A method for the preparation of a cationic liposome adjuvant and a veterinary vaccine

Abstract

The application discloses a preparation method of a cationic liposome adjuvant and a veterinary vaccine, relates to the technical field of veterinary vaccine adjuvants and preparation and application thereof. The preparation method of the cationic liposome adjuvant comprises the following steps: mixing phospholipid, a solid sterol compound, an ionizable cationic lipid and an organic solvent to obtain a lipid mixed solution; wherein the ionizable cationic lipid comprises 3-(aliphatic hydrocarbon amino)-1,2-propanediol; removing the organic solvent in the lipid mixed solution to form a lipid film; adding a buffer solution to the lipid film under a heating condition to perform hydration treatment, so as to obtain a crude liposome dispersion liquid; and performing high-pressure microjet treatment on the crude liposome dispersion liquid. By introducing 3-(aliphatic hydrocarbon amino)-1,2-propanediol, the surface positive electricity and the potential stability of the liposome are improved, the rigidity of the liposome structure is enhanced, the prepared liposome has uniform particle size distribution, low PDI, good storage stability and good biological safety.

Description

Technical Field

[0001] This invention relates to the field of veterinary vaccine adjuvants and their preparation and application technology, and particularly to a method for preparing a cationic liposome adjuvant and a veterinary vaccine. Background Technology

[0002] Vaccines are an effective means of preventing and controlling infectious diseases, and vaccine adjuvants play a crucial role in enhancing antigen immunogenicity, regulating the type of immune response, and reducing the amount of antigen used. Traditional adjuvants, such as aluminum salt adjuvants, have good safety profiles, but they mainly induce humoral immunity, resulting in a single type of response. Furthermore, they have limited delivery capabilities for protein, peptide, and nucleic acid antigens, making it difficult to meet the development needs of novel vaccines.

[0003] Liposomes are a class of nanocarriers composed of a phospholipid bilayer. Due to their excellent biocompatibility, structural designability, and superior antigen delivery capabilities, they are widely used in drug delivery and vaccine adjuvants. Compared to neutral or anionic liposomes, cationic liposomes, with their positively charged surface, can interact with negatively charged antigen molecules or the surface of antigen-presenting cells through electrostatic interactions, thereby improving antigen uptake efficiency and immune response levels.

[0004] However, existing cationic liposome adjuvants still generally suffer from the following technical problems: (1) cationic lipid molecules often lead to instability in the liposome system, easily causing particle size increase, aggregation and sedimentation, or zeta potential decay, thereby affecting the immunization effect and application stability; (2) excessively high positive charge density can easily cause non-specific damage to the cell membrane, leading to cytotoxicity and inflammatory response, limiting its application in vaccine adjuvants. Therefore, developing a novel cationic liposome vaccine adjuvant that ensures cationic properties while possessing high structural stability, low toxicity, and good immune enhancement effect is a key technical problem that urgently needs to be solved in this field. Summary of the Invention

[0005] The main objective of this invention is to propose a method for preparing cationic liposome adjuvants and a veterinary vaccine, aiming to solve the problems of poor stability, high toxicity, and poor immune enhancement effect of cationic liposome vaccine adjuvants.

[0006] To achieve the above objectives, this invention proposes a method for preparing a cationic liposome adjuvant, comprising the following steps: S10. Phospholipids, sterols, ionizable cationic lipids and organic solvents are mixed to obtain a lipid mixture solution; wherein the ionizable cationic lipids include 3-(aliphatic alkylamino)-1,2-propanediol. S20. Remove the organic solvent from the lipid mixture to form a lipid film; S30. Under heating conditions, a buffer solution is added to the lipid film for hydration treatment to obtain a crude liposome dispersion. S40. The crude liposome dispersion is processed by high-pressure microfluidic jet to obtain the cationic liposome adjuvant.

[0007] In one embodiment, in step S10, the aliphatic hydrocarbon group of the 3-(aliphatic alkylamino)-1,2-propanediol is a C12–C18 straight-chain alkyl or straight-chain alkenyl group.

[0008] In one embodiment, the aliphatic hydrocarbon group of the 3-(aliphatic alkylamino)-1,2-propanediol is hexadecyl or cis-9-octadecenyl.

[0009] In one embodiment, in step S10: The phospholipids include at least one of lecithin, soybean phospholipids, egg yolk phospholipids, and synthetic phospholipids; and / or, The sterol compounds include at least one of cholesterol, phytosterols, and cholanols; and / or, The mass ratio of the phospholipid to the sterol compound is (2~6):1; and / or, The amount of 3-(aliphatic amino)-1,2-propanediol used is 10% to 30% of the amount of phospholipid; and / or, The organic solvent includes at least one of ethanol, methanol, isopropanol, and chloroform.

[0010] In one embodiment, in step S20, the organic solvent in the lipid mixture is removed by rotary evaporation.

[0011] In one embodiment, in step S30: The heating conditions include heating at 35~40°C; and / or, Hydration treatment is performed under ultrasonic conditions, wherein the ultrasonic power is 350~400W and the ultrasonic time is 10~20 min; and / or, The buffer solution includes a PBS buffer solution with a pH range of 7.2 to 7.4.

[0012] In one embodiment, in step S40: The cationic liposome adjuvant has a particle size of 90-150 nm; and / or, The polydispersity index (PDI) of the cationic liposome adjuvant is ≤0.40; and / or, The cationic liposome adjuvant has a positive Zeta potential value and is ≤25 mV; and / or, The parameters for processing via high-pressure microjets include: extrusion twice at 10,000 to 15,000 psi.

[0013] This invention proposes a veterinary vaccine comprising a cationic liposome adjuvant, wherein the cationic liposome adjuvant is prepared by the cationic liposome adjuvant preparation method described in the foregoing technical solution.

[0014] In one embodiment, the veterinary vaccine further includes an antigen.

[0015] In one embodiment, the antigen comprises porcine circovirus CP08 strain antigen.

[0016] The present invention provides a method for preparing a cationic liposome adjuvant containing 3-(aliphatic amino)-1,2-propanediol. By introducing 3-(aliphatic amino)-1,2-propanediol as a key cationic lipid component, its unique molecular structure can maintain the stability of the liposome surface and give it a high positive charge density, significantly increasing the absolute value of the Zeta potential on the liposome surface, generating stronger electrostatic repulsion between liposome particles, thereby improving the dispersion stability of the system. At the same time, the secondary amine and hydroxyl groups in 3-(aliphatic amino)-1,2-propanediol form a hydrogen bond network with the phosphate groups in the lecithin molecule, forming stronger intermolecular forces, thereby enhancing the order and rigidity of the liposome bilayer at the molecular level, which is beneficial for the stable encapsulation of antigens. Furthermore, through the above-mentioned electrostatic interactions and hydrogen bond network, excessive positive charge can be structurally dispersed and shielded, thereby reducing the non-specific destructive effect of liposomes on cell membranes and reducing cytotoxicity. In addition, the diol moiety can improve the hydrophilicity of the compound, enhance hydration, and improve the overall water dispersibility of the liposomes. The cationic liposome adjuvant prepared by this invention has a uniform particle size distribution, low PDI, and good storage stability. While maintaining cationic properties, it can effectively reduce cytotoxicity and can be used to solve the problems of poor stability, high toxicity, and poor immune enhancement effect of existing cationic liposome vaccine adjuvants. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the structures shown in these drawings without creative effort.

[0018] Figure 1 TEM images of the cationic liposome adjuvants provided in Examples 1 and 2 of this invention; Figure 2The graph shows the liposome membrane fluidity test results of the cationic liposome adjuvants provided in Examples 1 and 2 of the present invention based on the DPH fluorescent probe method. Figure 3 Figure 1 shows the hydrophobicity test results of the cationic liposome adjuvants provided in Examples 1 and 2 of this invention based on the ANS fluorescent probe method for liposome membrane. Figure 4 The cytotoxicity test results of the cationic liposome adjuvants provided in Examples 1 and 2 of this invention are shown in the figure. Figure 5 The image shows the antibody response results induced by the veterinary vaccine prepared using the cationic liposome adjuvants provided in Examples 1 and 2 of this invention.

[0019] The realization of the objective, functional features and advantages of the present invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0020] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0021] It should be noted that if the embodiments of the present invention involve directional indicators (such as up, down, left, right, front, back, etc.), the directional indicators are only used to explain the relative positional relationship and movement of the components in a specific posture. If the specific posture changes, the directional indicators will also change accordingly.

[0022] Furthermore, if the embodiments of this invention involve descriptions such as "first" or "second," these descriptions are for descriptive purposes only and should not be construed as indicating or implying their relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined with "first" or "second" may explicitly or implicitly include at least one of those features. Additionally, the use of "and / or" or "and / or" throughout the text includes three parallel solutions. For example, "A and / or B" includes solution A, solution B, or a solution where both A and B are satisfied simultaneously. Furthermore, the technical solutions of the various embodiments can be combined with each other, but this must be based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or impossible to implement, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection claimed by this invention.

[0023] Liposomes are a class of nanocarriers composed of phospholipid bilayers. Due to their good biocompatibility, structural designability, and excellent antigen delivery capabilities, they are widely used in drug delivery and vaccine adjuvants. Compared to neutral or anionic liposomes, cationic liposomes, due to their positively charged surface, can interact with negatively charged antigen molecules or the surface of antigen-presenting cells through electrostatic interactions, thereby improving antigen uptake efficiency and immune response levels. However, existing cationic liposome adjuvants still generally suffer from the following technical problems: (1) cationic lipid molecules often lead to instability in the liposome system, easily resulting in increased particle size, aggregation and sedimentation, or zeta potential decay, thus affecting the immunization effect and application stability; (2) excessively high positive charge density can easily cause non-specific damage to the cell membrane, leading to cytotoxicity and inflammatory reactions, limiting their application in vaccine adjuvants. Therefore, developing a novel cationic liposome vaccine adjuvant that ensures cationic properties while possessing high structural stability, low toxicity, and good immune enhancement effects is a key technical problem that urgently needs to be solved in this field.

[0024] The structural formula of 3-alkylamino-1,2-propanediol is: There are currently no reports on the application of 3-alkylamino-1,2-propanediol in the preparation of cationic liposome adjuvants.

[0025] Based on the above background, this invention proposes a method for preparing a cationic liposome adjuvant containing 3-(aliphatic alkylamino)-1,2-propanediol. By introducing 3-(aliphatic alkylamino)-1,2-propanediol into a phospholipid-sterol compound system (such as lecithin / cholesterol liposomes), intermolecular hydrogen bonding is formed with lecithin, thereby achieving synergistic regulation of the surface charge, intermolecular interactions, and bilayer structure of liposomes. This significantly improves the dispersion stability and structural rigidity of liposomes and effectively reduces the cytotoxicity of cationic liposomes, making them more suitable for use as vaccine adjuvants.

[0026] The preparation method of the cationic liposome adjuvant includes the following steps: S10. Phospholipids, sterols, ionizable cationic lipids and organic solvents are mixed to obtain a lipid mixture solution; wherein the ionizable cationic lipids include 3-(aliphatic alkylamino)-1,2-propanediol. S20. Remove the organic solvent from the lipid mixture to form a lipid film; S30. Under heating conditions, a buffer solution is added to the lipid film for hydration treatment to obtain a crude liposome dispersion. S40. The crude liposome dispersion is processed by high-pressure microfluidic jet to obtain the cationic liposome adjuvant.

[0027] The present invention provides a method for preparing a cationic liposome adjuvant containing 3-(aliphatic amino)-1,2-propanediol. By introducing 3-(aliphatic amino)-1,2-propanediol as a key cationic lipid component, its unique molecular structure maintains the stability of the liposome surface and imparts a high positive charge density, significantly increasing the absolute value of the zeta potential on the liposome surface. This results in stronger electrostatic repulsion between liposome particles, thereby improving the dispersion stability of the system. Simultaneously, the secondary amine and hydroxyl groups in 3-(aliphatic amino)-1,2-propanediol form a hydrogen bond network with the phosphate groups in lecithin molecules, creating stronger intermolecular forces. This enhances the order and rigidity of the liposome bilayer at the molecular level, facilitating stable antigen loading. Furthermore, through the aforementioned electrostatic interactions and hydrogen bond network, excessively strong positive charges can be structurally dispersed and shielded, thereby reducing the non-specific destructive effect of liposomes on cell membranes and decreasing cytotoxicity. In addition, the diol moiety can improve the hydrophilicity of the compound, enhance hydration, and improve the overall water dispersibility of the liposomes. The cationic liposome adjuvant prepared by this invention exhibits uniform particle size distribution, low PDI, and good storage stability, effectively reducing cytotoxicity while maintaining cationic properties. Furthermore, the preparation method described in this application utilizes readily available raw materials and a mature preparation process, demonstrating promising prospects for industrial application.

[0028] It should be noted that in the technical solution of this invention, the introduction of 3-(aliphatic amino)-1,2-propanediol has multiple synergistic effects: (1) the secondary amine and alcohol hydroxyl group of 3-(aliphatic amino)-1,2-propanediol form a hydrogen bond network with the oxygen anion of the phosphate group and the carbonyl oxygen atom in the lecithin molecule, thereby forming stronger intermolecular forces and enhancing the order and rigidity of the liposome bilayer at the molecular level; (2) the oxygen anion of the phosphate group in the lecithin molecule forms a hydrogen bond with the secondary amine, making the lecithin... (3) It significantly increases the absolute value of the Zeta potential on the surface of liposomes, resulting in stronger electrostatic repulsion between liposome particles, thereby improving the dispersion stability of the system; (4) The diol moiety can improve the hydrophilicity of the compound, enhance hydration, and improve the water dispersibility of the entire liposome; (5) The above electrostatic effects and hydrogen bond network can structurally disperse and shield the excessive positive charge, thereby reducing the non-specific destructive effect of liposomes on cell membranes and reducing cytotoxicity.

[0029] In an embodiment of the present invention, in step S10, the aliphatic hydrocarbon group in the 3-(aliphatic amino)-1,2-propanediol is a C12-C18 straight-chain alkyl or straight-chain alkenyl group, that is, the aliphatic hydrocarbon group in the 3-(aliphatic amino)-1,2-propanediol is a straight-chain alkyl or straight-chain alkenyl group containing 12-18 carbon atoms. The aliphatic hydrocarbon group in the 3-(aliphatic amino)-1,2-propanediol is chosen to be a C12-C18 straight-chain alkyl or straight-chain alkenyl group because the fatty acids of lecithin are generally C16 or C18 acids. The closer the alkyl chain length of 3-alkylamino-1,2-propanediol is to that of lecithin, the better the interaction can be formed. Therefore, a C12-C18 chain length is selected. Too short or too long a chain may form unstable liposomes. Preferably, the aliphatic hydrocarbon group in the 3-(aliphatic amino)-1,2-propanediol is hexadecyl or cis-9-octadecenyl.

[0030] In an embodiment of the present invention, in step S10, the phospholipid includes at least one of lecithin, soybean lecithin, egg yolk lecithin, and synthetic phospholipids. The lecithin can be natural lecithin or synthetic lecithin. In one embodiment of the present invention, the phospholipid is lecithin, and the purity of natural lecithin is ≥90%.

[0031] In an embodiment of the present invention, in step S10, the sterol compound includes at least one selected from cholesterol, phytosterols, and cholanols. In one embodiment of the present invention, the sterol compound is cholesterol.

[0032] In an embodiment of the present invention, in step S10, the mass ratio of the phospholipid to the sterol compound is (2~6):1. Cholesterol and other sterol molecules have a unique rigid steroidal ring structure, which can be inserted into the phospholipid bilayer, playing a "bidirectional regulatory" role. An appropriate sterol ratio can significantly reduce membrane permeability and prevent leakage of encapsulated antigens or nucleic acid drugs before reaching the target cells. If there is too little sterol, the stability of liposomes will be poor, making them more prone to permeation; however, if the sterol ratio is too high, the excess sterol will crystallize and precipitate, leading to liposome aggregation, precipitation, or even rupture. In one embodiment of the present invention, the mass ratio of the phospholipid to the sterol compound is set to 4:1.

[0033] In an embodiment of the present invention, in step S10, the amount of 3-(aliphatic amino)-1,2-propanediol is 10% to 30% of the amount of phospholipid. Exemplarily, the amount of 3-(aliphatic amino)-1,2-propanediol can be 10%, 15%, 20%, 25%, or 30% of the amount of phospholipid, or any value within the range of the two values ​​described above. Setting the amount of 3-(aliphatic amino)-1,2-propanediol within the above range is beneficial for forming stable cationic liposomes and also ensures that the cationic liposomes do not have excessively high positive charge, thereby preventing increased toxicity.

[0034] In an embodiment of the present invention, in step S10, the organic solvent includes at least one selected from ethanol, methanol, isopropanol, and chloroform. In one embodiment of the present invention, ethanol is selected as the organic solvent.

[0035] In an embodiment of the present invention, in step S20, the organic solvent in the lipid mixture is removed by rotary evaporation.

[0036] In an embodiment of the present invention, step S30 includes heating at 35-40°C. Exemplarily, the heating temperature can be set to 35°C, 36°C, 37°C, 38°C, 39°C, or 40°C. Setting the heating temperature within this range, which is higher than the phase transition temperature of lipids, allows the lipid membrane to be in a liquid crystal state rather than a gel state. In the liquid crystal state, the lipid molecular chains are loosely arranged and have good fluidity, making it easier for water molecules to insert between the lipid bilayers, significantly improving hydration efficiency. In one embodiment of the present invention, the heating temperature is selected as 37°C.

[0037] In an embodiment of the present invention, in step S30, hydration treatment is performed under ultrasonic conditions, with an ultrasonic power of 350-400 W and an ultrasonic time of 10-20 min. For example, the ultrasonic power can be 350 W, 375 W, or 400 W, and the ultrasonic time can be 10 min, 15 min, or 20 min. Ultrasound can accelerate the hydration process, utilizing the microjets generated by the ultrasonic cavitation effect to eliminate particle agglomeration and form a colloidal system with uniform and stable particle size.

[0038] In an embodiment of the present invention, in step S30, the buffer solution includes a PBS buffer solution with a pH range of 7.2 to 7.4. PBS provides a physiologically isotonic environment and pH buffering capacity, preventing liposomes from rupturing or aggregating due to osmotic pressure differences during hydration.

[0039] In an embodiment of the present invention, in step S40, the average particle size of the cationic liposome adjuvant is 90-150 nm. When the aliphatic hydrocarbon group of the 3-(aliphatic alkylamino)-1,2-propanediol is hexadecyl or cis-9-octadecenyl, the average particle size of the prepared cationic liposome adjuvant is 90-150 nm.

[0040] In an embodiment of the present invention, in step S40, the polydispersity index (PDI) of the cationic liposome adjuvant is ≤0.40. When the aliphatic hydrocarbon group of the 3-(aliphatic alkylamino)-1,2-propanediol is hexadecyl or cis-9-octadecenyl, the polydispersity index (PDI) of the prepared cationic liposome adjuvant is ≤0.40. The cationic liposomes containing 3-(aliphatic alkylamino)-1,2-propanediol prepared by the present invention have uniform particle size distribution, low PDI, and good storage stability.

[0041] In an embodiment of the present invention, in step S40, the zeta potential of the cationic liposome adjuvant is positive and ≤25 mV. The cationic liposome adjuvant provided by the present invention can maintain positive charge and can structurally disperse and shield excessive positive charge through electrostatic interactions and hydrogen bond networks, reducing the problem of non-specific damage to cell membranes caused by excessive positive charge density, leading to cytotoxicity and inflammatory responses.

[0042] In an embodiment of the present invention, step S40, the parameters for processing by high-pressure microfluidics include: extrusion twice at 12000 psi. High-pressure microfluidic processing regulates the particle size of the liposomes, thereby obtaining a uniformly distributed cationic liposome adjuvant.

[0043] This invention proposes a veterinary vaccine comprising a cationic liposome adjuvant, which is prepared by the method described in the foregoing technical solution. The veterinary vaccine proposed in this invention includes a nanostructured lipid carrier adjuvant prepared by the method described above, and therefore possesses all the beneficial effects of the aforementioned preparation method for nanostructured lipid carrier adjuvants, which will not be elaborated further here.

[0044] In embodiments of the present invention, the veterinary vaccine further includes an antigen. The antigen is an inactivated antigen and / or a subunit antigen, which can effectively exert the vaccine's effect. The aforementioned cationic liposome adjuvant has good immunomodulatory activity, which is beneficial for inducing Th1-type responses related to cellular immunity.

[0045] In embodiments of the present invention, the antigen includes porcine circovirus CP08 strain antigen. The cationic liposome adjuvant provided by the present invention can be combined with porcine circovirus CP08 strain antigen to formulate a veterinary vaccine, which has good safety and immunomodulatory activity.

[0046] The technical solution of the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. It should be understood that the following embodiments are only used to explain the present invention and are not intended to limit the present invention.

[0047] The lecithin mentioned is soybean lecithin, with a purity of 90%, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., with product number L105733; The cholesterol in question was 95% pure and was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., product number C104028.

[0048] Example 1 A method for preparing a cationic liposome adjuvant includes the following steps: (1) Add 3 g lecithin, 0.75 g cholesterol, 0.9 g 3-hexadecylamino-1,2-propanediol (CAS No. 7517-27-3) and 200 mL ethanol to a 2 L round-bottom flask and mix with stirring at 40 °C to obtain a lipid mixture solution; (2) Ethanol was removed from the lipid mixture solution by rotary evaporation at 40°C and 0.098 MPa vacuum, and a uniform lipid film was formed at the bottom of the round-bottom flask. (3) At 37°C, 900 mL of PBS buffer solution (10 mM, pH 7.2-7.4) was added to the lipid film for 1 h of hydration treatment, and the film was sonicated at 360 W for 20 min to obtain a crude liposome dispersion. (4) The crude liposome dispersion was extruded twice under high pressure microjet at 12000 psi to obtain cationic liposome adjuvant, which was then sealed and stored at 4°C.

[0049] The cationic liposome adjuvant prepared in this embodiment has a particle size of 98.5 nm, a polydispersity index of 0.265, and a potential of +21.8 mV. During a 6-month storage period, the liposomes showed no bottom sedimentation and their appearance was indistinguishable from freshly prepared samples, indicating good stability.

[0050] Example 2 Compared to Example 1, the difference is that 3-hexadecaneamino-1,2-propanediol is replaced with an equal amount of 3-(cis-9-octadeceneamino)-1,2-propanediol.

[0051] The 3-(cis-9-octadecenamino)-1,2-propanediol is prepared by the following steps: 10 mmol of cis-9-octadecenamine (2.67 g) was dissolved in 20 mL of anhydrous ethanol and stirred at 30 °C until homogeneous. Then, 11 mmol of glycidyl ether (0.82 g) was slowly added dropwise over 10 minutes. After the addition was complete, the mixture was heated to 35 °C and stirred for 3 hours. After the reaction was complete, the ethanol was removed by rotary evaporation under reduced pressure to obtain 3-(cis-9-octadecenamino)-1,2-propanediol. This was then dissolved in 30 mL of ethyl acetate and washed twice with saturated brine (30 mL x 2). The ethyl acetate was dried over anhydrous sodium sulfate overnight, filtered, and then evaporated under reduced pressure to remove the ethyl acetate, yielding purified 3-(cis-9-octadecenamino)-1,2-propanediol.

[0052] The prepared materials were subjected to 1 Characterization by 1H NMR and high-resolution mass spectrometry (HRMS) yielded the following results: 1 H NMR (CDCl3, 400 MHz) δ: 0.88 (t, J = 8.0 Hz, 3H, CH3), 1.29 (s, 22H,CH2), 1.42-1.49 (m, 2H, CH2), 1.99-2.04 (m, 4H, allylic CH2), 2.46-2.82 (m,4H, CH2), 3.47-3.62 (m, 1H, glycerol CH), 3.67-3.79 (m, 2H,glycerol CH2), 5.35(t, J = 4.0 Hz, 2H, CH=CH).

[0053] HRMS: [M+H]+ calculated value is 342.33720, measured value is 342.33649.

[0054] The above results are consistent with the expected molecular structure of 3-(cis-9-octadecenamino)-1,2-propanediol, indicating that the target compound was successfully obtained in this embodiment. The structural formula of 3-(cis-9-octadecenamino)-1,2-propanediol is as follows: .

[0055] The cationic liposome adjuvant prepared in this embodiment has a particle size of 101.7 nm, a polydispersity index of 0.231, and a potential of +22.0 mV. During a 6-month storage period, the liposomes showed no bottom sedimentation and their appearance was indistinguishable from freshly prepared samples, indicating good stability.

[0056] Example 3 The difference compared to Example 1 is that the amount of 3-hexadecanoamino-1,2-propanediol used is 0.3 g.

[0057] The cationic liposome adjuvant prepared in this embodiment has a particle size of 93.8 nm, a polydispersity index of 0.216, and a potential of +10.5 mV. During a 3-month storage period, the liposomes showed no bottom sedimentation and their appearance was indistinguishable from freshly prepared samples, indicating good stability.

[0058] Example 4 The difference compared to Example 1 is that the amount of 3-hexadecanoamino-1,2-propanediol used is 0.6 g.

[0059] The cationic liposome adjuvant prepared in this embodiment has a particle size of 123.2 nm, a polydispersity index of 0.393, and a potential of +18.9 mV. During a 6-month storage period, the liposomes showed no bottom sedimentation and their appearance was indistinguishable from freshly prepared samples, indicating good stability.

[0060] Example 5 A method for preparing a veterinary vaccine includes the following steps: Take porcine circovirus CP08 strain antigen and calculate the required antigen content for vaccine preparation based on the final antigen content of 100 µg / ml in the vaccine; add the cationic liposome adjuvant prepared in Example 1 to the vaccine at a ratio of 1%, then make up the volume to 10 mL with physiological saline, and stir at low speed for 30 min with a magnetic stirrer to obtain veterinary vaccine.

[0061] Example 6 Compared to Example 1, the difference is that 3-hexadecaneamino-1,2-propanediol is replaced with an equal amount of 3-dodecaneamino-1,2-propanediol (CAS No. 821-91-0).

[0062] The cationic liposomes prepared in this embodiment have a hydrated particle size of 132.6 nm, a polydispersity index of 0.352, and a potential of +13.6 mV. These liposomes showed no bottom sedimentation during a 6-month storage period and their appearance was indistinguishable from freshly prepared samples, indicating good stability.

[0063] Example 7 Compared to Example 1, the difference is that 3-hexadecaneamino-1,2-propanediol is replaced with an equal amount of 3-octadecaneamino-1,2-propanediol (CAS No. 28900-95-0).

[0064] The cationic liposomes prepared in this embodiment have a hydrated particle size of 215.7 nm, a polydispersity index of 0.482, and a potential of +18.2 mV. These liposomes showed no bottom sedimentation during a 6-month storage period and their appearance was indistinguishable from freshly prepared samples, indicating good stability.

[0065] Comparative Example 1 The difference from Example 1 is that it does not contain 3-hexadecano-1,2-propanediol.

[0066] The hydrated particle size of the liposomes prepared in this comparative example was 151.9 nm, the polydispersity index was 0.512, and the potential was -3.57 mV. These liposomes showed bottom sedimentation after 7 days, indicating poor stability.

[0067] Comparative Example 2 Compared to Example 1, the difference is that 3-hexadecylamino-1,2-propanediol is replaced with an equal amount of hexadecyltrimethylammonium bromide.

[0068] The hydrated particle size of the liposomes prepared in this comparative example was 349.2 nm, the polydispersity index was 0.541, and the potential was +13.5 mV. These liposomes showed bottom sedimentation after 7 days, indicating poor stability.

[0069] Performance testing 1. Morphological observation: The cationic liposome adjuvant samples from Examples 1 and 2 were diluted 10-fold with deionized water, adsorbed onto 200-mesh copper carbide, and air-dried at 25°C. The morphology of the liposomes was observed using a transmission electron microscope at 10000× magnification. Transient images were inverted using ImageJ software.

[0070] Transmission electron microscopy results of two cationic liposome adjuvants are as follows: Figure 1 As shown in the figure, the results indicate that both cationic liposome adjuvants exhibited uniform spherical nanoparticles with smooth edges and good dispersibility at a 200 nm scale bar, and no obvious aggregation or collapse structures were observed. These results suggest that the introduction of 3-(aliphatic amino)-1,2-propanediol helps to regulate the self-assembly behavior of liposomes, thereby obtaining nanoliposomes with more uniform size and more regular morphology.

[0071] 2. Liposome membrane fluidity test: The liposome membrane fluidity of Examples 1-2 and Comparative Example 1 was studied using the 1,6-diphenyl-1,3,5-hexadetriene (DPH) fluorescent probe method. DPH is a classic hydrophobic fluorescent probe, mainly localized to the fatty chain regions of the lipid bilayer. Its fluorescence intensity and anisotropy are highly sensitive to the liposome membrane fluidity and the degree of ordering of the fatty chains. Enhancement of DPH fluorescence intensity is usually related to impaired fatty chain movement in the lipid bilayer, increased microenvironment rigidity, or increased local hydrophobicity. The specific procedure for DPH fluorescent probe detection is as follows: The liposomes to be tested were diluted 10-fold with PBS buffer and then mixed with DPH-DMSO (2 mM) solution at a volume ratio of 50:1, and incubated in the dark for 60 minutes. After incubation, the fluorescence intensity of the samples was recorded in the range of excitation wavelength 350 nm and emission wavelength 375–600 nm. The voltage was 600 V, and the slit width was 5 nm.

[0072] The DPH fluorescence spectra of the cationic liposome adjuvants in Examples 1-2 and Comparative Example 1 are as follows: Figure 2 As shown, the results indicate that Examples 1-2 exhibit higher fluorescence intensity compared to the cationic liposome adjuvant of Comparative Example 1. The introduction of 3-hexadecylamino-1,2-propanediol in Example 1 resulted in higher fluorescence intensity, suggesting that its adipose chain regions may have formed a more ordered or denser hydrophobic microenvironment. The fluorescence intensity of the cationic liposome adjuvant in Example 2, which introduced 3-oleoamino-1,2-propanediol, was slightly lower than that of Example 2, but higher than that of Comparative Example 1, indicating that it also stabilized the liposome bilayer structure and reduced the fluidity of the liposome membrane.

[0073] 3. Liposome membrane hydrophobicity test: The hydrophobicity of liposome membranes in Examples 1-2 and Comparative Example 1 was investigated using the sodium 8-anilino-1-naphthalenesulfonate (ANS) fluorescent probe method. ANS is an anionic fluorescent probe highly sensitive to environmental polarity; its fluorescence enhancement and blue shift of the emission peak are generally closely related to increased hydrophobicity at the liposome membrane interface and loosening of lipid arrangement. The specific procedure for the ANS fluorescent probe method is as follows: Liposomes were diluted 10-fold with PBS buffer and mixed with ANS (8 mM) solution at a volume ratio of 50:1, and incubated in the dark for 30 minutes. After incubation, the fluorescence intensity of the samples was recorded in the range of excitation wavelength 350 nm and emission wavelength 375–600 nm. The voltage was 580 V, and the slit width was 5 nm.

[0074] The ANS fluorescence spectra of the cationic liposome adjuvants in Examples 1-2 and Comparative Example 1 are as follows: Figure 3As shown, the ANS fluorescence intensity of Examples 1 and 2 was significantly higher than that of the liposomes in Comparative Example 1 (containing only lecithin and cholesterol), indicating that the introduction of 3-(aliphatic alkyl amino)-1,2-propanediol significantly altered the membrane interface properties of the liposomes, placing the ANS probe in a more hydrophobic or lower polarity environment and enhancing the hydrophobicity of the liposome membrane.

[0075] Comprehensive analysis of DPH and ANS fluorescence results showed that the introduction of 3-(aliphatic alkylamino)-1,2-propanediol resulted in a structural feature in liposomes characterized by "enhanced interfacial hydrophobicity and a relatively stable hydrophobic core," while DPH results indicated that the aliphatic chain region did not exhibit significant loosening or disorder. This "loose on the outside, stable on the inside" membrane structure is beneficial to the performance of vaccine adjuvants, ensuring the stability of the liposome structure while maintaining the integrity of the system after antigen loading, enhancing its interaction with immune cell membranes, and promoting the antigen presentation process.

[0076] 4. Cytotoxicity test: RAW264.7 cells were planted at a density of 2.0 × 10⁶ cells per well. 4 The culture medium was seeded at a density of [number] cells / wells in 96-well plates and incubated overnight at 37°C and 5% CO2. Subsequently, the medium was replaced with DMEM complete medium containing different concentrations of cationic liposome adjuvants from Examples 1 and 2, and incubated for another 24 h. Afterward, the original medium was discarded, and fresh DMEM medium containing 10 μL of CCK-8 reagent was added to each well, and the plates were incubated in the dark for 1–2 h under the same conditions. The optimal detection time point was determined based on the color change of the culture medium, and the absorbance (OD value) of each well was measured using a microplate reader at wavelengths of 450 nm and 630 nm. 450nm Used to reflect cell viability, OD 630nm It is used as a reference wavelength for background correction.

[0077] The cytotoxicity test results of the cationic liposome adjuvants in Examples 1 and 2 are as follows: Figure 4 As shown. Figure 4 The graph shows the cytotoxicity test results of the cationic liposome adjuvants from Examples 1 and 2, where the horizontal axis represents concentration (mg / mL) and the vertical axis represents cell viability (%). The results show that both cationic liposome adjuvants exhibited significant concentration-dependent cytotoxicity, with virtually no cytotoxicity at low concentrations (≤0.025 mg / mL), demonstrating good biocompatibility. Considering that the dosage used in immunologic assays is typically far below 0.05 mg / mL, both cationic liposome adjuvants are within the effective immunogenic concentration range and exhibit acceptable in vitro safety.

[0078] 5. Antigen-specific antibody titer analysis: The cationic liposome adjuvants from Examples 1 and 2 were prepared into veterinary vaccines according to the method in Example 5. Bal / c mice were randomly divided into five groups: a blank control group, an antigen group, Example 1 group, and Example 2 group, with five mice in each group. Each group received a subcutaneous injection (injection volume: 100 μL / mouse). Blood samples were collected 14 days post-immunization for antigen-specific antibody titer analysis. The blank control group received 100 μL of physiological saline, the antigen group received the same mass of antigen as the vaccine group (10 μg / mouse), and the Example 1 and Example 2 groups received subcutaneous injections of the veterinary vaccines prepared from the cationic liposome adjuvants of Examples 1 and 2, respectively.

[0079] Following a single immunization, mice were observed for 14 consecutive days, with daily observations and records of their mental state, appetite, and mortality. The safety test results showed that the mice had normal mental state and appetite and were all healthy. The injection site showed good vaccine absorption, with no redness, swelling, or nodules, indicating that the mice were safe at this dosage.

[0080] The results of the antigen-specific antibody titer test are as follows: Figure 5 As shown in the figure. Compared with the use of antigen alone, both Example 1 and Example 2 groups significantly enhanced total IgG levels, which was superior to the antigen-only groups. Both Example 1 and Example 2 groups showed a more significant increase in IgG2a, while the effect on IgG1 was not statistically significant. The increased IgG2a / IgG1 ratio suggests a shift in the immune response towards the Th1 type, and a greater tendency to induce a Th1-type response dominated by cell-mediated immunity. Example 1 group had a slightly higher absolute IgG2a level than Example 2 group, while Example 2 group had a slightly higher IgG2a / IgG1 ratio, but the overall difference was not significant. This indicates that compared with Example 1 group, the cationic liposome adjuvant in Example 2 group has good immunomodulatory activity and may be more conducive to inducing a Th1-type response related to cell-mediated immunity.

[0081] The above description is merely an exemplary embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural transformations made using the contents of the present invention specification and drawings under the technical concept of the present invention, or direct / indirect applications in other related technical fields, are included within the patent protection scope of the present invention.

Claims

1. A method for preparing a cationic liposome adjuvant, characterized in that, Includes the following steps: S10. Phospholipids, sterols, ionizable cationic lipids and organic solvents are mixed to obtain a lipid mixture solution; wherein the ionizable cationic lipids include 3-(aliphatic alkylamino)-1,2-propanediol. S20. Remove the organic solvent from the lipid mixture to form a lipid film; S30. Under heating conditions, a buffer solution is added to the lipid film for hydration treatment to obtain a crude liposome dispersion. S40. The crude liposome dispersion is processed by high-pressure microfluidic jet to obtain the cationic liposome adjuvant.

2. The method for preparing the cationic liposome adjuvant as described in claim 1, characterized in that, In step S10, the aliphatic hydrocarbon group in the 3-(aliphatic hydrocarbon amino)-1,2-propanediol is a C12-C18 straight-chain alkyl or straight-chain alkenyl group.

3. The method for preparing the cationic liposome adjuvant as described in claim 2, characterized in that, The aliphatic hydrocarbon group in the 3-(aliphatic hydrocarbon amino)-1,2-propanediol is hexadecyl or cis-9-octadecenyl.

4. The method for preparing the cationic liposome adjuvant as described in claim 1, characterized in that, In step S10: The phospholipids include at least one of lecithin, soybean phospholipids, egg yolk phospholipids, and synthetic phospholipids; and / or, The sterol compounds include at least one of cholesterol, phytosterols, and cholanols; and / or, The mass ratio of the phospholipid to the sterol compound is (2~6):1; and / or, The amount of 3-(aliphatic amino)-1,2-propanediol used is 10% to 30% of the amount of phospholipid; and / or, The organic solvent includes at least one of ethanol, methanol, isopropanol, and chloroform.

5. The method for preparing the cationic liposome adjuvant as described in claim 1, characterized in that, In step S20, the organic solvent in the lipid mixture is removed by rotary evaporation.

6. The method for preparing the cationic liposome adjuvant according to claim 1, characterized in that, In step S30: The heating conditions include heating at 35~40°C; and / or, Hydration treatment is performed under ultrasonic conditions, wherein the ultrasonic power is 350~400 W and the ultrasonic time is 10~20 min; and / or, The buffer solution includes a PBS buffer solution with a pH range of 7.2 to 7.

4.

7. The method for preparing the cationic liposome adjuvant according to claim 1, characterized in that, In step S40: The cationic liposome adjuvant has a particle size of 90-150 nm; and / or, The polydispersity index (PDI) of the cationic liposome adjuvant is ≤0.40; and / or, The cationic liposome adjuvant has a positive Zeta potential value and is ≤25 mV; And / or, The parameters for processing via high-pressure microjets include: extrusion twice at 10,000 to 15,000 psi.

8. A veterinary vaccine, characterized in that, The adjuvant includes a cationic liposome adjuvant, which is prepared by the method for preparing the cationic liposome adjuvant according to any one of claims 1 to 7.

9. The veterinary vaccine as described in claim 8, characterized in that, The veterinary vaccine also includes antigens.

10. The veterinary vaccine as described in claim 9, characterized in that, The antigens include porcine circovirus CP08 strain antigen.

Images