Pickering emulsion as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biomedical engineering, in particular to Pickering emulsion and a preparation method and application thereof. According to the Pickering emulsion provided by the invention, APS is selected as an immunologic stimulant and is combined with PLGA, so that the immunocompetence of PLGA as an adjuvant is enhanced; the PLGA-coated astragalus polysaccharide can prevent the antigen from being directly exposed in an organic solvent to influence the activity of the antigen; compared with PLGA nanoparticles, the Pickering emulsion has a larger specific surface area and can adsorb more antigens; the Pickering emulsion has good fluidity and can increase the interaction between the PLGA nanoparticles and cell membranes, when the PLGA nanoparticles are in contact with the APCs, a large number of antigens are absorbed by the APCs, and the activated APCs can carry a large number of nanoparticles to enter lymph nodes, so that the Pickering emulsion has good lymph node targeting property. Upon uptake of a large number of antigens, the immune cells induce strong cellular or humoral immune responses in various ways.

Description

Technical Field

[0001] This invention relates to the field of biomedical engineering technology, and in particular to a Pickering emulsion, its preparation method, and its application. Background Technology

[0002] Vaccination is one of the safest and most effective tools for preventing various infectious and chronic diseases, and has effectively reduced the incidence and mortality of many infectious diseases, such as smallpox and diphtheria. A vaccine consists of two parts: an antigen and an adjuvant. Because antigens alone have poor immunogenicity and are unlikely to elicit an immune response, adjuvants are needed to help the antigen stimulate the body's immune response. Currently researched adjuvants include mineral oil, biodegradable nanomaterials containing cytokines, and natural compounds that can enhance the immune response.

[0003] Recent studies have revealed that Astragalus contains various active ingredients such as polysaccharides, saponins, flavonoids, and amino acids. These active ingredients all promote antibody production and immune responses, with Astragalus polysacharin (APS) receiving the most research reports. Astragalus polysacharin is one of the main active components of Astragalus, obtained through high-tech extraction and separation, and is a widely used immunostimulant.

[0004] Poly(lactic-co-glycolic acid) (PLGA) is a biodegradable functional polymeric organic compound with good biocompatibility, non-toxicity, sustained antigen release capability, and adjuvant activity, making it widely used in pharmaceuticals, medical engineering materials, and modern industrial fields. Polyethyleneimine (PEI)-modified PLGA nanoparticles can serve as vaccine adjuvants, inducing humoral and cellular immune responses. However, during the preparation of antigen-loaded PLGA nanoparticles, the antigen is directly exposed to organic solvents, which may affect the antigen's activity. Summary of the Invention

[0005] The purpose of this invention is to provide a Pickering emulsion, its preparation method, and its application, wherein the Pickering emulsion can enhance antigen activity.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides a Pickering emulsion, the raw materials for which include PEI-modified APN, squalene, and water;

[0008] The APN in the PEI-modified APN is PLGA-encapsulated Astragalus polysaccharide.

[0009] Preferably, the mass ratio of the PEI-modified APN to the volume ratio of water is 10 mg:(1.6–3.6) mL.

[0010] Preferably, the mass ratio of PLGA to astragalus polysaccharide in the PLGA-encapsulated astragalus polysaccharide is (5-10):1.

[0011] Preferably, the mass ratio of PEI to APN in the PEI-modified APN is 1:(5-10).

[0012] Preferably, the preparation method of the PEI-modified APN includes the following steps:

[0013] A dichloromethane solution of PLGA and an aqueous solution of Astragalus polysaccharide were mixed to obtain a primary emulsion;

[0014] The primary emulsion and polyvinyl alcohol aqueous solution were mixed to obtain PLGA-encapsulated Astragalus polysaccharide nanoparticles.

[0015] The PLGA-encapsulated Astragalus polysaccharide nanoparticles were mixed with a polyethyleneimine aqueous solution to obtain the PEI-modified APN.

[0016] Preferably, the concentration of the dichloromethane solution of PLGA is 50–100 mg / mL;

[0017] The concentration of the Astragalus polysaccharide aqueous solution is 20–50 mg / mL;

[0018] The concentration of the polyvinyl alcohol aqueous solution is 5–20 mg / mL.

[0019] Preferably, the volume ratio of the PLGA dichloromethane solution to the Astragalus polysaccharide aqueous solution is (1-10):1;

[0020] The volume ratio of the primary emulsion to the polyvinyl alcohol aqueous solution is (1-3):1.

[0021] Preferably, the volume ratio of squalene to water is 1:(4-9).

[0022] This invention also provides a method for preparing the Pickering emulsion described above, comprising the following steps:

[0023] The Pickering emulsion was obtained by mixing PEI-modified APN, squalene, and water.

[0024] The present invention also provides the application of the Pickering emulsion described in the above technical solution in the preparation of vaccines.

[0025] This invention provides a Pickering emulsion, prepared from raw materials including PEI-modified APN, squalene, and water; the APN in the PEI-modified APN is PLGA-encapsulated Astragalus polysaccharide. This invention selects Astragalus polysaccharide as an immunostimulant, which binds to PLGA to enhance the immunomodulatory activity of PLGA as an adjuvant; the PLGA-encapsulated Astragalus polysaccharide avoids direct exposure of antigens to organic solvents, thus preventing interference with antigen activity; compared to PLGA nanoparticles, the Pickering emulsion has a larger specific surface area, allowing for the adsorption of more antigens; the Pickering emulsion has good fluidity, increasing the interaction between PLGA nanoparticles and cell membranes. When PLGA nanoparticles come into contact with antigen-presenting cells (APCs), a large amount of antigen is absorbed by the APCs, and the activated APCs can carry a large number of nanoparticles into lymph nodes, thus exhibiting good lymph node targeting. After taking up a large amount of antigen, immune cells induce strong cellular or humoral immune responses in various ways. Attached Figure Description

[0026] Figure 1 The results of the cytotoxicity test of P-APN described in Example 1;

[0027] Figure 2 After immunizing BALB / c mice with the OVA / P-APNE emulsion described in Example 1, the antibody level against OVA in the mouse serum was detected by ELISA.

[0028] Figure 3 After immunizing BALB / c mice with the OVA / P-APNE emulsion described in Example 1, the concentrations of IFN-γ and IL-4 in the supernatant were detected by ELISA.

[0029] Figure 4 To detect the proliferation of memory T cells using flow cytometry. Detailed Implementation

[0030] This invention provides a Pickering emulsion, the raw materials for which include PEI-modified APN, squalene, and water;

[0031] The APN in the PEI-modified APN is PLGA-encapsulated Astragalus polysaccharide.

[0032] In this invention, unless otherwise specified, all raw materials used in the preparation are commercially available products well known to those skilled in the art.

[0033] In this invention, the volume ratio of squalene to water is preferably 1:(4-9), more preferably 1:(5-8), and most preferably 1:(6-7).

[0034] In this invention, the mass ratio of the PEI-modified APN to the volume ratio of water is preferably 10 mg:(1.6-3.6) mL, more preferably 10 mg:(2.0-3.2) mL, and most preferably 10 mg:(2.3-2.6) mL.

[0035] In this invention, the APN in the PEI-modified APN is PLGA-encapsulated Astragalus polysaccharide; the mass ratio of PLGA to Astragalus polysaccharide in the PLGA-encapsulated Astragalus polysaccharide is preferably (5-10):1, more preferably (6-9):1, and most preferably (7-8):1.

[0036] In this invention, the mass ratio of PEI to APN in the PEI-modified APN is preferably 1:(5-10), more preferably 1:(6-9), and most preferably 1:(7-8).

[0037] In this invention, the preparation method of the PEI-modified APN includes the following steps:

[0038] A dichloromethane solution of PLGA and an aqueous solution of Astragalus polysaccharide were mixed to obtain a primary emulsion;

[0039] The primary emulsion and polyvinyl alcohol aqueous solution were mixed to obtain PLGA-encapsulated Astragalus polysaccharide nanoparticles.

[0040] The PLGA-encapsulated Astragalus polysaccharide nanoparticles were mixed with a polyethyleneimine aqueous solution to obtain the PEI-modified APN.

[0041] This invention involves mixing a dichloromethane solution of PLGA with an aqueous solution of Astragalus polysaccharide to obtain a primary emulsion.

[0042] In this invention, the concentration of the PLGA dichloromethane solution is preferably 50-100 mg / mL, more preferably 60-90 mg / mL, and most preferably 70-80 mg / mL. In this invention, the PLGA dichloromethane solution is preferably prepared by mixing polylactic-co-glycolic acid copolymer (PLGA) and dichloromethane; the mixing is preferably carried out under ultrasonic conditions. This invention does not impose any special limitations on the ultrasonic conditions; conditions well known to those skilled in the art are sufficient to completely dissolve the polylactic-co-glycolic acid copolymer (PLGA) in dichloromethane. In this invention, the PLGA is preferably obtained by polymerization of lactic acid and glycolic acid as monomers; the molar ratio of lactic acid to glycolic acid is preferably 50:50 or 75:25.

[0043] In this invention, the concentration of the Astragalus polysaccharide aqueous solution is preferably 20-50 mg / mL, more preferably 25-45 mg / mL, and most preferably 30-40 mg / mL. In this invention, the Astragalus polysaccharide aqueous solution is preferably prepared by mixing Astragalus polysaccharide and water; this invention does not impose any special limitations on the mixing conditions, and conditions well known to those skilled in the art can be used to ensure that the Astragalus polysaccharide is fully dissolved in water.

[0044] In this invention, the volume ratio of the PLGA dichloromethane solution to the Astragalus polysaccharide aqueous solution is preferably (1-10):1, more preferably (2-5):1, and most preferably (2.5-3.5):1.

[0045] In this invention, the mixing of the PLGA dichloromethane solution and the Astragalus polysaccharide aqueous solution is preferably carried out under ultrasonic conditions. This invention does not impose any special limitations on the ultrasonic conditions, and conditions well known to those skilled in the art can be used.

[0046] After obtaining the primary emulsion, the present invention mixes the primary emulsion with a polyvinyl alcohol aqueous solution to obtain PLGA-encapsulated astragalus polysaccharide nanoparticles.

[0047] In this invention, the concentration of the polyvinyl alcohol aqueous solution is preferably 5-20 mg / mL, more preferably 8-16 mg / mL, and most preferably 10-13 mg / mL.

[0048] In this invention, the volume ratio of the primary emulsion to the polyvinyl alcohol aqueous solution is preferably (1-3):1, more preferably (1.5-2.5):1, and most preferably (1.8-2.2):1.

[0049] In this invention, the mixing of the primary emulsion and the polyvinyl alcohol aqueous solution is preferably carried out under ultrasonic conditions. This invention does not impose any special limitations on the ultrasonic conditions, and conditions well known to those skilled in the art can be used.

[0050] After mixing, the present invention preferably includes the removal of dichloromethane and unbound PVA and APS; the removal of dichloromethane is preferably achieved by stirring; the stirring temperature is preferably room temperature, and the stirring time is preferably 4–24 h, more preferably 15–21 h, and most preferably 16–18 h; the present invention does not impose any special limitation on the stirring speed, and any speed known to those skilled in the art can be used to ensure complete removal of dichloromethane within the above time range. The removal of unbound PVA and APS is preferably achieved by washing with deionized water, and the number of washes is preferably 5; the present invention does not impose any special limitation on the washing process, and any process known to those skilled in the art can be used.

[0051] After obtaining PLGA-coated Astragalus polysaccharide nanoparticles, the present invention mixes the PLGA-coated Astragalus polysaccharide nanoparticles with a polyethyleneimine aqueous solution to obtain the PEI-modified APN.

[0052] In this invention, the concentration of the polyethyleneimine aqueous solution is preferably 0.1 to 5 mg / mL, more preferably 1 to 2 mg / mL, and most preferably 1 mg / mL.

[0053] In this invention, the mass ratio of the PLGA-encapsulated Astragalus polysaccharide nanoparticles to the polyethyleneimine is preferably (1-50):1, more preferably (5-20):1, and most preferably 10:1.

[0054] The present invention does not impose any special limitations on the mixing process. A process well known to those skilled in the art can be used to ensure that the mass of the PLGA-encapsulated Astragalus polysaccharide nanoparticles and the polyethyleneimine aqueous solution are mixed evenly.

[0055] After the mixing is completed, the present invention preferably includes sequential freezing and drying; the freezing temperature is preferably -80°C, and the freezing time is preferably 6 hours. In the present invention, the drying method is preferably freeze-drying. The present invention does not impose any special limitations on the freeze-drying process, and any process well known to those skilled in the art can be used.

[0056] After obtaining the PEI-modified APN, the present invention preferably freezes and stores the PEI-modified APN for later use; the preferred freezing and storage temperature is -20°C.

[0057] This invention also provides a method for preparing the Pickering emulsion described above, comprising the following steps:

[0058] The Pickering emulsion was obtained by mixing PEI-modified APN, squalene, and water.

[0059] In this invention, the mixing process preferably involves first mixing PEI-modified APN and water, then adding squalene and performing ultrasound. This invention does not impose any special limitations on the first mixing process; any process well-known to those skilled in the art can be used, provided that the PEI-modified APN is fully dissolved in the water. In this invention, the ultrasound power is preferably 60–80 W, more preferably 65–75 W, and most preferably 68–72 W; the ultrasound time is preferably 90–120 s, more preferably 95–115 s, and most preferably 100–110 s.

[0060] The present invention also provides the application of the Pickering emulsion described in the above technical solution in the preparation of vaccines.

[0061] In this invention, the preferred method of application is to load the antigen into the Pickering emulsion, and the preferred method of loading is to mix an aqueous solution of the antigen, PEI-modified APN and squalene to obtain a vaccine.

[0062] In this invention, the concentration of the antigen aqueous solution is preferably 0.1–1 mg / mL, more preferably 0.125–0.5 mg / mL, and most preferably 0.125 mg / mL. This invention does not impose any particular limitation on the type of antigen in the antigen aqueous solution; any type well-known to those skilled in the art can be used.

[0063] In this invention, the mass ratio of the antigen to the PEI-modified APN is preferably 1:(10-30), more preferably 1:(10-20), and most preferably 1:20.

[0064] In this invention, the mixing is preferably carried out by adding PEI-modified APN to the antigen aqueous solution until it is fully dissolved, followed by the addition of squalene and ultrasonication. In this invention, the ultrasonic power is preferably 60-80W, more preferably 65-75W, and most preferably 68-72W; the ultrasonic time is preferably 90-120s, more preferably 95-115s, and most preferably 100-110s.

[0065] The following detailed description of the Pickering emulsion, its preparation method, and its application provided by the present invention, with reference to specific embodiments, should not be construed as limiting the scope of protection of the present invention.

[0066] Example 1

[0067] 10 mg of Astragalus polysaccharide (APS) was mixed with 200 μL of deionized water to obtain an aqueous solution of Astragalus polysaccharide with a concentration of 50 mg / mL (as the internal aqueous phase);

[0068] 200 mg of polylactic acid glycolic acid copolymer (PLGA) and 2 mL of dichloromethane were mixed and sonicated until the PLGA was completely dissolved to obtain a PLGA solution with a concentration of 100 mg / mL.

[0069] The PLGA solution was added to the aqueous solution of Astragalus polysaccharide at a volume ratio of 5:1, and the mixture was sonicated to obtain a primary emulsion.

[0070] The primary emulsion was added to a 10 mg / mL polyvinyl alcohol (PVA) aqueous solution at a volume ratio of 1:1. After ultrasonication to form a stable PLGA-coated APS nanoparticle emulsion, the mixture was stirred at room temperature for 4 hours to allow dichloromethane to evaporate, resulting in solidified PLGA-coated APS nanoparticles (denoted as APN).

[0071] 100 mg of the PLGA-encapsulated APS nanoparticles were mixed with 10 mL of a 1 mg / mL polyethyleneimine aqueous solution, stirred at room temperature for 1 h, centrifuged at 12000 rpm for 30 min, washed with deionized water, repeated 3 times, frozen at -80℃ for 6 h, and then freeze-dried to obtain PEI-modified APN nanoparticles (P-APN), which were then stored frozen at -20℃.

[0072] After completely dissolving 10 mg of the P-APN in 1.6 mL of water, 0.4 mL of squalene was added, and the mixture was sonicated at 80 W for 90 s to obtain Pickering emulsion (P-APNE emulsion).

[0073] Using ovalbumin (OVA) as a model antigen, it was loaded into the P-APNE emulsion. The specific method was as follows: 10 mg of P-APN was added to 1.6 mL of OVA aqueous solution with a concentration of 0.125 mg / mL until completely dissolved, and then 0.4 mL of squalene was added. The mixture was sonicated at 80 W for 90 s to obtain a stable OVA / P-APNE emulsion.

[0074] Example 2

[0075] 10 mg of Astragalus polysaccharide (APS) was mixed with 200 μL of deionized water to obtain an aqueous solution of Astragalus polysaccharide with a concentration of 50 mg / mL (as the internal aqueous phase);

[0076] 200 mg of polylactic acid glycolic acid copolymer (PLGA) and 2 mL of dichloromethane were mixed and sonicated until the PLGA was completely dissolved to obtain a PLGA solution with a concentration of 100 mg / mL.

[0077] The PLGA solution was added to the aqueous solution of Astragalus polysaccharide at a volume ratio of 5:1, and the mixture was sonicated to obtain a primary emulsion.

[0078] The primary emulsion was added to a 10 mg / mL polyvinyl alcohol (PVA) aqueous solution at a volume ratio of 1:1. After ultrasonication to form a stable PLGA-coated APS nanoparticle emulsion, the mixture was stirred at room temperature for 4 hours to allow dichloromethane to evaporate, resulting in solidified PLGA-coated APS nanoparticles (denoted as APN).

[0079] 100 mg of the PLGA-encapsulated APS nanoparticles were mixed with 10 mL of a 0.5 mg / mL polyethyleneimine aqueous solution, stirred at room temperature for 1 h, centrifuged at 12000 rpm for 30 min, washed with deionized water, repeated 3 times, frozen at -80℃ for 6 h, and then freeze-dried to obtain PEI-modified APN nanoparticles (P-APN), which were then stored frozen at -20℃.

[0080] After completely dissolving 10 mg of the P-APN in 1.6 mL of water, 0.4 mL of squalene was added, and the mixture was sonicated at 80 W for 90 s to obtain Pickering emulsion (P-APNE emulsion).

[0081] Using OVA as the model antigen, it was loaded into the P-APNE emulsion. The specific method was as follows: 10 mg of P-APN was added to 1.6 mL of OVA aqueous solution with a concentration of 0.125 mg / mL until completely dissolved, and then 0.4 mL of squalene was added. The mixture was sonicated at 80 W for 90 s to obtain a stable OVA / P-APNE emulsion.

[0082] Test Example 1

[0083] The CCK8 assay was used to detect the cytotoxicity of the P-APN described in Example 1.

[0084] The P-APN was mixed with water to prepare a P-APN solution with a concentration of 1 mg / mL. Then, it was serially diluted to obtain a series of P-APN solutions with different concentrations (256 μg / mL, 128 μg / mL, 64 μg / mL, 32 μg / mL, 16 μg / mL, and 0 μg / mL (0 μg / mL is the blank control group)).

[0085] The series of P-APN solutions at different concentrations were added to cultured RAW cells and DC cells, and cultured for 0, 12, 24, and 48 hours, respectively. The test results are as follows: Figure 1 As shown, by Figure 1 It is known that P-APN does not affect cell proliferation and has no cytotoxicity at concentrations of 256 μg / mL or less.

[0086] Test Example 2

[0087] Immunological evaluation was performed on the OVA / P-APNE emulsion described in Example 1:

[0088] Animal immunization:

[0089] BALB / c mice were immunized with OVA / P-APNE emulsion and divided into four groups: PBS group, OVA group, OVA / P-APNE group, and A1 / OVA group. The immunization dose was 20 μg of OVA and 100 μg of P-APN, with an immunization volume of 100 μL. All mice were vaccinated at week 0 and week 3, respectively. Mice were euthanized at week 0 and week 8 post-immunization, and serum and spleen were collected for subsequent analysis.

[0090] ELISA detection of antibody levels against OVA in mouse serum, such as Figure 2 As shown, by Figure 2It can be seen that, compared with the OVA group, the OVA / P-APNE group produced a high level of antibodies against the OVA antigen;

[0091] IFN-γ is a major cytokine secreted by Th1 cells, inducing cell-mediated immune responses, while IL-4 is a major cytokine secreted by Th2 cells, inducing humoral immune responses. Mouse spleens were aseptically harvested and spleen cell suspensions were prepared. Spleen cells were seeded in 24-well plates and incubated with complete culture medium containing 10% OVA at 37°C for 70 hours. The supernatant was then collected, and the concentrations of IFN-γ and IL-4 in the supernatant were detected using ELISA. The results are shown below. Figure 3 As shown, by Figure 3 It was found that, compared with the OVA group, the secretion of IFN-γ and IL-4 in the OVA / P-APNE group was significantly increased, indicating that P-APNE can stimulate humoral and cellular immune responses.

[0092] While the secretion of specific antibodies plays a crucial role in the prevention and control of infectious diseases, antibody response alone is insufficient to resist pathogen invasion; cellular immunity is also important. Effector memory T cells (TEM, CD44) hi CD62L low It can quickly resist the reinvasion of pathogens, while central memory T cells (TCM, CD44) hi CD62L hi This can provide more effective feedback stimulation to DC cells and B cells, both of which are important for preventing pathogen reinfection. Therefore, flow cytometry was used to detect memory T cell proliferation (spleen). The test results are as follows: Figure 4 As shown, by Figure 4 It can be seen that, compared with the OVA group, the OVA / P-APNE group can significantly promote the proliferation of TEM and TCM.

[0093] In summary, the P-APNE emulsion described in this invention is safe and non-toxic, can effectively activate humoral and cellular immune responses, and can be used as a vaccine delivery system.

[0094] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A Pickering emulsion, characterized in that, The raw materials for preparation include PEI-modified APN, squalene, and water; The APN in the PEI-modified APN is PLGA-encapsulated Astragalus polysaccharide.

2. The Pickering emulsion as described in claim 1, characterized in that, The mass ratio of the PEI-modified APN to the volume ratio of water is 10 mg:(1.6–3.6) mL.

3. The Pickering emulsion as described in claim 2, characterized in that, The mass ratio of PLGA to Astragalus polysaccharide in the PLGA-encapsulated Astragalus polysaccharide is (5-10):

1.

4. The Pickering emulsion as described in claim 2, characterized in that, The mass ratio of PEI to APN in the PEI-modified APN is 1:(5-10).

5. The Pickering emulsion according to any one of claims 1 to 4, characterized in that, The preparation method of the PEI-modified APN includes the following steps: A dichloromethane solution of PLGA and an aqueous solution of Astragalus polysaccharide were mixed to obtain a primary emulsion; The primary emulsion and polyvinyl alcohol aqueous solution were mixed to obtain PLGA-encapsulated Astragalus polysaccharide nanoparticles. The PLGA-encapsulated Astragalus polysaccharide nanoparticles were mixed with a polyethyleneimine aqueous solution to obtain the PEI-modified APN.

6. The Pickering emulsion as described in claim 5, characterized in that, The concentration of the dichloromethane solution of the PLGA is 50–100 mg / mL; The concentration of the Astragalus polysaccharide aqueous solution is 20–50 mg / mL; The concentration of the polyvinyl alcohol aqueous solution is 5–20 mg / mL.

7. The Pickering emulsion as described in claim 5, characterized in that, The volume ratio of the PLGA dichloromethane solution to the Astragalus polysaccharide aqueous solution is (1-10):1; The volume ratio of the primary emulsion to the polyvinyl alcohol aqueous solution is (1-3):

1.

8. The Pickering emulsion as described in claim 1, characterized in that, The volume ratio of squalene to water is 1:(4-9).

9. A method for preparing the Pickering emulsion according to any one of claims 1 to 8, characterized in that, Includes the following steps: The Pickering emulsion was obtained by mixing PEI-modified APN, squalene, and water.

10. The use of the Pickering emulsion according to any one of claims 1 to 8 in the preparation of vaccines.

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