Preparation method and application of hyaluronic acid-glycyrrhetinic acid and hyaluronic acid-ferulic acid conjugate nanoparticle drug delivery system

Abstract

The invention discloses a preparation method and application of a hyaluronic acid-glycyrrhetinic acid and hyaluronic acid-ferulic acid conjugate nanoparticle drug delivery system, and relates to the technical field of biological pharmacy. The key points of the technical scheme are as follows: GA and FA are respectively connected with HA by using adipic acid dihydrazide as a connexon; and the hyaluronic acid-glycyrrhetinic acid and hyaluronic acid-ferulic acid polymer prodrugs are synthesized. An in-vitro release result shows that when the NPs are exposed in a physiological medium with slightly acidic pH, the drug release rate is higher than that in a blood environment with pH of 7.4. And the drug release rate and the drug release amount of the nanoparticles are superior to those of single drug release. The HA-ADH-GA and the HA-ADH-FANPs both have relatively low toxicity in vitro and a good effect of resisting the influenza A virus H1N1. Compared with the HA-ADH-FANPs, the HA-ADH-GANPs has the advantage that the HA-ADH-GANPs has a better protection effect on a mouse infected with the H1N1-UI182 in vivo.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical technology, and more specifically, to the preparation method and application of hyaluronic acid-glycyrrhetinic acid and hyaluronic acid-ferulic acid conjugate nanoparticle drug delivery systems. Background Technology

[0002] Influenza is a respiratory illness caused by the influenza virus. Seasonal influenza outbreaks cause epidemics every year, infecting millions globally and imposing health and economic burdens. Currently, influenza remains a significant risk to human health. The virus surface mainly contains two proteins: hemagglutinin, which agglutinates red blood cells, and neuraminidase, which releases viral particles from the surface of agglutinated red blood cells. Based on the structure and genetic characteristics of hemagglutinin and neuraminidase, it is further divided into various subtypes. New variants also emerge during each influenza pandemic. Small molecule drugs play an important role in treating influenza virus infections. Currently, clinically used drugs for treating influenza viruses include neuraminidase inhibitors (such as oseltamivir and zanamivir), M2 ion channel inhibitors (such as amantadine and rimantadine), and ribonucleic acid polymerase inhibitors (such as baloxavir). However, traditional antiviral drugs such as amantadine and oseltamivir, used to inhibit influenza viruses, are increasingly reported to have resistance. Therefore, the search for effective antiviral drugs remains of great practical significance. Highly effective and low-toxicity natural small molecule compounds can be innovatively used to treat the H1N1 influenza virus.

[0003] 18β-Glycyrrhetinic acid (GA) is the main bioactive hydrolysate of glycyrrhizic acid, the main component of licorice, produced by intestinal symbiotic bacteria. GA possesses a wide range of pharmacological activities, including anti-inflammatory, antioxidant, antitumor, antiviral, and neuroprotective effects. GA also exhibits antiviral properties and can improve symptoms caused by viral infections.

[0004] Ferulic acid (FA) is a phenolic acid extracted from the resin of Ferula assa- ...

[0005] In addition, further research into the other clinical pharmacological effects of glycyrrhetinic acid and ferulic acid is of great significance for the prevention and treatment of diseases and for future clinical development. Therefore, it is worthwhile for us to explore them in greater depth.

[0006] In recent years, researchers have explored the role of GA in the treatment of viral infections, discovering its antiviral effects against Zika virus, human immunodeficiency virus, viral hepatitis, respiratory syncytial virus, SARS-CoV-2, and influenza virus, exerting its effects by influencing viral entry into cells. Studies on FA have shown that its derivatives, including FA, possess antiviral activity against respiratory syncytial virus, SARS-CoV-2, porcine parvovirus, and influenza virus.

[0007] The very low water solubility of GA and FA reduces their bioavailability, limiting their application in disease treatment. To address this issue, this study designed and prepared two HA-based self-assembled nanoparticle drug delivery systems (HA-ADH-GA and HA-ADH-FA NPs) for drug delivery to influenza viruses. This not only solved the problem of poor water solubility of GA and FA, but also prolonged the duration of drug action in mice through sustained release of the nanoparticles; thus improving therapeutic efficacy and significantly enhancing the bioavailability of GA and FA. Summary of the Invention

[0008] The purpose of this invention is to provide a method for preparing and applying a hyaluronic acid-glycyrrhetinic acid and hyaluronic acid-ferulic acid conjugate nanoparticle drug delivery system.

[0009] The above-mentioned technical objective of the present invention is achieved through the following technical solution: a conjugate nanoparticle drug delivery system, wherein the drug delivery system is a hyaluronic acid-glycyrrhetinic acid conjugate nanoparticle drug delivery system (HA-ADH-GA NPs) or a hyaluronic acid-ferulic acid conjugate nanoparticle drug delivery system (HA-ADH-FA NPs);

[0010] The structure of the hyaluronic acid-glycyrrhetinic acid conjugate nanoparticles (HA-ADH-GA) is as follows:

[0011]

[0012] The structure of the hyaluronic acid-ferulic acid conjugate nanoparticles (HA-ADH-FA) is as follows:

[0013] B.

[0014]

[0015] The present invention is further configured such that: the drug delivery system HA-ADH-GA NPs is synthesized by combining hydrophilic material HA and hydrophobic drug GA by combining them separately with adipic acid dihydrazide and then self-assembling them into NPs;

[0016] The drug delivery system HA-ADH-FA NPs is synthesized by combining hydrophilic material HA and hydrophobic drug FA separately with adipic dihydrazide and then self-assembling them into NPs.

[0017] This invention further provides a method for preparing the above-mentioned conjugate: the synthesis of the hyaluronic acid-glycyrrhetinic acid conjugate includes the following steps:

[0018] (1) Synthesis of hyaluronic acid-adipate dihydrazide: Weigh HA with a molecular weight of 8 kDa and place it in a round-bottom flask. Add deionized water to dissolve it, then add activator EDC·HCl to activate it. Then add adipate dihydrazide (ADH) to the solution. After the solid dissolves, add HCl to adjust the pH to 4.7 and react under argon protection. Then, adjust the pH to neutral with NaOH solution and transfer the reaction solution to a dialysis bag. Dialyze with deionized water to remove unreacted EDC and ADH. Filter the solution with a microporous membrane and then transfer it to a dry glass dish. After pre-freezing overnight, freeze-dry to obtain the intermediate HA-ADH (8 kDa).

[0019] (2) Synthesis of hyaluronic acid-glycyrrhetinic acid: GA was weighed and dissolved in N,N-dimethylformamide (DMF), and EDC·HCl was added and placed on a magnetic stirrer for activation. The product HA-ADH (8kDa) in (1) was weighed and added to an appropriate amount of deionized water to completely dissolve it. The reaction flask after dissolution was then placed in an ice bath for pre-cooling. The activated GA was added dropwise to the HA-ADH solution. At this time, GA precipitated and the solution became turbid. An appropriate amount of DMF was added until the reaction solution became clear and transparent again. The reaction was carried out overnight under argon protection. The unreacted EDC and GA were removed by dialysis. The dialysis solution was deionized water. The unreacted GA precipitated at the bottom of the solution. To remove the precipitate, the final reaction solution was poured from the dialysis bag into a centrifuge tube. After centrifugation, the supernatant was filtered through a microporous membrane into a dry glass dish. After pre-freezing overnight, the supernatant was freeze-dried to obtain the final product HA-ADH-GA (8kDa).

[0020] In step (1), the molar ratio of HA, EDC·HCl and ADH is 1:1.2:3; in step (2), the molar ratio of GA, EDC·HCl and HA-ADH is 1:1.5:1.2.

[0021] Further: Dialysis uses 3600Da dialysis bags.

[0022] The present invention is further configured such that the synthesis of the hyaluronic acid-ferulic acid conjugate includes the following steps:

[0023] (1) Synthesis of hyaluronic acid-adipate dihydrazide: Weigh HA with a molecular weight of 98kDa and place it in a round-bottom flask. Add deionized water to dissolve it, then add activator EDC·HCl to activate it. Then add adipate dihydrazide (ADH) to the solution. After the solid dissolves, add HCl to adjust the pH to 4.7 and react under argon protection. Then, adjust the pH to neutral with NaOH solution and transfer the reaction solution to a dialysis bag. Dialyze with deionized water to remove unreacted EDC and ADH. Filter the solution with a microporous membrane and then transfer it to a dry glass dish. After pre-freezing overnight, freeze-dry to obtain the intermediate HA-ADH (98kDa).

[0024] (2) Synthesis of hyaluronic acid-glycyrrhetinic acid: FA was weighed and dissolved in N,N-dimethylformamide (DMF), EDC·HCl was added and placed on a magnetic stirrer for activation; HA-ADH (98kDa) from (1) was weighed and added to an appropriate amount of deionized water to completely dissolve it, and then the reaction flask after dissolution was placed in an ice bath for pre-cooling; the activated FA was added dropwise to the HA-ADH solution, at which point FA precipitated and the solution became turbid, and an appropriate amount of DMF was added until the reaction solution became clear and transparent again, and the reaction was carried out overnight under argon protection; the unreacted EDC and FA were removed by dialysis, and the dialysis solution was deionized water; the unreacted FA settled at the bottom of the solution in the form of a precipitate. In order to remove the precipitate, the final reaction solution was poured from the dialysis bag into a centrifuge tube, centrifuged and the supernatant was filtered through a microporous membrane into a dry glass dish, pre-frozen overnight, and then freeze-dried to obtain the final product HA-ADH-FA (98kDa).

[0025] In step (1), the molar ratio of HA, EDC·HCl and ADH is 1:1.2:3; in step (2), the molar ratio of FA, EDC·HCl and HA-ADH is 1:1.5:1.2.

[0026] Further: Dialysis is performed using 8000Da dialysis bags.

[0027] The present invention is further configured such that the synthesis process of the hyaluronic acid-glycyrrhetinic acid conjugate and the hyaluronic acid-ferulic acid conjugate is carried out at room temperature, and the diameter of the filter membrane is 0.45 μm.

[0028] The present invention also provides the use of hyaluronic acid-glycyrrhetinic acid conjugates and hyaluronic acid-ferulic acid conjugates in the preparation of drugs against type A H1N1 influenza virus.

[0029] In summary, the present invention has the following beneficial effects:

[0030] This invention utilizes chemical bonding to couple hyaluronic acid with glycyrrhetinic acid and ferulic acid, respectively, to improve the water solubility and bioavailability of glycyrrhetinic acid and ferulic acid. Two prodrug nanoparticles, HA-ADH-GA and HA-ADH-FA, were synthesized. 1H NMR spectroscopy showed that the chemical grafting rates of HA-ADH-GA and HA-ADH-FA were 25.5% and 37.2%, respectively, with calculated drug loadings of 24.6% and 32.9%, respectively. Infrared absorption spectroscopy confirmed the synthesis of the HA-ADH-GA and HA-ADH-FA grafts. Transmission electron microscopy revealed that the particle sizes of the HA-ADH-GA and HA-ADH-FA NPs were approximately 173.6±23.8 nm and 55.4±14.9 nm, respectively, both exhibiting a smooth spherical shape. In vitro release results showed that the drug release rate of the two nanoparticles was faster in a slightly acidic environment (pH 5.5) than in a blood environment (pH 7.4), and the drug release rate and amount of the nanoparticles were superior to those of single-drug release.

[0031] The hyaluronic acid-glycyrrhetinic acid and hyaluronic acid-ferulic acid conjugates of this invention exhibit inhibitory activity against H1N1-UI182 influenza virus in MDCK cells. Combined treatment with both nanoparticles showed a higher inhibitory effect, which was dose-dependent within safe concentrations. Immunofluorescence assays showed that HA-ADH-GA and HA-ADH-FA reduced vNP expression in virus-infected cells. RT-qPCR results showed that both nanoparticles inhibited viral RNA replication. Western blot results showed that both nanoparticles reduced viral nucleoprotein expression. These findings indicate that HA-ADH-GA and HA-ADH-FA exhibit antiviral activity by inhibiting the expression of virus-related gene proteins.

[0032] In mouse experiments, the hyaluronic acid-glycyrrhetinic acid and hyaluronic acid-ferulic acid conjugates of this invention showed that H&E results of lung tissue on day 3 (3 dpi) after infection indicated that HA-ADH-FA could delay pulmonary lesions and edema caused by viral infection in the early stages of viral infection. HA-ADH-GA administration effectively protected against pathological damage caused by viral infection. Compared with the Mock group mice, the HA-ADH-GA treatment group reduced the mRNA expression of inflammatory factors IL-6, IL-1β, TNF-α, and IFN-α. Western blot results showed that HA-ADH-GA treatment could inhibit the activation of the SAPK / JNK signaling pathway caused by influenza virus infection, reduce the expression of inflammatory factors, and exert antiviral activity. Attached Figure Description

[0033] Figure 1 Synthesis of HA-ADH-GA and HA-ADH-FA prodrug conjugates. Among them, (A) is the synthetic route of HA-ADH-GA conjugate; (B) is the synthetic route of HA-ADH-FA conjugate.

[0034] Figure 2 Characterization of HA-ADH-GA and HA-ADH-FA prodrug conjugates. (A) shows the 1H-NMR spectra of HA and HA-ADH-GA in D2O and GA in DMSOd6; (B) shows the 1H-NMR spectra of HA and HA-ADH-FA in D2O and FA in DMSOd6; (C) shows the FT-IR spectra of HA, HA-ADH, GA, and HA-ADH-GA; (D) shows the FT-IR spectra of HA-, HA-ADH, FA, and HA-ADH-FA.

[0035] Figure 3 Preparation and characterization of HA-ADH-GA and HA-ADH-FA NPs. (A) is a TEM image of HA-ADH-GA NPs [scale bar: 200 nm]; (B) is a TEM image of HA-ADH-FA NPs [scale bar: 100 nm]. (C) shows the release curves of HA-ADH-GA NPs and GA monotherapy in media with different pH values ​​(pH = 5.5, 7.4); (D) shows the release curves of HA-ADH-FA NPs and FA monotherapy in media with different pH values ​​(pH = 5.5, 7.4). Data are expressed as mean ± SEM (n = 3).

[0036] Figure 4 The efficacy of HA-ADH-GA and HA-ADH-FA nanoparticles against influenza A H1N1 virus in vitro was evaluated. (A) and (B) show the cytotoxicity and efficacy curves of HA-ADH-GA and HA-ADH-FA NPs, respectively; (C) and (D) show the effect of HA-ADH-GA and HA-ADH-FA NPs on influenza virus mRNA expression in infected cells; (E) and (F) show the status of HA-ADH-GA and HA-ADH-FA NPs 48 h after cell infection; (G) and (H) show the effect of HA-ADH-GA and HA-ADH-FA NPs on vNP expression in the nucleus of infected cells.

[0037] Figure 5 To validate the protective efficacy of HA-ADH-GA and HA-ADH-FA NPs against infected mice. (A) and (B) show the body weight and survival rate of infected mice treated with HA-ADH-GA NPs; (C) and (D) show the body weight and survival rate of infected mice treated with HA-ADH-FA NPs. (E) and (F) show the lung index of HA-ADH-GA NPs at 3 dpi and 5 dpi; (G) shows the lung index of HA-ADH-FA NPs at 3 dpi.

[0038] Figure 6The effects of HA-ADH-GA and HA-ADH-FA NPs on viral titers in the lungs of infected mice. (A)-(D) show viral titers of HA-ADH-GA NPs in the lungs and nasal turbinates at 3 dpi and 5 dpi; (E) and (F) show viral titers of HA-ADH-FA NPs in the lungs and nasal turbinates at 3 dpi.

[0039] Figure 7 To demonstrate the protective effect of HA-ADH-GA and HA-ADH-FA NPs against lung injury caused by influenza virus. (A) shows the lung condition and pathological sections of HA-ADH-GA NPs at 3 dpi and 5 dpi, and (B) shows the lung condition and pathological sections of HA-ADH-FA NPs at 3 dpi.

[0040] Figure 8 To demonstrate the protective effects of HA-ADH-GA and HA-ADH-FA NPs against other tissue damage caused by influenza virus. (A) Heart, liver, spleen, and kidney sections at 3 dpi and 5 dpi, and (B) Heart, liver, spleen, and kidney sections at 3 dpi.

[0041] Figure 9 The effects of HA-ADH-GA and HA-ADH-FA NPs on the expression of inflammatory factors in serum. (A)-(D) show the effects of HA-ADH-GA NPs on the expression of IFN-α, TNF-α, IL-6, and IL-1β; (E) and (F) show the effects of HA-ADH-FA NPs on the expression of IL-6 and TNF-α.

[0042] Figure 10 The effects of HA-ADH-GA NPs treatment on the expression levels of M gene and inflammatory cytokine mRNA in the lungs. (A) and (B) show the effects of HA-ADH-GA NPs on the expression of influenza virus M gene mRNA; (C)-(F) show the effects of HA-ADH-GA NPs on the expression of IFN-α, TNF-α, IL-6, and IL-1β verification factor mRNA.

[0043] Figure 11 The effects of HA-ADH-GA NPs treatment on the MAPK / JNK signaling pathway were investigated. (A) shows the proteomic imprinting analysis of viral nucleoprotein, MAPK / JNK, and p-MAPK / JNK; (B) and (C) show the quantitative analysis of viral nucleoprotein and MAPK / JNK, respectively. Detailed Implementation

[0044] The following is in conjunction with the appendix Figure 1-11 The present invention will be described in further detail below.

[0045] The main reagents, chemicals, and samples used in this application are as follows:

[0046] Hyaluronic acid (HA, Mw = 8kDa, 981kDa) was purchased from Shandong Freda Co., Ltd. 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Adipic acid dihydrazide (ADH) was purchased from Anaiji Chemical Reagent Co., Ltd. Concentrated hydrochloric acid (HCl) was purchased from Kaifeng Dongda Chemical Co., Ltd. Sodium hydroxide (NaOH) was purchased from Tianjin De'en Chemical Reagent Co., Ltd. Glycyrrhetinic acid (GA) was purchased from Shanghai Maclean Biochemical Technology Co., Ltd. Ferulic acid (FA) was purchased from Zhengzhou Alpha Chemical Co., Ltd. N,N-dimethylformamide (DMF) was purchased from Sinopharm Reagent Co., Ltd. Deuterated reagent (D2O) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Methanol (CH3OH) was purchased from Tianjin Fuyu Fine Chemical Co., Ltd. Argon gas was purchased from Henan Yuanzheng Technology Development Co., Ltd. Microporous filter membranes were purchased from Shanghai Xinya Purification Device Factory. Dialysis bags (MWCO 100035008000) were purchased from Beijing Aobo Tuoda Technology Co., Ltd. Potassium bromide (spectrally pure) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. DMEM, fetal bovine serum (FBS), and penicillin-streptomycin combined antibiotics were purchased from Gibco. Trypsin was purchased from Corning.

[0047] Six- to eight-week-old SPF-grade female BALB / c mice (16g-18g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0048] Example 1: Synthesis and Characterization of HA-ADH-GA and HA-ADH-FA

[0049] (1) Accurately weigh 600.0 mg (1.59 mmol) of HA (molecular weight of 8 kDa and 98 kDa) into a 100 mL round-bottom flask, add 40 mL of deionized water to dissolve, then add 429.5 mg (2.39 mmol) of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) to activate for 1 h, then add 830.9 mg (4.77 mmol) of adipic acid dihydrazide (ADH) to the solution. After the solid dissolves, add 1 mol / L HCl to adjust the pH to 4.75, and react for 4 h under argon protection. Then, the pH was adjusted to neutral with 1 mol / L NaOH solution and the reaction solution was transferred to a dialysis bag (MWCO 3500 Da). The solution was dialyzed with deionized water for 24 h to remove unreacted EDC and ADH and filtered through a microporous membrane (0.45 μm). The solution was then transferred to a dry glass dish, pre-frozen (-20 °C) overnight and then freeze-dried to obtain intermediates HA-ADH (8 kDa) and HA-ADH (98 kDa).

[0050] (2) Accurately weigh GA and FA 200.0 mg (0.42 mmol and 0.97 mmol) respectively, dissolve them in 4 mL of N,N-dimethylformamide (DMF), add 113.2 mg and 261.5 mg of EDC·HCl (0.63 mmol and 1.46 mmol) respectively, and stir on a magnetic stirrer for 4 h to activate. Weigh HA-ADH 233.4 mg (8 kDa) and 561.8 mg (98 kDa) (0.50 mmol and 1.16 mmol) respectively from section 2.2.1, add an appropriate amount of deionized water to completely dissolve them, and then pre-cool the reaction flask in an ice bath for 10 min. Add the activated GA and FA dropwise to the HA-ADH (8 kDa) and HA-ADH (98 kDa) solutions respectively. At this time, GA and FA precipitate and make the solution turbid. Add an appropriate amount of DMF until the reaction solution becomes clear and transparent again, and react overnight under argon protection. Unreacted EDC, GA, and FA were removed by dialysis for 24 hours using deionized water as the dialysate. Unreacted GA and FA precipitated at the bottom of the solution. To remove the precipitate, the final reaction solution was transferred from the dialysis bag to a centrifuge tube. After centrifugation, the supernatant was filtered through a 0.45 μm microporous membrane into a dry glass dish. The dish was pre-frozen (-20°C) overnight and then freeze-dried to obtain the final products HA-ADH-GA and HA-ADH-FA.

[0051] (3) Preparation of HA-ADH-GA and HA-ADH-FA nanoparticles: 5 mg of HA-ADH-GA and HA-ADH-FA sample powders were respectively placed in 5 mL of PBS solution and sonicated for 10 min to obtain uniformly dispersed amphiphilic HA-ADH-GA and HA-ADH-FA nanoparticles.

[0052] Structural characterization of HA-ADH-GA and HA-ADH-FA conjugates: Recorded using NMR spectroscopy (300 Hz, Bruker, Switzerland). 1 1H nuclear magnetic resonance (NMR) spectroscopy. Fourier transform infrared (FT-IR) spectroscopy was recorded on a Bruker IFS-55 (Switzerland). Enzyme-linked immunosorbent assay (ELISA) system (Thermo Fisher Scientific, USA).

[0053] The final product, HA-ADH-GA, is a white solid after lyophilization, such as... Figure 1As shown in Figure B, the characteristic peaks of HA at 1.99 ppm (N-acetylmethyl) and 4.4-4.6 ppm (hydroxyl) in the HA-ADH-GA spectrum are those of HA. The peaks at 5.77 ppm and 0.89-1.56 ppm are attributed to the allyl hydrogen and methylene and methyl groups on GA, respectively, confirming the successful synthesis of HA-ADH-GA. The final product, HA-ADH-FA, is a pale yellow solid after lyophilization. Figure 1 As shown in Figure C, the spectrum of HA is consistent with that of HA-ADH-GA. The peaks at 6.9 ppm and 7.1-7.3 ppm in the spectrum of the HA-ADH-FA conjugate are attributed to the allyl hydrogen group on FA and the hydrogen bond on the benzene ring of FA, respectively, proving that HA-ADH-FA was successfully prepared. Based on the integral ratio of characteristic hydrogens in the spectra of GA and HA, the grafting rate of HA-ADH-GA was calculated to be 25.5%, and the grafting rate of HA-ADH-FA was 37.2%.

[0054]

[0055] The drug loading was calculated using the ultraviolet absorbance method. The drug loadings of GA in HA-ADH-GA and FA in HA-ADH-FA prodrug were 24.6% and 32.9%, respectively.

[0056] like Figure 2 As shown in Figure B, HA, HA-ADH, GA, and HA-ADH-GA were analyzed using infrared absorption spectroscopy. Figure 2 Structural analysis of HA, HA-ADH, FA, and HA-ADH-FA shown in Figure C. FT-IR results show that HA has a density of 1045 cm⁻¹. -1 1616cm -1 and 3420cm- 1 An absorption peak appears at 1660 cm⁻¹, which can be attributed to the stretching vibration peak of the CO single bond, the stretching vibration peak of the carboxyl group (COOH), and the stretching vibration peak of the hydroxyl group (OH) in hyaluronic acid. Meanwhile, HA-ADH shows an absorption peak at 1660 cm⁻¹. -1 The presence of newly formed peaks at 1709 cm⁻¹ is attributed to the stretching vibration of amides (NC=O), indicating the formation of new amide bonds between HA and ADH. Furthermore, the HA-ADH-GA peak at 1709 cm⁻¹... -1 and 1659cm -1 An absorption peak appeared at 1655 cm⁻¹, indicating that the in-plane bending vibration of NH and stretching vibration of CN occurred due to the formation of a new amide bond, thus changing the position of the absorption peak. The infrared spectrum further confirms the successful synthesis of HA-ADH-GA. The infrared spectrum of HA-ADH-FA shows that the absorption peaks of HA and HA-ADH are roughly consistent with those described above, while HA-ADH-FA shows a peak at 1655 cm⁻¹. -1 and 1558cm- 1The appearance of new absorption peaks is likely due to the CN stretching vibration and NH in-plane bending vibration caused by the formation of new amide bonds. Similarly, the infrared spectrum confirms the successful preparation of HA-ADH-FA.

[0057] Morphological characterization of HA-ADH-GA and HA-ADH-FA NPs: 5 mg of HA-ADH-GA and HA-ADH-FA NPs were respectively dissolved in 5 mL of PBS solution and sonicated for 10 min to obtain uniformly dispersed amphiphilic HA-ADH-GA and HA-ADH-FA nanoparticles. The aqueous solution of HA-ADH-GA and HA-ADH-FA nanoparticles was dropped onto a 300-mesh copper grid, dried, and then the structure and morphology of the nanoparticles were observed using a JEM1400JEOL transmission electron microscope (Tokyo, Japan) at an accelerating voltage of 200 kV.

[0058] The formation of HA-ADH-GA and HA-ADH-FA NPs is attributed to the highly hydrophilic HA and the poorly soluble GA and FA. Due to the amphiphilic nature of the prodrugs, the HA-ADH-GA and HA-ADH-FA prodrug conjugates can self-assemble into nanoparticles in aqueous solution. The morphology and size distribution of the two nanoparticles were evaluated, and TEM results showed that... Figure 3 The AB, HA-ADH-GA, and HA-ADH-FA NPs are all spherical, uniformly distributed, and have smooth surfaces, without sticking together. The average particle size of HA-ADH-GA nanoparticles is 173.6±23.8 nm, and the average particle size of HA-ADH-FA nanoparticles is 55.4±14.9 nm.

[0059] Release curves of HA-ADH-GA and HA-ADH-FA NPs: The in vitro release rates were calculated by detecting the contents of HA-ADH-GA and HA-ADH-FA in the release medium prepared with PBS at different time points. The results are as follows: Figure 3 The drug release rate (CD) was high in the first 12 hours, followed by a rapid increase in the release rate, which then leveled off. The release of the single drug was rapid in the first 6 hours, followed by a slower release rate and a gradually decreasing amount of drug released. Overall, the release rate of both nanoparticles was faster in a slightly acidic environment (pH 5.5) than in a blood environment (pH 7.4), and the release rate and amount of the nanoparticles were superior to those of the single drug.

[0060] Example 2: Safety of HA-ADH-GA and HA-ADH-FA NPs in MDCK cells and their inhibitory effect on H1N1-UI182 in MDCK cells.

[0061] MDCK cells were preserved in the Laboratory of Animal Virology and Special Animal Diseases, Institute of Veterinary Medicine, Academy of Military Medical Sciences. The influenza virus strain H1N1-UI182 (A / Changchun / 01 / 2009) used was isolated and preserved by the Laboratory of Animal Virology and Special Animal Diseases, Academy of Military Medical Sciences. MDCK cells were cultured in DMEM complete medium supplemented with 10% fetal bovine serum (FBS) and 1% (w / v) penicillin-streptomycin; and grown in an incubator at 37°C and 5% CO2 with saturated humidity.

[0062] (1) Plating: Complete culture medium containing 10% fetal bovine serum and 1% (w / v) penicillin-streptomycin mixture was added to the digested MDCK cells, and 100 μL was seeded into each well of a 96-well plate. The plates were then incubated at 37°C in a 5% CO2 incubator until the cells adhered to the plate for drug cytotoxicity and efficacy assays. After 12 hours, the cells were observed under a microscope. When the cell density reached 80-90%, the drug was administered. In addition to the drug administration group, a Control group, a Mock group, and a positive control group (OSTA) were also set up, with 3 auxiliary wells in each group (n=3).

[0063] (2) Cytotoxicity assay: When the cell density reaches 80-90%, discard the original culture medium and add HA-ADH-GA and HA-ADH-FA at different concentrations diluted with the culture medium. Replace the Control group with double-free culture medium, 100 μL per well, and then place the culture plate in an incubator to continue culturing for 48 h. The Mock group and OSTA group are not set up for the cytotoxicity assay.

[0064] (3) Drug efficacy experiment: The thawed virus solution was diluted with double-free medium at a specified multiplicity of infection (MOI = 0.1) and mixed with equal volumes of different concentrations of drugs. The mixture was then incubated at 37°C in a 5% CO2 incubator for 1.5 h. The incubated mixture was then inoculated into MDCK cells that had grown into a monolayer. Control and Mock groups were set up. The medium in the Control group was changed to double-free medium. In the Mock group, the diluted virus solution was inoculated into the cells and incubated for 2 h. Then, the medium in the Mock group was changed to double-free medium and incubated for another 48 h.

[0065] (4) Observe the cytopathic effect after 48 hours and calculate the amount of reagent to be prepared according to the number of wells. Add 10 μL of CCK-8 solution and 90 μL of culture medium (the ratio of culture medium to CCK-8 is 10:1) to each well, discard the culture medium containing the drug, add 100 μL of the mixed CCK-8 reagent to each well, and then put it back into the incubator. Measure the OD value after 1 hour (note to avoid light).

[0066]

[0067] Cytotoxicity (%) = 1 - Cell viability (%)

[0068]

[0069] Graphpad 8.0 software was used to plot dose-dependent curves of cytotoxicity, and IBM SPSS Statistic 26 was used for regression analysis to calculate the half-maximal cytotoxicity concentration (CMC). 50 ) and half-maximum effective concentration (EC50) 50 ), and the selection index SI (SI = CC) 50 / EC 50 ).

[0070] To evaluate the safe concentrations of HA-ADH-GA and HA-ADH-FA nanoparticles for MDCK cells and their effective concentrations in preventing H1N1-UI182 influenza virus infection in MDCK cells, the cytotoxicity of different concentrations of HA-ADH-GA (1 μg / mL, 0.5 μg / mL, 0.1 μg / mL, 0.05 μg / mL, 0.01 μg / mL) and HA-ADH-FA (100 μg / mL, 10 μg / mL, 1 μg / mL, 0.1 μg / mL, 0.01 μg / mL) was detected using the CCK-8 assay. The results showed that HA-ADH-GA nanoparticles at the highest concentration of 1 μg / mL and HA-ADH-FA nanoparticles at the highest concentration of 100 μg / mL did not exhibit significant cytotoxicity. To understand the effects of HA-ADH-GA and HA-ADH-FA on different stages of the influenza virus life cycle, the virus was inoculated into cells using three different methods (pretreatment, combined treatment, and posttreatment) within the safe concentration range. The results showed that the combined treatment resulted in the highest cell survival rate. Therefore, HA-ADH-GA and HA-ADH-FA nanoparticles were co-treated with the virus at safe concentrations, followed by inoculation into MDCK cells. The relative absorbance (OD) at 48 h was measured using an automated microplate reader, and the inhibition rate was calculated. The inhibitory effect of HA-ADH-GA and HA-ADH-FA nanoparticles against H1N1-UI182 influenza virus gradually increased with increasing concentration. The cytotoxicity of HA-ADH-GA and HA-ADH-FA nanoparticles at concentrations of 1 μg / mL and 100 μg / mL were 11.2% and 28.3%, respectively, with inhibition rates of 89.3% and 98.3%. Their half-maximal inhibitory concentrations (EC50) were... 50 The concentrations were 0.24 μg / mL and 10.02 μg / mL, respectively, with a half-maximal toxicity concentration (CMC) of [missing value]. 50 The concentrations were 3.10 μg / mL and 236.85 μg / mL, respectively, with selectivity indices (SI) of 13.07 and 23.55, respectively. The results are as follows... Figure 4 As shown in AB. Figure 4The images show the microscopic appearance of MDCK cells in the HA-ADH-GA and HA-ADH-FA groups 36 hours after infection, representing the Mock group. It can be seen that the Mock group exhibited significant cytopathic effects, with cells showing swelling and rounding, and severely infected cells partially or completely detaching. The HA and HA-ADH groups were material groups and did not have a therapeutic effect; therefore, the cytopathic effects were equally severe. Treatment with GA and FA also resulted in some cytopathic effects. However, after treatment with HA-ADH-GA and HA-ADH-FA NPs, the number of diseased cells was reduced, most cells exhibited normal morphology, and cell state and activity were significantly improved.

[0071] Example 3: Effects of HA-ADH-GA and HA-ADH-FA NPs on the expression of vNPs in the nucleus of infected cells.

[0072] (1) Cell pretreatment: After stable passage, MDCK cells were seeded into 12-well cell culture plates and cultured in an incubator at 37°C and 5% CO2. In addition to the drug treatment group, a Control group (negative control), a Mock group (positive control), and a positive drug Osel group were also set up. When the cell density reached about 50-60%, the H1N1-UI182 virus was diluted with double-free medium and inoculated at the specified multiple of infection (MOI = 0.1, 100 TCID). 50 Working solutions of HA, HA-ADH, GA, HA-ADH-GA, and HA, HA-ADH, FA, and HA-ADH-FA were mixed with equal volumes of virus diluent and incubated at 37°C for 1.5 hours before being inoculated into MDCK cells. The control group received a double-free medium, while the Mock and OSTA groups received diluted virus solutions (100 TCID50). 50 The virus was inoculated into MDCK cells, and the virus solution was discarded after 2 hours. The Mock group was given double-free medium, and the Osel group was given 100 mg / mL Osel solution diluted in double-free medium. The cells were then incubated for 36 hours.

[0073] (2) Immunofluorescence staining: Discard the culture medium, wash 3 times with PBS, and fix with 4% paraformaldehyde on a shaker for 1 h at room temperature. Discard the fixative, wash 3 times with PBS, and permeabilize with 0.2% Triton-X100 (polyethylene glycol octylphenyl ether) for 20 min at room temperature. Discard the permeabilization solution, wash 3 times with PBS, and block with 2% BSA for 1 h at room temperature. Discard the blocking solution, wash 3 times with PBS, add 10% BSA diluted mouse monoclonal antibody (1:200 dilution), and incubate overnight at 4°C on a shaker. Wash 3 times with PBS, add 10% BSA diluted goat anti-mouse IgG (1:500) as a secondary antibody marker, and incubate for 2 h at room temperature. Nuclear staining: Wash 3 times with PBS, add 800 μL of Hoechst tetrahydrate (bisphenylimine) (10 μg / mL) to each well, and incubate for 20 min at room temperature. Wash 3 times with PBS, observe the fluorescence using a fluorescence microscope, and store at 4°C.

[0074] Influenza A virus nucleoproteins (NPs) are major protein components of the viral ribonucleoprotein (vRNP) complex in the genome, serving as the core units responsible for influenza virus replication and transcription. In the early stages of infection, NPs mediate the introduction of vRNPs into the host cell nucleus, where viral replication and transcription occur. Newly synthesized NP molecules are also targeted to the nucleus, enabling the core replication and assembly of progeny vRNPs. To investigate the effects of HA-ADH-GA and HA-ADH-FA nanoparticles on the localization and expression of vNPs in the nuclei of infected MDCK cells, indirect immunofluorescence was used for analysis. Compared to the untreated Mock group, the HA-ADH-GA and HA-ADH-FA NP groups significantly reduced the expression of influenza virus nucleoprotein (vNP) in cells. Based on experimental results of cell treatment with different infection modalities (combined treatment showed the highest cell survival rate) and experimental results of localization expression of vNPs in the nuclear cavity of infected cells (strong inhibitory effect on the expression of influenza virus nucleoproteins), it can be inferred that HA-ADH-GA and HA-ADH-FANPs effectively block early infection of cells by influenza virus by directly contacting the virus and inhibiting its entry into cells. Figure 4 As shown in GH.

[0075] Example 4: Effects of HA-ADH-GA and HA-ADH-FA NPs on viral RNA copy number in infected cells

[0076] Cell supernatants were extracted from the blank group, mock group, OSTA group, and the drug group after 48 hours of combined virus and drug treatment. Total RNA was extracted using the QIAGEN Universal RNA Kit. Influenza virus RNA copy number was detected using the Perfect Real Time One-Step Kit. The system is shown in Table 1. Data and results were analyzed using GraphPad Prism 8.0 (GraphPad Software, La Jolla, CA, USA) and are expressed as mean ± SEM. Unpaired Student's t-test was used to determine statistical significance. *P<0.05, **P<0.01, or ***P<0.001 indicated statistical significance. During each analysis, all experimental values ​​were calculated as the mean of three independent determinations and compared with the mock group.

[0077] Table 1. One-step PCR reaction amplification system

[0078]

[0079] Table 2. Specific conditions for one-step PCR amplification.

[0080]

[0081] To further investigate the expression of mRNA in host cells induced by H1N1-UI182 influenza virus, cellular RNA was extracted from cell supernatant 48 hours after infection. RT-qPCR analysis showed that, compared with the Mock group, H1N1-UI182 infection in both the HA-ADH-GA and HA-ADH-FA NPs groups significantly downregulated HA-ADH-GA (0.6 orders of magnitude) and HA-ADH-FA (0.9 orders of magnitude). Figure 4 Medium CD).

[0082] Example 5: Establishing an animal infection model

[0083] Before starting the experiment, 154 mice were fed for at least 3 days. The HA-ADH-GA NPs experiment was divided into 7 groups: Control group, Mock group (infected with virus + saline), HA group, HA-ADH group, GA group, HA-ADH-GA group, and oseltamivir phosphate (OSTA) group; the HA-ADH-FA NPs group was divided into: Control group, Mock group (infected with virus + saline), HA group, HA-ADH group, FA group, HA-ADH-FA group, and oseltamivir phosphate (OSTA) group. Except for the Control group mice, all other groups were anesthetized and given 50 μL of 5 times the MLD (medium-dose) intranasally. 50A lethal dose of the A / Changchun / 01 / 2009(H1N1) mouse-adapted strain of influenza virus was used to establish a lethal mouse model of influenza virus.

[0084] Example 6: Validation of the protective effect of HA-ADH-GA and HA-ADH-FA on infected mice and lung index

[0085] Mice were administered drugs via tail vein injection at 24 hours before infection, 2 hours after infection, 24 hours after infection, 48 hours after infection, and 72 hours after infection. The HA-ADH-GANPs group received a concentration of 20 mg / kg / day, the HA-ADH-FA NPs group received a concentration of 5 mg / kg / day, the control group received no treatment, and the mock group received an equal volume of physiological saline via tail vein injection. Injections were administered at fixed times daily, 100 μL each time, for 5 days. On days 3 and 5 (dpi), three mice from each group were randomly assigned to each group for euthanasia. Before dissection, the mice were weighed and blood was collected; lung lesions were examined, and lung weight was measured. The remaining mice were observed, and daily changes in weight and mortality were recorded until day 14 after infection.

[0086] By recording the weight changes and survival status of infected mice in each group over 14 days and performing statistical analysis, it was found that compared to the Mock group where all mice died (survival rate 0%), HA-ADH-GA improved the survival rate of mice and alleviated weight loss and infection status caused by severe infection (survival rate 80%). HA-ADH-GA began to restore weight at 10 dpi, compared to oseltamivir where weight recovery began at 9 dpi; the recovery time and extent of HA-ADH-GA were similar to oseltamivir. In contrast, HA-ADH-FA treatment only improved the survival rate of individual mice (survival rate 40%). These results indicate that HA-ADH-GA treatment significantly improved the overall survival rate of lethally infected mice (n=5) compared to HA-ADH-FA treatment. Figure 5 (Chinese AD).

[0087] Treatment with both HA-ADH-GA and HA-ADH-FA significantly improved the lung index in mice. Figure 5 This demonstrates that the infectivity of influenza virus in mice decreased after administration, a result attributable to the sustained therapeutic effect of HA-ADH-GA and HA-ADH-FA on infection.

[0088] Example 7: Detection of viral titer in mouse lungs

[0089] Lung index test: The body weight and lungs of euthanized mice were weighed, and the lung index (lung weight / body weight)*100 was calculated.

[0090] Viral titer detection: A suitable amount of lung tissue from each group of mice was added to Opti-MEM culture medium and thoroughly ground. The tissue supernatant was collected by centrifugation and then serially diluted 10-fold (1×10⁻⁶). -1 ×10 -8 Each serially diluted solution was inoculated into three 9-day-old SPF-grade chicken embryos. The embryos were incubated at 37°C for 48 hours, followed by a 1-hour cold treatment at 4°C. Simultaneously, allantoic fluid of each dilution was mixed with an equal volume of physiological saline to a final volume of 50 μL in a microplate, with duplicate wells. 50 μL of chicken blood containing 1% chicken erythrocytes was added, and hemagglutination changes were observed and measured. The viral titer was calculated using the Reed-Muench method, and the results are expressed as Log. 10 EID 50 .

[0091] Because the HA-ADH-FA group mice were in poor condition at 5 days post-contraction (dpi) and showed no significant difference compared to the Mock group, tissue samples were only collected at 3 days post-contraction (dpi) for further evaluation. Viral titer results showed that, compared to the Mock group, HA-ADH-GA significantly reduced viral titers in the lungs and nasal turbinate bones of mice at both 3 and 5 days post-contraction (dpi). Figure 6 (AD). Treatment with HA-ADH-FA at 3 dpi also reduced viral titers in the lungs and nasal turbinate bones. Figure 6 Medium EF).

[0092] Example 8: Pathological changes in mouse lungs and H&E staining

[0093] Mice were euthanized, lungs were carefully removed, and the tissue sections were preserved in 4.0% (w / v) paraformaldehyde, embedded in paraffin, and prepared for immunohistochemical sectioning. The sections were baked for 40 min, and then sequentially placed in reagent tanks for dewaxing and hydration. The specific steps were as follows: Dewaxing solution I for 15 min, Dewaxing solution II for 15 min, anhydrous ethanol for 2 min, 95% ethanol for 2 min, 80% ethanol for 2 min, 70% ethanol for 2 min, and water washing for 2 min. Hematoxylin counterstaining for 4 min, and tap water washing. Differentiation with hydrochloric acid alcohol for 2 s, and blueing for 30-40 min. Eosin staining for 2 min. 70% ethanol for 5 s, 95% ethanol for 5 s, anhydrous ethanol for 2 min, and anhydrous ethanol for 2 min. Xylene I (dewaxing solution) for 5 min, and xylene II (dewaxing solution) for 5 min. Finally, the sections were mounted with neutral resin.

[0094] Observation of the lungs and pathological examination of the heart, liver, spleen, lungs, and kidneys of mice treated with HA-ADH-GA (3 dpi and 5 dpi) and HA-ADH-FA (3 dpi) revealed pathological damage such as edema and lesions (indicated by black arrows) in the lungs of mice in the Mock group, HA group, HA-ADH group, GA monotherapy group, and FA monotherapy group. H&E staining showed severe conditions such as alveolar wall thickening and inflammatory cell infiltration (indicated by blue arrows). In contrast, the HA-ADH-GA and HA-ADH-FA treatment groups showed significant therapeutic effects, although not as significant as oseltamivir. These two nanoparticles alleviated the symptoms of pulmonary edema and reduced lung damage caused by inflammation. Figure 7 As shown. In addition to causing severe lung damage, lethal influenza virus infection also severely attacks the heart, liver, spleen, and kidneys. Therefore, we also performed H&E staining on the relevant organs of mice. The results showed that, compared with the organ tissues of mice infected with the virus alone, treatment with HA-ADH-GA and HA-ADH-FA alleviated the infection-induced peripheral inflammatory cell infiltration in cardiac tissue, loose and clear cytoplasm of liver cells, partial hepatocyte nuclear pyknosis and focal inflammatory cell infiltration, lymphocyte proliferation in the spleen, and perivascular inflammatory cell infiltration in the kidneys. Figure 8 (AC).

[0095] Example 9: Detection of inflammatory cytokine levels in mouse serum

[0096] Blood samples were collected from mice in each group on days 3 and 5 dpi. After standing at room temperature for 2 hours, the blood samples were centrifuged at 3000 rpm for 10 minutes, and serum was collected. The serum was stored at -80℃ or used directly for ELISA according to the manufacturer's instructions. The protein content of secreted inflammatory cytokines such as IL-6, TNF-α, IL-1β and IFN-α in the serum was detected using an ELISA kit.

[0097] Inflammatory factors serve as reliable biomarkers for IAV infection progression, correlated with infection severity and immune response, and severe viral infections can lead to elevated levels of pro-inflammatory factors. Interestingly, our analysis of serum inflammatory factor expression revealed that HA-ADH-GA treatment reduced the expression levels of secreted inflammatory cytokines IFN-α, TNF-α, IL-6, and IL-1β in serum. Figure 9 (AD). Treatment with HA-ADH-FA only revealed a decrease in IL-6 and TNF-α expression ( Figure 9 These results indicate that treatment with HA-ADH-GA and HA-ADH-FA significantly improved the immune function of mice during influenza virus infection, such as... Figure 9 As shown.

[0098] Example 10: Detection of viral RNA copy number in mouse lungs

[0099] Lung tissue was completely lysed using a strong lysis buffer. Total RNA was reverse transcribed into cDNA using a Perfect Real Time kit, and the cDNA was stored at -20°C or used directly for amplification. The reverse transcription process consisted of two steps: 1) removal of genomic DNA; 2) the reverse reaction. The reaction systems are shown in Tables 3, 4, and 5; PCR amplification conditions are shown in Table 6; primer sequences are shown in Table 7. The normalized gene β-actin was used as an internal control.

[0100] Table 3 Genomic DNA Removal Reactions

[0101]

[0102] After reacting the product to be reversed at room temperature for 15 minutes, reverse transcription was performed, as shown in the table below:

[0103] Table 4 Reverse reaction

[0104]

[0105] The prepared reverse PCR system was incubated in a 37°C water bath for 45 minutes to obtain cDNA. The RT-qPCR system was then prepared as shown in the table below:

[0106] Table 5 RT-qPCR amplification system

[0107]

[0108] Table 6. Specific conditions for RT-qPCR amplification.

[0109]

[0110] Table 7 RT-qPCR amplification system

[0111]

[0112] Compared to other influenza virus genes (such as PB2 and NP), the M gene, especially the M1 gene, has evolved relatively slowly. In other words, the influenza virus M gene is relatively conserved and less prone to mutation compared to other genes. Therefore, primers for the M gene were selected for RT-qPCR detection of influenza virus. Highly pathogenic influenza virus infection causes sustained release and accumulation of inflammatory factors. Therefore, to determine the expression of influenza virus and inflammatory factors in lung tissue, we used RT-qPCR. The results showed that infection induced increased mRNA expression levels of influenza virus at 3 dpi and 5 dpi in mouse lung tissue, as well as inflammatory cytokines IFN-α, TNF-α, IL-6, and IL-1β. The therapeutic effect of HA-ADH-GA was positive and significant; the reduction in influenza virus and cytokine mRNA expression demonstrated that HA-ADH-GA could alleviate severe influenza virus infection in mice. Figure 10 (A and B) and excessive immune stress in mice ( Figure 10 (CF).

[0113] Example 11: Detection of vNP protein expression in mouse lungs due to influenza virus

[0114] After treating the cell plates with the virus and drug for 48 hours, remove them from the incubator, discard the supernatant, and wash the cells three times with 1 mL of PBS. Add 200 μL of IRA strong lysis buffer to each well and lyse on ice for 45 min–1 h, shaking occasionally every ten minutes. After lysis, centrifuge at 4°C for 10 min to remove impurities, and transfer the supernatant to a new EP tube. Prepare the BCA working solution by thoroughly mixing solutions A and B of the BCA reagent as needed for the experiment. BSA protein standard solution (0.5 mg / mL) was added to the standard wells of a 96-well plate at concentrations of 0, 1, 2, 4, 8, 12, 16, and 20 μL, respectively, and then PBS was added to bring the total to 20 μL. The sample was then added to the sample wells of the 96-well plate. If the sample volume was less than 20 μL, PBS was added to bring the total to 20 μL. 200 μL of BCA working solution was added to each well, and the plate was incubated at 37°C for 60 min. The absorbance of the standard and sample was measured at 562 nm using a microplate reader to obtain a standard curve. The protein concentration of the corresponding sample was calculated based on the sample volume. After boiling the sample, it was added to the gel plate, and electrophoresis buffer was added to the appropriate level. Place the membrane under constant voltage (80V) and allow protein electrophoresis to reach the bottom of the separating gel. Transfer the membrane using a PVDF membrane at a constant current of 300mA for 90 minutes. Block with 5% BSA blocking buffer for 1 hour. After blocking, remove the PVDF membrane, cut and label the target bands according to the marker table. Incubate overnight at 4°C on a shaker / 4 hours at room temperature, washing the bands three times with TBST. After washing, place the bands in the secondary antibody incubator and incubate at room temperature for 2 hours, washing three times with TBST. Expose the membrane to the light using an automated fluorescence chemiluminescence imaging system at an appropriate exposure time and photograph the membrane. Use Image... J software was used for grayscale analysis of protein bands. Data and results were analyzed using GraphPadPrism 8.0 (GraphPad Software, LaJolla, CA, USA) and are expressed as mean ± SEM. Unpaired Student's t-test was used to determine statistical significance. *P<0.05, **P<0.01, or ***P<0.001 indicated statistical significance. At least three wells were used for each concentration during each analysis. All values ​​shown in the above experiments are calculated as the mean of three independent measurements and compared with the mock group.

[0115] The JNK / SAPK signaling pathway can be activated by stress stimuli (such as ultraviolet radiation, heat shock, hyperosmolar stimulation, and protein synthesis inhibitors), inflammatory cytokines (TNF-α), growth factors (EGF), and certain G protein-coupled receptors. Combining Examples 9 and 10, we found that HA-ADH-GA reduced the expression of inflammatory factors IL-6, IL-1β, TNF-α, and IFN-α in both serum and RNA. Therefore, we hypothesized that HA-ADH-GA might reduce the expression of inflammatory factors by activating the SAPK / JNK signaling pathway. Therefore, we analyzed the relevant proteins in this signaling pathway using Western blotting. The results showed that viral infection promoted the phosphorylation of MAPK / JNK and also increased the protein expression level of vNP. However, the opposite trend was observed after treatment with HA-ADH-GA. Figure 11 Therefore, it can be concluded that HA-ADH-GA can inhibit the activation of the SAPK / JNK signaling pathway mediated by influenza virus infection, thereby reducing the release of inflammatory factors.

[0116] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A conjugate nanoparticle drug delivery system, characterized by: The drug delivery system is either a hyaluronic acid-glycyrrhetinic acid conjugate nanoparticle drug delivery system (HA-ADH-GANPs) or a hyaluronic acid-ferulic acid conjugate nanoparticle drug delivery system (HA-ADH-FANPs). The structure of the hyaluronic acid-glycyrrhetinic acid conjugate nanoparticles (HA-ADH-GA) is as follows: The structure of the hyaluronic acid-ferulic acid conjugate nanoparticles (HA-ADH-FA) is as follows:

2. The conjugate nanoparticle drug delivery system according to claim 1, characterized in that: The drug delivery system HA-ADH-GANPs is synthesized by combining hydrophilic material HA and hydrophobic drug GA separately with adipic acid dihydrazide and then self-assembling them into NPs. The drug delivery system HA-ADH-FANPs is synthesized by combining hydrophilic material HA and hydrophobic drug FA separately with adipic dihydrazide and then self-assembling them into NPs.

3. The method for preparing the conjugate nanoparticle drug delivery system according to claim 1, characterized in that: The synthesis of the hyaluronic acid-glycyrrhetinic acid conjugate includes the following steps: (1) Synthesis of hyaluronic acid-adipate dihydrazide: Weigh HA with a molecular weight of 8 kDa and place it in a round-bottom flask. Add deionized water to dissolve it, then add activator EDC·HCl to activate it. Then add adipate dihydrazide (ADH) to the solution. After the solid dissolves, add HCl to adjust the pH to 4.7 and react under argon protection. Then, adjust the pH to neutral with NaOH solution and transfer the reaction solution to a dialysis bag. Dialyze with deionized water to remove unreacted EDC and ADH. Filter the solution with a microporous membrane and then transfer it to a dry glass dish. After pre-freezing overnight, freeze-dry to obtain the intermediate HA-ADH (8 kDa). (2) Synthesis of hyaluronic acid-glycyrrhetinic acid: GA was weighed and dissolved in N,N-dimethylformamide (DMF), and EDC·HCl was added and placed on a magnetic stirrer for activation. The product HA-ADH (8kDa) in (1) was weighed and added to an appropriate amount of deionized water to completely dissolve it. The reaction flask after dissolution was then placed in an ice bath for pre-cooling. The activated GA was added dropwise to the HA-ADH solution. At this time, GA precipitated and the solution became turbid. An appropriate amount of DMF was added until the reaction solution became clear and transparent again. The reaction was carried out overnight under argon protection. The unreacted EDC and GA were removed by dialysis. The dialysis solution was deionized water. The unreacted GA precipitated at the bottom of the solution. To remove the precipitate, the final reaction solution was poured from the dialysis bag into a centrifuge tube. After centrifugation, the supernatant was filtered through a microporous membrane into a dry glass dish. After pre-freezing overnight, the supernatant was freeze-dried to obtain the final product HA-ADH-GA (8kDa).

4. The method for preparing the conjugate according to claim 1, characterized in that: The synthesis of the hyaluronic acid-ferulic acid conjugate includes the following steps: (1) Synthesis of hyaluronic acid-adipate dihydrazide: Weigh HA with a molecular weight of 98kDa and place it in a round-bottom flask. Add deionized water to dissolve it, then add activator EDC·HCl to activate it. Then add adipate dihydrazide (ADH) to the solution. After the solid dissolves, add HCl to adjust the pH to 4.7 and react under argon protection. Then, adjust the pH to neutral with NaOH solution and transfer the reaction solution to a dialysis bag. Dialyze with deionized water to remove unreacted EDC and ADH. Filter the solution with a microporous membrane and then transfer it to a dry glass dish. After pre-freezing overnight, freeze-dry to obtain the intermediate HA-ADH (98kDa). (2) Synthesis of hyaluronic acid-glycyrrhetinic acid: FA was weighed and dissolved in N,N-dimethylformamide (DMF), EDC·HCl was added and placed on a magnetic stirrer for activation; HA-ADH (98kDa) from (1) was weighed and added to an appropriate amount of deionized water to completely dissolve it, and then the reaction flask after dissolution was placed in an ice bath for pre-cooling; the activated FA was added dropwise to the HA-ADH solution, at which point FA precipitated and the solution became turbid, and an appropriate amount of DMF was added until the reaction solution became clear and transparent again, and the reaction was carried out overnight under argon protection; the unreacted EDC and FA were removed by dialysis, and the dialysis solution was deionized water; the unreacted FA settled at the bottom of the solution in the form of a precipitate. In order to remove the precipitate, the final reaction solution was poured from the dialysis bag into a centrifuge tube, centrifuged and the supernatant was filtered through a microporous membrane into a dry glass dish, pre-frozen overnight, and then freeze-dried to obtain the final product HA-ADH-FA (98kDa).

5. The preparation method according to claim 3, characterized in that: In step (1), the molar ratio of HA, EDC·HCl and ADH is 1:1.2:3; in step (2), the molar ratio of GA, EDC·HCl and HA-ADH is 1:1.5:1.

2.

6. The preparation method according to claim 4, characterized in that: In step (1), the molar ratio of HA, EDC·HCl and ADH is 1:1.2:3; in step (2), the molar ratio of FA, EDC·HCl and HA-ADH is 1:1.5:1.

2.

7. The preparation method according to claim 3, characterized in that: Dialysis was performed using a 3600Da dialysis bag.

8. The preparation method according to claim 4, characterized in that: Dialysis was performed using an 8000Da dialysis bag.

9. The preparation method according to claim 3 or 4, characterized in that: The synthesis process is carried out at room temperature; the diameter of the filter membrane is 0.45 μm.

10. The use of the hyaluronic acid-glycyrrhetinic acid conjugate and the hyaluronic acid-ferulic acid conjugate according to 1 or 2 in the preparation of drugs against type A H1N1 influenza virus.

Images