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CN-121987773-A - Lipid particle adjuvant and preparation method and application thereof

CN121987773ACN 121987773 ACN121987773 ACN 121987773ACN-121987773-A

Abstract

The invention provides a lipid particle adjuvant and a preparation method and application thereof, wherein the lipid particle adjuvant comprises an adjuvant molecule, PEG lipid and other lipid components, the adjuvant molecule has a structure shown in a formula 1, and the lipid particle adjuvant is stable in structure, not easy to decompose, has a tumor immune effect with high specificity, has a strong lymph node targeting capability, and can fully meet the requirements of high efficiency and safety of immune adjuvants and vaccines.

Inventors

  • LIU JING
  • ZHANG XIAOYU
  • CHEN CHUNYING
  • ZHAO YULIANG

Assignees

  • 国家纳米科学中心

Dates

Publication Date
20260508
Application Date
20241104

Claims (10)

  1. 1. A lipid particle adjuvant comprising an adjuvanting molecule and a PEG lipid and other lipid component, the adjuvanting molecule having a structure according to formula 1: wherein R 1 is a nitrogen-containing six-membered heterocyclic ring or C1-C3 straight-chain alkane, R 2 is a C8-C22 unsaturated chain alkyl, a C8-C22 alkyl, cholesterol hemisuccinate or 1, 2-dioleoyl-SN-glycero-3-phosphorylethanolamine, and R3 is nitro or amino.
  2. 2. The lipid particle adjuvant of claim 1, wherein R1 is a nitrogen-containing hexacyclic ring or methylene; preferably, the adjuvanted molecule has any one of the structures shown in the following formulas 2-1 to 2-4: Wherein R 2 is a cholesterol hemisuccinate group or a 1, 2-dioleoyl-SN-glycerol-3-phosphorylethanolamine group; Preferably, the R 1 is a nitrogen-containing six-membered heterocyclic ring; Preferably, R 3 is amino.
  3. 3. A lipid particle adjuvant according to claim 1 or 2, wherein the adjuvanting molecule is any one of the following compounds: Further preferably, the adjuvanting molecule is a compound as follows: preferably, the adjuvanting molecule is any one of the following compounds:
  4. 4. A lipid particle adjuvant according to any one of claims 1-3, wherein the method of preparing the adjuvanted molecule comprises the steps of: The preparation method of the adjuvanted molecule comprises the following steps: (1) Reacting a compound of formula 2 with N-hydroxysuccinimide to obtain an acyloxy intermediate shown in formula 3; (2) Reacting the acyl oxide intermediate shown in the formula 3 obtained in the step (1) with a nitroimidazole compound containing primary amine shown in the formula 4 to obtain the adjuvant molecule with R3 as nitro shown in the formula 5, wherein the reaction formula is as follows: (3) Reacting an adjuvanted molecule of formula 5 wherein R3 is nitro with a reducing agent to obtain the adjuvanted molecule of formula 6 wherein R3 is amino, the reaction formula is as follows: Wherein R 1 and R 2 are as defined in formula 1; Preferably, the compound of formula 2 comprises 1, 2-dioleoyl-SN-glycerol-3-phosphorylethanolamine with carboxyl, 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-phosphorylethanolamine and succinic anhydride is carried out in an organic solvent, preferably dichloromethane; Preferably, the molar ratio of the 1, 2-dioleoyl-SN-glycerol-3-phosphorylethanolamine to the succinic anhydride is 1:1-1:1.5; 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 the group consisting of N, N-diisopropylethylamine; Preferably, the mol ratio of the catalyst to the 1, 2-dioleoyl-SN-glycerol-3-phosphorylethanolamine is (0.5-4): 1; Preferably, the reaction temperature of the 1, 2-dioleoyl-SN-glycerol-3-phosphorylethanolamine (DOPE) and succinic anhydride is 0-37 ℃ and the reaction time is 5-48 hours; Preferably, the molar ratio of the compound of formula 2 to N-hydroxysuccinimide in the step (1) is (0.5-2): 1; Preferably, the reaction of 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 of step (1) is carried out in the presence of a solvent; Preferably, the solvent comprises dichloromethane; preferably, the temperature of the reaction in the step (1) is 0-25 ℃, and the reaction time is 1-3 hours; preferably, after the reaction structure in step (1), a post-treatment is performed, wherein the post-treatment comprises extraction and drying; preferably, the extracted solvent comprises dichloromethane and water; Preferably, the volume ratio of dichloromethane to water in the extracted solvent is (3-5) 1; preferably, the number of extractions is 3-5; preferably, the reaction of 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 the formula 3 in the step (2) to the nitroimidazole compound containing primary amine shown in the 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 mol ratio of the acid binding agent to the nitroimidazole compound containing primary amine is (0.5-3): 1; Preferably, the reaction time in the step (2) is 12-24 hours; Preferably, after the reaction in the step (2) is finished, the purification treatment is carried out, wherein the purification method comprises the steps of drying a final reaction solution, adding methanol, collecting supernatant, vacuum drying a crude product, dissolving the crude product in a hydrochloric acid aqueous solution, collecting precipitated sediment, washing the pH value back to neutrality by using saturated saline water, and freeze-drying the product; preferably, the concentration of the aqueous hydrochloric acid solution is preferably 1moL/mL; preferably, the purification times with aqueous hydrochloric acid is 3-5 times; preferably, the reducing agent of step (3) is selected from sodium hydrosulfite, a combination of hydrazine hydrate and raney nickel, a combination of iron powder and ammonium chloride or a reducing metal chloride; preferably, the reaction of step (3) is carried out in a solvent comprising methanol and water; Preferably, the volume ratio of the methanol to the water in the solvent is (5-20): 1; Preferably, in the step (3), the mass ratio of the adjuvant molecule with the R 3 being nitro shown in the formula 5 to the reducing agent is 1 to (3:1); Preferably, the temperature of the reaction in the step (3) is 0-35 ℃ and the reaction time is 0.5-3 hours; preferably, after the reaction in the step (3) is finished, purifying to obtain a product; preferably, the purification treatment comprises drying the final reaction solution, adding methanol, collecting supernatant, vacuum drying the crude product, dissolving in ultrapure water solution, washing for 3 times, collecting precipitated precipitate, and freeze drying the product.
  5. 5. The lipid particle adjuvant according to any one of claims 1-4, wherein the PEG lipid comprises distearoyl phosphatidylethanolamine-polyethylene glycol, 1, 2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N- [ methoxy (polyethylene glycol) ], 1, 2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-methoxy (polyethylene glycol), 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine-N- [ methoxy (polyethylene glycol), and thiol-containing each of the above-described phospholipid polyethylene glycols, or a combination of at least two thereof; Preferably, the other lipid component comprises any one or a combination of at least two of dipalmitoyl phosphatidylcholine, a phospholipid poly (2-ethyl-2-oxazoline) or an ionizable lipid; Preferably, the molar ratio of the PEG lipid to the adjuvanted molecule is 1:1-1:6; Preferably, the molar ratio of the other lipid component to the PEG lipid is 1:1-1:8.
  6. 6. A method of preparing a lipid particle adjuvant according to any one of claims 1 to 5, comprising the steps of: and mixing the adjuvant molecule derivative, PEG lipid and other lipids to obtain the lipid particle adjuvant.
  7. 7. The method according to claim 6, wherein the method comprises dissolving the adjuvanted molecule in a first solvent to obtain solution A, mixing the PEG lipid and other lipid components with a second solvent to obtain solution B, mixing the solution A with the solution B, removing the solvent by rotary evaporation, and adding a third solvent for re-dissolution to obtain the cholesterol-modified lipid particle adjuvant.
  8. 8. The method according to claim 7, wherein the first solvent is selected from the group consisting of a mixed solvent of methanol and chloroform; Preferably, the volume ratio of the methanol to the chloroform is 20-5:1; Preferably, the concentration of the adjuvanted molecule in the first solvent is 0.5-4mg/mL; preferably, the second solvent comprises any one or a combination of methanol, ethanol or chloroform; preferably, the concentration of the PEG lipid and other lipid components in the second solvent is 5-20mg/mL, respectively; Preferably, the mode of reconstitution includes any one or a combination of at least two of stirring, vortexing or ultrasound; preferably, the third solvent comprises any one or a combination of at least two of water, phosphate buffer or physiological saline; preferably, the temperature of the reconstitution is 30-45 ℃; preferably, the redissolution time is 10-60min; preferably, the method further comprises probe ultrasonic and centrifugation after the reconstitution.
  9. 9. An adjuvant composition comprising a lipid particle adjuvant as described above and a further immunomodulatory adjuvant; preferably, the other immunomodulatory adjuvant comprises an organic molecule adjuvant and/or a metal salt adjuvant; preferably, the organic molecular adjuvant comprises any one or a combination of at least two of monophosphoryl lipid a, 3m052, raschimod, imiquimod or cytokine adjuvant; 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. 10. Use of a lipid particle adjuvant or adjuvant composition according to any one of claims 1-5 in a vaccine formulation.

Description

Lipid particle adjuvant and preparation method and application thereof Technical Field The invention belongs to the technical field of nano materials, and relates to a lipid particle adjuvant, a preparation method and application thereof. Background Most of novel adjuvants belong to small molecules, have the problems of entering blood to produce inflammatory reaction of a system, being easy to be degraded by in vivo enzymes, being difficult to enter cells and the like, and seriously influence the practical application. For vaccine delivery, the main challenge is to achieve direct efficient lymph node enrichment of antigens, thereby stimulating the onset and duration of adaptive immune responses with immune cells and cytokines in the lymph nodes. Vaccine adjuvants also often require immune cells in the lymph nodes to exert the greatest immune activating function. The immune adjuvant small molecules are not only difficult to enter lymph nodes after traditional interstitial injection, but also can be distributed to the whole body to cause systemic inflammatory response. The nano delivery technology is expected to solve the problems of the novel adjuvant, can improve the immunity stimulating capability of the adjuvant, and plays an important role in vaccine application. One common drawback of TLR agonists is the reactivity, and in recent years there has been extensive research attempting to overcome these side effects. Encapsulation of TLR7/8 agonists in cationic DOEPC-based liposome formulation nanoparticles, or covalent attachment of these small molecules to hyperbranched polymers, can avoid their detrimental systemic reactions while maintaining their impact on humoral immunity. The lymph node targeted delivery and responsive release of the adjuvant is achieved by utilizing the easily remodelled nature of the nanotechnology. Thus, further research into lipid particle adjuvant platforms to find simpler, efficient delivery systems remains of great interest in the art. Disclosure of Invention Aiming at the defects of the prior art, the invention aims to provide a lipid particle adjuvant, and a preparation method and application thereof. The lipid particle adjuvant disclosed by the invention has the advantages of stable structure, difficulty in decomposition, high specificity of tumor immunity effect, stronger lymph node targeting capability and capability of fully meeting the requirements of high efficiency and safety of an immune adjuvant and a vaccine. In order to achieve the aim of the invention, the invention adopts the following technical scheme: In one aspect, the present invention provides a 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: Wherein R 1 is a nitrogen-containing six-membered heterocycle or C1-C3 (e.g., C1, C2 or C3) straight chain alkane, R 2 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) alkyl group, cholesterol hemisuccinate or 1, 2-dioleoyl-SN-glycero-3-phosphorylethanolamine, and R 3 is nitro or amino. The lipid particle adjuvant provided by the invention has the advantages that the adjuvant molecules are used as important components of liposome or lipid nano particles, so that the lipid particle adjuvant has a stable platform structure and is not easy to decompose. The lipid particle adjuvant platform can exert high-specificity tumor immunity effect on tumor local part due to nitroimidazole. In addition, the lipid particle adjuvant platform can have stronger immune effect due to cholesterol derivative with an amino imidazole structure, and is not only applied to the field of tumor treatment. Meanwhile, the method has stronger lymph node targeting capability, and can fully meet the requirements of high efficiency and safety of immunoadjuvants and vaccines. In the present invention, the nitrogen-containing six-membered heterocyclic group may be Preferably, R 1 is a nitrogen-containing hexacyclic ring or methylene. Preferably, the adjuvanted molecule has any one of the structures shown in the following formulas 2-1 to 2-4: Wherein R 2 is Cholesterol Hemisuccinate (CHOL) or 1, 2-dioleoyl-SN-glycero-3-phosphoethanolamine (DOPE). Preferably, the R 1 is a nitrogen-containing six-membered heterocyclic ring; Preferably, R 3 is amino. Preferably, the adjuvanting molecule is any one of the following compounds: Further preferably, the adjuvanting molecule is a compound as follows: preferably, the adjuvanting molecule is any one of the following compounds: in the invention, the preparation method of the adjuvanted molecule comprises the following steps: (1) Reacting a compound of formula 2 with N-hydroxysuccinimide to obtain an acyloxy intermediate shown in formula 3; (2) Reacting the acyl oxide intermediate shown in the formula 3 obtained in the step (1) with a nitroimidazo