Multiple nano emulsion adjuvant and preparation method thereof

Abstract

The invention relates to the technical field of pharmaceutical preparations, in particular to a multiple nano emulsion adjuvant and a preparation method thereof. The multiple nano emulsion adjuvant is prepared from 2.73 to 6.37 g of an oil phase matrix based on 100 g of a raw material system, 0.10 to 0.25 g of a sterol compound; 0.212 to 0.424 g of a nonionic surfactant; 0.00050 to 0.00200 g of a triterpenoid saponin type active adjuvant; 0.010 to 0.020 g of an anionic polymer modifier; 0.005 to 0.015 g of a cationic polymer modifier; 70-90 g of a buffer solution; the total mass of the raw materials is 100 g. The nano emulsion adjuvant prepared by the invention can be stored for 6 months at 2-8 DEG C under a low sterol condition, the particle size change does not exceed + 5%, no crystallization or layering is generated, and the stability of the active adjuvant is remarkably improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of pharmaceutical preparations, and particularly relates to a multiple nanoemulsion adjuvant and a preparation method thereof. BACKGROUND

[0002] As a water-in-oil or oil-in-water colloidal dispersion system, nanoemulsion is widely used in drug delivery and vaccine adjuvant fields due to its small particle size, large specific surface area and the ability to simultaneously encapsulate hydrophilic and hydrophobic active ingredients. In the direction of immune adjuvant, nanoemulsion can improve the delivery efficiency of antigens, improve the in vivo distribution and enhance the immune response, and is particularly suitable for delivering heat-sensitive and easily degradable triterpene saponin active adjuvants such as QS-21.

[0003] In practical applications, the long-term storage stability of nanoemulsion at low temperature is a key indicator affecting its promotion and use. Vaccine adjuvants usually need to be stored at 2-8℃ for several months to several years. During this period, if the particle size drifts, crystallizes or stratifies, not only will the appearance change and the finished product be scrapped, but also the release of active adjuvants and the immune effect will be affected. Therefore, maintaining the particle size stability and preventing crystallization / stratification at low temperature for a long time is a core requirement that must be met by high-quality nanoemulsion adjuvants.

[0004] To improve the low-temperature storage stability, the prior art often adds a high proportion of sterol compounds such as cholesterol and phytosterol in the oil phase to enhance the rigidity of the oil core membrane and reduce oil droplet fusion. However, a high proportion of sterols is prone to crystallization under low-temperature conditions, resulting in a significant increase in particle size and even sedimentation and stratification. Although reducing the proportion of sterols can reduce crystallization, it will cause the particle size to drift over time and the dispersion to decrease, forming a technical contradiction between high sterol crystallization and low sterol instability.

[0005] Another existing solution attempts to improve low-temperature stability by constructing a single continuous high-molecular shell anion or cation on the surface of the nanoemulsion. However, a single high-molecular layer of a single electric property is prone to aggregation or covered by serum proteins in a protein or salt environment, changing the surface properties, and a thick high-molecular layer may reduce the effective interface interaction with antigens, affecting the immune activity. More importantly, this method fails to fundamentally solve the problem of long-term particle size drift at low sterol conditions.

[0006] To improve the long-term storage stability at low temperature, the controllability and repeatability of the preparation process also need to be considered. The existing technology lacks control over the type, sequence and amount of high-molecular modification, resulting in significant differences in low-temperature stability between different batches, and some batches even crystallize or have particle sizes exceeding the standard within 2-3 months. In addition, if the high-molecular modification sequence is unreasonable, it may cause the interface structure to be loose in a short period of time, reducing the long-term stability.

[0007] Therefore, there is an urgent need for a kind of nanoemulsion under low sterol conditions, combined with reasonable multiple interface structure design and high molecular order adding process, so that the nanoemulsion can still maintain ΔD50≤+5%, no crystallization or delamination when stored for 6 months or more under the condition of 2-8℃, so as to solve the technical contradiction between high sterol easy crystallization and low sterol instability, significantly improve the long-term storage stability at low temperature, and balance the active adjuvant retention rate and immune effect. SUMMARY

[0008] In order to solve the technical problems of existing nanoemulsion adjuvants under low sterol conditions, long-term storage at low temperature is easy to cause particle size drift, crystallization or delamination, the present application provides a preparation method of multiple nanoemulsion adjuvant, by introducing sequentially added anionic and cationic polymer modification layer on the basis of low sterol oil phase, and optimizing the design of surfactant and buffer system, the particle size uniformity is ensured, and the long-term storage stability at low temperature is significantly improved. The method can still maintain ΔD50≤+5% and no crystallization / delamination after being stored for 6 months under the condition of 2-8℃, so as to overcome the technical contradiction between high sterol easy crystallization and low sterol instability.

[0009] The present application provides a kind of multiple nanoemulsion adjuvant, including, based on 100g raw material system: 2.73-6.37g oil phase matrix; 0.10-0.25g sterol compound; 0.212-0.424g non-ionic surfactant; 0.00050-0.00200g triterpenoid active adjuvant; 0.010-0.020g anionic polymer modifier; 0.005-0.015g cationic polymer modifier; 70-90g buffer, pH 6.8-7.6, osmotic pressure 260-320mOsm / kg; Water is supplemented to a total mass of 100g.

[0010] Preferably, the oil phase matrix is ethyl linoleate, medium-chain triglyceride, squalene or a combination thereof.

[0011] Preferably, the sterol compound is campesterol, β-sitosterol, stigmasterol, cholesterol or a combination thereof.

[0012] Preferably, the non-ionic surfactant is polysorbate, polyoxyethylene fatty acid ester, polyoxyethylene castor oil derivative or a combination thereof.

[0013] Preferably, the triterpenoid active adjuvant is QS-21.

[0014] Preferably, the anionic high molecular modifier is sodium gamma-polyglutamate, hyaluronic acid, alginate, carrageenan or a combination thereof.

[0015] Further preferably, the anionic high molecular modifier is sodium gamma-polyglutamate.

[0016] Preferably, the cationic high molecular modifier is diethylaminoethyl dextran hydrochloride (DEAE-Dextran), polylysine, chitosan, polyguanidine salt or a combination thereof.

[0017] Further preferably, the cationic high molecular modifier is diethylaminoethyl dextran hydrochloride. Still further preferably, the cationic high molecular modifier is a combination of diethylaminoethyl dextran hydrochloride and polylysine.

[0018] Preferably, the buffer is HEPES buffer.

[0019] The present application also provides a preparation method of the above-mentioned multiple nanoemulsion adjuvant, comprising the following steps: (1) mixing and stirring to dissolve an oil phase matrix with a sterol compound to obtain an oil phase; (2) stirring to dissolve a triterpene saponin active adjuvant and a non-ionic surfactant in a buffer to obtain an aqueous phase; (3) adding the oil phase to the aqueous phase and stirring to form a primary emulsion; (4) performing emulsification treatment on the primary emulsion to obtain a nanoemulsion; (5) adding an anionic high molecular modifier to the nanoemulsion and mixing uniformly; (6) after step (5) is completed, adding a cationic high molecular modifier and mixing uniformly; (7) adding water to make up to a total mass of 100 g, mixing uniformly, filtering and obtaining the multiple nanoemulsion adjuvant.

[0020] In the present application, under low sterol conditions, an anionic high molecular modifier and a cationic high molecular modifier are sequentially introduced to modify the surface of a nanoemulsion particle to form a multiple interface structure with a distribution of double electricities. On the one hand, the anionic high molecular modifier can provide a stable negative potential and steric hindrance to inhibit the aggregation and crystallization of particles under low temperature conditions; on the other hand, the cationic high molecular modifier can form a local positive region on the surface of the particles to enhance the interaction with a negatively charged antigen or cell membrane and maintain good dispersibility in a salt or protein-containing environment. The synergistic effect of the double-layer modification maintains the uniformity of particle size and dispersibility while reducing the degradation rate of the active adjuvant during storage.

[0021] In the preferred embodiment, the present application further constructs a multi-interface structure by the order of high polymer modification of "first negative and then positive", so that the nanoemulsion has a particle size change rate (ΔD50) of no more than +5% after being stored at 2-8°C for 6 months, and no crystallization or delamination, and the long-term storage stability at low temperature is significantly improved. Experiments prove that the nanoemulsion prepared by this method has a higher active adjuvant retention rate under accelerated conditions. DETAILED DESCRIPTION

[0022] The endpoints of the ranges and any values disclosed in the specification are not limited to the precise values recited. The endpoints of the ranges and the values are approximations that are already "rounded", and thus the exact values are understood to be within the range. Any numerical range recited is intended to include all sub-ranges subsumed therein. For ranges including an ellipsis, any intervening value or values not recited are intended to be included in the range. For ranges including a first value and a second value, the first value and the second value are intended to be included in the range. For ranges including an ellipsis and a second value, the second value is intended to be included in the range. For ranges including a first value and an ellipsis, the first value is intended to be included in the range.

[0023] In the specification and claims, unless otherwise expressly stated, "multiple nanoemulsion adjuvant" does not refer to multiple emulsions (such as W / O / W or O / W / O structures) in the field of emulsion science, but refers to a single nanoemulsion droplet formed by an oil phase, an emulsifier and an aqueous phase, the surface of which is modified by two or more different types of materials in sequence, forming a nanoemulsion adjuvant with multiple functional interface layers. The "multiple" of the multiple nanoemulsion adjuvant of the present application refers to the multiplicity and diversity of the surface modification layer, rather than the multiplicity of the phase structure of the emulsion system.

[0024] The present application provides a preparation method of a multiple nanoemulsion adjuvant, comprising the following steps: (1) mixing 2.73-6.37 g of an oil phase matrix with 0.10-0.25 g of a sterol compound, heating and stirring until completely dissolved to obtain an oil phase; (2) adding 0.00050-0.00200 g of a triterpene saponin active adjuvant and 0.212-0.424 g of a non-ionic surfactant to 70-90 g of a buffer solution (pH 6.8-7.6, osmotic pressure 260-320 mOsm / kg) and stirring to dissolve to obtain an aqueous phase; (3) slowly adding the oil phase to the aqueous phase under stirring to form a primary emulsion; (4) high-energy emulsifying the primary emulsion to obtain a nanoemulsion with uniform particle size; (5) adding 0.010-0.020 g of an anionic high molecular modifier to the nanoemulsion and mixing uniformly to form a first layer of surface modification; (6) after step (5) is completed, adding 0.005-0.015 g of a cationic high molecular modifier and mixing uniformly to form a second layer of surface modification; (7) make up purified water to 100 g in total mass, mix well, filter, and the multiple nanoemulsion adjuvant is obtained.

[0025] The oil phase matrix is ethyl linoleate, medium-chain triglyceride, squalene or a combination thereof, as a hydrophobic inner core of the nanoemulsion particle, which can effectively dissolve and encapsulate hydrophobic or amphiphilic active ingredients, reduce their exposure in the aqueous phase, and thus reduce the influence of environmental factors such as temperature and oxidation on their stability. The sterol compound is campesterol, beta-sitosterol, stigmasterol, cholesterol or a combination thereof, which forms a complex structure with the matrix in the oil phase, increases the rigidity of the interfacial film, and inhibits the liquid-solid transition at low temperatures, thereby reducing the risk of crystallization; the sterol proportion is controlled in a low range of 15wt% of the oil phase mass, so as to balance the stability and avoid high sterol crystallization; The non-ionic surfactant is polysorbate, polyoxyethylene fatty acid ester, polyoxyethylene castor oil derivative or a combination thereof, which is used to reduce the oil / water interfacial tension, promote emulsion droplet refinement and prevent coalescence, and is preferably low in peroxide value to reduce oxidation of the active ingredient; The triterpenoid saponin active adjuvant is QS-21, which can induce both humoral immunity and cellular immunity, but is easily inactivated by temperature, oxidation and interfacial environment; the present application significantly improves the stability of QS-21 under low-temperature long-term storage and accelerated conditions through oil phase embedding and multi-layer interfacial modification; The anionic high molecular modifier is sodium gamma-polyglutamate, hyaluronic acid, alginate, carrageenan or a combination thereof, and sodium gamma-polyglutamate is preferred; this component forms a negatively charged first modification layer on the surface of the emulsion droplet, provides electrostatic repulsion and steric hindrance, prevents low-temperature aggregation and crystallization, and enhances dispersion stability; The cationic high molecular modifier is diethylaminoethyl dextran hydrochloride, polylysine, chitosan, polyguanidine salt or a combination thereof, and diethylaminoethyl dextran hydrochloride is preferred; this component forms a local positive modification layer outside the anionic high molecular modifier layer, which can enhance the binding with negatively charged antigens or cell membranes, and maintain the dispersibility in a salt-containing or protein-containing environment; The buffer is preferably HEPES buffer, which provides stable pH and osmotic pressure, maintains the physical stability of the nanoemulsion system, and prevents degradation of the active adjuvant due to acid-base fluctuations; purified water is used to make up the total mass, so as to ensure the accuracy of the concentration and avoid the introduction of impurities in the filtration and sterilization process.

[0026] Under low cholesterol conditions, stable double-electricity multi-layer interface structure can be formed by introducing anionic and cationic polymer modification layers on the surface of nanoemulsion particles in sequence. On the one hand, the stable negative potential and steric hindrance provided by the anionic polymer modifier can inhibit particle aggregation and crystallization at low temperatures; on the other hand, the local positive region formed by the cationic polymer modifier in the outer layer can enhance the binding with negatively charged antigens or cell membranes and maintain good dispersibility in salt or protein-containing environments. The process of the special order of "first anion and then cation" can form firm interlayer bonding and spatial arrangement at the interface, reduce the instability of the interface caused by the rearrangement of molecules, and thus significantly improve the long-term storage stability at low temperatures.

[0027] Further, when the combination of the anionic polymer modifier γ-polyglutamic acid sodium and the cationic polymer modifier diethylaminoethyl dextran hydrochloride is used, γ-polyglutamic acid sodium first forms a uniform, dense and highly hydrophilic negative layer on the surface of the emulsion droplets, which not only provides strong electrostatic repulsion and steric hindrance to inhibit close contact between the emulsion droplets, but also maintains the hydration film at low temperatures, thereby effectively preventing crystallization; the subsequently introduced diethylaminoethyl dextran hydrochloride forms a flexible positive region in the outer layer by local electrostatic complexing with the surface γ-polyglutamic acid sodium, which can not only enhance the interaction with negatively charged antigens or cell membranes, but also inhibit non-specific aggregation in salt and protein-containing environments. The inner layer of γ-polyglutamic acid sodium provides a durable interface stability foundation, and the outer layer of diethylaminoethyl dextran hydrochloride introduces moderate surface charge heterogeneity and a flexible protective layer, so that the entire multi-interface reaches a balance between physical stability, environmental adaptability and biological binding capacity, thereby significantly improving the long-term stability at low temperatures and the retention rate of active adjuvant of nanoemulsion under low cholesterol conditions.

[0028] Further, polylysine can quickly adsorb to the surface of the anionic polymer modifier layer to form a dense positive region and enhance the interface binding force; diethylaminoethyl dextran hydrochloride provides high hydrophilicity and flexibility in the outer layer to stabilize the local positive charge and resist salt ion shielding. The two work together to build a double-electricity outer layer with tightness and flexibility, thereby significantly improving the long-term stability at low temperatures and avoiding crystallization of nanoemulsion under low cholesterol conditions.

[0029] Introduction of raw materials: HEPES buffer, pH 7.3, osmotic pressure 290 mOsm / kg.

[0030] QS-21 adjuvant, product number: CFN91588, purchased from Wuhan Tianzhi Biotechnology Co., Ltd.

[0031] Tween 80, product number: T798873, purchased from Shanghai Macklin Biochemical Technology Co., Ltd.

[0032] Example 1 The preparation method of the multiple nano-emulsion adjuvant comprises the following steps: (1) Oil phase premixing In a beaker, 5.00 g of ethyl linoleate and 0.20 g of campesterol were placed in a water bath at 55°C, and stirred at 500 rpm by a magnetic stirrer until completely dissolved to obtain an oil phase solution.

[0033] (2) Water phase preparation In another beaker, 80.0 g of HEPES buffer (pH 7.3, osmotic pressure 290 mOsm / kg) was added, 1.00 mg of QS-21 adjuvant and 0.318 g of Tween 80 were added, and stirred at 200 rpm at room temperature until completely dissolved to obtain a water phase solution.

[0034] (3) Primary emulsion formation The oil phase solution was slowly added to the water phase at a rate of 0.5 g / min by a peristaltic pump, and the water phase was stirred at 600 rpm. After the oil phase was completely added, the stirring was continued for 10 min to form a primary emulsion.

[0035] (4) High-energy emulsification The primary emulsion was placed in an ice bath, and high-energy emulsification was performed using a probe-type ultrasonic instrument (power 200 W, amplitude 60%, intermittent mode 3 s on / 3 s off, total time 6 min) to obtain a nano-emulsion with uniform particle size.

[0036] (5) Anionic polymer modification To the nano-emulsion, 15.0 mg of γ-polyglutamic acid sodium was added, and stirred at 400 rpm at room temperature for 20 min to allow it to adsorb on the particle surface to form a modified layer.

[0037] (6) Cationic polymer modification After step (5) was completed, 10.0 mg of DEAE-Dextran was added to the system, and was slowly added at a rate of 0.5 g / min using a syringe pump, and stirred at 300 rpm for 15 min to form a surface modified layer again.

[0038] (7) Final treatment The purified water was added to a total mass of 100 g, and mixed well. The 0.22 μm PES filter membrane was used for positive pressure filtration, and the filtrate was collected to obtain the multiple nano-emulsion adjuvant finished product.

[0039] Example 2 The preparation method of the multiple nano-emulsion adjuvant was the same as in Example 1, and the adjustment was that 15.0 mg of γ-polyglutamic acid sodium in step (5) anionic polymer modification was replaced by 15.0 mg of hyaluronic acid. The others were the same.

[0040] Example 3 The preparation method of the multiple nanoemulsion adjuvant is the same as that of Example 1, with the exception that 10.0 mg of DEAE-Dextran in the cationic polymer modification of step (6) is replaced by 10.0 mg of polylysine. The others are the same.

[0041] Example 4 The preparation method of the multiple nanoemulsion adjuvant is the same as that of Example 1, with the exception that 15.0 mg of γ-polyglutamic acid sodium in the anionic polymer modification of step (5) is replaced by 15.0 mg of hyaluronic acid; and 10.0 mg of DEAE-Dextran in the cationic polymer modification of step (6) is replaced by 10.0 mg of polylysine. The others are the same.

[0042] Example 5: The preparation method of the multiple nanoemulsion adjuvant is the same as that of Example 1, with the exception that step (5) and step (6) are exchanged in position. The cationic polymer modification is performed first, and then the anionic polymer modification is performed. The others are the same.

[0043] Example 6: The preparation method of the multiple nanoemulsion adjuvant is the same as that of Example 1, with the exception that 10.0 mg of DEAE-Dextran in the cationic polymer modification of step (6) is replaced by 5.0 mg of polylysine and 5.0 mg of DEAE-Dextran. The others are the same.

[0044] Comparative Example 1: The preparation method of the multiple nanoemulsion adjuvant is the same as that of Example 1, with the exception that the original step (5) is not used. That is, the anionic polymer modification is not used.

[0045] Comparative Example 2: The preparation method of the multiple nanoemulsion adjuvant is the same as that of Example 1, with the exception that the original step (6) is not used. That is, the cationic polymer modification is not used.

[0046] Test Example 1: Long-term storage stability test of multiple nanoemulsion adjuvant at low temperature under the condition of 2-8℃ 1. Test sample and grouping Formulation: Each of the examples and comparative examples is independently prepared for 1 batch; each batch is divided into n=5 bottles (10 mL / bottle, headspace <10%), as parallel samples.

[0047] Identification and blind method: Each bottle is coded, and the detection personnel do not contact the formulation information (single-blind).

[0048] 2. Storage conditions and time points Conditions: 2-8℃, light-avoiding, and standing straight; the same refrigerator and the same shelf position are used to avoid temperature drift.

[0049] Time points: T0 (24h after preparation), 1 month, 3 months, 6 months. The main evaluation point of this project is 6 months.

[0050] 3. DLS particle size / dispersibility Instrument: DLS particle size analyzer, 25°C constant temperature cell, backscattering angle 173 ° .

[0051] Dilution: HEPES 10 mM (pH 7.3) at 1:50 volume dilution; the dilution medium is filtered with a 0.22 μm filter, and the sample is not filtered.

[0052] Parameters: medium viscosity 0.89 mPa·s; refractive index 1.59; each bottle is measured 3 times to take the average.

[0053] Index: D50 (Z-average can also be listed as a secondary index), PDI.

[0054] 4. Crystallization / lamination determination Polarizing microscope: 400x, random field ≥5; positive if bright crystal lines or birefringence appear.

[0055] DSC: heating 0→50℃, 10℃ / min; positive if a repeatable thermal effect peak appears.

[0056] Visual inspection: white flocculation / layering / sedimentation is positive. Any positive is recorded as "crystallization / layering positive".

[0057] 5. Main evaluation index and formula Main endpoint: ΔD50 (%) Calculate bottle by bottle: Intra-group report: mean ± SD (n=5), and give 95% CI.

[0058] By determination: ΔD50≤+5% and crystallization / layering = negative.

[0059] 6. Statistics Take Example 1 as a reference, and perform one-way ANOVA on the ΔD50 of other examples, followed by Dunnett's multiple comparison (α=0.05).

[0060] If not normal / homoscedastic, use Kruskal-Wallis + Dunn correction.

[0061] Report the Δ (%) of PDI of each group at the same time as auxiliary evidence (non-determination index).

[0062] 7. Quality control The instrument was routinely calibrated with 100 nm latex standards (CV≤3%).

[0063] Dilution was performed by the same person; samples were all done within 4h in one batch.

[0064] Test results ΔD50 (%) were calculated for each bottle according to the formula (D50 6mo −D50 T0 ) / D50 T0 ×100 and averaged ± SD, with 95% CI. Crystallization / delamination was determined by polarized light microscopy, DSC and visual inspection, and was considered positive if any of the three were positive. p values were derived from one-way ANOVA + Dunnett's multiple comparison, with Example 1 as the reference. The determination of pass or fail was based on the criteria of ΔD50≤+5% and crystallization / delamination = negative.

[0065] Table 1 After 6 months storage at 2-8°C, the ΔD50, 95% CI, crystallization / delamination determination and statistical results for each example are shown in the table above. The results show that Example 1, Example 2 and Example 6 all meet the determination criteria and pass the low temperature stability test, with Example 6 having the lowest ΔD50 (+2.6%) and a significant difference compared to Example 1 (p = 0.004). Example 3 and Example 4 have ΔD50 exceeding +5%, and are determined as fail; Example 5 has a ΔD50 as high as +12.8% and positive crystallization, with significantly reduced stability. The results show that the optimal preparation sequence of the present application is first negative then positive, and the use of polylysine and DEAE-Dextran in cation modification can further improve the long-term stability at low temperature.

[0066] The preferred embodiments of the present application are described in detail above. It should be understood that those of ordinary skill in the art can make modifications and variations without departing from the concept of the present application. Therefore, any technical solutions obtained by logical analysis, reasoning or limited experiments based on the prior art according to the concept of the present application should be within the scope of protection defined by the claims.

Claims

1. A multiple nanoemulsion adjuvant, characterized in that Based on 100g of the raw material system, it includes: 2.73–6.37 g oily matrix; 0.10–0.25 g sterols; 0.212–0.424 g nonionic surfactant; 0.00050–0.00200 g triterpenoid saponin active adjuvant; 0.010–0.020 g anionic polymer modifier; 0.005–0.015 g cationic polymer modifier; 70–90 g buffer, pH 6.8–7.6, osmolarity 260–320 mOsm / kg; Add water to make the total mass 100g.

2. The multiple nanoemulsion adjuvant according to claim 1, characterized in that The oil phase matrix is ​​ethyl linoleate, medium chain triglyceride, squalene or a combination thereof.

3. The multiple nanoemulsion adjuvant according to claim 1, characterized in that The sterol compound is campesterol, β-sitosterol, stigmasterol, cholesterol or a combination thereof.

4. The multiple nanoemulsion adjuvant according to claim 1, characterized in that The nonionic surfactant is polysorbate, polyoxyethylene fatty acid ester, polyoxyethylene castor oil derivative or a combination thereof.

5. The multiple nanoemulsion adjuvant according to claim 1, characterized in that The triterpenoid saponin active adjuvant is QS-21.

6. The multiple nanoemulsion adjuvant according to claim 1, characterized in that The anionic polymer modifier is sodium gamma-polyglutamate, hyaluronic acid, alginate, carrageenan or a combination thereof.

7. The multiple nanoemulsion adjuvant according to claim 1, characterized in that The cationic polymer modifier is diethylaminoethyl dextran hydrochloride, polylysine, chitosan, polyguanidine salt or a combination thereof.

8. The multiple nanoemulsion adjuvant according to claim 1, characterized in that The buffer is HEPES buffer.

9. The method for preparing the multiple nanoemulsion adjuvant according to any one of claims 1 to 8, characterized in that: The following steps are involved: (1) mixing the oil phase matrix and the sterol compound, stirring and dissolving them to obtain an oil phase; (2) adding a triterpenoid saponin active adjuvant and a nonionic surfactant into a buffer solution and stirring to dissolve the mixture to obtain an aqueous phase; (3) adding the oil phase to the water phase and stirring to form colostrum; (4) emulsifying the colostrum to obtain a nanoemulsion; (5) adding anionic polymer modifier to the nanoemulsion and mixing uniformly; (6) After step (5), add a cationic polymer modifier and mix well; (7) Add water to a total mass of 100 g, mix well, and filter.

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