Multiple nanoemulsion adjuvants and methods of making same

Abstract

The present application relates to the technical field of pharmaceutical preparation, and particularly relates to a multiple nano-emulsion adjuvant and a preparation method thereof. The multiple nano-emulsion adjuvant comprises, in terms of 100 g of a raw material system, 2.73-6.37 g of an oil phase matrix, 0.10-0.25 g of a sterol compound, 0.212-0.424 g of a non-ionic surfactant, 0.00050-0.00200 g of a triterpene saponin active adjuvant, 0.010-0.020 g of an anionic polymer modifier, 0.005-0.015 g of a cationic polymer modifier, 70-90 g of a buffer solution, and water to supplement the total mass to 100 g. The nano-emulsion adjuvant prepared by the present application can be stored at 2-8 DEG C for 6 months under low sterol conditions, with a particle size change of not more than +5% and no crystallization or delamination, and the stability of the active adjuvant is significantly improved.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical formulation technology, and in particular to a multiple nanoemulsion adjuvant and its preparation method. Background Technology

[0002] Nanoemulsions, as water-in-oil or oil-in-water colloidal dispersions, are widely used in drug delivery and vaccine adjuvants due to their small particle size, large specific surface area, and ability to simultaneously encapsulate both hydrophilic and hydrophobic active ingredients. In the field of immunoadjuvants, nanoemulsions can improve antigen delivery efficiency, enhance in vivo distribution, and strengthen immune responses, making them particularly suitable for delivering thermosensitive, easily degradable triterpenoid saponin-based active adjuvants such as QS-21.

[0003] In practical applications, the long-term low-temperature storage stability of nanoemulsions is a key indicator affecting their promotion and use. Vaccine adjuvants typically need to be stored at 2–8°C for months to years. During this period, if particle size drift, crystallization, or stratification occurs, it will not only lead to changes in appearance and product spoilage, but also affect the release of active adjuvants and immunization efficacy. Therefore, maintaining particle size stability without crystallization / stratification at low temperatures for long periods is a core requirement that high-quality nanoemulsion adjuvants must meet.

[0004] To improve low-temperature storage stability, existing technologies often add a high proportion of sterol compounds, such as cholesterol and phytosterols, to the oil phase to enhance the rigidity of the oil core film and reduce oil droplet fusion. However, high proportions of sterols are prone to crystallization at low temperatures, leading to a significant increase in particle size and even sedimentation and stratification. Although reducing the proportion of sterols can alleviate crystallization, it causes particle size drift over time and decreased dispersibility, creating a technical contradiction between the ease of crystallization with high sterol content and the instability with low sterol content.

[0005] Another existing approach attempts to improve low-temperature stability by constructing a single, continuous polymeric shell of anions or cations on the surface of nanoemulsions. However, a single electrically charged polymeric layer is prone to aggregation or coating by serum proteins in protein or salt environments, altering its surface properties. Furthermore, an excessively thick polymeric layer may reduce effective interfacial interaction with antigens, affecting immune activity. More importantly, this type of method fails to fundamentally solve the problem of long-term particle size drift under low-sterol conditions.

[0006] To improve long-term stability during low-temperature storage, the controllability and repeatability of the preparation process must also be considered. Current technologies lack sufficient control over the type, sequence, and amount of polymer modifications, leading to significant differences in low-temperature stability between different batches. Some batches even exhibit crystallization or exceed particle size limits within 2–3 months. Furthermore, an improper sequence of polymer modifications can easily result in a loose interfacial structure in the short term, reducing long-term stability.

[0007] Therefore, there is an urgent need for a method that, under low sterol conditions, combines a rational multi-interface structure design with a polymer sequential addition process, to enable nanoemulsions to maintain ΔD50≤+5%, without crystallization or stratification, when stored at 2–8℃ for 6 months or more. This would resolve the technical contradiction between the easy crystallization of high sterols and the instability of low sterols, significantly improve the stability of long-term storage at low temperatures, and balance the retention rate of active adjuvants and the immune effect. Summary of the Invention

[0008] To address the technical problem of particle size drift, crystallization, or stratification that occurs in existing nanoemulsion adjuvants during long-term low-temperature storage under low-sterol conditions, this invention provides a method for preparing multiple nanoemulsion adjuvants. By introducing sequentially added anionic and cationic polymeric modification layers onto a low-sterol oil phase, and through optimized design of the surfactant and buffer system, the method significantly improves long-term low-temperature storage stability while ensuring particle size uniformity. This method maintains ΔD50 ≤ +5% and exhibits no crystallization / stratification after 6 months of storage at 2–8℃, thus overcoming the technical contradiction between the ease of crystallization in high-sterol environments and the instability in low-sterol environments.

[0009] This invention provides a multiple nanoemulsion adjuvant, comprising, based on a 100g raw material system:

[0010] 2.73–6.37 g oil phase matrix;

[0011] 0.10–0.25 g of sterol compounds;

[0012] 0.212–0.424 g of nonionic surfactant;

[0013] 0.00050–0.00200g of triterpenoid saponin active adjuvant;

[0014] 0.010–0.020 g anionic polymeric modifier;

[0015] 0.005–0.015 g cationic polymeric modifier;

[0016] 70–90 g buffer solution, pH 6.8–7.6, osmotic pressure 260–320 mOsm / kg;

[0017] Add water to bring the total weight to 100g.

[0018] Preferably, the oil phase matrix is ​​ethyl linoleate, medium-chain triglycerides, squalene, or a combination thereof.

[0019] Preferably, the sterol compound is campesterol, β-sitosterol, stigmasterol, cholesterol, or a combination thereof.

[0020] Preferably, the nonionic surfactant is a polysorbate, a polyoxyethylene fatty acid ester, a polyoxyethylene castor oil derivative, or a combination thereof.

[0021] Preferably, the triterpenoid saponin active adjuvant is QS-21.

[0022] Preferably, the anionic polymeric modifier is sodium γ-polyglutamate, hyaluronic acid, alginate, carrageenan, or a combination thereof.

[0023] More preferably, the anionic polymeric modifier is sodium γ-polyglutamate.

[0024] Preferably, the cationic polymeric modifier is diethylaminoethyl dextran hydrochloride (DEAE-Dextran), polylysine, chitosan, polyguanidine salt, or a combination thereof.

[0025] More preferably, the cationic polymeric modifier is diethylaminoethyl dextran hydrochloride. Even more preferably, the cationic polymeric modifier is a combination of diethylaminoethyl dextran hydrochloride and polylysine.

[0026] Preferably, the buffer solution is a HEPES buffer solution.

[0027] This invention also provides a method for preparing the above-mentioned multiple nanoemulsion adjuvant, comprising the following steps:

[0028] (1) Mix the oil phase matrix with sterol compounds, stir to dissolve, and obtain the oil phase;

[0029] (2) Add the triterpenoid saponin active adjuvant and the nonionic surfactant to the buffer solution and stir to dissolve to obtain the aqueous phase;

[0030] (3) Add the oil phase to the aqueous phase and stir to form a primary emulsion;

[0031] (4) The proemulsion is emulsified to obtain a nanoemulsion;

[0032] (5) Add an anionic polymer modifier to the nanoemulsion and mix evenly;

[0033] (6) After step (5) is completed, add the cationic polymer modifier and mix well;

[0034] (7) Add water to a total mass of 100g, mix well, filter, and obtain the multi-nano emulsion adjuvant.

[0035] This invention, under low-sterol conditions, forms a multi-interface structure with a dual-electrical distribution by sequentially introducing anionic and cationic polymeric modifiers onto the surface of nanoemulsion particles. On one hand, the anionic polymeric modifier provides a stable negative potential and steric hindrance, inhibiting particle aggregation and crystallization at low temperatures. On the other hand, the cationic polymeric modifier forms locally positively charged regions on the particle surface, enhancing interactions with negatively charged antigens or cell membranes and maintaining good dispersibility in salt- or protein-containing environments. The synergistic effect of the bilayer modification maintains particle size uniformity and dispersibility while reducing the degradation rate of the active adjuvant during storage.

[0036] In a preferred embodiment, the present invention further constructs a multi-interface structure through a "cathioneous-cationeous" polymer modification sequence, enabling the nanoemulsion to maintain a particle size change rate (ΔD50) of no more than +5% after 6 months of storage at 2–8°C, without crystallization or stratification, and significantly improving its long-term low-temperature storage stability. Experiments have shown that the nanoemulsion prepared by this method has a higher retention rate of active adjuvants under accelerated conditions. Detailed Implementation

[0037] The endpoints and any values ​​of the ranges disclosed in this invention are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this invention.

[0038] Unless otherwise expressly stated in this specification and claims, "multiple nanoemulsion adjuvant" does not refer to multiple emulsions (such as W / O / W or O / W / O structures) in the field of emulsification, but rather to a nanoemulsion adjuvant formed from a single nanodroplet of an oil phase, an emulsifier, and an aqueous phase, the surface of which is sequentially modified with two or more different types of materials to form multiple functional interface layers. The "multiple" in the multiple nanoemulsion adjuvant described in this invention refers to the multiplicity and diversity of the surface modification layers, not the multiplicity of the phase structure of the emulsion system.

[0039] This invention provides a method for preparing multiple nanoemulsion adjuvants, comprising the following steps:

[0040] (1) Mix 2.73-6.37 g of oil phase matrix with 0.10-0.25 g of sterol compound, heat and stir until completely dissolved to obtain oil phase;

[0041] (2) Add 0.00050-0.00200g of triterpenoid saponin active adjuvant and 0.212-0.424g of nonionic surfactant to 70-90g of buffer solution (pH 6.8-7.6, osmotic pressure 260-320mOsm / kg) and stir to dissolve to obtain an aqueous phase;

[0042] (3) The oil phase is slowly added to the aqueous phase under stirring conditions to form a primary emulsion;

[0043] (4) The promulgated emulsion is subjected to high-energy emulsification to obtain nanoemulsion with uniform particle size;

[0044] (5) Add 0.010-0.020g of anionic polymer modifier to the nanoemulsion and mix evenly to form a first surface modification layer;

[0045] (6) After step (5) is completed, add 0.005-0.015g of cationic polymer modifier and mix evenly to form a second surface modification layer;

[0046] (7) Add purified water to a total mass of 100g, mix well, filter, and the multi-nano emulsion adjuvant is obtained.

[0047] Wherein: the oil phase matrix is ​​ethyl linoleate, medium-chain triglycerides, squalene or a combination thereof, which serves as the hydrophobic core of the nanoemulsion particles, effectively dissolving and encapsulating hydrophobic or amphiphilic active ingredients, reducing their exposure in the aqueous phase, thereby reducing the impact of environmental factors such as temperature and oxidation on their stability.

[0048] The sterol compounds are campesterol, β-sitosterol, stigmasterol, cholesterol, or combinations thereof. They form a complex structure with the matrix in the oil phase, increasing the rigidity of the interfacial film and inhibiting the liquid-solid transition at low temperatures, thereby reducing the risk of crystallization. In this invention, the proportion of sterols is controlled within a low range of 15 wt% of the oil phase mass to balance stability and avoid high sterol crystallization.

[0049] The nonionic surfactant is a polysorbate, a polyoxyethylene fatty acid ester, a polyoxyethylene castor oil derivative or a combination thereof, used to reduce the oil / water interfacial tension, promote droplet refinement and prevent aggregation, and preferably has a low peroxide value to reduce the oxidation of active ingredients;

[0050] The triterpenoid saponin active adjuvant is QS-21, which can induce both humoral and cellular immunity. However, it is easily inactivated by temperature, oxidation, and interfacial environment. This invention significantly improves its stability under low-temperature long-term storage and accelerated conditions through oil phase embedding and multilayer interfacial modification.

[0051] The anionic polymeric modifier is sodium γ-polyglutamate, hyaluronic acid, alginate, carrageenan or a combination thereof, preferably sodium γ-polyglutamate. This component forms a negatively charged first modification layer on the surface of the droplets, providing electrostatic repulsion and steric hindrance, preventing low-temperature aggregation and crystallization, and enhancing dispersion stability.

[0052] The cationic polymeric modifier is diethylaminoethyl dextran hydrochloride, polylysine, chitosan, polyguanidine salt or a combination thereof, preferably diethylaminoethyl dextran hydrochloride. This component forms a locally positively charged modification layer outside the anionic polymeric modifier layer, which can enhance the binding with negatively charged antigens or cell membranes and maintain dispersibility in salt-containing or protein-containing environments.

[0053] The preferred buffer solution is HEPES buffer, which provides stable pH and osmotic pressure, maintains the physical stability of the nanoemulsion system, and prevents the active adjuvant from being degraded due to acid-base fluctuations. Purified water is used to make up the total mass, ensuring accurate concentration and avoiding the introduction of impurities during the filtration and sterilization process.

[0054] Under low-sterol conditions, a stable, electrically dual-layered interfacial structure can be formed by sequentially introducing anionic and cationic polymer modification layers onto the surface of nanoemulsion particles. This structure utilizes anionic polymer modifiers to provide a stable negative potential and steric hindrance, inhibiting particle aggregation and crystallization at low temperatures. Simultaneously, cationic polymer modifiers create locally positively charged regions on the outer layer, enhancing binding with negatively charged antigens or cell membranes and maintaining good dispersibility in salt- or protein-containing environments. The specific "anionic-then-cationic" process enables the formation of strong interlayer bonds and spatial arrangements at the interface, reducing interfacial instability caused by intermolecular rearrangement, thereby significantly improving long-term low-temperature storage stability.

[0055] Furthermore, when using a combination of the anionic polymeric modifier sodium γ-polyglutamate and the cationic polymeric modifier diethylaminoethyl dextran hydrochloride, sodium γ-polyglutamate first forms a uniform, dense, and highly hydrophilic negatively charged layer on the surface of the emulsion droplets. This not only provides strong electrostatic repulsion and steric hindrance, inhibiting close contact between droplets, but also maintains the interfacial hydration film at low temperatures, effectively preventing crystallization. Subsequently, the introduced diethylaminoethyl dextran hydrochloride, through local electrostatic recombination with the surface sodium γ-polyglutamate, forms a flexible positively charged region on the outer layer. This positively charged region can enhance the interaction with negatively charged antigens or cell membranes and inhibit non-specific aggregation in saline and protein-containing environments. The inner layer of sodium γ-polyglutamate provides a durable interfacial stability basis, while the outer layer of diethylaminoethyl dextran hydrochloride introduces a moderate surface charge heterogeneity and a flexible protective layer, achieving a balance between physical stability, environmental adaptability, and biobinding capacity in the entire multi-interface. This significantly improves the long-term low-temperature stability and adjuvant retention rate of the nanoemulsion even under low-sterol conditions.

[0056] Furthermore, polylysine can be rapidly adsorbed onto the surface of the anionic polymer modifier layer, forming a dense positively charged region and enhancing interfacial bonding; diethylaminoethyl dextran hydrochloride provides highly hydrophilic and flexible protection on its outer layer, stabilizing local positive charges and resisting salt ion shielding. The two work synergistically to construct a dual-electrical outer layer that combines compactness and flexibility, thereby significantly improving the long-term low-temperature stability of the nanoemulsion under low-sterol conditions and preventing crystallization.

[0057] Raw material introduction:

[0058] HEPES buffer, pH 7.3, osmotic pressure 290 mOsm / kg.

[0059] QS-21 adjuvant, product number: CFN91588, was purchased from Wuhan Tianzhi Biotechnology Co., Ltd.

[0060] Tween80, product number: T798873, purchased from Shanghai McLean Biochemical Technology Co., Ltd.

[0061] Example 1

[0062] A method for preparing multiple nanoemulsion adjuvants includes the following steps:

[0063] (1) Oil phase premixing

[0064] Under 55℃ water bath conditions, 5.00g of ethyl linoleate and 0.20g of campesterol were placed in a beaker and magnetically stirred at 500rpm until completely dissolved to obtain an oil phase solution.

[0065] (2) Aqueous phase configuration

[0066] Add 80.0 g HEPES buffer (pH 7.3, osmotic pressure 290 mOsm / kg) to another beaker, add 1.00 mg QS-21 adjuvant and 0.318 g Tween 80, stir at room temperature for 200 rpm until completely dissolved to obtain an aqueous solution.

[0067] (3) Colostrum formation

[0068] The oil phase solution was slowly added to the aqueous phase at a rate of 0.5 g / min using a peristaltic pump, while the aqueous phase was stirred at 600 rpm. After all the oil phase was added, stirring was continued for 10 min to form the primary emulsion.

[0069] (4) High-energy emulsification

[0070] The colostrum was placed in an ice bath and emulsified using a probe-type ultrasonic instrument (power 200W, amplitude 60%, intermittent mode 3s on / 3s off, total time 6min) to obtain nanoemulsions with uniform particle size.

[0071] (5) Anionic polymer modification

[0072] 15.0 mg of sodium γ-polyglutamate was added to the nanoemulsion and stirred at 400 rpm for 20 min at room temperature to allow it to adsorb onto the particle surface and form a modification layer.

[0073] (6) Cationic polymer modification

[0074] After step (5) is completed, 10.0 mg of DEAE-Dextran is added to the system and added slowly at a rate of 0.5 g / min using a syringe pump. The mixture is stirred at 300 rpm for 15 min to form a surface modification layer again.

[0075] (7) Final processing

[0076] Add purified water to a total mass of 100g and mix well. Filter under positive pressure using a 0.22μm PES membrane, collect the filtrate, and obtain the multi-layer nanoemulsion adjuvant product.

[0077] Example 2

[0078] The preparation method of the multiple nanoemulsion adjuvant is the same as in Example 1, except that in step (5) the 15.0 mg of sodium γ-polyglutamate in the anionic polymer modification is replaced with 15.0 mg of hyaluronic acid. Everything else is the same.

[0079] Example 3

[0080] The preparation method of the multiple nanoemulsion adjuvant is the same as in Example 1, except that in step (6) the 10.0 mg DEAE-Dextran in the cationic polymer modification is replaced with 10.0 mg polylysine. All other steps are the same.

[0081] Example 4

[0082] The preparation method of the multiple nanoemulsion adjuvant is the same as in Example 1, except that: in step (5), 15.0 mg of sodium γ-polyglutamate in the anionic polymer modification is replaced with 15.0 mg of hyaluronic acid; and in step (6), 10.0 mg of DEAE-Dextran in the cationic polymer modification is replaced with 10.0 mg of polylysine. All other steps are the same.

[0083] Example 5:

[0084] The preparation method of the multiple nanoemulsion adjuvant is the same as in Example 1, except that steps (5) and (6) are swapped. First, cationic polymer modification is performed, followed by anionic polymer modification. Everything else is the same.

[0085] Example 6:

[0086] The preparation method of the multiple nanoemulsion adjuvant is the same as in Example 1, except that in step (6) the 10.0 mg DEAE-Dextran in the cationic polymer modification is replaced with 5.0 mg polylysine and 5.0 mg DEAE-Dextran. All other steps are the same.

[0087] Comparative Example 1:

[0088] The preparation method of the multiple nanoemulsion adjuvant is the same as in Example 1, except that step (5) is omitted. That is, anionic polymer modification is not used.

[0089] Comparative Example 2:

[0090] The preparation method of the multiple nanoemulsion adjuvant is the same as in Example 1, except that step (6) is omitted. That is, cationic polymer modification is not used.

[0091] Test Example 1:

[0092] Stability test of multiple nanoemulsion adjuvants under low temperature long-term storage at 2-8℃

[0093] 1. Samples and Grouping

[0094] Formulation: Each of the examples and comparative examples was prepared independently in one batch; each batch was dispensed into n=5 bottles (10mL / bottle, headspace <10%), as parallel samples.

[0095] Labeling and Blinding: Each bottle is coded, and the testing personnel do not have access to the formula information (single-blind).

[0096] 2. Storage conditions and time points

[0097] Conditions: 2–8℃, away from light, placed upright; in the same shelf within the same refrigerator to avoid temperature fluctuations.

[0098] Time points: T0 (24 hours after preparation), January, March, and June. June is the primary evaluation point for this project.

[0099] 3. DLS Particle Size / Dispersibility

[0100] Instruments: DLS particle size analyzer, 25℃ constant temperature cell, backscattering angle 173° ° .

[0101] Dilution: Dilute 10mM HEPES (pH 7.3) at a volume ratio of 1:50; filter the dilution medium using a 0.22μm filter, without filtering the sample.

[0102] Parameters: Medium viscosity 0.89 mPa·s; refractive index 1.59; each bottle was measured three times and the average value was taken.

[0103] Indicators: D50 (Z-average can also be considered a secondary indicator), PDI.

[0104] 4. Crystallization / Layering Determination

[0105] Polarizing microscope: 400×, random field of view ≥5; the presence of bright crystalline patterns or birefringence indicates a positive result.

[0106] DSC: Increase temperature from 0 to 50℃ at 10℃ / min; a positive result is indicated by the appearance of a repeatable thermal effect peak.

[0107] Visual inspection: White flocculent / layered / sedimented appearance indicates a positive result. Any positive result is recorded as "crystallization / layering positive".

[0108] 5. Main evaluation indicators and formulas

[0109] Primary endpoint: ΔD50 (%)

[0110] Calculate on a bottle-by-bottle basis:

[0111]

[0112] Within-group report: mean ± SD (n=5), with 95% CI.

[0113] The criteria are: ΔD50≤+5% and crystallization / delamination=negative.

[0114] 6. Statistics

[0115] Referring to Example 1, ΔD50 of other examples was subjected to one-way ANOVA and subsequent Dunnett multiple comparisons (α=0.05).

[0116] If normality / homogeneity of variance is not satisfied, use Kruskal–Wallis+Dunn correction.

[0117] The report also provides the Δ (%) of each group's PDI as supplementary evidence (not a judgment indicator).

[0118] 7. Quality Control

[0119] The instrument is routinely calibrated with 100nm standard latex (CV≤3%).

[0120] Dilution was performed by the same person; all samples were tested within 4 hours for the entire batch.

[0121] The test result ΔD50 (%) is calculated according to the formula (D50) 6mo -D50 T0 ) / D50 T0The mean ± SD was calculated after multiplying by 100 for each bottle, and the 95% CI was listed. Crystallization / stratification was determined based on polarized light microscopy, DSC, and visual inspection; any positive result was recorded as "positive". The p-value was obtained from one-way ANOVA + Dunnett multiple comparisons, with Example 1 as a reference. The judgment was based on the criteria of ΔD50 ≤ +5% and crystallization / stratification = negative.

[0122] Table 1

[0123]

[0124] After 6 months of storage at 2–8°C, the ΔD50, 95% CI, crystallization / stratification determination, and statistical results of each example are shown in the table above. The results show that Examples 1, 2, and 6 all met the determination criteria and passed the low-temperature stability test. Example 6 had the lowest ΔD50 (+2.6%), a significant difference compared to Example 1 (p=0.004). Examples 3 and 4 had ΔD50 exceeding +5%, and were deemed unsuccessful. Example 5 had a high ΔD50 of +12.8% and positive crystallization, indicating a significant decrease in stability. These results indicate that the optimal preparation sequence of this invention is anionic followed by cation modification, and that the combination of polylysine and DEAE-Dextran in the cationic modification can further improve long-term low-temperature stability.

[0125] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

Claims

1. A multi-nanoemulsion adjuvant, characterized in that, Based on a 100g raw material system, it includes: 2.73-6.37g ethyl linoleate; 0.10-0.25g campesterol; 0.212-0.424g Tween 80; 0.00050-0.00200g QS-21 adjuvant; 0.010-0.020g sodium γ-polyglutamate; 0.005-0.015g DEAE-Dextran; 70-90g buffer solution, pH 6.8-7.6, osmotic pressure 260-320mOsm / kg; Add water to bring the total weight to 100g; The method for preparing the aforementioned multiple nanoemulsion adjuvant is characterized by comprising the following steps: (1) Ethyl linoleate and campesterol were stirred and dissolved to obtain the oil phase; (2) Add QS-21 adjuvant and Tween 80 to the buffer solution and stir to dissolve, thus obtaining the aqueous phase; (3) Add the oil phase to the aqueous phase and stir to form a primary emulsion; (4) The proemulsion is emulsified to obtain a nanoemulsion; (5) Add sodium γ-polyglutamate to the nanoemulsion and mix well; (6) After step (5) is completed, add DEAE-Dextran and mix well; (7) Add water to a total weight of 100g, mix well, and filter.

2. A multi-nanoemulsion adjuvant, characterized in that, Based on a 100g raw material system, it includes: 2.73-6.37g ethyl linoleate; 0.10-0.25g campesterol; 0.212-0.424g Tween 80; 0.00050-0.00200g QS-21 adjuvant; 0.010-0.020g hyaluronic acid; 0.005-0.015g DEAE-Dextran; 70-90g buffer solution, pH 6.8-7.6, osmotic pressure 260-320mOsm / kg; Add water to bring the total weight to 100g; The preparation method of the aforementioned multiple nanoemulsion adjuvant includes the following steps: (1) Mix ethyl linoleate with campesterol and stir to dissolve, thus obtaining the oil phase; (2) Add QS-21 adjuvant and Tween 80 to the buffer solution and stir to dissolve, thus obtaining the aqueous phase; (3) Add the oil phase to the aqueous phase and stir to form a primary emulsion; (4) The proemulsion is emulsified to obtain a nanoemulsion; (5) Add hyaluronic acid to the nanoemulsion and mix well; (6) After step (5) is completed, add DEAE-Dextran and mix well; (7) Add water to a total weight of 100g, mix well, and filter.

3. A multi-nanoemulsion adjuvant, characterized in that, Based on a 100g raw material system, it includes: 2.73-6.37g ethyl linoleate; 0.10-0.25g campesterol; 0.212-0.424g Tween 80; 0.00050-0.00200g QS-21 adjuvant; 0.010-0.020g sodium γ-polyglutamate; A mixture of 0.005-0.015g polylysine and DEAE-Dextran; 70-90g buffer solution, pH 6.8-7.6, osmotic pressure 260-320mOsm / kg; Add water to bring the total weight to 100g; The preparation method of the aforementioned multiple nanoemulsion adjuvant includes the following steps: (1) Mix ethyl linoleate with campesterol and stir to dissolve, thus obtaining the oil phase; (2) Add QS-21 adjuvant and Tween 80 to the buffer solution and stir to dissolve, thus obtaining the aqueous phase; (3) Add the oil phase to the aqueous phase and stir to form a primary emulsion; (4) The proemulsion is emulsified to obtain a nanoemulsion; (5) Add sodium γ-polyglutamate to the nanoemulsion and mix well; (6) After step (5) is completed, add the mixture of polylysine and DEAE-Dextran and mix well; (7) Add water to a total weight of 100g, mix well, and filter.

4. The multiple nanoemulsion adjuvant according to claim 3, characterized in that, The buffer solution is HEPES buffer.

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