Chitosan modified radix cynanchi bungei polysaccharide liposome as well as preparation method and application thereof

Abstract

The invention relates to the field of immunologic adjuvant medicines, in particular to a chitosan modified radix cynanchi bungei polysaccharide liposome as well as a preparation method and application thereof. The bunge auriculate root polysaccharide with positive charges on the surface and the antigen liposome are coated with a layer with negative charges through electrostatic adsorption to form the vaccine. In the process, the chitosan-coated liposome is used as a carrier, so that not only can the radix cynanchi bungei polysaccharide and the antigen be delivered to a target spot, but also the release of the antigen and the radix cynanchi bungei polysaccharide can be retarded, and the action time of the medicine can be prolonged. The natural bunge auriculate root polysaccharide is used as the immunopotentiator, so that the immune effect of the mode antigen OVA can be improved, and the bunge auriculate root polysaccharide is safe and non-toxic, and provides experimental basis and theoretical guidance for the design of novel, safe and effective vaccine immunologic adjuvants.

Description

Technical Field

[0001] The invention relates to the field of immune adjuvant drugs, and in particular to a chitosan-modified Cynanchum multiflorum polysaccharide liposome and a preparation method and application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Cynanch auriculatum Royle ex wight is a perennial herb of the genus Cynanch in the family Asclepiadaceae. It has the effects of nourishing the liver and kidney, strengthening the tendons and bones, improving the essence and blood, and blackening the hair. It is a traditional tonic. Cynanch auriculatum contains active ingredients such as C21 steroidal glycosides, polysaccharides, and acetophenones, which are closely related to its pharmacological activities such as anti-tumor, liver protection, antioxidant and immunomodulatory.

[0004] As the main active substance of Radix Polygoni Multiflori, Polysaccharide of Radix Polygoni Multiflori has good immunomodulatory effect. However, most natural polysaccharides have problems such as being easily soluble in water, being absorbed and metabolized quickly, and having low utilization rate in the body, which results in a short duration of immune enhancement effect in the body.

[0005] Developing safe and effective vaccines is an effective means of preventing diseases. Although a lot of efforts have been made in vaccine development and preparation, the immune effects of some vaccines are not ideal. To overcome the shortcomings of the existing vaccines, such as the single immune response effect and weak immunogenicity, more effective immune adjuvants are needed to enhance the immune response of the vaccine. An ideal immune adjuvant can stimulate the body to produce an efficient immune response at a relatively low dosage, and has no toxic side effects, making it easy to produce and use. Existing immune adjuvants are increasingly unable to meet market requirements due to their shortcomings such as being difficult to metabolize or prone to causing adverse reactions. Therefore, developing new safe and effective vaccine immune adjuvants to stimulate the body to produce efficient natural and acquired immune responses, improve the levels of humoral and cellular immune responses, enrich the immune response types of vaccines, and give full play to the immune protection of vaccines is an important development direction for the future research and development of new immune adjuvants. Summary of the invention

[0006] In order to overcome the above problems, the present invention provides a chitosan-modified Cynomorium multiflorum polysaccharide liposome and a preparation method and application thereof.

[0007] In order to achieve the above technical objectives, the present invention adopts the following technical solutions:

[0008] The first aspect of the present invention provides a chitosan-modified Cynanchum multiflorum polysaccharide liposome, wherein the chitosan-modified Cynanchum multiflorum polysaccharide liposome is a composite nano-microsphere with a core-shell structure, with the Cynanchum multiflorum polysaccharide liposome as the core and chitosan coated on the surface of the core.

[0009] The positively charged surface of the Cynanchum multiflorum polysaccharide liposomes is coated with a negatively charged chitosan layer through electrostatic adsorption, which can slow down the release of drugs and prolong the duration of drug action.

[0010] The second aspect of the present invention provides a method for preparing the chitosan-modified Cynomorium multiflorum polysaccharide liposomes according to the first aspect, comprising the following steps:

[0011] (1) dissolving lipid substances in an organic solvent, removing the organic solvent, and forming a lipid film;

[0012] (2) mixing the lipid film obtained in step (1) with Radix Cynanchum multiflorum polysaccharide and a buffer solution, hydrating the lipid film to obtain Radix Cynanchum multiflorum polysaccharide liposomes;

[0013] (3) adding a carboxymethyl chitosan aqueous solution to the Cynomorium multiflorum polysaccharide liposomes prepared in step (2), and homogenizing to obtain chitosan-modified Cynomorium multiflorum polysaccharide liposomes.

[0014] The third aspect of the present invention provides the use of the chitosan-modified Cynomorium multiflorum polysaccharide liposomes described in the first aspect and / or the chitosan-modified Cynomorium multiflorum polysaccharide liposomes prepared by the preparation method described in the second aspect as vaccine immune adjuvants.

[0015] The fourth aspect of the present invention provides a vaccine immune adjuvant, comprising the chitosan-modified Cynomorium multiflorum polysaccharide liposomes described in the first aspect and / or the chitosan-modified Cynomorium multiflorum polysaccharide liposomes prepared by the preparation method described in the second aspect.

[0016] A fifth aspect of the present invention provides a vaccine, which includes composite nano-microspheres with a core-shell structure, with Cynanchum multiflorum polysaccharide and antigen liposomes as the core, and chitosan coated on the surface of the core.

[0017] The sixth aspect of the present invention provides a method for preparing the vaccine according to the fifth aspect, comprising the following steps:

[0018] S1, dissolving lipid substances in an organic solvent, removing the organic solvent, and forming a lipid film;

[0019] S2, mixing the lipid film obtained in step S1 with Radix Cynomorii multiflori polysaccharide, model antigen OVA, and buffer, and hydrating the lipid film to obtain Radix Cynomorii multiflori polysaccharide liposomes;

[0020] S3, adding carboxymethyl chitosan aqueous solution to the Cynomorium multiflorum polysaccharide liposomes in step S2, and homogenizing to obtain chitosan-modified Cynomorium multiflorum polysaccharide liposomes.

[0021] The seventh aspect of the present invention provides a vaccine composition, comprising the vaccine immune adjuvant described in the fourth aspect and / or the vaccine described in the fifth aspect.

[0022] The beneficial effects of the present invention are:

[0023] (1) The positively charged surface of Radix Polygoni Multiflori polysaccharides and antigen liposomes are coated with a negatively charged layer through electrostatic adsorption to form a vaccine. In this process, the chitosan-coated liposomes act as carriers, which can not only deliver Radix Polygoni Multiflori polysaccharides and antigens to the target, but also slow down the release of antigens and Radix Polygoni Multiflori polysaccharides, thereby prolonging the drug's action time.

[0024] (2) Using natural Polysaccharide from Radix Cynanchum multiflorum as an immune adjuvant can significantly increase the amount of antigen phagocytosis by macrophages, activate macrophages, and induce the M1 differentiation of macrophages, thereby enhancing the immune effect.

[0025] (3) Using natural Polysaccharide of Cynanchum multiflorum as an immune enhancer can improve the immune effect of the model antigen OVA, and it is safe and non-toxic, providing an experimental basis and theoretical guidance for the design of new safe and effective vaccine immune adjuvants. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0027] Figure 1 The elution curve of purified Cynanchum multiflorum polysaccharide through cellulose column;

[0028] Figure 2 The elution curve of Cynanchum multiflorum polysaccharide through dextran gel column;

[0029] Figure 3 This is the infrared spectrum of Cynomorium multiflorum polysaccharide, and the wave numbers shown from left to right are 3386.60, 2930.23, 1737.05, 1656.25, 1367.98, 1154.97, 1080.90, 1022.79, 932.71, 844.41, 761.26, 577.54, and 527.80;

[0030] Figure 4 This is the H-NMR spectrum of Cynanchum multiflorum polysaccharide;

[0031] Figure 5 This is the C-NMR spectrum of Cynanchum multiflorum polysaccharide;

[0032] Figure 6 This is the HPLC chromatogram of Cynanchum multiflorum polysaccharide;

[0033] Figure 7 The preparation process of the vaccine;

[0034] Figure 8 In vitro drug release of CRPS-OVA-Lip and CS-C-OVA-Lip;

[0035] Fig. 9 To detect the cytotoxicity of liposomes by CCK-8 method, the cell activities at drug concentrations of 250 μg / mL, 125 μg / mL, 62.5 μg / mL, 31.25 μg / mL, 15.62 μg / mL, 7.81 μg / mL, 3.90 μg / mL, and 1.95 μg / mL in the CRPS group, C-Lip group, CRPS-OVA-Lip group, and CS-C-OVA-Lip group from left to right were shown;

[0036] Fig.10 It is a statistical diagram of cell phagocytosis;

[0037] Fig.11 Differentiation into macrophage M1 type;

[0038] Fig.12 It is the fluorescence image of CY5.5 mixed with drugs. From left to right, the results of 6h, 24h, 48h, 96h, 168h and 336h in OVA group, CRPS-OVA-Lip group and CS-C-OVA-Lip group are shown;

[0039] Fig.13 The immune organ index of mice, from left to right in the Spleen group and the Thymus group are the results of the Control group, OVA group, CRPS group, OVA+CRPS group, OVA+CFA group, CRPS-OVA-Lip group and CS-C-OVA-Lip group;

[0040] Fig.14 The results of spleen lymphocyte proliferation in immunized mice, T cell group and B cell group from left to right are Control group, OVA group, CRPS group, OVA+CRPS group, OVA+CFA group, CRPS-OVA-Lip group and CS-C-OVA-Lip group;

[0041] Fig.15 A and B in the figure are the effects of T lymphocyte subsets in immunized mice;

[0042] Fig.16 A and B in the figure are the specific total IgG antibody levels in serum;

[0043] Fig.17 is the cytokine expression level. DETAILED DESCRIPTION

[0044] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0045] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0046] The first typical embodiment of the present invention provides a chitosan-modified Cynanchum multiflorum polysaccharide liposome, wherein the chitosan-modified Cynanchum multiflorum polysaccharide liposome is a composite nano-microsphere with a core-shell structure, with the Cynanchum multiflorum polysaccharide liposome as the core and chitosan coated on the surface of the core.

[0047] The particle size of Cynanchum multiflorum polysaccharide liposomes is 247.4667±3.88nm.

[0048] The positively charged surface of the Cynanchum multiflorum polysaccharide liposomes is coated with a negatively charged chitosan layer through electrostatic adsorption, which can slow down the release of drugs and prolong the duration of drug action.

[0049] In one or more embodiments, the method for preparing Cynomorium multiflorum polysaccharide comprises the following steps:

[0050] The crude sugar solution of Radix Cynanchum multiflorum was obtained by water extraction and alcohol precipitation.

[0051] Enzymes are added to the crude sugar solution of Radix Cynanchum multiflorum to remove water-insoluble starch, and then trichloroacetic acid is added to remove residual protein, and anhydrous ethanol is added to obtain the crude polysaccharide of Radix Cynanchum multiflorum.

[0052] The crude Cynanchum multiflorum polysaccharide is purified to obtain Cynanchum multiflorum polysaccharide.

[0053] Preferably, the method for obtaining the Cynanchum multiflorum crude sugar solution by water extraction and alcohol precipitation comprises:

[0054] Add 3 times the volume of anhydrous ethanol to the Radix Cynomorii multiflori powder, soak and reflux, and obtain the treated Radix Cynomorii multiflori powder after drying;

[0055] Ultrapure water is added to the processed Radix Cynomorii multiflori powder, and the mixture is soaked, heated for extraction, and centrifuged and concentrated to obtain a Radix Cynomorii multiflori crude sugar solution.

[0056] More preferably, the immersion reflux is performed 2 to 4 times, preferably 3 times; the immersion reflux temperature is 45 to 60° C., preferably 50° C.; and the immersion reflux time is 1.5 to 3 hours, preferably 2 hours.

[0057] More preferably, ultrapure water is added to the treated Cynomorium multiflorum powder, and the soaking time is 1.5 to 3 hours, preferably 2 hours.

[0058] More preferably, the temperature of the heating extraction is 75 to 85° C., preferably 80° C.; and the extraction time is 1.5 to 3 h, preferably 2 h.

[0059] Preferably, the enzymes include alpha-amylase and saccharifying enzyme.

[0060] Preferably, the method for obtaining Cynomorium multiflorum polysaccharide after purifying the crude Cynomorium multiflorum polysaccharide comprises:

[0061] Dissolving the crude polysaccharide of Radix Cynanchum multiflorum in water to obtain a crude polysaccharide solution of Radix Cynanchum multiflorum;

[0062] The crude polysaccharide solution of Radix Cynomorii multiflori was added to a DEAE anion exchange column prepared in advance, and a NaCl solution was used for gradient elution to obtain a preliminarily purified polysaccharide of Radix Cynomorii multiflori;

[0063] The initially purified Cynomorium multiflorum polysaccharide was dissolved in ultrapure water and then added to a dextran G-150 gel column. Ultrapure water was used as an eluent for elution, and the column was freeze-dried to obtain purified Cynomorium multiflorum polysaccharide.

[0064] A second typical embodiment of the present invention provides a method for preparing the chitosan-modified Cynomorium multiflorum polysaccharide liposomes according to the first aspect, comprising the following steps:

[0065] (1) dissolving lipid substances in an organic solvent, removing the organic solvent, and forming a lipid film;

[0066] (2) mixing the lipid film obtained in step (1) with Radix Cynanchum multiflorum polysaccharide and a buffer solution, hydrating the lipid film to obtain Radix Cynanchum multiflorum polysaccharide liposomes;

[0067] (3) adding a carboxymethyl chitosan aqueous solution to the Cynomorium multiflorum polysaccharide liposomes prepared in step (2), and homogenizing to obtain chitosan-modified Cynomorium multiflorum polysaccharide liposomes.

[0068] In one or more embodiments, in step (1), the lipid substance includes lecithin and cholesterol;

[0069] Preferably, the mass ratio of lecithin to cholesterol is 1:(0.2-0.3), preferably 1:0.25.

[0070] In one or more embodiments, in step (1), the organic solvent is chloroform.

[0071] In one or more embodiments, the mass ratio of Cynanchum multiflorum polysaccharide liposome to cholesterol is 1:(0.8-1.2), preferably 1:1.

[0072] In one or more embodiments, in step (2), the buffer solvent is Hepes buffer.

[0073] In one or more embodiments, in step (3), the mass fraction of the carboxymethyl chitosan aqueous solution is 1.5 wt % to 2.5 wt %, preferably 2 wt %.

[0074] A third typical embodiment of the present invention provides the use of the chitosan-modified Cynomorium multiflorum polysaccharide liposomes described in the first aspect and / or the chitosan-modified Cynomorium multiflorum polysaccharide liposomes prepared by the preparation method described in the second aspect as vaccine immune adjuvants.

[0075] A fourth typical embodiment of the present invention provides a vaccine immune adjuvant, comprising the chitosan-modified Cynomorium multiflorum polysaccharide liposomes described in the first aspect and / or the chitosan-modified Cynomorium multiflorum polysaccharide liposomes prepared by the preparation method described in the second aspect.

[0076] In one or more embodiments, the vaccine immunoadjuvant further includes a pharmaceutically acceptable excipient.

[0077] Preferably, the auxiliary materials include any one or a combination of at least two of carriers, diluents, excipients, fillers, binders, wetting agents, disintegrants, emulsifiers, cosolvents, solubilizers, osmotic pressure regulators, surfactants, coating materials, colorants, pH regulators, antioxidants, antibacterial agents or buffers.

[0078] A fifth typical embodiment of the present invention provides a vaccine, which includes composite nano-microspheres with a core-shell structure, with Cynanchum multiflorum polysaccharide and antigen liposomes as the core, and chitosan coated on the surface of the core.

[0079] In one or more embodiments, the antigen is the model antigen OVA.

[0080] Preferably, the particle sizes of Cynanchum multiflorum polysaccharide and antigen liposome are uniform, which is 260.9667±6.20 nm.

[0081] Preferably, the particle size of the composite nano-microspheres with a core-shell structure is uniform, which is 971.2±11.2 nm.

[0082] A sixth typical embodiment of the present invention provides a method for preparing the vaccine according to the fifth aspect, comprising the following steps:

[0083] S1, dissolving lipid substances in an organic solvent, removing the organic solvent, and forming a lipid film;

[0084] S2, mixing the lipid film obtained in step S1 with Radix Cynomorii multiflori polysaccharide, model antigen OVA, and buffer, and hydrating the lipid film to obtain Radix Cynomorii multiflori polysaccharide liposomes;

[0085] S3, adding carboxymethyl chitosan aqueous solution to the Cynomorium multiflorum polysaccharide liposomes in step S2, and homogenizing to obtain chitosan-modified Cynomorium multiflorum polysaccharide liposomes.

[0086] In one or more embodiments, in step S2, the mass ratio of Cynomorium multiflorum polysaccharide to model antigen OVA is 1:(0.8-1.2), preferably 1:1.

[0087] A seventh typical embodiment of the present invention provides a vaccine composition, comprising the vaccine immunoadjuvant described in the fourth aspect and / or the vaccine described in the fifth aspect.

[0088] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0089] Example 1

[0090] (1) Extraction of Cynanchum multiflorum polysaccharide:

[0091] The water extraction and alcohol precipitation method is used to obtain Cynomorium multiflorum polysaccharide. The specific steps are as follows:

[0092] Weigh 10g of Radix Cynanchum multiflorum powder in a flask, add 3 times the volume of anhydrous ethanol (30ml), soak and reflux 3 times (each reflux temperature is 50℃, time is 40min) to remove some impurities and grease, and dry the powder at 25℃ to constant weight. Add the treated Radix Cynanchum multiflorum powder to 200ml ultrapure water, soak at room temperature for 2h, heat the mixed solution at 80℃ water bath for 2h, centrifuge the mixed solution at 4000rpm for 10min, retain the supernatant, extract the precipitated residue repeatedly, combine the two extracts, and use a rotary evaporator to concentrate to 50ml to obtain a Radix Cynanchum multiflorum crude sugar solution.

[0093] Use enzymes to remove non-water-soluble starch, add α-amylase (dosage: 18μ / g) and saccharifying enzyme (dosage: 200μ / g) to the raw sugar solution of Radix Polygoni Multiflori, centrifuge the reaction solution at 4000rpm for 10min, collect the upper solution, and concentrate it under reduced pressure to 20ml. Add 60ml of trichloroacetic acid to the concentrate to remove the residual protein in the polysaccharide solution, slowly mix and let it stand overnight, centrifuge at 4000rpm for 10min, retain the supernatant, and concentrate it to a small volume of 10ml. Add 30ml of anhydrous ethanol for precipitation, let it stand at 4℃ for 12h, centrifuge, wash the precipitate twice with 80% ethanol, freeze-dry the precipitate, and obtain the raw polysaccharide of Radix Polygoni Multiflori.

[0094] 1 g of crude polysaccharide of Radix Cynomorii was accurately weighed and prepared into a 100 mg / mL aqueous solution, which was added to a DEAE anion exchange column prepared in advance, and a NaCl solution was used for gradient elution to obtain a preliminarily purified polysaccharide of Radix Cynomorii. The polysaccharide component treated with the DEAE fiber column was dissolved with ultrapure water, added to a dextran G-150 gel column, eluted with ultrapure water as an eluent, and freeze-dried to obtain purified polysaccharide of Radix Cynomorii (abbreviated as CRPS).

[0095] The dextran elution curve of purified polysaccharide is as follows Figure 1 As shown, from Figure 1 As can be seen from the figure, the dextran elution curve of the purified polysaccharide shows a single symmetrical main peak, proving that the purity of the polysaccharide is uniform.

[0096] The Cynomorium multiflorum polysaccharide obtained in this example was characterized:

[0097] (1) Determination of polysaccharide content: Take 10 mg of glucose and dry it until constant weight, and use ultrapure water to prepare a 0.1 mg / mL glucose standard solution. Take 6 dry and clean glass test tubes, add 0 mL, 0.4 mL, 0.8 mL, 1.2 mL, 1.6 mL and 2 mL of glucose standard solution to the test tubes numbered 1 to 6 in turn, and make up to 2 mL of each tube. Then, add 1 mL of 4% phenol solution to each tube, shake gently to mix, then slowly add 5 mL of concentrated sulfuric acid and shake well. React at room temperature for 10 minutes, then place in an 85°C water bath for 15 minutes, and place in ice water for 5 minutes to terminate the reaction. Use a UV spectrophotometer to measure its absorbance at 490 nm, and use the absorbance as the ordinate and the corresponding concentration as the abscissa to draw a glucose standard curve, and make a regression equation and correlation coefficient. Take a polysaccharide sample, dilute the volume to 50mL in a volumetric flask, take 1mL of the sample solution in a test tube, add 2mL of ultrapure water, then add 1mL of 5% phenol solution and 5mL of concentrated sulfuric acid. After the above steps, use a UV spectrophotometer to measure the absorbance at 490nm. Substitute into the formula to get the polysaccharide content, the formula is as follows:

[0098] Polysaccharide content (%) = (C × V × D) / (W × 106) × 100%;

[0099] Wherein, C: sample concentration (μg / mL); V: volume (mL); D: dilution factor; W: sample mass.

[0100] The results are as follows Figure 2 As shown, the elution rate of DEAE cellulose is calculated to be 36.7%. The crude polysaccharide content of Cynanchum multiflorum is 83%, and the polysaccharide content after purification is 92%.

[0101] (2) The infrared spectrum of Cynanchum multiflorum polysaccharide is as follows Figure 3As shown, it shows typical polysaccharide characteristic absorption peaks, specifically, 3336.5 cm -1 The characteristic broad peak at 2930.23 cm is the OH stretching vibration. -1 The vibration peak CH stretch, 1737.05cm -1 The stretching vibration peak of C=O is shown at 1154.97 cm -1 CO stretching vibration peak, 932.71 cm -1 It is the characteristic absorption peak of pyranose ring.

[0102] (3) The H-NMR and C-NMR spectra of Cynanchum multiflorum polysaccharide are as follows Figure 4 and 5 As shown in the H NMR spectrum, the chemical shifts in the 5.0-5.5 ppm and 4.4-5.0 ppm regions are usually assigned to the isomeric protons of the α and β glycosidic configurations, respectively. Therefore, CRPS contains both α and β configurations. The chemical shifts of the CH2-O and CH-O group protons on the sugar ring are between 3.20 and 4.50 ppm, indicating pyranose. In the C NMR spectrum, several resonance signals of isomeric carbons in the range of 98.04 to 101.67 ppm confirm the simultaneous existence of α-(90-100 ppm) and β-(100-110 ppm) glycosidic configurations in CRPS, which is consistent with the H NMR spectrum. The abnormal signals at 4.95 / 98.56 and 5.24 / 99.74 2 positions indicate the presence of (1→4)-linked-α-DGalpA and (1→2)-linked-α-L-Rhap, respectively. In summary, the complex signals of CRPS at 1H 3.0-4.7ppm and 13C 90-101ppm are consistent with the typical characteristic distribution of polysaccharide NMR signals.

[0103] (4) The homogeneity and average molecular weight of CRPS were determined by high performance liquid chromatography. Figure 6 As shown in the figure, at a retention time of 12.12 min, the HPLC spectrum showed a single and symmetrical narrow peak, indicating that a homogeneous polysaccharide with high purity was obtained after chromatography. According to the linear regression curve of the dextran standard, the weight average molecular weight of CRPS is 8.298×104Da. Monosaccharide analysis showed that the polysaccharide of Radix Cynomorii was composed of mannose, glucose, galactose, ribose, rhamnose, glucuronic acid, galacturonic acid, and arabinose, among which glucose had the highest content, followed by galactose and galacturonic acid.

[0104] Example 2

[0105] (1) 40 mg of egg yolk phosphatidylcholine, 10 mg of cholesterol and 0.1 mg of δ-tocopherol were dissolved in 10 mL of chloroform, transferred to a rotary evaporator at 100 rpm and rotated at 38°C under reduced pressure for 1.5 h until a light yellow uniform lipid film formed at the bottom of the bottle.

[0106] (2) After mixing 10 mg of Radix Polygoni Multiflori polysaccharide (CRPS) and 6 mL of Hepes buffer (pH 6.6), slowly add the mixture to the lipid film obtained in step (1), transfer the resulting solution to a 15 mL centrifuge tube, ultrasonicate at 120 W for 5 min, centrifuge at 14000 rpm for 20 min, and pass the supernatant through 0.45 μm and 0.22 μm microporous filters in turn. Transfer the liposome solution to a centrifuge tube, centrifuge at 16000 rpm for 1 h to remove unencapsulated free OVA and CRPS. Resuspend the lower precipitate in PBS buffer solution (pH 7.4) to obtain Radix Polygoni Multiflori polysaccharide liposome (CRPS-Lip).

[0107] The particle size of Cynanchum multiflorum polysaccharide liposomes is 247.4667±3.88nm.

[0108] Example 3

[0109] Figure 7 For the vaccine preparation process, refer to Figure 7 Synthetic vaccines.

[0110] Vaccine preparation:

[0111] (1) 40 mg of egg yolk phosphatidylcholine, 10 mg of cholesterol and 0.1 mg of δ-tocopherol were dissolved in 10 mL of chloroform, transferred to a rotary evaporator at 100 rpm and rotated at 38°C under reduced pressure for 1.5 h until a light yellow uniform lipid film formed at the bottom of the bottle.

[0112] (2) After mixing 10 mg of Radix Polygoni Multiflori polysaccharide (CRPS), 10 mg of model antigen OVA and 6 mL of Hepes buffer (pH 6.6), slowly add them to the lipid film obtained in step (1), transfer the resulting solution to a 15 mL centrifuge tube, ultrasonicate at 120 W for 5 min, centrifuge at 14000 rpm for 20 min, and pass the supernatant through 0.45 μm and 0.22 μm microporous filter membranes in turn, then transfer the liposome solution to a centrifuge tube, centrifuge at 16000 rpm for 1 h to remove unencapsulated free OVA and CRPS. The lower precipitate is resuspended in PBS buffer solution (pH 7.4) to obtain Radix Polygoni Multiflori polysaccharide and antigen liposome (CRPS-OVA-Lip).

[0113] (3) Carboxymethyl chitosan (CS) was prepared into a 2% aqueous solution. The CS solution and Radix Polysaccharides Liposomes were mixed using a magnetic stirrer. The previously prepared Radix Polysaccharides Liposomes were slowly added dropwise at a volume ratio of 4:1. The mixture was stirred at 200 rpm for 12 h. The stirred solution was collected and centrifuged at 8000 rpm for 30 min. The lower precipitate was collected and resuspended in PBS. The mixture was homogenized by squeezing it back and forth through a 200 nm filter membrane using a liposome extruder for 50 to 80 times to obtain the prepared vaccine (CS-C-OVA-Lip).

[0114] Comparative Example 1

[0115] Preparation of blank liposomes:

[0116] 40 mg of egg yolk lecithin, 10 mg of cholesterol and 0.1 mg of δ-tocopherol were dissolved in 10 mL of chloroform, transferred to a rotary evaporator at 100 rpm, and rotated at 38 ° C under reduced pressure for 1.5 h. When a light yellow uniform lipid film was formed at the bottom of the bottle, 6 mL of Hepes buffer (pH 6.6) was slowly added, and the mixture was rotated at 60 rpm for 2 h until the yellow film gradually swelled and hydrated, and the solution appeared to be a light yellow emulsion. The resulting solution was transferred to a 15 mL centrifuge tube, ultrasonicated at 120 W for 5 min, and ultracentrifuged at 14000 rpm for 20 min. The supernatant was passed through 0.45 μm and 0.22 μm microporous membranes in sequence to prepare blank liposomes (C-Lip).

[0117] Example 4

[0118] The Cynomorium multiflorum polysaccharide and antigen liposomes (CRPS-OVA-Lip), vaccine (CS-C-OVA-Lip) prepared in Example 3 and the blank liposomes (C-Lip) prepared in Comparative Example 1 were characterized.

[0119] Table 1 shows the particle sizes of CRPS-OVA-Lip, CS-C-OVA-Lip and C-Lip, Table 2 shows the polydispersity indexes PDI of CRPS-OVA-Lip, CS-C-OVA-Lip and C-Lip, and Table 3 shows the Zeta potential of CRPS-OVA-Lip, CS-C-OVA-Lip and C-Lip.

[0120] Table 1 Nanosphere particle size

[0121]

[0122] Table 2 Polydispersity coefficient PDI

[0123]

[0124]

[0125] Table 3 Zeta potential

[0126]

[0127] As can be seen from Table 3, the positively charged surface of Cynanchum multiflorum polysaccharide and antigen liposomes coated the negatively charged chitosan layer through electrostatic adsorption.

[0128] Example 5

[0129] In vitro drug release measurement: 5 mL of CRPS-OVA-Lip and CS-C-OVA-Lip liposome solutions were placed in dialysis bags with a molecular weight cutoff of 8000-10000 Da, and the dialysis bags were suspended in 100 mL of dialysate containing 5% PBST. The mixture was oscillated at 37°C for 2 weeks, and 3 mL of dialysate was drawn out at a fixed time period. At the same time, 3 mL of PBST solution was added to make up the balance. The release of the model antigen OVA was detected by the Coomassie Brilliant Blue method. The results were repeated three times, and the data were recorded to generate the release curve.

[0130] The results are as follows Figure 8 As shown in the figure, the in vitro drug release of CRPS-OVA-Lip and CS-C-OVA-Lip is similar, and the late release rate of liposome antigen after chitosan encapsulation is higher than that of liposome without encapsulation, indicating that chitosan encapsulation can play a certain role in sustained release of antigen. The cumulative release rate of antigens of CRPS-OVA-Lip and CS-C-OVA-Lip at 14 days is about 40%.

[0131] Example 6

[0132] The cytotoxicity of liposomes was detected by CCK-8 method. Three groups were set up: C-Lip, CRPS-OVA-Lip, and CS-C-OVA-Lip. Each group was set up with 8 drug concentrations of 250 μg / mL, 125 μg / mL, 62.5 μg / mL, 31.25 μg / mL, 15.62 μg / mL, 7.81 μg / mL, 3.90 μg / mL, and 1.95 μg / mL. In the logarithmic growth phase, cells were cultured at 1×10 4 Each well of the 96-well plate was inoculated with 1 mL of drug. A blank control group (serum-free culture medium) was set up. The cells were co-cultured at 37°C and 5% CO2 for 24 hours, and the cell viability was detected using the CCK-8 method. The optical density (OD) value of each well was measured using a multi-mode microplate detection system at a wavelength of 450 nm. The data were recorded and substituted into the formula to calculate the cell viability.

[0133]

[0134] Cytotoxicity studies were performed to elucidate the biocompatibility of the extracted polysaccharides and prepared liposomes. The cytotoxic effects of CRPS liposome nanosphere formulations (with or without CRPS) on RAW 264.7 cells were as follows: Fig. 9 As shown, CRPS and its formulations have no cytotoxicity to cells, and the cell survival rate exceeds 75% within 24 hours at a drug concentration of 0 to 250 μg / mL. This indicates that the CRPS liposome formulation (with or without CRPS) has no obvious toxicity to cells at the tested concentration and has good biocompatibility.

[0135] Flow cytometry characterization of endocytosis and cell phenotyping:

[0136] In flow cytometry, the cell concentration was adjusted to 2 × 10 6 / mL, prepare 6-well cell plates, add 1mL of different liposome drugs and culture for 48h, collect cells, stain macrophages with PE anti-mouse CD-80 and CD-86, and detect macrophage surface polarization by flow cytometry.

[0137] In the cell phagocytosis assay, 1 × 10 5 The density of cells was inoculated into 6-well plates. In order to study the uptake efficiency of different nanoparticles in macrophages, three liposomes were added to the culture medium: C-Lip, CRPS-OVA-Lip, and CS-C-OVA-Lip, and cultured for 24 hours. PBS was used to wash and prepare cell suspension, and the cell phagocytosis was statistically analyzed by flow cytometry, and the statistical data were obtained.

[0138] Fig.10 The cell phagocytosis statistics show that the fluorescence of CS-C-OVA-Lip is significantly higher than that of C-Lip and CRPS-OVA-Lip (P<0.05), indicating that CS-C-OVA-Lip can significantly increase the amount of antigen phagocytosis by macrophages.

[0139] Fig.11 As shown in the figure, the CD80 level of CS-C-OVA-Lip was significantly higher than that of other groups (P<0.05); the expression of CD86 was similar to that of CD80, and the expression of CD86 in CS-C-OVA-Lip was significantly higher than that in other groups (P<0.05). This indicates that CS-C-OVA-Lip can activate macrophages and induce macrophage M1 differentiation.

[0140] Example 7

[0141] Female ICR mice aged 4 to 6 weeks were adaptively fed for one week and randomly divided into 3 groups. CY5.5 was mixed with the drug, connected in the dark for 1 hour, and injected at a dose of 0.2 mL per mouse. The drug release in vivo was observed under a small animal in vivo imaging device at different times.

[0142] like Fig.12 As shown, the fluorescence value of OVA was higher in the early stage and lower in the later stage; the fluorescence value of CS-C-OVA-Lip was higher than that of the other two groups. The fluorescence of the OVA group was slightly higher than that of CRPS-OVA-Lip and CS-C-OVA-Lip at the beginning, and lower than that of the other two groups after 6 hours; the fluorescence of CS-C-OVA-Lip was higher than that of the other groups in the later stage, showing a certain sustained release effect, indicating that the chitosan modified liposome CS-C-OVA-Lip has the effect of slowing down the release of drugs and prolonging the drug release time.

[0143] Example 8

[0144] 8.1 Animal grouping and immunization procedures:

[0145] 105 4-6 week old ICR female mice (25-30 g / each) were purchased from Chengdu Dashuo Experimental Animal Company (SCXK (Chuan) 2022-065). The mice were divided into 7 groups and the immunization procedure was performed after one week of adaptive feeding.

[0146] The grouping and immunization procedures were as follows: the first group was the blank control group, and each mouse was subcutaneously injected with 0.2 mL of normal saline solution; the second group was the OVA group, and each mouse was subcutaneously injected with 0.2 mL (300 μg / mL) of OVA solution; the third group was the Cynomorium multiflorum polysaccharide (CRPS) group, and each mouse was subcutaneously injected with 0.2 mL (500 μg / mL) of Cynomorium multiflorum polysaccharide solution; the fourth group was the Cynomorium multiflorum polysaccharide + OVA (OVA + CRPS) group, and each mouse was subcutaneously injected with 0.2 mL (500 μg / mL + 300 μg / mL) of Cynomorium multiflorum polysaccharide + OVA solution. The fifth group was the positive control complete Freund's adjuvant (OVA+CFA) group, and each mouse was subcutaneously injected with 0.2mL (300μg / mL OVA+1mL Freund's adjuvant); the sixth group was the CRPS-OVA-Lip group, and each mouse was subcutaneously injected with 0.2mL (500μg / mL) of CRPS-OVA liposome solution; the seventh group was the chitosan-Calendula polysaccharide liposome (CS-C-OVA-Lip) group, and each mouse was subcutaneously injected with 0.2mL (500μg / mL) of chitosan-Calendula polysaccharide liposome. A booster immunization was performed once a week, and a total of 4 immunizations were completed, and the immunization dose for each vaccination remained the same.

[0147] 8.2 Determination of mouse immune organ index (spleen, thymus):

[0148] After the last immunization, the weight of the mice was recorded, the mice were anesthetized and killed, the spleen and thymus were separated from the body, the excess tissue was removed, the blood residue was washed with PBS buffer, the surface water was dried with absorbent paper, and then the weight of the thymus and spleen was weighed and the data was recorded. The immune organ index was calculated according to the following formula:

[0149] Immune organ index (mg / g) = organ mass (mg) / mouse body weight (g)

[0150] 8.3 Determination of mouse dendritic cell phenotype:

[0151] 48 hours after the first immunization (D3), 3 mice were randomly selected from each group and anesthetized and killed. The spleen was separated and placed in 2 mL PBS buffer, ground with a 1 mL syringe handle until there was no obvious tissue block to the naked eye, and the grinding solution was filtered with 3 layers of 200-mesh nylon mesh, and the plate was rinsed with 1 mL PBS buffer. After filtration, it was transferred into a 15 mL centrifuge tube and centrifuged at 4000 rpm for 2 minutes. The supernatant was poured out, and 2 mL of red blood cell lysis buffer was added to the bottom of the tube, mixed by pipetting, and lysed at room temperature for 2-4 minutes, and then an equal volume of PBS buffer was added to terminate the lysis reaction, and centrifuged again; the cells were resuspended with 1 mL PBS buffer, and then the cells were counted under a microscope using trypan blue staining solution, and the number of live cells was finally adjusted to 1.6×106 / mL.

[0152] Transfer 300 μL of cell suspension to a 2 mL Eppendorf tube and mix with an equal volume of 5% skimmed milk powder, shake on ice, and block for 30 min; wash twice with PBS buffer, centrifuge at 4000 rpm for 2 min, and discard the supernatant; stain the cells with anti-CD11C-FITC, CD80-PE, CD86-PE, and MHCⅡ-PE (dosage and usage in the instruction manual) antibodies, stain on ice for 30 min at 4°C in the dark; wash three times with PBS buffer, resuspend the cells in 500 μL PBS, pass the cell fluid through a cell sieve, and transfer to a flow tube for detection and analysis using a flow cytometer.

[0153] 8.4 Mouse spleen lymphocyte proliferation assay:

[0154] On the 21st day after immunization, three mice were randomly selected from each group and anesthetized and killed. The whole body was immersed in 75% alcohol for 5 minutes, and the mouse spleen tissue was completely separated by aseptic operation in the clean bench, and the spleen was rinsed with PBS (1% double antibody); the spleen was placed in a 200-mesh nylon mesh sieve, 1mL RPMI-1640 was used to keep the spleen moist, and it was ground until there was no obvious tissue block observed by the naked eye, and the grinding liquid was collected, centrifuged at 2000rpm for 10 minutes, and the supernatant was poured out; 2mL of red blood cell lysis buffer was added to the EP tube, the cell clumps were fully mixed, and the lysis was carried out at room temperature for 4-5min, and the reaction was terminated with two volumes of 1640, centrifuged again, the supernatant was discarded, and the PBS buffer was washed twice; the cells were resuspended in RPMI-1640 complete cell culture medium containing 12% fetal bovine serum, and the cell number was finally adjusted to 2×106 / mL.

[0155] The cells were divided into three groups, and 100 μL of cell suspension was added to each well. 100 μL of Con A (5 μg / mL), 100 μL of LPS (10 μg / mL), and 100 μL of RPMI-1640 culture medium were added respectively. The cells were cultured at 37°C and 5% CO2 for 48 h. Finally, 20 μL of CCK-8 was added for color development. After 4 h, the OD value at 450 nm was detected using a multi-mode microplate detection system, and the lymphocyte proliferation capacity was calculated according to the formula:

[0156] Lymphocyte proliferation capacity (SI) = OD1 / OD0

[0157] OD0 is the OD value of the control well, and OD1 is the OD value of the experimental well.

[0158] 8.5 T lymphocyte subset determination:

[0159] 28 days after immunization, 3 mice were randomly selected from each group, and the mice were killed after anesthesia to prepare spleen cell suspension. After centrifugation at 4000rpm for 2 minutes, the supernatant was discarded, and 6mL 5% skim milk was added to block on ice for 30min; after washing twice with pre-cooled PBS, the cells were transferred to a 2mL Ep tube, and the cells were stained with anti-CD3-FITC, CD4-PE, CD8-PE (dosage in the instruction manual) antibodies, placed on ice, and stained in the dark for 30min; washed twice with PBS, centrifuged and discarded the supernatant, 500μL PBS buffer was added to resuspend the cells, and the cells were filtered through a cell mesh and transferred to a flow cytometry tube for detection and analysis by flow cytometry.

[0160] 8.6 Determination of specific total IgG antibody content in serum:

[0161] After immunization of each group of mice, 3 mice were taken from each group for blood sampling from the retro-orbital venous plexus at 1 (D7), 2 (D14), 3 (D21), and 4 (D28) weeks. 0.2 mL of venous blood was collected from each mouse and collected in a 1.5 mL Eppendorf tube, placed flat in a 37°C constant temperature incubator for 30 min, placed in a 4°C refrigerator for 4 h, centrifuged (3000 rpm, 15 min), and the upper light yellow serum was separated and stored at -80°C for later use.

[0162] The total IgG content was detected by indirect ELISA: 100 μL of antigen coating solution containing 5 μg / mL 0.01 mol / L carbonate buffer was added to each well of a 96-well ELISA plate, and incubated at 4°C for 24 hours; 0.05% Tween-20 PBST washing solution was used to wash the ELISA plate three times, 300 μL per well, and each soaking was for 5 minutes; 300 μL of 3% blocking solution was added, and the plate was incubated at 37°C for 1 hour, washed, and patted dry; horseradish peroxidase-labeled anti-mouse IgG antibody was added, and the plate was incubated at 37°C for 30 minutes, then the plate was washed and patted dry; 100 μL of TMB substrate colorimetric solution was added, and the plate was incubated at 37°C for 15 minutes; 50 μL of 2M H2SO4 was added to each well to terminate the reaction; and the absorbance was measured at 450 nm using an ELISA reader.

[0163] 8.7 Antigen release in vivo detection:

[0164] Female ICR mice aged 4 to 6 weeks were adaptively fed for one week and randomly divided into 3 groups. CY5.5 was mixed with the drug and connected in the dark for 1 hour. The mice were injected with a dose of 0.2 mL per mouse, and the drug release in vivo was observed under a small animal in vivo imaging device at different times.

[0165] 8.8 Fluorescence quantitative detection of cytokines INF-γ, IL-4, IL-6, and CD44:

[0166] Quantitative Real-time PCR was used to detect the expression of interferon-γ (INF-γ), interleukin-4 (IL-4), interleukin-6 (IL-6) and glycoprotein CD44.

[0167] One week after the last immunization (D28), three mice in each group were randomly anesthetized and killed, and the spleens of the mice were collected aseptically, and the excess connective tissue was removed. The surface moisture was absorbed with absorbent paper and stored at -80°C for future use.

[0168] Total RNA extraction: Take spleen tissue, grind until the tissue is completely broken, add 700μL Trizol total RNA extraction reagent, and place on ice for 15min; then add 200μL chloroform solution, blow and mix, let stand for 5min, centrifuge at 4℃, 12000rpm for 5 minutes; use a pipette to absorb the top transparent liquid and transfer it to a new enzyme-free tube; then add 0.5mL of isopropanol solution, let stand for 10min; centrifuge at 7500rpm for 10 minutes, the white precipitate at the bottom of the test tube is RNA; wash twice with pre-cooled 75% alcohol, invert and dry; dissolve in 50μL DEPC, take a small amount of RNA to determine its concentration and purity. cDNA synthesis: according to the instructions of TaKaRa reverse transcription kit. PCR amplification: 25 μL reaction system, according to TB Green premix ExTaqⅡ12.5 μL, upstream primer 1 μL, downstream primer 1 μL, cDNA2 μL, enzyme-free water 8.5 μL; vortex to mix, remove bubbles; 95℃ pre-denaturation for 1 min, cycle 95℃ for half a minute, 60℃ for 30 seconds, perform 40 cycles, and end amplification. According to 2- △△ Ct algorithm was used to calculate the relative expression of cytokines.

[0169] result:

[0170] like Fig.13 As shown in the figure, after the immunization procedure, the spleen index of each test group increased compared with the blank group, and the spleen index of the OVA+CFA group was significantly higher than that of other treatment groups (P<0.05); the spleen index of the OVA group, CRPS group, OVA+CRPS group, CRPS-OVA-Lip group, and CS-C-OVA-Lip group was not significantly different (P>0.05). The results of mouse thymus index showed that there was no significant difference in the thymus index of the OVA+CRPS group, OVA+CFA group, CRPS-OVA-Lip group, and CS-C-OVA-Lip group (P>0.05); there was no significant difference between the OVA group and the CRPS group and the blank group (P>0.05). The results of immune organ index showed that the prepared chitosan nanoliposome CS-C-OVA-Lip had an immune-promoting effect. Chitosan nanoparticles were non-toxic to mouse organs.

[0171] On the 21st day of the immunization procedure, the spleen of mice was collected to prepare single cell suspension, and the proliferation level of spleen lymphocytes was detected by CCK-8 method. The results were as follows Fig.14 As shown. The T lymphocyte proliferation index was significantly higher than that of other experimental groups except the CRPS group (P<0.05); the B lymphocyte index increased and was significantly higher than that of other experimental groups (P<0.05). This shows that CRPS has a certain proliferation effect on T lymphocytes, and CS-C-OVA-Lip can promote the proliferation of T lymphocytes and B lymphocytes in the spleen of mice.

[0172] Effects of CS-C-OVA-Lip liposome vaccine on T lymphocyte subsets in mice Fig.15 As shown, compared with the blank group, the CS-C-OVA-Lip group can significantly increase the number of CD4 T+ cells (P<0.05), and the CRPS-OVA-Lip and complete Freund's adjuvant groups have the same effect on increasing the number of CD4 T+ cells (P>0.05); at the same time, the CS-C-OVA-Lip group can significantly increase the number of CD8 T+ cells (P<0.05).

[0173] On days 7, 14, 21, and 28 after immunization, the levels of total OVA-specific IgG antibodies in mouse sera were continuously measured. Fig.16 It can be seen that on the 7th and 14th days, the specific IgG antibody level of the CS-C-OVA-Lip group was significantly higher than that of other experimental treatment groups (P<0.05), and on the 21st and 28th days, the IgG antibody level of the CS-C-OVA-Lip group was significantly higher than that of other experimental groups except CRPS-OVA-Lip (P<0.05); during all the detection time, the specific IgG antibody levels of all experimental groups except the blank group showed a trend of first increasing and then decreasing, reaching a high level on the 21st day, and the CS-C-OVA-Lip group and the CRPS-OVA-Lip group still had a high level on the 28th day. This shows that both have good antigen sustained release effects and the sustained release effect of nanoparticles CS-C-OVA-Lip is better.

[0174] After the last immunization, the spleen of each group of mice was collected aseptically, total RNA was extracted, and the expression of cytokines INF-γ, IL-4, IL-6, and CD44 was detected by PCR technology. The results of each cytokine in the spleen are as follows Fig.17As shown in the results, the level of cytokine INF-γ in the CS-C-OVA-Lip group was significantly higher than that in the other experimental groups (P<0.05), there was no significant difference between the CRPS-OVA-Lip group and the OVA+CFA group (P>0.05), and there was no significant difference between the blank group and the free OVA group and the CRPS group (P>0.05). The level of cytokine IL-4 in the spleen in the CS-C-OVA-Lip group was significantly higher than that in the other experimental groups (P<0.05), and its cytokine level was similar to that of INF-γ; The expression level of cytokine IL-6 in the C-OVA-Lip group was significantly higher than that in the other experimental groups (P<0.05), and there was no significant difference between the blank group and the OVA group, CRPS group and OVA+CRPS group (P<0.05). As can be seen from the figure, the expression level of CD44 in the CS-C-OVA-Lip group was significantly higher than that in the other groups (P<0.05), and there were significant differences among the CRPS-OVA-Lip group, OVA+CFA group, OVA+CRPS group, CRPS group, OVA group and blank group (P<0.05).

[0175] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A chitosan-modified Cynanchum multiflorum polysaccharide liposome, characterized in that: The chitosan-modified Cynanchum multiflorum polysaccharide liposome is a composite nano-microsphere with a core-shell structure, with the Cynanchum multiflorum polysaccharide liposome as the core and chitosan coated on the surface of the core.

2. The chitosan-modified Cynomorium multiflorum polysaccharide liposome according to claim 1, characterized in that: The particle size of Cynanchum multiflorum polysaccharide liposomes is 247.4667±3.88nm.

3. The method for preparing chitosan-modified Cynanchum multiflorum polysaccharide liposomes according to claim 1 or 2, characterized in that: The following steps are involved: (1) dissolving lipid substances in an organic solvent, removing the organic solvent, and forming a lipid film; (2) mixing the lipid film obtained in step (1) with Radix Cynanchum multiflorum polysaccharide and a buffer solution, hydrating the lipid film to obtain Radix Cynanchum multiflorum polysaccharide liposomes; (3) adding a carboxymethyl chitosan aqueous solution to the Cynomorium multiflorum polysaccharide liposomes prepared in step (2), and homogenizing to obtain chitosan-modified Cynomorium multiflorum polysaccharide liposomes.

4. The preparation method according to claim 3, characterized in that: In step (1), the lipid substance includes lecithin and cholesterol; preferably, the mass ratio of lecithin to cholesterol is 1:(0.2-0.3), preferably 1:0.25; Or, in step (1), the organic solvent is chloroform; Or, the mass ratio of Cynanchum multiflorum polysaccharide liposome to cholesterol is 1:(0.8-1.2), preferably 1:1; Or, in step (2), the buffer solvent is Hepes buffer; Or, in step (3), the mass fraction of the carboxymethyl chitosan aqueous solution is 1.5wt% to 2.5wt%, preferably 2wt%.

5. Use of the chitosan-modified Cynanchum multiflorum polysaccharide liposomes according to claim 1 or 2 and / or the chitosan-modified Cynanchum multiflorum polysaccharide liposomes prepared by the preparation method according to claim 3 or 4 as vaccine immune adjuvants.

6. A vaccine immune adjuvant, characterized in that: The invention comprises providing the chitosan-modified Cynanchum multiflorum polysaccharide liposomes as claimed in claim 1 or 2 and / or the chitosan-modified Cynanchum multiflorum polysaccharide liposomes prepared by the preparation method as claimed in claim 3 or 4.

7. A vaccine, characterized in that The vaccine comprises composite nano-microspheres of a core-shell structure, with Radix Cynanchum multiflorum polysaccharide and antigen liposome as the core, and chitosan coated on the surface of the core.

8. The vaccine according to claim 7, characterized in that The antigen is the model antigen OVA; Preferably, the particle sizes of Cynanchum multiflorum polysaccharide and antigen liposome are uniform, which is 260.9667±6.20 nm. Preferably, the particle size of the composite nano-microspheres with a core-shell structure is uniform, which is 971.2±11.2 nm.

9. The method for preparing the vaccine according to claim 7 or 8, characterized in that: The following steps are involved: S1, dissolving lipid substances in an organic solvent, removing the organic solvent, and forming a lipid film; S2, mixing the lipid film obtained in step S1 with Radix Cynomorii multiflori polysaccharide, model antigen OVA, and buffer, and hydrating the lipid film to obtain Radix Cynomorii multiflori polysaccharide liposomes; S3, adding carboxymethyl chitosan aqueous solution to the Cynomorium multiflorum polysaccharide liposomes in step S2, and homogenizing to obtain chitosan-modified Cynomorium multiflorum polysaccharide liposomes.

10. A vaccine composition, characterized in that Comprising the vaccine immune adjuvant according to claim 6 and / or the vaccine according to claim 7.

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