Bacterial vaccine based on saponin microspheres

Abstract

The invention discloses a vaccine composition which comprises a bacterial antigen and at least one drug carrier, the drug carrier is a saponin-containing polymer microsphere which is formed by freezing and thawing a polymer microsphere with a cavity structure so as to form an opening or a pore on the surface of the polymer microsphere, the preparation method of the vaccine composition comprises the following steps: mixing the drug carrier with a solution containing bacterial antigens, preferably enabling the bacterial antigens to be loaded on the drug carrier in modes of infiltration and / or adsorption and the like, and then centrifuging to remove supernate so as to obtain the polymer microspheres loaded with the antigens.

Description

Technical Field

[0001] This invention relates to the field of biomedical particulate formulation technology, specifically to a bacterial vaccine based on saponin microspheres. Background Technology

[0002] Infectious diseases pose a serious threat to human health and economic development, and vaccines are a key weapon in the fight against them. However, effective protection requires multiple vaccinations, which not only disrupt daily life but also reduce willingness to be vaccinated and overall vaccination rates, severely hindering comprehensive prevention and control of infectious diseases. Therefore, there is still an urgent need to innovate vaccine formulations to reduce the number of doses required and establish long-term immune protection.

[0003] Polymer microsphere technology, dating back to the 1950s, encapsulates solids, liquids, or gases within cavities. Antigen release systems prepared in this way can protect antigens from rapid degradation and regulate their release behavior, leading to its widespread research in the vaccine field. Current polymer microsphere-loaded antigens largely replicate the release behavior of loaded drugs, exhibiting slow release characteristics. While this avoids burst release and achieves sustained immune stimulation, it neglects the self-enhancing effect of multiple vaccination strategies. There is an urgent need for a polymer microsphere preparation technology with adjustable drug release.

[0004] The information in the background section is merely intended to illustrate the general background of the invention and should not be construed as an admission or implication in any way that such information constitutes prior art known to those skilled in the art. Summary of the Invention

[0005] To address at least some of the technical problems in the prior art, this invention provides a drug carrier that uses a freeze-thaw process to create openings or pores on the surface of polymer microspheres. Drug loading is achieved through physical methods, such as drug diffusion or adsorption, and the loaded drug is released in stages, thus broadening the application of polymer microspheres in the biomedical field. Specifically, this invention includes the following:

[0006] This invention provides a vaccine composition comprising a bacterial antigen and at least one drug carrier, wherein the drug carrier is a polymer microsphere having a chamber structure that has undergone freeze-thaw treatment to form openings or pores on its surface. The preparation method of the vaccine composition comprises the following steps: mixing the drug carrier with a solution containing the antigen, such that the antigen is preferably loaded onto the drug carrier by means of infiltration and / or adsorption, and then removing the supernatant by centrifugation to obtain polymer microspheres loaded with the antigen.

[0007] In some embodiments, the bacterial antigen is a Pseudomonas aeruginosa antigen; further, the bacterial antigen is a Pseudomonas aeruginosa V antigen (PcrV antigen), or a Pseudomonas aeruginosa outer membrane lipoprotein I antigen (OprI antigen), or a combination of PcrV antigen and OprI antigen.

[0008] In some embodiments, the drug carrier has or substantially has a single-chamber structure.

[0009] In some embodiments, the polymer microspheres are prepared from natural or synthetic polymers, preferably, the polymer includes at least one of polylactic acid, polylactic acid-glycolic acid copolymer, polyglycolic acid, and polyethylene glycol-lactic acid copolymer.

[0010] In some embodiments, the drug carrier loaded with the antigen is further sealed to form a sealed microcapsule loaded with the antigen.

[0011] In some embodiments, the polymer microspheres or sealed microcapsules loaded with antigens are further subjected to a lyophilization process to produce a lyophilized formulation.

[0012] In some embodiments, the average particle size of the polymer microspheres is 10-30 μm, for example 15-30 μm, and the average surface wall thickness of the polymer microspheres is 0.1-10 μm, for example 0.2-5 μm, preferably 0.5-4 μm, for example 1.5-2.5 μm, for example 0.5-1.5 μm, for example 1-2 μm.

[0013] In some embodiments, the average particle size of the polymer microspheres is 1-10 μm, preferably 2-8 μm, and the average surface wall thickness of the polymer microspheres is 0.5-1.5 μm, for example 0.8-1.2 μm.

[0014] In some embodiments, the freeze-thaw process involves exposing a suspension containing the polymer microspheres to a cryogenic environment for freezing, and then thawing it by heating it to disrupt the surface structure of the polymer microspheres to create openings or pores, wherein the cryogenic environment is below the freezing point of the suspension system containing the polymer microspheres.

[0015] In some embodiments, the polymer microspheres are frozen to cause ice to form inside the polymer microspheres, such that the ice crystals formed during freezing puncture or burst the polymer microspheres.

[0016] In some embodiments, the polymer microspheres further contain saponins, such as ophiopogonin, more preferably ophiopogonin D'.

[0017] In some embodiments, the vaccine composition is an anti-Pseudomonas aeruginosa vaccine.

[0018] The present invention also provides the use of the vaccine composition of the present invention in medicaments for the prevention and / or treatment of bacterial infections, particularly Pseudomonas aeruginosa infections. Attached Figure Description

[0019] Figure 1 Laser confocal images and statistical graphs of wall thickness and particle size measurements of single-cavity microspheres prepared in Examples 1 and 2;

[0020] Figure 2 An optical microscope image of the single-cavity microspheres prepared in Example 3;

[0021] Figure 3 Optical microscope images of single-cavity microspheres prepared in Examples 4 and 5;

[0022] Figure 4 An optical microscope image of the multi-cavity microspheres prepared in Example 6;

[0023] Figure 5 Light mirror images taken by a high-speed camera during the freezing process of single-chamber microcapsules in different liquid systems;

[0024] Figure 6 Light micrographs and statistical graphs of the measured slit lengths after different freeze-thaw cycles in Examples 8 and 9;

[0025] Figure 7 Light microscopy and scanning electron microscopy images of the drug carrier prepared in Example 10;

[0026] Figure 8 Laser confocal images of proteins loaded after different freeze-thaw cycles in Examples 11 and 12, and a statistical graph of the measured loading positivity rate;

[0027] Figure 9 Scanning electron microscope image of the sealed polymer microspheres prepared in Example 13;

[0028] Figure 10 The images and quantitative curves of the laser released in vitro as measured in Example 14 are shown.

[0029] Figure 11 The images and quantitative curves of fluorescence released in mice as determined in Example 14 are shown.

[0030] Figure 12 The quantitative curve of release in mice as determined in Example 15;

[0031] Figure 13Light microscopy images and particle size distribution of the saponin microspheres prepared in Example 16;

[0032] Figure 14 The antibody titers against the two antigens in mouse serum as determined in Example 18;

[0033] Figure 15 The survival curve of mice after acute bacterial infection, as measured in Example 19. Detailed Implementation

[0034] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0035] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that the upper and lower limits of the range and each intermediate value between them are specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0036] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0037] Drug carrier

[0038] In one aspect, this invention provides a drug carrier. In a preferred embodiment, the drug carrier is a post-drug-loadable polymer microsphere, which enables drug loading after microsphere preparation, and the loaded drug exhibits adjustable or controllable release behavior. As used in this invention, "post-drug loading" refers to loading the drug after microsphere preparation. The term "adjustable or controllable release behavior" means that the drug release process is controlled by the microsphere structure, and different release stages can exist during the release process. There can be a plateau period between different release stages. There can be only one release stage or multiple release stages. It is understood that the number of release stages, plateau periods, etc., can be controlled by the structure.

[0039] The present invention provides a method for preparing a drug carrier, which includes the following steps: providing a biodegradable polymer microsphere with a chamber structure, and subjecting the polymer microsphere to freeze-thaw treatment to form openings or pores on the surface of the polymer microsphere.

[0040] In some preferred embodiments of the present invention, the polymer microspheres serving as drug carriers further contain saponins.

[0041] Saponins are a class of natural compounds widely found in plants, typically exhibiting strong surface activity and the ability to form foams with water molecules. Their chemical structures usually contain sugar molecules and steroidal or triterpenoid compounds, a structure that gives saponins certain immunostimulatory effects. Examples of plant saponins include Ophiopogon japonicus saponins, Fritillaria cirrhosa saponins, Paris polyphylla saponins, and Ophiopogon japonicus saponins, which can promote immune responses and activate the body's immune cells. In this invention, preferably, the saponin is Ophiopogon japonicus saponin, such as Ophiopogon japonicus saponin D'. Ophiopogon japonicus saponin (OPD) is derived from the roots of the plant Ophiopogon japonicus and is an important bioactive steroidal saponin. In this invention, the average particle size of the polymer microspheres is 0.5-500 μm, preferably 1-800 μm, even more preferably 1-600 μm, further preferably 1-400 μm, more preferably 1-200 μm, even more preferably 1-100 μm, and even more preferably 3-30 μm, for example, 10-30 μm, such as 3, 5, 10, 15, 20, 25, 30 μm, or even more preferably 1-10 μm, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 μm.

[0042] In some preferred embodiments, the internal structure of the polymer microspheres of the present invention comprises a single chamber, where a single chamber means that the microsphere has only one cavity inside. In another preferred embodiment, the internal structure of the polymer microspheres of the present invention comprises multiple chambers, such as 2, 3, 4, 5, 6, 7, 8, 9, 10 or more chambers. In one preferred embodiment, the internal structure of the polymer microspheres of the present invention is a single-chamber structure or substantially a single-chamber structure. The internal structure of the microspheres, such as single-chamber or multi-chamber structures, can be analyzed, determined and characterized by techniques such as optical microscopy, electron microscopy, scanning probe microscopy, X-ray diffraction and infrared spectroscopy, nuclear magnetic resonance (NMR) technology, microscopic hot stage observation, computed tomography (CT), and NMR imaging.

[0043] In some preferred embodiments of the present invention, for example, when the average particle size of the polymer spheres is large, such as 10-30 μm, the surface wall thickness of the polymer microspheres is 0.2-5 μm, preferably 0.5-4 μm, preferably 1-2 μm, preferably 1.5-2.5 μm, for example 1 μm, 1.5 μm, 1.8 μm, 2 μm, 2.5 μm, 3 μm, 4 μm.

[0044] In some preferred embodiments of the present invention, for example, when the average particle size of the polymer spheres is large, such as 1-10 μm, the surface wall thickness of the polymer microspheres is 0.5-1.5 μm, for example 0.8-1.2 μm, such as 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2 μm.

[0045] In some preferred embodiments of the present invention, for example, when the average particle size of the polymer spheres is small, such as 1-5 μm, the surface wall thickness of the polymer microspheres is 0.1-0.5 μm, for example 0.1-0.3 μm, such as 0.1, 0.15, 0.2, 0.25, 0.3, or 0.4 μm.

[0046] In this invention, the polymer used for the polymer microspheres can be a natural polymer or a synthetically produced biodegradable polymer, examples of which include, but are not limited to, at least one of polylactic acid, polylactic-co-glycolic acid copolymer, polyglycolic acid copolymer, polyethylene glycol-lactic acid copolymer, polycaprolactone, dextran, and chitosan. In a preferred embodiment, the polymer materials used for the polymer microspheres of this invention are polylactic-co-glycolic acid copolymer (PLGA) and polylactic acid (PLA).

[0047] In some preferred embodiments, the polymers used in the polymer microspheres of the present invention, such as PLGA or PLA, have a weight-average molecular weight of 20,000 to 150,000. For example, when using PLGA, the weight-average molecular weight of the polymer is 90,000 to 130,000, such as 90,000, 100,000, 110,000, 120,000, or 130,000. When using PLA, the weight-average molecular weight of the polymer is 30,000 to 70,000, such as 30,000, 40,000, 50,000, 60,000, or 70,000.

[0048] In some preferred embodiments, a method for preparing a drug carrier is provided, comprising the step of freeze-thawing chambered biodegradable polymer microspheres. Preferably, the method comprises: (a) preparing an emulsion containing the polymer, and then curing it to prepare polymer microspheres; (b) freeze-thawing the polymer microspheres to obtain polymer microspheres with openings or pores on their surface. Preferably, step (a) further comprises saponins.

[0049] In some preferred embodiments, according to the method for preparing the drug carrier of the present invention, the biodegradable polymer microspheres are prepared by the following method:

[0050] (1) Prepare an oil phase O, wherein the oil phase is a solution containing a polymer matrix and the solvent is an organic solvent; prepare an inner aqueous phase solution W1 and an outer aqueous phase solution W2, wherein a surfactant is added to the outer aqueous phase;

[0051] (2) Disperse the inner aqueous phase into the oil phase to form a water-in-oil W1 / O primary emulsion; then disperse the W1 / O primary emulsion into the outer aqueous phase to form a water-in-oil W1 / O / W2 double emulsion.

[0052] (3) The oil phase is solidified by solvent removal to obtain polymer microspheres;

[0053] The oil phase contains no or substantially no emulsifier, and the prepared polymer microspheres have or substantially have a single-chamber structure.

[0054] In this invention, preferably, the oil phase in the preparation of the drug carrier is a solution containing a polymer matrix and saponins. Preferably, in the preparation of the drug carrier, the polymer matrix and saponins are dissolved in an organic solvent to prepare the oil phase.

[0055] In this invention, preferably, in the preparation of polymer microspheres by the double emulsion solvent method, the volume ratio of the internal aqueous phase (W1) to the oil phase (O) will significantly affect the internal structure and the size of the internal chambers of the prepared polymer microspheres. Preferably, in this invention, the volume ratio of the internal aqueous phase (W1) to the oil phase (O) is 1:1 to 1:15, for example, 1:3 to 1:12, and more preferably 1:5 to 1:10.

[0056] In this invention, preferably, if the volume of the internal aqueous phase is larger than that of the oil phase, then during the preparation process using the double emulsion solvent method, more aqueous phase will be encapsulated within the polymer matrix. This will lead to the formation of more cavities or an increase in the size of existing cavities. Therefore, when the internal aqueous phase is larger, the internal chambers of the microspheres are usually larger or have more internal cavity structures. Furthermore, as the volume of the internal aqueous phase increases, the relatively large internal aqueous phase tends to aggregate in the dispersed oil phase (O phase), forming an unstable multi-cavity structure, causing the microspheres to be prone to structural collapse or morphological instability. If the volume of the internal aqueous phase is smaller than that of the oil phase, less water is encapsulated in the polymer matrix, resulting in smaller or fewer cavities. The presence of internal pores will be smaller, and the internal structure of the microspheres will be more compact and dense, thus generating more uniform and dense microspheres. A higher internal aqueous phase / oil phase ratio tends to form larger or more internal cavities, but it also increases the instability of the microspheres, potentially leading to cavity collapse or irregularly shaped microspheres. Lower internal aqueous / oil phase ratios result in smaller cavities or even denser microspheres with relatively stable internal cavity structures, but may lack the special properties of cavity structures (such as reduced load-bearing capacity). In a preferred embodiment of the invention, an internal aqueous / oil phase ratio of 1:5 yields relatively thin-walled single-cavity microspheres, while in another preferred embodiment, an internal aqueous / oil phase ratio of 1:10 yields relatively thick-walled single-cavity microspheres.

[0057] In this invention, preferably, during the preparation of polymer microspheres using the re-emulsification solvent method, the addition of an osmotic pressure regulator such as sodium chloride to the inner aqueous phase further affects the internal structure of the polymer microspheres, especially the size of the internal chambers and the wall thickness of the microspheres. This effect is mainly achieved by adjusting the osmotic pressure difference between the inner and outer aqueous phases, thus influencing the re-emulsification process. Adding an osmotic pressure regulator (such as sodium chloride) to the inner aqueous phase increases its osmotic pressure. This increase in osmotic pressure means a larger osmotic pressure difference between the inner and outer aqueous phases (W2 phase). Due to this osmotic pressure difference, during the re-emulsification process (W1 / O → W1 / O / W2) and subsequent solvent evaporation, water will permeate from the low-osmotic-pressure outer aqueous phase into the high-osmotic-pressure inner aqueous phase, particularly during the curing process. This significantly affects the morphology and internal structure of the microspheres.

[0058] Preferably, as those skilled in the art will understand, as the osmotic pressure of the inner aqueous phase increases, during the preparation process, due to the osmotic pressure difference between the inner and outer aqueous phases after solvent evaporation, more of the outer aqueous phase will permeate into the inner aqueous phase, causing the volume of the inner aqueous phase to expand. This will cause the cavities inside the microspheres to gradually increase. With the increase of the osmotic pressure regulator (such as the NaCl concentration) in the inner aqueous phase, the cavity expansion may be more obvious, forming larger cavities or more porous structures under certain conditions. This is because water continuously permeates into the inner aqueous phase, making the internal structure more "hollow".

[0059] Preferably, as those skilled in the art will understand, with the expansion of the internal aqueous phase volume, more of the internal aqueous phase will occupy the space originally occupied by the polymer, and the polymer material will form a coating layer around it. If the internal cavity volume increases significantly, the wall thickness of the microspheres will typically become thinner. The reduction in wall thickness is due to more polymer material participating in the formation of the outer shell, while the interior is an expanding cavity. Due to the osmotic pressure difference causing a large amount of water movement, combined with the dynamic synergy of polymer curing and solvent evaporation, the polymer wall structure may develop some porosity, making it thinner and more fragile.

[0060] This invention first prepares polymer microspheres or particles suitable for freeze-thaw processes. The term "freeze-thaw" as used herein should be interpreted broadly, meaning the process may involve exposing a suspension containing microspheres to a cryogenic environment for solidification, followed by thawing or refreezing by heating, thereby disrupting the surface structure of the polymer microspheres and creating pores. Cryogenic temperature refers to below the freezing point of the microsphere suspension system. Specifically, the freeze-thaw process may involve the following steps: (a) placing the microsphere suspension system in a cryogenic environment to achieve a frozen state. This step can be achieved using a cryogenic coolant, such as liquid nitrogen or dry ice, or any equipment or apparatus capable of providing cryogenic freezing. (b) heating the frozen microsphere suspension to a thawed or refreezed state. Heating can be achieved using any heating device known in the art, such as a hot plate or microwave. The heating temperature can be below, at, or above room temperature, and is not particularly limited thereto, and can be adjusted as needed.

[0061] In some preferred embodiments, according to the method for preparing a drug carrier according to the present invention, the freeze-thaw treatment involves exposing a suspension containing the polymer microspheres to a low-temperature environment for freezing, and then thawing it by heating it to destroy the surface structure of the polymer microspheres to create pores, wherein the low-temperature environment refers to a suspension system containing the polymer microspheres below the freezing point temperature.

[0062] In some preferred embodiments, according to the method for preparing a drug carrier according to the present invention, the polymer microspheres containing the polymer microspheres are frozen to cause the interior of the polymer microspheres to freeze, so that the ice crystals formed by the freezing can pierce the polymer microspheres. For example, if the polymer microspheres contain water, water molecules form ice crystals when frozen. These ice crystals can have a significant destructive effect on the structure of the polymer material, especially when the water content is high. Repeated freezing and thawing can further lead to physical cracking and changes in cracks.

[0063] In some preferred embodiments, common freeze-thaw procedures include a freezing step and a thawing step, and preferably, the thawing step can also be understood as a melting step.

[0064] In some preferred embodiments, the freezing step in this invention involves cooling the polymer microspheres to a low temperature, typically involving programmed cooling, rapid cooling, or slow cooling. In one preferred embodiment, the invention employs a programmed cooling process, gradually reducing the temperature by setting a suitable temperature profile, for example, rapid programmed cooling or a high-rate programmed cooling process, so that ice crystals quickly form to puncture the microspheres. Programmed cooling usually requires specialized programmed cooling equipment or a cryogenic freezer with controlled rates. When using rapid cooling, the polymer microspheres can be quickly placed in a liquid nitrogen (-196°C) or cryogenic (-80°C) cooling system, causing the moisture to freeze rapidly, promoting ice crystal formation and disrupting the original structure. When using slow freezing, the polymer microspheres are slowly cooled from room temperature to the freezing temperature, typically in a cryogenic chamber. In a preferred embodiment of the invention, the freezing step preferably employs programmed cooling, such as a rapid cooling program, for example, a programmed cooling process with a cooling rate of 30°C / min.

[0065] In some preferred embodiments, the thawing step in this invention includes, for example, room temperature thawing, which is relatively simple and involves slowly thawing the frozen sample at room temperature, ensuring that the moisture or structure gradually recovers. The thawing step can also employ water bath thawing, which involves rapidly thawing the frozen sample in a water bath (e.g., a constant temperature water bath) at, for example, 37°C. The thawing step can also employ cold room or low-temperature thawing: the sample is moved to a 4°C refrigerated environment for slow thawing, or thawing is performed using a cold table device. In a preferred embodiment of this invention, the thawing step preferably employs room temperature thawing.

[0066] In this invention, the method for preparing polymer microspheres is not particularly limited, as long as microspheres with chambers can be obtained. In a preferred embodiment, it includes the steps of preparing a premix and transforming the premix to produce polymer microspheres. The premix is ​​generally a water-in-oil-in-water composite emulsion, which includes an inner aqueous phase, an outer aqueous phase, and an oil phase. Exemplarily, the preparation of the premix includes preparing an inner aqueous phase solution W1, such as an aqueous sodium chloride solution; preparing an oil phase O, such as an ethyl acetate solution of polylactic acid-glycolic acid (PLGA) at a certain concentration; and preparing an outer aqueous phase W2, such as an aqueous solution of polyvinyl alcohol at a certain concentration. It should be noted that the oil phase of this invention does not contain any type of surfactant, thus enabling the controllable preparation of chambers.

[0067] In a preferred embodiment of the present invention, after preparing the water-in-oil-in-water composite emulsion, the oil phase is solidified and the residual surfactant is removed, preferably by sieving or centrifugal washing.

[0068] In this invention, the water-in-oil-in-water composite emulsion is obtained through homogenization or mechanical stirring. It generally includes the preparation of a primary emulsion and a secondary emulsion. Exemplarily, the primary emulsion is prepared using a traditional stirring method, such as mixing the oil phase and the inner aqueous phase in a centrifuge tube and obtaining the primary emulsion through mechanical shearing. Exemplarily, the secondary emulsion is prepared using a traditional stirring method, such as adding the primary emulsion to the outer aqueous phase and obtaining the secondary emulsion, i.e., the water-in-oil-in-water composite emulsion, through mechanical shearing.

[0069] In other preferred embodiments, the polymer microspheres of the present invention can also be obtained by membrane emulsification. An exemplary method includes passing an oil-in-water primary emulsion through a membrane tube having a micron-sized pore size, for example, 1-50 μm, preferably 15-30 μm, and passing it through the membrane multiple times to obtain a secondary emulsion, and then allowing the secondary emulsion to evolve over a suitable time to produce polymer microspheres.

[0070] In some preferred embodiments, the solvent of the inner aqueous phase is selected from at least one of water, water-in-injection buffer, sodium chloride aqueous solution, phosphate buffer, glucose solution, and sucrose solution, and sodium chloride can be used as an osmotic pressure regulator of the inner aqueous phase.

[0071] In some preferred embodiments, the solvent of the oil phase in this invention includes one or a combination of at least two of alcohols, ketones, esters, ethers, alkylbenzenes, haloalkanes, and haloaromatics that can dissolve polymers. More preferably, it is a volatile organic solvent, such as n-butanol, diethyl ether, chloroform, tetrachloromethane, etc. More preferably, it is a volatile organic solvent that is partially soluble in water, such as ethyl acetate, phenol, etc.

[0072] In some preferred embodiments, the organic solvent used in this invention has a solubility in water of less than 10%, more preferably less than 2%, and most preferably is an organic solvent insoluble in water. In one preferred embodiment, the organic solvent is selected from one or more of dichloromethane, trichloromethane, ethyl acetate, ethyl propionate, propyl acetate, or acetone, more preferably dichloromethane or ethyl acetate, and most preferably dichloromethane. It can also be any blend of the above-mentioned different organic solvents; the specific type or volume depends on the preparation parameters such as the membrane material used.

[0073] In some preferred embodiments, the external aqueous phase further comprises an emulsifier selected from at least one of sodium alginate, polyvinyl alcohol cellulose, Tween 80, methylcellulose, gelatin, polysorbate, lysine, gum arabic, and poloxamer 188.

[0074] In some preferred embodiments, the preparation of the colostrum in this invention can be achieved by mixing an internal aqueous phase with a salt concentration of 0.05-2 mg / mL (such as sodium chloride) with an oil phase of 20-100 mg / mL polymer at a mass ratio of 1:1-1:30. The salt concentration is preferably 0.06-1.9 mg / mL, more preferably 0.07-1.8 mg / mL, and even more preferably 0.08-1.7 mg / mL, for example: 0.08, 0.09, 0.10, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7 mg / mL. The polymer concentration is preferably 30-90 mg / mL, more preferably 40-80 mg / mL, and even more preferably 45-75 mg / mL, for example 45, 50, 55, 60, 65, 70, 75 mg / mL.

[0075] In some preferred embodiments of the present invention, during the preparation of the vaccine adjuvant, the mass ratio of polymer to saponin is 5000:1-10:1, preferably 1000:1-30:1, and even more preferably 300:1-50:1, for example 300:1, 200:1, 100:1, 50:1.

[0076] In some preferred embodiments, the present invention further includes a step of selecting polymer microspheres with appropriate wall thickness after step (a) of preparing polymer microspheres. The present invention has found through research that a suitable wall thickness is more beneficial for subsequent drug loading and drug release regulation. In one preferred embodiment, appropriate settling times are selected for different suspension systems to obtain polymer microspheres with different wall thicknesses. For example, when a polymer microsphere-water mixture is selected, polymer microspheres with a settling time of less than 30 minutes have a larger wall thickness, which is detrimental to the drug loading process. Polymer microspheres with a settling time of 30-120 minutes have an average wall thickness of 1-3 μm, preferably 1.5-2.5 μm, and exhibit excellent drug loading and drug release performance. Polymer microspheres with a settling time of more than 120 minutes have a smaller wall thickness; although they can be loaded with drugs, their drug release performance is not as good as that of polymer microspheres with a settling time of 30-120 minutes. It is understandable that when the sedimentation system is a salt ion or other type of solution, microspheres with a specific sedimentation time can be selected according to actual needs, or even microspheres with a suitable wall thickness can be obtained directly through operations such as density gradient centrifugation.

[0077] In some preferred embodiments, step (b) of the present invention is a step of preparing polymer microspheres with openings or pores on the surface using a freeze-thaw process. After the freeze-thaw treatment, the microsphere structure, such as the microsphere surface structure, will change, for example, pores will be generated, so that the microsphere can be used to load drugs by physical bonding methods, such as infiltration, adsorption and other methods.

[0078] In some preferred embodiments of the invention, the freezing temperature and freeze-thaw sequence in step (b) can vary depending on the suspension system. For example, the polymer microspheres and water mixture can be frozen at a low temperature and then thawed at room temperature. The freeze-thaw process can be performed once or multiple times, and the number of repetitions is not particularly limited, but preferably 2-10 times, and even more preferably 3-9 times, for example 3, 4, 5, 6, 7, 8, or 9 times. The present invention has found that increasing the number of freeze-thaw cycles significantly increases the number of cracks. Therefore, those skilled in the art can adjust the number of freeze-thaw cycles as needed, for example, according to the size of the pores required for drug loading.

[0079] In some preferred embodiments of the present invention, the low temperature during the freeze-thaw process refers to the temperature below the freezing point of the suspension system. For example, the aqueous suspension of polymer microspheres is selected to be 0 to -200°C, preferably -30 to -190°C, even more preferably -40 to -180°C, and further preferably -50 to -170°C, for example -60, -70, -80, -90, -100, -110, -120, -130, -140, -150, -160, and -170°C.

[0080] vaccine composition

[0081] This invention provides a vaccine composition comprising a bacterial antigen and at least one drug carrier, wherein the drug carrier is a polymer microsphere having a chamber structure that has undergone freeze-thaw treatment to form openings or pores on its surface. The preparation method of the vaccine composition comprises the following steps: mixing the drug carrier with a solution containing the antigen, preferably loading the antigen onto the drug carrier by means of infiltration and / or adsorption, and then removing the supernatant by centrifugation to obtain polymer microspheres loaded with the antigen.

[0082] In some preferred embodiments, the antigen is a Pseudomonas aeruginosa vaccine protein antigen, further, it is a single Pseudomonas aeruginosa V antigen (PcrV protein), or Pseudomonas aeruginosa outer membrane lipoprotein I (OprI protein), or a mixture of both PcrV protein and OprI protein.

[0083] In a preferred embodiment of the present invention, the method for preparing the vaccine composition includes: mixing the drug carrier with a solution containing antigen to allow the antigen to enter the interior of microspheres. For example, antigen loading after polymer microsphere preparation can be achieved by physical means, such as diffusion or adsorption. The antigen diffuses into the interior of the microspheres through infiltration, adsorption, etc., and the loaded antigen is released in stages. Preferably, the drug carrier is mixed with a solution containing antigen, and then the supernatant is removed by centrifugation to obtain polymer microspheres loaded with antigen.

[0084] In a preferred embodiment, the invention further includes: sealing the drug carrier loaded with antigen to form a sealed microcapsule loaded with antigen.

[0085] In this invention, those skilled in the art will understand that "sealing" means embedding, fixing, or retaining the antigen within the polymer microspheres. In this invention, sealing the microcapsule does not mean that the microcapsule surface has no openings, but simply that the antigen and / or other substances, such as other drugs, can be partially embedded, fixed, or retained within the microcapsule through the sealing process. The antigen and / or other substances, such as other drugs, may be released from the microcapsule through degradation.

[0086] In a preferred embodiment of the present invention, the sealing process of the drug carrier preferably includes solvent swelling, irradiation, and heating annealing. Those skilled in the art can also seal the drug carrier based on their professional knowledge / new technologies.

[0087] In a preferred embodiment of the present invention, the heating annealing method is a relatively ideal sealing method. For example, the unique self-healing sealing properties of biodegradable polymer blends such as polylactic acid can be utilized. This method involves irradiation or heating to cause the molecules on the surface of the microspheres to absorb energy and rearrange, thereby healing and sealing the pores on the surface. For example, by slowly heating the drug carrier carrying the drug to near the glass transition temperature of the microspheres, preferably 1 to 2°C below the glass transition temperature of the microspheres, and then slowly cooling it after a period of time, the pores on the surface of the open drug carrier are closed to prepare sealed microcapsules. Antigens and other drugs are effectively encapsulated in the microcapsules, and the loading rate and embedding rate are stable.

[0088] In a preferred embodiment of the present invention, a solvent-assisted heating annealing method can be used. For example, adding a small amount of organic solvent and surfactant that can dissolve the polymer microspheres during the heating annealing process can lower the required glass transition temperature and effectively encapsulate antigens and other drugs in microcapsules, resulting in stable loading and encapsulation rates. In one embodiment of the present invention, the small amount of organic solvent and surfactant is, for example, 4% ethyl acetate (by volume) and 0.375% PVA (by mass).

[0089] In a preferred embodiment of the present invention, the vaccine composition comprising the antigen and the drug carrier described in the present invention can be prepared into a freeze-dried formulation through a freeze-drying process.

[0090] In this invention, the vaccine composition further includes, optionally, a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known in the art and can be determined by those skilled in the art to meet clinical standards. Pharmaceutically acceptable carriers include diluents and excipients.

[0091] Examples of suitable pharmaceutically acceptable carriers include, but are not limited to: (1) Dulbecco phosphate buffered solution, pH about 7.4, containing or not containing about 1 mg / mL to 25 mg / mL human serum albumin; (2) 0.9% saline (0.9% w / v sodium chloride); and (3) 5% (w / v) glucose; which may also contain antioxidants such as tryptophan and stabilizers such as Tween20.

[0092] In a preferred embodiment, the vaccine composition of the present invention is for the prevention, treatment and / or improvement of disease, for the preventive or therapeutic treatment of tumors or infectious diseases in mammalian subjects.

[0093] The vaccine compositions of the present invention can be in any suitable dosage form, such as injections, suspensions, emulsions, etc. The vaccine compositions of the present invention can be administered into the body in known ways, such as by intramuscular injection to the tissue of interest, or optionally by intravenous, percutaneous, intranasal, oral, mucosal, or other delivery methods.

[0094] The present invention further provides a compound formulation according to the needs of vaccines, wherein the drug carrier has a single-chamber structure, or a multi-chamber structure, or different polymer microsphere wall thicknesses, or any combination thereof.

[0095] For example, regarding vaccines, based on immunodynamics, different types of the aforementioned drug carriers (polymer microspheres) can be compounded to achieve better pulsed release effects. Different types of microspheres can be compounded in any combination of polymer microspheres with a single chamber, polymer microspheres with a multi-chamber structure, and polymer microspheres with different wall thicknesses. For instance, the compounded microspheres can be a combination of polymer microspheres with a single chamber and polymer microspheres with a multi-chamber structure, a combination of polymer microspheres with different wall thicknesses, or even a combination of single-chamber and multi-chamber polymer microspheres with different wall thicknesses.

[0096] In this invention, the immunization with the vaccine can be therapeutic or preventative.

[0097] In some preferred embodiments, the post-drug-loaded pulse-release polymer microspheres of the present invention, i.e., the drug carriers of the present invention, can effectively achieve the phased release of drugs such as internal antigens. When internally loaded with antigens, they can be used as vaccines to induce a potent immune response and establish immune protection through subcutaneous or intramuscular injection.

[0098] In some embodiments, the drug carrier and vaccine composition provided by the present invention have at least the following advantages:

[0099] (1) The drug carrier of the present invention achieves surface openings or cracks through freeze-thaw cycles, allowing for post-loading of drugs such as antigens after microsphere preparation via spontaneous diffusion, effectively improving encapsulation efficiency and ensuring the structural and activity stability of antigens and other drugs. The drug carrier itself can be freeze-dried, and freeze-dried formulations of vaccine compositions can be prepared by selecting appropriate freeze-drying conditions depending on the different antigens and other drugs being encapsulated.

[0100] (2) The drug carrier of the present invention, as a drug delivery system, can control the release behavior of the vaccine composition by controlling the wall thickness of the polymer microspheres, the internal chamber structure of the polymer microspheres, and their compounding. For example, the drug release can have multiple stages, with a plateau period between the stages, or it can have a sustained release process.

[0101] (3) The drug carrier of the present invention, as a vaccine preparation, can effectively promote the local recruitment and activation of immune cells, induce efficient humoral and cellular immunity, establish an effective immune protection barrier, and enhance the protective ability of the vaccine.

[0102] The following embodiments are provided to illustrate the principles and practice of the embodiments disclosed herein more clearly to those skilled in the art, and should not be construed as limiting the scope of any claimed embodiments.

[0103] Example 1

[0104] This embodiment uses polylactic acid-glycolic acid copolymer as the material to prepare polymer microspheres, as detailed below.

[0105] Polylactic acid-glycolic acid copolymer (PLGA) labeled with Nile Red dye was dissolved in ethyl acetate to prepare a 50 mg / mL PLGA oil phase solution; a 1 mg / mL NaCl aqueous solution was prepared as the inner aqueous phase; polyvinyl alcohol (PVA) was placed in water and stirred in an 80°C water bath for 5 h to prepare a 15 mg / mL PVA aqueous solution as the outer aqueous phase.

[0106] 2 mL of oil phase was mixed with 200 μL of internal aqueous phase and stirred at 7000 rpm for 30 seconds in a homogenizer to obtain a primary emulsion. Then, a conventional mechanical stirring method can be used to prepare a W / O / W complex emulsion by adding the primary emulsion to 15 mL of external aqueous phase and stirring at 7000 rpm for 2 minutes in a homogenizer. The W / O / W complex emulsion was vortexed for 15 minutes to transform its oil phase into a single-chamber structure. Then, the complex emulsion was extracted with sufficient pure water or evaporated in a fume hood to remove organic solvents, resulting in solidified mixed polymer microspheres. The mixed polymer microspheres were mixed with 10 mL of water and placed in a 10 mL centrifuge tube. Microspheres with settling times between 30 minutes and 2 hours were collected. The microspheres were observed using laser confocal microscopy, their surface wall thickness was calculated, and their particle size was measured using a particle size analyzer. Figure 1 ).

[0107] Example 2

[0108] This embodiment uses polylactic acid-glycolic acid copolymer as the material to prepare polymer microspheres. The main difference from Example 1 is in the sedimentation time, as detailed below.

[0109] Polylactic acid-glycolic acid copolymer (PLGA) labeled with Nile Red dye was dissolved in ethyl acetate to prepare a 50 mg / mL PLGA oil phase solution; a 1 mg / mL NaCl aqueous solution was prepared as the inner aqueous phase; polyvinyl alcohol (PVA) was placed in water and stirred in an 80°C water bath for 5 h to prepare a 15 mg / mL PVA aqueous solution as the outer aqueous phase.

[0110] 2 mL of oil phase was mixed with 200 μL of internal aqueous phase and stirred at 7000 rpm for 30 seconds in a homogenizer to obtain a primary emulsion. Then, a conventional mechanical stirring method can be used to prepare a W / O / W complex emulsion by adding the primary emulsion to 15 mL of external aqueous phase and stirring at 7000 rpm for 2 minutes in a homogenizer. The W / O / W complex emulsion was vortexed for 15 minutes to transform its oil phase into a single-chamber structure. Then, the complex emulsion was extracted with sufficient pure water or evaporated in a fume hood to remove organic solvents, resulting in solidified mixed polymer microspheres. The mixed polymer microspheres were mixed with 10 mL of water and placed in a 10 mL centrifuge tube. Microspheres with a settling time within 30 minutes were collected. The obtained microspheres were observed using laser confocal microscopy, their surface wall thickness was calculated, and their particle size was measured using a particle size analyzer. Figure 1 ).

[0111] Example 3

[0112] This embodiment uses polylactic acid-glycolic acid copolymer as the material to prepare polymer microspheres. The main difference from Example 1 is in the final sedimentation time, as detailed below.

[0113] Polylactic acid-glycolic acid copolymer (PLGA) was dissolved in ethyl acetate to prepare a 50 mg / mL PLGA oil phase solution; a 1 mg / mL NaCl aqueous solution was prepared as the inner aqueous phase; polyvinyl alcohol (PVA) was placed in water and stirred in an 80°C water bath for 5 h to prepare a 15 mg / mL PVA aqueous solution as the outer aqueous phase.

[0114] 2 mL of oil phase was mixed with 200 μL of internal aqueous phase and stirred at 7000 rpm for 30 seconds in a homogenizer to obtain a primary emulsion. Then, a conventional mechanical stirring method can be used to prepare a W / O / W complex emulsion. The primary emulsion was added to 15 mL of external aqueous phase and stirred at 7000 rpm for 2 minutes in a homogenizer to obtain a W / O / W complex emulsion. The W / O / W complex emulsion was vortexed for 15 minutes to transform its oil phase into a single-chamber structure. Then, the complex emulsion was extracted with sufficient pure water or evaporated in a fume hood to remove organic solvents, yielding solidified mixed polymer microspheres. The mixed polymer microspheres were mixed with 10 mL of water and placed in a 10 mL centrifuge tube. Microspheres with a settling time of more than 2 hours were collected. The microspheres were observed under a light microscope. Figure 2 Its wall thickness is thinner than that of Example 1.

[0115] Example 4

[0116] This embodiment demonstrates a method for preparing single-cavity microspheres using polylactic acid-glycolic acid copolymer as the material, as detailed below.

[0117] Polylactic acid-glycolic acid copolymer (PLGA) was dissolved in ethyl acetate to prepare a 100 mg / mL PLGA oil phase solution; pure water was used as the inner aqueous phase; polyvinyl alcohol (PVA) was placed in water and stirred in an 80°C water bath for 5 h to prepare a 30 mg / mL PVA aqueous solution as the outer aqueous phase.

[0118] 1 mL of the oil phase was mixed with 200 μL of the internal aqueous phase and ultrasonically vibrated at 30% power for 12 seconds to obtain a primary emulsion. Then, using a conventional mechanical stirring method, the primary emulsion was added to 15 mL of the external aqueous phase and homogenized at 8000 rpm for 2 minutes to obtain a W / O / W complexed emulsion. This complexed emulsion was then immediately extracted with sufficient pure water or evaporated in a fume hood to remove organic solvents, yielding solidified single-cavity polymer microspheres.

[0119] Microspheres were observed using a light microscope, and their particle size was measured using dynamic light scattering. Figure 3 As shown, single-cavity microspheres can be prepared.

[0120] Example 5

[0121] This embodiment demonstrates a method for preparing single-cavity microspheres using polylactic acid-glycolic acid copolymer as the material, as detailed below.

[0122] Polylactic acid-glycolic acid copolymer (PLGA) was dissolved in ethyl acetate to prepare a PLGA oil phase solution of 100 mg / mL; pure water was used as the inner aqueous phase; polyvinyl alcohol (PVA) was placed in water and stirred in an 80°C water bath for 5 h to prepare a 30 mg / mL PVA aqueous solution as the outer aqueous phase.

[0123] Take 1 mL of the oil phase and mix it with 100 μL of the internal aqueous phase, then agitate at 30% ultrasonic power for 12 seconds to obtain the primary emulsion. Subsequently, a conventional mechanical stirring method can be used to prepare the emulsion: add the primary emulsion to 15 mL of the external aqueous phase and stir at 8000 rpm for 2 minutes using a homogenizer to obtain a W / O / W complex emulsion. Immediately afterward, extract the emulsion with sufficient pure water or evaporate it in a fume hood to remove the organic solvent, obtaining solidified mixed polymer microspheres.

[0124] Microspheres were observed using a light microscope, and their particle size was measured using dynamic light scattering. Figure 3 As shown, single-cavity microspheres were also prepared, but with thicker walls.

[0125] Example 6

[0126] This embodiment provides a method for preparing multi-cavity microspheres, characterized by the addition of PEG-PLA copolymer (PELA) to the oil phase.

[0127] Polylactic acid-glycolic acid copolymer (PLGA) and PELA were dissolved together in ethyl acetate at a mass ratio of 9:1 to prepare an oil phase solution with a total concentration of 50 mg / mL. A 1 mg / mL NaCl aqueous solution was prepared as the inner aqueous phase. Polyvinyl alcohol (PVA) was placed in water and stirred in an 80°C water bath for 5 h to prepare a 15 mg / mL PVA aqueous solution as the outer aqueous phase.

[0128] 2 mL of oil phase was mixed with 200 μL of internal aqueous phase and stirred at 7000 rpm for 30 seconds in a homogenizer to obtain a primary emulsion. Then, a conventional mechanical stirring method can be used to prepare a W / O / W complex emulsion by adding the primary emulsion to 15 mL of external aqueous phase and stirring at 7000 rpm for 2 minutes in a homogenizer. This complex emulsion was then directly extracted with sufficient pure water or evaporated in a fume hood to remove organic solvents, yielding solidified multi-cavity polymer microspheres. Microscopic observation of the obtained microspheres revealed a distinct multi-cavity structure. Figure 4 ).

[0129] Example 7

[0130] This embodiment describes the programmed cooling of the polymer microspheres from Example 4, and the cooling process is observed using an optical microscope and a high-speed camera, as detailed below.

[0131] The polymer microspheres prepared in Example 4 were mixed with a 3% PVA aqueous solution to form a suspension, and then cooled to -80°C at a cooling rate of 30°C / min. It was observed that no ice formed inside the microspheres, nor did any ice crystals puncture the microspheres. Figure 5 ).

[0132] The polymer microspheres prepared in Example 4 were mixed with pure water to form a suspension, and then cooled to -80°C at a rate of 30°C / min. It was observed that ice crystals pierced the microspheres instantly after freezing inside, resulting in cracks and pores in the polymer microspheres. Figure 5 This embodiment illustrates that the freeze-thaw loading method of the present invention can only be carried out based on the formation of ice crystals.

[0133] Example 8

[0134] This embodiment describes a freeze-thaw process for loading the polymer microspheres of Example 4 with a drug, as detailed below.

[0135] The polymer microspheres prepared in Example 4 were mixed with an equal volume of water to form a suspension, which was then placed in a -80°C freezer for rapid cooling to -80°C. After freezing at -80°C for 2 hours, the suspension was thawed at room temperature. This freeze-thaw process was repeated 0, 2, 4, and 8 times to obtain polymer microspheres with different drug loading. The polymer microspheres after the freeze-thaw process were characterized using an optical microscope, and the cracks appearing on their surface were measured. Figure 6 Clear cracks can be seen on the surface of the polymer microspheres.

[0136] Example 9

[0137] This embodiment describes a freeze-thaw process for loading the polymer microspheres of Example 5 with a drug, as detailed below.

[0138] The polymer microspheres prepared in Example 5 were mixed with an equal volume of water to form a suspension, which was then placed in a -80°C freezer for rapid cooling to -80°C. After freezing at -80°C for 2 hours, the suspension was thawed at room temperature. This freeze-thaw process was repeated 0, 2, 4, and 8 times to obtain polymer microspheres with different drug loading. The polymer microspheres after the freeze-thaw process were characterized using an optical microscope, and the cracks appearing on their surface were measured. Figure 6 It can be seen that under the same freeze-thaw process, the number and length of cracks in the polymer microspheres of Example 5 are less than those in the polymer microspheres of Example 4.

[0139] Example 10

[0140] This embodiment illustrates a vaccine adjuvant based on polymer microspheres prepared according to Example 4 and a freeze-thaw process.

[0141] Polylactic acid-glycolic acid copolymer (PLGA) was dissolved in ethyl acetate to prepare a 100 mg / mL PLGA oil phase solution; pure water was used as the inner aqueous phase; polyvinyl alcohol (PVA) was placed in water and stirred in an 80°C water bath for 5 h to prepare a 30 mg / mL PVA aqueous solution as the outer aqueous phase.

[0142] 1 mL of the oil phase was mixed with 200 μL of the internal aqueous phase and ultrasonically vibrated at 30% power for 12 seconds to obtain a primary emulsion. Then, using a conventional mechanical stirring method, the primary emulsion was added to 15 mL of the external aqueous phase and homogenized at 8000 rpm for 2 minutes to obtain a W / O / W complexed emulsion. This complexed emulsion was then immediately extracted with sufficient pure water or evaporated in a fume hood to remove organic solvents, yielding solidified single-cavity polymer microspheres.

[0143] Microspheres were mixed with an equal volume of pure water to form a suspension, which was then rapidly cooled to -80°C in a freezer. The suspension was frozen at -80°C for 2 hours, then thawed at room temperature. This freeze-thaw process was repeated four times to obtain a drug carrier based on single-cavity polymer microspheres. Observation using optical and scanning electron microscopy revealed obvious cracks on the surface of the drug carrier. Figure 7 Drugs can be loaded into the microspheres through the cracks.

[0144] Example 11

[0145] This embodiment illustrates the drug loading of the post-drug-loaded polymer microspheres obtained in Example 4.

[0146] Cy5-labeled bovine serum albumin (BSA) was prepared into a 1 mg / mL solution and mixed with the microspheres from Example 4 after different freeze-thaw cycles in Example 8. The mixture was then swirled in a 4°C freezer for 3 days to allow the protein to be loaded via diffusion.

[0147] By examining drug loading using a laser confocal microscope, it can be observed that different freeze-thaw cycles affect drug loading. For example... Figure 8 As shown, the positive rate of drug loading increased continuously when the freeze-thaw cycles were repeated 0, 2, and 4 times. When the freeze-thaw cycles were repeated 8 times, the positive rate of drug loading remained consistent with that of 4 times, reaching its peak.

[0148] Example 12

[0149] This embodiment illustrates the drug loading of the post-drug-loaded polymer microspheres obtained in Example 5.

[0150] Cy5-labeled bovine serum albumin (BSA) was prepared into a 1 mg / mL solution and mixed with the microspheres from Example 5 after different freeze-thaw cycles in Example 9. The mixture was then swirled in a 4°C freezer for 3 days to allow the protein to be loaded via diffusion.

[0151] Examining drug loading using laser confocal microscopy revealed that the number of freeze-thaw cycles affected drug loading. The positive drug loading rate was lower than that shown in Example 11 when freeze-thaw cycles were repeated 0, 2, 4, and 8 times. Figure 8 This shows that the wall thickness of the microspheres affects the protein drug loaded onto the microspheres after freeze-thaw treatment.

[0152] Example 13

[0153] This embodiment illustrates a post-drug-loaded polymer microsphere prepared by sealing polymer microspheres.

[0154] Based on Example 11, after suspension, the sample tube was removed, and the bottom deposited microspheres were collected by centrifugation. A healing solution of 4% (v / v) ethyl acetate and 0.375% PVA aqueous solution was used, mixed at a microsphere volume to healing solution volume ratio of 1:25, and resuspended uniformly in a centrifuge tube. After sealing, the tube was placed in a 40°C constant temperature water bath, and the centrifuge tube was kept rotating to prevent microsphere aggregation. Heating was carried out for 30 minutes to obtain polymer microspheres with sealed surface cracks. Figure 9 ).

[0155] Example 14

[0156] This example illustrates the drug release behavior after the drug carrier loaded with protein in Example 10.

[0157] The microspheres prepared in Example 10 were used as drug carriers and mixed with a Cy5-labeled BSA solution at 1 mg / mL. The mixture was then vortexed at 4°C for 3 days to allow drug loading. The bottom microspheres were collected by centrifugation and washed 2–3 times with PBS to obtain the drug carrier for detecting in vitro and in vivo release.

[0158] The formulation was placed in PBS and subjected to simulated in vitro release in a 37°C constant-temperature shaker. A portion of the formulation was collected daily for laser confocal microscopy to determine the in vitro release behavior. Figure 10 As shown, the drug carrier involves two release processes, which can significantly prolong the drug residence time.

[0159] The formulation was mixed with physiological saline and injected subcutaneously into mice. Mice were then placed in an anesthesia chamber for gas anesthesia (isoflurane) at different time points. After complete anesthesia, they were placed in a small animal in vivo imaging chamber for imaging to detect the in vivo drug release behavior of the drug carrier in mice. Figure 11 As shown, this drug carrier also exhibits a two-stage sustained-release behavior in mice, which can significantly prolong the drug residence time.

[0160] Example 15

[0161] This embodiment proposes a drug carrier composed of microspheres with multiple structures, which can regulate the drug release behavior through compounding.

[0162] The microspheres prepared in Examples 3 and 4 were mixed 1:1 to form composite microspheres. The composite microspheres were then mixed with an equal volume of pure water to form a suspension, which was rapidly cooled to -80°C in a freezer. The suspension was frozen at -80°C for 2 hours and then thawed at room temperature. This freeze-thaw process was repeated four times to obtain the drug carrier based on the composite microspheres.

[0163] The aforementioned compounded microspheres were used as drug carriers and mixed with a Cy5-labeled 1 mg / mL BSA solution. The mixture was then vortexed at 4°C for 3 days to allow drug loading. The bottom microspheres were collected by centrifugation and washed 2–3 times with PBS to obtain the drug carrier for detecting in vitro and in vivo release.

[0164] The formulation was mixed with physiological saline and injected subcutaneously into mice. Mice were then placed in an anesthesia chamber for gas anesthesia (isoflurane) at different time points. After complete anesthesia, they were placed in a small animal in vivo imaging chamber for imaging to detect the in vivo drug release behavior of the drug carrier in mice. Figure 12 As shown, this drug carrier exhibits similar sustained-release behavior in mice, but the drug release is faster than that of the single-cavity microsphere drug carrier in Example 14.

[0165] Example 16

[0166] In this embodiment, polymer microspheres were prepared using polylactic acid-glycolic acid copolymer as the material, and saponin molecules were doped during the process, as detailed below.

[0167] Polylactic acid (PLA) with a weight-average content of 50,000 (Jinan Daigang Company, DG-DLH050) was dissolved in ethyl acetate to prepare a 100 mg / mL PLA solution. Simultaneously, 0.05% (v / v) of Span80 (Guoyao Reagent, 30170828) was mixed in. A 0.2% NaCl aqueous solution was used as the internal aqueous phase. Polyvinyl alcohol (PVA) was dissolved in water and stirred in an 80°C water bath for 5 hours to prepare a 15 mg / mL PVA aqueous solution as the external aqueous phase.

[0168] Take 15 mL of the oil phase and add 300 μl of a 100 mg / mL DMSO solution of Ophiopogon japonicus saponin D' (OPD', Chengdu Puryfa, BP1036) to make the final polylactic acid to Ophiopogon japonicus saponin D' mass ratio 50:1. Mix thoroughly by ultrasonic oscillation in a water bath. Add 15 mL of the inner aqueous phase to the oil phase and stir at 24000 rpm for 5 min using a T25 homogenizer to obtain the primary emulsion. Add the primary emulsion to 225 mL of the outer aqueous phase and stir at 13000 rpm for 1 min using a homogenizer to obtain the secondary emulsion. Immediately extract the emulsion with sufficient pure water or evaporate it in a fume hood to remove the organic solvent, obtaining solidified polylactic acid microspheres containing Ophiopogon japonicus saponin D'.

[0169] Saponin microspheres observed under a light microscope, such as Figure 13 As shown, the polylactic acid microspheres containing ophiopogonin D' have a single-cavity structure and a wall thickness of approximately 1 μm. Their particle size can be detected by dynamic light scattering, and the average particle size is 5.3 μm.

[0170] Example 17

[0171] This embodiment uses the microspheres prepared in Example 16 as a basis to prepare a bacterial vaccine based on saponin microspheres.

[0172] The saponin microspheres obtained in Example 1 were mixed with 4 times their volume of pure water in a regular centrifuge tube to prepare a suspension with a microsphere content of 20%. The suspension was then rapidly frozen at -80°C and thawed at room temperature after 2 hours. This freeze-thaw cycle was repeated 3 times, for a total of 4 cycles, to prepare freeze-thawed saponin microspheres that could be loaded with antigens.

[0173] After freeze-thawing, saponin microspheres were mixed with equal volumes of 2 mg / mL Pseudomonas aeruginosa PcrV antigen (purchased from Wuhan Huamei Biotechnology Co., Ltd., CSB-EP6908EZW) and 2 mg / mL Pseudomonas aeruginosa OprI antigen (purchased from Wuhan Huamei Biotechnology Co., Ltd., CSB-EP319977FQE) in centrifuge tubes. The centrifuge tubes were placed in a vacuum drying oven with a vacuum level of 0.1 Pa and incubated for 1 hour after the vacuum level was reached. The centrifuge tubes were then removed, and the microspheres were washed twice with physiological saline to obtain saponin microspheres successfully loaded with bacterial antigens. The microspheres were resuspended in physiological saline so that each 100 μL of physiological saline contained 5 mg of polylactic acid, 100 μg of Ophiopogon japonicus saponin D' (OPD'), 20 μg of PcrV protein, and 20 μg of OprI protein.

[0174] Example 18

[0175] This embodiment describes the animal experiments and immunization effects of the saponin microsphere-based bacterial vaccine, as detailed below.

[0176] BALB / c mice were injected with blank phosphate-buffered saline (PBS) on days 0 and 14 as blank control groups. Other control groups included: PcrV antigen and OprI antigen dissolved in PBS (Ag1+Ag2, each dose containing 20 μg PcrV antigen and 20 μg OprI antigen); OPD' dissolved in DMSO and mixed with PcrV antigen and OprI antigen dissolved in PBS (Ag1+Ag2+OPD', each dose containing 5 μl DMSO, 20 μg PcrV antigen, 20 μg OprI antigen, and 100 μg OPD'); and PcrV antigen and OprI antigen adsorbed with aluminum adjuvant [Ag1+Ag2+AL(OH)], each dose containing 100 μg Alhydrogel (purchased from InvivoGen, vac-alu-50), 20 μg PcrV antigen, and 20 μg... OprI antigen】, polylactic acid microspheres without added saponins (prepared according to the method of Example 16) were loaded with PcrV antigen and OprI antigen [Ag1+Ag2+MC(Blank), each dose containing 5 mg polylactic acid, 20 μg PcrV antigen, and 20 μg OprI antigen]. Polylactic acid microspheres with added saponins prepared in Example 18 were used as the experimental group [Ag1+Ag2+MC(OPD'), each dose containing 5 mg polylactic acid, 20 μg PcrV antigen, 20 μg OprI antigen, and 100 μg OPD']. Seven days after the second immunization, serum was collected, and the levels of PcrV antigen and OprI antigen-specific IgG antibodies in the serum of immunized mice were detected by ELISA. In this application, MC refers to microspheres.

[0177] The results showed that the saponin microsphere group induced the highest levels of specific IgG antibodies against PcrV and OprI antigens in mouse serum. Figure 14 The levels of Ophiopogon japonicus saponin D' (OPD') were significantly higher than those in the saponin-only group and the blank microsphere group, and also higher than those in the commercially available aluminum adjuvant group. The use of Ophiopogon japonicus saponin D' (OPD') further enhanced the immune response in the saponin microsphere group. This indicates that the bacterial vaccine based on saponin microspheres induced an effective immune response against bacteria, which is beneficial for establishing bacterial protection.

[0178] Example 19

[0179] This embodiment describes the protective effect of a saponin microsphere-based bacterial vaccine on animals infected with Pseudomonas aeruginosa lung infection, as detailed below.

[0180] Referring to the dosage and method described in Example 18, Pseudomonas aeruginosa was challenged via the airway 7 days after the second immunization (1×10⁻⁶). 7 A mouse model of acute pneumonia was constructed using CFU / mouse to investigate bacterial infection of the mouse lungs, and the survival rate of the mice was continuously monitored.

[0181] like Figure 15The results showed that the saponin microsphere-based bacterial vaccine effectively protected mice from acute lung infection, with a 7-day survival rate of 60%, far exceeding the protective effect of other groups. This indicates that the bacterial vaccine administered with saponin microspheres can establish sufficiently effective protection against acute Pseudomonas aeruginosa infection, preventing life-threatening bacterial infections.

[0182] Although the invention has been described with reference to exemplary embodiments, it should be understood that the invention is not limited to the disclosed exemplary embodiments. Various adjustments or changes may be made to the exemplary embodiments described in this specification without departing from the scope or spirit of the invention. The scope of the claims should be interpreted in the broadest possible sense to cover all modifications and equivalent structures and functions.

Claims

1. A vaccine composition comprising a bacterial antigen and at least one drug carrier, wherein, The drug carrier is a polymer microsphere with a chamber structure that has undergone freeze-thaw treatment to form openings or pores on its surface. The preparation method of the vaccine includes the following steps: mixing the drug carrier with a solution containing bacterial antigen, preferably loading the bacterial antigen onto the drug carrier by means of infiltration and / or adsorption, and then removing the supernatant by centrifugation to obtain polymer microspheres loaded with antigen.

2. The vaccine composition of claim 1, wherein, The bacterial antigen is Pseudomonas aeruginosa antigen, preferably Pseudomonas aeruginosa V antigen (PcrV antigen) or Pseudomonas aeruginosa outer membrane lipoprotein I (OprI antigen), or a combination thereof.

3. The vaccine composition of claim 1, wherein, The drug carrier has or substantially has a single-chamber structure.

4. The vaccine composition of claim 1, wherein, The polymer microspheres are prepared from natural or artificially synthesized polymers. Preferably, the polymer includes at least one of polylactic acid, polylactic acid-glycolic acid copolymer, polyglycolic acid, and polyethylene glycol-lactic acid copolymer.

5. The vaccine composition of claim 1, wherein, Furthermore, the drug carrier loaded with antigens is sealed to form sealed microcapsules loaded with antigens.

6. The vaccine composition according to claim 1 or 3, wherein, Furthermore, polymer microspheres or sealed microcapsules loaded with antigens are subjected to a lyophilization process to produce lyophilized formulations.

7. The vaccine composition of claim 1, wherein, The average particle size of the polymer microspheres is 1-10 μm, preferably, for example, 2-8 μm, or 3-7 μm, and the average surface wall thickness of the polymer microspheres is 0.5-1.5 μm, for example, 0.8-1.2 μm.

8. The vaccine composition of claim 1, wherein, The freeze-thaw process refers to exposing a suspension containing the polymer microspheres to a low-temperature environment for freezing, and then thawing it by heating it, thereby destroying the surface structure of the polymer microspheres to create openings or pores, wherein the low-temperature environment refers to a suspension system containing polymer microspheres below the freezing point temperature.

9. The vaccine composition of claim 6, wherein, The polymer microspheres are subjected to freezing treatment to cause ice to form inside the polymer microspheres, so that the ice crystals formed by the freezing will pierce or burst the polymer microspheres.

10. The vaccine composition of claim 1, wherein, The polymeric microspheres further contain saponins, such as ophiopogonin saponins, more preferably ophiopogonin D'.

11. The vaccine composition of claim 2, wherein, The vaccine composition is an anti-Pseudomonas aeruginosa vaccine.

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