoHV-1. After 2 h of infection, the supernatant was discarded and the cells were washed twice with PBS. Then, 25 μg / mL Acyclovir (ACY) and 25 μg / mL Acyclovir encapsulated in exosomes (EDV-ACY) were added to treat the cells. Cells treated with exosomes secreted by cells not infected with the virus were used as controls.

[0061] (3) 24 h after infection, the cell culture plate and the culture medium were placed in a -80°C refrigerator and frozen and thawed twice. The cells were centrifuged at 12,000 rpm for 5 min and the cell supernatant was collected to determine the TCID 50 To determine the change in viral titer, the TCID of the obtained viral titer was calculated. 50 Get log 10 .

[0062] The results are as follows Figure 6 As shown in the figure, although 25 μg / mL of Acyclovir (ACY) cannot effectively inhibit viral replication, the prepared nanomedicine can produce a good inhibitory effect after being encapsulated by exosomes, indicating that the encapsulation enhances the antiviral effect of the drug ACY and achieves the expected purpose. In addition, the exosomes can also induce the expression of alpha interferon protein and mRNA, which may be an important molecular mechanism for enhancing the antiviral effect. This characteristic also gives the exosomes a natural advantage as a delivery carrier for antiviral or anti-tumor drugs.

[0063] 3. In vivo evaluation of the therapeutic outcome of nano-Acyclovir against BoHV-1 infection The New Zealand white rabbit model was used to evaluate the therapeutic effect of nano-Acyclovir on BoHV-1 infection in vivo. The specific steps were as follows: (1) Prepare nano-Acyclovir (Exo-ACY) according to the method in Example 3-1.

[0064] (2) Animal experiments were conducted using New Zealand white rabbits as model animals: Based on the principle of minimizing the number of experimental animals, the animals were divided into two groups, with 4 in each group. The control group was treated with untreated Acyclovir (ACY). The animals were infected with New Zealand white rabbits through the eyes and nose, and the infection dose was 10 7 PFU / mL, 2 days after infection, treatment was carried out through the eyes and nasal cavity. Both Exo-ACY and ACY were used by intravenous injection, once a day, with a dosage of 300 μg, for 3 consecutive days.

[0065] (3) After the animal is anesthetized and killed, the trigeminal nerve and brain stem can be removed and mixed, and the viral content can be measured using fluorescent quantitative PCR.

[0066] (4) Data analysis: The conservative gene GAPDH was used as an internal reference and the relative quantitative calculation formula was used. -ΔΔCt The control group was artificially set as 100%, and the fold change of viral genome after drug Exo-ACY treatment relative to the control was expressed as a percentage.

[0067] The results are as follows Figure 7 As shown, the results showed that the drug delivered by the exosomes can effectively reduce the level of viral replication in the trigeminal ganglion and brainstem. Quantitative analysis found that if the viral genome copies measured after ACY treatment were set to 100% and used as a control, the number of viral copies was reduced to 38.82% after treatment with the drug encapsulated in exosomes, indicating that during in vivo drug treatment, the exosome-encapsulated drug can still enhance the antiviral effect.

[0068] Although some preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present application.

[0069] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the inventive concept. Thus, if these modifications and variations of the present application fall within the scope of the claims of the present application and their equivalents, the present application is also intended to include these modifications and variations.

Claims

1. An exosome, characterized in that: It is a lipid body or / and its analogue obtained by separation and purification of A549 cell metabolites after infection with BoHV-1 virus.

2. Use of the exosomes according to claim 1 in any one of the following (1) to (6): (1) Inhibiting the binding of BoHV-1 virus to cell receptors or preparing products that inhibit the binding of BoHV-1 virus to cell receptors; (2) Inhibiting the replication of BoHV-1 virus or preparing products that inhibit the replication of BoHV-1 virus; (3) Treating BoHV-1 virus infection or preparing products for treating BoHV-1 virus infection; (4) Drug products that cross the blood-brain barrier or are prepared to cross the blood-brain barrier; (5) Inducing the expression of interferon-alpha protein and / or mRNA or preparing products that induce the expression of interferon-alpha protein and / or mRNA; (6) Products formed in combination with Acyclovir.

3. The method for preparing exosomes according to claim 1, characterized in that: The following steps are involved: (1) Cultivate A549 cells to a cell density of 70-80%, and add BoHV-1 virus solution to the A549 cells at an infection dose of 1-2 for infection treatment; (2) After the infection, the BoHV-1 virus solution was discarded and the residual virus was washed away with PBS, and then serum-free DMEM was added and cultured for 45-55 h; (3) After the culture is completed, the cell supernatant is collected and centrifuged at 3500-4500 rpm at 3-5°C to remove cell debris; Then centrifuge at 15,000-25,000 rpm at 3-5°C to remove the virus particles, and collect the supernatant after centrifugation; (4) Mix 3×PEG8000 and 3×NaCl solution with the supernatant in a volume ratio of 1:0.8~1.2:0.8~1.2 to form a mixed solution, and then shake the mixed solution at 3~4°C for 10~12 h; (5) The mixture obtained after the shaking in the previous step was centrifuged at 16,000-18,000 g at 3-5°C, the precipitate was collected, and PBS was added thereto for resuspending to obtain a resuspension, and then the resuspension was centrifuged at 16,000-18,000 g at 3-5°C to remove PEG8000 and NaCl; (6) After removing PEG8000 and NaCl, the precipitate is resuspended in PBS or physiological saline and then stored at -70 to -90°C.

4. A product containing or compounded with the exosomes according to claim 1, which has at least one of the following functions (1) to (5): (1) Inhibit the binding of BoHV-1 virus to cell receptors; (2) Inhibit BoHV-1 virus replication; (3) Treatment of BoHV-1 virus infection; (4) Crossing the blood-brain barrier; (5) Induce the expression of alpha interferon protein and / or mRNA.

5. The product according to claim 4, characterized in that The nanometer Acyclovir drug is formed by compounding the exosomes and Acyclovir.

6. A method for preparing a nano-Acyclovir drug, characterized in that: The following steps are involved: (1) Mixing the exosomes described in claim 1, PBS solution and Acyclovir solution in a volume ratio of 1:45-50:45-50 to obtain a mixed solution; (2) subjecting the mixed solution to ultrasonic treatment; (3) Incubate the ultrasonic mixed solution at 35-40°C. After the incubation, separate it by ultracentrifugation at 13,000-17,000g at 2-4°C and discard the supernatant.

7. The preparation method according to claim 6, characterized in that: In the step (1), the concentration of exosomes is 8-12 mg / mL; the concentration of Acyclovir solution is 48-52 μM.

8. The preparation method according to claim 6, characterized in that: In step (2), the ultrasonic treatment process is: run for 25 to 30 seconds, stop for 25 to 30 seconds, continue for 2.5 to 3 minutes, and then cool for 1 to 2 minutes. The above operation process is regarded as one cycle, and 5 to 6 cycles are performed.

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