Application of drug-loaded extracellular vesicles in the preparation of drugs for treating inflammation caused by viral infections

Abstract

This invention discloses the application of drug-loaded extracellular vesicles in the preparation of drugs for treating inflammation caused by viral infections, belonging to the field of biomedical technology. The drug comprises a protein fragment capable of competitively binding to a substrate by a protein expressed by the Trim21 gene. In vitro and in vivo experiments have demonstrated that mEVs / TRIM21-ΔRING can achieve precise targeting of macrophages in liver, lung, and kidney tissues, alleviating viral-induced inflammatory diseases and inhibiting viral infection by competitively binding to substrate proteins by TRIM21.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of drug-loaded extracellular vesicles in the preparation of drugs for treating inflammation caused by viral infections. Background Technology

[0002] Extracellular vesicles (EVs) are small vesicles with a lipid bilayer structure secreted by living cells, and can be divided into four subgroups: exosomes, extranuclear granules, apoptotic bodies, and cancer bodies. As a natural endogenous drug carrier, it has unique advantages: its diameter is at the nanometer level, allowing it to penetrate various barriers, including the blood-brain barrier; its complex protein and phospholipid bilayer structure better protects drugs from being cleared by the body; and it has low immunogenicity, meaning it does not induce an immune response in the host when used as a carrier to deliver drugs or medicinal proteins for treatment. Milk, as a natural and nutritious beverage, contains abundant exosomes and is closely related to our lives. According to literature reports, oral administration of milk-derived exosomes (mEVs) to mice can target multiple organs throughout the body, such as the liver, spleen, and lungs. Viral infections can damage multiple organs throughout the body, while milk-derived exosomes can serve as carriers to deliver drugs or small molecules that regulate the tissue immune microenvironment mediated by immune cells such as macrophages in various organs throughout the body, and can be used for targeted therapy or to alleviate viral infection-related inflammatory diseases.

[0003] Viral infections are a serious global problem threatening human health and life. Many large-scale viral outbreaks in recent years have been caused by RNA viruses, such as the common influenza virus. Because RNA viruses have a high mutation rate, vaccine development is difficult, posing a significant obstacle to disease prevention.

[0004] Antibodies and immune sera have long been used to treat pathogen infections. For example, equine antiserum was used to treat tetanus and diphtheria in the 1890s. However, antiserum is perceived as exogenous by the human immune system, which responds by producing antibodies against it, especially at repeated doses. For much of the 20th century, the side effects of animal antibodies spurred the use of human antiserum from donors who had recovered from disease, typically for the prevention of respiratory and hepatitis B infections. After the popularity of antibody therapy declined due to toxicity issues, humanization and human antibodies eliminated these problems and led to a resurgence of these treatments. See Casadevall et al., Nature Reviews Microbiology 2, 695-703 (September 2004) for a review. Diseases targeted by antibody therapy include anthrax, pertussis, cough, tetanus, botulism, cryptococcosis, cryptosporidiosis, enterovirus gastrointestinal infections, streptococcal infections, necrotizing fasciitis, hepatitis B, measles, tuberculosis, meningitis, aplastic anemia, rabies, RSV infection, pneumonia, herpes zoster, chickenpox and VZV-induced pneumonia, and smallpox. Despite these advancements, antibody therapy is only considered when no other suitable treatments exist, as it requires high doses of antibodies and can produce unpredictable results.

[0005] Viral infection is a highly contagious and rapidly spreading process with numerous complications. Therefore, finding a safe and effective strategy to quickly alleviate inflammatory diseases caused by viral infections is currently one of the hot topics in the research on the treatment of viral infection-related diseases. Summary of the Invention

[0006] The applicant's research found that TRIM21 molecules can inhibit the expression of antiviral infection functional proteins, thereby promoting viral immune evasion. By loading TRIM21 protein fragments, which can competitively bind to substrate proteins, onto milk-derived exosomes, viral infection can be inhibited by regulating the expression of antiviral infection functional proteins in macrophages of lung and liver tissues.

[0007] This invention provides the application of drug-loaded extracellular vesicles in the preparation of drugs for treating viral infection-related indicators, with the aim of finding a safe and effective strategy to alleviate viral infection-related diseases.

[0008] The specific technical solution is as follows:

[0009] This invention provides the application of the Trim21 gene or expressed protein as a target in the preparation of drugs for treating inflammation caused by viral infections.

[0010] Preferably, the virus is a VSV virus, an H1N1 virus, a SeV virus, or an RSV virus.

[0011] Indicators of viral infection inflammation include the viral load in various tissue pathological sections, serum or tissue, or the levels of interferon and various inflammatory factors in serum or tissue.

[0012] The present invention also provides a medicament for treating inflammation caused by viral infection, comprising a protein fragment capable of competitively binding a substrate to a protein expressed by the Trim21 gene.

[0013] Preferably, the protein fragment is a TRIM21 protein lacking the RING domain.

[0014] The nucleotide sequence of the human TRIM21 protein-coding gene is available in GenBank: NC 000011.10.

[0015] The present invention also provides an extracellular vesicle loaded with a drug, wherein the drug is the drug used to treat inflammation caused by viral infection.

[0016] Preferably, the extracellular vesicles used for drug delivery are exosomes, apoptotic bodies, carcinoma bodies, or extranuclear particles.

[0017] Specifically, the extracellular vesicles used to carry drugs are derived from the milk, blood, saliva, urine, or cerebrospinal fluid of humans or other mammals.

[0018] The present invention also provides the use of the drug-loaded extracellular vesicles in the preparation of medicaments for treating inflammation caused by viral infections.

[0019] Preferably, the virus is a VSV virus, an H1N1 virus, a SeV virus, or an RSV virus.

[0020] The present invention also provides a medicament for treating inflammation caused by viral infection, comprising extracellular vesicles carrying the drug.

[0021] The beneficial effects of this invention are:

[0022] Both in vivo and in vitro experiments of this invention have demonstrated that mEVs / TRIM21-ΔRING can achieve precise targeting of macrophages in liver, lung, and kidney tissues, and alleviate inflammatory diseases caused by viral infection and inhibit viral infection by competitively binding to substrate proteins of TRIM21. Attached Figure Description

[0023] Figure 1 Figure 1 shows the preparation and identification results of milk-derived exosomes loaded with TRIM21-ΔRING (mEVs / TRIM21-ΔRING); where A is the exosome morphology image detected by electron microscopy, and B is the exosome particle size image analyzed by NTA particle size distribution.

[0024] Figure 2 Figure 1 shows the preparation and identification results of milk-derived exosomes loaded with TRIM21-ΔRING (mEVs / TRIM21-ΔRING); A is a Western blotting graph of classic markers of milk exosomes, and B is a flow cytometry graph of the efficiency of milk exosomes loaded with TRIM21-ΔRING; "ns" indicates no statistical significance, and "***" indicates P < 0.001.

[0025] Figure 3 Figure 1 shows the in vitro targeting function validation results of mEVs / TRIM21-ΔRING; where A is a Western blot diagram of the interaction between TRIM21 and UBE2M in macrophages; B is a Western blot diagram of the K48 ubiquitination modification of UBE2M in macrophages; and C is a Western blot diagram of the expression of the macrophage protein UBE2M.

[0026] Figure 4 Figure A shows the results of the in vivo distribution verification of mEVs; Figure B shows the distribution of mEVs in various organs of mice after gavage, as detected by a small animal imaging analyzer; Figure C shows the uptake of mEVs by monocytes in mouse blood after gavage, as analyzed by flow cytometry.

[0027] Figure 5 Image showing morphological changes in the heart, liver, spleen, lungs, and kidneys after HE staining.

[0028] Figure 6 The graphs show the expression distribution of UBE2M and TRIM21-ΔRING in liver and lung macrophages in vivo. A represents the expression distribution of UBE2M and TRIM21-ΔRING in liver macrophages detected by immunofluorescence; B represents the expression distribution of UBE2M and TRIM21-ΔRING in lung macrophages detected by immunofluorescence; and C represents the correlation between the expression of UBE2M and TRIM21-ΔRING in macrophages. "*" indicates P < 0.05, and "***" indicates P < 0.001.

[0029] Figure 7 Figure A shows the results of mEVs / TRIM21-ΔRING in alleviating VSV virus infection-related diseases; Figure B shows the changes in lung tissue morphology detected by HE staining.

[0030] Figure 8The graph shows the results of mEVs / TRIM21-ΔRING in alleviating VSV virus infection-related diseases. A shows the viral load in lung, liver, and spleen tissues detected by RT-qPCR (left) and the serum IFN-β, IL-6, and TNF levels detected by ELISA (right). B shows the expression of Ifna and Ifnb in lung, liver, and spleen tissues detected by RT-qPCR. "ns" indicates no statistical significance, "*" indicates P < 0.05, "**" indicates P < 0.01, and "***" indicates P < 0.001.

[0031] Figure 9 Figure A shows the results of mEVs / TRIM21-ΔRING in alleviating H1N1 virus infection-related diseases; Figure B shows the changes in lung tissue morphology detected by HE staining.

[0032] Figure 10 Figure A shows the results of mEVs / TRIM21-ΔRING alleviating H1N1 virus infection-related diseases; Figure B shows the serum IFN-β, IL-6 and TNF levels detected by ELISA; Figure C shows the expression of Ifna and Ifnb in lung, liver and spleen tissues detected by RT-qPCR; “ns” indicates no statistical significance, “*” indicates P<0.05, and “**” indicates P<0.01.

[0033] Figure 11 Figure A shows the results of mEVs / TRIM21-ΔRING in alleviating RSV virus infection-related diseases; Figure B shows the histological changes in the lungs detected by HE staining; Figure C shows the serum IFN-β, IL-6 and TNF levels detected by ELISA, and the expression of Ifna and Ifnb in lung, liver and spleen tissues detected by RT-qPCR; “ns” indicates no statistical significance, “*” indicates P<0.05, and “**” indicates P<0.01.

[0034] Figure 12 Figure A shows the results of mEVs / TRIM21-ΔRING in alleviating SeV virus infection-related diseases; Figure B shows the histological changes in the lungs detected by HE staining; Figure C shows the serum IFN-β, IL-6 and TNF levels detected by ELISA, and the expression of Ifna and Ifnb in lung, liver and spleen tissues detected by RT-qPCR; “ns” indicates no statistical significance, “*” indicates P<0.05, and “**” indicates P<0.01. Detailed Implementation

[0035] The protein fragment TRIM21-ΔRING of TRIM21 (Gene ID: 20821 encoding gene sequence) was synthesized by General Biotechnology (Anhui) Co., Ltd., with glycosylphosphatidylinositol added to the C-terminus. The amino acid sequence of the synthesized protein fragment TRIM21-ΔRING is shown in SEQ ID NO.2, and the nucleotide sequence encoding the protein fragment TRIM21-ΔRING is shown in SEQ ID NO.1.

[0036] Example 1

[0037] Preparation and identification of milk-derived exosomes loaded with TRIM21-ΔRING (mEVs / TRIM21-ΔRING).

[0038] (1) Collect fresh raw milk and centrifuge it at 10000×g at 4℃ for 20 minutes to remove lipids;

[0039] (2) Mix the supernatant obtained by centrifugation with an equal volume of ddH2O, and acidify with 6M HCl until pH = 4.5;

[0040] (3) After centrifuging at 6000×g and 4℃ for 20 minutes twice, the supernatant was filtered through 0.45μm and 0.22μm sterile filters in sequence;

[0041] (4) The supernatant obtained by filtration was centrifuged at 100,000×g and 4℃ for 90 minutes, and the precipitate was washed once with sterile PBS. Finally, the precipitate was resuspended in an appropriate volume of sterile PBS to obtain milk-derived exosomes mEVs.

[0042] (5) The TRIM21 protein fragment TRIM21-ΔRING and mEVs were co-incubated at 37℃ for 3h, and the free protein was removed by washing and centrifugation to obtain milk-derived exosomes loaded with TRIM21-ΔRING (mEVs / TRIM21-ΔRING).

[0043] (6) The morphology and diameter of mEVs and mEVs / TRIM21-ΔRING were detected by electron microscopy and nanoparticle tracer analysis (NTA), respectively. The classical markers of extracellular vesicles were detected by Western blot, and the loading efficiency of TRIM21-ΔRING was detected by flow cytometry.

[0044] The results are as follows Figure 1 and Figure 2 As shown, the morphology, size, particle diameter, and expression of classical markers of mEVs and mEVs / TRIM21-ΔRING were identified, the loading efficiency of TRIM21-ΔRING was detected, and mEVs / TRIM21-ΔRING was successfully constructed.

[0045] Example 2

[0046] In vitro targeting functional validation of mEVs / TRIM21-ΔRING.

[0047] Macrophages were treated with mEVs and mEVs / TRIM21-ΔRING in vitro. Western blot was used to detect the effects on the interaction between TRIM21 and UBE2M in macrophages, the level of K48 ubiquitination of UBE2M in macrophages, and the expression of UBE2M protein in macrophages.

[0048] The results are as follows Figure 3 As shown, mEVs / TRIM21-ΔRING significantly inhibited the interaction between TRIM21 and UBE2M in macrophages in vitro; reduced the level of ubiquitination at K48 of UBE2M; and promoted the stability of UBE2M protein in macrophages.

[0049] Example 3

[0050] In vivo distribution verification of mEVs.

[0051] Mice were orally administered mEVs labeled with VivoTrack 680 once by gavage. 24 hours later, various organs and peripheral blood were collected. The distribution of mEVs in the organs was observed using a small animal imaging system; the uptake and phagocytosis of mEVs by peripheral blood mononuclear cells were analyzed by flow cytometry.

[0052] The results are as follows Figure 4 As shown, mEVs are mainly distributed in the gastrointestinal tract and liver after oral gavage, with a small portion also found in the lungs and kidneys; and blood mononuclear cells take up some mEVs.

[0053] Example 4

[0054] In vivo toxicity and side effects detection of mEVs / TRIM21-ΔRING.

[0055] Mice were given mEVs / TRIM21-ΔRING orally once by gavage. After 48 hours, tissues were taken, fixed, and stained with hematoxylin and eosin (HE) to observe whether mEVs / TRIM21-ΔRING caused damage to organs such as the heart, liver, spleen, lungs, and kidneys in vivo.

[0056] The results are as follows Figure 5 Oral administration of mEVs / TRIM21-ΔRING via gavage has no obvious toxic side effects on various tissues and organs.

[0057] Example 5

[0058] In vivo targeting functional validation of mEVs / TRIM21-ΔRING.

[0059] Mice were orally administered mEVs and mEVs / TRIM21-ΔRING once by gavage, and lung and liver tissues were collected and fixed 48 hours later. Immunofluorescence was used to detect the expression of macrophage UBE2M, macrophage marker F4 / 80, and TRIM21-ΔRING (with a tag) in the lungs and liver.

[0060] The results are as follows Figure 6 As shown, TRIM21-ΔRING can be delivered to the liver and lungs and taken up by macrophages in the tissues, ultimately increasing the expression level of UBE2M protein in macrophages.

[0061] Example 6

[0062] mEVs / TRIM21-ΔRING improves survival rate and reduces lung damage in mice infected with VSV virus.

[0063] Mice were infected with VSV virus via tail vein infusion. Three hours later, the mice were orally administered the same dose of mEVs and mEVs / TRIM21-ΔRING once by gavage, and the survival status of the mice was observed and recorded. Lung tissue from both groups of mice was fixed and lung damage was detected by HE staining.

[0064] The results are as follows Figure 7 As shown, oral administration of mEVs / TRIM21-ΔRING improved the survival rate of mice infected with VSV virus and reduced lung damage.

[0065] Example 7

[0066] mEVs / TRIM21-ΔRING reduces viral load in mice infected with VSV and promotes interferon production.

[0067] Mice were infected with VSV virus via tail vein infusion. Three hours later, the mice were orally administered the same dose of mEVs and mEVs / TRIM21-ΔRING once by gavage. Forty-eight hours later, peripheral blood, liver, lung, and spleen samples were collected. Serum IFN-β, IL-6, and TNF levels were detected by ELISA; viral load and Ifna and Ifnb expression in lung, liver, and spleen tissues were detected by RT-qPCR.

[0068] The results are as follows Figure 8 As shown, oral administration of mEVs / TRIM21-ΔRING reduced VSV viral load in mice and promoted the production of interferon and inflammatory factors.

[0069] Example 8

[0070] mEVs / TRIM21-ΔRING improves survival rate and reduces lung damage in mice infected with H1N1 virus.

[0071] Mice were infected with H1N1 via tail vein infusion. Three hours later, the mice were orally administered the same dose of mEVs and mEVs / TRIM21-ΔRING once by gavage, and the survival status of the mice was observed and recorded. Lung tissue from both groups of mice was fixed and lung damage was detected by HE staining.

[0072] The results are as follows Figure 9 As shown, oral administration of mEVs / TRIM21-ΔRING improved the survival rate of mice infected with H1N1 virus and reduced lung damage.

[0073] Example 9

[0074] mEVs / TRIM21-ΔRING promotes the production of interferon in mice induced by H1N1 virus infection.

[0075] Mice were infected with H1N1 virus via tail vein infusion. Three hours later, the mice were orally administered the same dose of mEVs and mEVs / TRIM21-ΔRING once. Forty-eight hours later, peripheral blood, liver, lung, and spleen samples were collected. Serum IFN-β, IL-6, and TNF levels were detected by ELISA; the expression of Ifna and Ifnb in lung, liver, and spleen tissues was detected by RT-qPCR.

[0076] The results are as follows Figure 10 As shown, oral administration of mEVs / TRIM21-ΔRING promoted the production of interferon and inflammatory factors.

[0077] Example 10

[0078] mEVs / TRIM21-ΔRING alleviated lung tissue damage in mice induced by RSV virus infection and promoted interferon production.

[0079] Mice were infected with RSV virus. Three hours later, the mice were orally administered the same dose of mEVs and mEVs / TRIM21-ΔRING once by gavage. Lung tissues from both groups of mice were fixed, and lung damage was detected by HE staining. Peripheral blood, liver, lungs, and spleen were collected. Serum IFN-β, IL-6, and TNF levels were detected by ELISA; the expression of Ifna and Ifnb in lung, liver, and spleen tissues was detected by RT-qPCR.

[0080] The results are as follows Figure 11 As shown, oral administration of mEVs / TRIM21-ΔRING reduced lung tissue damage induced by RSV virus infection in mice and promoted the production of interferon and inflammatory factors.

[0081] Example 11

[0082] mEVs / TRIM21-ΔRING alleviates lung tissue damage in mice induced by SeV virus infection and promotes interferon production.

[0083] Mice were infected with SeV virus. Three hours later, the mice were orally administered the same dose of mEVs and mEVs / TRIM21-ΔRING once by gavage. Lung tissues from both groups of mice were fixed, and lung damage was detected by HE staining. Peripheral blood, liver, lungs, and spleen were collected. Serum IFN-β, IL-6, and TNF levels were detected by ELISA; the expression of Ifna and Ifnb in lung, liver, and spleen tissues was detected by RT-qPCR.

[0084] The results are as follows Figure 12 As shown, oral administration of mEVs / TRIM21-ΔRING reduced lung tissue damage induced by SeV virus infection in mice and promoted the production of interferon and inflammatory factors.

Claims

1. The application of drug-loaded extracellular vesicles in the preparation of drugs for treating inflammation caused by viral infections, wherein the drug-loaded component comprises a protein fragment capable of competitively binding a substrate to a protein expressed by the Trim21 gene, wherein the protein fragment is a TRIM21 protein lacking the RING domain; The virus is VSV, H1N1, SeV, or RSV. The amino acid sequence of the protein fragment is shown in SEQ ID NO.

2.

2. The application as described in claim 1, characterized in that, Extracellular vesicles used for drug delivery include exosomes, apoptotic bodies, carcinoma bodies, or extranuclear particles.

3. The application as described in claim 1, characterized in that, Extracellular vesicles used to load drugs are derived from human or other mammalian milk, blood, saliva, urine, or cerebrospinal fluid.

4. A drug for treating inflammation caused by viral infection, characterized in that, The component comprises extracellular vesicles carrying a drug, wherein the drug-carrying component comprises a protein fragment capable of competitively binding a substrate to a protein expressed by the Trim21 gene, and the protein fragment is a TRIM21 protein lacking the RING domain. The amino acid sequence of the protein fragment is shown in SEQ ID NO.

2.

5. The medicament for treating inflammation caused by viral infection as described in claim 4, characterized in that, Extracellular vesicles used for drug delivery include exosomes, apoptotic bodies, carcinoma bodies, or extranuclear particles.

6. The medicament for treating inflammation caused by viral infection as described in claim 4, characterized in that, Extracellular vesicles used to load drugs are derived from human or other mammalian milk, blood, saliva, urine, or cerebrospinal fluid.

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