Drug carrier as well as preparation method and application thereof

Abstract

The invention discloses a drug carrier as well as a preparation method and application thereof. According to the invention, freeze thawing treatment is carried out on the biodegradable polymer microspheres with the cavity structures, so that openings or pores are formed in the surfaces of the polymer microspheres, and the loading process of the drug on the drug carrier is mild. The prepared drug carrier can be used for vaccine adjuvants and can promote collection and activation of immune cells, improve the antibody level and quality, induce efficient cellular immunity and improve the vaccine protection rate.

Description

Technical Field

[0001] 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

[0002] Infectious diseases seriously threaten human health and economic development, and vaccines are a key weapon to defeat infectious diseases. However, good protection requires multiple vaccinations. Frequent vaccinations not only affect people's daily lives, but also reduce people's willingness to be vaccinated and the full vaccination rate, which seriously hinders the comprehensive prevention and control of infectious diseases. Therefore, there is still an urgent need to innovate vaccine formulations to reduce the number of vaccination doses and establish long-term immune protection.

[0003] Polymer microsphere technology can be traced back to the 1950s. It can embed and seal solids, liquids or gases in cavities. The antigen release system prepared in this way can protect antigens from rapid degradation and regulate the release behavior of antigens, and has been widely studied in the vaccine field. Current polymer microspheres loaded with antigens mostly continue the release behavior after drug loading and exhibit slow release characteristics. Although it avoids sudden release and can achieve long-lasting immune stimulation, it ignores the self-enhancement effect of multiple vaccination strategies. At present, there is an urgent need for a polymer microsphere preparation technology with adjustable drug release.

[0004] The information in the background technology is only for illustrating the general background of the present invention and should not be regarded as admitting or suggesting in any form that such information constitutes the prior art known to a person skilled in the art. Summary of the invention

[0005] In order to solve at least some of the technical problems in the prior art, the present invention provides a drug carrier and a preparation method and use thereof, which realizes the formation of openings or pores on the surface of polymer microspheres through a freeze-thaw procedure, realizes the loading of drugs after microsphere preparation by physical means, such as drug diffusion or adsorption, and realizes the staged release of the loaded drugs, thereby broadening the application of polymer microspheres in the field of biomedicine. Specifically, the present invention includes the following contents.

[0006] 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 chamber structure, and subjecting the polymer microsphere to freeze-thaw treatment to form openings or pores on the surface of the polymer microsphere.

[0007] In certain embodiments, according to the method for preparing a drug carrier of the present invention, the interior of the polymer microsphere comprises a single chamber and / or a multi-chamber structure.

[0008] 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.

[0009] 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.

[0010] In certain embodiments, according to the method for preparing a 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.

[0011] 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, such as 0.1-10 μm, such as 0.2-5 μm, preferably 0.5-5 μm, such as 0.5-4 μm, preferably 1.5-2.5 μm, such as 0.5-1.5 μm, such as 1-2 μm.

[0012] In certain embodiments, the biodegradable polymer microspheres are prepared by the following method:

[0013] (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;

[0014] (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;

[0015] (3) using a solvent removal method to solidify the oil phase to obtain polymer microspheres;

[0016] 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.

[0017] 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 unclosed pores.

[0018] 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. The preferred internal aqueous phase is, for example, purified water, water for injection, sodium chloride aqueous solution, phosphate buffer, glucose solution, and sucrose solution.

[0019] In certain embodiments, according to the method for preparing the drug carrier of the present invention, the method further comprises the step of selecting a specific sedimentation time for the suspension system containing the polymer microspheres to obtain polymer microspheres with different wall thicknesses.

[0020] In certain embodiments, according to the method for preparing the drug carrier of the present invention, the freeze-thaw treatment refers to 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.

[0021] 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 inside of the polymer microspheres, so that ice crystals generated by the freezing pierce or burst the polymer microspheres.

[0022] 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, which is used for preventive or therapeutic treatment of tumors or infectious diseases in mammalian subjects.

[0023] 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.

[0024] In certain embodiments, the pharmaceutical composition according to the present invention further comprises: sealing the drug carrier loaded with the drug to form a sealed microcapsule loaded with the drug.

[0025] 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 has different wall thicknesses of polymer microspheres, or any combination thereof.

[0026] In certain embodiments, according to the pharmaceutical composition of the present invention, the pharmaceutical composition is an influenza vaccine, a herpes vaccine, a new coronavirus vaccine, or an anti-tumor vaccine. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Laser confocal images of single-cavity microspheres prepared in Example 1 and Example 2 and statistical graphs of measured wall thickness and particle size;

[0028] Figure 2 This is an optical microscope image of the single-cavity microsphere prepared in Example 3;

[0029] Figure 3 Optical microscope pictures of single-cavity microspheres prepared in Example 4 and Example 5;

[0030] Figure 4 This is an optical microscope image of the multi-cavity microsphere prepared in Example 6;

[0031] Figure 5 Light microscopy pictures taken by a high-speed camera of the freezing process of single-chamber microcapsules in different liquid systems;

[0032] Figure 6 The optical microscopic images and the statistical graphs of the measured gap lengths after different freeze-thaw times of Example 8 and Example 9;

[0033] Figure 7 Light microscopy and scanning electron microscopy images of the drug carrier prepared in Example 10;

[0034] Figure 8 The laser confocal images of the loaded proteins after different freeze-thaw times in Example 11 and Example 12 and the statistical graph of the measured loading positive rate;

[0035] Fig. 9 This is a scanning electron microscope image of the sealed polymer microspheres prepared in Example 13;

[0036] Fig.10 The laser confocal microscopy images and quantitative curves of the in vitro release measured in Example 14;

[0037] Fig.11 The fluorescence images and quantitative curves of the release in mice measured in Example 14;

[0038] Fig.12 The quantitative curve of release in mice measured in Example 15;

[0039] Fig.13 Schematic diagram of the preparation process of a lyophilized drug carrier;

[0040] Fig.14 The figure is a comparison of the morphology and particle size of the microspheres before and after freeze-drying as measured in Example 16, and the circular dichroism spectra of the antigen protein before and after freeze-drying;

[0041] Fig.15Immune cells recruited by the vaccine adjuvant measured in Example 17 at the injection site of mice;

[0042] Fig.16 The types and functions of dendritic cells recruited by the vaccine adjuvants at the injection site of mice as determined in Example 17;

[0043] Fig.17 The number of germinal center B cells induced by the vaccine adjuvant in the draining lymph nodes of mice determined in Example 17;

[0044] Fig.18 The transcriptome results of dendritic cells recruited by the vaccine adjuvant at the injection site of mice measured in Example 17;

[0045] Fig.19 The results of the cellular immunity of mice induced by the vaccine adjuvant determined in Example 18;

[0046] Fig. 20 The results of T cell memory in mice induced by vaccine adjuvants determined in Example 18;

[0047] Fig.21 The results of the vaccine adjuvant-induced mouse antibody titer and the pseudo-virus neutralizing antibody titer determined in Example 19;

[0048] Fig. 22 The results of BCR sequencing of lymph nodes of mice induced by vaccine adjuvants determined in Example 19;

[0049] Fig.23 The results of the vaccine adjuvant-induced T cell response in cynomolgus monkeys determined in Example 20;

[0050] Fig.24 The influenza virus protection effect of the vaccine adjuvant in the mouse model determined in Example 21;

[0051] Fig.25 These are the results of the safety evaluation of the vaccine adjuvants determined in Example 22 in mouse and cynomolgus monkey models. DETAILED DESCRIPTION

[0052] 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 should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0053] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that the upper and lower limits of the scope and each intermediate value therebetween are specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0054] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings 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 a conflict with any incorporated document, the content of this specification shall prevail.

[0055] Drug carrier

[0056] 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 drug after the microspheres are prepared. The term "adjustable or controllable release behavior" means that the drug release process is controlled by the microsphere structure, and there can be different release stages in the release process. There can be a plateau period between different release stages. There can be only one release stage or there can be multiple release stages. It can be understood that the number of release stages, the plateau period, etc. can be controlled by the structure.

[0057] The invention provides a method for preparing a drug carrier, which comprises the following steps: providing a biodegradable polymer microsphere with a chamber structure, and subjecting the polymer microsphere to freeze-thaw treatment to form openings or pores on the surface of the polymer microsphere.

[0058] 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, for example 3, 5, 10, 15, 20, 25, 30 μm.

[0059] In a preferred embodiment, the internal structure of the polymer microspheres of the present invention includes a single chamber, and a single chamber 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 chamber 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, computer tomography and nuclear magnetic resonance imaging.

[0060] 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.

[0061] In the present invention, the polymer used for 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 selected for the polymer microspheres of the present invention is polylactic acid-co-glycolic acid (PLGA).

[0062] In a preferred embodiment, a method for preparing a drug carrier is provided, which comprises the step of subjecting biodegradable polymer microspheres containing chambers to freeze-thaw treatment. Preferably, the method comprises: (a) preparing an emulsion containing the polymer, and preparing polymer microspheres by solidification; (b) subjecting the polymer microspheres to freeze-thaw treatment to obtain polymer microspheres having openings or pores on the surface.

[0063] 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:

[0064] (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;

[0065] (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;

[0066] (3) using a solvent removal method to solidify the oil phase to obtain polymer microspheres;

[0067] 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 chamber structure.

[0068] 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 chamber 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.

[0069] In the present invention, preferably, if the volume of the inner water phase is larger than that of the oil phase, more water phase will be wrapped in the polymer matrix during the preparation process of the double emulsion solvent method, which will lead to the formation of more cavities or the enlargement of existing cavities. Therefore, when the inner water phase is larger, the internal chamber of the microsphere is usually larger or there are more cavity structures inside; in addition, as the volume of the inner water phase increases, the relatively large inner water phase is easy to aggregate in the dispersed oil phase (O phase) to form an unstable multi-cavity structure, which makes the microsphere prone to structural collapse or morphological instability. If the volume of the inner water phase is smaller than that of the oil phase, the amount of water wrapped in the polymer matrix is ​​less, and the cavities formed will be smaller or fewer in number. The presence of internal pores will be smaller, and the internal structure of the microsphere will be more compact and dense, thereby generating more uniform and dense microspheres. A higher ratio of inner water phase / oil phase will tend to form larger or more internal cavities, but will also increase the instability of the microspheres, which may lead to the collapse of the cavity or irregular morphology of microspheres. A lower internal water phase / oil phase ratio will form a smaller cavity or even a denser microsphere, and the internal cavity structure is relatively stable, but may lack the special properties of the cavity structure (such as 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.

[0070] In the present invention, preferably, in the process of preparing polymer microspheres by the double emulsion solvent method, adding an osmotic pressure regulator such as sodium chloride to the inner water phase will further affect the internal structure of the polymer microspheres, especially the size of the internal chamber and the wall thickness of the microspheres, among which the double emulsion process is mainly affected by adjusting the osmotic pressure difference between the inner and outer water phases. When an osmotic pressure regulator (such as sodium chloride) is added to the inner water phase, the osmotic pressure of the inner water phase will be increased. The increase in osmotic pressure means that the osmotic pressure difference between the inner water phase and the outer water phase (W2 phase) becomes larger. Due to the osmotic pressure difference, during the double emulsion process (W1 / O→W1 / O / W2) and the subsequent solvent volatilization process, water will penetrate from the outer water phase with low osmotic pressure into the inner water phase with high osmotic pressure, especially during the curing process, which will significantly affect the morphology and internal structure of the microspheres.

[0071] Preferably, it can be understood by those skilled in the art that as the osmotic pressure of the inner water phase increases, during the preparation process, due to the osmotic pressure difference between the inner and outer water phases after the solvent evaporates, more outer water phase will penetrate into the inner water phase, resulting in the volume expansion of the inner water phase. This will gradually increase the cavity inside the microsphere. As the osmotic pressure regulator (such as NaCl concentration) in the inner water phase increases, the cavity expansion may be more obvious, forming a larger cavity or more porous structure under certain conditions. This is because the water osmosis continues to move to the inner water phase, making the internal structure more "empty".

[0072] Preferably, it will be appreciated by those skilled in the art that as the volume of the inner water phase expands, more of the inner water phase will occupy the space originally occupied by the polymer, and the polymer material will form a coating around it. If the volume of the inner cavity increases significantly, the wall thickness of the microsphere will usually become thinner. The reduction in wall thickness is due to more polymer material participating in the formation of the outer shell, while the interior is an expanded cavity. Due to the osmotic pressure difference of water causing a large amount of water to move, 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.

[0073] 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 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 a 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.

[0074] In a preferred embodiment, according to the method for preparing the drug carrier of the present invention, the freeze-thaw treatment refers to 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.

[0075] 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 inside 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 rupture and crack changes.

[0076] In a preferred embodiment, the common freeze-thaw treatment procedure comprises a freezing step and a thawing step. Preferably, the thawing step can also be understood as a melting step.

[0077] In a preferred embodiment, wherein, in the present invention, the freezing step is to cool the polymer microspheres to a low temperature, generally involving several processes such as programmed cooling, rapid cooling or slow cooling. In a preferred embodiment, the present invention adopts a programmed cooling process, for example, a rapid programmed cooling or a programmed cooling process with a high cooling rate, so that ice crystals will be generated quickly to pierce the microspheres. . Programmed cooling usually requires a special programmed cooling device or a low-temperature refrigerator with a controlled rate. When rapid cooling is adopted, the polymer microspheres can be quickly placed in a liquid nitrogen (-196°C) or low-temperature (-80°C) cooling system to quickly freeze the water, promote the formation of ice crystals and destroy the original structure. When slow freezing is adopted, the polymer microspheres can be slowly cooled from room temperature to a freezing temperature, generally in a cryopreservation box. In a preferred embodiment of the present invention, preferably, the freezing step adopts programmed cooling, such as a rapid cooling program, for example, programmed cooling at a rate of 30°C / min.

[0078] In a preferred embodiment, in the present invention, the thawing step includes, for example, room temperature thawing. Room temperature thawing is relatively simple, and the method is to slowly thaw the frozen sample at room temperature 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 thaw by heating the cold table equipment. In a preferred embodiment of the present invention, preferably, the thawing step uses room temperature thawing.

[0079] 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 an oil phase O: such as an ethyl acetate solution of polylactic acid-glycolic acid (PLGA) of a certain concentration. Configuring an outer aqueous phase W2: such as a polyvinyl alcohol aqueous solution of a certain concentration. 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 chamber.

[0080] In a preferred embodiment of the present invention, after preparing the water-in-oil-in-water composite emulsion, the oil phase is solidified and then the residual surfactant in the water phase is removed. It is particularly preferred to remove the residual surfactant by sieving or centrifugal washing.

[0081] In the present invention, the water-in-oil-in-water composite emulsion is obtained by homogenization or mechanical stirring. It generally includes the preparation of colostrum and the preparation of double emulsion. Exemplarily, the preparation of colostrum adopts a traditional stirring method, for example, the oil phase is mixed with the inner water phase and placed in a centrifuge tube, and the colostrum is obtained by mechanical shearing. Exemplarily, the preparation of double emulsion adopts a traditional stirring method, for example, colostrum is added to the outer water phase, and the double emulsion is obtained by mechanical shearing, i.e., the water-in-oil-in-water composite emulsion.

[0082] In another preferred embodiment, the polymer microspheres of the present invention can also be obtained by membrane emulsification. An exemplary method includes passing the water-in-oil colostrum through a membrane tube having a micron pore size, such as 1-50 μm, preferably 15-30 μm, to obtain a double emulsion after multiple membrane passes, and the double emulsion is evolved for a suitable time to produce polymer microspheres.

[0083] In a preferred embodiment, in the present invention, the solvent of the inner 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 inner aqueous phase.

[0084] In a preferred embodiment, in the present invention, the solvent of the oil phase includes 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, and is more preferably a volatile organic solvent, such as n-butanol, ether, chloroform, tetrachloromethane, etc., and is further preferably a volatile and partially water-soluble organic solvent, such as ethyl acetate, phenol, etc.

[0085] In a preferred embodiment, as preferred, in the present invention, the organic solvent is an organic solvent with a solubility in water of less than 10%, preferably an organic solvent with a solubility in water of less than 2%, and most preferably an organic solvent insoluble in water. In a preferred embodiment, as preferred, the organic solvent is 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. It can also be selected from any combination of the above different organic solvents, and the specific type or volume depends on the preparation parameters such as the membrane material used.

[0086] In a preferred embodiment, in the present invention, the external aqueous phase further comprises an emulsifier, which is selected from at least one of sodium alginate, polyvinyl alcohol fiber, Tween 80, methyl cellulose, gelatin, polysorbate, lysine, gum arabic, and poloxamer 188.

[0087] In a preferred embodiment, in the present invention, colostrum can be prepared by mixing an internal aqueous phase with a salt concentration of 0.05-2 mg / mL such as sodium chloride and a polymer with a concentration of 20-100 mg / mL as an oil phase in 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.

[0088] In a preferred embodiment, in the present invention, after the step (a) of preparing polymer microspheres, a step of selecting polymer microspheres with suitable wall thickness is also included. The present invention has found through research that the 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 a polymer microsphere and water mixing system is selected, the 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. The 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. The polymer microspheres with a sedimentation time of more than 120 minutes have a smaller wall thickness. Although they can be loaded with drugs, the drug release performance is not as good as the polymer microspheres with a sedimentation time of less than 30-120 minutes. It is understood that when the selected sedimentation system is salt ions or other types of solutions, 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.

[0089] 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 by physical binding methods, such as infiltration, adsorption, etc.

[0090] 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 vary. For example, the polymer microspheres and water mixture are frozen at low temperatures and melted at room temperature, and freeze-thaw can be performed once or multiple times. The number of repeated freeze-thaw processes is not particularly limited, but preferably 2-10 times, and preferably 3-9 times, such as 3, 4, 5, 6, 7, 8, 9 times. The present invention has been found through research that the number of freeze-thaw times increases significantly, and therefore, those skilled in the art can adjust the number of freeze-thaw times as needed, for example, according to the size of the pores required for drug loading.

[0091] In a preferred embodiment, in the present invention, low temperature during freeze-thaw process refers to below the freezing point of the suspension system. For example, the water suspension of polymer microspheres is selected to be 0 to -200°C, preferably -30 to -190°C, also preferably -40 to -180°C, further preferably -50 to -170°C, for example -60, -70, -80, -90, -100, -110, -120, -130, -140, -150, -160, -170°C.

[0092] Pharmaceutical composition

[0093] 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 antigens, protein drugs or other drugs, etc., and can be antigenic proteins of infectious pathogens, such as antigens of new crown vaccines, such as new crown BRD proteins, for example, antigens of influenza vaccines, such as influenza HA proteins, or can be protein therapeutic agents used clinically, such as monoclonal antibodies, protein hormones, etc., and can also be water-soluble chemical drugs, such as goserelin, minoxidil, etc., without particular limitation.

[0094] 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.

[0095] 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.

[0096] In a preferred embodiment, in the present invention, according to the pharmaceutical composition of the present invention, the preparation method of the pharmaceutical composition includes: mixing the drug carrier with a solution containing the drug so that the drug enters the microspheres, for example, the drug loading after the polymer microspheres are prepared can be achieved by physical means, such as drug diffusion or adsorption, 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 a solution containing the drug, and then the supernatant is removed by centrifugation to obtain polymer microspheres loaded with the drug.

[0097] In a preferred embodiment, in the present invention, the pharmaceutical composition according to the present invention further comprises: sealing the drug carrier loaded with the drug to form a sealed microcapsule loaded with the drug.

[0098] In the present invention, those skilled in the art can understand that the "sealing" means embedding or fixing or retaining the drug inside the polymer microsphere. In the present invention, the sealed microcapsule does not mean that there are no openings on the surface of the microcapsule, but only means that the antigen and / or other substances such as other drugs can be partially embedded or fixed or retained inside the microcapsule through the sealing process, and the antigen and / or other substances such as other drugs can be released from the microcapsule by degradation of the microcapsule.

[0099] 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 rising annealing method, and technicians in the relevant field can also seal the drug carrier according to their professional knowledge / new technology.

[0100] In a preferred embodiment of the present invention, the temperature rise 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 to heal and seal the pores on the surface. 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, and after a period of time, the temperature is slowly lowered to close 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.

[0101] In a preferred embodiment of the present invention, a solvent-assisted temperature rise annealing method can be used, for example, a small amount of organic solvent and surfactant that can dissolve the polymer microspheres can be added during the temperature rise annealing process, which can reduce the required glass transition temperature, and can also effectively encapsulate drugs such as antigens in microcapsules, and the loading rate and embedding rate are stable. Among them, in one embodiment of the present invention, the small amount of organic solvent and surfactant, for example, is ethyl acetate with a volume ratio of 4% and PVA with a mass volume ratio of 0.375%.

[0102] 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.

[0103] 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 those of ordinary skill in the art can determine that they meet clinical standards. Pharmaceutically acceptable carriers include diluents and excipients.

[0104] 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) glucose; antioxidants such as tryptamine and stabilizers such as Tween 20 may also be included.

[0105] In a preferred embodiment, the pharmaceutical composition of the present invention is a therapeutic agent for preventing, treating and / or improving a disease, preferably an infectious disease. In another embodiment, the pharmaceutical composition of the present invention is a vaccine for preventing, treating and / or improving 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 rubella, echinococcosis, filariasis, epidemic hemorrhagic conjunctivitis, bacillary dysentery, typhoid and paratyphoid fever, etc.

[0106] It is understood that the pharmaceutical composition of the present invention is not limited to the above 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.

[0107] The pharmaceutical composition of the present invention can be any suitable dosage form. For example, injection, suspension, emulsifier, etc. The pharmaceutical composition of the present invention can be applied to the body by known means. For example, delivered to the tissue of interest by intramuscular injection, optionally administered via intravenous, percutaneous, intranasal, oral, mucosal or other delivery methods. Such administration can be carried out via a single dose or multiple doses. It is understood by those skilled in the art that the actual dose to be administered herein can vary depending on a variety of factors to a great extent, such as target cells, biological types or their tissues, the general condition of the subject to be treated, the route of administration, the mode of administration, etc.

[0108] It is understood that the pharmaceutical composition is given to an individual in a preventive effective amount or a therapeutically effective amount (depending on the circumstances, although prevention can be considered as treatment), which is sufficient to show benefit to the individual. Typically, this will cause therapeutically useful activity that is beneficial to the individual. The actual amount of the 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 doctors, and typically take into account the condition being treated, the individual patient's condition, the delivery site, the method of administration, and other factors known to the doctor.

[0109] In a preferred embodiment, in the present invention, the pharmaceutical composition is a vaccine, which is used for preventive or therapeutic treatment of tumors or infectious diseases in mammalian subjects.

[0110] 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 has different wall thicknesses of polymer microspheres, or any combination thereof.

[0111] 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.

[0112] For a specific vaccine, the ratio of different types of microspheres can be adjusted to achieve the purpose of the present invention. Based on the teachings of the present invention, any combination of compound preparations can be obtained by adjusting the evolution time and the sedimentation time, thereby obtaining a pulse release polymer microsphere with multiple release stages. Therefore, the polymer microspheres of the present invention can have multiple, for example, at least 2, such as 3, 4, 5, 6, 7, 8, 9, 10, or even more release processes.

[0113] Therefore, the present invention also provides a method for regulating drug release behavior, which comprises the step of using the drug carrier (polymer microsphere) or compound preparation of the present invention. Preferably, by selecting appropriate evolution time and sedimentation time, any combination of polymer microspheres with a single chamber, polymer microspheres with a multi-chamber structure, and polymer microspheres with different wall thicknesses is obtained.

[0114] Vaccine adjuvants

[0115] In one aspect of the present invention, a vaccine adjuvant is provided, which includes the drug carrier described herein, such as the polymer microspheres of the present invention. The term "vaccine" as used herein refers to a preventive biological product for preventing and controlling the occurrence and spread of infectious diseases and for human vaccination, including those prepared using microorganisms or their toxins, enzymes, human or animal sera, cells, etc. for prevention, diagnosis and / or treatment. 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, etc. can be used.

[0116] In a preferred embodiment, the vaccine is an influenza vaccine, a herpes vaccine, a COVID-19 vaccine, or an anti-tumor vaccine.

[0117] In the present invention, the vaccination of the vaccine may be therapeutic or prophylactic. For example, it may be possible to achieve prophylactic protection against the onset of cancer disease by immunizing individuals who do not suffer from cancer. Examples of individuals for whom such prophylactic vaccination may be used are individuals with an increased risk of developing cancer, although such use is not limited to such individuals. Patients at risk of cancer may have already developed a tumor, either as a primary tumor or a metastasis, or have shown a predisposition to cancer.

[0118] 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 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.

[0119] 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 a polymer microsphere having or substantially having a single chamber structure, and freeze-thaw the polymer microsphere to form openings or pores on the surface of the polymer microsphere, wherein the polymer microsphere is prepared from polylactic acid, polylactic acid-glycolic acid copolymer and / or polyglycolic acid, and the average particle size of the polymer microsphere 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 hemagglutinin (HA) is mixed with the drug carrier described in the present invention in a solution so that influenza hemagglutinin 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 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 process to prepare a freeze-dried preparation.

[0120] 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 sealed microcapsules 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 procedure to form a freeze-dried preparation.

[0121] In a preferred embodiment, the present invention provides a herpes zoster vaccine, which comprises 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 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, 2-20 μm, the Span value of the polymer microspheres is preferably, for example, less than 1.5, and the average particle size 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 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 herpes zoster virus antigen gE protein. Preferably, the drug carrier loaded with herpes zoster virus antigen gE protein can be sealed to form a sealed microcapsule 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.

[0122] 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 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 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 a sealed microcapsule loaded with prostate cancer neoantigens. Preferably, the drug carrier loaded with prostate cancer neoantigens can be made into a lyophilized preparation by 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.

[0123] 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.

[0124] 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, so the therapeutic dose of the present invention can vary widely. Generally speaking, the dosage of the vaccine adjuvant used in the present invention is well known to those skilled in the art. The dosage applied is within the predictable range of the clinician or laboratory personnel, and the dosage can be appropriately adjusted, for example, through effectiveness and safety tests, to obtain the optimal dosage.

[0125] use

[0126] The present invention provides the use of a drug carrier in the preparation of a drug. Preferably, the polymer microspheres that can be loaded with drugs are used as delivery carriers for vaccines, and the loaded substances may include hydrophobic or hydrophilic antigens and / or adjuvants.

[0127] In a preferred embodiment, the pulse-release polymer microspheres loaded with drugs in the present invention can effectively achieve the staged release of internal drugs. When loaded with antigens, they can be used as vaccines to induce potent immune responses and establish immune protection through subcutaneous or intramuscular injection.

[0128] In certain embodiments, the drug carrier provided by the present invention has at least the following advantages:

[0129] (1) The drug carrier of the present invention can realize surface opening or cracks by freeze-thawing, and can realize drug post-loading by spontaneous diffusion of drugs after microsphere preparation, effectively improving the encapsulation rate and ensuring the stability of drug structure and activity. The drug carrier itself can be freeze-dried, and freeze-dried preparations can be prepared by selecting appropriate freeze-drying conditions according to different encapsulated drugs.

[0130] (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 a plateau between the stages, or it can have a sustained release process.

[0131] (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.

[0132] 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.

[0133] The following examples are set forth in order to more clearly illustrate the principles and practices of the embodiments disclosed herein to those skilled in the art, and should not be construed as limiting the scope of any claimed embodiments.

[0134] Example 1

[0135] In this embodiment, polymer microspheres are prepared using poly(lactic acid-co-glycolic acid) copolymer as a material, as follows.

[0136] The polylactic acid-glycolic acid copolymer (PLGA) labeled with Nile red dye was dissolved in ethyl acetate to prepare a 50 mg / mL PLGA oil phase solution; a 1 mg / mL NaCl aqueous solution was prepared as the inner aqueous phase; polyvinyl alcohol (PVA) was placed in water, 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.

[0137] Take 2mL of oil phase and mix it with 200μL of inner water phase, stir it at 7000rpm in a homogenizer for 30 seconds to obtain colostrum. After that, you can choose the traditional mechanical stirring method to prepare it. Add colostrum to 15mL of outer water phase and stir it at 7000rpm in a homogenizer for 2 minutes to obtain a W / O / W double emulsion. Vortex the W / O / W double emulsion for 15 minutes to make its oil phase evolve into a single-chamber structure, and then add the double emulsion to a sufficient amount of pure water for extraction or evaporate it in a fume hood to remove the organic solvent to obtain solidified mixed polymer microspheres. Mix the mixed polymer microspheres with 10mL of water and place them in a 10mL centrifuge tube, and take microspheres with a sedimentation time between 30 minutes and 2h. The microspheres obtained were observed by laser confocal microscopy, and their surface wall thickness was counted, and their particle size was measured using a particle size analyzer ( Figure 1 ).

[0138] Example 2

[0139] This example uses polylactic acid-glycolic acid copolymer as a material to prepare polymer microspheres, which is different from Example 1 mainly in the sedimentation time, as follows.

[0140] The polylactic acid-glycolic acid copolymer (PLGA) labeled with Nile red dye was dissolved in ethyl acetate to prepare a 50 mg / mL PLGA oil phase solution; a 1 mg / mL NaCl aqueous solution was prepared as the inner aqueous phase; polyvinyl alcohol (PVA) was placed in water, 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.

[0141] Take 2mL of oil phase and mix it with 200μL of inner water phase, and stir it at 7000rpm in a homogenizer for 30 seconds to obtain colostrum. After that, you can choose the traditional mechanical stirring method to prepare it. Add colostrum to 15mL of outer water phase and stir it at 7000rpm in a homogenizer for 2 minutes to obtain a W / O / W double emulsion. Vortex the W / O / W double emulsion for 15 minutes to allow its oil phase to evolve into a single-chamber structure, and then add the double emulsion to a sufficient amount of pure water for extraction or evaporate it in a fume hood to remove the organic solvent to obtain solidified mixed polymer microspheres. Mix the mixed polymer microspheres with 10mL of water and place them in a 10mL centrifuge tube, and take the microspheres with a sedimentation time of less than 30 minutes. The microspheres obtained were observed by laser confocal microscopy, and their surface wall thickness was counted, and their particle size was measured using a particle size analyzer ( Figure 1 ).

[0142] Example 3

[0143] This example uses polylactic acid-glycolic acid copolymer as a material to prepare polymer microspheres, which is different from Example 1 mainly in the final sedimentation time, as follows.

[0144] 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.

[0145] Take 2mL of oil phase and mix it with 200μL of inner water phase, and stir it at 7000rpm in a homogenizer for 30 seconds to obtain colostrum. After that, you can choose the traditional mechanical stirring method to prepare it. Add colostrum to 15mL of outer water phase and stir it at 7000rpm in a homogenizer for 2 minutes to obtain a W / O / W double emulsion. Vortex the W / O / W double emulsion for 15 minutes to allow its oil phase to evolve into a single-chamber structure. Then add the double emulsion to a sufficient amount of pure water for extraction or evaporate it in a fume hood to remove the organic solvent to obtain solidified mixed polymer microspheres. Mix the mixed polymer microspheres with 10mL of water and place them in a 10mL centrifuge tube. Take the microspheres with a sedimentation time of more than 2 hours. The microspheres obtained by optical microscopy ( Figure 2 ), the wall thickness of which is thinner than that of Example 1.

[0146] Example 4

[0147] This embodiment demonstrates a method for preparing single-cavity microspheres using polylactic acid-glycolic acid copolymer as a material, as follows.

[0148] 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.

[0149] 1 mL of the oil phase was mixed with 200 μL of the inner aqueous phase and oscillated at 30% ultrasonic power for 12 seconds to obtain colostrum. Then, the traditional mechanical stirring method was selected to prepare the colostrum, add the colostrum to 15 mL of the outer aqueous phase, and stir at 8000 rpm for 2 minutes in a homogenizer to obtain a W / O / W double emulsion. Then, the double emulsion was immediately added to a sufficient amount of pure water for extraction or evaporated in a fume hood to remove the organic solvent to obtain solidified single-cavity polymer microspheres.

[0150] The obtained microspheres were observed by light microscopy, and the particle size was measured by dynamic light scattering, e.g. Figure 3 As shown, single-cavity microspheres can be prepared.

[0151] Example 5

[0152] This example demonstrates a method for preparing single-cavity microspheres using polylactic acid-glycolic acid copolymer as a material, as follows.

[0153] 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.

[0154] Take 1mL of oil phase and mix it with 100μL of inner water phase, and oscillate it at 30% ultrasonic power for 12 seconds to obtain colostrum. Then, you can choose the traditional mechanical stirring method to prepare it. Add colostrum to 15mL of outer water phase and stir it at 8000rpm for 2 minutes to obtain W / O / W emulsion. Then immediately add enough pure water to extract or evaporate it in a fume hood to remove organic solvent to obtain solidified mixed polymer microspheres.

[0155] The obtained microspheres were observed by light microscopy, and the particle size was measured by dynamic light scattering, e.g. Figure 3 As shown, single-cavity microspheres were also prepared, but with thicker walls.

[0156] Example 6

[0157] 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.

[0158] Polylactic acid-glycolic acid copolymer (PLGA) and PELA were dissolved in ethyl acetate with 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 hours, and a 15 mg / mL PVA aqueous solution was prepared as the outer aqueous phase.

[0159] Take 2mL of oil phase and mix it with 200μL of inner water phase, and stir it at 7000rpm for 30 seconds in a homogenizer to obtain colostrum. After that, the traditional mechanical stirring method can be selected to prepare it. Add colostrum to 15mL of outer water phase and stir it at 7000rpm for 2 minutes in a homogenizer to obtain W / O / W double emulsion. Directly add the double emulsion into sufficient pure water for extraction or evaporate it in a fume hood to remove the organic solvent to obtain solidified multi-cavity polymer microspheres. The obtained microspheres were observed under a light microscope and it can be seen that they have obvious multi-cavity structure ( Figure 4 ).

[0160] Example 7

[0161] 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.

[0162] The polymer microspheres prepared in Example 4 were mixed with 3% PVA aqueous solution to form a suspension, and the temperature was cooled to -80°C at a cooling rate of 30°C / min. It can be observed that there was no ice formation inside the microspheres, and no ice crystals pierced the microspheres ( Figure 5 ).

[0163] The polymer microspheres prepared in Example 4 were mixed with pure water to form a suspension, and the temperature was lowered to -80°C at a cooling rate of 30°C / min. It can be observed that ice crystals pierced the microspheres instantly after the polymer microspheres were frozen, and the polymer microspheres showed cracks and holes ( Figure 5 ). This embodiment illustrates that the freeze-thaw loading method of the present invention can be performed based on the formation of ice crystals.

[0164] Example 8

[0165] In this example, the polymer microspheres of Example 4 were subjected to a freeze-thaw process for drug loading, as follows.

[0166] The polymer microspheres prepared in Example 4 were mixed with an equal volume of water to form a suspension, and placed in a -80°C refrigerator to quickly cool to -80°C. Freeze at -80°C for 2h, and then dissolve at room temperature. The freeze-thaw process was repeated 0, 2, 4, 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 to measure the cracks on their surface ( Figure 6 ), clear cracks can be seen on the surface of the polymer microspheres.

[0167] Example 9

[0168] In this example, the polymer microspheres of Example 5 were subjected to a freeze-thaw process for drug loading, as follows.

[0169] The polymer microspheres prepared in Example 5 were mixed with an equal volume of water to form a suspension, and placed in a -80°C refrigerator to quickly cool to -80°C. Freeze at -80°C for 2h, and then dissolve at room temperature. The freeze-thaw process was repeated 0, 2, 4, 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 to measure the cracks on their surface ( Figure 6 ), it can be seen that under the same freeze-thaw process, the number and length of cracks in the polymer microspheres of Example 5 are smaller than those in the polymer microspheres of Example 4.

[0170] Example 10

[0171] This example shows a vaccine adjuvant based on polymer microspheres prepared by Example 4 and a freeze-thaw process.

[0172] 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.

[0173] 1 mL of the oil phase was mixed with 200 μL of the inner aqueous phase and oscillated at 30% ultrasonic power for 12 seconds to obtain colostrum. Then, the traditional mechanical stirring method was selected to prepare the colostrum, add the colostrum to 15 mL of the outer aqueous phase, and stir at 8000 rpm for 2 minutes in a homogenizer to obtain a W / O / W double emulsion. Then, the double emulsion was immediately added to a sufficient amount of pure water for extraction or evaporated in a fume hood to remove the organic solvent to obtain solidified single-cavity polymer microspheres.

[0174] The microspheres were mixed with an equal volume of pure water to form a suspension, and then placed in a -80°C refrigerator to quickly cool to -80°C. Freeze at -80°C for 2 hours, and then dissolve at room temperature. The freeze-thaw process was repeated 4 times to obtain a drug carrier based on single-cavity polymer microspheres. Optical microscopy and scanning electron microscopy showed that the drug carrier had obvious cracks on its surface ( Figure 7 ), drugs can be loaded into the microspheres through the cracks.

[0175] Embodiment 11

[0176] This example shows the drug loading of the post-drug loaded polymer microspheres obtained in Example 4.

[0177] Cy5-labeled bovine serum albumin (BSA) was prepared into a 1 mg / mL solution, mixed with the microspheres of Example 4 after different freeze-thaw times in Example 8, and vortexed in a refrigerator at 4 degrees for 3 days to allow the protein to be loaded by diffusion.

[0178] By observing the drug loading through laser confocal microscopy, it can be found that different freeze-thaw times affect the drug loading. Figure 8 As shown, when the repeated freezing and thawing was performed 0 times, 2 times, and 4 times, the drug loading positive rate continued to increase. When the repeated freezing and thawing was performed 8 times, the drug loading positive rate remained consistent with the 4th time and reached a peak value.

[0179] Example 12

[0180] This example shows the drug loading of the post-drug loaded polymer microspheres obtained in Example 5.

[0181] Cy5-labeled bovine serum albumin (BSA) was prepared into a 1 mg / mL solution, mixed with the microspheres of Example 5 after different freeze-thaw times in Example 9, and vortexed in a refrigerator at 4 degrees for 3 days to allow the protein to be loaded by diffusion.

[0182] By checking the drug loading through laser confocal microscopy, it can be found that different freeze-thaw times affect the drug loading. When repeated freeze-thaw times 0, 2, 4, and 8 times, the drug loading positive rate is lower than the result shown in Example 11 ( Figure 8 ), which shows that the wall thickness of microspheres affects the loading of protein drugs into microspheres after freeze-thaw treatment.

[0183] Embodiment 13

[0184] This embodiment shows a polymer microsphere prepared by sealing the polymer microsphere and then loading the drug thereon.

[0185] On the basis of Example 11, after the suspension is completed, the sample tube is taken out, the microspheres deposited at the bottom are collected by centrifugation, 4% (volume ratio) ethyl acetate and 0.375% PVA aqueous solution are used as the healing liquid, the volume of the microspheres: the volume of the healing liquid is 1:25, and the microspheres are evenly resuspended in the centrifuge tube. After sealing, put it in a 40°C constant temperature water bath, keep the centrifuge tube rotating to prevent the microspheres from aggregating, and heal for 30 minutes to obtain polymer microspheres with closed surface cracks ( Fig. 9 ).

[0186] Embodiment 14

[0187] This example shows the drug release behavior of the drug carrier of Example 10 after loading the protein.

[0188] The microspheres prepared in Example 10 were used as drug carriers, mixed with 1 mg / mL BSA solution labeled with Cy5, and vortexed in a refrigerator at 4°C for 3 days to load the drug. The microspheres at the bottom were collected by centrifugation, and the microspheres were washed 2 to 3 times with PBS to obtain drug carriers for detecting in vitro and in vivo release.

[0189] The preparation was placed in PBS and placed in a 37°C constant temperature shaker to simulate in vitro release. A portion of the preparation was taken every day for laser confocal microscopy to determine the in vitro release behavior. Fig.10 As shown, the drug carrier has two release processes in succession, which can significantly delay the drug residence time.

[0190] Another preparation was mixed with normal saline and injected subcutaneously into mice. The mice were placed in an anesthesia box for gas anesthesia (isoflurane) at different time points. After being fully anesthetized, the mice were placed in a small animal in vivo imaging chamber for imaging to detect the in vivo drug release behavior of the drug carrier in mice. Fig.11 As shown, the drug carrier also exhibits a two-stage sustained-release behavior in mice, which can significantly delay the drug residence time.

[0191] Embodiment 15

[0192] This embodiment proposes a drug carrier of compounded microspheres of various structures, through which the release behavior of the drug can be adjusted.

[0193] The microspheres prepared in Example 3 and Example 4 were mixed in a ratio of 1:1 to form composite microspheres. The composite microspheres were compounded with an equal volume of pure 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, and then dissolve at room temperature. Repeat the freeze-thaw process 4 times to obtain a drug carrier based on composite microspheres.

[0194] The above-prepared microspheres were used as drug carriers, mixed with 1 mg / mL BSA solution labeled with Cy5, and vortexed in a refrigerator at 4°C for 3 days to load the drug. The microspheres at the bottom were collected by centrifugation, and the microspheres were washed 2 to 3 times with PBS to obtain drug carriers for testing in vitro and in vivo release.

[0195] The preparation was mixed with normal saline and injected subcutaneously into mice. The mice were placed in an anesthesia box for gas anesthesia (isoflurane) at different time points. After being fully anesthetized, the mice were placed in a small animal in vivo imaging chamber for imaging to detect the in vivo drug release behavior of the drug carrier in mice. Fig.12 As shown, the drug carrier has a similar sustained-release behavior in mice, but the drug release is faster than that of the drug carrier of the single-cavity microspheres in Example 14.

[0196] Example 16

[0197] This example proposes a freeze-dried vaccine preparation based on Example 10. The preparation process diagram is as follows: Fig.13 As shown, the protein function of the antigen and the adjuvant morphology are still maintained after lyophilization.

[0198] 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, 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-chamber microspheres.

[0199] The SARS-CoV-2 RBD is compounded with a certain lyophilization protectant, 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, and mixed in a 4°C refrigerator for 3 days. The bottom microspheres are collected by centrifugation, washed 2 to 3 times with PBS, and frozen in a -80°C refrigerator overnight. The next day, they are taken out and freeze-dried in a freeze dryer to obtain a freeze-dried vaccine preparation. The overall process is shown in Figure x.

[0200] The morphology of the freeze-dried and non-freeze-dried microsphere preparations was observed by scanning electron microscopy, and the distribution was measured by an ergodic analyzer. The results showed that the morphology and particle size distribution did not change before and after freeze-drying ( Fig.14The freeze-dried and non-freeze-dried microsphere preparations were ultrasonically broken, and the supernatant was collected by centrifugation to separate the protein. The protein structure was detected by circular dichroism. The results showed that the protein structure did not change before and after freeze-drying ( Fig.14 ).

[0201] Embodiment 17

[0202] This example shows the immune activation effect of the lyophilized vaccine adjuvant described in Example 16 on mice.

[0203] Based on the vaccine adjuvant described in Example 16 and physiological saline, the new coronavirus subunit vaccine was prepared. Mice were immunized by intramuscular injection, with 3 μg equivalent of the dimer prototype strain RBD (obtained from Zhifei Company) / mouse and 500 μg equivalent of polymer microspheres / mouse. Mice vaccinated with PBS were used as blank controls, and mice vaccinated with 3 μg equivalent of the dimer prototype strain RBD / mouse and 50 μg equivalent of Al aluminum adjuvant (purchased from Invivogen Company, ) / only as experimental control, mice were killed on day 0, day 1, day 3, and day 5, and muscles at the injection site were sliced ​​and stained with H&E. It can be seen that LyoC can recruit more monocytes on the third day ( Fig.15 ). At the same time, muscle tissue was taken on the third day after injection to prepare single cell suspension and flow staining was performed to mark the number of various immune cells. It can be seen that the monocytes recruited by LyoC on the third day were mainly dendritic cells, and the cell ratio was significantly higher than that of the Al group ( Fig.15 ).

[0204] 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 + ) recruitment and expression of activation markers ( Fig.16 ), and both types of dendritic cells, cDC1 and cDC2, were increased ( Fig.16 ), and also increased the number of germinal center B cells (CD19 + FAS + GL-7 + )quantity( Fig.17 ). Rapid recruitment and activation of dendritic cells is beneficial for antigen presentation, while more germinal center B cells are beneficial for the subsequent production of higher antibody levels.

[0205] By sorting DCs at the injection site with 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 ( Fig.18 ).

[0206] Embodiment 18

[0207] This example shows that the lyophilized vaccine adjuvant described in Example 16 induces higher levels of cellular immunity and cellular immune memory.

[0208] Reference Example 17: Two doses of the above drug were given 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 red lysis, the cells were cultured under antigen stimulation for 2 days, and then the cells were collected for flow staining. It was found that LyoC could induce stronger CD4 + T cells and CD8 + T cell immune response ( Fig.19 ), with higher levels of cytokine secretion and activation marker levels.

[0209] Staining of memory-related markers in splenocytes extracted on day 28 revealed that LyoC could induce higher 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, and 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 )( Fig. 20 ).

[0210] Embodiment 19

[0211] This example shows that the lyophilized vaccine adjuvant described in Example 16 induces higher levels of antibodies.

[0212] Referring to Example 17, mice were immunized twice on days 0 and 14 (LyoC group, Al group), and the levels of RBD (antigen)-specific IgG antibodies in the serum of mice in each group were continuously detected at different time points after the first immunization. The results showed that LyoC could induce a strong humoral immune response in mice ( Fig.21At the same time, pseudoviruses of various variants of the new coronavirus were co-incubated with mouse serum obtained in the 4th week to infect hACE-2 transgenic HEK-293T cells. The infection of the pseudovirus was detected by the Luc gene reporter system, and the half-inhibitory titer of the serum was used as an indicator of antibody quality. The results indicated that the vaccine adjuvant can induce higher antibody quality ( Fig.21 ).

[0213] The mice were killed one month after the two immunizations, and the lymph nodes were taken for BCR sequencing to evaluate the maturity of B cells in each group. It was found that the polymer microsphere group induced more diverse BCR sequences, higher mutation rates and longer CDR3 sequences, indicating that the B cells in the polymer microsphere group were more mature. This may be the reason for the high antibody quality ( Fig. 22 ).

[0214] Embodiment 20

[0215] This example shows that the lyophilized vaccine adjuvant described in Example 16 induces a potent immune response in the cynomolgus monkey model.

[0216] Cynomolgus monkeys were injected with aluminum adjuvant (purchased from Invivogen, ) was used as the experimental group (LyoC), and on the 21st day, the mice were injected with the polymer microspheres compounded with the BA.4 / 5 strain vaccine prepared based on the scheme of Example 15 (25 μg equivalent of BA.4 / 5 strain RBD (purchased from Sino Biological) / mouse and 2500 μg equivalent of post-drug-loaded polymer microspheres / mouse) as the experimental group (LyoC), and on the 21st day, the mice were injected with the aluminum adjuvant (purchased from Invivogen, ) 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 both antigens ( Fig.23 ).

[0217] Embodiment 21

[0218] This example shows that the lyophilized vaccine adjuvant described in Example 16 induces effective immune protection in a mouse influenza challenge model.

[0219] 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 of influenza virus antigen PR8 strain (purchased from Sino Biological) was prepared, and mice were immunized by intramuscular injection, with 5 μg equivalent of HA / mouse and 500 μg equivalent of polymer microspheres loaded with drugs / mouse. The mice vaccinated with PBS 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, AddaVax TM ) / only as experimental control 2 (Addavax). After two doses on days 0 and 14, 10 4 PFU of PR8 influenza virus, and the weight and survival of mice were tested within 14 days. The mice were killed on the 7th day, and the lungs and noses were taken for detection of viral load by real-time quantitative PCR. The lung lesions were observed by H&E sections, and the presence of the virus was determined 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 challenge. At the same time, the viral load in the lungs and noses of the mice was significantly lower than that in the Al-adjuvant group, comparable to that in the Addavax group, and had a lower level of lung inflammation, and no obvious viral sites were observed ( Fig.24 ).

[0220] Embodiment 22

[0221] This example demonstrates the safety of the lyophilized vaccine adjuvant described in Example 16 in mouse and cynomolgus monkey models.

[0222] Mice and cynomolgus monkeys were immunized with reference to Example 17 and Example 20, respectively. At the end of immunization, mice were killed and the heart, liver, spleen, lung, and kidney were taken for H&E sections to observe the inflammation level. At the end of immunization, peripheral blood of cynomolgus monkeys was taken for blood routine and blood biochemical tests. It was found that there were no abnormal changes in biochemical parameters in the LyoC group, and no obvious pathological characteristics were found in the sections of various organs of mice, which fully demonstrated the safety of LyoC as a vaccine adjuvant ( Fig.25 ).

[0223] Although the present invention has been described with reference to exemplary embodiments, it should be understood that the present invention is not limited to the disclosed exemplary embodiments. Various adjustments or changes 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 should be based on the broadest interpretation to cover all 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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