Cationic lipid nanovaccine adjuvants and methods of making same

Abstract

This application provides a cationic lipid nanovaccine adjuvant and its preparation method, which can be applied to the field of pharmaceutical formulation in pharmaceutical engineering. The method includes: dissolving polylactic acid materials and cationic surfactants in an organic solvent, and ultrasonically mixing them to obtain an oil phase; using ultrapure water as the aqueous phase, uniformly adding the oil phase to the aqueous phase under stirring at a predetermined speed, and continuously stirring until an oil-in-water emulsion is formed; solidifying the oil-in-water emulsion by vacuum evaporation for 3-6 hours at 10-70℃ and -0.01-0.8 Pa to obtain a cationic lipid nanosphere suspension; and circulating and washing the cationic lipid nanosphere suspension with a tangential flow ultrafiltration membrane with a molecular weight cutoff of 100 kDa to obtain the cationic lipid nanovaccine adjuvant.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical formulation in pharmaceutical engineering, and more particularly to a cationic lipid nanovaccine adjuvant and its preparation method, which can be scaled up and is easy to industrialize. Background Technology

[0002] Adjuvants are substances injected into the body before or simultaneously with vaccines to enhance the body's immune response, overcoming the problem of weak immunogenicity in traditional vaccines. Aluminum salt adjuvants are currently the most widely used FDA-approved human vaccine adjuvants in clinical practice; however, as their use expands, their shortcomings have become increasingly apparent—they tend to form hard, non-degradable nodules at the injection site and cannot induce an effective cellular immune response. Biodegradable polymer nanoparticles have the advantages of being non-toxic, having controllable particle size, and being able to maximize the body's immune response. Cationic lipid nanovaccine adjuvants can significantly improve humoral and cellular immunity levels. Cationic nanovaccine adjuvants are typically prepared using a nanoprecipitation method, followed by high-speed centrifugation, repeated ultrasonic washing, and concentration after complete solidification.

[0003] The traditional nanoprecipitation method for preparing cationic lipid nanovaccine adjuvants relies on solvent evaporation and diffusion. This process often requires prolonged curing at room temperature. Incompletely cured cationic lipid nanovaccine adjuvants exhibit poor surface morphology, uneven particle size distribution, and reduced antigen loading. Therefore, the curing time at room temperature must be at least 10 hours for the traditional nanoprecipitation method to prepare cationic lipid nanovaccine adjuvants. During the centrifugation process for washing and collecting nanospheres, the limited centrifugation speed fails to remove most of the extremely small nanospheres, resulting in significant nanosphere loss. Furthermore, high-speed centrifugation causes nanospheres to clump together, making uniform ultrasonic dispersion difficult. Excessive ultrasonic power also affects the sphericity and potential of the nanospheres, further limiting the adsorption rate and yield. Consequently, the final yield of nanospheres obtained using the traditional centrifugation method is extremely low (<15%).

[0004] In summary, current methods for preparing cationic lipid nanovaccine adjuvants suffer from problems such as long preparation time, low yield, and low efficiency. Summary of the Invention

[0005] In view of this, the main objective of this invention is to provide a cationic lipid nanovaccine adjuvant and its preparation method, in order to at least partially solve at least one of the aforementioned technical problems. To achieve the above objective, the technical solution of this invention is as follows:

[0006] As one aspect of the present invention, a method for preparing a cationic lipid nanovaccine adjuvant is provided, comprising the following steps:

[0007] (1) Polylactic acid materials and cationic surfactants are dissolved in an organic solvent as the oil phase, and water is used as the aqueous phase;

[0008] (2) The oil phase is slowly and uniformly added to the aqueous phase under stirring to form an oil-in-water emulsion;

[0009] (3) The water-in-oil emulsion is solidified by vacuum evaporation to obtain a suspension of cationic lipid nanospheres;

[0010] (4) The suspension of the cationic lipid nanospheres is washed by tangential flow ultrafiltration to collect the cationic lipid nanovaccine adjuvant.

[0011] As another aspect of the present invention, a cationic lipid nanovaccine adjuvant prepared by the preparation method described above is provided, which is regularly spherical and has a uniform particle size distribution.

[0012] Based on the above technical solution, the beneficial effects of the present invention are as follows:

[0013] (1) The boiling point of organic reagents decreases under vacuum. Vacuum evaporation can accelerate the volatilization process of organic reagents and shorten the curing time. Tangential flow ultrafiltration is relatively mild. After multiple washing and concentration, the organic reagents and surfactants that have not been completely volatilized in the fully cured nano-vaccine adjuvant can be continuously replaced, and most of the small-sized nanospheres can be retained.

[0014] (2) By optimizing the dosage, oil-water ratio, rotary evaporation time and water circulation times, the preparation time of cationic lipid nanovaccine adjuvants by combining rotary evaporation and tangential flow ultrafiltration can be reduced from more than 10 h to 3-6 h, and the yield can be increased from less than 15% to more than 90%. The operation is simple, the batch-to-batch repeatability is good, and it is easy to industrialize.

[0015] (3) The cationic lipid nanovaccine adjuvant prepared by this method has the characteristics of small particle size, stable positive charge and ability to induce high levels of cellular and humoral immune response, and is universally applicable to a variety of antigens. Attached Figure Description

[0016] Figure 1 This is a schematic diagram of the preparation apparatus and process of the cationic lipid nanovaccine adjuvant according to an embodiment of the present invention;

[0017] Figure 2-A This is a scanning electron microscope image of the cationic lipid nanovaccine adjuvant obtained in Example 3 of the present invention;

[0018] Figure 2-B This is a scanning electron microscope image of the cationic lipid nanovaccine adjuvant obtained in Comparative Example 1 of the present invention;

[0019] Figure 3 The IgG antibody titer in the serum of animals immunized in Example 16 of this invention;

[0020] Figure 4 This refers to the amount of interferon-gamma (IFN-γ) secreted by mouse spleen cells after immunization in Example 16 of this invention.

[0021] Figure 5 This refers to the amount of interleukin-4 (IL-4) secreted by mouse spleen cells after immunization in Example 16 of this invention. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0023] This invention discloses a cationic lipid nanovaccine adjuvant and its preparation method. The preparation method has the advantages of shortening the preparation time, high product yield, good batch-to-batch reproducibility, and ease of industrial production. The cationic lipid nanovaccine adjuvant prepared by this method has the characteristics of small particle size, stable positive charge, and the ability to effectively induce high levels of cellular and humoral immune responses, and it is universally applicable to a variety of antigens.

[0024] This invention provides a method for preparing a cationic lipid nanovaccine adjuvant, comprising the following steps:

[0025] (1) Polylactic acid materials and cationic surfactants are dissolved in an organic solvent as the oil phase, and water is used as the aqueous phase;

[0026] (2) The oil phase is slowly and uniformly added to the aqueous phase under stirring to form an oil-in-water emulsion;

[0027] (3) The water-in-oil emulsion is solidified by vacuum evaporation to obtain a suspension of cationic lipid nanospheres;

[0028] (4) The suspension of the cationic lipid nanospheres is washed by tangential flow ultrafiltration to collect the cationic lipid nanovaccine adjuvant.

[0029] In step (1), the polylactic acid material is any one or a mixture of at least two of polylactic acid copolymer, polylactic acid-polyglycolic acid copolymer, and polylactic acid-polyethylene glycol copolymer; the molecular weight of the polylactic acid material is 6000~30000, for example, it can be 6000, 6500, 7000, 8000, 8200, 9000, 10000, 12000, 15000, 16000, 19000, 19500, 20000, 25000, 30000, etc.; preferably 8000~20000.

[0030] The organic solvent is specifically a mixture of ethanol and acetone, but it is not limited to this and can also be other water-soluble or volatile solvents. In this invention, ultrapure water is used as the aqueous phase. "Ultrapure water" in this field refers to water that is almost free of any impurities, specifically water with a resistivity of 18 MΩ∙cm at 25°C.

[0031] The cationic surfactant is specifically any one or a mixture of at least two of the following: dioctadecyl dimethyl ammonium bromide, didodecyl dimethyl ammonium bromide, and didecyl dimethyl ammonium bromide.

[0032] The total mass of polylactic acid material and cationic surfactant is 100%. The amount of polylactic acid material added is 74% to 91% of the total mass, for example, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 85%, 87%, 89%, 91%, etc., preferably 77% to 83%. The amount of cationic surfactant added is 9% to 26% of the total mass, for example, 9%, 11%, 13%, 15%, 17%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, preferably 17% to 23%.

[0033] In step (2), the volume ratio of the oil phase to the aqueous phase is 1:4 to 1:7.3, for example, it can be 1:4, 1:4.5, 1:5, 1:5.5, 1:6, 1:6.5, or 1:7.3; preferably 1:5 to 1:6; the dropping time is 0.5 to 3 h, for example, it can be 0.5 h, 0.6 h, 0.7 h, 1 h, 1.1 h, 1.5 h, 2 h, 2.1 h, 2.5 h, or 3 h; preferably 0.6 to 2 h. The dropping rate has little effect on the particle size, but it does affect the antigen adsorption rate. Too fast a dropping rate will lead to a decrease in the antigen adsorption rate.

[0034] In step (3), the reduced-pressure evaporation time is 0.5~10 h, for example, it can be 0.5 h, 1 h, 1.5 h, 2 h, 2.5 h, 3 h, 3.5 h, 4 h, 5 h, 5.5 h, 6 h, 6.5 h, 7 h, 7.5 h, 8 h, 8.5 h, 9 h, 9.5 h, or 10 h; preferably 3~6 h; preferably, the reduced-pressure evaporation temperature is 10~70℃; and the reduced-pressure evaporation pressure is -0.01~0.8 Pa. It can be understood that the temperature and pressure of the reduced-pressure evaporation are adjusted according to the specific solvent selected, so as to better control the reduced-pressure evaporation time.

[0035] In step (4), the tangential flow ultrafiltration method is cyclically washed 1 to 6 times, for example, 1, 2, 3, 4, 5, or 6 times; preferably 3 to 4 times. Preferably, the molecular weight cutoff of the tangential flow ultrafiltration membrane is 100 kDa.

[0036] The present invention also provides a cationic lipid nanovaccine adjuvant prepared by the method described above, which is regularly spherical and has a uniform particle size distribution. Specifically, its particle size is between 120 and 200 nm.

[0037] The following are specific embodiments to illustrate the technical solution of the present invention in detail:

[0038] Example 1

[0039] like Figure 1 As shown, 900 ml of ultrapure water was accurately measured and placed in a medium-sized reactor as the aqueous phase. 0.3 g of dioctadecyldimethylammonium bromide and 1 g of PLA with a molecular weight of 10 kDa were accurately weighed and dissolved in 150 ml of organic solvent (ethanol:acetone = 1:1). After ultrasonication to ensure uniform mixing, the mixture was placed in the titration apparatus of the reactor as the oil phase. Under constant stirring speed of 210~260 rpm / min, the oil phase was added dropwise to the aqueous phase at a uniform rate for 1 h. After the addition was completed, the liquid in the reactor was immediately transferred to a 2 L flask. After rotary evaporation under reduced pressure of 25℃ and 0.08 Pa for 10 h, a cationic lipid nanosphere suspension was obtained. The suspension was washed three times using a Vivaflow 50 membrane with a molecular weight cutoff of 100 kDa and concentrated to obtain the cationic lipid nanovaccine adjuvant with a particle size of 140~160 nm and a yield of 95%.

[0040] The prepared cationic lipid nanosphere suspension was mixed with an equal volume of antigen solution, with a final antigen concentration of 250 μg / ml and a final nanosphere concentration of 5 mg / ml. The mixture was vertically suspended at room temperature for 3 h. The vaccine formulation was centrifuged at 16000 g for 5 min, and the supernatant was collected. The concentration of free antigen in the supernatant was measured using a micro BCA kit, and the content of antigen adsorbed on the surface of the nanospheres was indirectly calculated. The adsorption rate of cationic lipid nanospheres for OVA antigen was found to be 75%.

[0041] Example 2

[0042] 900 ml of ultrapure water was accurately measured and placed in a medium-sized reactor as the aqueous phase. 0.3 g of dioctadecyldimethylammonium bromide and 1 g of PLA with a molecular weight of 10 kDa were accurately weighed and dissolved in 150 ml of organic solvent (ethanol:acetone = 1:1). After ultrasonication to ensure homogeneity, the mixture was placed in the titration apparatus of the reactor as the oil phase. The oil phase was gradually added dropwise to the aqueous phase under constant stirring speed of 210–260 rpm / min for 1 h. After the addition was complete, the liquid in the reactor was immediately transferred to a 2 L flask. The flask was then rotary evaporated for 0.5 h under reduced pressure conditions of 25°C and 0.08 Pa to obtain a cationic lipid nanosphere suspension. This suspension was washed three times using a Vivaflow 50 membrane with a molecular weight cutoff of 100 kDa and concentrated to obtain the cationic lipid nanovaccine adjuvant with a particle size of 190–220 nm and a yield of 93%. Using the same adsorption operation as in Example 1, an adsorption rate of 60% for OVA antigen was obtained.

[0043] Example 3

[0044] 900 ml of ultrapure water was accurately measured and placed in a medium-sized reactor as the aqueous phase. 0.3 g of dioctadecyldimethylammonium bromide and 1 g of PLA (10 kDa) were accurately weighed and dissolved in 150 ml of organic solvent (ethanol:acetone = 1:1). After ultrasonication to ensure homogeneity, the mixture was placed in the titration apparatus of the reactor as the oil phase. The oil phase was gradually added dropwise to the aqueous phase over a constant stirring speed of 210–260 rpm for 1 hour. After the addition was complete, the liquid in the reactor was immediately transferred to a 2 L flask. The flask was then rotary evaporated under reduced pressure (25°C, 0.08 Pa) for 6 hours to obtain a cationic lipid nanosphere suspension. This suspension was washed three times using a Vivaflow 50 membrane with a molecular weight cutoff of 100 kDa and concentrated to obtain the cationic lipid nanovaccine adjuvant. Electron micrographs are shown below. Figure 2-A As shown, the particle size was 120–140 nm, and the yield was 97%. Using the same adsorption operation as in Example 1, an adsorption rate of 85% for OVA antigen was obtained.

[0045] Example 4

[0046] 900 ml of ultrapure water was accurately measured and placed in a medium-sized reactor as the aqueous phase. 0.3 g of dioctadecyldimethylammonium bromide and 1 g of PLA (molecular weight 20 kDa) were accurately weighed and dissolved in 150 ml of organic solvent (ethanol:acetone = 1:1). The mixture was ultrasonically mixed and then placed in the titration apparatus of the reactor as the oil phase. Under constant stirring at 210–260 rpm / min, the oil phase was gradually added dropwise to the aqueous phase over 1 hour. After addition, the liquid in the reactor was immediately transferred to a 2 L flask and evaporated under reduced pressure (25°C, 0.08 Pa) for 6 hours to obtain a cationic lipid nanosphere suspension. This suspension was washed three times using a Vivaflow 50 membrane with a molecular weight cutoff of 100 kDa and then concentrated to obtain the cationic lipid nanovaccine adjuvant. The particle size was 150–160 nm, and the yield was 94%. Using the same adsorption operation as in Example 1, an adsorption rate of 80% for OVA antigen was obtained.

[0047] Example 5

[0048] 900 ml of ultrapure water was accurately measured and placed in a medium-sized reactor as the aqueous phase. 0.3 g of dioctadecyldimethylammonium bromide and 1 g of PLA with a molecular weight of 10 kDa were accurately weighed and dissolved in 150 ml of organic solvent (ethanol:acetone = 1:1). The mixture was ultrasonically stirred until homogeneous and then placed in the titration apparatus of the reactor as the oil phase. The oil phase was gradually added dropwise to the aqueous phase over a constant stirring speed of 210–260 rpm for 0.5 h. After the addition was complete, the liquid in the reactor was immediately transferred to a 2 L flask. The flask was then rotary evaporated under reduced pressure (25°C, 0.08 Pa) for 6 h to obtain a cationic lipid nanosphere suspension. This suspension was washed three times using a Vivaflow 50 membrane with a molecular weight cutoff of 100 kDa and concentrated to obtain the cationic lipid nanovaccine adjuvant. The particle size was 150–170 nm, and the yield was 95%. Using the same adsorption operation as in Example 1, an adsorption rate of 55% for OVA antigen was obtained.

[0049] Example 6

[0050] 900 ml of ultrapure water was accurately measured and placed in a medium-sized reactor as the aqueous phase. 0.35 g of dioctadecyldimethylammonium bromide and 1 g of PLA with a molecular weight of 10 kDa were accurately weighed and dissolved in 150 ml of organic solvent (ethanol:acetone = 1:1). After ultrasonication to ensure homogeneity, the mixture was placed in the titration apparatus of the reactor as the oil phase. Under constant stirring at 210–260 rpm / min, the oil phase was gradually added dropwise to the aqueous phase over a period of 1 h. After the addition was complete, the liquid in the reactor was immediately transferred to a 2 L flask. The flask was then rotary evaporated under reduced pressure (25°C, 0.08 Pa) for 6 h to obtain a cationic lipid nanosphere suspension. This suspension was washed three times using a Vivaflow 50 membrane with a molecular weight cutoff of 100 kDa and concentrated to obtain the cationic lipid nanovaccine adjuvant. The particle size was 140–150 nm, and the yield was 98%. Using the same adsorption operation as in Example 1, an adsorption rate of 80% for OVA antigen was obtained.

[0051] Example 7

[0052] 900 ml of ultrapure water was accurately measured and placed in a medium-sized reactor as the aqueous phase. 0.1 g of dioctadecyldimethylammonium bromide and 1 g of PLA with a molecular weight of 10 kDa were accurately weighed and dissolved in 150 ml of organic solvent (ethanol:acetone = 1:1). The mixture was ultrasonically stirred until homogeneous and then placed in the titration apparatus of the reactor as the oil phase. Under constant stirring speed of 210–260 rpm / min, the oil phase was gradually added dropwise to the aqueous phase over a period of 1 h. After the addition was complete, the liquid in the reactor was immediately transferred to a 2 L flask. The flask was then rotary evaporated under reduced pressure of 25°C and 0.08 Pa for 6 h to obtain a cationic lipid nanosphere suspension. This suspension was washed three times using a Vivaflow 50 membrane with a molecular weight cutoff of 100 kDa and concentrated to obtain the cationic lipid nanovaccine adjuvant. The particle size was 110–130 nm, and the yield was 96%. Using the same adsorption operation as in Example 1, an adsorption rate of 73% for OVA antigen was obtained.

[0053] Example 8

[0054] 900 ml of ultrapure water was accurately measured and placed in a medium-sized reactor as the aqueous phase. 0.3 g of dioctadecyldimethylammonium bromide and 1 g of PLA with a molecular weight of 10 kDa were accurately weighed and dissolved in 200 ml of organic solvent (ethanol:acetone = 1:1). The mixture was ultrasonically stirred until homogeneous and then placed in the titration apparatus of the reactor as the oil phase. Under constant stirring speed of 210–260 rpm / min, the oil phase was gradually added dropwise to the aqueous phase over a period of 1 h. After the addition was complete, the liquid in the reactor was immediately transferred to a 2 L flask. The flask was then rotary evaporated under reduced pressure of 25°C and 0.08 Pa for 6 h to obtain a cationic lipid nanosphere suspension. This suspension was washed three times using a Vivaflow 50 membrane with a molecular weight cutoff of 100 kDa and concentrated to obtain the cationic lipid nanovaccine adjuvant. The particle size was 120–140 nm, and the yield was 95%. Using the same adsorption operation as in Example 1, an adsorption rate of 76% for OVA antigen was obtained.

[0055] Example 9

[0056] 1000 ml of ultrapure water was accurately measured and placed in a medium-sized reactor as the aqueous phase. 0.3 g of dioctadecyldimethylammonium bromide and 1 g of PLA with a molecular weight of 10 kDa were accurately weighed and dissolved in 200 ml of organic solvent (ethanol:acetone = 1:1). The mixture was ultrasonically mixed and then placed in the titration apparatus of the reactor as the oil phase. Under constant stirring at 210–260 rpm / min, the oil phase was gradually added dropwise to the aqueous phase over a period of 1 h. After the addition was complete, the liquid in the reactor was immediately transferred to a 2 L flask. The flask was then rotary evaporated under reduced pressure (25 °C, 0.08 Pa) for 6 h to obtain a cationic lipid nanosphere suspension. This suspension was washed three times using a Vivaflow 50 membrane with a molecular weight cutoff of 100 kDa and concentrated to obtain the cationic lipid nanovaccine adjuvant. The particle size was 120–130 nm, and the yield was 96%. Using the same adsorption operation as in Example 1, an adsorption rate of 82% for OVA antigen was obtained.

[0057] Example 10

[0058] 1050 ml of ultrapure water was accurately measured and placed in a medium-sized reactor as the aqueous phase. 0.3 g of dioctadecyldimethylammonium bromide and 1 g of PLA with a molecular weight of 10 kDa were accurately weighed and dissolved in 150 ml of organic solvent (ethanol:acetone = 1:1). The mixture was ultrasonically stirred until homogeneous and then placed in the titration apparatus of the reactor as the oil phase. The oil phase was gradually added dropwise to the aqueous phase over a constant stirring speed of 210–260 rpm for 1 h. After the addition was complete, the liquid in the reactor was immediately transferred to a 2 L flask. The flask was then rotary evaporated under reduced pressure (25 °C, 0.08 Pa) for 6 h to obtain a cationic lipid nanosphere suspension. This suspension was washed three times using a Vivaflow 50 membrane with a molecular weight cutoff of 100 kDa and concentrated to obtain the cationic lipid nanovaccine adjuvant. The particle size was 120–130 nm, and the yield was 97%. Using the same adsorption operation as in Example 1, an adsorption rate of 75% for OVA antigen was obtained.

[0059] Example 11

[0060] 900 ml of ultrapure water was accurately measured and placed in a medium-sized reactor as the aqueous phase. 0.3 g of dioctadecyldimethylammonium bromide and 1 g of PLA (10 kDa) were accurately weighed and dissolved in 150 ml of organic solvent (ethanol:acetone = 1:1). The mixture was ultrasonically mixed and then placed in the titration apparatus of the reactor as the oil phase. Under constant stirring at 210–260 rpm / min, the oil phase was gradually added dropwise to the aqueous phase over 1 hour. After addition, the liquid in the reactor was immediately transferred to a 2 L flask. The flask was then rotary evaporated under reduced pressure (25°C, 0.08 Pa) for 6 hours to obtain a cationic lipid nanosphere suspension. This suspension was washed three times using a Vivaflow 50 membrane with a molecular weight cutoff of 100 kDa and concentrated to obtain the cationic lipid nanovaccine adjuvant. The particle size was 130–140 nm, and the yield was 96%. Using the same adsorption operation as in Example 1, except that the antigen was replaced with recombinant hepatitis B antigen, the adsorption rate of recombinant hepatitis B antigen was 92%.

[0061] Example 12

[0062] 900 ml of ultrapure water was accurately measured and placed in a medium-sized reactor as the aqueous phase. 0.3 g of dioctadecyldimethylammonium bromide and 1 g of PLA (10 kDa) were accurately weighed and dissolved in 150 ml of organic solvent (ethanol:acetone = 1:1). The mixture was ultrasonically mixed and then placed in the titration apparatus of the reactor as the oil phase. Under constant stirring at 210–260 rpm / min, the oil phase was gradually added dropwise to the aqueous phase over 1 hour. After addition, the liquid in the reactor was immediately transferred to a 2 L flask and rotary evaporated under reduced pressure (25°C, 0.08 Pa) for 6 hours to obtain a cationic lipid nanosphere suspension. This suspension was washed three times using a Vivaflow 50 membrane with a molecular weight cutoff of 100 kDa and then concentrated to obtain the cationic lipid nanovaccine adjuvant. The particle size was 130–140 nm, and the yield was 97.5%. Using the same adsorption operation as in Example 1, except that the antigen was replaced with influenza virus NM2e, an adsorption rate of 80% for influenza virus NM2e was obtained.

[0063] Example 13

[0064] 900 ml of ultrapure water was accurately measured and placed in a medium-sized reactor as the aqueous phase. 0.3 g of dioctadecyldimethylammonium bromide and 1 g of PLA (10 kDa) were accurately weighed and dissolved in 150 ml of organic solvent (ethanol:acetone = 1:1). The mixture was ultrasonically mixed and then placed in the titration apparatus of the reactor as the oil phase. Under constant stirring at 210–260 rpm / min, the oil phase was gradually added dropwise to the aqueous phase over 1 hour. After addition, the liquid in the reactor was immediately transferred to a 2 L flask. The flask was then rotary evaporated under reduced pressure (25°C, 0.08 Pa) for 6 hours to obtain a cationic lipid nanosphere suspension. This suspension was washed three times using a Vivaflow 50 membrane with a molecular weight cutoff of 100 kDa and concentrated to obtain the cationic lipid nanovaccine adjuvant. The particle size was 130–140 nm, and the yield was 97%. Using the same adsorption operation as in Example 1, except that the antigen was replaced with the MERS virus antigen, the adsorption rate of the MERS virus antigen was 90%.

[0065] Example 14

[0066] 900 ml of ultrapure water was accurately measured and placed in a medium-sized reactor as the aqueous phase. 0.3 g of dioctadecyldimethylammonium bromide and 1 g of PLA with a molecular weight of 10 kDa were accurately weighed and dissolved in 150 ml of organic solvent (ethanol:acetone = 1:1). The mixture was ultrasonically stirred until homogeneous and then placed in the titration apparatus of the reactor as the oil phase. The oil phase was gradually added dropwise to the aqueous phase over a constant stirring speed of 210–260 rpm for 1 h. After the addition was complete, the liquid in the reactor was immediately transferred to a 2 L flask. The flask was then rotary evaporated under reduced pressure of 25°C and 0.08 Pa for 6 h to obtain a cationic lipid nanosphere suspension. This suspension was washed once using a Vivaflow 50 membrane with a molecular weight cutoff of 100 kDa and concentrated to obtain the cationic lipid nanovaccine adjuvant. The particle size was 140–150 nm, and the yield was 96%. Using the same adsorption operation as in Example 1, the adsorption rate for OVA antigen was 73%.

[0067] Example 15

[0068] 900 ml of ultrapure water was accurately measured and placed in a medium-sized reactor as the aqueous phase. 0.3 g of dioctadecyldimethylammonium bromide and 1 g of PLA with a molecular weight of 10 kDa were accurately weighed and dissolved in 150 ml of organic solvent (ethanol:acetone = 1:1). The mixture was ultrasonically stirred until homogeneous and then placed in the titration apparatus of the reactor as the oil phase. Under constant stirring speed of 210–260 rpm / min, the oil phase was gradually added dropwise to the aqueous phase over a period of 1 h. After the addition was complete, the liquid in the reactor was immediately transferred to a 2 L flask. The flask was then rotary evaporated under reduced pressure of 25°C and 0.08 Pa for 6 h to obtain a cationic lipid nanosphere suspension. This suspension was washed 6 times using a Vivaflow 50 membrane with a molecular weight cutoff of 100 kDa and concentrated to obtain the cationic lipid nanovaccine adjuvant. The particle size was 140–150 nm, and the yield was 96%. Using the same adsorption operation as in Example 1, an adsorption rate of 82% for OVA antigen was obtained.

[0069] Comparative Example 1

[0070] 90 ml of ultrapure water was accurately measured and placed in a 250 ml beaker as the aqueous phase. The beaker was placed on a magnetic stirrer, and the stirring speed was adjusted to 500 rpm. 0.03 g of dioctadecyldimethylammonium bromide and 0.1 g of PLA with a molecular weight of 10 kDa were accurately weighed and dissolved in 15 ml of organic solvent (ethanol:acetone = 1:1). The mixture was ultrasonically stirred until homogeneous, and this was used as the oil phase. Using a dropper, the oil phase was slowly added dropwise to the aqueous phase while stirring at a constant speed of 500 rpm. Ten batches were prepared using the same method, and all were placed in a fume hood for overnight evaporation for 10 h. The solidified nanospheres were centrifuged at 20,000 g for 15 min, washed three times, and a small amount of ultrapure water was added to the washed nanospheres for ultrasonic resuspending. The supernatant was collected by centrifugation at 5,000 g for 10 min, yielding the cationic lipid nanovaccine adjuvant prepared by the traditional nanoprecipitation method and washed by centrifugation. Scanning electron micrographs are shown below. Figure 2-B As shown, the particle size was 170~180 nm, the yield was 15%, and the adsorption operation was the same as in Example 1, resulting in an adsorption rate of 84% for OVA antigen.

[0071] Comparative Example 2

[0072] Accurately measure 90 ml of ultrapure water and place it in a 250 ml beaker as the aqueous phase. Place the beaker on a magnetic stirrer and adjust the stirring speed to 500 rpm. Accurately weigh 0.03 g of dioctadecyldimethylammonium bromide and 0.1 g of PLA with a molecular weight of 10 kDa and dissolve them in 15 ml of organic solvent (ethanol:acetone = 1:1). Sonicate the mixture to ensure uniform mixing and use it as the oil phase. Using a dropper, slowly add the oil phase to the aqueous phase while stirring at a constant speed of 500 rpm / min. Prepare 10 batches using the same preparation method and place them in a fume hood overnight for 10 h to evaporate. The solidified nanospheres were centrifuged at 20,000 g for 15 min, washed three times, and then a small amount of ultrapure water was added to the washed nanospheres for ultrasonic resuspending. The supernatant was collected by centrifugation at 5,000 g for 10 min. The cationic lipid nanovaccine adjuvant prepared by the traditional nanoprecipitation method and washed by centrifugation was obtained with a yield of 14%. The same adsorption operation as in Example 1 was used, except that the antigen was replaced with hepatitis B recombinant antigen, and the adsorption rate of hepatitis B recombinant antigen was 95%.

[0073] Comparative Example 3

[0074] Accurately measure 90 ml of ultrapure water and place it in a 250 ml beaker as the aqueous phase. Place the beaker on a magnetic stirrer and adjust the stirring speed to 500 rpm. Accurately weigh 0.03 g of dioctadecyldimethylammonium bromide and 0.1 g of PLA with a molecular weight of 10 kDa and dissolve them in 15 ml of organic solvent (ethanol: acetone = 1:1). Sonicate the mixture to ensure uniform mixing and use it as the oil phase. Use a dropper to slowly add the oil phase to the aqueous phase while stirring at a constant speed of 500 rpm / min. Prepare 10 batches using the same preparation method and place them in a fume hood overnight for 10 h to evaporate. The solidified nanospheres were centrifuged at 20,000 g for 15 min, washed three times, and then a small amount of ultrapure water was added to the washed nanospheres for ultrasonic resuspending. The supernatant was collected by centrifugation at 5,000 g for 10 min. The cationic lipid nanovaccine adjuvant prepared by the traditional nanoprecipitation method and washed by centrifugation was obtained with a yield of 15%. The same adsorption operation as in Example 1 was used, except that the antigen was replaced with influenza virus NM2e, and the adsorption rate of influenza virus NM2e was 79%.

[0075] Comparative Example 4

[0076] Accurately measure 90 ml of ultrapure water and place it in a 250 ml beaker as the aqueous phase. Place the beaker on a magnetic stirrer and adjust the stirring speed to 500 rpm. Accurately weigh 0.03 g of dioctadecyldimethylammonium bromide and 0.1 g of PLA with a molecular weight of 10 kDa and dissolve them in 15 ml of organic solvent (ethanol: acetone = 1:1). Sonicate the mixture to ensure uniform mixing and use it as the oil phase. Use a dropper to slowly add the oil phase to the aqueous phase while stirring at a constant speed of 500 rpm / min. Prepare 10 batches using the same preparation method and place them in a fume hood overnight for 10 h to evaporate. The solidified nanospheres were centrifuged at 20,000 g for 15 min, washed three times, and then resuspended in a small amount of ultrapure water by sonication. The supernatant was collected after centrifugation at 5,000 g for 10 min, yielding a cationic lipid nanovaccine adjuvant prepared by the traditional nanoprecipitation method and washed by centrifugation, with a yield of 16%. The same adsorption operation as in Example 1 was used, except that the antigen was replaced with MERS virus antigen, resulting in an adsorption rate of 86% for the MERS virus antigen.

[0077] Example 16

[0078] Female BALB / c mice aged 6-8 weeks were selected and immunized via intramuscular injection in the leg at days 0, 14, and 28. The cationic nanovaccine adjuvant prepared in Example 3 served as the experimental group, while the aluminum salt adjuvant and pure antigen groups served as the control groups. OVA was used as the antigen. The immunization dose per mouse for each vaccine preparation was 100 μL, and the corresponding antigen amount was 25 μg. Serum was collected from mice on days 14, 21, 28, and 35 post-immunization to detect serum IgG antibody titers. Mice were sacrificed on day 35, and the spleen was aseptically harvested and prepared into a single-cell suspension. The cell culture supernatant was collected after in vitro stimulation culture to determine the cytokine secretion level of spleen cells. The immunization results showed that the cationic nanovaccine adjuvant prepared using a vacuum evaporation combined with tangential flow ultrafiltration significantly increased the serum specific IgG antibody titer compared to the aluminum adjuvant and pure antigen. Figure 3 ), and at the same time, the levels of cellular immune-related cytokines such as interferon-gamma (IFN-γ) Figure 4 ) and interleukin-4 (IL-4) Figure 5 At the level of secretion, it also showed a better immune response than pure antigen and aluminum salt adjuvant.

[0079] Example 17

[0080] Female BALB / c mice aged 6-8 weeks were selected and immunized via intramuscular injection in the leg at days 0, 14, and 28. The cationic nanovaccine adjuvant prepared in Example 10 served as the experimental group, while the aluminum salt adjuvant and pure antigen groups served as the control groups. Recombinant hepatitis B was used as the antigen. The immunization dose per mouse for each vaccine preparation was 100 μL, and the corresponding antigen amount was 2 μg. Serum was collected from mice on days 14, 21, 28, and 35 post-immunization to detect serum IgG antibody titers. Mice were sacrificed on day 35, and spleens were aseptically harvested. Single-cell suspensions were prepared, and the cell culture supernatant was collected after in vitro stimulation culture to determine the cytokine secretion levels of spleen cells. The immunization results confirmed that the cationic nanovaccine adjuvant prepared using a method combining reduced pressure evaporation and tangential flow ultrafiltration significantly increased the serum specific IgG antibody titer and significantly enhanced the secretion of cellular immunity-related cytokines compared to the aluminum adjuvant and pure antigen.

[0081] Example 18

[0082] Female BALB / c mice aged 6-8 weeks were selected and immunized via intramuscular injection in the leg, once on day 0 and again on day 28. The cationic nanovaccine adjuvant prepared in Example 11 served as the experimental group, while the aluminum salt adjuvant and pure antigen groups served as the control groups. Influenza virus NM2e was used as the antigen. The immunization dose per mouse for each vaccine preparation was 100 μL, and the corresponding antigen amount was 10 μg. Serum was collected from mice on days 14, 35, and 42 post-immunization to detect serum IgG antibody titers. Mice were sacrificed on day 42, and spleens were aseptically harvested. Single-cell suspensions were prepared, and the cell culture supernatant was collected after in vitro stimulation culture to determine the cytokine secretion levels of spleen cells. The immunization results confirmed that the cationic nanovaccine adjuvant prepared using a method combining reduced pressure evaporation and tangential flow ultrafiltration significantly enhanced humoral and cellular immunity levels, especially cellular immunity, compared to the aluminum adjuvant and pure antigen.

[0083] Example 19

[0084] Female BALB / c mice aged 6-8 weeks were selected and immunized via intramuscular injection in the leg, once on day 0 and again on day 28. The cationic nanovaccine adjuvant prepared in Example 12 served as the experimental group, while the aluminum salt adjuvant and pure antigen groups served as the control groups. MERS influenza virus was used as the antigen. The immunization dose per mouse for each vaccine preparation was 100 μL, and the corresponding antigen amount was 10 μg. Serum was collected from mice on days 14, 35, and 42 post-immunization to detect serum IgG antibody titers. Mice were sacrificed on day 42, and spleens were aseptically harvested. Single-cell suspensions were prepared, and the cell culture supernatant was collected after in vitro stimulation culture to determine the cytokine secretion levels of spleen cells. The immunization results confirmed that the cationic nanovaccine adjuvant prepared using a method combining reduced pressure evaporation and tangential flow ultrafiltration significantly enhanced humoral and cellular immunity levels compared to the aluminum adjuvant and pure antigen.

[0085] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a cationic lipid nanovaccine adjuvant, characterized in that, The method includes: Polylactic acid materials and cationic surfactants are dissolved in an organic solvent and ultrasonically mixed to obtain an oil phase; Using ultrapure water as the aqueous phase, the oil phase is added dropwise to the aqueous phase at a predetermined stirring speed, and stirring is continued until an oil-in-water emulsion is formed. The oil-in-water emulsion was solidified by vacuum evaporation for 3-6 hours at 10-70℃ and -0.01-0.8Pa to obtain a cationic lipid nanosphere suspension. The cationic lipid nanosphere suspension was circulated and washed using a tangential flow ultrafiltration membrane with a molecular weight cutoff of 100 kDa to obtain a cationic lipid nanovaccine adjuvant.

2. The method according to claim 1, characterized in that, The tangential flow ultrafiltration method involves 3 to 4 cycles of cyclic washing.

3. The method according to claim 1, characterized in that, The molecular weight of the polylactic acid material is 8000~20000.

4. The method according to claim 1, characterized in that, The predetermined speed ranges from 210 to 260 rpm / min.

5. The method according to claim 1, characterized in that, The organic solvent is a mixture of ethanol and acetone.

6. The method according to claim 1, characterized in that, The total mass of the polylactic acid material and the cationic surfactant is 100%, the amount of polylactic acid material added is 74% to 91% of the total mass, and the amount of cationic surfactant added is 9% to 26% of the total mass.

7. The method according to claim 1, characterized in that, The polylactic acid materials include any one or a mixture of at least two of polylactic acid copolymers, polylactic acid-polyglycolic acid copolymers, and polylactic acid-polyethylene glycol copolymers; The cationic surfactant includes bis(octadecyldimethylammonium bromide).

8. The method according to claim 1, characterized in that, When the oil-in-water emulsion is formed, the volume ratio of the oil phase to the water phase is 1:4 to 1:

7.

9. A cationic lipid nanovaccine adjuvant prepared by the method according to any one of claims 1 to 8, characterized in that, The particle size of the cationic lipid nanovaccine adjuvant is between 120 and 200 nm.

10. The cationic lipid nanovaccine adjuvant as described in claim 9, characterized in that, The particle size of the cationic lipid nanovaccine adjuvant is between 140 and 160 nm.

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