Lipid particle adjuvant as well as preparation method and application thereof

Abstract

The invention provides a lipid particle adjuvant and a preparation method and application thereof.The lipid particle adjuvant comprises an adjuvant molecule, PEG lipid and other lipid components, and the adjuvant molecule is of a structure shown in the formula I. The lipid particle adjuvant is stable in structure and not prone to decomposition, has a high-specificity tumor immune effect, and can be used for preparing the lipid particle adjuvant. The immunologic adjuvant has strong lymph node targeting ability, and can fully meet the requirements of high efficiency and safety of immunologic adjuvants and vaccines.

Description

Technical Field

[0001] This invention belongs to the field of nanomaterials technology, and relates to a lipid particle adjuvant, its preparation method and application. Background Technology

[0002] Most novel adjuvants are small molecules, which present challenges such as inducing systemic inflammatory responses upon entering the bloodstream, being easily degraded by enzymes in the body, and having difficulty penetrating cells, severely impacting their practical application. For vaccine delivery, the main challenge is achieving direct and efficient lymph node enrichment of antigens, thereby utilizing immune cells and cytokines in the lymph nodes to stimulate and sustain adaptive immune responses. Vaccine adjuvants often need to act on immune cells in the lymph nodes to exert maximum immune activation. However, small molecules of immune adjuvants, when injected via traditional interstitial injection, not only have difficulty entering the lymph nodes but may even distribute throughout the body, causing systemic inflammatory responses.

[0003] Nanoparticle delivery technology holds promise for solving these problems with novel adjuvants and enhancing their immunostimulatory capabilities, playing a crucial role in vaccine applications. A common drawback of TLR agonists is their reactivity, and numerous studies in recent years have attempted to overcome these side effects. Encapsulating TLR7 / 8 agonists in cationic DEOEPC-based liposomal formulation nanoparticles, or covalently linking these small molecules to hyperbranched polymers, can avoid their harmful systemic reactions while maintaining their effect on humoral immunity. Utilizing the easily modifiable nature of nanotechnology, lymph node-targeted delivery and responsive release of adjuvants can be achieved. Therefore, further research into lipid particle adjuvant platforms to find simpler and more efficient delivery systems remains of great significance in this field. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention aims to provide a lipid particle adjuvant, its preparation method, and its applications. The lipid particle adjuvant of the present invention has a stable structure, is not easily decomposed, exhibits highly specific tumor immune effects, and possesses strong lymph node targeting capabilities, thus fully meeting the requirements for high efficiency and safety in immune adjuvants and vaccines.

[0005] To achieve this objective, the present invention adopts the following technical solution:

[0006] On one hand, the present invention provides a lipid particle adjuvant, the lipid particle adjuvant comprising an adjuvanted molecule and neutral lipids and other lipid components, the adjuvanted molecule having a structure as shown in Formula 1:

[0007]

[0008] Wherein, R1 is a nitrogen-containing six-membered heterocyclic ring or a C1-C3 (e.g., C1, C2, or C3) straight-chain alkane; R2 is a C8-C22 (e.g., C8, C10, C12, C14, C16, C18, C20, or C22) unsaturated chain hydrocarbon group, a C8-C22 (e.g., C8, C10, C12, C14, C16, C18, C20, or C22) alkane group, cholesterol hemisuccinate, or 1,2-dioleoyl-SN-glycerol-3-phosphoethanolamine; and R3 is a nitro or amino group.

[0009] In this invention, the adjuvanted molecule in the lipid particle adjuvant serves as a crucial component of liposomes or lipid nanoparticles, resulting in a stable lipid particle adjuvant platform structure that is resistant to decomposition. This lipid particle adjuvant platform, due to the presence of nitroimidazole, can exert a highly specific tumor immune effect at the tumor site. Furthermore, this lipid particle adjuvant platform also exhibits a stronger immune effect due to the presence of cholesterol derivatives with an aminoimidazole structure, extending its application beyond tumor treatment. Simultaneously, it possesses strong lymph node targeting capability, fully meeting the requirements for high efficiency and safety in immune adjuvants and vaccines.

[0010] In this invention, the nitrogen-containing six-membered heterocyclic group can be...

[0011] Preferably, R1 is a nitrogen-containing hexagonal heterocycle or methylene group.

[0012] Preferably, the adjuvanted molecule has any one of the structures shown in Formula 2-1 to Formula 2-4:

[0013]

[0014] R2 is cholesterol hemisuccinate (CHOL) or 1,2-dioleoyl-SN-glycerol-3-phosphoethanolamine (DOPE).

[0015] Preferably, R1 is a nitrogen-containing six-membered heterocycle;

[0016] Preferably, R3 is an amino group.

[0017] Preferably, the adjuvanting molecule is any one of the following compounds:

[0018]

[0019]

[0020] More preferably, the adjuvanting molecule is a compound of the following type:

[0021]

[0022] Preferably, the adjuvanting molecule is any one of the following compounds:

[0023]

[0024] In this invention, the method for preparing the adjuvanted molecule includes the following steps:

[0025] (1) The compound of formula 2 was reacted with N-hydroxysuccinimide to obtain the acyl oxide intermediate shown in formula 3;

[0026]

[0027] (2) The acyl oxide intermediate of formula 3 obtained in step (1) is reacted with the nitroimidazolium compound containing a primary amine of formula 4 to obtain the adjuvanted molecule of formula 5, where R3 is a nitro group; the reaction formula is as follows:

[0028]

[0029] (3) The adjuvanted molecule with nitro group R3 shown in Formula 5 reacts with a reducing agent to obtain the adjuvanted molecule with amino group R3 shown in Formula 6. The reaction formula is as follows:

[0030]

[0031] The constraints for R1 and R2 are the same as in Equation 1.

[0032] In this invention, the DOPE-COOH structure included in the compound of formula 2 can be prepared by reacting 1,2-dioleoyl-SN-glycerol-3-phosphorylethanolamine (DOPE) and succinic anhydride; preferably, the reaction is carried out in an organic solvent, preferably dichloromethane.

[0033] Preferably, the molar ratio of 1,2-dioleoyl-SN-glycerol-3-phosphoethanolamine (DOPE) to succinic anhydride is 1:1 to 1:1.5; for example, it can be 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5; and specific values ​​between the above values. Due to space limitations and for the sake of brevity, the present invention will not exhaustively list the specific values ​​included in the range.

[0034] Preferably, the reaction of 1,2-dioleoyl-SN-glycerol-3-phosphorylethanolamine (DOPE) and succinic anhydride is carried out in the presence of a catalyst;

[0035] Preferably, the catalyst is selected from N,N-diisopropylethylamine;

[0036] Preferably, the molar ratio of the catalyst to DOPE is (0.5 to 4):1, for example, it can be 0.5:1, 1:1, 1.5:1, 2:1, 3:1, 4:1, and specific values ​​between the above points. Due to space limitations and for the sake of brevity, the present invention will not exhaustively list the specific values ​​included in the range.

[0037] Preferably, the reaction temperature of 1,2-dioleoyl-SN-glycerol-3-phosphorylethanolamine (DOPE) and succinic anhydride is 0-37°C, for example, 0°C, 25°C, 30°C, or 37°C, and the reaction time is 5-48 hours, for example, 5h, 12, 24, or 48 hours, as well as specific values ​​between the above values. Due to space limitations and for the sake of brevity, the present invention will not exhaustively list the specific values ​​included in the range.

[0038] Preferably, the molar ratio of the compound of formula 2 in step (1) to N-hydroxysuccinimide is (0.5 to 2):1, for example, it can be 0.5:1, 1:1, 1.5:1, 2, and specific values ​​between the above points. Due to space limitations and for the sake of brevity, the present invention will not exhaustively list the specific values ​​included in the range.

[0039] Preferably, the reaction in step (1) is carried out in the presence of a condensing agent.

[0040] Preferably, the condensing agent is a carbodiimide condensing agent;

[0041] Preferably, the carbodiimide condensing agent is selected from 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide.

[0042] Preferably, the molar ratio of the compound of formula 2 to the condensing agent in step (1) is 1:1.2-2, for example 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.8 or 1:2, and specific values ​​between the above values ​​are not exhaustively listed in this invention due to space limitations and for the sake of brevity.

[0043] Preferably, the reaction described in step (1) is carried out in the presence of a solvent;

[0044] Preferably, the solvent includes dichloromethane.

[0045] Preferably, the reaction time in step (1) is 1 to 3 hours, and the reaction endpoint is determined by TLC. For example, it can be 1h, 2h, or 3h. The specific endpoint value needs to be determined based on TLC.

[0046] Preferably, after the reaction structure described in step (1), post-processing is performed, including extraction and drying.

[0047] Preferably, the solvent for extraction includes dichloromethane and water;

[0048] Preferably, the volume ratio of dichloromethane to water in the extraction solvent is 3 to 5:1, for example, 3:1, 4:1, 5:1, and specific values ​​between the above-mentioned values. Due to space limitations and for the sake of brevity, the present invention will not exhaustively list the specific values ​​included in the range.

[0049] Preferably, the extraction is performed 3-5 times, for example, 3, 4, or 5 times.

[0050] Preferably, the reaction in step (2) is carried out in the presence of a solvent.

[0051] Preferably, the mixed solvent includes dichloromethane and methanol.

[0052] Preferably, the volume ratio of dichloromethane to methanol in the mixed solvent is 5 to 15:1, for example, it can be 5:1, 6:1, 7:1, 8:1, 10:1, 12:1, 15:1, and specific values ​​between the above values. Due to space limitations and for the sake of brevity, the present invention will not exhaustively list the specific values ​​included in the range.

[0053] Preferably, the molar ratio of the acyl oxide intermediate shown in Formula 3 to the nitroimidazole compound containing a primary amine shown in Formula 4 in step (2) is preferably 1:1 to 2, for example, it can be 1:1, 1.2:1, 1.5:1, 1.8:1, 2:1, and specific values ​​between the above values. Due to space limitations and for the sake of brevity, the present invention will not exhaustively list the specific values ​​included in the range.

[0054] Preferably, the reaction in step (2) is carried out in the presence of an acid-binding agent.

[0055] Preferably, the acid-binding agent is triethylamine;

[0056] Preferably, the molar ratio of the acid-binding agent to the nitroimidazole compound containing a primary amine is 0.5 to 3:1, for example, it can be 0.5:1, 0.8:1, 1:1, 1.3:1, 1.5:1, 1.8:1, 2:1, 2.5:1, 2.8:1 or 3:1, as well as specific values ​​between the above-mentioned values. Due to space limitations and for the sake of brevity, the present invention will not exhaustively list the specific values ​​included in the range.

[0057] Preferably, the reaction time in step (2) is 12 to 24 hours. The reaction endpoint is determined by TLC. For example, it can be 12h, 14h, 16h, 18h, 20h, 22h, 24h, and specific point values ​​between the above points. Due to space limitations and for the sake of brevity, the present invention will not exhaustively list the specific point values ​​included in the range.

[0058] Preferably, after the reaction in step (2) is completed, purification is performed. The purification method includes: drying the final reaction solution, adding methanol, collecting the supernatant, drying the crude product under vacuum, dissolving it in hydrochloric acid aqueous solution, collecting the precipitate, washing the pH value back to neutral with saturated saline, and freeze-drying the product.

[0059] Preferably, the concentration of the hydrochloric acid aqueous solution is 1 mol / mL.

[0060] Preferably, the purification process using hydrochloric acid aqueous solution is performed 3-5 times, for example, 3, 4 or 5 times.

[0061] The purified products from steps (1) and (2) can be purified by rotary evaporation to remove the organic solvent and freeze-drying to obtain a pure product.

[0062] Preferably, the reducing agent in step (3) is selected from a combination of sodium dithionite, hydrazine hydrate and Raney nickel, a combination of iron powder and ammonium chloride, or a reducing metal chloride (such as tin dichloride, titanium trichloride, etc.).

[0063] Preferably, the reaction in step (3) is carried out in a solvent, which includes methanol and water.

[0064] Preferably, the volume ratio of methanol to water in the solvent is 5 to 20:1, for example, 5:1, 10:1, 15:1, or 20:1; and specific values ​​between the above-mentioned values ​​are not exhaustively listed here for space limitations and for the sake of brevity.

[0065] Preferably, in step (3), the mass ratio of the adjuvant molecule with nitro group R3 shown in Formula 5 to the reducing agent is 1 to 3:1, for example, it can be 1:1, 1.5:1, 2:1, or 3:1; and the specific point values ​​between the above point values ​​are not exhaustively listed in this invention due to space limitations and for the sake of brevity.

[0066] Preferably, the reaction temperature in step (3) is 0-35℃, for example, 0℃, 3℃, 5℃, 8℃, 10℃, 15℃, 18℃, 20℃, 23℃, 25℃, 28℃, 30℃ or 35℃, and the reaction time is 0.5-3 hours, for example, 0.5h, 1h, 2h or 3h, and the reaction endpoint is determined by the value at the absorption point of 320nm.

[0067] Preferably, after the reaction in step (3) is completed, purification is performed to obtain the product.

[0068] Preferably, the purification process includes: drying the final reaction solution, adding methanol, collecting the supernatant, vacuum drying the crude product, dissolving it in an ultrapure aqueous solution, washing it three times, collecting the precipitate, and freeze-drying the product.

[0069] In this invention, the purified product is subjected to rotary evaporation to remove the organic solvent and freeze-dried to obtain a pure product.

[0070] This invention introduces cholesterol and phospholipid structures with lymph node targeting function into the structure of pipemnidazole (PMND), a commonly used radiosensitizer in clinical practice, and reduces nitroimidazole to an aminoimidazole derivative with a significant adjuvant effect. The derivatives with cholesterol or phospholipid structures can participate in the construction of a lipid particle adjuvant platform as part of a lipid carrier.

[0071] Preferably, the PEG lipid comprises any one or a combination of at least two of the above phospholipid polyethylene glycols containing distearylphosphatidylethanolamine-polyethylene glycol (DSPE-mPEG), 1,2-dimyristoyl-sn-glycero-3-phosphate ethanolamine-N-[methoxy(polyethylene glycol)] (DMPE-PEG), 1,2-dipalmitoyl-sn-glycero-3-phosphate ethanolamine-N-methoxy(polyethylene glycol) (DPPE-PEG2000), 1,2-dioleoyl-sn-glycero-3-phosphate ethanolamine-N-[methoxy(polyethylene glycol)] (DOPE-PEG2000), and thiol groups.

[0072] Preferably, the other lipid components include any one or a combination of at least two of dipalmitoylphosphatidylcholine (DPPC), phospholipid poly(2-ethyl-2-oxazoline) (DSPE-PEOz), and ionizable lipids (e.g., D-Lin-MC3-DMA, ALC-0315, SM-102).

[0073] Preferably, the molar ratio of the PEG lipid to the adjuvanted molecule is 1:1 to 1:6, for example, it can be 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4 or 1:6, as well as specific values ​​between the above-mentioned values. Due to space limitations and for the sake of brevity, the present invention will not exhaustively list the specific values ​​included in the range.

[0074] Preferably, the molar ratio of the other lipid components to the PEG lipid is 1:1 to 1:8, for example, it can be 1:5, 1:5.5, 1:6, 1:6.5, 1:7, 1:7.5 or 1:8, as well as specific values ​​between the above-mentioned values. Due to space limitations and for the sake of brevity, the present invention will not exhaustively list the specific values ​​included in the range.

[0075] On the other hand, the present invention provides a method for preparing the lipid particle adjuvant as described above, the method comprising the following steps:

[0076] The adjuvanted molecule, PEG lipid, and other lipid components are mixed to obtain the lipid particle adjuvant.

[0077] Preferably, the preparation method specifically includes the following steps: dissolving the adjuvanted molecules in a first solvent to obtain solution A; mixing PEG lipids and other lipid components with a second solvent to obtain solution B; mixing solution A and solution B; removing the solvent by rotary evaporation; and re-dissolving in a third solvent to obtain the lipid particle adjuvant.

[0078] Preferably, the first solvent is selected from a mixture of methanol and chloroform.

[0079] Preferably, the volume ratio of methanol to chloroform is 20-5:1, for example, it can be 20:1, 18:1, 15:1, 13:1, 10:1, 8:1 or 5:1, as well as specific values ​​between the above points. Due to space limitations and for the sake of brevity, the present invention will not exhaustively list the specific values ​​included in the range.

[0080] Preferably, the concentration of the adjuvanting molecule in the first solvent is 0.5-4 mg / mL, for example 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL; and specific values ​​between the above values. Due to space limitations and for the sake of brevity, the present invention will not exhaustively list the specific values ​​included in the range.

[0081] Preferably, the second solvent includes any one or a combination of methanol, ethanol, or chloroform.

[0082] Preferably, the concentration of each of the PEG lipids and other lipid components in the second solvent is 5-20 mg / mL, for example, 5 mg / mL, 8 mg / mL, 10 mg / mL, 12 mg / mL, 15 mg / mL, 18 mg / mL or 20 mg / mL; and specific values ​​between the above values. Due to space limitations and for the sake of brevity, the present invention will not exhaustively list the specific values ​​included in the range.

[0083] Preferably, the reconstitution method includes any one or a combination of at least two of stirring, vortexing, or ultrasound.

[0084] Preferably, the third solvent includes any one or a combination of at least two of water, phosphate buffer, or physiological saline.

[0085] Preferably, the resolution temperature is 30-45℃, for example, it can be 30℃, 32℃, 35℃, 38℃, 40℃, 42℃, 45℃, and specific values ​​between the above points. Due to space limitations and for the sake of brevity, the present invention will not exhaustively list the specific values ​​included in the range.

[0086] Preferably, the reconstitution time is 10-60 min, for example, it can be 10 min, 15 min, 20 min, 25 min, 30 min, 40 min, 60 min, and specific values ​​between the above values. Due to space limitations and for the sake of brevity, the present invention will not exhaustively list the specific values ​​included in the range.

[0087] Preferably, the process after reconstitution further includes probe ultrasound and centrifugation.

[0088] Preferably, the preparation method further includes using ultraviolet spectroscopy and differential method, or high performance liquid chromatography, to quantitatively analyze the cholesterol derivatives of the adjuvant linker carried by the lipid particle adjuvant.

[0089] On the other hand, the present invention provides an adjuvant composition comprising, as described above, lipid particle adjuvants and other immunomodulatory adjuvants.

[0090] In this invention, the lipid particle adjuvant can be used as a carrier in combination with other immunomodulatory adjuvants.

[0091] Preferably, the other immunomodulatory adjuvants include organic molecular adjuvants and / or metal salt adjuvants.

[0092] Preferably, the organic molecular adjuvant includes any one or a combination of at least two of monophosphoryl lipid A (MPL), 3m052, rethimod (R848), imiquimod (R837), or cytokine adjuvants.

[0093] Preferably, the metal in the metal salt adjuvant is selected from any one or a combination of at least two of aluminum, manganese, zinc, or iron.

[0094] On the other hand, the present invention provides the use of the lipid particle adjuvant as described above in vaccine formulations.

[0095] In this invention, the lipid particle adjuvant is intended for application in the medical field, particularly in tumor treatment and influenza. Specifically, this lipid particle adjuvant uses adjuvanted molecules as key adjuvant components, combined with various lipid components, to achieve highly efficient lymph node targeting and immune activation effects. Furthermore, adjuvanted molecules with different structures can be prepared according to different treatment scenarios and needs, achieving optimal therapeutic goals through appropriate administration methods.

[0096] It has the following application prospects:

[0097] Lymph node targeting: This lipid particle adjuvant platform is prepared by combining adjuvant linker cholesterol derivatives or adjuvant linker lipid derivatives with lipid components, which can achieve lymph node targeting and thus achieve immune activation.

[0098] Wide range of applications: This lipid particle adjuvant platform can be prepared into various dosage forms to meet the needs of different application models, based on the characteristics of different adjuvant derivatives and application requirements.

[0099] Excellent biocompatibility: This lipid particle adjuvant platform is designed based on an adjuvanted molecule, specifically pimonidazole, a commonly used radiosensitizer. This strategy repurposes an "old drug" with clinically acceptable safety profile.

[0100] In summary, this invention demonstrates lymph node targeting capabilities, a wide range of application scenarios, and good biosafety, showing broad market application prospects and enormous clinical potential in the vaccine field.

[0101] Compared with the prior art, the present invention has the following beneficial effects:

[0102] The lipid particle adjuvant prepared in this invention can target lymph nodes, thereby activating lymph node immune cells. Among them, the adjuvant linker cholesterol derivative with a nitro group exhibits tumor targeting, and an adjuvant linker cholesterol derivative with an amino group, which has a higher adjuvant effect, can be obtained under hypoxic conditions. This is suitable for tumor models. Furthermore, the lipid particle adjuvant platform constructed from the amino group adjuvant linker cholesterol derivative has a wider range of application models. The preparation method of the lipid particle adjuvant platform is simple, the conditions are mild, the cost is low, and it is easy to promote and apply. Attached Figure Description

[0103] Figure 1 This is a high-resolution electrospray ionization mass spectrum of the adjuvant linker cholesterol derivative CHOL-PMND(CP) with a nitro structure obtained in Example 1;

[0104] Figure 2 This is a high-resolution electrospray ionization mass spectrum of the adjuvant linker cholesterol derivative NH2-CHOL-PMND(NCP) with an amino structure obtained in Example 1.

[0105] Figure 3 The images show the 1H NMR spectra of the immune agonists CHOL-PMND (CP) and CHOL-NMA (CN) obtained in Example 1, where Figure A is the 1H NMR spectrum of CHOL-PMND and Figure B is the 1H NMR spectrum of CHOL-NMA.

[0106] Figure 4The images show high-resolution electrospray mass spectra of the immune agonists DOPE-PMND (DP) and DOPE-NMA (DN) obtained in Example 2; where Figure A is the high-resolution electrospray mass spectrum of DOPE-PMND and Figure B is the high-resolution electrospray mass spectrum of DOPE-NMA.

[0107] Figure 5 The images show the 1H NMR spectra of the immune agonists DOPE-PMND (DP) and DOPE-NMA (DN) obtained in Example 2, where Figure A is the 1H NMR spectrum of DOPE-PMND and Figure B is the 1H NMR spectrum of DOPE-NMA.

[0108] Figure 6 High-resolution electrospray mass spectra of the amino-structured Toll-like receptor agonists (NH2-CHOL-PMND (NCP, Figure A), NH2-DOPE-PMND (NDP, Figure B), NH2-CHOL-NMA (NCN, Figure C), NH2-DOPE-NMA (NDN, Figure D)) obtained in Example 3;

[0109] Figure 7A DLS diagram of the hypoxia-responsive lipid particle adjuvant platform obtained in Example 4;

[0110] Figure 7B TEM image of the hypoxia-responsive lipid particle adjuvant platform obtained in Example 4;

[0111] Figure 8A This is a DLS diagram of the lipid particle adjuvant platform with an amino structure obtained in Example 5;

[0112] Figure 8B This is a TEM image of the lipid particle adjuvant platform with an amino structure obtained in Example 5;

[0113] Figure 9 The high-resolution electrospray mass spectrum of adjuvant molecules with amino structures released by the hypoxia-responsive lipid particle adjuvant platform obtained in Example 6 is shown in Figure 6. A is the mass spectrum of NCP, B is the mass spectrum of NCN, C is the mass spectrum of NDP, and D is the mass spectrum of NDN.

[0114] Figure 10 The high-resolution mass spectra of drug release from the amino-structured lipid particle adjuvant platform obtained in Example 7 under different acid conditions are shown in Figure 7. A is the mass spectrum of NCP, B is the mass spectrum of NCN, C is the mass spectrum of NDP, and D is the mass spectrum of NDN.

[0115] Figure 11 Example 8 shows the activation effect of two lipid particle adjuvant platforms on primary dendritic cells;

[0116] Figure 12The image shows the fluorescence intensity test results of mouse lymph nodes after subcutaneous injection of free lipid particle adjuvant in Example 9.

[0117] Figure 13 This is a curve showing the inhibition of tumor growth in the hypoxia-responsive lipid particle adjuvant antitumor model of Example 10.

[0118] Figure 14 This is a graph showing the test results of the activation effect of cholesterol-containing lipid particle adjuvant platform with amino groups in the influenza model in Example 11 on lymph node activation.

[0119] Figure 15 The diagram shows the activation effect of lipid particle adjuvant combined with monophospholipid A on antigen-presenting cells.

[0120] Figure 16 Figure showing the activation effect of combined lipid particle adjuvant and aluminum salt adjuvant on antigen-presenting cells. Detailed Implementation

[0121] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0122] Example 1

[0123] This embodiment provides the synthesis of adjuvant linker cholesterol derivatives CP and NCP;

[0124]

[0125] The specific preparation method is as follows:

[0126] (1) Activation of cholesterol hemisuccinate, the specific preparation method is as follows:

[0127] Cholesterol hemisuccinate (100 mg, 102 mmol) and 1,3-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxysuccinimide (16.6 g, 158 mmol) were added to a 50 mL round-bottom flask and 5 mL of dichloromethane were added. The mixture was reacted at room temperature for 24 h. The sample was purified by extraction and impurities were removed by filtration through a quinolone funnel to obtain pure activated cholesterol hemisuccinate.

[0128] (2) Activated cholesterol hemisuccinate (100 mg, 102 mmol) and pipemnidazole (PMND) were added to a 50 mL round-bottom flask, followed by 5 mL of a mixed solvent of dichloromethane and methanol (v / v = 5:1). Then, 2 eq of triethylamine was added, and the reaction was carried out at room temperature for 24 h. The reaction progress was monitored by TLC. The sample was evaporated to dryness by rotation, reconstituted with methanol, centrifuged, concentrated, and dried. The sample was washed with 1 N HCl, and the precipitate obtained by centrifugation was freeze-dried to obtain the pure target product (CP).

[0129] (3) Accurately weigh 5.00 mg of CHOL-PMND compound and dissolve it in 1 mL of methanol solution; dissolve 5.00 mg of sodium dithionite, the reducing agent, in 100 μL of ultrapure water. React in a mixed solvent at a mass ratio of 1:1.

[0130] (4) During the stirring process, sodium dithionite solution was added to the CP compound solution, and stirring was continued for 1 hour. The endpoint was monitored using an enzyme-linked immunosorbent assay (ELISA) reader.

[0131] (5) The final reaction solution was dried by rotary evaporation, methanol was added, the supernatant was collected, the crude product was dried under vacuum and dissolved in ultrapure water, washed 3 times, the precipitate was collected, and the product was freeze-dried to obtain the final product NCP.

[0132] The adjuvant linker cholesterol derivative in Example 1 was detected by high-resolution electrospray mass spectrometry (LCMS-8040, Shimadzu Corporation (Japan)), and the results are as follows: Figure 1 and Figure 2 As shown. Figure 1 High-resolution mass spectrometry showed that the molecular weight of the prepared CP agonist was 752 (theoretical prediction + H), which was completely consistent with the theoretical prediction of 751.51, proving the successful preparation of the product. Figure 2 High-resolution mass spectrometry showed that the molecular weight of the prepared NH2-CHOL-PMND compound was 722.5 (theoretical prediction + H), which was completely consistent with the theoretical prediction of 721.52.

[0133] Figure 3 The 1H NMR spectrum results showed that the hydrogen atom peak shift of the prepared molecular agonist was in perfect agreement with the theoretical prediction, proving that the molecule was successfully prepared; the clean spectrum proved that the product had high purity.

[0134] Example 2

[0135] This embodiment provides the synthesis of the Toll-like receptor molecular agonists DOPE-PMND and DOPE-NMA;

[0136]

[0137] (1) Synthesis of DOPE-COOH structure: 1,2-dioleoyl-SN-glycerol-3-phosphorylethanolamine (DOPE) and succinic anhydride were reacted in DMF at a molar ratio of 1:1, and DIPEA was added to the reaction system.

[0138] (2) Following the DOPE-COOH activation step and subsequent reference to Example 1, the Toll-like receptor agonists DOPE-PMND and DOPE-NMA were prepared.

[0139] The Toll-like receptor agonist in Example 2 was detected by high-resolution electrospray mass spectrometry (LCMS-8040, Shimadzu Corporation (Japan)), and the results are as follows: Figure 4 As shown. Figure 4 The high-resolution mass spectrometry of Figure A shows that Figure 4 The high-resolution mass spectrometry of Figure B shows that the molecular weight of the prepared DOPE-PMND agonist is 1110 (theoretical prediction + H), which is completely consistent with the theoretical prediction of 1108.72, proving the successful preparation of the product; the molecular weight of the prepared DOPE-NMA agonist is 983 (theoretical prediction + H), which is completely consistent with the theoretical prediction of 981.62.

[0140] Figure 5 The 1H NMR spectrum results showed that the hydrogen atom peak shift of the prepared molecular agonist was in perfect agreement with the theoretical prediction, proving that the molecule was successfully prepared; the clean spectrum proved that the product had high purity.

[0141] Example 3

[0142] This embodiment provides an amino-structured Toll-like receptor agonist (NH2-CHOL-PMND).

[0143]

[0144] (1) Accurately weigh 5.00 mg of CHOL-PMND compound and dissolve it in 1 mL of methanol solution; dissolve 5.00 mg of sodium dithionite, the reducing agent, in 100 μL of ultrapure water. React in a mixed solvent at a mass ratio of 1:1.

[0145] (2) During the stirring process, sodium dithionite solution was added to CHOL-PMND compound solution, and stirring was continued for 1 hour. The endpoint was monitored using an enzyme-linked immunosorbent assay (ELISA) reader.

[0146] (3) The final reaction solution was dried by rotary evaporation, methanol was added, the supernatant was collected, the crude product was dried under vacuum and dissolved in ultrapure water, washed 3 times, the precipitate was collected, and the product was freeze-dried.

[0147] Following the experimental method described in Example 3, amino-structured Toll-like receptor agonists (NH2-DOPE-PMND, NH2-CHOL-NMA, and NH2-DOPE-NMA) were prepared.

[0148]

[0149] High-resolution electrospray ionization mass spectrometry was performed on the adjuvant linker cholesterol compound with an amino structure prepared in Example 3, and the results are as follows: Figure 6 As shown, the molecular weight measurements of the prepared adjuvant linker cholesterol compounds containing amino structures are completely consistent with the theoretical predictions.

[0150] Example 4

[0151] This embodiment provides a method for preparing hypoxia-responsive lipid particle adjuvants using adjuvanted molecules containing nitro groups as components:

[0152] The specific preparation method of hypoxia-responsive liposomes is as follows:

[0153] (1) Accurately weigh 10 mg of DPPC and DSPEG-mPEG2000 and prepare a 10 mg / mL solution using anhydrous ethanol. Accurately weigh 2 mg of nitro-structured adjuvanted molecules (CP, CN, DP, or DN) and dissolve them in anhydrous methanol. Prepare a mixed solution of DSPEG-mPEG2000, DPPC, and CP solution in a molar ratio of 1:7:2. Add the solution to a 50 mL round-bottom flask and obtain a thin film using a rotary evaporator. Then add pure water and hydrate by rotating for 40 min at 37°C. An opalescence can be observed in the solution. Sonicate the solution for 2 min (30%, 2 min, 3 s on, 3 s off) and then ultrafilter and centrifuge to obtain hypoxia-responsive liposomes.

[0154] The specific preparation method of hypoxia-responsive lipid nanoparticles is as follows:

[0155] (1) Accurately weigh 5 mg each of DPPC, DSPE-mPEG2000 / SH-DSPE-mPEG2000, and nitro-structured adjuvant molecules (CP, CN, DP, or DN), and dissolve them in anhydrous methanol. Prepare a mixed solution of DSPE-mPEG2000 / SH-DSPE-mPEG2000, DPPC, and CP in a molar ratio of 1:7:2 as the oil phase; use acetate buffer as the aqueous phase, and prepare the solution using a microfluidic instrument (FluidicLab, LNP-S1). The preparation conditions are oil phase:water phase = 1:3. Hypoxia-responsive lipid nanoparticles were obtained.

[0156] (2) Specific method for loading antigen: The two prepared lipid particle adjuvants were co-incubated with the antigen OVA protein for 12 h at 4℃, and the free antigen protein was removed by ultrafiltration and centrifugation. The antigen loading efficiency was calculated to be 65% using the BCA protein kit.

[0157] The four hypoxia-responsive lipid particle adjuvants in Example 4 were analyzed by DLS (Malvern Instruments Ltd., UK, Zetasizer Nano ZS) and transmission electron microscopy (FEI Ltd., USA, Tecnai G2 20S-TWIN). The results are as follows: Figure 7A and Figure 7B As shown. Figure 7A and Figure 7B The results showed that the particle size of the two hypoxia-responsive lipid adjuvants was approximately 100-200 nm, and they exhibited good dispersibility.

[0158] Example 5

[0159] This embodiment provides a method for preparing lipid particle adjuvants using adjuvanted molecules containing amino structures as components:

[0160] The specific preparation method of liposome adjuvant is as follows:

[0161] (1) Accurately weigh 10 mg each of DPPC, DSPE-PEOz, or DSPE-mPEG, and prepare a 10 mg / mL solution using anhydrous ethanol. Accurately weigh 2 mg of an amino-structured adjuvanted molecule (NCP, NCN, NDP, or NDN) and dissolve it in anhydrous methanol and chloroform (v / v = 5:1). Prepare a mixed solution of DSPE-PEOz, DPPC, and the amino-structured adjuvanted molecule solution at a molar ratio of 1:7:2, add it to a 50 mL round-bottom flask, and obtain a thin film using a rotary evaporator. Then add pure water, hydrate and rotate for 40 min at 37°C. An opalescence can be observed in the solution. Sonicate the probe for 2 min (30%, 2 min, 3 s on, 3 s off), and obtain hypoxia-responsive liposomes by ultrafiltration and centrifugation.

[0162] The specific preparation method of lipid nanoparticle adjuvant is as follows:

[0163] (1) Accurately weigh 5 mg each of DPPC, DSPE-mPEG2000 or DSPE-PEOz, and nitro-structured adjuvant molecules (NCP, NCN, NDP, or NDN), and dissolve them in anhydrous ethanol. Prepare a mixed solution of DSPE-PEOz or DSPE-mPEG2000, DPPC, and NCP in a molar ratio of 1:7:2 as the oil phase; use acetate buffer as the aqueous phase, and prepare the solution using a microfluidic instrument. The preparation conditions are oil phase:water phase = 1:3. Hypoxia-responsive lipid nanoparticles are obtained.

[0164] (2) Specific method for loading antigen: The prepared lipid particle adjuvant was co-incubated with the antigen OVA / HA protein for 12 h at 4℃. The free antigen protein was removed by ultrafiltration and centrifugation. The antigen loading efficiency was calculated to be 65-70% using the BCA protein kit.

[0165] The four lipid particle adjuvants with amino groups in Example 5 were analyzed by DLS and transmission electron microscopy, and the results are as follows: Figure 8A and Figure 8B As shown. Figure 8A and Figure 8B The results showed that the particle size of the two hypoxia-responsive lipid adjuvants was approximately 100-200 nm, and they exhibited good dispersibility.

[0166] Example 6

[0167] Hypoxia-responsive release validation of lipid particle adjuvants prepared from adjuvanted molecules with nitro groups.

[0168] The specific experimental method is as follows:

[0169] Prepare a 15 mL centrifuge tube containing PBS buffer, NADPH (2 mg / mL, 0.1 mL), and nitroreductase (1 mg / mL, 10 μL). Bubble the tube with argon gas for 60 min, then stop the argon flow. Add the prepared adjuvanted lipid particles with nitro groups to the 15 mL centrifuge tube. Incubate overnight at 37°C. Separate the sample by HPLC.

[0170] The sample obtained in Example 6 was analyzed by high-resolution electrospray mass spectrometry (Thermo Fisher Scientific, Q-Exactive). The results are as follows: Figure 9 As shown. Adjuvanted molecules containing amino groups were obtained under hypoxic conditions and in the presence of nitroreductase. Figure 9 High-resolution mass spectrometry revealed that lipid particle adjuvants with nitro groups can be reduced to amino-structured derivatives with stronger adjuvant effects under hypoxic conditions and nitro reductase conditions. Figure 9 A and D represent the amino-form adjuvanted molecules obtained after treatment with nitroreductase. (A: NCP mass spectrometry, B: NCN mass spectrometry, C: NDP mass spectrometry, D: NDN mass spectrometry) This demonstrates that lipid particle adjuvants with nitro groups can respond to hypoxia and successfully prepare adjuvant linker cholesterol derivatives with amino groups.

[0171] Example 7

[0172] Validation of acid-responsive release of lipid particle adjuvants prepared from adjuvanted molecules with amino groups.

[0173] Lipid particles with amino groups were added to PBS at different pH values ​​(pH 7.4 and pH 5.5). At different time points, the mixture was ultrafiltered (MW: 3000) and centrifuged. The filtrate was collected, and the released adjuvanted molecules with amino groups were determined by HPLC.

[0174] The release behavior of amino-group adjuvanted molecules carried by this lipid particle adjuvant, such as Figure 10 As shown, at pH 4.5, the adjuvant molecules undergo rapid release, with a cumulative release exceeding 30% within 2 hours, while at pH 7.4, this value is only about 6% (where A: NCP, B: NCN, C: NDP, D: NDN). These results demonstrate that the lipid particle adjuvant of the present invention possesses acid-responsive drug release properties, thereby increasing the rate of drug release in lysosomes.

[0175] Example 8

[0176] The activation effect of eight lipid particle adjuvant platforms on primary dendritic cells was verified.

[0177] The specific preparation method is as follows:

[0178] BALB / c mice were euthanized by cervical dislocation. The femur and tibia were dissected, and the bone marrow was washed with a syringe containing PBS. The cell suspension containing bone marrow was centrifuged at 1200 rpm for 5 min to collect the cell pellet. Red blood cell lysis buffer was added, and the cells were incubated at 4°C for 5 min, then centrifuged at 1200 rpm for 5 min to collect the cell pellet. RPMI-1640 complete culture medium was added, the cells were counted, cytokines were added, and the cells were seeded into culture dishes at a density of 2 × 10⁶ cells / mL as required for the experiment. 6 Cells / well. Add medium on day 3, and change medium halfway on day 6. Collect suspension cells on day 8 of BMDC culture, place them in fresh medium, count them, and seed them in six-well plates. On day 9, when cells adhere to the bottom of the dish, add the cholesterol-modified lipid particle adjuvant provided in Example 4 (this condition requires nitroimidazole reductase and a hypoxic environment) and the cholesterol-modified lipid particle adjuvant provided in Example 5, respectively, and treat for 12 hours. Empty medium, free CP, and free NCP are used as controls. Then, label cells with dendritic cell surface maturation factor antibody, and use flow cytometry (Beckman, Cytoflex LX) to detect the maturation status of dendritic cells after lipid particle adjuvant treatment, obtaining the activation results of antigen-presenting cells by lipid particle adjuvant, as shown in the figure. Figure 11 As shown, the eight lipid particle adjuvants provided by this invention can significantly stimulate the maturation of dendritic cells, thereby further promoting antigen presentation.

[0179] Example 9

[0180] Targeted lymph node verification

[0181] Experimental animals: C57 / BL6 mice, male, 6 weeks old, weighing approximately 20g.

[0182] Blank control group 1: 3 mice were injected subcutaneously into the back with PBS buffer.

[0183] Experimental group 1: 12 mice were injected subcutaneously into the back of their bodies with the four lipid particle adjuvants containing nitro adjuvant molecules provided in Example 4.

[0184] Experimental group 2: 12 mice were subcutaneously injected in the back with the four lipid particle adjuvants containing amino adjuvant molecules provided in Example 5.

[0185] The eight molecules prepared above were linked with fluorescent molecules to prepare cholesterol-modified lipid particle adjuvants according to the methods provided in Examples 4 and 5. A subcutaneous injection of 50 μg per mouse was administered via the tail. Lymph nodes were harvested 12 hours later, and fluorescence signals from the injected lymph nodes were captured using small animal imaging to determine the lymph node targeting efficiency of the cholesterol-modified lipid particle adjuvant. Figure 12 The results showed that all eight lipid particles exhibited strong fluorescence signals at the drainage lymph nodes, indicating that these eight lipid particle adjuvants have a strong targeting ability for lymph nodes.

[0186] Example 10

[0187] Application of lipid particle adjuvant platform in antitumor therapy

[0188] 1×10⁻⁶ mice were subcutaneously injected into the left posterior dorsal region of C57BL / 6 mice. 6 One B16-OVA cell was used to treat mouse tumors reaching 80 mm in size. 3 Mice were randomly divided into 6 groups of 8 mice each: Group 1, saline; Group 2, CP lipid adjuvant + OVA; Group 3, CN lipid adjuvant + OVA; Group 4, DP lipid adjuvant + OVA; Group 5, DN lipid adjuvant + OVA; Group 6, NCP lipid adjuvant + OVA; Group 7, NCN lipid adjuvant + OVA; Group 8, NDP lipid adjuvant + OVA; Group 9, NDN lipid adjuvant + OVA. Mice in all 9 groups were subcutaneously administered 0.5 mg / kg of the drug every 7 days for a total of 3 administrations. Tumor volume was measured daily using calipers and calculated using the formula: Volume = Length × Width × Width / 2. A graph was plotted with tumor volume on the vertical axis and the number of experimental days on the horizontal axis to obtain the in vivo tumor inhibition experiment results of the lipid adjuvant, as shown below. Figure 13As shown, the lipid particle adjuvant + OVA provided in Examples 4 and 5 effectively inhibited tumor growth. The second and sixth groups showed more significant inhibitory effects, indicating that the lipid particle adjuvant of the present invention has hypoxia and acid responses, effectively releases amino molecules with more obvious activation effects, effectively activates immune cells, and thus effectively inhibits tumor growth.

[0189] Example 13: Lymph node activation effect of a cholesterol-modified lipid particle adjuvant platform with amino groups.

[0190] Beyond its application in tumor treatment platforms, cholesterol-modified lipid particle adjuvant platforms with amino groups have a wider range of applications. Therefore, this cholesterol-modified lipid particle adjuvant was loaded with HA protein (H1N1 viral protein) to verify its cholesterol-activating effect.

[0191] Six C57BL / 6 mice (6-8 weeks old) were randomly selected and injected subcutaneously into the back of the neck into each experimental group (Group 1: saline; Group 2: NCP lipid adjuvant + HA; Group 3: NCN lipid adjuvant + HA; Group 4: NDP lipid adjuvant + HA; Group 5: NDN lipid adjuvant + HA). For H1N1 vaccine immunization, mice were immunized every 7 days for a total of 2 doses. Seven days after the last immunization, the mice were sacrificed, and inguinal lymph node cells were collected, processed into single-cell suspensions, and the maturation status of dendritic cells was analyzed by flow cytometry.

[0192] Example 11

[0193] Verification of the activation effect of NCP lipid particle adjuvant in combination with other molecular adjuvants on primary dendritic cells.

[0194] Monophosphoryl lipid A (MPLA) molecules were used as the oil phase to prepare a combined adjuvant plate according to the method in Example 5. Bone marrow-derived BMDCs were cultured to day 8, and suspension cells were collected, placed in fresh culture medium, counted, and seeded into six-well plates. On day 9, cells adhered to the bottom of the plate. NCP lipid particle adjuvant, NCP lipid particle adjuvant + MPL, and commercially available AS01 adjuvant were added, respectively. Treatment lasted 12 hours. Empty culture medium was used as a control. Cell maturation status was then assessed according to the method in Example 8.

[0195] The activation results of lipid particle adjuvant on antigen-presenting cells were obtained, as shown in the figure. Figure 15 As shown, the NCP lipid particle adjuvant + MPL platform provided by this invention has a maturation effect on dendritic cells comparable to that of the commercially available AS01 adjuvant.

[0196] Example 12

[0197] Verification of the activation effect of NCP lipid particle adjuvant combined with immune metals on primary dendritic cells.

[0198] A particulate adjuvant platform was prepared according to the method in Example 5 and mixed with an aluminum salt adjuvant (aluminum phosphate). BMDCs extracted from bone marrow were cultured until day 8, at which point suspension cells were collected, placed in fresh culture medium, counted, and seeded into six-well plates. On day 9, cells adhered to the bottom of the plate. NCP lipid particle adjuvant, aluminum adjuvant, NCP lipid particle adjuvant + Al, and commercially available ASO4 adjuvant were added, respectively. Treatment lasted 12 hours. Empty culture medium was used as a control. Cell maturation status was then assessed according to the method in Example 8.

[0199] The activation results of lipid particle adjuvant on antigen-presenting cells were obtained, as shown in the figure. Figure 16 As shown, the NCP lipid particle adjuvant + Al platform provided by this invention has a significantly better effect than the aluminum adjuvant group, and has a maturation effect on dendritic cells comparable to that of commercially available ASO4 adjuvant.

[0200] The applicant declares that this invention illustrates the cholesterol-modified lipid particle adjuvant, its preparation method, and its application through the above embodiments. However, this invention is not limited to the above embodiments, meaning that this invention does not necessarily rely on the above embodiments for implementation. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of the raw materials used in this invention, additions of auxiliary components, and selection of specific methods all fall within the protection and disclosure scope of this invention.

Claims

1. A lipid particle adjuvant, characterized in that, The lipid particle adjuvant comprises an adjuvanted molecule and PEG lipids and other lipid components, wherein the adjuvanted molecule has a structure as shown in Formula 1: Wherein, R1 is a nitrogen-containing six-membered heterocycle or a C1-C3 straight-chain alkane; R2 is a C8-C22 unsaturated chain hydrocarbon, a C8-C22 alkane, a cholesterol hemisuccinate, or 1,2-dioleoyl-SN-glycerol-3-phosphoethanolamine; and R3 is a nitro or amino group.

2. The lipid particle adjuvant according to claim 1, characterized in that, R1 is a nitrogen-containing hexacyclic heterocycle or methylene group; Preferably, the adjuvanted molecule has any one of the structures shown in Formula 2-1 to Formula 2-4: R2 is a cholesterol hemisuccinate group or a 1,2-dioleoyl-SN-glycerol-3-phosphoethanolamine group; Preferably, R1 is a nitrogen-containing six-membered heterocycle; Preferably, R3 is an amino group.

3. The lipid particle adjuvant according to claim 1 or 2, characterized in that, The adjuvanting molecule is any one of the following compounds: More preferably, the adjuvanting molecule is a compound of the following type: Preferably, the adjuvanting molecule is any one of the following compounds:

4. The lipid particle adjuvant according to any one of claims 1-3, characterized in that, The method for preparing the adjuvanted molecule includes the following steps: The method for preparing the adjuvanted molecule includes the following steps: (1) The compound of formula 2 was reacted with N-hydroxysuccinimide to obtain the acyl oxide intermediate shown in formula 3; (2) The acyl oxide intermediate of formula 3 obtained in step (1) is reacted with the nitroimidazolium compound containing a primary amine of formula 4 to obtain the adjuvanted molecule of formula 5, where R3 is a nitro group; the reaction formula is as follows: (3) The adjuvanted molecule with nitro group R3 shown in Formula 5 reacts with a reducing agent to obtain the adjuvanted molecule with amino group R3 shown in Formula 6. The reaction formula is as follows: The constraints for R1 and R2 are the same as in Equation 1; Preferably, the compound of Formula 2 includes 1,2-dioleoyl-SN-glycerol-3-phosphorylethanolamine with a carboxyl group, which is prepared by reacting 1,2-dioleoyl-SN-glycerol-3-phosphorylethanolamine with succinic anhydride; Preferably, the reaction of 1,2-dioleoyl-SN-glycerol-3-phosphoethanolamine and succinic anhydride is carried out in an organic solvent, preferably dichloromethane; Preferably, the molar ratio of 1,2-dioleoyl-SN-glycerol-3-phosphoethanolamine to succinic anhydride is 1:1 to 1:1.5; Preferably, the reaction of 1,2-dioleoyl-SN-glycerol-3-phosphorylethanolamine and succinic anhydride is carried out in the presence of a catalyst; Preferably, the catalyst is selected from N,N-diisopropylethylamine; Preferably, the molar ratio of the catalyst to 1,2-dioleoyl-SN-glycerol-3-phosphoethanolamine is (0.5-4):1; Preferably, the reaction temperature of 1,2-dioleoyl-SN-glycerol-3-phosphorylethanolamine (DOPE) and succinic anhydride is 0-37°C, and the reaction time is 5-48 hours. Preferably, the molar ratio of the compound of formula 2 in step (1) to N-hydroxysuccinimide is (0.5-2):1; Preferably, the reaction in step (1) is carried out in the presence of a condensing agent; Preferably, the condensing agent is a carbodiimide condensing agent; Preferably, the carbodiimide condensing agent is selected from 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide; Preferably, the molar ratio of the compound of formula 2 to the condensing agent in step (1) is 1:1.2-2; Preferably, the reaction described in step (1) is carried out in the presence of a solvent; Preferably, the solvent includes dichloromethane; Preferably, the temperature of the reaction in step (1) is 0–25°C and the reaction time is 1–3 hours; Preferably, after the reaction structure described in step (1), post-processing is performed, including extraction and drying; Preferably, the solvent for extraction includes dichloromethane and water; Preferably, the volume ratio of dichloromethane to water in the extraction solvent is (3-5):1; Preferably, the extraction is performed 3-5 times; Preferably, the reaction in step (2) is carried out in the presence of a solvent; Preferably, the mixed solvent comprises dichloromethane and methanol; Preferably, the volume ratio of dichloromethane to methanol in the mixed solvent is (5-15):1; Preferably, the molar ratio of the acyl oxide intermediate shown in Formula 3 in step (2) to the nitroimidazole compound containing a primary amine shown in Formula 4 is preferably 1:(1-2); Preferably, the reaction in step (2) is carried out in the presence of an acid-binding agent; Preferably, the acid-binding agent is triethylamine; Preferably, the molar ratio of the acid-binding agent to the nitroimidazole compound containing a primary amine is (0.5-3):1; Preferably, the reaction time in step (2) is 12 to 24 hours; Preferably, after the reaction in step (2) is completed, purification is performed. The purification method includes: drying the final reaction solution, adding methanol, collecting the supernatant, drying the crude product under vacuum, dissolving it in hydrochloric acid aqueous solution, collecting the precipitate, washing the pH value back to neutral with saturated saline, and freeze-drying the product. Preferably, the concentration of the hydrochloric acid aqueous solution is 1 mol / mL; Preferably, the purification process using hydrochloric acid aqueous solution is performed 3-5 times; Preferably, the reducing agent in step (3) is selected from a combination of sodium dithionite, hydrazine hydrate and Raney nickel, a combination of iron powder and ammonium chloride, or a reducing metal chloride; Preferably, the reaction in step (3) is carried out in a solvent, which includes methanol and water; Preferably, the volume ratio of methanol to water in the solvent is (5-20):1; Preferably, in step (3), the mass ratio of the adjuvant molecule with a nitro group R3 shown in Formula 5 to the reducing agent is 1 to (3:1); Preferably, the reaction temperature in step (3) is 0-35°C and the reaction time is 0.5-3 hours; Preferably, after the reaction in step (3) is completed, purification is performed to obtain the product; Preferably, the purification process includes: drying the final reaction solution, adding methanol, collecting the supernatant, vacuum drying the crude product, dissolving it in an ultrapure aqueous solution, washing it three times, collecting the precipitate, and freeze-drying the product.

5. The lipid particle adjuvant according to any one of claims 1-4, characterized in that, The PEG lipids include distearate phosphatidylethanolamine-polyethylene glycol, 1,2-dimyristoyl-sn-glycero-3-phosphate ethanolamine-N-[methoxy(polyethylene glycol)], 1,2-dipalmitoyl-sn-glycero-3-phosphate ethanolamine-N-methoxy(polyethylene glycol), 1,2-dioleoyl-sn-glycero-3-phosphate ethanolamine-N-[methoxy(polyethylene glycol)], and any one or a combination of at least two of the above phospholipid polyethylene glycols containing a thiol group; Preferably, the other lipid components include any one or a combination of at least two of dipalmitoylphosphatidylcholine, phospholipid poly(2-ethyl-2-oxazoline), or ionizable lipids; Preferably, the molar ratio of the PEG lipid to the adjuvanted molecule is 1:1 to 1:6; Preferably, the molar ratio of the other lipid components to the PEG lipid is 1:1 to 1:

8.

6. The method for preparing the lipid particle adjuvant according to any one of claims 1-5, characterized in that, The preparation method includes the following steps: The lipid particle adjuvant is obtained by mixing adjuvanted molecular derivatives, PEG lipids, and other lipids.

7. The preparation method according to claim 6, characterized in that, The preparation method specifically includes the following steps: dissolving the adjuvanted molecules in a first solvent to obtain solution A; mixing PEG lipids and other lipid components with a second solvent to obtain solution B; mixing solution A and solution B; removing the solvent by rotary evaporation; and adding a third solvent to re-dissolve to obtain the cholesterol-modified lipid particle adjuvant.

8. The preparation method according to claim 7, characterized in that, The first solvent is selected from a mixture of methanol and chloroform; Preferably, the volume ratio of methanol to chloroform is 20-5:1; Preferably, the concentration of the adjuvanting molecule in the first solvent is 0.5-4 mg / mL; Preferably, the second solvent includes any one or a combination of methanol, ethanol, or chloroform; Preferably, the concentrations of the PEG lipid and other lipid components in the second solvent are 5-20 mg / mL, respectively; Preferably, the resolution method includes any one or a combination of at least two of stirring, vortexing, or ultrasound; Preferably, the third solvent includes any one or a combination of at least two of water, phosphate buffer, or physiological saline; Preferably, the resolution temperature is 30-45℃; Preferably, the reconstitution time is 10-60 min; Preferably, the process after reconstitution further includes probe ultrasound and centrifugation.

9. An adjuvant composition, characterized in that, The adjuvant composition includes the lipid particle adjuvant and other immunomodulatory adjuvants as described above; Preferably, the other immunomodulatory adjuvants include organic molecular adjuvants and / or metal salt adjuvants; Preferably, the organic molecular adjuvant includes any one or a combination of at least two of monophosphoryl lipid A, 3m052, rethimod, imiquimod, or cytokine adjuvants; Preferably, the metal in the metal salt adjuvant is selected from any one or a combination of at least two of aluminum, manganese, zinc, or iron.

10. The use of the lipid particle adjuvant or adjuvant composition according to any one of claims 1-5 in a vaccine formulation.

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