GSH / pH double-response arctigenin nanoparticles as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of antivirus, and particularly relates to GSH / pH double-response arctigenin nanoparticles as well as a preparation method and application thereof. According to the invention, traditional Chinese medicine active components arctigenin and carboxymethyl chitosan are connected through a disulfide bond, and a series of reaction treatments are carried out to prepare the arctigenin nanoparticles with GSH / pH double response. Results of the embodiment of the invention show that the dual-response nanoparticles can selectively release the drug under specific conditions, and more accurate drug release can be realized, so that the treatment effect is improved, the influence on normal cells can be reduced, the biocompatibility of the drug is improved, and the side effect is reduced.

Description

Technical Field

[0001] The invention belongs to the field of antiviral technology, and specifically relates to an antiviral GSH / pH dual-responsive arctiin nanoparticle and a preparation method and application thereof. Background Art

[0002] Arctigenin is a natural lignan compound extracted from burdock (Arctium lappa). It is used in traditional medicine to treat a variety of diseases, such as rheumatism, cough, and indigestion. Modern research has found that arctigenin has antioxidant, anti-inflammatory, immunomodulatory, and antiviral biological activities. Arctigenin can inhibit viral replication by affecting viral protein expression and replication pathways, and eliminate viruses by enhancing the host's immune response. Its antioxidant properties can also reduce cell damage caused by viral infection. Arctigenin's broad-spectrum antiviral activity, immunomodulatory ability, and relatively low toxicity make it a potential candidate for antiviral drugs.

[0003] Carboxymethyl chitosan is a chitosan derivative obtained through carboxymethylation reaction. Compared with chitosan, it has good water solubility in neutral and slightly acidic environments, which helps the drug release through diffusion mechanism and water molecules can quickly penetrate into the carrier to promote the detachment of drug molecules. In addition, due to the presence of carboxyl groups, carboxymethyl chitosan has higher hydrophilicity, larger specific surface area, stronger drug loading capacity and controlled release characteristics, which is suitable for intelligent drug delivery systems.

[0004] Porcine circovirus (PCV) is a small single-stranded circular DNA virus belonging to the Circoviridae family. Among them, porcine circovirus type 2 (PCV2) is an important viral pathogen present in major pig-raising countries around the world, causing huge economic losses to major pig-raising countries around the world every year. Porcine circovirus type 2 (PCV2) mainly infects pigs, often causing symptoms such as slow growth, immunosuppression, respiratory problems and skin lesions in pig herds. In addition, porcine circovirus type 2 can be complicated or secondary to swine fever, porcine epidemic diarrhea, multiple systemic atrophy, etc., which complicates the epidemic and aggravates economic losses. Effective measures to prevent and control PCV-2 infection include vaccination, strengthening biosecurity measures and good feeding management. Vaccination is the key to preventing infection and can significantly reduce clinical symptoms and economic losses. In addition, regular monitoring of the health status of the pig herd and providing balanced nutrition are also crucial. Since there is currently no specific antiviral treatment, supportive treatment plays an important role in infection management. The control of PCV2 is of great significance for improving the economic benefits of farming and ensuring the health of pigs.

[0005] Targeted drug delivery is a very important part of modern drug delivery systems. Its purpose is to accurately deliver drugs to the lesion site, maximize the therapeutic effect and reduce damage to normal tissues. Commonly used targeted drug delivery carriers mainly include nanoparticles and gels. Nanoparticles usually refer to tiny solid particles with a diameter between 1 and 1000 nanometers. The smaller size of nanoparticles can significantly increase the specific surface area of ​​the drug, thereby improving the solubility and bioavailability of poorly soluble drugs; nanoparticles can be administered through a variety of routes such as oral administration and injection, and have a wide range of applications; they have strong biological barrier penetration ability and are suitable for targeted therapy. Compared with nanoparticles, when gels are used as drug delivery carriers, their drug release mainly depends on the infiltration and diffusion of the structure, which makes them have certain controlled release characteristics but not as accurate as nanoparticles; in addition, drug release is also affected by environmental factors such as temperature and pH, resulting in uneven release. Although gels also show advantages in local administration, nanoparticles are obviously more flexible and effective in systemic treatment, targeting and controlled release.

[0006] Therefore, there is an urgent need to develop an antiviral drug that uses nanoparticles as targeted drug delivery carriers to treat PCV2. Summary of the invention

[0007] The purpose of the present invention is to provide a GSH / pH dual-responsive arctiin nanoparticle (PGA). The GSH / pH dual-responsive arctiin nanoparticle prepared by the present invention can not only effectively resist viruses, but also achieve more accurate drug release, improve the biocompatibility of drugs, and thus reduce side effects.

[0008] In order to achieve the above object, the present invention provides the following technical solutions:

[0009] The invention provides a GSH / pH dual-response arctiin nanoparticle. The nanoparticle is prepared from carboxymethyl chitosan and arctiin.

[0010] Preferably, the particle size of the nanoparticles is 500 nm; and the drug loading of the nanoparticles is 12.38%.

[0011] The present invention also provides a method for preparing the above-mentioned nanoparticles, which specifically comprises the following steps:

[0012] S1. Weigh a disulfide bond-containing carboxylic acid compound and dissolve it in a mixed organic solvent, add a catalyst until it is completely dissolved, add an activator and stir to activate it, to obtain an activated reaction system;

[0013] S2, dissolving arctiin in N,N-dimethylformamide to obtain an arctiin solution, and adding the solution dropwise to the reaction system of step S1 for reaction, analyzing and extracting the reaction solution, and then drying it to obtain an intermediate product;

[0014] S3, dissolving the intermediate product of step S2 in N,N-dimethylformamide, adding 1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide hydrochloride for activation, and dropping it into a 2% carboxymethyl chitosan aqueous solution for reaction, freeze-drying, dispersing the freeze-dried product in PBS, and obtaining nanoparticles by ultrasound;

[0015] The disulfide bond-containing carboxylic acid compound is 3,3'-dithiodipropionic acid;

[0016] The mixed organic solvent is a mixture of N,N-dimethylformamide solution and dichloromethane.

[0017] Preferably, in step S1, the concentration of the carboxylic acid compound after being dissolved in the mixed organic solvent is 0.1-0.25 mol / ml; the catalyst is triethylamine, and the amount of triethylamine added is 0.1-0.5 eq of the amount of the carboxylic acid compound substance; the activating agents are 1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide hydrochloride and 4-(dimethylamino)pyridine, and the amounts of 1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide hydrochloride and 4-(dimethylamino)pyridine added are 1.0-2.0 eq and 0.2-1.0 eq of the amount of the carboxylic acid compound substance.

[0018] Preferably, the concentration of the arctigenin solution in step S2 is 0.2-0.5 mol / ml; and the reaction conditions of the reaction solution are 60° C. for 12-48 hours.

[0019] Preferably, in step S3, the concentration of the intermediate product after being dissolved in N,N-dimethylformamide is 0.2 mol / ml; the amount of 1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide hydrochloride added is 1.0 to 2.0 eq of the amount of the intermediate product substance; and the ratio of the amount of carboxymethyl chitosan added to the amount of the intermediate product substance is 1:(1-5).

[0020] Preferably, the step S3 further comprises dispersing the lyophilized product in a buffer solution at pH 7.4 and ultrasonically treating the product for 10 to 30 minutes; the concentration of the lyophilized product in the buffer solution is 1 mg / ml.

[0021] The present invention also provides the use of the nanoparticles or the nanoparticles prepared by the above preparation method in the preparation of antiviral products or medicines.

[0022] Preferably, the virus comprises PCV-2 virus.

[0023] The present invention also provides the use of the nanoparticles or the nanoparticles prepared by the above preparation method in the preparation of anti-inflammatory products or medicines.

[0024] Beneficial effects of the present invention:

[0025] The GSH / pH dual-responsive arctiin nanoparticles of the present invention can selectively release drugs under specific conditions, can achieve more accurate drug release, thereby improving the therapeutic effect, and can also reduce the impact on normal cells, improve the biocompatibility of the drug, thereby reducing side effects.

[0026] The drug loading amount of the GSH / pH dual-responsive arctiin nanoparticles of the present invention is 12.38%, the antiviral and anti-inflammatory effects of the nanoparticles are better than those of naked drug arctiin, and the cell uptake effect is also better than that of the naked drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0028] Figure 1 This is a comparison diagram of infrared characterization;

[0029] Figure 2 This is a comparison diagram of NMR characterization;

[0030] Figure 3 is the scanning electron microscope image of the nanoparticles;

[0031] Figure 4 It is the particle size analysis diagram of nanoparticles;

[0032] Figure 5 This is the arctigenin liquid phase standard curve;

[0033] Figure 6 This is the UV standard curve of arctigenin;

[0034] Figure 7 It is the cumulative release curve of the drug;

[0035] Figure 8 This is a diagram of safe cell concentrations, with the left diagram showing ACT and the right diagram showing PGA;

[0036] Fig. 9 This is a graph showing the antiviral effects of ACT and PGA;

[0037] Fig.10 This is a graph showing the anti-inflammatory effects of ACT and PGA;

[0038] Fig.11 This is a comparison chart of the anti-inflammatory effects of carboxymethyl chitosan and chitosan;

[0039] Fig.12 Figure 2 shows the uptake of ACT and PGA by cells. DETAILED DESCRIPTION

[0040] Based on the difference between the PCV2 infection microenvironment and the normal site, the invention uses a compound with a disulfide bond as a connecting arm to connect carboxymethyl chitosan and arctiin to prepare GSH / pH dual-responsive arctiin nanoparticles.

[0041] The preparation method of the GSH / pH dual-responsive arctiin nanoparticles provided by the present invention specifically comprises the following steps:

[0042] S1. Weigh a disulfide bond-containing carboxylic acid compound and dissolve it in a mixed organic solvent, add a catalyst until it is completely dissolved, add an activator and stir to activate, to obtain an activated reaction system; the mixed organic solvent is a mixture of N,N-dimethylformamide solution and dichloromethane.

[0043] S2. Dissolve arctiin in N,N-dimethylformamide to obtain arctiin solution, and add it dropwise to the reaction system of step S1 for reaction. After analyzing and extracting the reaction solution, spin-drying to obtain an intermediate product, the reaction solution is analyzed by thin layer chromatography, and the reaction result is extracted with ethyl acetate; the chromatographic column is Agilent ZORBAX SB-C18 (250×4.6mm, 5μm); the detection wavelength is 280nm; the mobile phase is methanol-acetonitrile-water (31:20:49, V:V:V); the tassel is 1.0mL / min; the injection volume is 30μL; and the column temperature is (30±0.1)℃.

[0044] S3, dissolving the intermediate product of step S2 in N,N-dimethylformamide, adding 1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide hydrochloride for activation, and adding dropwise to a 2% carboxymethyl chitosan aqueous solution for reaction, and after freeze-drying, dispersing the freeze-dried product in PBS, and obtaining nanoparticles by ultrasound; the freeze-dried product is a yellow to white powder product.

[0045] Furthermore, in step S1, the concentration of the carboxylic acid compound after being dissolved in the mixed organic solvent is 0.1 to 0.25 mol / ml; the catalyst is triethylamine, and the amount of triethylamine added is 0.1 to 0.5 eq of the amount of the carboxylic acid compound substance; the activating agents are 1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide hydrochloride and 4-(dimethylamino)pyridine, and the amounts of 1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide hydrochloride and 4-(dimethylamino)pyridine added are 1.0 to 2.0 eq and 0.2 to 1.0 eq of the amount of the carboxylic acid compound substance.

[0046] Furthermore, the concentration of the arctigenin solution in step S2 is 0.2-0.5 mol / ml; and the reaction conditions of the reaction solution are 60° C. for 12-48 hours.

[0047] Furthermore, the step S3 also includes dispersing the freeze-dried product in a buffer solution with a pH of 7.4 and ultrasonically treating it for 10 to 30 minutes; the concentration of the freeze-dried product in the buffer solution is 1 mg / ml; the concentration of the intermediate product in the step S3 after being dissolved in N,N-dimethylformamide is 0.2 mol / ml; the amount of 1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide hydrochloride added is 1.0 to 2.0 eq of the amount of the intermediate product substance; the ratio of the amount of carboxymethyl chitosan added to the amount of the intermediate product substance is 1:(1-5).

[0048] In the present invention, eq (equivalent) refers to an amount equivalent to a specific or conventional value; a chemistry term used to refer to the mass ratio of substances when they interact. In chemical reactions, equivalent is an important concept used to express the quantitative relationship between reactants.

[0049] The disulfide bond described in the present invention is formed by two sulfur atoms connected by a covalent bond, and is widely present in many proteins and small molecules. In glutathione, disulfide bonds mainly exist in the oxidized form (GSSG), which can help stabilize the three-dimensional structure of proteins. Disulfide bonds produce a reduced state through glutathione catalysis, directly removing oxidative substances in cells; disulfide bonds participate in the repair and stabilization of proteins, help maintain the reducing environment of cells, and protect cells from oxidative damage; disulfide bonds have good stability in biological environments, are easily reduced to sulfhydryl groups, and promote the reduction reaction. Compared with similar diselenide bonds, the latter has poor stability in vivo, and mainly exhibits a strong antioxidant effect by directly neutralizing ROS, but the reaction is relatively slow and can cause cytotoxicity and adverse reactions in some cases.

[0050] The microenvironment responsive nanoparticles of the present invention refer to nanoparticles that can undergo physical or chemical property changes under specific biological or chemical microenvironment conditions (such as pH value, temperature, enzyme concentration, oxygen level, etc.). They can adjust their release characteristics or morphology according to changes in their environment (such as changes in pH value, temperature increase or the presence of specific enzymes, etc.). This intelligent property enables them to achieve precise release of drugs under specific conditions. Microenvironment responsive nanoparticles are usually synthesized by self-assembly, polymer modification and specific functionalization of nanomaterials. Polymer nanoparticles, metal nanoparticles and liposomes can all be designed into responsive nanoparticles.

[0051] The GSH / pH dual response of the present invention refers to the dual response characteristics of a material or system to changes in glutathione (GSH) concentration and pH value under specific conditions. This characteristic is of great significance in drug delivery, nanotechnology and biomedical applications.

[0052] Experiments have shown that the GSH / pH dual-responsive arctiin nanoparticles provided by the present invention have a drug loading of 12.38%, the antiviral and anti-inflammatory effects of the nanoparticles are better than those of the naked drug, and the cellular uptake effect is also better than that of the naked drug. Furthermore, the GSH / pH dual-responsive arctiin nanoparticles can be used to prepare anti-inflammatory or antiviral products or medicines.

[0053] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0054] The production processes, experimental methods or detection methods involved in the embodiments of the present invention, unless otherwise specified, are all conventional methods in the prior art, and their names and / or abbreviations are all conventional names in the field, and are very clear and unambiguous in the relevant application fields. Technical personnel in the field can understand the conventional process steps based on the names and apply the corresponding equipment, and implement them according to conventional conditions or the conditions recommended by the manufacturer.

[0055] Arctigenin, with a content of 99%, was prepared by the Animal Disease Prevention and Control Laboratory of Wuhan Institute of Animal Husbandry and Veterinary Sciences; solvents such as dichloromethane, dimethyl sulfoxide, N,N-dimethylformamide, acetonitrile, methanol, triethylamine, and hydrochloric acid, and catalysts such as 1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide hydrochloride and 4-(dimethylamino)pyridine were all purchased from Sinopharm Chemical Reagent Co., Ltd.

[0056] SCIENTZ-IID ultrasonic cell disruptor (Ningpo Xinzhi Biotechnology Co., Ltd.); ZetasizerNANO-ZS 90 nanometer particle size surface potential analyzer (Malvern, UK); JEM-2100PLUS transmission electron microscope (JEOL Ltd.); ME104E analytical balance (Mettler, USA); D3024R small high-speed low-temperature centrifuge (SCILOGEX); IQ-7000 ultrapure water instrument (Millipore, France); 1260 high-performance liquid chromatograph (Agilent, USA), Agilent ZORBAX SB-C18 chromatographic column (250×4.6mm, 5μm); Cary 630FTIR Fourier transform infrared spectrometer (Agilent, USA); RE-52A rotary evaporator (Shanghai Lichen Instrument Technology Co., Ltd.), ZLGJ-10 freeze dryer (Zhengzhou Kewanda Biological Instrument Co., Ltd.), UV-1800 ultraviolet-visible spectrophotometer (Shimadzu, Japan); microplate reader, ThermoMultiskan FC; nucleic acid concentration meter, Thermo Fisher (NanoDrop One); DELI digital caliper (DL91150), QuantStudio real-time quantitative polymerase chain reaction (q-PCR) instrument (Thermo Fisher).

[0057] The other instruments, equipment, raw materials or reagents used in the embodiments of the present invention are not particularly limited in their sources, and are all conventional products that can be purchased through regular commercial channels, or can be prepared according to conventional methods well known to those skilled in the art.

[0058] Example 1 Preparation of dual-responsive arctiin nanoparticles (PGA)

[0059] (1) Weigh 1-5 mol of 3,3'-dithiodipropionic acid; dissolve it in 5-10 mL of N,N-dimethylformamide solution and 5-10 mL of dichloromethane, and add a catalytic amount of triethylamine (0.1-0.5 eq) until it is completely dissolved.

[0060] Under ice bath, add 1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide hydrochloride (1.0-2.0 eq) and 4-(dimethylamino)pyridine (0.2-1.0 eq) in sequence, stir and activate for 30-60 min to activate the carboxyl group in the disulfide bond-containing carboxylic acid compound; then weigh 1-5 mol of arctigenin and dissolve it in 5-10 mL of N,N-dimethylformamide, slowly add it dropwise to the above reaction system, and react at 60°C for 12-48 h.

[0061] (2) The reaction solution was analyzed by thin layer chromatography, extracted with ethyl acetate, and then dried by spin drying to obtain an intermediate product.

[0062] The chromatographic column was Agilent ZORBAX SB-C18 (250×4.6mm, 5μm); the detection wavelength was 280nm; the mobile phase was methanol-acetonitrile-water (31:20:49, V:V:V); the flow rate was 1.0mL / min; the injection volume was 30μL; and the column temperature was (30±0.1)℃.

[0063] (3) Add 5-10 mL N,N-dimethylformamide to dissolve 1-2 mol of the intermediate product, add 1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide hydrochloride (1.0-2.0 eq) again to activate the carboxyl group, slowly add it dropwise to the 2% carboxymethyl chitosan aqueous solution, react at 40°C for 12-48 hours to obtain the final product. According to the molecular weight and substitution amount of carboxymethyl chitosan, the designed feed molar ratio is 1:1, 1:2, 1:3; 1:4, 1:5.

[0064] (4) The above product is freeze-dried to obtain a light yellow to white powder product.

[0065] (5) Disperse 10 mg of the product in 10 mL of PBS (pH 7.4) and treat with an ultrasonic cell disruptor for 10 to 30 min to allow it to self-assemble into nanoparticles (PGA).

[0066] Example 2 is a comparison diagram of NMR characterization

[0067] HNMR analysis: 10 mg of product (PGA), carboxymethyl chitosan (O-CMCS), intermediate product (SSA), and arctigenin (ACT) were weighed and placed in a vacuum drying oven for 24 h, and then dissolved in 500 μL of deuterated water and deuterated DMSO, respectively.

[0068] The nuclear magnetic resonance hydrogen spectrum (1HNMR) of each sample is measured, such as Figure 1 As shown. Among them, the hydroxyl peak at 8.7ppm on ACT disappeared, indicating that an esterification reaction occurred. The characteristic signal at 2.8ppm on the nanoparticles is the amide bond produced by the acylation reaction between the amino group on the carboxymethyl chitosan and the intermediate product. This shows that the PGA amphiphilic molecule has been successfully prepared.

[0069] Example 3 Infrared Characterization Comparison

[0070] Weigh 10 mg of ACT, SSA, O-CMCS, and PGA powder respectively, dry them thoroughly in a vacuum drying oven, and mix them with dry potassium bromide powder in a ratio of 1:10. Grind them thoroughly and dry them thoroughly under a mercury lamp, and take an appropriate amount of powder to make tablets in a mold.

[0071] Fourier transform infrared spectrometer (FTIR) is mainly used to detect and analyze the chemical structure and composition of substances.

[0072] The infrared spectra (FT-IR) of each sample were recorded by Fourier transform infrared spectrometer. The strong peak at 1761cm-1 should be attributed to the stretching vibration of the ester bond (C=O) generated by the esterification reaction, the peak at 1180cm-1 should be attributed to the stretching vibration peak of the corresponding ester bond (COC), the peak at 1640cm-1 should be attributed to the stretching vibration peak of the amide (C=O) of PGA, which is the absorption peak of the amide I band, and the peak at 1592cm-1 is the stretching vibration peak of the amide (NH), which is the absorption peak of the amide II band. The results are as follows Fig.11 As shown, it further indicates that the PGA substance was successfully synthesized.

[0073] Example 4 Nanoparticles and Particle Size Determination

[0074] Weigh an appropriate amount of nanoparticles and dissolve them in pure water to make the final concentration of the nanoparticles 5 mg / mL. Ultrasonicate for 15 minutes to evenly distribute the particles. Use a micropipette to take out the dispersed liquid and drop it on a clean glass slide, evenly distribute it, and vacuum dry it. Since the sample is a non-conductive material, the sample is sprayed with gold.

[0075] The morphology was observed by scanning electron microscopy. Figure 3 As shown. Through particle size measurement and analysis, the results are as follows Figure 4 As shown, the particle size of the nanoparticles is about 500 nm.

[0076] Example 5 Determination of arctigenin content in nanoparticles

[0077] Weigh two portions of PGA, 1 mg each. Dissolve each in 5 mL of methanol. Adjust the pH of one portion to about 5.4 with hydrochloric acid, and add GSH to create a GSH response environment. Prepare PGA with concentrations of 0, 10, 20, 30, 40, and 50 μg / ml and incubate overnight. Measure the peak area, substitute it into the standard curve, and calculate the concentration of free arctigenin. The results are as follows: Figure 5 As shown, the drug loading was measured to be 12.38%.

[0078] Example 6 Determination of nanoparticle responsive release

[0079] Weigh 4 portions of 5 mg PGA nanoparticles, transfer them to activated dialysis bags, and place them in 50 mL of release systems of pH 7.4 GSH 0 mM, pH 7.4 GSH 10 mM, pH 5.5 GSH 10 mM, and pH 5.5 GSH 0 mM for reaction.

[0080] Samples were taken at 1, 3, 6, 10, 24, and 48 hours of reaction, and the absorbance measured by UV was substituted into the standard curve to calculate the final ACT arctigenin concentration. The results are as follows: Figure 6 shown.

[0081] The results of the drug cumulative release curve are as follows Figure 7 As shown, arctigenin release was optimal at pH 5.5 and GSH 10 mM, that is, the product had a GSH / pH dual response.

[0082] Example 7 Safety Study of Nanoparticles

[0083] The CCK-8 method was used to detect and compare the maximum safe concentrations of ACT and PGA in cells.

[0084] The results are as follows Figure 8 As shown in the results, the maximum safe concentration of ACT was 15.625 μg / mL, at which the cell survival rate was 102.9%. The maximum safe concentration of PGA was 62.5 μg / mL (because the drug loading of PGA was 12.38%, equivalent to an ACT concentration of 7.74 μg / mL), at which the cell survival rate was 102.3% (P < 0.05), further indicating that the synthetic PGA nanoparticles were safe.

[0085] Example 8 Nanoparticle Antiviral Test

[0086] The standard plasmid of PCV-2 virus was extracted and tested by RT-qPCR. The result was 2×10 10 Take 9 EP tubes (3μL to 27μL) and dilute with DEPC water, and do 3 replicates. Set up four groups, one of which is a blank control group, and the other three experimental groups are ACT+PCV-2, PGA+PCV-2 and PCV-2 groups.

[0087] The standard curve is established with Log as the horizontal axis and Ct value as the vertical axis: y = -2.8348x + 35.422R 2 =0.9981.

[0088] Different concentrations of ACT (1000, 500, 250, 125, 62.5, 31.25, 15.625, 7.8125 μg / mL) and PGA (500, 250, 125, 62.5, 31.25, 15.625, 7.8125, 3.90625 μg / mL) were diluted. The Ct values ​​of different concentrations of ACT and PGA were substituted into the formula, and the results were plotted using GraphPad. The results are shown in Figure 2. Fig. 9As shown, the anti-PCV-2 effect of ACT and PAG increases with the increase of concentration. ACT has the strongest anti-PCV-2 effect at a concentration of 15.625μg / mL; PGA has the strongest anti-PCV-2 effect at a concentration of 62.5μg / mL. When PGA is 31.25μg / mL and the drug loading is 3.86875μg / mL, which is approximately equal to the content when ACT is 3.98125μg / mL, a comparison of the two shows that PGA has a better anti-PCV-2 effect than ACT.

[0089] Example 9 Nanoparticle Anti-Inflammation Test

[0090] PK-15 cells were inoculated in 24-well cell culture plates and cultured for 24 hours. The PCV-2 virus solution diluted with DMEM was inoculated in a 24-well culture plate with a monolayer of PK-15 cells, placed in an incubator for incubation for 2 hours, and the supernatant was discarded. Different concentrations of ACT and PGA were diluted, ACT (1000, 500, 250, 125, 62.5, 31.25, 15.625, 7.8125 μg / mL) and PGA (500, 250, 125, 62.5, 31.25, 15.625, 7.8125, 3.90625 μg / mL), and inoculated in cell culture plates, with 3 replicates in each group. The experiment was divided into four groups, one of which was a blank control group, and the other three experimental groups were ACT+PCV-2, PGA+PCV-2 and PCV-2 groups.

[0091] The cells were cultured in a 37°C CO2 constant temperature cell culture incubator for 6 h. Single-stranded DNA was extracted using a nucleic acid extraction kit, and the expression levels of IL-6, TNF-α, and IL-1β in the cells were measured using RT-qPCR.

[0092] The results are as follows Fig.10 As shown in the figure, after PK-15 cells were infected with PCV-2 cells for 6 hours, the expression of IL-6, TNF-α and IL-1β mRNA was significantly increased compared with the blank group, indicating that PCV-2 can induce inflammatory response in cells, and ACT and PGA can significantly reduce the expression of IL-6, TNF-α and IL-1β mRNA after acting on cells. This shows that ACT and PGA can inhibit the occurrence of inflammatory response, and the anti-inflammatory effect of PGA is significantly better than ACT.

[0093] Example 10 Comparison of anti-inflammatory effects of chitosan and carboxymethyl chitosan

[0094] PK-15 cells were inoculated into a 24-well cell culture plate and cultured for 24 hours. PCV-2 virus solution diluted with DMEM was inoculated into a 24-well culture plate with a monolayer of PK-15 cells, incubated in an incubator for 2 hours, and the supernatant was discarded.

[0095] Chitosan and carboxymethyl chitosan with different concentrations (0.1% to 1%) were inoculated into cell culture plates, with 3 replicates in each group. The experiment was divided into four groups, one of which was a blank control group, and the other three experimental groups were chitosan + PCV-2, carboxymethyl chitosan + PCV-2 and PCV-2 groups. The cells were cultured in a 37℃ CO2 constant temperature cell culture incubator for 6 hours. Single-stranded DNA was extracted using a nucleic acid extraction kit, and the expression of IL-6, TNF-α and IL-1β in the cells was measured by RT-qPCR.

[0096] The results are as follows Fig.11 As shown, the anti-inflammatory effect of carboxymethyl chitosan is significantly better than that of chitosan.

[0097] Example 11 Cellular uptake assay

[0098] 1 mg of arctigenin and 8 mg of PGA were dissolved in 5 mL of methanol, and then diluted to 10 mL with PBS to obtain an ACT solution with a concentration of 100 μg / mL (drug loading 12.38%).

[0099] Take 1 mL of each of the above samples, dilute with an equal amount of DMEM medium, and add to pk-15 cells in the logarithmic growth phase. Take samples at 2, 10, and 24 hours of reaction. Add GSH to the sample and adjust the pH. Then extract with dichlorodichlorobenzene, discard the aqueous phase, wait for the dichlorodichlorobenzene to evaporate, and re-dissolve in methanol. Substitute the absorbance measured by ultraviolet into the standard curve to calculate the final ACT concentration (not taken up). The results are as follows Fig.12 As shown, the cellular uptake of PGA was significantly better than that of naked ACT.

[0100] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A GSH / pH dual-responsive arctiin nanoparticle, characterized in that: The nanoparticles are prepared from carboxymethyl chitosan and arctigenin.

2. The nanoparticle according to claim 1, characterized in that: The particle size of the nanoparticles is 500 nm.

3. The method for preparing nanoparticles according to claim 1, characterized in that: The specific steps include: S1. Weigh a disulfide bond-containing carboxylic acid compound and dissolve it in a mixed organic solvent, add a catalyst until it is completely dissolved, add an activator and stir to activate it, to obtain an activated reaction system; S2, dissolving arctiin in N,N-dimethylformamide to obtain an arctiin solution, and adding the solution dropwise to the reaction system of step S1 for reaction, analyzing and extracting the reaction solution, and then drying it to obtain an intermediate product; S3, dissolving the intermediate product of step S2 in N,N-dimethylformamide, adding 1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide hydrochloride for activation, and dropping it into a 2% carboxymethyl chitosan aqueous solution for reaction, freeze-drying, dispersing the freeze-dried product in PBS, and obtaining nanoparticles by ultrasound; The disulfide bond-containing carboxylic acid compound is 3,3'-dithiodipropionic acid; The mixed organic solvent is a mixture of N,N-dimethylformamide solution and dichloromethane.

4. The preparation method according to claim 3, characterized in that: In the step S1, the concentration of the carboxylic acid compound after being dissolved in the mixed organic solvent is 0.1-0.25 mol / ml; the catalyst is triethylamine, and the amount of triethylamine added is 0.1-0.5 eq of the amount of the carboxylic acid compound substance; the activating agents are 1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide hydrochloride and 4-(dimethylamino)pyridine, and the amounts of 1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide hydrochloride and 4-(dimethylamino)pyridine added are 1.0-2.0 eq and 0.2-1.0 eq of the amount of the carboxylic acid compound substance.

5. The preparation method according to claim 3, characterized in that: The concentration of the arctigenin solution in step S2 is 0.2-0.5 mol / ml; the reaction conditions of the reaction solution are 60° C. for 12-48 hours.

6. The preparation method according to claim 3, characterized in that: The concentration of the intermediate product after being dissolved in N,N-dimethylformamide in step S3 is 0.2 mol / ml; the amount of 1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide hydrochloride added is 1.0 to 2.0 eq of the intermediate product substance; the ratio of the amount of carboxymethyl chitosan added to the amount of the intermediate product substance is 1:(1-5).

7. The preparation method according to claim 3, characterized in that: The step S3 further comprises dispersing the lyophilized product in a buffer solution at pH 7.4 and ultrasonically treating the product for 10 to 30 minutes; the concentration of the lyophilized product in the buffer solution is 1 mg / ml.

8. Use of the nanoparticles according to any one of claims 1 to 2 or the nanoparticles prepared by the preparation method according to claim 3 in the preparation of antiviral products or medicines.

9. The use according to claim 8, characterized in that: The virus includes PCV-2 virus.

10. Use of the nanoparticles according to any one of claims 1 to 2 or the nanoparticles prepared by the preparation method according to claim 3 in the preparation of anti-inflammatory products or medicines.

Images