Polysaccharide modified liposome nano vaccine as well as preparation method and application thereof

Abstract

The invention belongs to the field of biological medicine, and particularly relates to a polysaccharide modified liposome nano vaccine as well as a preparation method and application thereof. The polysaccharide modified lipidosome nano vaccine is composed of an anionic polysaccharide shell containing a D-glucuronic acid skeleton and a drug-loading cationic lipidosome inner core coated in the polysaccharide shell, and the cationic lipidosome inner core is loaded with an anionic antigen OVA. The polysaccharide-modified lipidosome nano vaccine containing the sulfuric acid acetyl structure has more excellent capability of overcoming inherent barriers of lung mucosa vaccines, and has the advantages of good stability in a lung microenvironment, high biological safety, capability of enhancing in-vivo triple immunity (cell, body fluid and mucosa reaction) and the like; the method has a wide prospect in lung vaccine development and design, and can also be used for vaccine delivery of other administration routes.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a polysaccharide-modified liposome nano-vaccine and a preparation method and application thereof. Background Art

[0002] Globally, infectious diseases caused by respiratory mucosal pathogens still have high mortality and morbidity, and the situation is more serious in low-income areas. Vaccines are one of the greatest achievements of modern medicine. Vaccination can effectively reduce the morbidity and mortality of infectious diseases. However, the vaccines currently on the market do not effectively cover the prevention of all respiratory pathogens. For example, although vaccines for preventing influenza virus, Mycobacterium tuberculosis, Streptococcus pneumoniae and Bordetella pertussis infections are on the market, most of them are injected, which makes it difficult to induce antigen-specific immune responses on the mucosal surface invaded by pathogens, resulting in less than ideal prevention effects. Except for a few reports that retinoic acid can be used as an adjuvant to induce mucosal tissue to secrete IgA, vaccines administered through conventional intramuscular injection can only induce systemic immune responses characterized by serum IgG, and have limited ability to clear viral particles. Compared with injection vaccines, pulmonary inhalation vaccines can not only stimulate humoral immunity and cellular immunity, but also stimulate antibodies in nasal and airway tissues, achieving the purpose of efficiently inducing mucosal immunity, blocking the infection of pathogens and their further spread and transmission at the infection site, and ultimately achieving triple immune protection. In addition, the antigen dose and vaccination frequency are reduced and protective immunity with the same effectiveness as injected vaccines can be achieved, greatly improving safety, convenience and compliance.

[0003] Traditional vaccines have drawbacks in terms of safety and preparation cycle. Technological advances and the pursuit of safety, harmlessness and cost-effectiveness have led to the development of subunit vaccines. Subunit vaccines generally have poor immunogenicity and require the addition of adjuvants to induce strong and lasting protective immune responses. According to the different modes of action, adjuvants are divided into delivery systems and immune stimulators / regulators / enhancers. However, there is currently a lack of safe and effective inhalable immune adjuvants. In contrast, nanodelivery systems, with their clear composition, stable structure and special physicochemical properties, greatly promote the uptake of antigens in mucosal sites, and deliver vaccine components to key immune cells or lymphoid tissues, stimulate immune cell maturation, and ultimately achieve efficient immune protection. In the lungs, nanoparticles provide a potential solution for overcoming biological barriers and targeting dendritic cells (DCs). In addition, nanoparticles with inherent adjuvant effects can easily mimic the physical and chemical properties of natural pathogens to enhance immune responses, such as morphology, size, charge, flexibility and other physicochemical properties. Among them, liposomes are currently considered to be one of the most flexible and successful delivery systems because they have many advantages, including biocompatibility, high loading capacity and easy surface modification to achieve functional design. Their inherent immunostimulatory properties are attributed to the protection of antigens, the promotion of DC maturation and the induction of a series of cytokines and chemokines. Currently approved liposome injection vaccines include hepatitis A (Epaxal), influenza (Inflexal V), herpes zoster (Shingrix), and malaria vaccine (Mosquirix). Although liposomes have the potential for rapid clinical transformation, especially the adjuvant effect of cationic liposomes has been widely studied and applied in various preclinical experiments, showing good immune induction potential, the effect after delivery by pulmonary inhalation is not very ideal, mainly due to the presence of mucus clearance mechanism in mucosal sites, lack of DC targeting and poor physical stability, resulting in premature release of antigens and lung safety issues.

[0004] Many polysaccharides have the function of promoting specific and nonspecific immunity of the body, and have the characteristics of low toxicity and safety, and may become ideal vaccine adjuvants. As a delivery system, it helps antigen recognition and uptake by antigen presenting cells. At the same time, as a pattern recognition receptor (PRRs) agonist, it activates and initiates cascade immune responses. In addition, polysaccharides and other immune adjuvants are made into composite adjuvants to produce a synergistic effect, thereby inducing a stronger and more comprehensive immune effect. Through simple complexation with anionic polysaccharides, the immunostimulatory properties of liposomes can be changed, their stability can be increased, and the problems of the above-mentioned liposomes can be overcome. Studies have shown that the conformation, molecular weight, certain functional groups present, and degree of branching of polysaccharides may be related to their immunostimulatory function. The conformation of polysaccharides can also affect their interaction with immune molecules or cells, thereby affecting their immunoregulatory effects.

[0005] In view of this, there is a need to develop a safe and effective inhalable polysaccharide adjuvant system and to solve various problems in the lung delivery of subunit antigens, so as to ultimately apply universal polysaccharide adjuvants that optimally activate immune effects to the immunotherapy of lung diseases. Summary of the invention

[0006] The present invention provides a polysaccharide-modified liposome nanovaccine, in particular, provides an inhalable polysaccharide-modified liposome nanovaccine for enhancing pulmonary mucosal immunity, so as to solve the problems of unstable pulmonary delivery of subunit antigens or adjuvants, poor immunogenicity, toxicity of cationic liposomes and pulmonary delivery barrier. The present invention provides an inhalable polysaccharide-modified liposome nanovaccine for enhancing pulmonary mucosal immunity, which is composed of a polysaccharide shell with different structures and a cationic liposome core; the cationic liposome core carries an anionic antigen OVA or an adjuvant.

[0007] A polysaccharide modified liposome nano vaccine, the nano vaccine consists of an anionic polysaccharide shell containing a D-glucuronic acid skeleton and a drug-loaded cationic liposome core coated in the polysaccharide shell, wherein the cationic liposome core carries anionic antigen OVA.

[0008] In the above technical solution, the nano vaccine is in the form of particles with an average particle size of 100 to 650 nm and a surface charge of -10 to -40 mV.

[0009] In the above technical solution, the polysaccharide containing a D-glucuronic acid skeleton is selected from one or more of hyaluronic acid (HA), chondroitin sulfate (SC), and heparan sulfate (SH).

[0010] In the above technical solution, the raw materials for preparing cationic liposomes include endogenous phospholipids in the lungs, cationic lipids and cholesterol.

[0011] Furthermore, the endogenous phospholipids in the lungs are one or more of phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine, and phosphatidylglycerol.

[0012] Furthermore, the cationic lipid is selected from one or more of 2,3-dioleyloxypropyl)trimethylammonium chloride (DOTAP), dimethyldioctadecylammonium (DDA), and 3β-[N-(N',N'-dimethylaminoethyl)carbamoyl]cholesterol (DC-Chol).

[0013] Furthermore, the molar ratio of the lung endogenous phospholipids, cationic lipids and cholesterol is 120-40:40-20:10.

[0014] The ratio of the anionic polysaccharide to the cationic lipid in the present invention is based on the surface charge of the final polysaccharide-modified liposome nanovaccine being -10 to -40 mV.

[0015] Another object of the present invention is to provide a method for preparing the above-mentioned polysaccharide-modified liposome nanovaccine.

[0016] A method for preparing a polysaccharide-modified liposome nanovaccine comprises mixing a dissolved lipid membrane material with an anionic antigen OVA, performing high-speed shearing, rotary evaporation, and hydration to obtain drug-loaded cationic liposomes; dissolving a polysaccharide in ultrapure water, and adding the drug-loaded cationic liposome solution dropwise to obtain a polysaccharide-modified liposome nanovaccine.

[0017] In the above technical solution, the concentration of the lipid solution is 2-15 mg / mL, and the concentration of the OVA solution is 0.01-5 mg / mL ， The volume ratio of the lipid solution to the anionic antigen OVA solution is 1:1 to 10:1.

[0018] In the above technical solution, the concentration of the polysaccharide solution is 0.1-2 mg / mL, and the volume ratio of the drug-loaded cationic liposome solution to the polysaccharide solution is 0.2:1-5:1.

[0019] Taking DPPC as an example, the drug-loaded cationic liposomes are prepared by the following method: DPPC, DOTAP and cholesterol are fully dissolved in chloroform at a certain molar ratio to prepare a lipid solution as an oil phase. Under room temperature, OVA aqueous phase is added and high-speed shear treatment is performed to obtain water-in-oil colostrum; the colostrum is rotary evaporated and fully hydrated under certain conditions, and then further ultrasonically treated to obtain drug-loaded cationic liposomes (abbreviated as DPPC / DOTAP Liposome).

[0020] Furthermore, the DPPC / DOTAP Liposome and the polysaccharide solution are fully mixed, incubated at room temperature for 15 minutes, and ultrafiltration is performed to remove free OVA and polysaccharides, or the free OVA and polysaccharides are not removed.

[0021] Another object of the present invention is to provide the use of the above-mentioned polysaccharide-modified liposome nanovaccine in the preparation of subunit vaccines or adjuvant carriers for delivery to the lungs, nasal cavity, eyes, oral cavity, and vaginal mucosa.

[0022] Furthermore, the invention provides the use of the polysaccharide-modified liposome nanovaccine in the preparation of an inhaled vaccine for enhancing lung mucosal immunity.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The present invention covers the positive charge on the surface of the cationic liposome by encapsulating anionic polysaccharides based on D-glucuronic acid outside the cationic liposome, reduces the biological toxicity of the cationic liposome, and has excellent biosafety performance. The anionic polysaccharides based on D-glucuronic acid selected by the present invention are easier to complex with cationic liposomes than other cationic polysaccharides (chitosan, guar gum), and compared with other structural anionic polysaccharides (sodium alginate, dextran, pectin), the nano vaccine formed by the polysaccharide of the present invention has stronger physical stability, and is widely available and easier to obtain.

[0025] The liposome nanovaccine modified with anionic polysaccharides based on D-glucuronic acid of different structures of the present invention improves the ability of cationic liposomes to overcome the lung clearance barrier and the stability in the lung microenvironment, improves the macrophage escape and mucus permeability of cationic liposomes and enhances the immune response (cellular, humoral, mucosal response) in vivo. Compared with other cationic polysaccharides (chitosan, guar gum), it is superior in the ability to overcome the liposome lung delivery barrier and is suitable for the delivery of subunit antigen vaccines and adjuvants in the lungs and other mucosal sites. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 (a) and (b) show the cell viability of polysaccharide-modified liposome nanovaccines in BEAS-2b and DC 2.4 cells in Examples 1, 2, 3, and 4 (n=3).

[0027] Figure 2 The polysaccharide-modified liposome nanovaccine overcomes the clearance ability of lung macrophages in Examples 1, 2, 3, and 4 (n=3).

[0028] Figure 3 (a), (b), (c) and (d) are the permeability of the polysaccharide-modified liposome nanovaccines in artificial mucus in Examples 1, 2, 3 and 4 (n=3).

[0029] Figure 4 The stability of polysaccharide-modified liposome nanovaccine in BALF in Examples 1, 2, 3, and 4 (n=3).

[0030] Figure 5 (a), (b), (c) and (d) show the production of specific antibodies in mice by the polysaccharide-modified liposome nanovaccines in Examples 1, 2, 3 and 4 (n=6).

[0031] Figure 6 This is a schematic diagram of the structure of the polysaccharide-modified liposome nanovaccine of the present invention. DETAILED DESCRIPTION

[0032] The present invention is further described below in conjunction with specific embodiments, but the present invention is not limited in any way.

[0033] In order to facilitate the understanding of the present invention, the present invention will be described more fully below with reference to the relevant drawings. The preferred embodiments of the present invention are shown in the drawings. However, the present invention can be implemented in many different forms, but is not limited to the embodiments described herein. The purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0034] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0035] The experimental methods in the examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are commercially available products unless otherwise specified.

[0036] Comparative Example 1

[0037] (1) DPPC, DOTAP and cholesterol were fully dissolved in chloroform at a molar ratio of 75:25:10 (the ratio of DPPC to chloroform was 15:1 (mmol / L)) to prepare a lipid solution as the oil phase. At room temperature, 2 mg / mL of OVA was added to the aqueous phase and high-speed shear treatment was performed at a speed of 8000 rpm, a time of 90 s, and a volume ratio of the oil phase to the aqueous phase of 3:1. At this time, it was an oil-in-water colostrum.

[0038] (2) Colostrum was rotary evaporated under certain conditions (30 rpm, 40°C) and fully hydrated, and then further subjected to probe ultrasonic treatment at a power of 200 W, ultra-sonication for 3 s, stop for 3 s, and time for 3 min to obtain a drug-loaded cationic liposome solution (abbreviated as DPPC / DOTAPLiposome).

[0039] Example 1

[0040] (1) DPPC, DOTAP and cholesterol were fully dissolved in chloroform at a molar ratio of 75:25:10 (the ratio of DPPC to chloroform was 15:1 (mmol / L)) to prepare a lipid solution as the oil phase. At room temperature, 2 mg / mL of OVA was added to the aqueous phase and high-speed shear treatment was performed at a speed of 8000 rpm, a time of 90 s, and a volume ratio of the oil phase to the aqueous phase of 3:1. At this time, it was an oil-in-water colostrum.

[0041] (2) Colostrum was rotary evaporated under certain conditions (30 rpm, 40°C) and fully hydrated, and then further subjected to probe ultrasonic treatment at a power of 200 W, ultra-sonication for 3 s, stop for 3 s, and time for 3 min to obtain a drug-loaded cationic liposome solution (abbreviated as DPPC / DOTAPLiposome).

[0042] (3) DPPC / DOTAP Liposome was added dropwise to a 1 mg / mL hyaluronic acid solution, incubated at room temperature for 15 min, and free OVA and hyaluronic acid were removed by ultrafiltration. The concentration of all lipids (including DPPC and DOTAP) in the drug-loaded cationic liposome solution was 1 mg / mL, and the volume ratio of the drug-loaded cationic liposome solution to the hyaluronic acid solution was 0.2:1. The hyaluronic acid polysaccharide-modified liposome nanovaccine was prepared, which is referred to as HA-Liposome for the convenience of subsequent description.

[0043] Example 2

[0044] The same method as in Example 1 was used, except that DPPC / DOTAP Liposome was added dropwise to a 1 mg / mL chondroitin sulfate solution, incubated at room temperature for 15 min, and free OVA and chondroitin sulfate were removed by ultrafiltration. The concentration of all lipids (including DPPC and DOTAP) in the drug-loaded cationic liposome solution was 5 mg / mL, and the volume ratio of the drug-loaded cationic liposome solution to the chondroitin sulfate solution was 5:1, and a chondroitin sulfate polysaccharide-modified liposome nanovaccine was obtained, which is referred to as SC-Liposome for convenience of subsequent description.

[0045] Example 3

[0046] The same method as in Example 1 was used, except that DPPC / DOTAP Liposome was added dropwise to a 2 mg / mL heparan sulfate solution, incubated at room temperature for 15 min, and free OVA and heparan sulfate were removed by ultrafiltration. The concentration of all lipids (including DPPC and DOTAP) in the drug-loaded cationic liposome solution was 15 mg / mL, and the volume ratio of the drug-loaded cationic liposome solution to the heparan sulfate solution was 1:1, and a heparan sulfate polysaccharide-modified liposome nanovaccine was obtained, which was referred to as SH-Liposome for convenience of subsequent description.

[0047] Example 4

[0048] According to the same method as Example 1, the difference is that DPPC, phosphatidylinositol, DOTAP and cholesterol are fully dissolved in chloroform with a molar ratio of 50:50:30:10 (the ratio of DPPC, phosphatidylinositol and chloroform is 5:5:1 (mmol / L)) and are prepared into a lipid solution as an oil phase. Under room temperature, 5mg / mL of OVA aqueous phase is added and high-speed shearing is performed to obtain cationic liposomes. The above-mentioned cationic liposomes are added dropwise to 1mg / mL hyaluronic acid solution, incubated at room temperature for 10min, the concentration of all lipids in the drug-loaded cationic liposome solution is 15mg / mL, and the volume ratio of the drug-loaded cationic liposome solution to the hyaluronic acid solution is 3:1, and a liposome nano vaccine modified with hyaluronic acid polysaccharide is obtained.

[0049] Example 5

[0050] According to the same method as Example 1, the difference is that distearoylcholine (DSPC), DOTAP and cholesterol are fully dissolved in chloroform with a molar ratio of 40:25:10 (the ratio of DSPC to chloroform is 7:1 (mmol / L)) and are prepared into a lipid solution as an oil phase. Under room temperature, 0.01mg / mL of OVA aqueous phase is added and high-speed shearing is performed. The above-mentioned cationic liposome is added dropwise to 0.5mg / mL hyaluronic acid solution, incubated at room temperature for 10min, the concentration of all lipids in the drug-loaded cationic liposome solution is 7mg / mL, and the volume ratio of the drug-loaded cationic liposome solution to the hyaluronic acid solution is 2:1, and a liposome nano vaccine modified with hyaluronic acid polysaccharide is obtained.

[0051] Example 6

[0052] The same method as in Example 2 is used, except that DPPC, DOTAP and cholesterol are fully dissolved in chloroform at a molar ratio of 120:40:10 (the ratio of DPPC to chloroform is 9:1 (mmol / L)) to prepare a lipid solution as an oil phase. At room temperature, 0.05 mg / mL of OVA aqueous phase is added and high-speed shear treatment is performed. The above liposomes are added dropwise to a 1 mg / mL chondroitin sulfate solution and incubated at room temperature for 15 min. The concentration of all lipids in the drug-loaded cationic liposome solution is 5 mg / mL, and the volume ratio of the drug-loaded cationic liposome solution to the chondroitin sulfate solution is 4:1, and a chondroitin sulfate polysaccharide-modified liposome nanovaccine is obtained.

[0053] Example 7

[0054] According to the same method as Example 2, the difference is that DPPC, DSPC, DOTAP and cholesterol are fully dissolved in chloroform with a molar ratio of 20:20:20:10 (the ratio of DPPC, DSPC to chloroform is 10:1 (mmol / L)) to prepare a lipid solution as an oil phase. At room temperature, 1 mg / mL of OVA aqueous phase is added and high-speed shear treatment is performed. The above-mentioned liposomes are added dropwise to a 2 mg / mL chondroitin sulfate solution and incubated at room temperature for 10 min. The concentration of all lipids in the drug-loaded cationic liposome solution is 10 mg / mL, and the volume ratio of the drug-loaded cationic liposome solution to the chondroitin sulfate solution is 2:1, and a chondroitin sulfate polysaccharide-modified liposome nanovaccine is obtained.

[0055] Example 8

[0056] The same method as in Example 3 was used, except that DPPC, DOTAP and cholesterol were fully dissolved in chloroform at a molar ratio of 110:25:10 (the ratio of DPPC to chloroform was 15:1 (mmol / L)) to prepare a lipid solution as the oil phase. At room temperature, 3 mg / mL of OVA aqueous phase was added and high-speed shear treatment was performed. The above cationic liposomes were added dropwise to a 2 mg / mL heparan sulfate solution and incubated at room temperature for 10 min. The concentration of all lipids in the drug-loaded cationic liposome solution was 5 mg / mL, and the volume ratio of the drug-loaded cationic liposome solution to the heparan sulfate solution was 4:1, and a heparan sulfate polysaccharide-modified liposome nanovaccine was obtained.

[0057] Example 9

[0058] The same method as in Example 3 was used, except that DPPC, DOTAP and cholesterol were fully dissolved in chloroform at a molar ratio of 60:30:10 (the ratio of DPPC to chloroform was 15:1 (mmol / L)) to prepare a lipid solution as an oil phase. At room temperature, 2 mg / mL of OVA aqueous phase was added and high-speed shearing was performed. The above-mentioned cationic liposomes were added dropwise to a 1 mg / mL heparan sulfate solution and incubated at room temperature for 10 min. The concentration of all lipids in the drug-loaded cationic liposome solution was 10 mg / mL, and the volume ratio of the drug-loaded cationic liposome solution to the heparan sulfate solution was 5:1, and a liposome nanovaccine modified with heparan sulfate polysaccharide was obtained.

[0059] Experimental Example 1: Study on Physical and Chemical Properties

[0060] The particle size and potential of the nanoparticles were measured by dynamic light scattering (DLS) using a Malvern laser particle size analyzer. The measurement temperature was 25°C, the instrument scattering angle was 90°, and the equilibrium time was 30 seconds. Table 1 shows the relevant properties of the representative examples.

[0061] Experimental Example 2: Cytotoxicity Study

[0062] Dendritic cells DC 2.4 and lung bronchial epithelial cells BEAS-2b were used as models and CCK-8 method was used for investigation. 4 The cells were seeded at a density of 100 μL / well in a 96-well plate. After 24 hours of adherence culture, the culture medium was discarded and the cells were rinsed once with PBS. 100 μL of nano-prescription culture medium containing drug concentrations of 10, 20, 40, and 80 μg / mL was used for 24 hours of culture. After the incubation, the samples were removed and rinsed once with PBS. 100 μL of culture medium containing 10% CCK8 was added to the cells. After incubation in a cell culture incubator for 1 hour, the absorbance of each well at 450 nm was measured using an ELISA reader. Figure 1 As shown, when the drug concentration was greater than 20ug / mL, the cell viability of the cationic liposome group decreased significantly, as low as 60%, indicating its potential cytotoxicity. In contrast, the cell viability after treatment with polysaccharide-modified liposome nanovaccine was above 80%, showing good biosafety.

[0063] Experimental Example 3: Alveolar macrophage escape study

[0064] RAW 264.7 cells were cultured at 1 × 10 5 The cells were inoculated in a 6-well plate at a density of 1.54 × 10 cells / well. The cells were cultured in a 37°C, 5% CO2 incubator with 2.0 mL of DMEM complete culture medium containing 10% FBS for 24 h. The original culture medium was then replaced with 2.0 mL of fresh culture medium containing different volumes of polysaccharide-modified lipid nanocomplexes (containing FITC-OVA 40 μg / well). After culturing for another 4 h, the cell surface was gently rinsed with cold sterile PBS, the cells were digested and collected with trypsin, and the cells were resuspended in 500 μL of PBS and tested on the machine. Figure 2 As shown, RAW 264.7 took up the positively charged DOTAP the best, while the uptake of the three negatively charged and smaller polysaccharide-modified lipid nanovaccines was much lower than that of cationic liposomes, indicating a good ability to escape macrophage capture.

[0065] Experimental Example 4: Mucus Permeability Study

[0066] 1. Multiple particle tracking method to observe the mucus penetration behavior of single particles

[0067] 50uL of FITC-labeled OVA-loaded nanoformulation was mixed with artificial lung mucus and incubated at 37°C for 30 minutes. The tracking of nanoparticles was recorded using a nanoparticle tracking analysis system (NTA, NanoSight LM10, UK), that is, the Brownian motion trajectory of the particles was measured under a fluorescence module (excitation 488nm, emission 525nm). Finally, the mean square displacement (MSD) of the particles was calculated by the following equation:

[0068] MSD=[x(t+τ)-x(t)]2+[y(t+τ)-y(t)]2

[0069] Among them, x and y are the coordinates of the particle at a fixed time scale, and τ is the time interval. Figure 3 As shown in A, the trajectory span of particles modified with different polysaccharides is larger and the diffusion ability is stronger. On a time scale of 1 s, the MSD of SH-Liposome is always greater than that of other groups ( Figure 3 B), which is about 2.75 times that of DOTAP liposomes. The MSD of SC-Liposome ranks second, which is 2.18 times that of DOTAP. In summary, SH-Liposome modified with sulfated acetyl polysaccharide has excellent diffusion efficiency in mucus.

[0070] 2. Transwell model to investigate the overall mucus penetration efficiency of nanovaccines

[0071] 200 μL of simulated lung mucus was slowly added to a Transwell-24 membrane nested chamber (5 μm diameter) and placed in a 37°C air bath for 1 h. Then, 100 μL of the nanoformulation containing FITC-labeled OVA was slowly added above the mucus layer, and 300 μL of PBS buffer (10 mM; pH 7.4) was added to the receiving pool below and incubated at 37°C. The receiving pool samples were taken out at fixed time points (0.5 h, 1 h, 1.5 h, 2 h and 4 h), and the fluorescence intensity was measured by an ELISA instrument (excitation wavelength 485 nm, emission wavelength 525 nm). The apparent permeability coefficient P was obtained by the slope of the mucus permeability versus time curve. app .

[0072]

[0073] Where, dQ / dt represents the penetration rate of FITC-OVA; A represents the surface area of ​​the chamber; C0 represents the initial concentration. Figure 3 C and D show the results. Figure 3 B, the overall penetration capacity of polysaccharide-modified liposome nanovaccine was significantly higher than that of cationic liposomes, and the P app The highest, an increase of about 2.73 times, has excellent mucus penetration ability.

[0074] Experimental Example 5: Study on the stability of bronchoalveolar lavage fluid (BALF)

[0075] Balb / c mice were killed by overdose of urethane, and the rat cervical trachea was surgically isolated and the rat lungs were perfused with saline buffer to obtain BALF. 300 μL DPPC / DOTAP Liposome, HA-Liposome, SC-Liposome, and SH-Liposome were incubated with 1 mL BALF at 37°C for 4 h. The particle size and polydispersity index (PDI) were measured by dynamic light scattering. Figure 4 As shown in the figure, the particle size of cationic liposomes increased immediately after contact with BALF. However, the particle size of polysaccharide-modified liposome nanovaccines did not change significantly within 4 hours after contact with BALF, indicating that they have good stability in BALF.

[0076] Experimental Example 6: In vitro immune response study

[0077] Balb / c mice were intrapulmonary injected with free OVA or various OVA-loaded nanoformulations twice, with an interval of 1 week, and the ELISA method was used to detect the relevant antibodies in the blood of mice on the seventh day after the second lung inoculation. Figure 5 As shown, the serum IgG1 and IgG2a titers of mice after immunization with SH-modified liposome nanovaccine were significantly higher than those of free OVA and higher than those of the other two polysaccharide-modified groups. In addition, the ratio of IgG2a to IgG1 was close to 1, indicating that polysaccharide adjuvants containing acetyl sulfate groups can induce a strong triple immune response.

[0078] Table 1 shows the physical and chemical properties of the drug-loaded liposomes in Comparative Example 1 and the polysaccharide-modified liposome nanovaccines in Examples 1 to 9 (n=3).

[0079] Table 1 Properties of Nanovaccines in Examples

[0080] Example Particle size (nm) Particle size distribution (PDI) Potential (mV) Comparative Example 1 179.1±5.3 0.46±0.04 32.00±0.98 Example 1 525.1±6.3 0.24±0.03 -15.10±0.06 Example 2 209.0±1.8 0.17±0.05 -21.40±1.96 Example 3 152.9±0.4 0.25±0.02 -38.60±0.59 Example 4 482.2±7.0 0.25±0.08 -14.20±0.10 Example 5 355.8±4.4 0.30±0.12 -34.20±0.55 Example 6 425.7±3.3 0.24±0.07 -17.20±0.40 Example 7 305.1±4.9 0.28±0.10 -34.80±0.70 Example 8 477.1±9.4 0.22±0.05 -20.20±0.30 Example 9 285.1±3.8 0.26±0.07 -34.20±0.61

[0081] The above is only a preferred embodiment of the present invention. It should be understood that the present invention is not limited to the form disclosed herein, and should not be regarded as excluding other embodiments, but can be used in various other combinations, modifications and environments, and can be modified within the scope of the concept described herein through the above teachings or the technology or knowledge of the relevant field. The changes and modifications made by those skilled in the art shall not deviate from the spirit and scope of the present invention, and shall be within the scope of protection of the claims attached to the present invention.

Claims

1. A polysaccharide-modified liposome nanovaccine, characterized in that: The nano vaccine consists of an anionic polysaccharide shell containing a D-glucuronic acid skeleton and a drug-loaded cationic liposome core coated in the polysaccharide shell, wherein the cationic liposome core carries an anionic antigen OVA.

2. The polysaccharide-modified liposome nanovaccine according to claim 1, characterized in that: The polysaccharide containing a D-glucuronic acid skeleton is selected from one or more of hyaluronic acid (HA), chondroitin sulfate (SC), and heparan sulfate (SH).

3. The polysaccharide-modified liposome nanovaccine according to claim 1, characterized in that: The raw materials for preparing the cationic liposomes include endogenous phospholipids in lungs, cationic lipids and cholesterol.

4. The polysaccharide-modified liposome nanovaccine according to claim 3, characterized in that: The endogenous phospholipids in the lungs are one or more of phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine and phosphatidylglycerol.

5. The polysaccharide-modified liposome nanovaccine according to claim 3, characterized in that: The cationic lipid is selected from one or more of 2,3-dioleyloxypropyl)trimethylammonium chloride (DOTAP), dimethyldioctadecylammonium (DDA), and 3β-[N-(N',N'-dimethylaminoethyl)carbamoyl]cholesterol (DC-Chol).

6. The polysaccharide-modified liposome nanovaccine according to claim 1, characterized in that: The average particle size of the nano vaccine is 100 to 650 nm, and the surface charge is -10 to -40 mV.

7. The method for preparing the polysaccharide-modified liposome nanovaccine according to claim 1, characterized in that: The dissolved lipid membrane material is mixed with the anionic antigen OVA, subjected to high-speed shearing, rotary evaporation and hydration to obtain drug-loaded cationic liposomes; polysaccharides are dissolved in ultrapure water, and the drug-loaded cationic liposome solution is added dropwise to obtain polysaccharide-modified liposome nanovaccines.

8. The method according to claim 6, characterized in that The concentration of the lipid solution is 2-15 mg / mL, the concentration of the OVA solution is 0.01-5 mg / mL, and the volume ratio of the lipid solution to the anionic antigen OVA solution is 1:1-10:1; the concentration of the polysaccharide solution is 0.1-2 mg / mL, and the volume ratio of the drug-loaded cationic liposome solution to the polysaccharide solution is 0.2:1-5:

1.

9. Use of the polysaccharide-modified liposome nanovaccine according to claim 1 in the preparation of a subunit vaccine or adjuvant carrier for delivery to the pulmonary, nasal, ocular, oral, or vaginal mucosa.

10. The use according to claim 9, characterized in that The polysaccharide-modified liposome nano-vaccine is used in the preparation of an inhaled vaccine for enhancing pulmonary mucosal immunity.

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