Nanoemulsion vaccine adjuvants and methods of making same

Abstract

The application discloses a nano-emulsion vaccine adjuvant and a preparation method thereof, and belongs to the technical field of vaccine adjuvants.The preparation method comprises the following steps: (1) mixing and heating medium-chain triglyceride, cholesterol and QS-21 to obtain an oil phase; (2) mixing an emulsifier with water to obtain an aqueous phase; (3) adding the oil phase into the aqueous phase, and shearing or ultrasonicating to obtain a primary nano-emulsion; (4) adding a solution containing a cationic polymer into the primary nano-emulsion, and stirring; adding a crosslinking agent, and stirring; and (5) purifying and drying.Compared with the prior art, the nano-emulsion vaccine adjuvant prepared by the application is of a core-shell structure of nano-emulsion oil droplets and a crosslinking modified cationic polymer, can effectively encapsulate and stabilize QS-21, and effectively improves the application prospect of the QS-21 in vaccines.

Description

Technical Field

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

[0002] Vaccine adjuvants, as important excipients for enhancing antigen immunogenicity, have received widespread attention in the development of novel vaccines in recent years. QS-21 is a natural triterpenoid saponin derived from soap bark, which has significant immunomodulatory activity, especially in activating Th1-type cellular immunity, promoting antigen presentation, and inducing cytotoxic T lymphocyte (CTL) responses. It has become a key component in many vaccines under development (such as tumor vaccines, malaria vaccines, and shingles vaccines).

[0003] In applications, QS-21 typically requires encapsulation using carrier systems such as emulsions or nanoparticles to mitigate its hemolytic toxicity, prolong in vivo release time, and improve delivery efficiency. However, QS-21 itself is a typical amphoteric molecule containing a hydrophobic triterpenoid backbone and multiple hydrophilic glycoside side chains. Its overall structure is asymmetrical and its surface activity is high, which can easily affect the physical stability of the carrier system, becoming a core technical bottleneck in formulation development.

[0004] Specifically, current emulsion or nanoparticle systems based on QS-21 exhibit significant physical instability. Common manifestations include gradually increasing particle size, precipitation or flocculation, phase separation, and a "bimodal particle size" phenomenon in dynamic light scattering (DLS) analysis, indicating low encapsulation efficiency or uneven distribution. Under freeze-thaw cycles, slight pH fluctuations, or low-concentration electrolyte interference, these systems are highly susceptible to disintegration, precipitation, or inactivation, severely limiting their industrial application in vaccine formulations.

[0005] Studies have found that although QS-21 can form complexes with cholesterol, lecithin, etc., it still struggles to cope with the disruption of nanostructure integrity caused by various disturbances due to the lack of a stable and continuous "protective shell" structure. Furthermore, the membrane structure of traditional liposome configurations is susceptible to oxidation, hydration, and phase transitions during long-term storage or freeze-drying, further exacerbating the instability of the QS-21 encapsulation system.

[0006] Therefore, there is an urgent need to develop a novel technical solution that can effectively enhance the structural stability of QS-21 nanoemulsions or nanoparticles, so as to improve their adaptability in complex vaccine environments and their prospects for industrial transformation. Summary of the Invention

[0007] The purpose of this invention is to solve the problem of physical instability of QS-21 as a vaccine adjuvant in emulsion or nanoparticle encapsulation systems in the prior art, and to provide a nanoemulsion vaccine adjuvant with significantly improved stability and its preparation method. Through the core-shell structure of nanoemulsion oil droplets and cross-linked modified cationic polymers, QS-21 is effectively encapsulated and stabilized, thereby improving its application prospects in vaccines.

[0008] This invention provides a method for preparing a nanoemulsion vaccine adjuvant, comprising the following steps:

[0009] (1) Medium-chain triglycerides, cholesterol and QS-21 were mixed and heated to obtain an oil phase;

[0010] (2) Mix the emulsifier with water to obtain an aqueous phase;

[0011] (3) Add the oil phase to the aqueous phase and shear or sonicate to obtain a primary nanoemulsion;

[0012] (4) Add the solution containing cationic polymers to the primary nanoemulsion and stir; add the crosslinking agent and stir;

[0013] (5) Purification and drying.

[0014] Preferably, the preparation method of the nanoemulsion vaccine adjuvant is as follows:

[0015] (1) Mix 4.5-5.5g of medium-chain triglycerides, 0.4-0.6g of cholesterol and 0.08-0.12g of QS-21, and heat and stir in a water bath at 800-1200rpm and 45-55℃ for 15-30min until completely dissolved and mixed to form a homogeneous and transparent oil phase;

[0016] (2) Mix 1.2-1.6g of emulsifier with 92-96mL of deionized water, then make up the volume with deionized water, and stir at 30-35℃ and 500-800rpm for 15-30min until completely dissolved and mixed to obtain the aqueous phase;

[0017] (3) Add the oil phase to the aqueous phase at a dropping rate of 0.5-2 mL / min, and then mix and emulsify at 10000-30000 rpm for 3-8 min to obtain a primary nanoemulsion;

[0018] (4) Add 0.1-0.3g of cationic polymer to 99.5-99.9mL of 1-3wt% acetic acid aqueous solution, then mix and stir at 200-500rpm for 8-12min, then adjust the pH to 4.8-5.2 with 0.5-2mol / L NaOH aqueous solution and continue stirring for 20-40min to obtain cationic polymer solution; then add cationic polymer solution dropwise to primary nanoemulsion at a rate of 0.5-2mL / min; then place in an ice bath at 1-5℃ and stir magnetically for 15-30min to obtain mixed emulsion;

[0019] Alternatively, add 0.1-0.3 g of cationic polymer to 99.5-99.9 mL of 1-3 wt% acetic acid aqueous solution, then mix and stir at 200-500 rpm for 8-12 min. Adjust the pH to 4.8-5.2 with 0.5-2 mol / L NaOH aqueous solution and continue stirring for 20-40 min to obtain a cationic polymer solution. Then, add the cationic polymer solution dropwise to the primary nanoemulsion at a rate of 0.5-2 mL / min. Next, place the mixture in an ice bath at 1-5℃ and magnetically stir for 15-30 min to obtain a mixed emulsion system. Dissolve the crosslinking agent in deionized water to prepare a 5-20 mmol / L crosslinking agent solution. Then, add the crosslinking agent solution dropwise to the mixed emulsion system at a rate of 0.5-2 mL / min. Continue stirring and reacting for 40-50 min to obtain a mixed emulsion.

[0020] (5) The mixed emulsion is concentrated by ultrafiltration and dialyzed to obtain nanoemulsion; sucrose is added to the nanoemulsion and stirred evenly; then freeze-dried to obtain loose powder, which is nanoemulsion vaccine adjuvant.

[0021] Preferably, the emulsifier in step (2) is selected from Tween 80 and Poloxamer 188.

[0022] Preferably, the primary nanoemulsion in step (3) has a particle size distribution of 110±10nm and a PDI of less than 0.3.

[0023] Preferably, in step (4), the cationic polymer solution is slowly added dropwise to the primary nanoemulsion at a volume ratio of 1:5-7; the cationic polymer is selected from maleimide-modified chitosan, quaternized chitosan, and guanidine-modified chitosan.

[0024] Preferably, the crosslinking agent in step (4) is selected from at least one of glutathione, L-cysteine, and dithiothreitol.

[0025] Preferably, the final concentration of the crosslinking agent in the mixed emulsion system in step (4) is 0.5-2 mmol / L.

[0026] Preferably, in step (5), the mixed emulsion is passed through an ultrafiltration tube and centrifuged at 1-5℃ and 3000-6000 rpm for 15-30 min to collect the retentate; the retentate is transferred to a dialysis bag for dialysis for 6 h; the dialysis solution is concentrated to 1 / 4-1 / 5 of its original volume to obtain a nanoemulsion.

[0027] Preferably, the final concentration of sucrose in the nanoemulsion in step (5) is 5-7% w / v.

[0028] The present invention also provides a nanoemulsion vaccine adjuvant, which is prepared by the above method.

[0029] The beneficial effects of this invention are:

[0030] Compared with existing technologies, this invention introduces cationic polymers containing positively charged groups into the preparation method of nanoemulsion vaccine adjuvants. These polymers spontaneously adsorb onto the negatively charged emulsion surface via electrostatic attraction, forming a preliminary shell to improve the stability of QS-21 in formulations. Furthermore, glutathione, L-cysteine, and dithiothreitol are selected as cross-linking agents to covalently cross-link with maleimide-modified chitosan, constructing a composite network cross-linked shell. This structure achieves highly efficient loading of QS-21, imparts higher structural strength to the particles, forms a stable antigen or adjuvant delivery reservoir, significantly improves the stability and practicality of QS-21, and is suitable for highly stable multivalent vaccines, possessing good industrial feasibility and patent prospects. Detailed Implementation

[0031] The parameters and sources of the specific chemical substances used.

[0032] QS-21, analytical grade, purity ≥95%, sourced from Chengdu Changguang Biotechnology;

[0033] Cholesterol, purity ≥99%, brand: Sigma;

[0034] Medium-chain triglycerides (MCT oil) Captex 300, ABITEC;

[0035] Twain 80, CP grade, brand: Aladdin;

[0036] Sucrose, sterile injection grade, commercially available;

[0037] Deionized water is used after being filtered through a 0.22μm filter.

[0038] Dithiothreitol, purity ≥99%, brand: Sigma;

[0039] Glutathione, pharmaceutical grade, CAS No.: 70-18-8;

[0040] L-cysteine, pharmaceutical grade, CAS No.: 52-90-4;

[0041] Maleimide-modified chitosan, product number: Q-0148660, brand: Xi'an Qiyue Biotechnology;

[0042] Guanidino-modified chitosan, catalog number: WH250113307G, is sourced from Guangzhou Weihua Biotechnology Co., Ltd.

[0043] Preparation of quaternized chitosan: 1 g of chitosan was dissolved in 72 mL of dilute acetic acid solution (0.5% v / v) and stirred continuously for 2 h; then 2.82 g of 2,3-epoxypropyltrimethylammonium chloride was added to the chitosan solution and reacted at 70 °C for 12 h. Subsequently, the product was purified by dialysis with water for 3 days (MWCO: 8000 Da), and then freeze-dried to obtain quaternized chitosan.

[0044] Example 1

[0045] A method for preparing a nanoemulsion vaccine adjuvant includes the following steps:

[0046] (1) Mix 5.0g medium chain triglycerides, 0.5g cholesterol and 0.1g QS-21, heat and stir at 1000rpm and 50℃ water bath for 30min until completely dissolved and mixed to form a homogeneous transparent oil phase;

[0047] (2) Mix 1.5g Tween 80 and 94 mL of deionized water, then dilute to a total volume of 100 mL with deionized water. Mix and stir at 33℃ and 600 rpm for 20 min until completely dissolved and mixed to obtain an aqueous phase.

[0048] (3) The oil phase prepared in step (1) above is added dropwise to the aqueous phase prepared in step (2) above at a drop rate of 1 mL / min; then, a high-speed shear emulsifier (IKA T25) is used to mix and emulsify at 15000 rpm for 5 min to obtain a primary nanoemulsion with a particle size distribution of 110±10 nm and a PDI of 0.24.

[0049] (4) Add 0.2g of maleimide-modified chitosan to 99.8mL of 1wt% acetic acid aqueous solution, then mix and stir at 200rpm for 10min, then adjust the pH to 5.0 with 1mol / L NaOH aqueous solution and continue stirring for 30min to obtain maleimide-modified chitosan solution; then add maleimide-modified chitosan solution to primary nanoemulsion at a volume ratio of 1:5 at a dropping rate of 1mL / min, and complete the addition within 30min; then place in an ice bath at 4℃ and stir magnetically for 20min to obtain a mixed emulsion system; dissolve dithiothreitol in deionized water to prepare 10 A mmol / L dithiothreitol solution was added; then the dithiothreitol solution was added dropwise to the mixed emulsion system at a rate of 1 mL / min, and the addition was completed within 30 min; then the reaction was stirred for another 45 min to obtain a mixed emulsion, wherein the final concentration of dithiothreitol added to the mixed emulsion system was 1 mmol / L;

[0050] (5) The mixed emulsion was passed through a 50 kDa ultrafiltration tube (Amicon) and centrifuged at 4000 rpm for 30 min at 4℃. The retentate was collected. The retentate was transferred to a dialysis bag (molecular weight cutoff 10 kDa) and placed in 500 mL PBS buffer (pH 7.4). Dialysis was performed at 4℃ for 6 h, and the buffer was changed every 2 h. The dialysis solution was concentrated to 1 / 5 of the original volume to obtain nanoemulsion.

[0051] (6) Add sucrose to the nanoemulsion until the final sucrose concentration is 5% w / v and stir evenly; then pre-freeze at -80℃ for 4 hours and freeze-dry in a freeze dryer for 24 hours to obtain loose powder, i.e. nanoemulsion vaccine adjuvant.

[0052] Example 2

[0053] A method for preparing a nanoemulsion vaccine adjuvant differs from Example 1 only in that step (4) is:

[0054] Add 0.2 g of maleimide-modified chitosan to 99.8 mL of 1 wt% acetic acid aqueous solution, then mix and stir at 200 rpm for 10 min, and then use 1 mol / L... The pH of the NaOH aqueous solution was adjusted to 5.0 and stirred for 30 min to obtain a maleimide-modified chitosan solution. Then, the maleimide-modified chitosan solution was added dropwise to the primary nanoemulsion at a volume ratio of 1:5 (maleimide-modified chitosan solution: primary nanoemulsion) at a rate of 1 mL / min, and the addition was completed within 30 min. After that, the mixture was placed in an ice bath at 4 °C and magnetically stirred for 20 min to obtain a mixed emulsion system. L-cysteine ​​was dissolved in deionized water to prepare a 10 mmol / L L-cysteine ​​solution. Then, the L-cysteine ​​solution was added dropwise to the mixed emulsion system at a rate of 1 mL / min, and the addition was completed within 30 min. The reaction was then stirred for 45 min to obtain a mixed emulsion, wherein the final concentration of L-cysteine ​​added to the mixed emulsion system was 1 mmol / L.

[0055] Example 3

[0056] A method for preparing a nanoemulsion vaccine adjuvant differs from Example 1 only in that step (4) is:

[0057] 0.2 g of maleimide-modified chitosan was added to 99.8 mL of 1 wt% acetic acid aqueous solution, and then mixed and stirred at 200 rpm for 10 min. The pH was then adjusted to 5.0 with 1 mol / L NaOH aqueous solution, and stirring was continued for 30 min to obtain a maleimide-modified chitosan solution. The maleimide-modified chitosan solution was then added dropwise to the primary nanoemulsion at a volume ratio of 1:5 (maleimide-modified chitosan solution: primary nanoemulsion) at a rate of 1 mL / min, and the addition was completed within 30 min. The mixture was then placed in an ice bath at 4 °C and magnetically stirred for 20 min to obtain a mixed emulsion system. Glutathione was dissolved in deionized water to prepare a 10 mmol / L glutathione solution. The glutathione solution was then added dropwise to the mixed emulsion system at a rate of 1 mL / min, and the addition was completed within 30 min. The reaction was then continued to be stirred for 45 min to obtain a mixed emulsion, wherein the final concentration of glutathione added to the mixed emulsion system was 1 mmol / L.

[0058] Example 4

[0059] A method for preparing a nanoemulsion vaccine adjuvant differs from Example 1 only in that step (4) is:

[0060] 0.2 g of maleimide-modified chitosan was added to 99.8 mL of 1 wt% acetic acid aqueous solution, and then mixed and stirred at 200 rpm for 10 min. The pH was then adjusted to 5.0 with 1 mol / L NaOH aqueous solution, and stirring was continued for 30 min to obtain a maleimide-modified chitosan solution. The maleimide-modified chitosan solution was then added dropwise to the primary nanoemulsion at a volume ratio of 1:5 (maleimide-modified chitosan solution: primary nanoemulsion) at a rate of 1 mL / min, and the addition was completed within 30 min. The mixture was then placed in an ice bath at 4 °C and magnetically stirred for 20 min to obtain a mixed emulsion system. The crosslinking agent was dissolved in deionized water to prepare a 10 mmol / L crosslinking agent solution. The crosslinking agent solution was then added dropwise to the mixed emulsion system at a rate of 1 mL / min, and the addition was completed within 30 min. The reaction was then continued to be stirred for 45 min to obtain a mixed emulsion, wherein the final concentration of the crosslinking agent added to the mixed emulsion system was 1 mmol / L.

[0061] The crosslinking agent is a mixture of glutathione and L-cysteine ​​in a mass ratio of 1:1.

[0062] Example 5

[0063] A method for preparing a nanoemulsion vaccine adjuvant differs from Example 1 only in that step (4) is:

[0064] 0.2 g of maleimide-modified chitosan was added to 99.8 mL of 1 wt% acetic acid aqueous solution, and then mixed and stirred at 200 rpm for 10 min. The pH was then adjusted to 5.0 with 1 mol / L NaOH aqueous solution, and stirring was continued for 30 min to obtain a maleimide-modified chitosan solution. The maleimide-modified chitosan solution was then added dropwise to the primary nanoemulsion at a volume ratio of 1:5 at a rate of 1 mL / min, and the addition was completed within 30 min. After that, the mixture was placed in an ice bath at 4 °C and magnetically stirred for 20 min to obtain a mixed emulsion.

[0065] Example 6

[0066] A method for preparing a nanoemulsion vaccine adjuvant differs from Example 1 only in that step (4) is:

[0067] 0.2 g of quaternized chitosan was added to 99.8 mL of 1 wt% acetic acid aqueous solution, and then mixed and stirred at 200 rpm for 10 min. The pH was then adjusted to 5.0 with 1 mol / L NaOH aqueous solution, and stirring was continued for 30 min to obtain a quaternized chitosan solution. The quaternized chitosan solution was then added dropwise to the primary nanoemulsion at a volume ratio of 1:5 at a rate of 1 mL / min, and the addition was completed within 30 min. After that, the mixture was placed in an ice bath at 4 °C and magnetically stirred for 20 min to obtain a mixed emulsion.

[0068] Example 7

[0069] A method for preparing a nanoemulsion vaccine adjuvant differs from Example 1 only in that step (4) is:

[0070] 0.2 g of guanidinyl-modified chitosan was added to 99.8 mL of 1 wt% acetic acid aqueous solution, and then mixed and stirred at 200 rpm for 10 min. The pH was then adjusted to 5.0 with 1 mol / L NaOH aqueous solution, and stirring was continued for 30 min to obtain a guanidinyl-modified chitosan solution. The guanidinyl-modified chitosan solution was then added dropwise to the primary nanoemulsion at a volume ratio of 1:5 at a rate of 1 mL / min, and the addition was completed within 30 min. After that, the mixture was placed in an ice bath at 4 °C and magnetically stirred for 20 min to obtain a mixed emulsion.

[0071] Comparative Example 1

[0072] A method for preparing a nanoemulsion vaccine adjuvant differs from Example 1 only in that step (4) is:

[0073] 0.2 g of chitosan was added to 99.8 mL of 1 wt% acetic acid aqueous solution, and then mixed and stirred at 200 rpm for 10 min. The pH was then adjusted to 5.0 with 1 mol / L NaOH aqueous solution and the mixture was stirred for another 30 min to obtain a chitosan solution. The chitosan solution was then added dropwise to the primary nanoemulsion at a volume ratio of 1:5 at a rate of 1 mL / min, and the addition was completed within 30 min. The mixture was then placed in an ice bath at 4 °C and magnetically stirred for 20 min to obtain a mixed emulsion.

[0074] Comparative Example 2

[0075] A method for preparing a nanoemulsion vaccine adjuvant includes the following steps:

[0076] (1) Mix 5.0g medium chain triglycerides, 0.5g cholesterol and 0.1g QS-21, heat and stir at 1000rpm and 50℃ water bath for 30min until completely dissolved and mixed to form a homogeneous transparent oil phase;

[0077] (2) Mix 1.5g Tween 80 and 94 mL of deionized water, then dilute to a total volume of 100 mL with deionized water. Mix and stir at 33℃ and 600 rpm for 20 min until completely dissolved and mixed to obtain an aqueous phase.

[0078] (3) The oil phase prepared in step (1) above is added dropwise to the aqueous phase prepared in step (2) above at a drop rate of 1 mL / min; then, a high-speed shear emulsifier (IKA T25) is used to mix and emulsify at 15000 rpm for 5 min to obtain a mixed emulsion with a particle size distribution of 110±10 nm and a PDI of 0.24.

[0079] (4) The mixed emulsion was passed through a 50 kDa ultrafiltration tube (Amicon) and centrifuged at 4000 rpm for 30 min at 4℃. The retentate was collected. The retentate was transferred to a dialysis bag (molecular weight cutoff 10 kDa) and placed in 500 mL PBS buffer (pH 7.4). Dialysis was performed at 4℃ for 6 h, and the buffer was changed every 2 h. The dialysis solution was concentrated to 1 / 5 of the original volume to obtain nanoemulsion.

[0080] (5) Add sucrose to the nanoemulsion until the final sucrose concentration is 5% w / v and stir evenly; then pre-freeze at -80℃ for 4 hours and freeze-dry in a freeze dryer for 24 hours to obtain loose powder, i.e. nanoemulsion vaccine adjuvant.

[0081] Test Example 1

[0082] Encapsulation efficiency test

[0083] Take 1 mL of the nanoemulsions prepared in step (5) of Examples 1-7 and Comparative Example 1 respectively as samples and record them as Group 1-7 and Group 1 of Examples 1-7 and Comparative Example 1 respectively; take 100 μL of each group as the original nanoemulsion sample, add 4 mL of PBS buffer (pH 7.4) to the remaining nanoemulsion, vortex for 30 s to obtain a mixture; centrifuge the mixture at 100000 rpm for 60 min (4℃); after centrifugation, take the supernatant and filter it with a 0.22 μm filter membrane for sterilization;

[0084] The absorbance (A) of the supernatant at 280 nm was measured using UV-Vis, and the concentration of free QS-21 (C) was calculated using a QS-21 standard curve (0.1–1 mg / mL). 游离 ); Add 1 mL of 1% SDS solution to 100 μL of the original nanoemulsion sample, shake to mix, centrifuge at 12000 rpm for 10 min, take the supernatant and detect A using UV-Vis, calculate the total QS-21 concentration (C). 总 Then, based on the total QS-21 concentration and the free QS-21 concentration, the encapsulation efficiency (EE, %) is calculated. The specific calculation formula is as follows:

[0085] EE (%) = (1-C 游离 / C 总 ) × 100%

[0086] Freeze-thaw resistance test

[0087] Take 3 mL of the nanoemulsions prepared in Examples 1-7 and Comparative Example 1 as samples, and record them as Groups 1-7 and 1, respectively. Then, divide each group of samples into 3 equal parts and perform 1, 3, and 5 freeze-thaw cycles, respectively, and label them as F1 (1 cycle), F3 (3 cycles), and F5 (5 cycles). Then, test the encapsulation efficiency of each group of nanoemulsion samples after freeze-thaw cycles according to the above method. The freeze-thaw cycle operation is as follows: place the sample at -80℃ for pre-freezing for 4 h, and then transfer it to a 37℃ water bath for rehydration for 30 min.

[0088] The specific test results are shown in Table 1 below.

[0089] Table 1

[0090]

[0091] As shown in Table 1, Examples 1-7 exhibited significantly higher initial encapsulation efficiencies and encapsulation efficiencies after freeze-thaw cycles compared to Comparative Example 1. Furthermore, Examples 1-4 were generally superior to Examples 5-7, demonstrating a smaller decrease in encapsulation efficiency after freeze-thaw cycles. This indicates that relying solely on simple cationic polymer electrostatic adsorption or physical coating results in a shell layer primarily maintained by weak interactions, which is prone to loosening and desorption under freeze-thaw cycles and slight acid-base fluctuations. This leads to high porosity and insufficient mechanical strength, resulting in severe exudation of QS-21. However, the introduction of a crosslinking agent allows maleimide-modified chitosan to undergo an addition reaction with the thiol groups in the crosslinking agent, forming a chemically cured network shell at the interface. This enhances both charge shielding and structural strength, thereby significantly improving freeze-thaw resistance. Comparing Examples 1-3, Example 2 showed the best encapsulation efficiency and freeze-thaw retention rate. This is because L-cysteine ​​molecules are small and have no steric hindrance, allowing them to react rapidly with maleimide groups to form a more uniform and dense cured layer. Example 1, due to the high degree of crosslinking using dithiothreitol, had an excessively rigid shell that was prone to microcracks under freeze-thaw stress, resulting in a lower retention rate than Example 2. Example 3, due to the large size of glutathione molecules and significant steric hindrance, had insufficient crosslinking efficiency, resulting in a loose cured layer that was more prone to disintegration under freeze-thaw stress, also had a lower retention rate than Example 2. Example 4 showed higher encapsulation efficiency and freeze-thaw retention rate than the best single agent in Examples 2 and 3, indicating that the rapid densification provided by L-cysteine ​​and the complementary effects of glutathione in terms of interchain spacing regulation and antioxidant buffering achieve a better balance between shell density and toughness.

[0092] Test Example 2

[0093] Long-term stability test

[0094] Take 5 mL of the nanoemulsions prepared in steps (5) of Examples 1-7 and (4) of Comparative Example 1 and Comparative Example 2 respectively as samples and record them as Examples 1-7 and Comparative Example 1-2 groups; disperse 1 mL of each group of nanoemulsion samples in 9 mL of PBS buffer (pH 7.4), and set 5 parallel samples for each group; store at room temperature (25℃) for long-term stability test, and take samples of each group at day 0, 3 months, 6 months and 12 months respectively for testing;

[0095] Detection method: Take 50 μL of sample, add 1 mL of 1% SDS solution, shake to mix, centrifuge at 12000 rpm for 10 min, take the supernatant and detect the peak area of ​​QS-21 by HPLC, calculate the QS-21 residual rate by comparing with the original peak area (before the start of long-term stability experiment); the test results are averaged.

[0096] The specific test results are shown in Table 2 below.

[0097] Table 2

[0098]

[0099] As shown in Table 2, after 12 months of storage, the QS-21 residue rate of Examples 1-7 was significantly higher than that of Comparative Examples 1-2, with Examples 1-4 generally outperforming Examples 5-7. This indicates that after the introduction of the crosslinking agent, maleimide-modified chitosan can undergo an addition reaction with thiol groups to form stable thioether bonds, thereby constructing a dense crosslinked shell at the emulsion interface. This shell exhibits high chemical stability and hydrolysis resistance, effectively resisting ionic interference and delaying the aging process, thus significantly improving the long-term stability of the system. Further comparing Examples 1-3, the QS-21 residue rate of Example 2 was consistently higher than that of Examples 1 and 3, indicating that the cured layer formed by L-cysteine ​​is more uniform and dense, exhibiting superior stability. Example 4 still showed a higher residual rate than the best single agent in Examples 2 and 3 at 12 months, indicating that the rapid densification provided by L-cysteine ​​and the antioxidant buffering effect of glutathione complement each other in the system, further enhancing the shell integrity and durability, thus demonstrating the synergistic effect of combination over single agents.

[0100] Test Example 3

[0101] Biosafety testing

[0102] The biosafety of the nanoemulsion vaccine adjuvants prepared in Examples 1-7 and Comparative Examples 1-2 of this invention were tested, and the specific test methods are as follows:

[0103] Animal preparation: 6-8 week old SPF grade BALB / c female mice (weighing 18-22g) were selected, with half males and half females; the mice were placed in a breeding cage and kept at a temperature of 22±2℃, humidity of 50±10%, with a 12h light cycle, free access to water and food, for 7 days for acclimatization.

[0104] Test sample: Nanoemulsion vaccine adjuvants prepared in Examples 1-7 and Comparative Examples 1-2 of this invention (the nanoemulsion vaccine adjuvants were reconstituted with 0.9% sodium chloride injection 20 minutes before the experiment and stored at 4°C for later use).

[0105] Reference standard: physiological saline (0.9% sodium chloride injection)

[0106] I. Acute toxicity test

[0107] After 7 days of acclimatization, mice were observed for any abnormalities (such as diarrhea or hair loss) before the experiment was conducted. Thirty mice were randomly divided into three groups of 10 each (half male and half female): a high-dose group, a low-dose group, and a blank control group. The dosages were as follows: high-dose group: 1000 mg / kg (8 times the clinical dose); low-dose group: 250 mg / kg (2 times the clinical dose); control group: an equal volume of physiological saline. Mice in each group were administered the medication subcutaneously at a dose of 20 mL / kg. After administration, the mice were placed in cages, fed normally, and observed and recorded.

[0108] Within 2 hours after administration, the behavior and mortality of mice were observed and recorded every 15 minutes. Specifically, the behavior of mice was observed to see if they exhibited tremors, convulsions, rapid breathing (>80 breaths / minute), piloerection, or diarrhea. If a mouse died, the time of death was recorded (accurate to the minute).

[0109] Within 2 hours after administration, the weight and condition of the mice were observed and recorded daily; specifically, the mice were weighed fasting at 9:00 am each day (accurate to 0.1g); the daily food intake of the mice was recorded; the activity level (active / listless), coat (smooth / fluffy), and eye and nasal secretions (absent / present) of the mice were recorded.

[0110] Fourteen days after administration, the mice were euthanized, then dissected, and the thoracic and abdominal cavities were opened. The heart, liver, spleen, lungs, and kidneys were visually inspected for congestion, edema, or necrosis. The organs were weighed and the organ coefficient was calculated (organ coefficient = organ weight / body weight × 100%).

[0111] Test Results and Analysis: During the acute toxicity test, no mice died within 14 days after administration. Their behavior remained normal and without abnormalities, and no acute toxic symptoms were observed. Weight gain and food intake were normal in all groups. Autopsy revealed no abnormalities; upon opening the thoracic and abdominal cavities, no congestion, edema, necrosis, or nodules were found in major organs such as the heart, liver, spleen, lungs, and kidneys. No abnormal lesions were observed at the injection sites (skin, muscles, etc.). There were no significant differences in organ coefficients compared to the control group. The acute toxicity test results showed no deaths, no acute toxic reactions, and normal weight and organ function, indicating that a single administration posed no overall toxic risk to the mice.

[0112] II. Local Irritation Test

[0113] After 7 days of acclimatization, mice were observed for any abnormalities (such as diarrhea or hair loss) before the experiment was conducted. Twelve mice were randomly divided into two groups of six each, with half males and half females, designated as the experimental group and the blank control group. The dosage was 1000 mg / kg (8 times the clinical dose) for the experimental group and an equal volume of physiological saline for the control group. Mice in each group were administered the drug subcutaneously at a dose of 20 mL / kg. After administration, the mice were placed in cages, fed normally, and observed and recorded.

[0114] The injection sites of mice were observed at 1h, 24h, 48h and 72h after administration; the mice were observed for redness, swelling and induration of the skin, and the results were judged and scored according to Table 3 below.

[0115] Table 3

[0116]

[0117] 72 hours after administration, the mice were euthanized, and the skin and subcutaneous tissue (1cm×1cm) at the injection site were collected, fixed with 10% formalin, stained with HE, and observed for keratinocyte necrosis and shedding in the epidermis, inflammatory cell infiltration in the dermis (neutrophils predominant, indicating acute inflammation), and edema and fibrin exudation in the subcutaneous tissue.

[0118] Test Results and Analysis: During the local irritation test, mice in the experimental group showed no edema or induration at the injection site at all time points, indicating no irritation. Observation of the skin and subcutaneous tissue at the injection site revealed no keratinocyte necrosis or shedding in the epidermis, maintaining its intact structure; no obvious inflammatory cell infiltration in the dermis; and no edema, fibrin exudation, or necrosis in the subcutaneous tissue. The results of the local irritation test showed no local reaction and no irreversible tissue damage, indicating good safety at the injection site and meeting the local tolerance requirements for vaccine adjuvants.

[0119] In summary, the results of both the acute toxicity test and the local irritation test simultaneously met the criteria of no acute death or toxic reaction, normal body weight and organ function, and no significant local irritation or tissue damage, indicating that the biocompatibility of the nanoemulsion vaccine adjuvant prepared in this invention in mice was deemed acceptable.

Claims

1. A method for preparing a nanoemulsion vaccine adjuvant, characterized in that, Includes the following steps: (1) Mix 4.5-5.5g of medium-chain triglycerides, 0.4-0.6g of cholesterol and 0.08-0.12g of QS-21, and heat and stir in a water bath at 800-1200rpm and 45-55℃ for 15-30min until completely dissolved and mixed to form a homogeneous and transparent oil phase; (2) Mix 1.2-1.6g of emulsifier with 92-96mL of deionized water, then make up the volume with deionized water, and stir at 30-35℃ and 500-800rpm for 15-30min until completely dissolved and mixed to obtain the aqueous phase; (3) Add the oil phase to the aqueous phase at a dropping rate of 0.5-2 mL / min, and then mix and emulsify at 10000-30000 rpm for 3-8 min to obtain a primary nanoemulsion; (4) Add 0.1-0.3g of cationic polymer to 99.5-99.9mL of 1-3wt% acetic acid aqueous solution, then mix and stir at 200-500rpm for 8-12min, then adjust the pH to 4.8-5.2 with 0.5-2mol / L NaOH aqueous solution and continue stirring for 20-40min to obtain cationic polymer solution; then add cationic polymer solution dropwise to primary nanoemulsion at a rate of 0.5-2mL / min; then place in an ice bath at 1-5℃ and stir magnetically for 15-30min to obtain mixed emulsion system; add crosslinking agent to deionized water to dissolve and prepare a 5-20mmol / L crosslinking agent solution; then add crosslinking agent solution dropwise to mixed emulsion system at a rate of 0.5-2mL / min; then continue stirring and reacting for 40-50min to obtain mixed emulsion; (5) The mixed emulsion was concentrated by ultrafiltration and dialyzed to obtain nanoemulsion; sucrose was added to the nanoemulsion and stirred evenly; then freeze-dried to obtain loose powder, which is nanoemulsion vaccine adjuvant; The emulsifier used in step (2) is Tween 80; In step (4), the cationic polymer solution is slowly added dropwise to the primary nanoemulsion at a volume ratio of 1:5-7 between the cationic polymer and the primary nanoemulsion; the cationic polymer is selected from maleimide-modified chitosan. The crosslinking agent in step (4) is selected from at least one of glutathione, L-cysteine, and dithiothreitol.

2. The method for preparing the nanoemulsion vaccine adjuvant according to claim 1, characterized in that: In step (3), the primary nanoemulsion has a particle size distribution of 110±10nm and a PDI of less than 0.

3.

3. The method for preparing the nanoemulsion vaccine adjuvant according to claim 1, characterized in that: In step (4), the final concentration of the crosslinking agent in the mixed emulsion system is 0.5-2 mmol / L.

4. The method for preparing the nanoemulsion vaccine adjuvant according to claim 1, characterized in that: In step (5), the mixed emulsion is passed through an ultrafiltration tube and centrifuged at 1-5℃ and 3000-6000 rpm for 15-30 min to collect the retentate; the retentate is transferred to a dialysis bag for dialysis for 6 h; the dialysis solution is concentrated to 1 / 4-1 / 5 of the original volume to obtain nanoemulsion.

5. The method for preparing the nanoemulsion vaccine adjuvant according to claim 1, characterized in that: In step (5), the final concentration of sucrose in the nanoemulsion is 5-7% w / v.

6. A nanoemulsion vaccine adjuvant, characterized in that: Prepared by the method described in any one of claims 1-5.

Images