A Pickering emulsion composite adjuvant and its preparation method and application

Abstract

The present invention relates to a Pickering emulsion composite adjuvant and a preparation method and application thereof. The composite adjuvant comprises aluminum hydroxide, an oil phase, a particle wetting agent, polysialic acid and an aqueous phase. The adjuvant has excellent thermal stability and long-term storage stability and a high antigen loading rate. The Pickering emulsion modified with polysialic acid has higher antigen uptake on cells, which is beneficial to antigen presenting cells to process antigens and further enhance immune response.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine and relates to a Pickering emulsion composite adjuvant and a preparation method and application thereof. Background Art

[0002] Vaccination is the most cost-effective way to prevent various infectious diseases. Compared with traditional vaccines, new-generation vaccines, especially recombinant protein or synthetic peptide vaccines, are safer than crude inactivated and attenuated microbial vaccines, but have lower immunogenicity. The use of appropriate adjuvants can enhance the immunogenicity of these vaccines. Adjuvants are important components of vaccines that enhance the magnitude, breadth, and durability of immune responses. Advances in immune mechanism research and delivery technology have promoted the optimization of traditional adjuvants and the construction of a variety of new adjuvant carriers, which are conducive to the generation of effective or enhanced immune protection against infectious diseases such as complex pathogens or outbreaks.

[0003] Aluminum hydroxide adjuvant is the first approved human vaccine adjuvant. It is widely used due to its excellent safety, low reactogenicity and low price. However, it has great limitations in inducing T cell-mediated immune activation and is incompatible with some antigens. It is no longer suitable for new pathogens to induce strong immune protection. MF59 is the second human vaccine adjuvant after aluminum hydroxide adjuvant. It is an oil-in-water emulsion composed of squalene, Tween 80 and Span 85, and is mainly used in influenza vaccines. MF59 mainly produces an "immune microenvironment" at the injection site and then induces a strong and lasting germinal center reaction in the draining lymph nodes. As a traditional emulsion, it is a thermodynamically unstable system, and its stability is easily challenged under external interference; and the emulsion stabilized by surfactants will form a water layer on the surface of the droplets, preventing the droplets from adhering to the cell membrane surface or interacting with the antigen, and it is not easily internalized by antigen-presenting cells, making it difficult to control antigen delivery and activate cellular immune responses.

[0004] Pickering emulsions are stabilized by solid particles. Compared with traditional molecular surfactant-stabilized emulsions, Pickering emulsions have low toxicity, high stability, large specific surface area and good biocompatibility. Therefore, Pickering emulsions are widely used in the chemical industry, food industry, agriculture and biomedicine. Due to the low immunogenicity, superior safety and low price of commercial aluminum hydroxide microgels, it has also attracted much attention as a particle stabilizer for Pickering emulsions. Polysialic acid (PSA) is a negatively charged linear homogeneous polysaccharide formed by NeuAc connected by α-2, 8 and / or α-2, 9 glycosidic bonds. PSA plays an important role in neuronal signal transduction and the immune system. It can be used as a substitute for PEG to modify protein and peptide drugs to prolong systemic circulation time or achieve targeted delivery. It can also be used as a new biomaterial for tissue engineering and composite biology. At present, research based on PSA mainly focuses on delivery carriers and new materials. There are few studies on PSA in immunity, and there is no research to prove that PSA can act as an immunostimulant.

[0005] The invention discloses a stable PSA-modified Pickering emulsion composite adjuvant with outstanding immune effect. Summary of the invention

[0006] In view of the deficiencies of the prior art, the object of the present invention is to provide a Pickering emulsion composite adjuvant and a preparation method and application thereof.

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

[0008] In a first aspect, the present invention provides a Pickering emulsion composite adjuvant, wherein the composite adjuvant comprises aluminum hydroxide, an oil phase, a particle wetting agent, polysialic acid and an aqueous phase.

[0009] The purpose of the present invention is to design a new composite adjuvant combining a delivery carrier and an immunostimulant to address the defects of traditional single adjuvants, such as aluminum hydroxide, MF59, etc., which cannot effectively induce cellular immune responses and cannot produce the immune responses required for lasting protective immunity. Combining the advantages of the delivery carrier and the polysaccharide immunostimulant, the new composite adjuvant can induce a sustained mixed Th1 / Th2 type immune response, a higher antibody titer, and a cellular immunity level that is significantly higher than commercially available adjuvants. The aluminum hydroxide in the composite adjuvant is nanometer-scale, stabilized by nano aluminum hydroxide, and the Pickering emulsion that adds trace surfactant molecules to improve the wettability of nano aluminum hydroxide particles shows excellent thermal stability and long-term storage stability, and a high antigen loading rate compared to traditional emulsions (MF59) or other Pickering emulsions. Moreover, the PSA-modified Pickering emulsion has a higher antigen uptake on cells, which is beneficial to the processing of antigens by antigen-presenting cells and further enhancing immune responses.

[0010] Preferably, the composite adjuvant comprises, by mass percentage, 0.025%-0.8% aluminum hydroxide, 1.6%-8.3% oil phase, 0.02-0.5% particle wetting agent, 90.3-98.4% aqueous phase, and comprises, by concentration, 0.001-0.06 mg / mL polysialic acid.

[0011] The mass percentage of the aluminum hydroxide can be selected from 0.025%, 0.05%, 0.08%, 0.1%, 0.12%, 0.15%, 0.18%, 0.2%, 0.22%, 0.25%, 0.28%, 0.3%, 0.32%, 0.35%, 0.38%, 0.4%, 0.42%, 0.45%, 0.48%, 0.5%, 0.52%, 0.55%, 0.58%, 0.6%, 0.62%, 0.65%, 0.68%, 0.7%, 0.75%, 0.80%, 0.81%, 0.82%, 0.83%, 0.84%, 0.86%, 0.87%, 0.88%, 0.89%, 0.90%, 0.91%, 0.92%, 0.93%, 0.94%, 0.95%, 0.96%, 0.97%, 0.98%, 0.99%, 0.91%, 0.99%, 0.91%, 0.99%, 0.91%, 0.99%, 0.91%, 0.99%, 0.99%, 0.91%, 0.93%, 0.99%, 0.99%, 0.91%, 0.99%, 0.99%, 0.99%, 0.91%, 0.99%, .8%, etc., the mass percentage of the oil phase can be selected from 1.6%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.3%, etc., the mass percentage of the granular wetting agent can be selected from 0.02%, 0.024%, 0.03%, 0.048%, 0.24%, 0.3%, 0.32%, 0.35%, 0.38%, 0.4%, 0.42%, 0.45%, 0.48%, 0.5%, etc., the mass percentage of the water phase The content can be selected from 90.3%, 90.5%, 90.8%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.4%, etc., and the concentration of polysialic acid can be selected from 0.001mg / mL, 0.002mg / mL, 0.005mg / mL, 0.008mg / mL, 0.01mg / mL, 0.012mg / mL, 0.015mg / mL, 0.018mg / mL, 0.02mg / mL, 0.022mg / mL, 0.02 5mg / mL, 0.028mg / mL, 0.03mg / mL, 0.032mg / mL, 0.035mg / mL, 0.038mg / mL, 0.04mg / mL, 0.042mg / mL, 0.045mg / mL, 0.048mg / mL, 0.05mg / mL, 0.052mg / mL, 0.055mg / mL, 0.058mg / mL, 0.06mg / mL, etc. Other specific point values ​​within the above numerical range can be selected, so they will not be described one by one here.

[0012] Preferably, the oil phase comprises any one of squalene, soybean oil, olive oil, coconut oil, isopropyl myristate, medium chain triglycerides, and paraffin oil, or a combination of at least two thereof.

[0013] Preferably, the particle wetting agent includes any one of soybean lecithin, nonionic surfactant, anionic surfactant, cationic surfactant, zwitterionic surfactant, or a combination of at least two thereof.

[0014] Preferably, the non-ionic surfactant comprises Tween 80.

[0015] Preferably, the source of polysialic acid includes Neisseria meningitidis B polysaccharide B, Neisseria meningitidis C polysaccharide C or Escherichia coli K92 polysaccharide.

[0016] Preferably, the average molecular weight of the polysialic acid is 300-100000 Da, for example 300 Da, 500 Da, 800 Da, 1000 Da, 5000 Da, 10000 Da, 20000 Da, 30000 Da, 40000 Da, 50000 Da, 60000 Da, 70000 Da, 80000 Da, 90000 Da, 100000 Da, etc. Other specific values ​​within the above numerical range can be selected, which will not be described one by one here.

[0017] Preferably, the particle size of the aluminum hydroxide is 200-3000nm, for example, 200nm, 500nm, 800nm, 1000nm, 1500nm, 2000nm, 2500nm, 3000nm, etc. Other specific point values ​​within the above numerical range can be selected, which will not be described here one by one.

[0018] Preferably, the particle size of the aluminum hydroxide is 200-600nm, for example, 200nm, 250nm, 300nm, 350nm, 400nm, 450nm, 500nm, 550nm, 600nm, etc. Other specific point values ​​within the above numerical range can be selected, which will not be described here one by one.

[0019] The present invention finds that the particle size of aluminum hydroxide is very important for the stability of the adjuvant, and too high a particle size of aluminum hydroxide will affect the stability of the adjuvant.

[0020] Preferably, the aqueous phase comprises any one of ultrapure water, citric acid buffer, 0.9% sodium chloride solution, phosphate buffer or histidine buffer, or a combination of at least two thereof.

[0021] In a second aspect, the present invention provides a method for preparing the Pickering emulsion composite adjuvant according to the first aspect, the preparation method comprising:

[0022] (1) mixing an aqueous phase, a particle wetting agent, and aluminum hydroxide, and homogenizing by water bath ultrasound, probe ultrasound, or high pressure to obtain a nano aluminum hydroxide solution;

[0023] (2) mixing the oil phase with the nano aluminum hydroxide solution;

[0024] (3) The product obtained in step (2) is subjected to probe ultrasound or homogenization to obtain a Pickering emulsion, which is then mixed with polysialic acid to obtain the product.

[0025] The novel composite adjuvant of the present invention is composed of a polysaccharide immunostimulant and a Pickering emulsion carrier, wherein the Pickering emulsion is an O / W type Pickering emulsion prepared by a one-step method of probe ultrasound by nano-forming an aluminum hydroxide adjuvant as a particle stabilizer to replace the molecular surfactant in the MF59 emulsion; finally, the immunostimulant, the antigen or the mixture of the immunostimulant and the antigen are mixed with the Pickering emulsion in a certain proportion to obtain a composite adjuvant or vaccine composition solution; and the immune level is evaluated after percutaneous immunization.

[0026] The mixing with polysialic acid can also be completed in step (1), that is, the polysialic acid is dissolved in the aqueous phase and then used as a diluent for the aluminum hydroxide gel, and a nano aluminum hydroxide solution containing polysialic acid is prepared by probe ultrasound, which is then mixed with the oil phase and a Pickering emulsion composite adjuvant containing polysialic acid is prepared by probe ultrasound.

[0027] In step (1), the particle size of aluminum hydroxide can be reduced by ultrasonic or high-pressure homogenization.

[0028] Preferably, the power of the ultrasound in step (1) is 60-120 W, the temperature is 0-4° C., and the time is 5-20 min.

[0029] The power can be selected as 60W, 70W, 80W, 90W, 100W, 110W, 120W, etc. The temperature can be selected as 0℃, 0.5℃, 1℃, 1.5℃, 2℃, 2.5℃, 3℃, 3.5℃, 4℃, etc. The time can be selected as 5min, 8min, 10min, 12min, 15min, 18min, 20min, etc. Other specific point values ​​within the above numerical range can be selected, so they will not be elaborated here.

[0030] Preferably, the pressure of the high-pressure homogenization is 600-2000 bar, and the time is 5-15 cycles.

[0031] The pressure can be selected as 600bar, 800bar, 1000bar, 1200bar, 1500bar, 2000bar, etc. The number of cycles is 5 times, 6 times, 8 times, 10 times, 12 times, 15 times, etc. Other specific point values ​​within the above numerical range can be selected, so they will not be described here one by one.

[0032] Preferably, the oil phase is mixed with the nano aluminum hydroxide solution and then vortexed.

[0033] Preferably, the vortex time is 30-60s, for example 30s, 35s, 40s, 45s, 50s, 55s, 60s, etc. Other specific point values ​​within the above numerical range can be selected and will not be described here one by one.

[0034] Preferably, the ultrasonic power in step (3) is 30-105W, the temperature is 0-4°C, and the time is 1-2min.

[0035] The power can be selected as 30W, 40W, 50W, 60W, 70W, 80W, 90W, 100W, 105W, etc. The temperature can be selected as 0℃, 0.5℃, 1℃, 1.5℃, 2℃, 2.5℃, 3℃, 3.5℃, 4℃, etc. The time can be selected as 1min, 1.2min, 1.4min, 1.6min, 1.8min, 2min, etc. Other specific point values ​​within the above numerical range can be selected, so they will not be elaborated here.

[0036] In a third aspect, the present invention provides a use of the Pickering emulsion composite adjuvant according to the first aspect in preparing a vaccine.

[0037] Pickering emulsion composite adjuvant is mainly combined with different antigens to form a vaccine composition solution, which is used to enhance the immune effect after in vivo immunization.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] 1. The Pickering emulsion stabilized by nano-aluminum hydroxide and added with a trace amount of particle wetting agent Tween 80 showed excellent stability. There was no significant change in appearance, particle size and potential after being placed under accelerated conditions for 10 months.

[0040] 2. After the Pickering emulsion is combined with different proteins, the system is stable, the particle size distribution diagram is a single peak distribution, and the protein binding rate is as high as over 90%.

[0041] 3. PSA-modified Pickering emulsion showed higher OVA antigen uptake on RAW264.7 cells.

[0042] 4. Nano-aluminum hydroxide-stabilized Pickering emulsion or nano-aluminum hydroxide-stabilized Pickering emulsion modified with PSA can induce a sustained mixed Th1 / Th2-type specific immune response, and can induce higher antibody titers and cellular immune response levels compared with commercially available aluminum hydroxide and MF59 emulsion adjuvants. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 It is a schematic diagram of the structure of the nano-aluminum hydroxide / PSA-stabilized Pickering emulsion composite adjuvant.

[0044] Figure 2Optical microscopy and transmission electron microscopy images of Pickering emulsion morphology.

[0045] Figure 3 It is the particle size result of Pickering emulsion under accelerated conditions.

[0046] Figure 4 It is the potential result under accelerating conditions.

[0047] Figure 5 These are the toxicity results of different prescriptions on RAW264.7.

[0048] Figure 6 These are laser confocal uptake images of FITC-labeled antigen OVA on RAW264.7 with different formulations, where the scale bar is 10 μm. DETAILED DESCRIPTION

[0049] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0050] The hydrodynamic particle size, polydispersity index (PDI) and zeta potential of nano-aluminum hydroxide or Pickering emulsion droplets were determined by dynamic light scattering (DLS) using a Malvern Zetasizer Nano ZS instrument (ZEN3600). Nano-aluminum hydroxide or Pickering emulsion was diluted 8 times and 100 times with ultrapure water to measure particle size and zeta potential, respectively.

[0051] The maximum power of the ultrasonic instrument used in this application is 150W, and the energy percentages involved in the following embodiments are all percentages of this power, such as 70% energy means 70% of 150W, that is, 105W.

[0052] Example 1

[0053] This example provides a 3 mg / mL Pickering emulsion, and the sources of raw materials for preparing the Pickering emulsion are as follows:

[0054]

[0055]

[0056] Its preparation method is:

[0057] (1) Preparation of nano-aluminum hydroxide (4 mg / mL): (10 mg / mL) was mixed and then 4 mL was taken and mixed with 5.94 mL ultrapure water and 60 μL 5% Tween 80 solution; 1.2 mL of the above solution was transferred into 2 mL centrifuge tubes (8 in total), and 8 3 mm probes were inserted into appropriate positions below the liquid level of the centrifuge tubes under the condition of an ice water bath, and the energy was 85%, and the ultrasound was performed for 10 minutes with an ultrasound interval of 5 seconds to prepare a nano aluminum hydroxide solution.

[0058] (2) Preparation of Pickering emulsion: 900 μL of nanoaluminum hydroxide solution was transferred from the centrifuge tube to 8 new 2 mL centrifuge tubes, 300 μL of ultrapure water and 60 μL of squalene were added to each centrifuge tube, and after vortexing for 30 seconds, 8 3 mm probes were inserted below the liquid surface of the centrifuge tube in an ice water bath, and ultrasonic treatment was performed at 70% energy for 2 minutes with an ultrasonic interval of 5 seconds to prepare the Pickering emulsion.

[0059] The particle size, PDI and potential of nano-aluminum hydroxide and Pickering emulsion were measured using a dynamic light scattering instrument, and the results are shown in Table 1.

[0060] Table 1

[0061]

[0062] Example 2

[0063] This example provides an 8 mg / mL Pickering emulsion, and the sources of raw materials for preparing the Pickering emulsion are as follows:

[0064]

[0065] Its preparation method is:

[0066] (1) Preparation of nano-aluminum hydroxide (8 mg / mL): (10 mg / mL) and then 8 mL is mixed with 2 mL ultrapure water; 1.2 mL of the above solution is transferred into a 2 mL centrifuge tube (8 in total), and under the condition of an ice water bath, 8 3 mm probes are inserted below the liquid surface of the centrifuge tube, and the energy is 85%, and the ultrasound is performed for 10 minutes with an ultrasound interval of 5 seconds to prepare a nano aluminum hydroxide solution.

[0067] (2) Preparation of Pickering emulsion: 60 μL of squalene was added to each centrifuge tube, vortexed for 30 seconds, and then eight 3 mm probes were inserted below the liquid surface of the centrifuge tube in an ice water bath. The Pickering emulsion was prepared by ultrasonication at 70% energy for 2 minutes with an ultrasonic interval of 5 seconds.

[0068] The particle size, PDI and potential of nano-aluminum hydroxide and Pickering emulsion were measured using a dynamic light scattering instrument, and the results are shown in Table 2.

[0069] Table 2

[0070]

[0071] Example 3

[0072] This embodiment provides 5 kinds of Pickering emulsions, and the sources of raw materials for preparing the Pickering emulsions are as follows:

[0073]

[0074] Its preparation method is:

[0075] (1) Preparation of nano-aluminum hydroxide (4 mg / mL): After mixing, 8 mL of the mixture was mixed with 11.88 mL of aqueous phase (F1 was ultrapure water, F2 was citric acid buffer, F3 was phosphate buffer, F4 was 0.9% sodium chloride aqueous solution, and F5 was histidine buffer) and 120 μL of 5% Tween 80 solution. The mixed solution was placed under an 8 mm probe, an ice water bath was added, the ultrasonic energy was set to 40%, the ultrasonic interval was set to 5 seconds, and the ultrasonic was performed for 5 minutes to prepare a nano aluminum hydroxide solution.

[0076] (2) Preparation of Pickering emulsion: Add 1000 μL of squalene solution to the above nano-aluminum hydroxide solution, sonicate in a water bath for 30 seconds (shake the bottle at the same time) to mix the oil and water phases evenly, sonicate under an 8 mm probe, add an ice water bath, set the ultrasonic energy to 20%, the ultrasonic interval to 5 seconds, and the ultrasonic time to 2 minutes.

[0077] The particle size and PDI of the Pickering emulsion were measured using a dynamic light scattering instrument. The results are shown in Table 3.

[0078] Table 3

[0079] serial number Sample name Particle size (nm) PDI F1 Pickering emulsion (aqueous phase: ultrapure water) 1013.8±19.3 0.22±0.0 F2 Pickering emulsion (aqueous phase: citrate) 875.6±5.7 0.24±0.0 F3 Pickering emulsion (water phase: phosphate) 866.2±23.2 0.22±0.0 F4 Pickering emulsion (aqueous phase: 0.9% sodium chloride) 848.5±30.1 0.09±0.1 F5 Pickering emulsion (aqueous phase: histidine salt) 926.5±13.9 0.21±0.0

[0080] Example 4

[0081] This embodiment provides a Pickering emulsion, and the sources of raw materials for preparing the Pickering emulsion are as follows:

[0082]

[0083] Its preparation method is:

[0084] (1) Preparation of soybean lecithin (10%)-squalene solution: Accurately weigh 0.2 g soybean lecithin and 1.8 g squalene into a 15 mL centrifuge tube and sonicate in a 40°C water bath for 1 h to completely dissolve the soybean lecithin.

[0085] (2) Preparation of nano-aluminum hydroxide (4 mg / mL): After mixing (10 mg / mL), 8 mL was taken and mixed with 12 mL ultrapure water, and the mixed solution was placed under an 8 mm probe, an ice water bath was added, the ultrasonic energy was set to 40%, the ultrasonic interval was set to 5 seconds, and the ultrasonic was performed for 5 minutes to prepare a nano aluminum hydroxide solution.

[0086] (3) Preparation of Pickering emulsion: 60 μL of soybean lecithin-squalene solution and 1000 μL of squalene solution were added to the nano-aluminum hydroxide solution, and the oil and water phases were mixed evenly by water bath sonication for 30 seconds (while shaking the bottle), and then sonication was performed under an 8 mm probe with an ice water bath. The ultrasonic energy was set to 20%, the ultrasonic interval was 5 seconds, and the ultrasonic time was 2 minutes.

[0087] The particle size and PDI of the Pickering emulsion at T0 and room temperature for 30 days were measured using a dynamic light scattering instrument. The results are shown in Table 4.

[0088] Table 4

[0089]

[0090] Example 5

[0091] This embodiment provides a prescription composition and preparation method of Pickering emulsion, and the composition of Pickering emulsion is as follows:

[0092]

[0093] Its preparation method is:

[0094] (1) (4 mg / mL) solution: After mixing well, 8 mL of the mixture was taken out and mixed with 11.88 mL of ultrapure water and 120 μL of 5% Tween 80 solution to serve as the aqueous phase of the Pickering emulsion.

[0095] (2) Preparation of Pickering emulsion: Add 1000 μL of squalene solution to the solution, perform water bath sonication for 30 seconds (shake the bottle at the same time) to mix the oil and water phases evenly, perform sonication under an 8 mm probe, add an ice water bath, set the ultrasonic energy to 20%, the ultrasonic interval to 5 seconds, and the ultrasonic time to 2 minutes.

[0096] The particle size and PDI of the Pickering emulsion were measured using a dynamic light scattering instrument. The results are shown in Table 5.

[0097] Table 5

[0098]

[0099] Example 6

[0100] This embodiment provides a composition and preparation method of a polysialic acid-modified Pickering emulsion or vaccine composition. The composition of the Pickering emulsion is as follows:

[0101]

[0102] Its preparation method is:

[0103] (1) Preparation of nano-aluminum hydroxide (4 mg / mL): After mixing well, 8 mL of the mixture was taken and mixed with 11.88 mL of ultrapure water and 120 μL of 5% Tween 80 solution. The mixed solution was placed under an 8 mm probe, an ice water bath was added, the ultrasonic energy was set to 40%, the ultrasonic interval was set to 5 seconds, and the ultrasonic was performed for 5 minutes to prepare a nano aluminum hydroxide solution.

[0104] (2) Preparation of Pickering emulsion: Add 1000 μL of squalene solution to the nano-aluminum hydroxide solution, sonicate in a water bath for 30 seconds (shake the bottle at the same time) to mix the oil and water phases evenly, sonicate under an 8 mm probe, add an ice water bath, set the ultrasonic energy to 20%, the ultrasonic interval to 5 seconds, and the ultrasonic time to 2 minutes.

[0105] (3) Polysialic acid / OVA loaded Pickering emulsion:

[0106] Preparation of OVA solution: Accurately weigh 0.36 mg of OVA powder and 2.64 g (20 mM His, pH=6.5) of buffer solution into a 5 mL centrifuge tube to completely dissolve OVA.

[0107] Preparation of polysialic acid solution: 6 mg of polysialic acid powder and 0.994 g (20 mM His, pH=5.5) of buffer solution were accurately weighed into a 2 mL centrifuge tube to completely dissolve the polysialic acid.

[0108] Use a pipette to transfer 400 μL of Pickering emulsion, 400 μL of OVA solution, 4 μL of polysialic acid solution and 396 μL of ultrapure water into a 2 mL centrifuge tube, and use a pipette to pipette back and forth to mix the solution evenly.

[0109] After standing for 30 minutes, the particle size, PDI and potential of the mixed formulation were measured. The results are shown in Table 6.

[0110] Table 6

[0111]

[0112] By comparing the emulsions prepared by F2 in Example 6 with those prepared in Example 5, it can be seen from the results that the particle size and PDI of the Pickering emulsion prepared in Example 5 are higher than those of sample F2 in Example 6, indicating that the particle size of aluminum hydroxide has a great influence on the particle size and stability of the Pickering emulsion.

[0113] Example 7

[0114] This embodiment provides a polysialic acid-modified Pickering emulsion, the composition of the Pickering emulsion is as follows:

[0115]

[0116]

[0117] Its preparation method is:

[0118] 1. Preparation of polysialic acid (5 μg / mL) / nanoaluminum hydroxide solution (4 mg / mL) (aqueous phase):

[0119] Preparation of polysialic acid (8.5 μg / mL)-Tween 80 (0.05%) solution: Use a pipette to transfer 100 μL of 5% Tween 80 solution and 170 μL of 0.05% polysialic acid solution into a 15 mL centrifuge tube, and then add 9.73 mL of ultrapure water and mix well.

[0120] Polysialic acid (5 μg / mL) / Alhydrogel (4 mg / mL) solution: Pipette 4 mL (10 mg / mL) in a 15 mL centrifuge tube. Alhydrogel needs to be fully shaken before sampling. Then add 6 mL of polysialic acid-Tween 80 mixture into the centrifuge tube and mix well.

[0121] Polysialic acid (5 μg / mL) / nano-aluminum hydroxide solution (4 mg / mL): 1.2 mL of polysialic acid / Alhydrogel solution was transferred into 2 mL centrifuge tubes (8 in total) using a pipette. Under the condition of an ice-water bath, 8 3 mm probes were inserted below the liquid level of the centrifuge tubes. The energy was 85%, and the ultrasound was performed for 10 minutes with an interval of 5 seconds to prepare a polysialic acid / nano-aluminum hydroxide solution. The particle size, PDI and potential of the polysialic acid / nano-aluminum hydroxide were measured using a dynamic light scattering instrument.

[0122] 2. Preparation of polysialic acid modified Pickering:

[0123] 60 μL of squalene was added to the 8 centrifuge tubes of the prepared polysialic acid / nanoaluminum hydroxide, and the mixture was vortexed for 30 seconds to mix the oil and water phases evenly.

[0124] Under the condition of an ice-water bath, 8 3mm probes were inserted into the appropriate position below the liquid level of the centrifuge tube, and the polysialic acid-modified Pickering emulsion was prepared by ultrasonication at 70% energy for 2 minutes with an ultrasonication interval of 5 seconds. The particle size, PDI and potential of the Pickering emulsion were measured using a dynamic light scattering instrument.

[0125] The results are shown in Table 7.

[0126] Table 7

[0127] Sample name Particle size (nm) PDI Potential (mV) Polysialic acid / nano aluminum hydroxide 332.5±4.5 0.22±0.01 23.3±0.7 Polysialic acid modified Pickering emulsion 1392.3±239.6 0.40±0.03 27.3±4.3

[0128] Example 8

[0129] This embodiment provides a polysialic acid-modified Pickering emulsion, and the composition of the polysialic acid-modified Pickering emulsion is as follows:

[0130]

[0131] Its preparation method is:

[0132] 1. Preparation of nano aluminum hydroxide solution (4 mg / mL) (aqueous phase):

[0133] Pipette 4mL (10 mg / mL) in a 15 mL centrifuge tube. Alhydrogel needs to be fully shaken before sampling. Then, 5.94 mL of ultrapure water and 60 μL of 5% Tween 80 solution are added to the centrifuge tube and mixed evenly.

[0134] The nano aluminum hydroxide solution was prepared using the probe ultrasound (Ningbo Xinzhi) method. 1.2 mL of the above solution was transferred to a 2 mL centrifuge tube (8 in total) with a pipette. Under the condition of an ice-water bath, 8 3 mm probes were inserted into the appropriate position below the liquid level of the centrifuge tube. The energy was 85%, and the ultrasound was performed for 10 minutes with an ultrasound interval of 5 seconds to prepare the nano aluminum hydroxide solution. The particle size, PDI and potential of the nano aluminum hydroxide were measured using a dynamic light scattering instrument.

[0135] 2. Preparation of Pickering emulsion modified with polysialic acid:

[0136] Preparation of polysialic acid (0.02%) solution: accurately weigh 1 mg of polysialic acid powder into a 15 mL centrifuge tube, add 4.999 g of 20 mM His, pH = 6.0 buffer to completely dissolve the polysialic acid.

[0137] 1200 μL of the nano-aluminum hydroxide solution was transferred to 8 new 2 mL centrifuge tubes, 32 μL of 0.02% polysialic acid solution and 60 μL of squalene were added to each centrifuge tube, and the solution was vortexed for 30 seconds to mix evenly.

[0138] Under the condition of an external ice-water bath, 8 3mm probes were inserted into appropriate positions below the liquid level of the centrifuge tube, and the Pickering emulsion was prepared by ultrasonication for 2 minutes at 70% energy and an ultrasonication interval of 5 seconds. The particle size, PDI and potential of the Pickering emulsion were measured using a dynamic light scattering instrument, and the results are shown in Table 8.

[0139] Table 8

[0140] Sample name Particle size (nm) PDI Potential (mV) Nano aluminum hydroxide (4 mg / mL) 416.7±1.9 0.24±0.01 20.2±0.64 Polysialic acid (0.02%)-His solution 14.4±0.4 0.51±0.05 -30.7±2.1 Pickering emulsion modified with polysialic acid (5 μg / mL) 1632.0±114.5 0.21±0.02 NA

[0141] Example 9

[0142] This embodiment provides a sialic acid-modified Pickering emulsion, and the composition of the sialic acid-modified Pickering emulsion is as follows:

[0143]

[0144] The only difference between it and the F3 sample in Example 6 is that the molecular weight of sialic acid is different. The molecular weight of sialic acid in this example is about 300 Da, while the molecular weight of sialic acid in Example 6 is 50-60 kDa. The preparation method is consistent with the preparation method of the F3 sample in Example 6.

[0145] After standing for 30 minutes, the particle size, PDI and potential of the mixed formulation were measured. The results are shown in Table 9.

[0146] Table 9

[0147]

[0148] Test Example 1

[0149] Morphological identification:

[0150] Optical microscope: After 4 mg / mL nano-aluminum hydroxide stabilized Pickering emulsion (sample F2 in Example 6) was diluted 50 times with ultrapure water, 2 μL was dripped onto a glass slide, covered with a cover glass, and the structure of the Pickering emulsion was observed under a polarized field under an Olympus optical microscope at 40X magnification to observe whether the nano-aluminum hydroxide was on the surface of the droplet. The results are as follows Figure 2 As shown in the left figure, under the polarized field, the Pickering emulsion stabilized by nano-aluminum hydroxide is a hollow bright circle, which is completely different from the aluminum hydroxide distributed in the form of particle luminescence. This shows that the nano-aluminum hydroxide is adsorbed on the oil-water interface to form a Pickering emulsion stabilized by nano-aluminum hydroxide.

[0151] Transmission electron microscopy: 4 mg / mL nano-aluminum hydroxide stabilized Pickering emulsion (sample F2 in Example 6) was diluted 50 times with ultrapure water, and the Pickering emulsion droplets were captured using a copper mesh. The morphology of the Pickering emulsion was observed by transmission electron microscopy. Figure 2As shown in the right figure, the Pickering emulsion is a regular sphere with irregular protrusions on the surface. These protrusions are mainly caused by the uneven distribution of nano-aluminum hydroxide with a lamellar structure; nano-aluminum hydroxide not only adsorbs on the surface of squalene oil droplets, but also forms a chain structure to connect with the droplets.

[0152] Test Example 2

[0153] Stability study: The Pickering emulsion (sample F1 in Example 3) was placed in stability boxes at 5°C, 25°C and 40°C, and samples were taken at 1W, 2W, 3W, 5W, 7W, 8W, 9W, 11W, 13W, 16W, 22W, 26W and 52W to test the appearance, particle size, PDI and potential of the Pickering emulsion. The results are shown in Figure 3 and Figure 4 As shown in the figure, it can be seen that the emulsion prepared in the present application has high stability.

[0154] Test Example 3

[0155] Cytotoxicity assay:

[0156] The proliferation of RAW264.7 cells treated with different agents was studied by cell counting kit (CCK-8, Bio-Tech) to evaluate cytotoxicity. RAW264.7 cells were cultured at 5×10 4 The cells were seeded into 96-well plates (Corning, USA) at a density of 10 cells / well and cultured in an incubator for 12 hours.

[0157] F1, F2, F3 in Example 6, aluminum hydroxide, polysialic acid and commercially available MF59 emulsion were diluted to different concentrations (5 μg / mL, 10 μg / mL, 15 μg / mL, 20 μg / mL, 40 μg / mL, 80 μg / mL, 160 μg / mL) with cell culture medium (DMEM (Zhongqiao Xinzhou)), and incubated with RAW264.7 cells for 24 hours, respectively, and the DMEM wells without samples were used as the control group. After 24 hours, the culture medium was discarded and the cells were washed three times with PBS to remove the residual preparation. Afterwards, 100 μL of fresh culture medium containing 10 μL of CCK-8 solution was added to each well, and the cells were incubated for another 2 hours. The absorbance of each well was tested at 450 nm with an ELISA reader (SpectraMax M5e, Molecular Devices, USA), and the OD value was recorded. Each sample was repeated three times.

[0158]

[0159] The results are as follows Figure 5 shown.

[0160] Test Example 4

[0161] Cellular uptake assay:

[0162] RAW264.7 cells were cultured at 2 × 10 5 The density of 1000 / well was inoculated on a 24-well glass slide (Corning, USA) and cultured for 12 hours. F1 in Example 4 and F2 and F3 in Example 6 were mixed with OVA-FITC solution at a volume ratio of 2:1, placed in a refrigerator at 4 degrees overnight for cell uptake evaluation, and the particle size, PDI and potential of the mixed samples are shown in Table 10.

[0163] Table 10

[0164] Sample name Particle size (nm) PDI Potential (mV) F1 (Pickering emulsion / soy lecithin) / OVA-FITC 1410.3±268.1 0.30±0.06 25.9±0.3 F2 (Pickering emulsion) / OVA-FITC 1582.7±432.0 0.19±0.03 21.8±0.3 F3 (Pickering emulsion / polysialic acid) / OVA-FITC 1975.7±110.8 0.17±0.05 17.5±1.3

[0165] After culturing RAW464.7 cells for 12 hours, the Pickering emulsion loaded with OVA-FITC was diluted with fresh DMEM medium (1:20) and the primary culture medium was replaced; DMEM medium containing PBS and OVA-FITC was used as the control group, and the cells were cultured for 4 hours. After 4 hours of co-incubation, the cells were fixed with 4% PFA for 30 minutes and then washed twice with PBS. Then, F-actin and nuclei of RAW264.7 cells were stained with TRITC Phalloidin (Yisheng Bio) and Hoechst 33258 (Biyuntian) at 37°C for 30 minutes in the dark. The cell slides were stored at 4°C until observed with a confocal laser scanning microscope (CLSM).

[0166] The results are as follows Figure 6 As shown by Figure 6 It can be seen that the antigen prepared by the composite adjuvant obtained in the present application has the best cellular uptake result, and the effect of Tween 80 as a particle wetting agent is better than that of soybean lecithin.

[0167] The applicant declares that the present invention illustrates a Pickering emulsion composite adjuvant and its preparation method and application through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0168] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0169] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A Pickering emulsion composite adjuvant, characterized in that The composite adjuvant comprises aluminum hydroxide, an oil phase, a particle wetting agent, polysialic acid and an aqueous phase.

2. The Pickering emulsion composite adjuvant according to claim 1, characterized in that The composite adjuvant comprises, by weight percentage, 0.025%-0.8% aluminum hydroxide, 1.6%-8.3% oil phase, 0.02%-0.5% particle wetting agent, 90.3%-98.4% water phase, and comprises, by concentration, 0.001-0.06 mg / mL polysialic acid.

3. The Pickering emulsion composite adjuvant according to claim 1 or 2, characterized in that: The oil phase includes any one of squalene, soybean oil, olive oil, coconut oil, isopropyl myristate, medium chain triglyceride, and paraffin oil, or a combination of at least two of them.

4. The Pickering emulsion composite adjuvant according to any one of claims 1 to 3, characterized in that The particle wetting agent includes any one or a combination of at least two of soybean lecithin, nonionic surfactant, anionic surfactant, cationic surfactant, and zwitterionic surfactant; Preferably, the non-ionic surfactant comprises Tween 80.

5. The Pickering emulsion composite adjuvant according to any one of claims 1 to 4, characterized in that The source of the polysialic acid includes Neisseria meningitidis B polysaccharide B, Neisseria meningitidis C polysaccharide C or Escherichia coli K92 polysaccharide; Preferably, the average molecular weight of the polysialic acid is 300-100000 Da.

6. The Pickering emulsion composite adjuvant according to any one of claims 1 to 5, characterized in that The particle size of the aluminum hydroxide is 200-3000nm; Preferably, the aqueous phase comprises any one of ultrapure water, citric acid buffer, 0.9% sodium chloride solution, phosphate buffer or histidine buffer, or a combination of at least two thereof.

7. A method for preparing a Pickering emulsion composite adjuvant according to any one of claims 1 to 6, characterized in that: The preparation method comprises: (1) mixing an aqueous phase, a particle wetting agent, and aluminum hydroxide, and homogenizing by water bath ultrasound, probe ultrasound, or high pressure to obtain a nano aluminum hydroxide solution; (2) mixing the oil phase with the nano aluminum hydroxide solution; (3) The product obtained in step (2) is subjected to probe ultrasound or homogenization to obtain a Pickering emulsion, which is then mixed with polysialic acid to obtain the product.

8. The preparation method according to claim 7, characterized in that: The ultrasonic power of step (1) is 60-120W, the temperature is 0-4°C, and the time is 5-20min; Preferably, the pressure of the high-pressure homogenization is 600-2000 bar, and the time is 5-15 cycles.

9. The preparation method according to claim 7 or 8, characterized in that: The oil phase is mixed with the nano-aluminum hydroxide solution and then vortexed; Preferably, the vortex time is 25-60s; Preferably, the ultrasonic power in step (3) is 30-105W, the temperature is 0-4°C, and the time is 1-2min.

10. Use of the Pickering emulsion composite adjuvant according to any one of claims 1 to 6 in the preparation of a vaccine.

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