A TRIF-loaded MOF nanomaterial and its preparation method and application

Abstract

The present invention discloses a MOF nanomaterial loaded with TRIF, a preparation method and an application thereof, and belongs to the field of biomedical technology. The present invention provides a MOF nanomaterial loaded with TRIF (TRIF@MOF), the preparation method of which is simple and the raw material source is wide. Experiments have shown that the TRIF@MOF cell of the present invention has high safety, and with MOF as a carrier, the presentation and controllable release of TRIF are realized, which can effectively inhibit the replication of H1N1 influenza virus and reduce the viral load of mouse lung tissue. At the same time, TRIF@MOF enhances the protective efficiency of the body and alleviates the symptoms such as lung inflammation and weight fluctuation in mice caused by H1N1 influenza virus infection. The TRIF@MOF provided by the present invention can be used to prepare drugs for treating H1N1 influenza virus infection, and has broad application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a MOF nanomaterial loaded with TRIF, and a preparation method and application thereof. Background Art

[0002] H1N1 is a highly contagious respiratory pathogen that poses a serious threat to global public health. It is crucial to prevent and control seasonal influenza epidemics and influenza pandemics. Traditional treatments mainly use antiviral drugs and vaccines, but due to the variability and drug resistance of the virus, the treatment effect is not ideal. Therefore, the development of new virus blocking strategies and treatment methods is of great significance for the prevention and treatment of H1N1 influenza virus. In recent years, as people have paid close attention to virus-infected controllable treatment systems, nanotherapeutic systems have received widespread attention due to their excellent targeting and controllability.

[0003] Whether a virus can survive and replicate inside a cell depends on the host's antiviral mechanism. Innate immunity is an important line of defense against the invasion of foreign pathogens formed by biological individuals during a long period of evolution, and plays an important role in the process of antiviral infection. It is generally triggered by the recognition of a wide range of pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs) by the body's pattern recognition receptors (PRRs), activating receptor-related intracellular signaling pathways, thereby inducing the expression of interferon (IFN) and inflammatory factors and producing antiviral effects. Among them, Toll-like receptors (TLRs) play an important role in sensing the invasion of pathogenic microorganisms, triggering cytokine secretion and promoting host cell defense. When TLR3 is activated by PAMP, it sends signals through the downstream adaptor protein TRIF (Toll-interleukin-1 receptor-domain-containing adaptor-inducing IFN-β) to activate protein kinases TBK1 / IKK-ε and IKK-α / IKK-β, which in turn activate the corresponding transcription regulators IRF3 and NF-κB, respectively, inducing the production of antiviral type I IFN (IFN-I) and pro-inflammatory cytokines, etc., to resist pathogen infection.

[0004] The study reported that TRIF-dependent TLR3 signaling is essential for reducing viral load in the central nervous system during West Nile virus infection. TRIF-mediated TLR3 signaling also plays a role in regulating the immune system and central nervous system diseases caused by viruses. The study found that TLR3-deficient mice were highly sensitive to EMCV and CVB3 viruses, showing suppressed cardiac proinflammatory responses and increased viral loads, and increased mortality. Similar impaired proinflammatory responses and pancreatic beta cell damage were observed in the pancreas of TLR3-deficient mice infected with mutant EMCV, leading to diabetes. TRIF-dependent signaling pathways are also important for efficient clearance of lung infection. Compared with wild-type mice, TRIF-deficient mice had reduced survival and increased bacterial loads in the lungs when infected with Klebsiella pneumoniae. Outer membrane vesicles secreted by Gram-negative bacteria induce antiviral immunity in macrophages through TRIF signaling to reduce viral replication in macrophages, alveolar epithelial cells, and lung tissue.

[0005] Influenza virus infection remains the leading cause of acute respiratory illness, posing a huge burden on public health. Symptoms associated with influenza virus infection range from mild to severe. Influenza virus infection can cause severe pneumonia and even acute respiratory distress syndrome, which is reported to be the main cause of death. Although vaccination is the most effective measure to prevent infection and its related complications. However, current seasonal influenza vaccines are designed to induce immune responses against the highly variable hemagglutinin (HA) head domain (HA1), thereby providing only strain-specific protection. Therefore, there is an urgent need to develop a safe, effective and easy-to-produce material with long-lasting cross-protection. Summary of the invention

[0006] The purpose of the present invention is to provide a TRIF-loaded MOF nanomaterial and a preparation method and application thereof to solve the problems existing in the above-mentioned prior art. The present invention uses MOF material as a carrier to enhance the presentation and sustained release of TRIF, realize the controlled release of drugs, and achieve the purpose of inhibiting viruses.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The present invention provides a method for preparing a TRIF-loaded MOF nanomaterial, comprising the following steps:

[0009] Mix the 2-methylimidazole solution and TRIF solution evenly, add Zn(NO3)2·6H2O solution dropwise, and stir at room temperature;

[0010] The mixture was centrifuged, the precipitate was collected, and vacuum dried to obtain the TRIF-loaded MOF nanomaterial.

[0011] Preferably, the mass ratio of the 2-methylimidazole to the TRIF is 33:1.

[0012] Preferably, the mass ratio of the Zn(NO3)2·6H2O to the TRIF is 24:1.

[0013] Preferably, the stirring time is 12 hours.

[0014] Preferably, the vacuum drying time is 24 hours.

[0015] The invention provides a TRIF-loaded MOF nano material prepared by the preparation method.

[0016] The present invention also provides the use of the TRIF-loaded MOF nanomaterial in preparing a medicine for treating influenza virus infection.

[0017] The present invention also provides the use of the TRIF-loaded MOF nanomaterial in preparing a drug for inhibiting influenza virus replication.

[0018] The present invention also provides the use of the TRIF-loaded MOF nanomaterial in preparing a drug for alleviating lung tissue inflammation caused by influenza virus infection.

[0019] Preferably, the influenza virus is an H1N1 influenza virus.

[0020] The present invention discloses the following technical effects:

[0021] The present invention provides a MOF nanomaterial loaded with TRIF, which has a simple preparation method and a wide range of raw materials. Experiments have shown that the TRIF@MOF cell of the present invention has high safety, uses MOF as a carrier, realizes the presentation and controllable release of TRIF, can effectively inhibit the replication of H1N1 influenza virus, and reduce the viral load of mouse lung tissue. At the same time, TRIF@MOF enhances the body's protective efficiency and alleviates symptoms such as lung inflammation and weight fluctuation in mice caused by H1N1 influenza virus infection. The TRIF@MOF provided by the present invention can be used to prepare drugs for treating H1N1 influenza virus infection, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0023] Figure 1 is the X-ray diffraction spectrum of TRIF@MOF; 2-theta is the incident angle of X-ray;

[0024] Figure 2 This is a scanning electron microscope image of TRIF@MOF; the scale length is 500nm;

[0025] Figure 3 This is a statistical chart of the effect of nanomaterials on cell activity; where Concentration is the concentration and CellViability is the cell activity;

[0026] Figure 4 The figure is a statistical chart of the daily weight gain of mice; where Days post infection is the number of days after injection, and DailyWeight gain is the daily weight gain;

[0027] Figure 5 Statistical graph of viral load in mouse lung tissue; Days post infection is the number of days after injection, Relative expression of M gene is the expression level of M gene; "**" indicates P<0.01;

[0028] Figure 6 This is the pathological section of lung tissue of mice in the PBS group;

[0029] Figure 7 This is a picture of the lung tissue pathological section of mice in the pcDNA3.1@MOF group;

[0030] Figure 8 Pathological sections of lung tissue of mice in the TRIF@MOF group. DETAILED DESCRIPTION

[0031] 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 should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0032] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0033] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. 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 associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0034] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0035] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0036] Example 1 Preparation and characterization of TRIF@MOF

[0037] Add 2-methylimidazole to 27.4 mg / mL in 0.3 mL of water and sonicate until dissolved; add 0.3 mL of 0.82 mg / mL TRIF solution, mix well, add 0.1 mL of 59.5 mg / mL Zn(NO3)2·6H2O aqueous solution dropwise, and stir at room temperature for 12 h.

[0038] After stirring, the precipitated product was collected by centrifugation and dried in vacuum for 24 h to finally obtain TRIF@MOF powder.

[0039] The XRD powder diffraction spectrum confirmed that TRIF@MOF has a high degree of crystallinity (e.g. Figure 1 As shown). Through scanning electron microscopy images, it was observed that TRIF@MOF is a nanoparticle with an average particle size of 200nm (as shown Figure 2 shown).

[0040] Example 2 Cytotoxicity Experiment

[0041] CCK-8 cell counting kit (Boster Biotechnology Co., Ltd., Wuhan) was used to detect cytotoxicity, and three parallel samples were taken from each group to eliminate experimental errors.

[0042] At 37°C, MDCK cells were treated with different concentrations of sterilized TRIF@MOF (12.5-200 μg / mL) for 24 h, with untreated MDCK cells as the control. Then, 10 μL of CCK-8 solution was added to each well and incubated for 30 min. Each group was expressed as the percentage of live cells relative to the control group. The results showed that within a concentration of 50 μg / mL, TRIF@MOF cells were highly safe (e.g. Figure 3 shown).

[0043] Example 3 In vivo experiment

[0044] 1. Experimental Methods

[0045] Eight-week-old female BALB / c mice were randomly divided into three groups (PBS group, pcDNA3.1@MOF group and TRIF@MOF group), with 6 mice in each group. At the beginning of the experiment, all mice in each group were intranasally inoculated with 80 μL H1N1. On the first and third days after inoculation, all mice in each group were subcutaneously injected with 100 μL PBS, 100 μL pcDNA3.1@MOF (concentration of 50 μg / mL), and 100 μL TRIF@MOF (concentration of 50 μg / mL) on the back.

[0046] The preparation method of pcDNA3.1@MOF is as follows: add 2-methylimidazole to 27.4 mg / mL in 0.3 mL of water, and ultrasonicate until dissolved; add 0.3 mL of pcDNA3.1 solution with a concentration of 0.35 mg / mL, mix evenly, dropwise add 0.1 mL of Zn(NO3)2·6H2O aqueous solution with a concentration of 59.5 mg / mL, and stir at room temperature for 12 hours. After stirring, centrifuge and collect the precipitate, vacuum dry for 24 hours, and finally obtain pcDNA3.1@MOF powder.

[0047] The mice were observed and weighed every day, and the deaths were recorded. In addition, three mice were randomly killed in each group on the 7th and 14th days after inoculation. A portion of the lung tissue was collected to detect the replication of the virus in the mice, and the other portion was stored in 4% paraformaldehyde for histopathological examination.

[0048] 2. Experimental results

[0049] 2.1 Daily weight change results

[0050] like Figure 4 As shown, the results showed that the daily body weight changes of virus-infected mice in the TRIF@MOF group were slowed down compared with those in the PBS group.

[0051] 2.2 Histopathological examination results

[0052] like Figure 6 , Figure 7and Figure 8 As shown, the results showed that the inflammation of lung tissues of mice in the TRIF@MOF group was milder compared with those in the PBS group and the pcDNA3.1@MOF group.

[0053] Example 4 RT-qPCR detection of viral load in mouse lung tissue

[0054] 1. Experimental Methods

[0055] The frozen lung tissue samples isolated in Example 3 were weighed and homogenized using a mortar and pestle to obtain 25-30 mg of tissue for total RNA extraction. Total RNA was extracted using an RNA kit according to the manufacturer's instructions. 500 ng of the isolated RNA was reverse transcribed into cDNA using a HiScript III RT SuperMix for qPCR kit, and the M gene transcription level was detected to examine H1N1 replication.

[0056] The primer sequences used are as follows:

[0057] H1N1-MF: 5'-TGATTCTTCTTGAAAATTTGCAG-3' (SEQ ID NO. 1);

[0058] H1N1-MR: 5'-CCGTAGAAGGCCCTCTTTTAA-3' (SEQ ID NO. 2).

[0059] 2. Experimental results

[0060] like Figure 5 As shown, on the 14th day after injection, the viral load in the lung tissue of mice in the TRIF@MOF group was significantly decreased compared with that in the PBS group and the pcDNA3.1@MOF group.

[0061] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for preparing a TRIF-loaded MOF nanomaterial, characterized in that: The following steps are involved: The 2-methylimidazole solution and the TRIF solution are mixed evenly, and a Zn(NO3)2·6H2O solution is added dropwise, stirred at room temperature, centrifuged, and the precipitate is collected and vacuum dried to obtain the TRIF-loaded MOF nanomaterial.

2. The preparation method according to claim 1, characterized in that The mass ratio of the 2-methylimidazole to the TRIF is 33:

1.

3. The preparation method according to claim 1, characterized in that: The mass ratio of the Zn(NO3)2·6H2O to the TRIF is 24:

1.

4. The preparation method according to claim 1, characterized in that: The stirring time is 12h.

5. The preparation method according to claim 1, characterized in that: The vacuum drying time is 24 hours.

6. A TRIF-loaded MOF nanomaterial prepared by the preparation method according to any one of claims 1 to 5.

7. Use of the TRIF-loaded MOF nanomaterial as claimed in claim 6 in the preparation of a drug for treating influenza virus infection.

8. Use of the TRIF-loaded MOF nanomaterial as claimed in claim 6 in the preparation of a drug for inhibiting influenza virus replication.

9. Use of the TRIF-loaded MOF nanomaterial according to claim 6 in the preparation of a drug for alleviating lung tissue inflammation caused by influenza virus infection.

10. The use according to any one of claims 7 to 9, characterized in that: The influenza virus is an H1N1 influenza virus.

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