Nanoparticle HA-ADH-GL for inhibiting influenza virus as well as preparation method and application of nanoparticle HA-ADH-GL

Abstract

The invention belongs to the field of medicine, and particularly relates to a nanoparticle HA-ADH-GL for inhibiting influenza virus as well as a preparation method and application of the nanoparticle HA-ADH-GL. The nanoparticle is a HA-ADH-GL conjugate synthesized by connecting glycyrrhizic acid (GL) with hyaluronic acid (HA) and a linker adipic dihydrazide (ADH), wherein the hyaluronic acid (HA) and the linker are good in biocompatibility and hydrophilicity. The nanoparticles can inhibit influenza virus replication and cytokine storm. The preparation method of the nanoparticles is simple, the cost is low, the water solubility of glycyrrhizic acid is improved, no biological safety risk and other problems exist, and reference is provided for research and development of antiviral drugs in the future.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and particularly relates to nanoparticles HA-ADH-GL for inhibiting influenza virus, and a preparation method and application thereof. Background Art

[0002] Influenza viruses belong to the Orthomyxoviridae family and have an envelope structure. They possess a genome consisting of eight single-stranded, negative-sense RNA segments encoding 17 proteins, including hemagglutinin (HA), neuraminidase (NA), matrix protein (M1), nucleoprotein (NP), polymerase basic protein 1 (PB1), polymerase basic protein 2 (PB2), polymerase acidic protein (PA), nonstructural protein (NS1), and nuclear transport protein (NEP). This genome is associated with the viral RNA-dependent RNA polymerase (RdRp) and viral nucleoprotein (NP), forming a viral ribonucleoprotein (vRNP) complex. Influenza viruses are classified into four types: A (A), B (B), C (C), and D (D), based on the antigenic properties of their NP and matrix protein (M1). Influenza A and B viruses are particularly prone to causing pandemics. Influenza A viruses are classified into subtypes based on the structure of their surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA), with H1N1 and H3N2 subtypes being the most common.

[0003] The influenza virus life cycle includes attachment, entry and uncoating, replication and transcription, assembly and release. First, influenza A virus attaches to the host cell surface sialic acid receptor through its surface glycoprotein HA1, and then activates extracellular Ca 2+ The virus enters the host cell through clathrin-mediated endocytosis. The M2 ion channel mediates internal acidification of the influenza virus, causing the matrix protein M1 to dissolve at a pH of 5.5-6.0. Under the acidic conditions of the late endosome, hemagglutinin (HA2) undergoes an irreversible conformational change, exposing the fusion peptide and activating fusion of the viral envelope with the endosomal membrane, releasing the vRNP into the cytoplasm. NP, through its nuclear localization signal (NLS), mediates the entry of the vRNP complex into the nucleus. Replication and transcription occur in the nucleus, and the transcribed mRNA is exported to the nucleus. Protein translation occurs in ribosomes, followed by protein processing in the endoplasmic reticulum and Golgi apparatus, and transported to the cell membrane. The processed NP, PA, PB1, and PB2 proteins enter the nucleus, assemble with the replicated viral RNA into vRNP, and are exported from the nucleus. At the cell membrane, viral proteins such as HA, NA, and M1 are assembled, and the virion buds off of the vesicle. The progeny virions are released by cleavage of the terminal sialic acid residues from the surface NA glycans.

[0004] Due to the continuous evolution of influenza A viruses, chemically synthesized drugs may develop antiviral resistance during or after treatment. There is an urgent need to develop drugs that can replace or supplement the antiviral effects of small molecule chemically synthesized drugs. Natural polymers are gradually being recognized due to their good biocompatibility and certain antiviral effects. Summary of the Invention

[0005] To improve the water solubility of glycyrrhizic acid (GL) and enhance its antiviral effects, the present invention provides a HA-ADH-GL conjugate as a nanoparticle for inhibiting influenza viruses. The HA-ADH-GL conjugate provided by the present invention is inexpensive and has promising development and application prospects. It aims to enhance the anti-influenza virus effects of natural chemical products, leveraging the advantages of natural products to inhibit influenza viruses.

[0006] In order to achieve the above object, the present invention specifically adopts the following technical solutions:

[0007] The influenza virus-inhibiting nanoparticles HA-ADH-GL provided by the present invention are HA-ADH-GL conjugates obtained by linking the natural product GL with hyaluronic acid (HA), which has excellent biocompatibility and hydrophilicity, via the water-soluble bifunctional cross-linking reagent adipic acid dihydrazide (ADH). Specifically, the nanoparticles HA-ADH-GL are a conjugate formed by linking GL and HA via ADH.

[0008] A method for preparing nanoparticles HA-ADH-GL for inhibiting influenza virus comprises the following steps:

[0009] Step 1: Synthesis of hyaluronic acid-adipic acid dihydrazide (HA-ADH):

[0010] The hyaluronic acid solution after activation of the carboxyl groups was reacted with adipic acid dihydrazide at room temperature under inert gas protection to obtain hyaluronic acid-adipic acid dihydrazide. Furthermore, the reaction time was 2 hours. The molar ratio of the hyaluronic acid to adipic acid dihydrazide was 0.5:1.58.

[0011] The method for activating the carboxyl group of hyaluronic acid comprises:

[0012] A hyaluronic acid solution is reacted with 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide at 0°C under an inert gas atmosphere to activate the carboxyl groups on the hyaluronic acid. Furthermore, the reaction time is 30 minutes; the hyaluronic acid solution is an aqueous hyaluronic acid solution. The molar ratio of the hyaluronic acid to 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide is 0.5:0.63:0.63.

[0013] Step 2: Synthesis of hyaluronic acid-adipic acid dihydrazide-glycyrrhizic acid (HA-ADH-GL):

[0014] The carboxyl-activated glycyrrhizic acid was added dropwise to hyaluronic acid-adipic acid dihydrazide, and the mixture was reacted at room temperature under an inert gas atmosphere to produce hyaluronic acid-adipic acid dihydrazide-glycyrrhizic acid nanoparticles. The reaction time was 12 hours or longer. The molar ratio of hyaluronic acid-adipic acid dihydrazide to glycyrrhizic acid was 0.61:0.64.

[0015] The method for activating the carboxyl group of glycyrrhizic acid comprises:

[0016] A glycyrrhizic acid solution was reacted with 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide at 0°C under an inert gas atmosphere to activate the carboxyl groups on the glycyrrhizic acid. Furthermore, the reaction time was 30 minutes; the glycyrrhizic acid solution was a DMSO solution of glycyrrhizic acid. The molar ratio of glycyrrhizic acid to 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide was 1:1:1.

[0017] The purification steps of the reaction solution of the above step 1 and step 2 are as follows: the reaction solution is dialyzed and then centrifuged, the supernatant is collected and freeze-dried.

[0018] Subsequently, we evaluated the inhibitory effects of the nanoparticles HA-ADH-GL on influenza virus infection through both in vitro and in vivo experiments. The experimental results demonstrated that the nanoparticles reduced the cytotoxicity of glycyrrhizic acid in vitro, increased its water solubility, and inhibited influenza virus infection in a dose-dependent manner. The nanoparticles effectively improved the survival rate of mice infected with the virus, improved lung lesions after infection, effectively inhibited inflammatory factors, and reduced viral NP levels in the lungs. Therefore, the present invention provides the use of the nanoparticles HA-ADH-GL in the preparation of a drug for treating influenza virus infection.

[0019] As an inventive point of the present invention, the nanoparticle HA-ADH-GL can reduce the viral titer in lung tissue and nasal turbinates, and reduce the level of viral protein and viral M gene in the lungs.

[0020] The beneficial effects of the present invention are:

[0021] This invention uses hyaluronic acid (HA) as a drug carrier to prepare HA-adipic acid dihydrazide (ADH). This overcomes the technical bottlenecks that limit the clinical application of glycyrrhizic acid (GL) due to its poor solubility and low absorption rate, significantly enhancing the antiviral efficacy of GL. This provides a new, highly effective and safe formulation for the treatment of influenza virus, laying a solid foundation for the industrial production and clinical application of GL. It also provides new ideas and methods for natural product drug delivery technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 , synthesis route of self-assembled nanoparticles HA-ADH-GL.

[0023] Figure 2 , (A)-(D) are the H NMR spectra (500 MHz) of HA(D2O), HA-ADH(D2O), HA-ADH-GL(D2O), and GL(DMSO-d6), respectively.

[0024] Figure 3 (A) is the infrared spectra of self-assembled nanoparticles GL, HA-ADH-GL, HA-ADH, and HA. (B) is the UV spectra of self-assembled nanoparticles HA-ADH-GL and GL.

[0025] Figure 4 (A)-(C) are the transmission electron micrographs of HA-ADH-GL and the local magnified images, particle size images, and potential images, respectively.

[0026] Figure 5 (A) and (B) are the cytotoxicity of MDCK cells incubated with different drugs for 24 h and 48 h, respectively.

[0027] Figure 6 , cytotoxicity of MDCK cells incubated with HA-ADH-GL for 48 hours and inhibition rate of influenza virus infection.

[0028] Figure 7 、100 times TCID 50 Immunofluorescence image of H1N1-challenged MDCK cells treated with HA-ADH-GL for 24 hours.

[0029] Figure 8 , (A) and (B) are H1N1 (5 times MLD 50 ) Body weight and survival rate of mice challenged with HA-ADH-GL on day 14 after treatment with different administration methods.

[0030] Figure 9 , (A) and (B) are H1N1 (3 times MLD 50 ) Body weight and survival rate of challenged mice 14 days after oral gavage treatment.

[0031] Figure 10 、(A)-(C) are H1N1(3 times MLD 50 ) Virus titers in the lungs of challenged mice on days 3 and 5 after drug treatment, and in the nasal turbinates on day 3 after drug treatment.

[0032] Figure 11 、H1N1(3 times MLD 50 ) Images of lung tissues of challenged mice on days 3 and 5 of treatment and lung index on day 5.

[0033] Figure 12 、H1N1(3 times MLD 50 ) Hematoxylin-eosin staining results of challenged mice on days 3 and 5 after drug treatment.

[0034] Figure 13 、H1N1(3 times MLD 50 ) Immunohistochemistry of lung tissues of challenged mice on days 3 and 5 after drug treatment.

[0035] Figure 14 、(A)-(C) are H1N1(3 times MLD 50 ) Viral gene levels and pro-inflammatory factors IL-6 and IL-1β levels in the lungs of challenged mice 5 days after drug treatment. DETAILED DESCRIPTION

[0036] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly described below in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0037] The main experimental reagents and instruments used in the examples of the present invention are briefly introduced as follows: hyaluronic acid (HA, 7 kDa), adipic acid dihydrazide (ADH), glycyrrhizic acid (GL), N-hydroxysuccinimide (NHS), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, CAS: 25952-53-8), dialysis bag (MW 3.6 kDa); fully digital superconducting nuclear magnetic resonance hydrogen spectrometer, freeze dryer, etc.

[0038] The bioavailability of GL can be improved by increasing its water solubility, such as by coupling GL with the hydrophilic carrier hyaluronic acid. However, direct coupling of GL and hyaluronic acid requires an esterification reaction, which requires relatively high conditions. Therefore, the present invention uses adipic acid dihydrazide as a linker to connect GL and hyaluronic acid. During the connection process, an amidation reaction occurs, which reduces the severity of the reaction conditions. The hyaluronic acid-adipic acid dihydrazide-glycyrrhizic acid structure was confirmed by nuclear magnetic resonance spectroscopy and infrared spectroscopy, the drug loading of glycyrrhizic acid was measured by ultraviolet light, and its morphology, particle size, and charge were observed using transmission electron microscopy and Malvern particle size potentiometer.

[0039] Example 1

[0040] The synthetic route of hyaluronic acid-adipic acid dihydrazide-glycyrrhizic acid (HA-ADH-GL) in this embodiment is as follows: Figure 1 The specific steps are as follows:

[0041] 1.1. Synthesis of hyaluronic acid-adipic acid dihydrazide (HA-ADH)

[0042] Weigh 200.0 mg of HA (0.50 mmol) into a reaction flask, add an appropriate amount of distilled water, and stir until completely dissolved. Then, add 120.1 mg of EDCI (0.63 mmol) and 72.8 mg of NHS (0.63 mmol), and react at 0°C under argon for 30 minutes to activate the carboxyl groups on HA. After activation, add 275.5 mg of ADH (1.58 mmol), and react at room temperature under argon for 2 hours. After the reaction, dialyze in ddH2O using a 3.6 kDa dialysis bag for 24 hours. After dialysis, centrifuge the supernatant and lyophilize to obtain the white solid product HA-ADH.

[0043] 1.2 Synthesis of hyaluronic acid-adipic acid dihydrazide-glycyrrhizic acid (HA-ADH-GL)

[0044] 300.0 mg of lyophilized HA-ADH (0.61 mmol) was weighed into a reaction flask and dissolved in distilled water. Meanwhile, 529.7 mg of glycyrrhizic acid (0.64 mmol) was placed in another reaction flask and dissolved in DMSO. Then, 123.4 mg of EDCI (0.64 mmol) and 74.0 mg of NHS (0.64 mmol) were added and reacted at 0°C under argon for 0.5 h. After the reaction, the carboxyl-activated glycyrrhizic acid was slowly added dropwise to the HA-ADH reaction flask at room temperature under argon with stirring. The reaction was allowed to proceed overnight. The reaction solution was added to a 3.6 kDa dialysis bag, dialyzed against ddH2O for 12 h, and then lyophilized to obtain self-assembled nanoparticles HA-ADH-GL.

[0045] 1.3 Characterization methods and result analysis

[0046] (1) Use nuclear magnetic resonance to verify that the HA-ADH-GL connection is successful

[0047] Dissolve 5-10 mg of GL in DMSO-d6, and dissolve HA, HA-ADH, and HA-ADH-GL in D2O. Use nuclear magnetic resonance to verify that the HA-ADH-GL connection is successful, as shown in Figure 2. Figure 2 shown.

[0048] (2) Verification of HA and GL structures using Fourier transform infrared spectroscopy

[0049] Take appropriate amounts of the solid samples HA, HA-ADH, GL, and HA-ADH-GL to be tested, mix them with dry potassium bromide at a ratio of 1:100, grind them, press them into tablets, and use a Fourier transform infrared spectrometer (FT-IR) to measure the infrared spectrum, such as Figure 3 As shown in A, the results showed that HA-ADH-GL contained GL and HA-ADH and GL were linked by an amide bond.

[0050] (3) Calculation of GL drug loading using UV-visible spectrometer

[0051] Dissolve HA-ADH-GL in water to prepare a 110 μg / mL solution in a cuvette and measure the OD value at 249 nm using a UV-visible spectrophotometer. Substitute this OD value into the GL standard curve to calculate the drug loading. Substitute the absorbance value of HA-ADH-GL at 249 nm into the GL methanol solution standard curve to obtain a GL drug loading of 46%. Figure 3 As shown in B.

[0052] (4) Observation of HA-ADH-GL structure using transmission electron microscopy

[0053] Dissolve 3-4 mg of HA-ADH-GL in 4 mL of ddH2O, ultrasonicate for 20 min, and drop 5 μL of the solution onto a copper grid. Dry the solution and perform transmission electron microscopy. Take 1 mL of the remaining HA-ADH-GL solution and add it to a cuvette to measure the particle size. Take another 1 mL and add it to a potentiometer to measure the potential. Figure 4 As shown in Figure 2, transmission electron microscopy images and particle size images indicate that HA-ADH-GL is a spherical nanoparticle structure with a hydrophobic structure on the inside and a hydrophilic structure on the outside. Potential maps indicate that the nanoparticles are negatively charged.

[0054] Example 2 Comparative Example

[0055] This example plans to synthesize hyaluronic acid-adipic acid dihydrazide-ursolic acid (HA-ADH-UI) and hyaluronic acid-phloretin. The results show that ursolic acid and HA-ADH are not successfully connected, and the drug loading of phloretin is lower than that of glycyrrhizic acid. The details are as follows:

[0056] 2.1 Synthesis of hyaluronic acid-adipic acid dihydrazide-ursolic acid (HA-ADH-UI)

[0057] 2.1.1 Synthesis of hyaluronic acid-adipic acid dihydrazide (HA-ADH)

[0058] Hyaluronic acid-adipic acid dihydrazide was synthesized according to the synthesis method of hyaluronic acid-adipic acid dihydrazide in Example 1.

[0059] 2.1.2 Synthesis of hyaluronic acid-adipic acid dihydrazide-ursolic acid (HA-ADH-UI)

[0060] Weigh 200.0 mg of lyophilized HA-ADH (0.41 mmol) into a reaction flask and add an appropriate amount of distilled water to dissolve it. At the same time, place 207 mg of ursolic acid in another reaction flask and add DMSO to dissolve it. Then add 130 mg of EDCI and 78.3 mg of NHS and stir at 0°C under argon protection for 0.5 h. After the reaction is completed, the ursolic acid with activated carboxyl groups is slowly added dropwise to the HA-ADH reaction flask at room temperature under argon protection, stirring while adding dropwise. After the addition is complete, the reaction is allowed to react overnight. The reaction solution is added to a 3.6 kDa dialysis bag, dialyzed in ddH2O for 12 h, and then freeze-dried. No nanoparticles HA-ADH-UI are obtained, indicating that the ursolic acid and HA-ADH are not successfully connected.

[0061] 2.2 Hyaluronic acid-phloretin synthesis

[0062] Weigh 500 mg of hyaluronic acid and dissolve it in 5 mL of water, stirring until completely dissolved. Then, add 575.1 mg of EDCI and 345.3 mg of NHS and react at 0°C under argon for 2 hours to activate the carboxyl groups on HA.

[0063] Weigh 411.4 mg of phloretin and dissolve it in dimethyl sulfoxide (DMSO). After complete dissolution, add it to the carboxyl-activated hyaluronic acid. Stir the mixture at room temperature under argon with a magnetic stirrer at 300 rpm for 24 hours. Add the reaction solution to a 3.6 kDa dialysis bag, dialyze it in ddH2O for 24 hours, and then lyophilize it to obtain the hyaluronic acid-phloretin conjugate.

[0064] Example 3

[0065] 1. Experimental Materials

[0066] The following materials were selected to study the level of inhibition of influenza A virus infection by the present invention:

[0067] The mouse-adapted H1N1 influenza virus strain (A / Changchun / 01 / 2009) and the mouse-adapted H3N2 influenza virus strain (A / Baikalteal / Shanghai / SH-89 / 2013) were maintained in the laboratory. CCK-8 kits were purchased from MCE. Antibodies against viral nucleoprotein (Ab128193) and related signaling pathway proteins were purchased from Abcam and CST. Fluorescent secondary antibodies were purchased from Beyotime. Primers were synthesized by Changchun Kumeihe Biotechnology. RNA extraction kits were manufactured by Magen. RT-qPCR kits were from TaKaRa (RR047A). Fluorescent dyes were purchased from TaKaRaA (RR082A). Six- to eight-week-old female BALB / c mice were purchased from Jintai Meditech.

[0068] 2. Experimental methods

[0069] 2.1. CCK-8 kit to measure the toxicity of HA-ADH-GL to MDCK cells

[0070] MDCK cells grown to 80%-90% of their basal area in a culture flask were washed with PBS, trypsinized, and resuspended. DMEM complete medium was added to obtain a cell suspension, which was then pipetted evenly and counted. 7,000 cells were plated per well in a 96-well cell culture plate and incubated in a humidified incubator for 12 hours. Drugs (HA, HA-ADH, GL, or HA-ADH-GL) were prepared in basal medium (purchased from Gibico) at concentrations of 10, 50, 100, 150, 200, 300, 400, 500, or 600 μg / mL. When cells reached 60-70% of their basal area, 100 μL of each drug concentration was added to each well. The cells were incubated in a 37°C, 5% CO2 incubator for 24 and 48 hours, with triplicate wells for each concentration. The toxicity of the drugs to MDCK cells was assessed using a CCK-8 assay. A blank control group consisted of 100 μL of basal medium alone, without MDCK cells. Control group cells: MDCK cells + basal culture medium (100 μL).

[0071] Cell activity (%) = (OD value of cells in the drug administration group - OD value of cells in the blank group) / (OD value of cells in the control group - OD value of cells in the blank group) × 100%.

[0072] 2.2. CCK-8 kit to measure the inhibition rate of HA-ADH-GL on influenza virus H1N1 infection of MDCK cells

[0073] MDCK cells grown to 80% to 90% in a culture flask were washed with PBS, digested with trypsin, and resuspended. DMEM complete medium was added to obtain a cell suspension, which was evenly pipetted and counted. 7,000 cells were added to each well of a 96-well cell culture plate and cultured in an incubator for 10 hours. H1N1 was diluted with basal medium to a TCID of 100. 50 Discard the culture medium in the 96-well plate, add basal culture medium to the first column as a blank control group, and add 100 μL of 100 times TCID to each well of the remaining wells. 50The virus dilution was placed in a virus incubator and incubated for 2 hours, and shaken once at regular intervals in a secondary biosafety cabinet. HA-ADH-GL was added to the basal culture medium to prepare HA-ADH-GL drugs at 0.0316, 0.1, 0.316, 1, 3.16, 10, 31.6, 100, and 316 μg / mL. This experiment set up a blank control group (Control group), a negative control group (Mock group), and the above-mentioned drug experimental groups with different concentrations. Three replicates were set for each concentration. Placed in a virus incubator and incubated for 48 hours. The virus inhibition rate was determined using a CCK-8 kit. The MDCK cells in the negative control group were given basal culture medium after the virus was attacked. The MDCK cells in the blank control group were not attacked or given drugs.

[0074] Virus inhibition rate (%) = (OD value of drug experimental group - average OD value of negative control group) / (average OD value of blank control group - average OD value of negative control group).

[0075] 2.3. Indirect immunofluorescence detection of H1N1 virus nucleoprotein levels after HA-ADH-GL treatment

[0076] MDCK cells were resuspended in DMEM complete medium and plated in 24-well cell culture plates at a cell density of 20% per well. When the cells grew to 50% to 60%, the venom of H1N1 and H3N2 was diluted by logarithmic dilution and the TCID of 100 was used. 50 After 2 hours of infection, the medium was changed and the positive drug (Oseltamivir, 60 μg / mL), HA-ADH-GL (100 μg / mL), and HA, HA-ADH, and GL at equivalent concentrations of HA-ADH-GL were given and incubated in a 37°C, 5% CO2 virus incubator for 24 hours. Fixation, membrane permeabilization, blocking, incubation with antibodies, nuclei staining, photography, and fluorescence intensity analysis were performed. A blank control group (Control group) and a negative control group (Mock group) were also set up. The MDCK cells in the negative control group were given basal culture medium after infection. The MDCK cells in the blank control group were not infected and not given drugs.

[0077] 2.5 Protection level of mice after challenge with HA-ADH-GL by different administration methods

[0078] The mice were raised for 3 days before exposure to the poison, and were marked with ear tags and randomly divided into groups (gavage group, intraperitoneal injection group, and tail vein injection group). 50Twelve hours after challenge, HA-ADH-GL was administered via gavage (ig), intraperitoneal injection (ip), and caudal vein injection (iv). HA-ADH-GL was administered at a dose of 50 mg / kg twice daily (ig); 20 mg / kg twice daily (ip); and 20 mg / kg once daily (iv). A positive drug group (oseltamivir, gavage, twice daily, 20 mg / kg per dose, for a total of 40 mg / kg) was also established. The body weights of mice treated for 0 to 14 days were measured and recorded, and survival rates were calculated.

[0079] 2.6. Protection of mice from virus attack

[0080] The mice were raised for 3 days before exposure to the poison, marked with ear tags, and randomly divided into six groups, namely, negative control group (Mock group), positive drug group, HA, HA-ADH, HA-ADH-GL, and GL administration group. The initial weight of the mice was weighed and recorded.

[0081] Mice were anesthetized in a biosafety cabinet and challenged with 50 μL of the drug (3 times the MLD) in one nostril. 50 The mice were challenged with nasal drops and then gavage administered 12 hours after challenge, twice daily for 5 consecutive days. The nanoparticle group was formulated with a HA-ADH-GL concentration of 50 mg / kg, while the remaining groups were formulated with GL loading. The negative control group was treated with PBS, and the positive drug group was treated with oseltamivir. The mice were observed, their weights were measured, and their survival rates were calculated from day 0 to 14 of treatment.

[0082] 2.7. Determination of viral load in mouse lung tissue and nasal turbinates using the chick embryo method

[0083] Grind samples: Place lung tissue and nasal turbinates from mice on day 5 post-challenge (as described in 2.6) into separate 2 mL grinding tubes containing steel balls. Add 1 mL of Opti-MEM to 80 mg of lung tissue, and 500 μL of Opti-MEM to the nasal turbinates of individual mice. Grind in a grinder at 4°C, 50 Hz, 180 min, for four cycles. Centrifuge at 12,000 rpm, 4°C for 10 min. Transfer the supernatant to a new centrifuge tube. Dilute the sample: Dilute the lung and nasal turbinates separately using a logarithmic dilution method. Use a syringe to draw up the lung and nasal turbinates from the infected mouse, and inoculate each chick embryo with 100 μL of the lung and nasal turbinates solution. Seal the tubes with adhesive after inoculation. Place in a 37°C incubator for 48 hours. The air chamber of the chicken embryo was opened and 50 μL of the allantoic fluid of the chicken embryo was harvested with a pipette. The hemagglutination assay was performed and the chicken embryo median infectious dose (EID) of the abrasive fluid of the lung tissue and nasal turbinate of the infected mice was measured. 50 ).

[0084] 2.8 Pathological changes in the lungs

[0085] On days 3 and 5 after the challenge (2.6), mice were autopsied and lung tissue was taken for photographs. Lung lesions and lung viral load were analyzed by hematoxylin-eosin staining and immunohistochemistry.

[0086] 2.9. RT-qPCR analysis of HA-ADH-GL inhibition of proinflammatory factors and viral M gene levels in the lungs of infected mice

[0087] For mice 5 days after the challenge under item 2.6, RNA extraction and quantification of lung tissue were performed according to the instructions of the Magen kit. After 1 μL of protein eluate was taken to calibrate the instrument, ssRNA in the tissue was detected after loading. The system was prepared according to the instructions of the RT-qPCR kit, and the program was set on the Real Time PCR amplification instrument. After the reaction was completed, a melting curve was performed to verify the specificity of the qPCR amplification product, and the obtained Ct value was substituted into 2 -ΔΔct calculate.

[0088] All animal experimental conditions and procedures described above adhered to the ethical guidelines of the International Pain Society and were approved by the ethics committee. All experiments were conducted in a biosafety level 2 laboratory, using approved viral strains and autoclaving medical instruments that came into contact with viruses.

[0089] 3. Results Analysis

[0090] 3.1. CCK-8 kit to measure HA-ADH-GL cytotoxicity and influenza virus H1N1 infection inhibition rate

[0091] After 24h or 48h of administration of 100 μg / mL glycyrrhizic acid, cytotoxicity was observed. Figure 5 、 Figure 6 As shown, HA-ADH-GL has no obvious cytotoxicity in the concentration range of 50μg / mL to 600μg / mL, indicating that after GL is modified with HA, its water solubility increases and its cytotoxicity to MDCK cells decreases. In order to verify the safety and efficacy of HA-ADH-GL in a larger concentration range, the above concentration range was expanded to 1000μg / mL, the drug was diluted by logarithmic dilution method, and incubated with MDCK cells for 48h. The results showed that the HA-ADH-GL nanoparticle group was safer at 100μg / mL, and the inhibition rate reached a peak of 43.89%, and the IC 50 =0.2957 μg / mL, and the selectivity index SI was 3304.13.

[0092] 3.2. HA-ADH-GL treatment reduces H1N1 viral nucleoprotein levels in infected cells

[0093] Indirect immunofluorescence assay was used to determine the level of viral nucleoprotein in cells infected with influenza virus. Figure 7 Compared with the untreated Mock group, the drug-treated group had a certain inhibitory effect on viral infection in vitro; compared with the other drug-treated groups, the effect of the HA-ADH-GL-treated group was comparable to that of the positive drug and was better than that of the single-drug group.

[0094] 3.3 Protection level of mice challenged with HA-ADH-GL by different administration methods

[0095] After the mice were challenged with the poison, the treatment was administered by gavage, intraperitoneal cavity and tail vein. Figure 8 As shown in the results, comprehensive safety and efficacy results showed that oral administration of nanoparticles HA-ADH-GL was better than intraperitoneal and tail vein administration, so oral administration was used in the subsequent studies.

[0096] 3.4. HA-ADH-GL challenge protection study

[0097] like Figure 9 As shown, compared with the negative control group (Mock group), HA-ADH-GL can improve the survival rate of infected mice and reduce the weight loss of mice to a certain extent. The survival rate of infected mice treated with HA-ADH-GL is 60%, which is higher than the survival rate of the single-drug GL group. The weight of mice recovered on the 9th day after being challenged with HA-ADH-GL and GL group drugs.

[0098] 3.5. HA-ADH-GL reduces virus titers in lung tissue and nasal turbinates of H1N1-infected mice

[0099] Compared with the viral load in lung tissue of the Mock group, HA and HA-ADH had no significant effect on the viral load in the lungs of mice. Figure 10 As shown, the single-drug GL group and the HA-ADH-GL group significantly reduced the lung viral load on days 3 and 5 of treatment, and the effect of the HA-ADH-GL group was better than that of the single-drug GL group.

[0100] Compared with the viral load in the nasal turbinates of the Mock group, HA-ADH-GL treatment reduced the viral titer in the nasal turbinates of infected mice 3 days later.

[0101] 3.6. HA-ADH-GL treatment significantly improved lung lesions in H1N1-infected mice

[0102] In the Mock group, a large area of ​​alveolar septum thickening (indicated by the black arrow) was accompanied by inflammatory cell infiltration (indicated by the blue arrow). After treatment with drugs HA-ADH-GL and GL, the alveolar septum thickening was improved, the infiltration of inflammatory factors was reduced, and bleeding and edema were reduced. Figure 11 、 Figure 12 In addition, the immunohistochemical results showed that Figure 13 As shown, nanoparticle HA-ADH-GL treatment significantly reduced the viral load in the mouse lungs.

[0103] 3.7. HA-ADH-GL treatment inhibits proinflammatory gene levels and viral gene replication in the lungs

[0104] The results are as follows Figure 14 As shown, for mice 5 days after infection treatment, the HA-ADH-GL group and the GL group could reduce the level of viral M gene replication in the lungs; for mice on the 5th day of infection treatment, the HA-ADH-GL group and the GL group could reduce the levels of lung inflammatory factors IL-6 and IL-1β.

[0105] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. Nanoparticles HA-ADH-GL for inhibiting influenza virus, characterized in that The nanoparticle HA-ADH-GL is a conjugate obtained by linking glycyrrhizic acid and hyaluronic acid via adipic acid dihydrazide.

2. The method for preparing the influenza virus-inhibiting nanoparticle HA-ADH-GL according to claim 1, characterized in that: The method includes the following steps: Step 1: reacting the hyaluronic acid solution after carboxyl group activation with adipic acid dihydrazide at room temperature under inert gas protection to obtain hyaluronic acid-adipic acid dihydrazide; Step 2: adding the glycyrrhizic acid after activation of the carboxyl group to hyaluronic acid-adipic acid dihydrazide, reacting at room temperature under inert gas protection to obtain hyaluronic acid-adipic acid dihydrazide-glycyrrhizic acid nanoparticles.

3. The method according to claim 2, characterized in that The reaction time in step 1 is 2 h.

4. The method according to claim 2, characterized in that The method for activating the carboxyl group of hyaluronic acid in step 1 comprises: The hyaluronic acid solution was reacted with 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide at 0°C under inert gas protection to activate the carboxyl groups on the hyaluronic acid.

5. The method according to claim 4, characterized in that The hyaluronic acid solution is a hyaluronic acid aqueous solution.

6. The method according to claim 2, characterized in that The reaction time in step 2 is more than 12 hours.

7. The method according to claim 2, characterized in that The method for activating the carboxyl group of glycyrrhizic acid in step 2 comprises: The glycyrrhizic acid solution was reacted with 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide at 0°C under inert gas protection to activate the carboxyl group on the glycyrrhizic acid.

8. The method according to claim 7, characterized in that The glycyrrhizic acid solution is a DMSO solution of glycyrrhizic acid.

9. The method according to claim 2, characterized in that The purification steps of the reaction solution of step 1 and step 2 are as follows: the reaction solution is dialyzed and then centrifuged, the supernatant is collected, and lyophilized.

10. Use of the nanoparticle HA-ADH-GL according to claim 1 in preparing a drug for treating influenza virus.

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