Drug carrier, preparation method therefor, and use thereof

Abstract

A drug carrier, a preparation method therefor, and use thereof. The carrier is obtained by subjecting a biodegradable polymer microsphere with a chambered structure to a freeze-thaw treatment, resulting in the formation of openings or pores on the surface of the polymer microsphere, thereby enabling a gentle drug loading process. The prepared drug carrier can be used as a vaccine adjuvant, and is capable of promoting immune cell recruitment and activation, improving antibody levels and quality, inducing efficient cellular immunity, and thus improving the vaccine protection rate.

Description

A drug carrier and its preparation method and use

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to application number CN202311446363.3, filed on November 2, 2023, entitled “A drug carrier, its preparation method and use”, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] The present invention relates to the technical field of biomedical granule preparations, and in particular to a drug carrier and a preparation method and application thereof. Background Art

[0004] Infectious diseases pose a serious threat to human health and economic development, and vaccines are a key weapon in defeating them. However, effective protection requires multiple vaccinations. Frequent vaccinations not only disrupt daily life but also reduce vaccination willingness and full vaccination rates, severely hindering comprehensive infectious disease prevention and control. Therefore, there is an urgent need for innovative vaccine formulations to reduce the number of doses and establish long-term immune protection.

[0005] Polymer microsphere technology dates back to the 1950s. It can encapsulate solids, liquids, or gases within cavities, creating antigen-delivery systems that protect antigens from rapid degradation and regulate their release, leading to extensive research in the vaccine field. Current polymer microspheres loaded with antigens often exhibit a slow-release behavior similar to that of drug loading. While this avoids burst release and enables sustained immune stimulation, it neglects the self-enhancing effects of multiple vaccination strategies. There is an urgent need for a polymer microsphere preparation technology with adjustable drug release.

[0006] The information in the background technology is only intended to illustrate the general background of the invention and should not be regarded as an admission or any form of suggestion that this information constitutes the prior art known to a person skilled in the art.

[0007] Summary of the Invention

[0008] To address at least some of the technical problems in the prior art, the present invention provides a drug carrier, a preparation method, and uses thereof. By creating openings or pores on the surface of polymer microspheres through a freeze-thaw process, the microspheres are loaded with drugs using physical methods such as drug diffusion or adsorption, and the loaded drugs are released in a phased manner, thereby broadening the application of polymer microspheres in the biopharmaceutical field. Specifically, the present invention includes the following.

[0009] The first aspect of the present invention provides a method for preparing a drug carrier, which comprises the following steps: providing a biodegradable polymer microsphere having a cavity structure, and subjecting the polymer microsphere to freeze-thaw treatment to form openings or pores on the surface of the polymer microsphere.

[0010] In certain embodiments, according to the method for preparing the drug carrier of the present invention, the interior of the polymer microsphere comprises a single chamber and / or a multi-chamber structure.

[0011] In certain embodiments, according to the method for preparing the drug carrier of the present invention, the polymer microspheres are prepared from natural high molecular polymers or artificially synthesized high molecular polymers.

[0012] In certain embodiments, according to the method for preparing a drug carrier of the present invention, the polymer includes at least one of polylactic acid, polylactic acid-glycolic acid copolymer, polyethylene glycol-lactic acid copolymer, polyglycolic acid copolymer, polycaprolactone, dextran, chitosan, and trehalose.

[0013] In certain embodiments, according to the method for preparing the drug carrier of the present invention, the average particle size of the polymer microspheres is 0.5-500 μm, preferably 1-200 μm, and further preferably 3-30 μm.

[0014] In certain embodiments, according to the method for preparing a drug carrier of the present invention, the average wall thickness of the surface layer of the polymer microspheres is 0.05-20 μm, for example, 0.1-10 μm, for example, 0.2-5 μm, preferably 0.5-5 μm, for example, 0.5-4 μm, preferably 1.5-2.5 μm, for example, 0.5-1.5 μm, for example, 1-2 μm.

[0015] In certain embodiments, the biodegradable polymer microspheres are prepared by the following method:

[0016] (1) preparing an oil phase O, wherein the oil phase is a solution containing a polymer matrix, wherein the solvent is an organic solvent; preparing an inner aqueous phase solution W1 and an outer aqueous phase solution W2, wherein a surfactant is added to the outer aqueous phase;

[0017] (2) Dispersing the inner aqueous phase into the oil phase to form a water-in-oil (W1 / O) primary emulsion; then dispersing the W1 / O primary emulsion into the outer aqueous phase to form a water-in-oil-in-water (W1 / O / W2) secondary emulsion;

[0018] (3) using a solvent removal method to solidify the oil phase to obtain polymer microspheres;

[0019] The oil phase does not contain or substantially does not contain a surfactant, and the prepared polymer microspheres have or substantially have a single cavity structure.

[0020] In certain embodiments, according to the method for preparing a drug carrier of the present invention, the surface of the drug carrier contains closed or non-closed pores.

[0021] In certain embodiments, according to the preparation method of the drug carrier of the present invention, when the polymer microspheres are prepared by the double emulsion solvent method, the internal aqueous phase is an aqueous solution, or further contains an osmotic pressure regulator. Preferred internal aqueous phases are, for example, purified water, water for injection, sodium chloride aqueous solution, phosphate buffer, glucose solution, and sucrose solution.

[0022] In certain embodiments, the method for preparing the drug carrier according to the present invention further comprises the step of selecting a specific sedimentation time for the suspension system containing the polymer microspheres, thereby obtaining polymer microspheres with different wall thicknesses.

[0023] In certain embodiments, according to the method for preparing the drug carrier of the present invention, the freeze-thaw treatment involves exposing the suspension containing the polymer microspheres to a low-temperature environment for freezing treatment, and then heating it to melt it, thereby destroying the surface structure of the polymer microspheres to produce openings or pores, wherein the low-temperature environment refers to a temperature below the freezing point of the suspension system containing the polymer microspheres.

[0024] In certain embodiments, according to the method for preparing the drug carrier of the present invention, the polymer microspheres are subjected to a freezing treatment to freeze the interior of the polymer microspheres, so that ice crystals generated by the freezing pierce or burst the polymer microspheres.

[0025] The second aspect of the present invention provides a pharmaceutical composition comprising a drug and at least one drug carrier according to the present invention. Preferably, the pharmaceutical composition is a vaccine for the preventive or therapeutic treatment of tumors or infectious diseases in mammalian subjects.

[0026] In certain embodiments, according to the pharmaceutical composition of the present invention, preferably, the preparation method of the pharmaceutical composition comprises: mixing the drug carrier with a solution containing the drug.

[0027] In certain embodiments, the pharmaceutical composition according to the present invention further comprises: sealing the drug carrier loaded with the drug to form sealed microcapsules loaded with the drug.

[0028] In certain embodiments, according to the pharmaceutical composition of the present invention, the drug carrier has a single-chamber structure, or a multi-chamber structure, or polymer microspheres with different wall thicknesses, or any combination thereof.

[0029] In certain embodiments, the pharmaceutical composition according to the present invention is an influenza vaccine, a herpes vaccine, a COVID-19 vaccine, or an anti-tumor vaccine. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] FIG1 is a laser confocal micrograph of single-cavity microspheres prepared in Example 1 and Example 2 and a statistical graph of the measured wall thickness and particle size;

[0031] FIG2 is an optical microscope image of the single-cavity microspheres prepared in Example 3;

[0032] FIG3 is an optical microscope image of single-cavity microspheres prepared in Example 4 and Example 5;

[0033] FIG4 is an optical microscope image of the multi-cavity microspheres prepared in Example 6;

[0034] FIG5 is a light microscope image taken by a high-speed camera of the freezing process of single-cavity microcapsules in different liquid systems;

[0035] FIG6 is an optical microscopic image and a statistical graph of the measured gap lengths after different freeze-thaw times in Example 8 and Example 9;

[0036] FIG7 is a light microscopy and scanning electron microscopy image of the drug carrier prepared in Example 10;

[0037] FIG8 is a laser confocal micrograph of protein loading after different freeze-thaw times in Example 11 and Example 12 and a statistical graph of the measured loading positive rate;

[0038] FIG9 is a scanning electron microscope image of the sealed polymer microspheres prepared in Example 13;

[0039] FIG10 is a laser confocal micrograph and quantitative curve of the in vitro release measured in Example 14;

[0040] FIG11 is a fluorescence image and quantitative curve of the release in mice measured in Example 14;

[0041] FIG12 is a quantitative curve of in vivo release in mice measured in Example 15;

[0042] FIG13 is a schematic diagram of the preparation process of a lyophilized drug carrier;

[0043] FIG14 is a comparison of the morphology and particle size of the microspheres before and after freeze-drying as measured in Example 16, as well as circular dichroism spectra of the antigen protein before and after freeze-drying;

[0044] FIG15 shows the immune cells recruited by the vaccine adjuvant at the injection site of mice as determined in Example 17;

[0045] FIG16 shows the types and functions of dendritic cells recruited by the vaccine adjuvant at the injection site in mice as determined in Example 17;

[0046] FIG17 shows the number of germinal center B cells induced by the vaccine adjuvant in the draining lymph nodes of mice measured in Example 17;

[0047] FIG18 shows the transcriptome results of dendritic cells recruited by vaccine adjuvants at the injection site of mice as determined in Example 17;

[0048] FIG19 shows the results of cellular immunity in mice induced by vaccine adjuvants as determined in Example 18;

[0049] FIG20 shows the results of T cell memory in mice induced by vaccine adjuvants as determined in Example 18;

[0050] Figure 21 shows the results of the vaccine adjuvant-induced mouse antibody titers and pseudo-virus neutralizing antibody titers measured in Example 19;

[0051] Figure 22 shows the results of BCR sequencing of lymph nodes of mice induced by vaccine adjuvants as determined in Example 19;

[0052] FIG23 shows the results of the vaccine adjuvant-induced T cell responses in cynomolgus monkeys measured in Example 20;

[0053] FIG24 shows the protective effect of the vaccine adjuvants tested in Example 21 against influenza in a mouse model;

[0054] FIG25 shows the results of the safety evaluation of the vaccine adjuvants determined in Example 22 in mouse and cynomolgus monkey models. DETAILED DESCRIPTION

[0055] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0056] It should be understood that the terms described in the present invention are only for describing particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges in the present invention, it should be understood that the upper and lower limits of the ranges and each intermediate value therebetween are specifically disclosed. Each smaller range between any stated value or intermediate value within a stated range and any other stated value or intermediate value within the stated range is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0057] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the invention belongs. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In the event of any conflict with any incorporated document, the content of this specification shall prevail.

[0058] Drug carrier

[0059] One aspect of the present invention provides a drug carrier. In a preferred embodiment, the drug carrier is a polymer microsphere that can be post-loaded with drugs, which can achieve drug loading after the microspheres are prepared, and the loaded drugs have adjustable or controllable release behavior. The term "post-drug loading" used in the present invention refers to loading the drugs after the microspheres are prepared. The term "adjustable or controllable release behavior" means that the drug release process is controlled by the structure of the microspheres, and there can be different release stages in the release process. There can be plateaus between different release stages. There can be only one release stage or multiple release stages. It can be understood that the number of release stages, plateaus, etc. can be controlled by the structure.

[0060] The present invention provides a preparation method of a drug carrier, which comprises the following steps: providing a biodegradable polymer microsphere with a cavity structure, and subjecting the polymer microsphere to freeze-thaw treatment to form openings or pores on the surface of the polymer microsphere.

[0061] In the present invention, the average particle size of the polymer microspheres is 0.5-500 μm, preferably 1-800 μm, further preferably 1-600 μm, further preferably 1-400 μm, more preferably 1-200 μm, further more preferably 1-100 μm, most preferably 3-30 μm, for example, 15-30 μm, such as 3, 5, 10, 15, 20, 25, 30 μm.

[0062] In a preferred embodiment, the internal structure of the polymer microspheres of the present invention includes a single cavity, and a single cavity refers to only one cavity inside the microsphere. In another preferred embodiment, the internal structure of the polymer microspheres of the present invention includes multiple chambers, such as 2, 3, 4, 5, 6, 7, 8, 9, 10 or more chambers. In a preferred embodiment, the internal structure of the polymer microspheres of the present invention is or substantially is a single cavity structure. The internal structure of the microspheres, such as a single cavity or a multi-cavity structure, can be analyzed, determined and characterized by optical microscopy, electron microscopy, scanning probe microscopy, X-ray diffraction and infrared spectroscopy, nuclear magnetic resonance technology, microscopic hot stage observation, computed tomography and nuclear magnetic resonance imaging.

[0063] In a preferred embodiment, in the present invention, the surface wall thickness of the polymer microspheres is 0.05-20 μm, preferably 0.1-15 μm, for example 0.1-10 μm, preferably 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.

[0064] In the present invention, the polymer used in the polymer microspheres can be a natural high molecular polymer or a synthetic degradable high molecular polymer, examples of which include but are not limited to at least one of polylactic acid, polylactic acid-co-glycolic acid, polyglycolic acid copolymer, polyethylene glycol-lactic acid copolymer, polycaprolactone, dextran, and chitosan. In a preferred embodiment, the polymer material used in the polymer microspheres of the present invention is polylactic acid-co-glycolic acid (PLGA).

[0065] In a preferred embodiment, a method for preparing a drug carrier is provided, comprising subjecting biodegradable polymer microspheres containing chambers to a freeze-thaw process. Preferably, the method comprises: (a) preparing an emulsion containing the polymer, and curing the emulsion to prepare polymer microspheres; and (b) subjecting the polymer microspheres to a freeze-thaw process to obtain polymer microspheres having openings or pores on their surfaces.

[0066] In a preferred embodiment, according to the method for preparing the drug carrier of the present invention, the biodegradable polymer microspheres are prepared by the following method:

[0067] (1) preparing an oil phase O, wherein the oil phase is a solution containing a polymer matrix, wherein the solvent is an organic solvent; preparing an inner aqueous phase solution W1 and an outer aqueous phase solution W2, wherein a surfactant is added to the outer aqueous phase;

[0068] (2) Dispersing the inner aqueous phase into the oil phase to form a water-in-oil (W1 / O) primary emulsion; then dispersing the W1 / O primary emulsion into the outer aqueous phase to form a water-in-oil-in-water (W1 / O / W2) secondary emulsion;

[0069] (3) using a solvent removal method to solidify the oil phase to obtain polymer microspheres;

[0070] The oil phase does not contain or substantially does not contain surfactants such as emulsifiers, and the prepared polymer microspheres have or substantially have a single cavity structure.

[0071] In the present invention, preferably, in the preparation of polymer microspheres by the double emulsion solvent method, the volume ratio of the inner water phase (W1) to the oil phase (O) will significantly affect the internal structure and the size of the internal cavity of the prepared polymer microspheres. Preferably, in the present invention, the volume ratio of the inner water phase (W1) to the oil phase (O) is 1:1-1:15, for example, 1:3-1:12, preferably 1:5-1:10.

[0072] In the present invention, preferably, if the volume of the internal aqueous phase is larger than that of the oil phase, more of the aqueous phase will be encapsulated within the polymer matrix during the double emulsion solvent method preparation process, resulting in the formation of more cavities or the enlargement of existing cavities. Therefore, when the internal aqueous phase is larger, the internal cavities of the microspheres are generally larger or contain more internal cavities. Furthermore, as the volume of the internal aqueous phase increases, the relatively large internal aqueous phase tends to aggregate within the dispersed oil phase (O phase), forming an unstable multi-cavity structure, which can lead to structural collapse or morphological instability in the microspheres. If the volume of the internal aqueous phase is smaller than that of the oil phase, the amount of water encapsulated in the polymer matrix is ​​less, and the resulting cavities are smaller or fewer in number. The presence of internal pores is reduced, and the internal structure of the microspheres is more compact and dense, resulting in 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 microsphere instability, potentially leading to cavity collapse or irregular microsphere morphology. A lower internal water phase / oil phase ratio results in smaller cavities and even denser microspheres with relatively stable internal cavity structures, but may lack the special properties of cavity structures (e.g., reduced load capacity). In a preferred embodiment of the present invention, an internal water phase / oil phase ratio of 1:5 produces relatively thin-walled single-cavity microspheres, and in a preferred embodiment of the present invention, an internal water phase / oil phase ratio of 1:10 produces relatively thick-walled single-cavity microspheres.

[0073] In the present invention, preferably, in the process of preparing polymer microspheres by the emulsion solvent method, an osmotic pressure regulator such as sodium chloride is added in the inner aqueous phase, which can further affect the internal structure of the polymer microspheres, especially the size of the inner chamber and the wall thickness of the microspheres, wherein the emulsion process is mainly affected by the osmotic pressure difference between the inner and outer aqueous phases. When an osmotic pressure regulator (such as sodium chloride) is added in the inner aqueous phase, the osmotic pressure of the inner aqueous phase can be increased. The increase in osmotic pressure means that the osmotic pressure difference between the inner aqueous phase and the outer aqueous phase (W2 phase) becomes larger. Due to the osmotic pressure difference, in the emulsion process (W1 / O→W1 / O / W2) and in the solvent volatilization process thereafter, moisture can penetrate into the inner aqueous phase of high osmotic pressure from the outer aqueous phase of low osmotic pressure, particularly in the curing process, which will significantly affect the morphology and the internal structure of the microspheres.

[0074] Preferably, it will be understood by those skilled in the art that 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 the solvent evaporates, more of the outer aqueous phase will penetrate into the inner aqueous phase, resulting in volume expansion of the inner aqueous phase. This will gradually increase the cavity inside the microspheres. As the osmotic pressure regulator (such as NaCl concentration) in the inner aqueous phase increases, the cavity expansion may become more obvious, forming a larger cavity or more porous structure under certain conditions. This is because the water osmotic pressure continues to move into the inner aqueous phase, making the internal structure become more "empty".

[0075] Preferably, those skilled in the art will understand that as the volume of the internal aqueous phase expands, 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 volume of the internal cavity increases significantly, the wall thickness of the microsphere will generally become thinner. The reduction in wall thickness is due to the fact that more polymer material is involved in forming the outer shell, while the interior is an expanded cavity. Due to the osmotic pressure difference of water, a large amount of water moves, combined with the dynamic synergistic effect of polymer curing and solvent volatilization, the structure of the polymer wall may form some porosity, making it thinner and more fragile.

[0076] The present invention first prepares high molecular polymer microspheres or particles that can be used for freeze-thaw procedures. The term "freeze-thaw" used herein should be understood in a broad sense, that is, the process may involve exposing a suspension containing microspheres to a low temperature environment for solidification, and then melting or thawing it by heating, so that the surface structure of the polymer microspheres is destroyed to produce pores. Low temperature refers to below the freezing point of the microsphere suspension system. Specifically, the freeze-thaw procedure may involve the following steps: (a) placing the suspension system containing microspheres in a low temperature environment to make it reach a frozen state. This step can be achieved by using a low-temperature coolant, such as liquid nitrogen or dry ice, or any equipment or device that can provide low-temperature freezing. (b) heating the frozen microsphere suspension to a melted or thawed state. Heating can be achieved by using any heating device known in the art, such as a hot plate or microwave. The heating temperature can be lower than room temperature, room temperature or higher than room temperature, which is not particularly limited and can be adjusted as needed.

[0077] In a preferred embodiment, according to the method for preparing the drug carrier of the present invention, the freeze-thaw treatment involves exposing the suspension containing the polymer microspheres to a low-temperature environment for freezing treatment, and then heating it to melt it, thereby destroying the surface structure of the polymer microspheres to generate pores, wherein the low-temperature environment refers to a temperature below the freezing point of the suspension system containing the polymer microspheres.

[0078] In a preferred embodiment, according to the preparation method of the drug carrier of the present invention, the polymer microspheres are subjected to a freezing treatment to freeze the interior of the polymer microspheres so that ice crystals generated by the freezing pierce the polymer microspheres. For example, the polymer microspheres contain water, and water molecules form ice crystals when frozen. These ice crystals will have a greater destructive force on the structure of the polymer material. Especially when the water content is high, repeated freezing and thawing will further lead to physical breakage and crack changes.

[0079] In a preferred embodiment, the common freeze-thaw processing procedure includes a freezing step and a thawing step. Preferably, the thawing step can also be understood as a melting step.

[0080] In a preferred embodiment, the present invention comprises a step of freezing the polymer microspheres to a low temperature, which typically involves several processes such as programmed cooling, rapid cooling, or slow cooling. In a preferred embodiment, the present invention utilizes a programmed cooling process, such as rapid programmed cooling or a high cooling rate programmed cooling process, so that ice crystals rapidly form to puncture the microspheres. Programmed cooling typically requires specialized programmed cooling equipment or a controlled-rate cryogenic refrigerator. When rapid cooling is employed, the polymer microspheres can be rapidly placed in a liquid nitrogen (-196°C) or low-temperature (-80°C) cooling system to rapidly freeze the water, promote ice crystal formation, and destroy the original structure. When slow freezing is employed, the polymer microspheres can be slowly cooled from room temperature to a freezing temperature, typically in a cryogenic storage box. In a preferred embodiment of the present invention, the freezing step preferably utilizes programmed cooling, such as a rapid cooling program, for example, programmed cooling at a rate of 30°C / min.

[0081] In a preferred embodiment, wherein, in the present invention, the thawing step includes, for example, room temperature thawing, and room temperature thawing is relatively simple, and the method is to place the frozen sample at room temperature and slowly thaw to ensure that the moisture or structure is gradually restored. The thawing step can also use water bath thawing, which means placing the frozen sample in a water bath at, for example, 37°C (such as a constant temperature water bath) for rapid thawing. The thawing step can also use cold room or low temperature thawing: move the sample to a 4°C refrigerated environment, slowly thaw, or heat and thaw with a cold table device. In a preferred embodiment of the present invention, preferably, the thawing step uses room temperature thawing.

[0082] In the present invention, the preparation method of polymer microspheres is not particularly limited, as long as microspheres with cavities can be obtained. In a preferred embodiment, it includes the steps of preparing a premix and evolving 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 configuring an inner aqueous phase solution W1: such as a sodium chloride aqueous solution. Configuring the oil phase O: such as a certain concentration of polylactic acid-glycolic acid (PLGA) ethyl acetate solution. Configuring the outer aqueous phase W2: such as a certain concentration of polyvinyl alcohol aqueous solution. It should be noted that the oil phase of the present invention does not contain any type of surfactant, but can achieve controllable preparation of the cavity.

[0083] In a preferred embodiment of the present invention, after the water-in-oil-in-water composite emulsion is prepared, the oil phase is solidified and then the residual surfactant in the water phase is removed. It is particularly preferred that the residual surfactant be removed by sieving or centrifugal washing.

[0084] In the present invention, the water-in-oil-in-water composite emulsion is obtained by homogenization or mechanical stirring. This generally includes the preparation of colostrum and the preparation of a double emulsion. For example, colostrum is prepared using a conventional stirring method, for example, by mixing the oil phase with the inner aqueous phase and placing them in a centrifuge tube, followed by mechanical shearing to obtain the colostrum. For example, a double emulsion is prepared using a conventional stirring method, for example, by adding colostrum to the outer aqueous phase and mechanically shearing to obtain the double emulsion, i.e., a water-in-oil-in-water composite emulsion.

[0085] In another preferred embodiment, the polymer microspheres of the present invention can also be obtained by membrane emulsification. An exemplary method includes passing water-in-oil colostrum through a membrane tube having a micron pore size, for example, 1-50 μm, preferably 15-30 μm, to obtain a double emulsion after multiple membrane passages, and then evolving the double emulsion for a suitable time to produce polymer microspheres.

[0086] In a preferred embodiment, in the present invention, the solvent of the internal aqueous phase is selected from at least one of water, water for 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 internal aqueous phase.

[0087] In a preferred embodiment, in the present invention, the solvent of the oil phase comprises one or a combination of at least two of alcohols, ketones, esters, ethers, alkylbenzenes, halogenated alkanes, and halogenated aromatic hydrocarbons that can dissolve high molecular weight polymers, more preferably a volatile organic solvent, such as n-butanol, ether, chloroform, tetrachloromethane, etc., more preferably a volatile and partially water-soluble organic solvent, such as ethyl acetate, phenol, etc.

[0088] In a preferred embodiment, the organic solvent used in the present invention preferably has a solubility in water of less than 10%, preferably less than 2%, and most preferably is a water-insoluble organic solvent. In a preferred embodiment, the organic solvent is preferably selected from one or more of dichloromethane, chloroform, ethyl acetate, ethyl propionate, propyl acetate, or acetone, more preferably dichloromethane or ethyl acetate, and most preferably dichloromethane. Any combination of the aforementioned organic solvents may also be used, with the specific type and volume depending on the membrane material used and other preparation parameters.

[0089] In a preferred embodiment, in the present invention, the external aqueous phase further comprises an emulsifier selected from at least one of sodium alginate, polyvinyl alcohol fiber, Tween 80, methylcellulose, gelatin, polysorbate, lysine, gum arabic, and poloxamer 188.

[0090] In a preferred embodiment, in the present invention, colostrum can be prepared by mixing an internal aqueous phase having a salt concentration of 0.05-2 mg / mL such as sodium chloride with an oil phase having a polymer concentration of 20-100 mg / mL at a mass ratio of 1:1-1:30, wherein the salt concentration is preferably 0.06-1.9 mg / mL, further preferably 0.07-1.8 mg / mL, and further 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, further preferably 40-80 mg / mL, further preferably 45-75 mg / mL, for example 45, 50, 55, 60, 65, 70, 75 mg / mL.

[0091] In a preferred embodiment, in the present invention, after the step (a) of preparing polymer microspheres, the step of selecting polymer microspheres with suitable wall thickness is also included. The present invention has found through research that wall thickness is more conducive to subsequent drug loading and drug release regulation within a suitable range. In a preferred embodiment, suitable sedimentation time is selected for different suspension systems to obtain polymer microspheres with different wall thicknesses. For example, when selecting a polymer microsphere and water mixing system, polymer microspheres with a sedimentation time of less than 30 minutes have a larger wall thickness, which is not conducive to the drug loading process. Polymer microspheres with a sedimentation time of less than 30-120 minutes have an average wall thickness of 1-3 μm, preferably 1.5-2.5 μm, and have excellent drug loading and drug release performance. Polymer microspheres with a sedimentation time of more than 120 minutes have a smaller wall thickness. Although they can be drug loaded, their drug release performance is not as good as polymer microspheres with a sedimentation time of less than 30-120 minutes. It is understood that when the selected sedimentation system is a salt ion or other type of solution, microspheres within a specific sedimentation time can be selected according to actual needs, and microspheres with appropriate wall thickness can even be directly obtained by operations such as density gradient centrifugation.

[0092] In a preferred embodiment, in the present invention, step (b) of the present invention is a step of preparing polymer microspheres having openings or pores on the surface using a freeze-thaw procedure. After the freeze-thaw treatment, the microsphere structure, such as the surface structure of the microspheres, will change, for example, pores will be generated, so that the microspheres can be used for drug loading through physical binding methods, such as infiltration, adsorption, etc.

[0093] In a preferred embodiment, in the present invention, in step (b), the freezing temperature and freeze-thaw program selected according to different suspension systems can be changed. For example, polymer microspheres and water mixture are frozen at low temperatures, and are melted at room temperature, and freeze-thaw can be performed once or multiple times, and the repeated freeze-thaw process number of times is not particularly limited, but preferably 2-10 times, also preferably 3-9 times, such as 3,4,5,6,7,8,9 times. The present invention has been found through research that freeze-thaw number of times increases cracks and also significantly increases. Therefore, those skilled in the art can adjust freeze-thaw number of times as needed, such as according to the size of the pores of required drug loading.

[0094] In a preferred embodiment, in the present invention, low temperature during the freeze-thaw process refers to 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, further 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, -170°C.

[0095] Pharmaceutical composition

[0096] In one aspect of the present invention, a pharmaceutical composition is provided, which comprises a drug and the drug carrier described in the present invention, such as the polymer microspheres of the present invention, wherein the drug can be any water-soluble macromolecule or small molecule therapeutic agent, such as an antigen, protein drug or other drug, etc., and can be an antigen protein of an infectious pathogen, such as an antigen of a new crown vaccine, such as a new crown BRD protein, or an antigen of an influenza vaccine, such as an influenza HA protein, or can be a protein therapeutic agent used clinically, such as a monoclonal antibody, a protein hormone, etc., or can be a water-soluble chemical drug, such as goserelin, minoxidil, etc., without particular limitation.

[0097] Those skilled in the art know that the new coronavirus RBD protein refers to the receptor binding domain (RBD) of the spike protein of the new coronavirus (SARS-CoV-2). Those skilled in the art know that influenza vaccines usually contain the following types of antigens, such as influenza virus surface proteins. Influenza viruses mainly have two surface proteins, namely hemagglutinin (HA) and neuraminidase (NA). Hemagglutinin (HA): HA protein is the key for the virus to enter the host cell. It can recognize and bind to specific receptors on the cell surface. The antigenicity of HA protein varies greatly between influenza virus strains, so the vaccine needs to be updated according to the main influenza virus strains that are prevalent each year. Neuraminidase (NA): NA protein works after the virus is released from the host cell. It can cut sialic acid on the surface of the host cell, thereby helping the virus particles fall off the cell surface.

[0098] In a preferred embodiment of the present invention, the tumor antigen is, for example, mucin (MUC1), tumor membrane antigen, tumor whole cell antigen, or tumor neoantigen.

[0099] In a preferred embodiment, the pharmaceutical composition according to the present invention is prepared by a method comprising: mixing the drug carrier with a solution containing the drug to allow the drug to enter the interior of the microspheres. For example, drug loading can be achieved by physical means, such as drug diffusion or adsorption, after the polymer microspheres are prepared. The drug diffuses into the microspheres by infiltration, adsorption, etc., and the loaded drug is released in stages. Preferably, the drug carrier is mixed with the solution containing the drug, and then centrifuged and the supernatant is removed to obtain the drug-loaded polymer microspheres.

[0100] In a preferred embodiment, the pharmaceutical composition according to the present invention further comprises: sealing the drug carrier loaded with the drug to form sealed microcapsules loaded with the drug.

[0101] In the present invention, those skilled in the art will understand that "sealing" means embedding, fixing, or retaining the drug within the polymer microspheres. In the present invention, sealed microcapsules do not mean that there are no openings on the surface of the microcapsules. Instead, it simply means that antigens and / or other substances, such as other drugs, can be partially embedded, fixed, or retained within the microcapsules through the sealing process, and the antigens and / or other substances, such as other drugs, can be released from the microcapsules through degradation of the microcapsules.

[0102] In a preferred embodiment of the present invention, preferably, the sealing process of the drug carrier includes a solvent swelling method, an irradiation method and a temperature-raising annealing method. Those skilled in the art can also seal the drug carrier according to their professional knowledge / new technology.

[0103] In a preferred embodiment of the present invention, the temperature-raising annealing method is a more ideal sealing method. For example, the unique self-healing sealing properties of biodegradable polymer blend matrices such as polylactic acid can be utilized. It uses irradiation or heating methods to cause the molecules on the surface of the microspheres to absorb energy and rearrange, so that the pores on the surface are healed and sealed. For example, the drug carrier loaded with drugs is slowly heated to a temperature close to the glass transition temperature of the microspheres, preferably 1 to 2°C lower than the glass transition temperature of the microspheres. After a period of time, the temperature is slowly lowered to seal the pores on the surface of the open-pore drug carrier to prepare sealed microcapsules. Antigens and other drugs are effectively encapsulated in the microcapsules, and the loading rate and embedding rate are stable.

[0104] In a preferred embodiment of the present invention, a solvent-assisted temperature-increasing annealing method can be employed. For example, a small amount of an organic solvent and a surfactant capable of dissolving the polymer microspheres can be added during the temperature-increasing annealing process to lower the required glass transition temperature, thereby effectively encapsulating drugs such as antigens in the microcapsules with stable loading and embedding rates. In one embodiment of the present invention, the small amount of organic solvent and surfactant is, for example, 4% by volume of ethyl acetate and 0.375% by weight of PVA.

[0105] In a preferred embodiment of the present invention, the pharmaceutical composition comprising the drug and the drug carrier of the present invention can be prepared into a lyophilized preparation through a lyophilization process.

[0106] In the present invention, the pharmaceutical composition further includes an optional pharmaceutically acceptable carrier. In the present invention, pharmaceutically acceptable carriers are well known in the art and can be determined by a person of ordinary skill in the art to meet clinical criteria. Pharmaceutically acceptable carriers include diluents and excipients.

[0107] Examples of suitable pharmaceutically acceptable carriers include, but are not limited to: (1) Dulbecco's phosphate buffered saline, pH about 7.4, with or without approximately 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) dextrose; antioxidants such as tryptamine and stabilizers such as Tween 20 may also be included.

[0108] In a preferred embodiment, the pharmaceutical composition of the present invention is a therapeutic agent for preventing, treating and / or ameliorating a disease, preferably an infectious disease. In another embodiment, the pharmaceutical composition of the present invention is a vaccine for preventing, treating and / or ameliorating a disease, preferably an infectious disease. Infectious diseases include bacterial infectious diseases, viral infectious diseases, parasitic infectious diseases, etc., examples of which include but are not limited to AIDS, viral hepatitis, pneumonia, avian influenza, measles, rabies, hemorrhagic fever, tuberculosis, scarlet fever, gonorrhea, syphilis, epidemic encephalitis B, dengue fever, anthrax bacteria and amebic dysentery, brucellosis, leptospirosis, schistosomiasis, malaria, poliomyelitis, human infection with highly pathogenic avian influenza, influenza, mumps and rubella, echinococcosis, filariasis, epidemic hemorrhagic conjunctivitis, bacillary dysentery, typhoid and paratyphoid fever, etc.

[0109] It is understood that the pharmaceutical composition of the present invention is not limited to the above-mentioned infectious diseases. When the drug carrier is loaded with tumor or cancer therapeutic drugs, the pharmaceutical composition of the present invention can be used for any solid tumor or blood disease.

[0110] The pharmaceutical composition of the present invention can be in any suitable dosage form. For example, an injection, a suspension, an emulsifier, etc. The pharmaceutical composition of the present invention can be administered to the body in a known manner. For example, it can be delivered to the tissue of interest by intramuscular injection, optionally administered intravenously, transdermally, intranasally, orally, through the mucosa, or other delivery methods. Such administration can be carried out via a single dose or multiple doses. It will be appreciated by those skilled in the art that the actual dose to be administered herein can vary to a large extent depending on a variety of factors, such as the target cell, the type of organism or its tissue, the general condition of the subject to be treated, the route of administration, the mode of administration, etc.

[0111] It will be appreciated that the pharmaceutical composition is administered to an individual in a prophylactically effective amount or a therapeutically effective amount (as appropriate, although prophylaxis may be considered treatment) sufficient to demonstrate benefit to the individual. Typically, this will result in therapeutically useful activity that is beneficial to the individual. The actual amount of compound administered, as well as the rate and time course of administration, will depend on the nature and severity of the condition being treated. Prescriptions for treatment, such as dosage decisions, are within the responsibility of general practitioners and other physicians, and typically take into account the condition being treated, the individual patient's condition, the site of delivery, the method of administration, and other factors known to the physician.

[0112] In a preferred embodiment, in the present invention, the pharmaceutical composition is a vaccine, which is used for the preventive or therapeutic treatment of tumors or infectious diseases in mammalian subjects.

[0113] The present invention further provides a compound preparation according to the needs of vaccines or drugs, wherein the drug carrier has a single chamber structure, or a multi-chamber structure, or polymer microspheres with different wall thicknesses, or any combination thereof.

[0114] For example, for vaccines, different types of the above-mentioned drug carriers (polymer microspheres) can be compounded according to immunodynamics to achieve a better pulse release effect. Different types of microspheres can be compounded to have any combination of polymer microspheres with a single chamber, polymer microspheres with a multi-chamber structure, and polymer microspheres with different wall thicknesses. For example, the compounded microspheres can be a combination of polymer microspheres with a single chamber and polymer microspheres with a multi-chamber structure, or a combination of polymer microspheres with different wall thicknesses, or even a combination of polymer microspheres with a single chamber and a multi-chamber structure with different wall thicknesses.

[0115] For a specific vaccine, the ratio of different types of microspheres can be adjusted to achieve the objectives of the present invention. Based on the teachings of the present invention, any combination of compound formulations can be obtained by adjusting the evolution time and sedimentation time, thereby obtaining pulsatile release polymer microspheres with multiple release stages. Therefore, the polymer microspheres of the present invention can have multiple release processes, for example at least 2, such as 3, 4, 5, 6, 7, 8, 9, 10, or even more.

[0116] The present invention also provides a method for regulating drug release behavior, comprising the step of using the drug carrier (polymer microsphere) or compound formulation described herein. Preferably, appropriate evolution time and sedimentation time are selected to obtain any combination of polymer microspheres having a single cavity, polymer microspheres having a multi-cavity structure, and polymer microspheres having different wall thicknesses.

[0117] vaccine adjuvants

[0118] One aspect of the present invention provides a vaccine adjuvant comprising the drug carrier described herein, such as the polymer microspheres of the present invention. As used herein, the term "vaccine" refers to a prophylactic biological product used for human vaccination to prevent or control the occurrence and spread of infectious diseases. This includes preparations prepared using microorganisms or their toxins, enzymes, human or animal sera, cells, and the like for preventive, diagnostic, and / or therapeutic purposes. The specific type of vaccine adjuvant that can be used in the present invention is not particularly limited, and subunit vaccines, inactivated vaccines, live attenuated vaccines, and the like can be used.

[0119] In a preferred embodiment, the vaccine is an influenza vaccine, a herpes vaccine, a COVID-19 vaccine, or an anti-tumor vaccine.

[0120] In the present invention, vaccination with a vaccine can be therapeutic or prophylactic. For example, it may be possible to provide prophylactic protection against the development of a cancerous disease by immunizing an individual who does not suffer from cancer. Examples of individuals for whom such prophylactic vaccination may be used are individuals at increased risk of developing cancer, although such use is not limited to such individuals. Patients at risk for cancer may already have a tumor, either as a primary tumor or a metastasis, or may otherwise exhibit a predisposition to cancer.

[0121] In a preferred embodiment, the vaccine that can be used in the present invention is a recombinant protein vaccine, for example, including but not limited to the new crown (COVID-19) recombinant protein vaccine. In another preferred embodiment, the vaccine that can be used in the present invention is an influenza vaccine. In yet another preferred embodiment, the vaccine that can be used in the present invention is an mRNA vaccine. In yet another preferred embodiment, the vaccine that can be used in the present invention is a hepatitis B vaccine.

[0122] In a preferred embodiment, the present invention provides an influenza vaccine comprising an influenza virus surface protein, preferably influenza hemagglutinin (HA), and the drug carrier of the present invention, wherein the preparation method of the drug carrier is to provide polymer microspheres having or substantially having a single chamber structure, and subjecting the polymer microspheres to freeze-thaw treatment to form openings or pores on the surface of the polymer microspheres, wherein the polymer microspheres are prepared from polylactic acid, polylactic-co-glycolic acid and / or polyglycolic acid, and the average particle size of the polymer microspheres is preferably, for example, 15-30 μm, and the The Span value of the polymer microspheres is preferably, for example, less than 1.5, and the average surface wall thickness of the polymer microspheres is preferably, for example, 1-2 μm, wherein the hemagglutinin (HA) is mixed with the drug carrier described in the present invention in a solution so that the influenza hemagglutinin is loaded on the drug carrier by infiltration and / or adsorption, and then the supernatant is removed by centrifugation to obtain polymer microspheres loaded with hemagglutinin. Preferably, the drug carrier loaded with hemagglutinin can be sealed to form sealed microcapsules loaded with hemagglutinin. Preferably, the drug carrier loaded with hemagglutinin can be subjected to a freeze-drying procedure to prepare a freeze-dried preparation.

[0123] In a preferred embodiment, the present invention provides a new crown vaccine, which comprises a new crown RBD protein and the drug carrier of the present invention, wherein the preparation method of the drug carrier is to provide polymer microspheres having or substantially having a single chamber structure, and freeze-thaw the polymer microspheres to form openings or pores on the surface of the polymer microspheres, wherein the polymer microspheres are prepared from polylactic acid, polylactic acid-glycolic acid copolymer and / or polyglycolic acid, and the average particle size of the polymer microspheres is preferably, for example, 15-30 μm, and the Span value of the polymer microspheres is preferably, for example, 1.5 or less, the average surface wall thickness of the polymer microspheres is preferably, for example, 1-2 μm, wherein the new crown RBD protein is mixed with the drug carrier described in the present invention in a solution, so that the new crown RBD protein can be loaded on the drug carrier by infiltration and / or adsorption, and then the supernatant is removed by centrifugation to obtain polymer microspheres loaded with the new crown RBD protein. Preferably, the drug carrier loaded with the new crown RBD protein can be sealed to form a sealed microcapsule loaded with the new crown RBD protein. Preferably, the drug carrier loaded with the new crown RBD protein can be subjected to a freeze-drying treatment procedure to prepare a freeze-dried preparation.

[0124] In a preferred embodiment, the present invention provides a herpes zoster vaccine comprising herpes zoster virus antigen gE protein and the drug carrier of the present invention, wherein the preparation method of the drug carrier is to provide polymer microspheres having or substantially having a single chamber structure, and subjecting the polymer microspheres to freeze-thaw treatment to form openings or pores on the surface of the polymer microspheres, wherein the polymer microspheres are prepared from polylactic acid, polylactic acid-glycolic acid copolymer and / or polyglycolic acid, and the average particle size of the polymer microspheres is preferably, for example, 2-20 μm, the Span value of the polymer microspheres is preferably, for example, less than 1.5, and the flatness of the polymer microspheres is preferably, for example, less than 1.5. The average surface wall thickness is preferably, for example, 0.5-1.5 μm, wherein the herpes zoster virus antigen gE protein is mixed with the drug carrier of the present invention in a solution so that the herpes zoster virus antigen gE protein is loaded on the drug carrier by infiltration and / or adsorption, and then centrifuged to remove the supernatant to obtain polymer microspheres loaded with herpes zoster virus antigen gE protein. Preferably, the drug carrier loaded with herpes zoster virus antigen gE protein can be sealed to form sealed microcapsules loaded with herpes zoster virus antigen gE protein. Preferably, the drug carrier loaded with herpes zoster virus antigen gE protein can be made into a lyophilized preparation through a lyophilization process. Preferably, molecular adjuvants, such as MPLA, QS-21 and other molecules, can be added to the drug-loaded solution containing the drug carrier to construct a composite vaccine adjuvant.

[0125] In a preferred embodiment, the present invention provides a prostate cancer vaccine comprising a prostate cancer neoantigen and the drug carrier of the present invention, wherein the preparation method of the drug carrier is to provide polymer microspheres having or substantially having a single chamber structure, and subjecting the polymer microspheres to freeze-thaw treatment to form openings or pores on the surface of the polymer microspheres, wherein the polymer microspheres are prepared from polylactic acid, polylactic acid-glycolic acid copolymer and / or polyglycolic acid, and the average particle size of the polymer microspheres is preferably, for example, 15-30 μm, and the Span value of the polymer microspheres is preferably, for example, 1.5 or less, the average surface wall thickness of the polymer microspheres is preferably, for example, 1-2 μm, wherein the prostate cancer neoantigen is mixed with the drug carrier of the present invention in a solution so that the prostate cancer neoantigen is loaded on the drug carrier by infiltration and / or adsorption, and then the supernatant is removed by centrifugation to obtain polymer microspheres loaded with prostate cancer neoantigens. Preferably, the drug carrier loaded with prostate cancer neoantigens can be sealed to form sealed microcapsules loaded with prostate cancer neoantigens. Preferably, the drug carrier loaded with prostate cancer neoantigens can be made into a lyophilized preparation through a lyophilization procedure. Preferably, molecular adjuvants such as MPLA, QS-21 and other molecules can be added to the drug-carrier-containing drug solution to construct a composite vaccine adjuvant. Preferably, immune checkpoint inhibitors such as PD-1 antibodies can be added to the drug-carrier-containing drug solution to improve the immune microenvironment to promote the function of tumor vaccines.

[0126] As used herein, the term "subject" refers to any animal (eg, mammal), including but not limited to humans, non-human primates, rodents, and the like, that is to receive a particular treatment.

[0127] The dosage of the vaccine adjuvant of the present invention depends on many factors, such as the nature and severity of the disease to be prevented or treated, the sex, age, weight, personality and individual response of the patient or animal, the route of administration, the number of administrations, and the purpose of treatment. Therefore, the therapeutic dose of the present invention can vary widely. Generally speaking, the dosage of the vaccine adjuvant in the present invention is well known to those skilled in the art. The dosage administered is within the expected range of the clinician or laboratory personnel, and the dosage can be appropriately adjusted, for example, through effectiveness and safety testing, to obtain the optimal dosage.

[0128] use

[0129] The present invention provides the use of drug carriers in the preparation of drugs. Preferably, polymer microspheres capable of post-loading drugs are used as vaccine delivery carriers, and the substances that can be loaded may include hydrophobic or hydrophilic antigens and / or adjuvants.

[0130] In a preferred embodiment, the post-drug-loaded pulse-release polymer microspheres of the present invention can effectively achieve a phased release of the internal drug. When loaded with antigens, they can be used as vaccines to induce a potent immune response and establish immune protection through subcutaneous or intramuscular injection.

[0131] In certain embodiments, the drug carrier provided by the present invention has at least the following advantages:

[0132] (1) The drug carrier of the present invention creates surface openings or cracks through freeze-thaw cycles, allowing for post-loading of the drug by spontaneous diffusion after microsphere preparation, effectively improving the encapsulation efficiency and ensuring the stability of the drug structure and activity. The drug carrier itself can be lyophilized, and lyophilized preparations can be prepared by selecting appropriate lyophilization conditions based on the type of drug being encapsulated.

[0133] (2) The drug carrier of the present invention as a drug delivery system can achieve controllable drug release behavior by controlling the wall thickness of the polymer microspheres, the internal cavity structure of the polymer microspheres, and their compounding. For example, the drug release can have multiple stages with plateaus between the stages, or a sustained release process.

[0134] (3) The drug carrier of the present invention can be used as a vaccine preparation to effectively promote the local recruitment and activation of immune cells, induce efficient humoral immunity and cellular immunity levels, establish an effective immune protection barrier, and enhance the protective ability of the vaccine.

[0135] Those skilled in the art will appreciate that the drug carrier described herein, after being loaded with drugs, can also be used in combination with other drugs for the prevention, treatment and / or improvement of diseases or conditions. The other drugs can be any therapeutic agent that is beneficial to the disease, such as solid tumors or blood diseases, and there is no particular limitation on this.

[0136] The following examples are set forth in order to more clearly illustrate to those skilled in the art the principles and practice of the embodiments disclosed herein and should not be construed as limiting the scope of any claimed embodiments.

[0137] Example 1

[0138] In this embodiment, polymer microspheres were prepared using poly(lactic acid-co-glycolic acid) copolymer as the material, as follows.

[0139] Polylactic acid-co-glycolic acid (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, stirred and dissolved in an 80°C water bath for 5 hours, and a 15 mg / mL PVA aqueous solution was prepared as the outer aqueous phase.

[0140] Colostrum is obtained by mixing 2 mL of the oil phase with 200 μL of the inner aqueous phase and stirring at 7000 rpm for 30 seconds in a homogenizer. Alternatively, a conventional mechanical stirring method can be used to prepare colostrum: add the colostrum to 15 mL of the outer aqueous phase and stir at 7000 rpm for 2 minutes in a homogenizer to obtain a W / O / W emulsion. The W / O / W emulsion is vortexed for 15 minutes to allow the oil phase to develop a single-chamber structure. The emulsion is then extracted with sufficient pure water or evaporated in a fume hood to remove the organic solvent, yielding solidified hybrid polymer microspheres. The hybrid polymer microspheres are then mixed with 10 mL of water and placed in a 10 mL centrifuge tube. Microspheres are collected after sedimentation for between 30 minutes and 2 hours. Laser confocal microscopy is used to analyze the resulting microspheres, calculate their surface wall thickness, and measure their particle size using a particle size analyzer (Figure 1).

[0141] Example 2

[0142] In this example, polymer microspheres were prepared using poly(lactic acid-co-glycolic acid) copolymer as the material. The main difference from Example 1 is the sedimentation time, as follows.

[0143] Polylactic acid-co-glycolic acid (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, stirred and dissolved in an 80°C water bath for 5 hours, and a 15 mg / mL PVA aqueous solution was prepared as the outer aqueous phase.

[0144] Colostrum is obtained by mixing 2 mL of the oil phase with 200 μL of the inner aqueous phase and stirring at 7000 rpm for 30 seconds in a homogenizer. Alternatively, a conventional mechanical stirring method can be used to prepare colostrum: add the colostrum to 15 mL of the outer aqueous phase and stir at 7000 rpm for 2 minutes in a homogenizer to obtain a W / O / W emulsion. The W / O / W emulsion is vortexed for 15 minutes to allow the oil phase to develop a single-chamber structure. The emulsion is then extracted with sufficient pure water or evaporated in a fume hood to remove the organic solvent, yielding solidified hybrid polymer microspheres. The hybrid polymer microspheres are then mixed with 10 mL of water and placed in a 10 mL centrifuge tube. Microspheres that have settled within 30 minutes are collected. Laser confocal microscopy is used to analyze the resulting microspheres, calculate their surface wall thickness, and measure their particle size using a particle size analyzer (Figure 1).

[0145] Example 3

[0146] This example uses poly(lactic acid-co-glycolic acid) copolymer as the material to prepare polymer microspheres. The main difference from Example 1 is the final sedimentation time, as follows.

[0147] 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, stirred and dissolved in an 80°C water bath for 5 hours, and a 15 mg / mL PVA aqueous solution was prepared as the outer aqueous phase.

[0148] Take 2mL of oil phase and mix it with 200μL of inner aqueous phase, stir it at 7000rpm of homogenizer for 30 seconds to obtain colostrum. Afterwards, traditional mechanical stirring method can be selected to prepare, colostrum is added to 15mL of outer aqueous phase, and stir it at 7000rpm of homogenizer for 2 minutes to obtain W / O / W emulsion. The W / O / W emulsion is vortexed for 15 minutes to make its oil phase evolve into a single cavity structure, and then the emulsion is added into sufficient pure water for extraction or evaporated in a fume hood to remove organic solvent to obtain solidified mixed polymer microspheres. The mixed polymer microspheres are mixed with 10mL of water and placed in a 10mL centrifuge tube, and the microspheres with sedimentation time of more than 2 hours are taken. The microspheres obtained by optical microscopy (Figure 2) are thinner than those in Example 1.

[0149] Example 4

[0150] This example demonstrates a method for preparing single-cavity microspheres using poly(lactic acid-co-glycolic acid) copolymer as a material, as follows.

[0151] 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, stirred and dissolved in an 80°C water bath for 5 hours, and a 30 mg / mL PVA aqueous solution was prepared as the outer aqueous phase.

[0152] 1 mL of the oil phase was mixed with 200 μL of the inner aqueous phase and ultrasonically vibrated at 30% power for 12 seconds to produce colostrum. Then, using conventional mechanical stirring, the colostrum was added to 15 mL of the outer aqueous phase and stirred at 8000 rpm for 2 minutes in a homogenizer to produce a W / O / W double emulsion. This double emulsion was then immediately extracted with sufficient pure water or evaporated in a fume hood to remove the organic solvent, yielding solidified single-cavity polymer microspheres.

[0153] The obtained microspheres were observed by optical microscopy and the particle size was measured by dynamic light scattering, as shown in FIG3 , indicating that single-cavity microspheres were obtained.

[0154] Example 5

[0155] This example demonstrates a method for preparing single-cavity microspheres using poly(lactic acid-co-glycolic acid) as a material, as follows.

[0156] 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, stirred and dissolved in an 80°C water bath for 5 hours, and a 30 mg / mL PVA aqueous solution was prepared as the outer aqueous phase.

[0157] Colostrum is obtained by mixing 1 mL of the oil phase with 100 μL of the inner aqueous phase and oscillating at 30% ultrasonic power for 12 seconds. Alternatively, a conventional mechanical stirring method can be used to prepare the colostrum: add the colostrum to 15 mL of the outer aqueous phase and stir at 8000 rpm in a homogenizer for 2 minutes to produce a W / O / W emulsion. The emulsion is then immediately extracted with sufficient pure water or evaporated in a fume hood to remove the organic solvent, yielding solidified hybrid polymer microspheres.

[0158] The obtained microspheres were observed by optical microscopy and the particle size was measured by dynamic light scattering. As shown in FIG3 , single-cavity microspheres were also prepared, but with thicker walls.

[0159] Example 6

[0160] This embodiment provides a method for preparing multi-cavity microspheres, which is characterized in that PEG-PLA copolymer (PELA) is added to the oil phase.

[0161] Polylactic acid-co-glycolic acid (PLGA) and PELA were dissolved in ethyl acetate at a mass ratio of PLGA to PELA 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, stirred and dissolved in an 80°C water bath for 5 h, and a 15 mg / mL PVA aqueous solution was prepared as the outer aqueous phase.

[0162] Colostrum is obtained by mixing 2 mL of the oil phase with 200 μL of the inner aqueous phase and stirring at 7000 rpm for 30 seconds in a homogenizer. Alternatively, a conventional mechanical stirring method can be used to prepare the colostrum: add the colostrum to 15 mL of the outer aqueous phase and stir at 7000 rpm for 2 minutes in a homogenizer to obtain a W / O / W emulsion. This emulsion is then extracted directly into sufficient pure water or evaporated in a fume hood to remove the organic solvent, yielding solidified multi-cavity polymer microspheres. Light microscopy of the resulting microspheres reveals a distinct multi-cavity structure (Figure 4).

[0163] Example 7

[0164] In this example, the polymer microspheres of Example 4 were subjected to programmed cooling, and the cooling process was observed using an optical microscope and a high-speed camera, as follows.

[0165] The polymer microspheres prepared in Example 4 were mixed with a 3% PVA aqueous solution to form a suspension, and the temperature was cooled to -80°C at a rate of 30°C / min. It was observed that no ice formed inside the microspheres, and no ice crystals pierced the microspheres (Figure 5).

[0166] 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. Ice crystals were observed to pierce the polymer microspheres immediately after freezing, revealing cracks and holes (Figure 5). This example demonstrates that the freeze-thaw loading method of the present invention relies on the formation of ice crystals.

[0167] Example 8

[0168] In this example, the polymer microspheres of Example 4 were subjected to a freeze-thaw process for drug loading, as follows.

[0169] The polymer microspheres prepared in Example 4 were reconstituted with an equal volume of water to form a suspension, and placed in a -80 degree Celsius refrigerator to quickly cool to -80 ° C. Freeze at -80 ° C for 2 hours, then dissolve at room temperature, and repeat the freeze-thaw process 0 times, 2 times, 4 times and 8 times to obtain different post-drug-loaded polymer microspheres. The polymer microspheres after the freeze-thaw process were characterized using an optical microscope, and the cracks appearing on their surfaces were measured (Figure 6). It can be seen that clear cracks exist on the surface of the polymer microspheres.

[0170] Example 9

[0171] In this example, the polymer microspheres of Example 5 were subjected to a freeze-thaw process for drug loading, as follows.

[0172] The polymer microspheres prepared in Example 5 were reconstituted with an equal volume of water to form a suspension, and placed in a -80 degrees Celsius refrigerator to quickly cool to -80°C. Freeze at -80°C for 2h, then dissolve at room temperature, and repeat the freeze-thaw process 0 times, 2 times, 4 times, and 8 times to obtain different post-drug-loaded polymer microspheres. The polymer microspheres after the freeze-thaw process were characterized using an optical microscope, and the cracks appearing on their surfaces were measured (Figure 6). It can be seen that under the same freeze-thaw process, the number and length of cracks appearing in the polymer microspheres of Example 5 are less than those in the polymer microspheres of Example 4.

[0173] Example 10

[0174] This example shows a vaccine adjuvant based on polymer microspheres prepared according to Example 4 and the freeze-thaw process.

[0175] 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, stirred and dissolved in an 80°C water bath for 5 hours, and a 30 mg / mL PVA aqueous solution was prepared as the outer aqueous phase.

[0176] 1 mL of the oil phase was mixed with 200 μL of the inner aqueous phase and ultrasonically vibrated at 30% power for 12 seconds to produce colostrum. Then, using conventional mechanical stirring, the colostrum was added to 15 mL of the outer aqueous phase and stirred at 8000 rpm for 2 minutes in a homogenizer to produce a W / O / W double emulsion. This double emulsion was then immediately extracted with sufficient pure water or evaporated in a fume hood to remove the organic solvent, yielding solidified single-cavity polymer microspheres.

[0177] The microspheres were mixed with an equal volume of pure water to form a suspension, which was then rapidly cooled to -80°C in a -80°C freezer. The suspension was frozen at -80°C for 2 hours and then dissolved at room temperature. This freeze-thaw process was repeated four times to obtain a drug carrier based on single-cavity polymer microspheres. Optical microscopy and scanning electron microscopy revealed distinct cracks on the surface of the drug carrier (Figure 7), allowing the drug to be loaded into the microspheres through these cracks.

[0178] Example 11

[0179] This example shows the drug loading of the post-drug-loaded polymer microspheres obtained in Example 4.

[0180] Cy5-labeled bovine serum albumin (BSA) was prepared into a 1 mg / mL solution and mixed with the microspheres of Example 4 after different freeze-thaw cycles in Example 8. The microspheres were vortexed in a refrigerator at 4°C for 3 days to allow the protein to be loaded by diffusion.

[0181] Laser confocal microscopy revealed that different freeze-thaw cycles affected drug loading. As shown in Figure 8, the drug loading positive rate increased after 0, 2, and 4 freeze-thaw cycles. After 8 freeze-thaw cycles, the drug loading positive rate remained consistent with that after 4 cycles, reaching a peak.

[0182] Example 12

[0183] This example shows the drug loading of the post-drug-loaded polymer microspheres obtained in Example 5.

[0184] Cy5-labeled bovine serum albumin (BSA) was prepared into a 1 mg / mL solution and mixed with the microspheres of Example 5 after different freeze-thaw cycles in Example 9. The microspheres were vortexed in a refrigerator at 4°C for 3 days to allow the protein to be loaded by diffusion.

[0185] Laser confocal microscopy revealed that different freeze-thaw cycles affected drug loading. The drug loading positive rate was lower than that shown in Example 11 ( Figure 8 ) after 0, 2, 4, and 8 freeze-thaw cycles, indicating that microsphere wall thickness affected protein drug loading in the microspheres after freeze-thaw treatment.

[0186] Example 13

[0187] This embodiment shows a type of polymer microspheres that are sealed to prepare drug-loaded polymer microspheres.

[0188] Following Example 11, the sample tube was removed from the sample tube and centrifuged to collect the microspheres deposited at the bottom. A healing solution was then added, using 4% (v / v) ethyl acetate and 0.375% PVA aqueous solution, at a volume ratio of 1:25 (microsphere volume: healing solution). The tube was then resuspended evenly in a centrifuge tube. After sealing, the tube was placed in a 40°C water bath, rotating the tube to prevent microsphere aggregation. Healing was continued for 30 minutes to obtain polymer microspheres with sealed surface cracks (Figure 9).

[0189] Example 14

[0190] This example shows the drug release behavior of the drug carrier of Example 10 after loading the protein.

[0191] The microspheres prepared in Example 10 were used as drug carriers and mixed with a 1 mg / mL Cy5-labeled BSA solution. The mixture was vortexed in a refrigerator at 4°C for 3 days to allow drug loading. The microspheres at the bottom were collected by centrifugation and washed 2-3 times with PBS to obtain drug carriers for in vitro and in vivo release testing.

[0192] The formulation was placed in PBS and incubated in a 37°C incubator to simulate in vitro release. A portion of the formulation was taken daily for laser confocal microscopy to determine the in vitro release behavior. As shown in Figure 10, this drug carrier exhibits two sequential release processes, significantly delaying drug residence time.

[0193] The formulation was mixed with saline and injected subcutaneously into mice. At different time points, the mice were placed in an anesthesia box for gas anesthesia (isoflurane). After complete anesthesia, the mice were placed in a small animal in vivo imaging chamber to monitor the in vivo drug release behavior of the drug carrier in mice. As shown in Figure 11, the drug carrier also exhibited a two-stage sustained release behavior in mice, significantly delaying the drug's residence time.

[0194] Example 15

[0195] This embodiment proposes a drug carrier comprising microspheres of various structures, the release behavior of the drug can be adjusted through compounding.

[0196] The microspheres prepared in Example 3 and Example 4 were mixed in a 1:1 ratio to form composite microspheres. The composite microspheres were then mixed with an equal volume of pure water to form a suspension, which was then rapidly cooled to -80°C in a -80°C freezer. Freeze at -80°C for 2 hours, then thaw at room temperature. This freeze-thaw process was repeated four times to obtain a drug carrier based on the composite microspheres.

[0197] The prepared microspheres were mixed with a 1 mg / mL Cy5-labeled BSA solution as drug carriers and incubated in a 4°C refrigerator for 3 days to allow drug loading. The microspheres were collected by centrifugation and washed 2–3 times with PBS to obtain drug carriers for in vitro and in vivo release testing.

[0198] The formulation was mixed with normal saline and injected subcutaneously into mice. At different time points, the mice were placed in an anesthesia box for gas anesthesia (isoflurane). After complete anesthesia, the mice were placed in a small animal in vivo imaging chamber to monitor the in vivo drug release behavior of the drug carrier in mice. As shown in Figure 12, the drug carrier exhibited similar sustained-release behavior in mice, but the drug release was faster than that of the single-cavity microsphere drug carrier in Example 14.

[0199] Example 16

[0200] This example proposes a freeze-dried vaccine preparation based on Example 10. The preparation process diagram is shown in FIG13 . After freeze-drying, the protein function of the antigen and the adjuvant morphology are still maintained.

[0201] The SARS-CoV-2 RBD protein (at a concentration of 1 mg / mL) was mixed with the drug carrier described in Example 10, vortexed in a 4°C refrigerator for 3 days, and the bottom microspheres were collected by centrifugation and washed 2 to 3 times with PBS to obtain an unfreeze-dried vaccine preparation based on single-cavity microspheres.

[0202] The SARS-CoV-2 RBD is compounded with a certain lyoprotectant, preferably a PBS solution of 5% trehalose and 10% glycerol, so that the final concentration of the antigen protein is 1 mg / mL, and mixed with the drug carrier described in Example 10. The mixture is vortexed in a refrigerator at 4°C for 3 days, and the bottom microspheres are collected by centrifugation. The microspheres are washed 2 to 3 times with PBS and frozen in a -80°C refrigerator overnight. The microspheres are taken out and freeze-dried in a freeze dryer the next day to obtain a freeze-dried vaccine preparation. The overall process is shown in Figure x.

[0203] The morphology of the freeze-dried and unfreeze-dried microsphere preparations was observed by scanning electron microscopy, and the distribution was measured using a chromaticity analyzer. The results showed that the morphology and particle size distribution did not change before and after lyophilization (Figure 14). The freeze-dried and unfreeze-dried microsphere preparations were ultrasonically disrupted, and the supernatant was collected by centrifugation to separate the protein. The protein structure was analyzed by circular dichroism spectroscopy, which showed that the protein structure did not change before and after lyophilization (Figure 14).

[0204] Example 17

[0205] This example shows the immune activation effect of the freeze-dried vaccine adjuvant described in Example 16 on mice.

[0206] Based on the vaccine adjuvant described in Example 16 and compounded with physiological saline to prepare the new coronavirus subunit vaccine, mice were immunized by intramuscular injection, with 3 μg equivalent of the prototype dimeric strain RBD (obtained from Zhifei Company) per mouse and 500 μg equivalent of polymer microspheres per mouse. Mice vaccinated with PBS served as blank controls, and mice vaccinated with 3 μg equivalent of the prototype dimeric strain RBD per mouse and 50 μg equivalent of Al aluminum adjuvant (purchased from Invivogen Company, ) / mice served as experimental controls. Mice were sacrificed on days 0, 1, 3, and 5, and muscle tissue from the injection site was sectioned and stained with H&E. LyoC recruited more monocytes on day 3 (Figure 15). Muscle tissue from day 3 post-injection was also prepared into a single-cell suspension and flow cytometry-stained to identify various immune cell types. The monocytes recruited by LyoC on day 3 were primarily dendritic cells, with a significantly higher proportion than in the Al group (Figure 15).

[0207] The mice were killed on day 7, and the muscles and lymph nodes at the injection site were removed to prepare single cell suspensions. The results of flow cytometry showed that LyoC could effectively increase the number of dendritic cells (CD45 + CD11c + MHC-II + ) and the expression of activation markers ( FIG16 ), and both types of dendritic cells, cDC1 and cDC2, were increased ( FIG16 ), and the number of germinal center B cells (CD19 + FAS + GL-7 + ) number (Figure 17). Rapid recruitment and activation of dendritic cells facilitates antigen presentation, while more germinal center B cells contribute to the subsequent production of higher antibody levels.

[0208] By sorting DCs at the injection site using CD11c magnetic beads and performing transcriptome sequencing analysis, it was found that the polymer microsphere group induced higher DC function, with upregulation of activation molecules, cytokine secretion, MHC-I and MHC-II complexes, and chemokine receptors ( Figure 18 ).

[0209] Example 18

[0210] This example shows that the lyophilized vaccine adjuvant described in Example 16 induces higher levels of cellular immunity and cellular immune memory.

[0211] Reference Example 17: Two doses of the above-mentioned drug were administered to mice on day 0 and day 14. The mice were killed on day 28, and the spleen was taken to extract single cell suspension. After erythrocyte lysis, the cells were cultured under antigen stimulation for 2 days. After that, the cells were collected for flow cytometry staining, and it was found that LyoC could induce stronger CD4 + T cells and CD8 + T cell immune response (Figure 19), with higher levels of cytokine secretion and activation marker levels.

[0212] The memory-related marker staining of splenocytes extracted on day 28 revealed that LyoC could induce a higher expression of CD8 central memory T cells (CD8 + T cm At the same time, mice were killed on the 28th day to extract peripheral blood mononuclear cells, and the same flow cytometry staining was performed. It was found that LyoC could induce higher levels of CD4 effector memory T cells (CD4 + T em ) and CD8 effector memory T cells (CD8 + T em )(Figure 20).

[0213] Example 19

[0214] This example shows that the lyophilized vaccine adjuvant described in Example 16 induces higher levels of antibodies.

[0215] With reference to Example 17, mice were immunized twice on days 0 and 14 (LyoC group, Al group). The RBD (antigen)-specific IgG antibody levels in the serum of mice in each group were continuously detected at different time points after the first immunization. The results showed that LyoC can induce a strong humoral immune response in mice (Figure 21). At the same time, pseudoviruses of various variants of the new coronavirus were co-incubated with mouse serum obtained at week 4 to infect hACE-2 transgenic HEK-293T cells. The infection of the pseudovirus was detected by the Luc gene reporter system, and the half-maximal inhibition titer of the serum was used as an antibody quality indicator. The results indicated that the vaccine adjuvant can induce higher antibody quality (Figure 21).

[0216] One month after the second immunization, mice were sacrificed and lymph nodes were harvested for BCR sequencing to assess the maturity of B cells in each group. The polymer microsphere group induced more diverse BCR sequences, a higher mutation rate, and longer CDR3 sequences, indicating more mature B cells in the polymer microsphere group. This may be the reason for the high antibody quality (Figure 22).

[0217] Example 20

[0218] This example shows that the lyophilized vaccine adjuvant described in Example 16 induces a potent immune response in a cynomolgus monkey model.

[0219] Cynomolgus monkeys were injected with aluminum adjuvant (purchased from Invivogen, ) was injected on day 21 with the novel coronavirus prototype vaccine (25 μg equivalent of dimer prototype strain RBD / mouse and 250 μg equivalent of Al of aluminum adjuvant / mouse), and the polymer microspheres compounded novel coronavirus BA.4 / 5 strain vaccine (25 μg equivalent of BA.4 / 5 strain RBD (purchased from Yiqiao Shenzhou) / mouse and 2500 μg equivalent of post-drug-loaded polymer microspheres / mouse) prepared based on the scheme of Example 15 were injected as the experimental group (LyoC), and the aluminum adjuvant (purchased from Invivogen) was injected on day 21. ) was used as the control group (Al). Peripheral blood mononuclear cells were extracted from peripheral blood at week 5 and stimulated with the prototype strain RBD protein or BA.4 / 5 RBD protein. The prototype strain RBD-specific T cells or BA.4 / 5 RBD-specific IFN-γ secretion were measured by ELISPOT. The results showed that the polymer microsphere group could induce a higher level of T cell response against the two antigens (Figure 23).

[0220] Example 21

[0221] This example shows that the lyophilized vaccine adjuvant described in Example 16 induces effective immune protection in a mouse influenza challenge model.

[0222] Based on the method described in Example 16 (only the type of antigen was changed to influenza antigen), a freeze-dried polymer microsphere vaccine (LyoC) loaded with HA (purchased from Yiqiao Shenzhou) of the influenza virus antigen PR8 strain was prepared and mice were immunized by intramuscular injection. 5 μg equivalent of HA / mouse and 500 μg equivalent of drug-loaded polymer microspheres / mouse were used as blank controls, 5 μg equivalent of HA / mouse and 50 μg equivalent of Al aluminum adjuvant / mouse were used as experimental control 1 (Al), and 5 μg equivalent of HA / mouse and 50 μl equivalent of oil emulsion adjuvant AddaVax (purchased from Invivogen, AddaVaxTM) / mouse were used as experimental control 2 (Addavax). After two doses on day 0 and day 14, 10 mice were injected intranasally on day 28. 4PFU of PR8 influenza virus, and the weight and survival of the mice were detected within 14 days. The mice were killed on the 7th day, and the lungs and noses were taken to detect the viral load by real-time quantitative PCR. The lung lesions were observed by H&E sections, and the presence of the virus was confirmed by immunohistochemical staining of the NA protein of the virus. The results showed that the LyoC group induced more effective vaccine protection, the mice had lower weight loss, and no mice died during the attack. At the same time, the viral load in the lungs and noses of the mice was significantly lower than that in the aluminum adjuvant group, comparable to that in the Addavax group, and had a lower level of lung inflammation, and no obvious viral sites were observed (Figure 24).

[0223] Example 22

[0224] This example demonstrates the safety of the lyophilized vaccine adjuvant described in Example 16 in mouse and cynomolgus monkey models.

[0225] Mice and cynomolgus macaques were immunized with reference to Examples 17 and 20, respectively. At the end of immunization, the mice were sacrificed and the hearts, livers, spleens, lungs, and kidneys were removed for H&E sections to observe the level of inflammation. Peripheral blood was collected from the cynomolgus macaques at the end of immunization for routine blood tests and blood biochemical analysis. It was found that there were no abnormal changes in biochemical parameters in the LyoC group, and no obvious pathological features were found in the sections of the mouse organs, which fully demonstrated the safety of LyoC as a vaccine adjuvant ( Figure 25 ).

[0226] Although the present 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 modifications and variations may be made to the exemplary embodiments of the present specification without departing from the scope or spirit of the present invention. The scope of the claims is to be given the broadest interpretation so as to encompass all such modifications and equivalent structures and functions.

Claims

1. A method for preparing a drug carrier, comprising the following steps: Provided is a biodegradable polymer microsphere with a chamber structure. The polymer microsphere is subjected to freeze-thaw treatment to form openings or pores on the surface of the polymer microsphere.

2. The method for preparing a drug carrier according to claim 1, wherein: The polymer microspheres are prepared from natural high molecular polymers or artificially synthesized high molecular polymers. Preferably, the polymer includes at least one of polylactic acid, polylactic acid-glycolic acid copolymer, polyglycolic acid, polyethylene glycol-lactic acid copolymer, polycaprolactone, dextran, and chitosan.

3. The method for preparing a drug carrier according to claim 1, wherein: The biodegradable polymer microspheres have a single chamber and / or a multi-chamber structure.

4. The method for preparing a drug carrier according to claim 1, wherein: The biodegradable polymer microspheres are prepared by the following method: (1) preparing an oil phase O, wherein the oil phase is a solution containing a polymer matrix, wherein the solvent is an organic solvent; preparing an inner aqueous phase solution W1 and an outer aqueous phase solution W2, wherein a surfactant is added to the outer aqueous phase; (2) dispersing the inner water phase into the oil phase to form a water-in-oil W1 / O primary emulsion; and then dispersing the W1 / O primary emulsion into the outer water phase to form a water-in-oil-in-water W1 / O / W2 secondary emulsion; (3) using a solvent removal method to solidify the oil phase to obtain polymer microspheres; The oil phase does not contain or substantially does not contain a surfactant, and the prepared polymer microspheres have or substantially have a single chamber structure.

5. The method for preparing a drug carrier according to claim 4, wherein the internal aqueous phase is an aqueous solution, or further contains an osmotic pressure regulator, and a preferred internal aqueous phase is, for example, purified water, water for injection, sodium chloride aqueous solution, phosphate buffer, glucose solution, or sucrose solution.

6. The method for preparing a drug carrier according to claim 1, wherein the freeze-thaw treatment involves exposing the suspension containing the polymer microspheres to a low temperature environment for freezing treatment, and then heating it to melt it, thereby destroying the surface structure of the polymer microspheres to generate openings or pores, wherein, The low temperature environment refers to a temperature below the freezing point of the suspension system containing polymer microspheres.

7. The method for preparing a drug carrier according to claim 6, wherein: The polymer microspheres are subjected to freezing treatment to freeze the inside of the polymer microspheres, so that ice crystals generated by freezing pierce or burst the polymer microspheres.

8. The method for preparing a drug carrier according to claim 1, wherein: The average wall thickness of the surface layer of the polymer microspheres is 0.1-10 μm, such as 0.2-5 μm, preferably 0.5-4 μm, such as 1.5-2.5 μm, such as 0.5-1.5 μm, such as 1-2 μm.

9. A pharmaceutical composition comprising a drug and at least one drug carrier according to any one of claims 1 to 7, preferably, the pharmaceutical composition is a vaccine for preventive or therapeutic treatment of tumors or infectious diseases in mammalian subjects.

10. The pharmaceutical composition according to claim 9, wherein Preferably, the method for preparing the pharmaceutical composition comprises: mixing the drug carrier with a solution containing the drug.

11. The pharmaceutical composition according to claim 10, wherein The method further comprises: sealing the drug carrier loaded with the drug to form a sealed microcapsule loaded with the drug.

12. The pharmaceutical composition according to claim 9, wherein The drug carrier has a single chamber structure, or a multi-chamber structure, or has different average wall thicknesses of polymer microspheres, or any combination thereof.

13. The pharmaceutical composition according to claim 8, wherein The pharmaceutical composition is an influenza vaccine, a shingles vaccine, a COVID-19 vaccine or an anti-tumor vaccine.

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