Nanoparticle immunologic adjuvant with immune synergistic effect and preparation method of nanoparticle immunologic adjuvant

Abstract

The preparation method comprises the following steps: selecting PLGA (poly (lactic-co-glycolic acid)) with biocompatibility and biodegradability as a carrier material, loading an immunopotentiator alpha-tocopherol on PLGA nano-particles, and preparing the nano-particle immunologic adjuvant by using a nano-precipitation method, so as to obtain the nano-particle immunologic adjuvant with an immune synergistic effect. By controlling the polymer concentration and the drug concentration in the preparation process, the average particle size of the drug-loaded nano-particles is 90-120nm, the monodispersion coefficient is less than 0.2, the particle size of the product is uniform and stable, the encapsulation efficiency reaches 80% or more, the content of alpha-tocopherol in the nano-particles is 1-30%, and the drug-loaded nano-particles have a sustained-release effect of one month or more. The PLGA nanoparticles loaded with alpha-tocopherol can promote proliferation of mouse macrophages and enhance secretion of cell inflammatory cytokines and expression of surface costimulatory molecules, have a synergistic enhancement effect of the PLGA nanoparticles and alpha-tocopherol, and can be used as an effective immunologic adjuvant.

Description

Technical Field

[0001] The present invention relates to the technical field of immunity and pharmaceutical preparations, and in particular to a nanoparticle immune adjuvant with immune synergistic enhancement effect and a preparation method thereof. Background Art

[0002] At present, only a few adjuvants are used in human vaccines, including aluminum salt adjuvants, MF59, AS01 and AS03. Traditional immune adjuvants such as aluminum salt adjuvants and oil emulsion adjuvants have been widely used. However, aluminum salt adjuvants have certain safety issues, cannot enhance cell-mediated Th1 or cytotoxic T lymphocyte immune responses, and are not suitable for the mucosal immune system. Oil emulsion adjuvants have the problem of local inflammatory response at the injection site and have large side effects. In addition, in order to cope with the threats posed by infectious diseases and tumors to humans and animals, new vaccines are constantly being developed, and there is still a need to develop safer and more effective immune adjuvants to induce immune responses.

[0003] The use of combined adjuvants (usually a combination of an immunostimulant and a delivery vehicle) can often produce better immune responses and vaccine efficacy to vaccine antigens. Currently, the use of combined adjuvants has become increasingly important. By inducing complementary or synergistic effects on the body's immune response to vaccines, new vaccine adjuvants other than traditional adjuvants can be developed.

[0004] Nanoparticles are similar in size and structure to pathogens and can be easily recognized by immune cells. As delivery carriers, they can improve the stability of antigens, deliver antigens in a targeted manner, and slowly release antigens. As immunostimulants, they can also activate certain immune pathways and enhance the response of the immune system. Common preparation methods for nanoparticles include emulsification-solvent evaporation, salting out, dialysis, membrane emulsification, spray drying, and nanoprecipitation. Among them, the nanoprecipitation method has the advantages of simple preparation method and low cost. Generally, the size of nanoparticles and the drug encapsulation efficiency are affected by factors such as solvent, polymer concentration, surfactant, and the ratio of drug to polymer.

[0005] Poly(lactic acid-glycolic acid) (PLGA) is a polymer material formed by the ring-opening copolymerization of lactic acid (LA) and glycolic acid (GA). It has been widely used in the biomedical field, such as drug delivery, medical imaging, tissue engineering, cancer treatment and vaccine adjuvant. PLGA has excellent biocompatibility and good biodegradability. It can load hydrophilic or hydrophobic molecules, protect drugs and antigens from degradation and release them continuously, and can also target specific organs and cells through modification.

[0006] α-Tocopherol is the most biologically active substance in vitamin E, has certain immune-enhancing properties, and has been widely used in the field of emulsion vaccine adjuvants. Due to its high lipophilicity and low solubility in water, the development of α-tocopherol as an immunostimulant is limited to oil-emulsion adjuvants, and there are few reports on the loading and sustained release of α-tocopherol and the construction of nanoparticle immune adjuvants.

[0007] Therefore, the present invention discloses a nanoparticle immune adjuvant with immune synergistic enhancing effect and a preparation method thereof. It is the first time that the high molecular polymer PLGA is used as a carrier material, and the immune enhancer α-tocopherol is loaded on the PLGA nanoparticles, thereby realizing the controllable preparation of PLGA nanoparticles loaded with α-tocopherol with a high encapsulation rate. Summary of the invention

[0008] The purpose of the present invention is to provide a nanoparticle immune adjuvant with immune synergistic enhancing effect and a preparation method thereof. In view of the demand for the development of new vaccine nanoadjuvants, it is proposed to use PLGA as a carrier and α-tocopherol as an immunostimulant to develop a PLGA nanoparticle loaded with α-tocopherol, so as to synergize the immunoregulatory effects of the sustained-release α-tocopherol and PLGA nanoparticles, thereby serving as a new nanoparticle immune adjuvant with good stability and sustained immune synergistic effect.

[0009] The purpose of the present invention is achieved through the following technical solutions:

[0010] A nanoparticle immune adjuvant with immune synergistic enhancement effect and a preparation method thereof. The nanoparticle immune adjuvant with immune synergistic enhancement effect is composed of a spherical main body of a high molecular polymer and an immunopotentiator loaded in the structure.

[0011] Preferably, calculated based on the total mass percentage of the polymer drug-loaded nanoparticles, the mass percentage of the high molecular weight polymer of the spherical body is 70% to 99%, and the mass percentage of the immunopotentiator α-tocopherol loaded in the structure is 1% to 30%.

[0012] Preferably, the high molecular polymer is polylactic acid-glycolic acid copolymer (PLGA), and the molar ratio of lactic acid (LA) to glycolic acid (GA) in the polylactic acid-glycolic acid copolymer (PLGA) is 90:10 to 10:90.

[0013] More preferably, the molar ratio of lactic acid (LA) to glycolic acid (GA) in the polylactic acid-glycolic acid copolymer (PLGA) is 50:50.

[0014] The method for preparing the nanoparticle immune adjuvant with immune synergistic effect, the method uses the principle of nanoprecipitation, and specifically comprises the following steps by adjusting the concentration of PLGA, the concentration of α-tocopherol and the preparation conditions:

[0015] (1) dissolving a surfactant in deionized water, and the resulting solution is used as the aqueous phase W;

[0016] (2) dissolving poly(lactic acid-co-glycolic acid) (PLGA) and α-tocopherol in a volatile organic solvent, and the resulting solution is used as the organic phase O;

[0017] (3) adding the O phase to the W phase while it is stirred, and the resulting solution is used as a suspension of drug-loaded nanoparticles;

[0018] (4) The drug-loaded nanoparticle suspension obtained in step (3) is repeatedly centrifuged and washed, concentrated to obtain a nanoparticle suspension with synergistic immune enhancement effect, and stored at low temperature.

[0019] Preferably, in step (1), the concentration of the surfactant is 0.1 mg / mL to 4 mg / mL, and the surfactant is selected from one of poloxamer 188, polyvinyl alcohol (PVA), sodium dodecyl sulfate (SDS), and Tween, but is not limited to the above.

[0020] Preferably, in step (2), the concentration of the polylactic acid-co-glycolic acid (PLGA) is 1 mg / mL to 25 mg / mL, the concentration of the α-tocopherol is 0.1 mg / mL to 10 mg / mL, and the volatile organic solvent is selected from one of acetone, methanol, acetonitrile, toluene, and ethyl acetate, but is not limited to the above.

[0021] Preferably, in step (3), the particle size, dispersibility and encapsulation efficiency of the drug-loaded nanoparticles are controlled by regulating the volume ratio of phase O and phase W or the droplet velocity of phase O and the stirring conditions. The volume ratio of phase O to phase W is 1:2 to 1:50, the droplet velocity of phase O is 0.5 mL / min to 3 mL / min, the stirring speed is 100 to 1000 rpm, and the stirring time is 2 min to 30 min.

[0022] Preferably, in step (4), the centrifugal speed is 8000 r / min to 20000 r / min, and the centrifugal time is 10 min to 30 min.

[0023] Preferably, the nanoparticle immune adjuvant with immune synergistic enhancing effect is used in the field of vaccine adjuvant.

[0024] Working mechanism:

[0025] The invention adopts a nano-precipitation method, uses a degradable polymer PLGA as a carrier material, loads an immunopotentiator alpha-tocopherol, and completes the preparation by adjusting the concentrations of PLGA and alpha-tocopherol, the amount of surfactant, the volume ratio of the organic phase and the aqueous phase, and the stirring conditions. The raw materials for preparing the drug-loaded nanoparticle adjuvant include PLGA and alpha-tocopherol.

[0026] Due to the application of the above technical solution, the present invention has the following beneficial effects compared with the prior art:

[0027] 1. The present invention utilizes the principle of nanoprecipitation, and by adjusting the PLGA concentration, α-tocopherol concentration and the method of preparation conditions, the particle size of the prepared PLGA nanoparticles loaded with α-tocopherol is controllable and has a good monodispersity coefficient (PDI<0.2). The process is simple and the repeatability is good, which is conducive to the production of PLGA nanoparticles loaded with α-tocopherol as an immune adjuvant. The average particle size of the PLGA nanoparticles loaded with α-tocopherol is 90-120nm, the PDI is less than 0.2, the Zeta potential is about -13mV, the encapsulation rate can reach more than 80%, the drug loading amount is 1%-30%, the product is uniform and stable, and has a drug sustained release effect;

[0028] 2. The preparation method of the present invention is simple to operate, and the prepared PLGA nanoparticles loaded with α-tocopherol have excellent comprehensive properties. In vitro cell experiments show that the preparation method can promote the proliferation of mouse RAW264.7 macrophages, promote the secretion of pro-inflammatory cytokine IL-6 of RAW264.7 cells, enhance the expression of RAW264.7 cell surface molecule CD80, and have an immunopotentiating effect. It has a more effective synergistic stimulation effect than unloaded PLGA nanoparticles and direct use of α-tocopherol, and provides a choice for new nanoparticle vaccine adjuvants. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, some of the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be made based on these drawings without paying any creative work.

[0030] Figure 1 is a particle size distribution diagram of drug-loaded nanoparticles prepared in Example 1 of the present invention;

[0031] Figure 2 This is a scanning electron microscope image of the drug-loaded nanoparticles prepared in Example 1 of the present invention;

[0032] Figure 3 This is a graph showing the in vitro release results of the drug-loaded nanoparticles prepared in Example 1 of the present invention;

[0033] Figure 4 This is a particle size distribution diagram of blank PLGA nanoparticles prepared in Comparative Example 1 of the present invention;

[0034] Figure 5 This is a scanning electron microscope image of blank PLGA nanoparticles prepared in Comparative Example 1 of the present invention;

[0035] Figure 6 The figure is a result of the two nanoparticles in Example 1 of the present invention and Comparative Example 1 stimulating the proliferation of RAW264.7 macrophages;

[0036] Figure 7 The graphs are the effects of the nanoparticles of Example 1 and Comparative Example 1 of the present invention, and α-tocopherol (TOC) on the secretion of IL-6 by RAW264.7 macrophages;

[0037] Figure 8 The graphs show the effects of the nanoparticles of Example 1 and Comparative Example 1 of the present invention, and α-tocopherol (TOC) on the expression of the surface molecule CD80 of RAW264.7 macrophages;

[0038] Among them, Figure 1 The horizontal axis is the diameter size, and the vertical axis is the volume percentage; Figure 3 The horizontal axis is the number of days, and the vertical axis is the cumulative release rate; Figure 4 The horizontal axis is the diameter size, and the vertical axis is the volume percentage; Figure 6 The horizontal axis is the concentration, and the vertical axis is the cell viability. Figure 7 The horizontal axis is the concentration, and the vertical axis is the content of IL-6 in the supernatant; Figure 8 The horizontal axis is concentration and the vertical axis is percentage. DETAILED DESCRIPTION

[0039] In order to have a clearer understanding of the technical features, purposes and effects of the present invention, a specific implementation scheme is now described in detail.

[0040] The present invention is further described below in conjunction with the examples, but the present invention is not limited to the following examples. The implementation conditions used in the examples can be further adjusted according to the different requirements of specific use, and the implementation conditions not indicated are conventional conditions in the industry. The technical features involved in each embodiment of the present invention can be combined with each other as long as they do not conflict with each other.

[0041] Example 1

[0042] As attached Figure 1-3 And attached Figure 6-8 As shown, this embodiment provides a nanoparticle immune adjuvant with immune synergistic effect and a preparation method thereof.

[0043] Preparation of PLGA nanoparticles loaded with α-tocopherol, the specific steps include:

[0044] (1) dissolving polyvinyl alcohol in deionized water to prepare a 0.5 wt % polyvinyl alcohol solution as phase W;

[0045] (2) dissolving PLGA and α-tocopherol in acetonitrile to prepare an acetonitrile solution containing 6 mg / mL PLGA and 1 mg / mL α-tocopherol as phase O;

[0046] (3) The W phase was stirred at 750 rpm, the O phase was collected with a syringe, and was uniformly added to the W phase at a dropping speed of about 1 mL / min. The volume ratio of the O phase to the W phase was set to 1:10 to obtain a drug-loaded nanoparticle suspension;

[0047] (4) repeatedly centrifuging and washing the drug-loaded nanoparticle suspension obtained in step (3) with ultrapure water at 18,000 rpm for 20 min to obtain PLGA nanoparticles loaded with α-tocopherol;

[0048] According to the nanoparticle size and Zeta potential analyzer, the particle size of the PLGA nanoparticles loaded with α-tocopherol is 110.8 nm, and the PDI is 0.042. See the particle size distribution diagram of the drug-loaded nanoparticles (see Figure 1 ), with monodispersity and a Zeta potential of -13.26 mV;

[0049] Scanning electron microscopy showed that the PLGA nanoparticles loaded with α-tocopherol were spherical structures with smooth surfaces, as shown in the scanning electron microscopy image of the drug-loaded nanoparticles ( Figure 2 );

[0050] PBS solution (pH = 7.4) was used as the drug release medium in vitro. The collected drug-loaded particle suspension was fixed to volume. 1 mL of the nanoparticle suspension was placed in a dialysis bag (MWCO = 14 kD), sealed with a sealing clip and placed in a centrifuge tube containing 30 mL of PBS. The centrifuge tube was placed in a water bath constant temperature oscillator and dialyzed at 37°C and 80 rpm. An equal volume of fresh PBS solution was replaced daily. Samples were recovered at specific time intervals (3, 5, 10, 15, 20, 25, 30 and 35 d). After the samples were freeze-dried, a mixed solution of acetonitrile and methanol was used to dissolve the drug-loaded particles. The residual amount of α-tocopherol in the samples was analyzed by high performance liquid chromatography. The cumulative release amount of α-tocopherol was obtained after conversion, and the in vitro release result graph of the drug-loaded nanoparticles was drawn (as shown in FIG. 1 ). Figure 3 ).

[0051] Comparative Example 1

[0052] As attached Figure 4-5As shown, this comparative example 1 is to prepare blank PLGA nanoparticles, and the specific steps are:

[0053] (1) dissolving polyvinyl alcohol in deionized water to prepare a 0.5 wt % polyvinyl alcohol solution as phase W;

[0054] (2) dissolving PLGA in acetonitrile to prepare an acetonitrile solution containing 6 mg / mL PLGA as phase O;

[0055] (3) The W phase was stirred at 750 rpm, the O phase was collected with a syringe, and was uniformly added to the W phase at a dropping speed of about 1 mL / min. The volume ratio of the O phase to the W phase was set to 1:10 to obtain a blank PLGA nanoparticle suspension;

[0056] (4) repeatedly centrifuging and washing the blank PLGA nanoparticle suspension obtained in step (3) with ultrapure water at 18,000 rpm for 20 min to obtain blank PLGA nanoparticles;

[0057] The particle size of blank PLGA nanoparticles was 100.8 nm and the PDI was 0.054 (Fig. Figure 4 ), with monodispersity and a Zeta potential of -18.03 mV;

[0058] Scanning electron microscopy showed that blank PLGA nanoparticles were spherical structures with smooth surfaces (e.g. Figure 5 ).

[0059] Cell viability assay:

[0060] In a 96-well plate, 8 × 10 4 RAW264.7 macrophages were inoculated at a density of 10 cells / well for cell activity experiments.

[0061] The blank PLGA nanoparticles (PLGANPs) obtained in Comparative Example 1 and the α-tocopherol-loaded PLGA nanoparticles (TOC-PLGANPs) suspension obtained in Example 1 were diluted from 300 μg / mL to 8 concentrations (300, 150, 75, 37.5, 18.75, 9.375, 4.688 and 2.344 μg / mL) in an isocratic manner, and complete cell culture medium and nanoparticle suspensions of various concentrations (100 μL / well in total) were added to a 96-well plate, 4 replicate wells were set for each sample concentration, and a cell control group and a blank control group were set up;

[0062] RAW264.7 cells were cultured in a carbon dioxide incubator at 37°C, 5% CO2 and 95% humidity. After incubation for 48 h, the old culture medium was removed, and 100 μL of basal DMEM culture medium and 10 μL of CCK-8 solution were added to each well and incubated for another 1 h. The optical density (OD value) of each well at 450 nm was then measured using a microplate reader, and the cell survival rate of each group was calculated, as shown in the figure showing the proliferation of RAW264.7 macrophages stimulated by PLGA-based nanoparticles (see Figure 1). Figure 6 );

[0063] RAW264.7 macrophages were cultured at 8 × 10 6 The cells were inoculated in a 6-well plate at a density of 10 cells / well, and the cells were incubated for 24 hours to allow the macrophages to adhere to the wall. The old culture medium was aspirated from each well, and 200 μL of nanoparticle suspension (the final concentrations of PLGANPs and TOC-PLGANPs were 50, 100, and 200 μg / mL, respectively) and 1800 μL of complete culture medium were added. A negative control group (Control group), a drug group (the final concentration of TOC was 5 μg / mL) and a positive control group (the final concentration of LPS was 5 μg / mL) were set up. The incubation was continued for 24 hours. After the incubation, the cell supernatant was collected and centrifuged at 1200 rpm for 3 minutes. 1 mL of the supernatant was used for ELISA kit to detect IL-6 and cytokines, and statistical software was used for analysis, see the effect of PLGA-based nanoparticles on the secretion of IL-6 by RAW264.7 macrophages (see Figure 2). Figure 7 );

[0064] RAW264.7 macrophages were cultured at 8 × 10 4 cells / cm 2 The cells were inoculated at a density of 1.50 μg / mL in a T25 cell culture flask and cultured for 24 h to allow the cells to adhere to the wall. The old culture medium was discarded and 4.5 mL of cell suspension and 0.5 mL of nanoparticle suspension (the final concentrations of PLGANPs and TOC-PLGANPs were 50, 100 and 200 μg / mL, respectively) were added. An LPS positive control group (final concentration of 5 μg / mL), a negative control group (Control group) and a drug group (TOC final concentration of 5 μg / mL) were also set up. The cells were incubated for another 24 h and stained with FITC-CD80 antibody. The cells were then detected by flow cytometry to compare the activation levels of macrophages. See the effect of PLGA-based nanoparticles on the expression of surface molecule CD80 of RAW264.7 macrophages (see Figure 2). Figure 8 );

[0065] It can be seen from the in vitro release results of drug-loaded nanoparticles that the release curve of TOC-PLGANPs within 35 days has two stages: the first stage is from 0 to 15 days, and the drug release rate is slow (the cumulative release amount on the 15th day is 9.7%); the second stage is from 15 to 35 days, and the drug release rate is accelerated. Within 35 days, a total of 47.2% of the drug is released from the sample. TOC-PLGANPs has the characteristics of slow release without burst release.

[0066] The cell survival rate of RAW264.7 cells was analyzed by the results of PLGA-based nanoparticles stimulating the proliferation of RAW264.7 macrophages. It can be seen from the figure that TOC-PLGANPs have a stronger macrophage proliferation-promoting effect than PLGANPs when the concentration is equal to or higher than 18.75 μg / mL.

[0067] The effect of PLGA-based nanoparticles on the secretion of IL-6 by RAW264.7 macrophages showed that compared with the Control group, the TOC group had no significant stimulating effect on the secretion of IL-6. At a low concentration of PLGANPs (50 μg / mL), the expression of IL-6 was not statistically different from that of the Control group. However, with the increase of PLGANPs concentration, the amount of IL-6 secreted by macrophages increased. At a concentration of 200 μg / mL, the IL-6 secretion of the PLGANPs group was about 20 times that of the Control group. The TOC-PLGANPs group had a significant effect on the secretion of IL-6. The stimulating effect on macrophage secretion of IL-6 is strong. TOC-PLGANPs can stimulate macrophages to secrete IL-6 at 50μg / mL, 100μg / mL and 200μg / mL, which are 98.3 times, 111.5 times and 120.6 times that of the Control group, respectively. Compared with the TOC group and the PLGANPs group, TOC-PLGANPs has a stronger effect on stimulating macrophages to secrete cytokines, which may be due to the immune synergistic stimulation of PLGANPs and released TOC, and the secretion of IL-6 further increases with the increase of particle concentration.

[0068] The effect of PLGA-based nanoparticles on the expression of surface molecule CD80 of RAW264.7 cells showed that: compared with the Control group, TOC alone could promote the expression of macrophage surface molecule CD80, with an expression level of 58.4%; with the increase of PLGANPs concentration, the expression level of CD80 also increased from 29.8% (50μg / mL) to 42.5% (200μg / mL); TOC-PLGANPs group at 50μg / mL, 100μg / mL and 200μg / mL The expression levels of CD80 molecules expressed by macrophages were 76.2%, 88.1% and 91.1%, respectively, indicating that TOC-PLGANPs had a significant stimulating effect on the expression of CD80 molecule on the surface of RAW264.7 cells; compared with the TOC group and PLGANPs group, TOC-PLGANPs had a stronger effect in stimulating macrophage activation, which may be due to the immune synergistic stimulation of PLGA nanoparticles and released TOC, and the expression of CD80 was further upregulated with the increase of particle concentration.

[0069] Therefore, the preparation method of the present invention can prepare PLGA-based nanoparticles with a particle size control within the range of 60 to 250 nm, and the polydispersity coefficient is less than 0.2; the PLGA nanoparticles loaded with α-tocopherol prepared by the preparation method of the present invention have uniform product particle size and good monodispersity. Since α-tocopherol is a fat-soluble drug, it is dialyzed and released in 37°C phosphate buffer (PBS) and has a long-term sustained release effect; in vitro investigation of the immunoenhancing effect of PLGA nanoparticles loaded with α-tocopherol on mouse RAW264.7 macrophages, a concentration range that stimulates the proliferation of macrophages is selected, and compared with other blank groups and control groups, the PLGA nanoparticles loaded with α-tocopherol can significantly promote the proliferation of RAW264.7 cells at various concentrations, indicating that it has the function of stimulating the proliferation of macrophages; in vitro investigation of the negative The immunopotentiating effect of α-tocopherol-loaded PLGA nanoparticles on mouse RAW264.7 macrophages was studied. The concentration range that stimulated the proliferation of macrophages was selected. Compared with other blank groups and control groups, α-tocopherol-loaded PLGA nanoparticles could significantly promote the secretion of IL-6 cytokines by RAW264.7 cells at various concentrations, indicating that it has a sustained immunopotentiating function. The immunopotentiating effect of α-tocopherol-loaded PLGA nanoparticles on mouse RAW264.7 macrophages was studied in vitro. The concentration range that stimulated the proliferation of macrophages was selected. It was found that compared with other blank groups and control groups, α-tocopherol-loaded PLGA nanoparticles could significantly promote the expression of CD80 on the surface molecule of RAW264.7 cells at various concentrations, indicating that it has an immunopotentiating effect.

[0070] In summary, the present invention utilizes the principle of nanoprecipitation, and by adjusting the PLGA concentration, the α-tocopherol concentration and the preparation conditions, the particle size of the prepared PLGA nanoparticles loaded with α-tocopherol is controllable and has a good monodispersity coefficient (PDI<0.2). The process is simple and the repeatability is good, which is conducive to the production of PLGA nanoparticles loaded with α-tocopherol as an immune adjuvant. The average particle size of the PLGA nanoparticles loaded with α-tocopherol is 90-120 nm, the PDI is less than 0.2, the Zeta potential is about -13 mV, the encapsulation rate can reach more than 80%, and the drug loading is 1% to The product is uniform and stable, and has a drug sustained-release effect. The PLGA nanoparticles loaded with α-tocopherol prepared by the present invention have excellent comprehensive performance. In vitro cell experiments show that the PLGA nanoparticles can promote the proliferation of mouse RAW264.7 macrophages, promote the secretion of pro-inflammatory cytokine IL-6 of RAW264.7 cells, enhance the expression of RAW264.7 cell surface molecule CD80, have an immunopotentiating effect, and have a more effective synergistic stimulation effect than unloaded PLGA nanoparticles and direct use of α-tocopherol, thereby providing a choice for a new nanoparticle vaccine adjuvant.

[0071] The above-mentioned embodiment only expresses one implementation mode of the present invention, and its description is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached claims.

Claims

1. A nanoparticle immune adjuvant with immune synergistic effect, characterized in that: The nanoparticles are composed of a spherical main body of high molecular polymer and an immunopotentiator loaded in the structure.

2. The nanoparticle immune adjuvant with immune synergistic effect according to claim 1, characterized in that: Calculated based on the total mass percentage of polymer drug-loaded nanoparticles, the mass percentage of the high molecular weight polymer of the spherical body is 70% to 99%, and the mass percentage of the immunopotentiator alpha-tocopherol loaded in the structure is 1% to 30%.

3. The nanoparticle immune adjuvant with immune synergistic effect according to claim 1, characterized in that: The high molecular polymer is a polylactic acid-glycolic acid copolymer, and the molar ratio of lactic acid to glycolic acid in the polylactic acid-glycolic acid copolymer is 90:10-10:

90.

4. The method for preparing the nanoparticle immune adjuvant with immune synergistic effect according to any one of claims 1 to 3, characterized in that: The specific steps include: (1) dissolving a surfactant in deionized water, and the resulting solution is used as the aqueous phase W; (2) dissolving poly(lactic acid-co-glycolic acid) copolymer and α-tocopherol in a volatile organic solvent, and the resulting solution is used as an organic phase O; (3) adding the O phase to the W phase while it is in a stirring state, and the resulting solution is used as a suspension of drug-loaded nanoparticles; (4) The drug-loaded nanoparticle suspension obtained in step (3) is repeatedly centrifuged and washed, concentrated to obtain a nanoparticle suspension with synergistic immune enhancement effect, and stored at low temperature.

5. The method for preparing a nanoparticle immune adjuvant having an immune synergistic enhancing effect according to claim 4, characterized in that: In step (1), the concentration of the surfactant is 0.1 mg / mL to 4 mg / mL, and the surfactant is selected from one of poloxamer 188, polyvinyl alcohol (PVA), sodium dodecyl sulfate (SDS), and Tween, but is not limited to the above.

6. The method for preparing a nanoparticle immune adjuvant having an immune synergistic enhancing effect according to claim 4, characterized in that: In step (2), the concentration of the polylactic acid-co-glycolic acid is 1 mg / mL to 25 mg / mL, the concentration of the α-tocopherol is 0.1 mg / mL to 10 mg / mL, and the volatile organic solvent is selected from one of acetone, methanol, acetonitrile, toluene, and ethyl acetate, but is not limited to the above.

7. The method for preparing a nanoparticle immune adjuvant with immune synergistic effect according to claim 4, characterized in that: In step (3), the particle size, dispersibility and encapsulation efficiency of the drug-loaded nanoparticles are controlled by regulating the volume ratio of the O phase and the W phase or the O phase droplet velocity and stirring conditions. The volume ratio of the O phase to the W phase is 1:2 to 1:50, the O phase droplet velocity is 0.5 mL / min to 3 mL / min, the stirring speed is 100 to 1000 rpm, and the stirring time is 2 min to 30 min.

8. The method for preparing a nanoparticle immune adjuvant with immune synergistic effect according to claim 4, characterized in that: In step (4), the centrifugal speed is 8000 r / min to 20000 r / min, and the centrifugal time is 10 min to 30 min.

9. Application of the nanoparticle immune adjuvant with immune synergistic effect prepared by the nanoparticle immune adjuvant with immune synergistic effect according to any one of claims 1 to 3 and the method for preparing the nanoparticle immune adjuvant with immune synergistic effect according to any one of claims 4 to 9 in the field of vaccine adjuvants.

Images