A microcapsule-based vaccine

Abstract

The present application relates to a vaccine comprising an antigen and a biodegradable polymer blend matrix, the polymer blend containing a hydrophobic polymer and an amphiphilic block copolymer, the vaccine being in the form of microcapsules, the microcapsules having a multi-chamber structure inside, the average particle size of the microcapsules being preferably 10-100 μm, more preferably 30-60 μm, the microcapsules being prepared by the following method: first, open microspheres are prepared from the polymer blend, then mixed with an antigen-containing solution, and then the open microspheres loaded with the antigen solution are sealed to form sealed microcapsules loaded with the antigen.

Description

Technical Field

[0001] This invention relates to a microencapsulated vaccine comprising an antigen and a biodegradable polymer blend matrix, such as an antitumor vaccine. Technical Background

[0002] With the development of tumor biology and immunology, immunotherapy, which primarily modulates the body's own immune system, has opened up a new avenue for cancer treatment. Among the many immunotherapy strategies, tumor vaccines can mimic the body's way of fighting pathogens, restoring the body's natural "recognition" and response to tumor cells, thereby specifically eliminating tumor cells, and have therefore attracted increasing attention.

[0003] One of the challenges in achieving high efficacy of anti-tumor vaccines is that tumor antigens alone are difficult to elicit a sustained and effective immune response. Using nanoscale carriers to load tumor antigens can effectively enhance the endocytosis and maturation of immune cells, thereby stimulating a stronger immune response. However, due to their small size, nanocarriers are easily cleared by the body, making it difficult to produce a sustained anti-tumor effect. Preparing micron-sized carriers by encapsulating antigens using a double emulsion method effectively avoids this problem; however, it faces the dilemma of antigen release being too slow to elicit an effective immune response.

[0004] CN101601860A discloses a polymer particle-based vaccine, wherein the antigen composition consists of two forms: antigen adsorbed on biodegradable polymer particles and unadsorbed antigen (free antigen). The average particle size of the particles is 0.1-20 μm. The vaccine is prepared by mixing polymer particles with a hepatitis B surface antigen solution. The suspension obtained by the adsorption of the polymer particles and the antigen solution is the vaccine product. The vaccine of this invention can rapidly induce an immune response in the body, inducing the body to produce a high level of humoral immunity and cellular immunity.

[0005] CN102489230A discloses a method for preparing biodegradable microcapsules, including the preparation of open-pore microspheres, the filling of core material, and the sealing of the open-pore microspheres. Compared with traditional double emulsion encapsulation methods, this method is gentler and avoids damage to bioactive substances; the remaining core material in the solution after encapsulation can be recycled; it is more environmentally friendly, achieving the same particle size and pore size levels as previously reported, and has a larger internal volume, which is more conducive to the loading of core material. However, this patent document only mentions that the biodegradable microcapsules can be used to encapsulate small molecules and biomacromolecules, and can also load nano- and micron-sized particles.

[0006] Despite the various studies mentioned above, the development of vaccines that can better stimulate the body to produce specific immunity, such as those that kill tumor cells using cytotoxic T cells, is of great significance for their clinical application. Invention Overview

[0007] The inventors of this application discovered that an open-pore microsphere made of biodegradable polymer was prepared using a double emulsion-solvent removal method. Tumor antigens were then filled into these microspheres, which were subsequently sealed to create larger-sized, locally residing microcapsules in mice. Because these microcapsules are not easily phagocytosed or metabolized, they are readily degraded. During degradation, they stimulate a sustained inflammatory response in the body. As an antigen reservoir, the microcapsules continuously recruit antigen-presenting cells, such as dendritic cells (DCs), to the injection site to phagocytose antigens. The acidic microenvironment generated by the polymer matrix metabolism significantly increases the number of recruited cells, enhances endocytosis of antigens, stimulates DC maturation and differentiation, and ultimately leads to DC homing to lymph nodes. This stimulates a highly efficient and sustained immune response, promoting continuous proliferation and differentiation of T cells, resulting in specific and persistent killing. This significantly inhibits tumor growth and prolongs the survival time of mice.

[0008] Based on this, the present invention provides a vaccine comprising an antigen and a biodegradable polymer blend matrix, wherein the polymer blend contains a hydrophobic polymer and an amphiphilic block copolymer, the vaccine being in the form of microcapsules, the microcapsules containing a multi-chamber structure, the average particle size of the microcapsules preferably being 10-100 μm, more preferably 30-60 μm, the microcapsules being prepared by the following method: firstly, open-pore microspheres are prepared from the polymer blend, then the microspheres are mixed with a solution containing the antigen, and then the open-pore microspheres loaded with the antigen solution are sealed to form sealed microcapsules loaded with the antigen.

[0009] Preferably, in the vaccine of the present invention, through optimization of the preparation process parameters, the release of antigen and the generation of cell recruitment behavior when the vaccine is locally retained in the body have a synergistic effect, so that the released antigen is phagocytosed by the recruited immature DCs, which significantly improves the utilization rate of antigen.

[0010] Preferably, the vaccine is a therapeutic vaccine, such as an anti-tumor vaccine or a therapeutic hepatitis B vaccine, and the preferred antigen is a tumor antigen or hepatitis B surface antigen.

[0011] In the vaccine of the present invention, a certain amount of chemokines, such as granulocyte colony-forming factor (GM-CSF), macrophage inflammatory protein 3α (MIP-3α), and monocyte chemoattractant protein 1 (MCP-1), can also be loaded into the porous microsphere system to enhance the recruitment of cells by the microcapsules, further improve antigen utilization, and enhance the immune response. Attached Figure Description

[0012] Figure 1 SEM image of microspheres prepared from PLA

[0013] Figure 2 SEM images of microspheres prepared from PLA and PELA

[0014] Figure 3 Particle size distribution of microspheres prepared from PLA and PELA

[0015] Figure 4 SEM images of the internal structure of microspheres prepared from PLA and PELA

[0016] Figure 5 Internal pore size distribution of microspheres prepared from PLA and PELA

[0017] Figure 6 SEM image of the sealing microcapsules of the present invention

[0018] Figure 7 Internal structure of the sealing microcapsule of the present invention

[0019] Figure 8 The loading and encapsulation rates of the OVA antigen in the sealed microcapsules of this invention are improved.

[0020] Figure 9 The loading rate and encapsulation rate of peptide MUC1 in the sealed microcapsules of this invention

[0021] Figure 10 Quantitative fluorescence intensity graphs of antigens at the injection site for different vaccine formulations

[0022] Figure 11 The recruitment behavior of the microcapsules of this invention on inflammatory cells: (a) representative tissue section images of recruited cells from tissues containing sealed microcapsules; (b) quantitative analysis of the number of cells recruited by each microcapsule.

[0023] Figure 12 Comparison of antigen utilization rates of different vaccine formulations: (a) OVA + Cell number, (b) OVA + (c) Antigen endocytosis by cells (expressed as mean fluorescence intensity), antigen utilization rate

[0024] Figure 13 pH change curve in the local microenvironment during the degradation of the microcapsules of this invention

[0025] Figure 14 CD86 in acidic and neutral microenvironments + The ratio of MHC-I to MHC-II in DC

[0026] Figure 15Tumor growth curves of E.G7 tumor-bearing mice immunized with different vaccine formulations

[0027] Figure 16 Tumor growth curves of B16 tumor-bearing mice immunized with different vaccine formulations

[0028] Figure 17 Quantitative analysis of metastatic lesions in lung (a) and kidney (b) tissues after treatment with different vaccine formulations

[0029] Figure 18 Tumor growth curves of 4T1 tumor-bearing mice immunized with different vaccine formulations

[0030] Figure 19 Tumor growth curves after postoperative recurrence following treatment with different vaccine formulations

[0031] Figure 20 Bioluminescence imaging images and quantification of postoperative recurrence model

[0032] Figure 21 H&E staining of recruited cells in subcutaneous tissue using blank sealed microcapsules (a) and sealed microcapsules loaded with the chemokine GM-CSF (b). Invention Details

[0033] vaccine

[0034] This invention relates to a vaccine for the preventive or therapeutic treatment of diseases such as tumors in mammalian subjects. The vaccine comprises an antigen and a biodegradable polymer blend matrix containing a hydrophobic polymer and an amphiphilic block copolymer. The vaccine is in the form of microcapsules with a multi-chamber structure. The average particle size of the microcapsules is preferably 10-100 μm, more preferably 30-60 μm. The microcapsules are prepared by: first, preparing open-cell microspheres with through-channels from the polymer blend; then mixing the microspheres with a solution containing the antigen; and finally sealing the open-cell microspheres containing the antigen solution to form sealed microcapsules loaded with the antigen. The vaccine of this invention may also contain a pharmaceutically acceptable carrier, salt, or diluent.

[0035] In a preferred embodiment of the present invention, the vaccine is an anti-tumor vaccine. The vaccine is simple to prepare and has universal applicability for the prevention of recurrence, suppression of metastasis, and treatment of various types of tumors, and exhibits excellent anti-tumor efficacy. Upon vaccination with the anti-tumor vaccine, antigen-presenting cells (APCs) first take up and process tumor antigens, converting them into antigenic peptides, which then bind to major histocompatibility complex (MHC) molecules on the cell surface. The APCs further present the peptide information to T cells, thereby generating a subsequent immune response. During this process, most antigens, after being processed into peptides in lysosomes, bind to MHC-II molecules and are presented to CD4 cells. + T cells; more importantly, a small portion of antigens can also be processed into peptides by the proteasome in the cytoplasm, which bind to MHC-I molecules and are presented to CD8 cells. + T cells are transformed into cytotoxic T lymphocytes (CTLs). On one hand, CTLs directly bind to tumor cells to produce lysis and killing effects; on the other hand, CD4+... + T cells and CD8 + T cells work together to secrete cytokines to kill tumors, ultimately leading to tumor apoptosis and lysis.

[0036] In a preferred embodiment of the present invention, the antigen is a tumor antigen. Tumor antigens play an important role in tumor occurrence, development, and inducing anti-tumor immune responses in the body, and are target molecules for tumor immunotherapy. Tumor antigens are divided into two main categories according to their specificity: (1) tumor-associated antigens, which are antigens that are highly expressed in tumor tissues and also expressed to a certain extent in normal tissues; (2) tumor-specific antigens, which are antigens that are expressed only in tumor tissues but not in normal tissues, and therefore have better safety as tumor antigens. However, in the early stages of tumor vaccine development, due to technological limitations, it was difficult to discover and purify tumor-specific antigens, which limited their widespread application. In recent years, with the development of gene editing technology, researchers have successively discovered some specific antigens and neoantigens as new targets for tumor vaccine immunization, thereby specifically preparing personalized tumor antigens to improve the immune effect of vaccines, and finally solving the problem of antigen selection. However, suitable tumor antigens are still difficult to elicit an effective immune response, mainly because simple tumor antigens have a short half-life and are rapidly degraded and metabolized after injection, making it difficult for the body's immune system to be effectively activated.

[0037] In some embodiments of the present invention, the antitumor antigen is obtained by collecting peptides of proteins on the surface of live tumor cells, which are obtained by periodically adding a protease that does not cause cell death to primary cultured live tumor cells. For example, live tumor cells pre-eluted from growth medium are subjected to primary culture, and the tumor cells are treated with a protease without causing cell death, and the released surface tumor antigen is collected. Then, after a period of time, the primary cultured live tumor cells are repeatedly treated with the protease, with intervals sufficient to allow the activity of the surface tumor antigen to be restored by the cells. The surface tumor antigen is accumulated (enriched) until a dose sufficient for vaccination is reached, and the composition of the obtained surface tumor antigen is controlled.

[0038] 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.

[0039] The antitumor vaccine of the present invention may contain one or more tumor antigens, with the aim of generating one or more cytotoxic T lymphocyte clones, each recognizing a specific antigenic peptide to produce a more effective immune response. When several tumor antigens are present, the tumor antigen is preferably selected to induce an immune response against a type of tumor or tumor cells.

[0040] In some embodiments of the present invention, the antitumor vaccine of the present invention is basically composed of a mixture of different antigens or a mixture of antigens and different immune stimulating substances.

[0041] For cancer treatment, significant activation of CTLs and secretion of Th1 cytokines are essential for the body to attack and eliminate tumors. Similarly, the recruitment of sufficient APCs to engulf the vaccine during vaccine component release is crucial for eliciting an effective immune response. APCs are widely distributed throughout the body's tissues, playing a vital role in detecting pathogens. In peripheral tissues like the skin, Langerhans cells, other types of dendritic cells (DCs), and macrophages can detect danger signals and antigens, and upon activation, they home to lymphoid organs to present antigen information and activate the immune response. The migration of these immune cells from peripheral tissues to lymphoid organs is complex and primarily regulated by numerous chemokines.

[0042] In a preferred embodiment of the present invention, the antigen is hepatitis B surface antigen, and the vaccine is a therapeutic hepatitis B vaccine.

[0043] Hepatitis B is a global infectious disease caused by the hepatitis B virus (HBV). To date, 2 billion people worldwide have been infected with the hepatitis B virus, of whom approximately 350 million are chronic hepatitis B carriers. There is currently no effective treatment for hepatitis B. The interferon and lamivudine commonly used in antiviral therapy can rapidly inhibit viral replication, but rebound is common after discontinuation of the drugs, and they can also easily lead to the emergence of mutant strains.

[0044] In individuals with chronic HBV infection, the presence of immune tolerance leads to a weakened specific T cell response. The therapeutic vaccine aims to utilize the cellular immune response that primarily relies on the cytolytic activity of specific cytotoxic T lymphocytes (CTLs) to clear the virus. This involves CTLs releasing perforin and granzymes to kill infected hepatocytes, or inducing apoptosis of infected hepatocytes through a Fas-mediated pathway. However, recent studies have shown that non-cytolytic mechanisms involving Th1 cells and cytokines secreted by CTLs play a dominant role in antiviral immunity. Cytokines such as IFN-α can inhibit viral replication and even clear viral DNA through non-cytopathic mechanisms. In particular, studies in various animal models have shown that IFN-γ inhibits HBV replication intermediates and HBV-specific mRNA, playing a crucial role in viral clearance by stimulating the body's own cellular immune response and promoting the secretion of cytokines, primarily IFN-γ, to clear the virus.

[0045] Regarding hepatitis B surface antigen (HBsAg), there are several antigen types to choose from, including HBsAg expressed by *Saccharomyces cerevisiae* and *Hansenula polymorpha*, as well as CHO HBsAg expressed in mammalian cell systems. However, the preferred antigen type is HBsAg obtained through the *Hansenula polymorpha* expression system. HBsAg from different sources differs significantly in structure and properties. Even with the same or similar gene coding, HBsAg particles expressed by different systems may still have different molecular sizes, molecular weights, and subunit numbers, thus exhibiting different charges, hydrophobicities, and immunogenicity.

[0046] In a preferred embodiment of the present invention, the vaccine further contains chemokines. Chemokines are a class of polypeptides with a molecular weight of 10 kDa that primarily act on receptors associated with G glycoproteins on the cell surface. Chemokines and other chemotactic molecules are produced locally and then diffuse to form a soluble or solid concentration gradient. Cells expressing chemokine receptors then rely on this concentration gradient information to reach the source of the chemotactic signal.

[0047] Many chemokines are involved in the migration of dendritic cells (DCs) and monocytes, including monocyte chemoattractant protein 1 (MCP-1), monocyte chemoattractant protein 2 (MCP-2), macrophage inflammatory protein 1α (MIP-1α), macrophage inflammatory protein 1β (MIP-1β), chemokines that regulate the expression and secretion of activated normal T cells (RANTES), complement C5a, β-defensins, and bacterial-derived formate peptides. Since DCs normally constitute only 1% or less of the content in peripheral tissues and blood, if a vaccine can create a local chemokine hub, continuously releasing chemokines to mimic and enhance the recruitment of DCs to the injection site, it can significantly enhance the immune response.

[0048] In a preferred embodiment of the present invention, the chemokine is, for example, macrophage inflammatory protein 3α (MIP-3α), monocyte chemoattractant protein 1 (MCP-1), and GM-CSF, preferably GM-CSF. The chemokine is contained in the antigen-containing solution and is filled into sealed microcapsules via open-cell microspheres. In a preferred embodiment of the present invention, the closed-chamber structure of the microcapsule of the vaccine ensures that the molecules loaded in the sealed microcapsules are effectively encapsulated within the cavity, achieving sustained and effective release. Throughout the release process, there is no significant burst release, no plateau phase, and the metabolic rate is slow and efficient, ultimately achieving an effective metabolic rate of up to 90%, meaning the antigen is essentially completely released.

[0049] The excellent antigen retention capacity of microcapsules ensures a more sustained release of antigens in vivo. Accompanying antigen release, the in-situ retention effect after microcapsule injection triggers an inflammatory response, thereby recruiting inflammatory-associated phagocytic vectors (APCs) and the released antigens. The kinetics of antigen and adjuvant polymer matrix release play a crucial role in controlling the immune response and affect the final immune effect of the vaccine. When their release kinetics are asynchronous—that is, when the antigen is released slowly and the adjuvant is released too quickly or too slowly—it will affect the level of activation of APCs that locally phagocytose the vaccine, thus making it difficult to elicit an effective immune response.

[0050] Among the many APCs, DCs (dentrilic cells) are the most powerful professional APCs in the body. Based on their maturation and differentiation states, they are divided into: DC precursor cells, immature DCs, and mature DCs. DC precursor cells are cells that do not yet possess the DC phenotype or function under normal mechanistic conditions. When the body faces pathogenic microbial infection or inflammatory stimulation, they are rapidly mobilized, differentiated, and developed into immature DCs. Immature DCs represent the majority of DCs under normal mechanistic conditions; they can efficiently take up, process, and present antigens, and possess strong migration capabilities. Mature DCs, due to activation, have a weaker ability to take up antigens, but their ability to present antigen information and stimulate naïve T cell activation is enhanced. The sealing microcapsules of this invention locally generate a large number of DC chemokines, which is conducive to recruiting more DCs, enabling antigens to be processed and presented more effectively, and inducing a more effective immune response in the body.

[0051] In a preferred embodiment of the present invention, the locally generated inflammatory microenvironment continuously recruits inflammation-related cells and APCs to the injection site to phagocytose antigens. During this process, the secretion of chemokines plays a crucial role in the types and numbers of recruited cells. The degradation rate of the polymer matrix as a carrier and the antigen release behavior significantly affect the amount of chemokines secreted.

[0052] In a preferred embodiment of the present invention, antigen release and cell recruitment work synergistically to achieve the best effect, so that the antigens released throughout the entire stage are phagocytosed by the cells with the highest antigen presentation efficiency, namely immature dendritic cells (DCs). These DCs have the function of phagocytosing large amounts of antigens, which further improves the utilization rate of antigens.

[0053] During the metabolism of the polymer matrix as a carrier, the locally generated inflammatory microenvironment continuously recruits inflammation-related cells and APCs to the injection site to phagocytose antigens. In this process, the secretion of chemokines plays a crucial role in the types and numbers of recruited cells, and the rate of carrier degradation and antigen release behavior significantly affect the amount of chemokines secreted. In a preferred embodiment of the present invention, the acidic microenvironment generated by the metabolism of the polymer matrix as a carrier can significantly increase the number of recruited cells. It has also been found that the acidic microenvironment significantly enhances the endocytosis of antigens by cells. The acidic microenvironment constructed from polylactic acid and other microcapsules possesses strong immune adjuvant properties. This acidic microenvironment is also an important factor in improving antigen utilization, serving as a retention site for activated dendritic cells (DCs). That is, without the need for additional vaccine adjuvants or multiple immunizations, it can continuously provide activated APCs with a home to lymph nodes. The acidic microenvironment can also significantly enhance antigen cross-presentation and Th1 cytokine secretion.

[0054] In a preferred embodiment of the present invention, the vaccine of the present invention contains an immunostimulatory enhancer, such as monophosphate A (MPLA), cytosine-guanine oligodeoxynucleotide and / or polyinosinic acid.

[0055] Microcapsule preparation

[0056] In a preferred embodiment of the present invention, the preparation of microcapsules refers to the process of preparing particulate composites by encapsulating or dispersing functional materials in a shell material through a certain method. Preferably, the microcapsules of the present invention contain a multi-chamber structure, and the average particle size of the microcapsules is preferably 10-100 μm, more preferably 20-80 μm, and more preferably 30-60 μm.

[0057] In a preferred embodiment of the present invention, the porosity of the open-cell microspheres is at least 40%, for example, at least 50%, preferably at least 60%, and more preferably at least 70%, and has channels with a pore size of, for example, 1-5 μm. Preferably, the open-cell microspheres are prepared by a re-emulsification and solvent removal method, which facilitates the use of biodegradable polymers as shell materials and is suitable for the development of drug-loaded microcapsules. The open-cell microspheres have a through-pore structure, a multi-cavity internal structure, and a porous shell. By controlling two dynamic processes, re-emulsification evolution and re-emulsification solidification, the pore structure and number of pores of the open-cell microspheres can be easily controlled. Subsequently, the open-cell microspheres are immersed in a solution containing antigen, and the antigen diffuses into the interior of the microspheres. The open-cell microspheres can be sealed using three methods: solvent swelling, irradiation, and temperature annealing.

[0058] In a preferred embodiment of the present invention, the preparation of microcapsules mainly includes the preparation of open-cell microspheres, the filling of antigens, and the sealing of the open-cell microspheres. Preferably, the method specifically includes the following steps:

[0059] (1) Prepare an oil phase, wherein the oil phase is a polymer matrix solution and the solvent is an organic solvent; prepare an inner aqueous phase solution and an outer aqueous phase solution, wherein a surfactant is added to the outer aqueous phase;

[0060] (2) The inner aqueous phase is dispersed into the oil phase to form a water-in-oil primary emulsion; the primary emulsion is then dispersed into the outer aqueous phase to form a water-in-oil secondary emulsion.

[0061] (3) The oil phase is solidified by solvent removal to obtain open-pore microspheres with through channels;

[0062] (4) The open-pore microspheres are mixed with a solution containing antigen. The antigen enters the internal cavity from the surface of the porous microspheres through diffusion mass transfer in the solution, thus obtaining open-pore microspheres loaded with antigen.

[0063] (5) Sealing of the perforated microspheres to form sealed microcapsules loaded with antigens.

[0064] In a preferred embodiment of the present invention, after the complex emulsion is prepared, during the evolution process, the small droplets of the inner aqueous phase in the complex emulsion gradually fuse and grow larger. Simultaneously, due to the salt concentration difference between the inner and outer aqueous phases, the inner aqueous phase escapes. Therefore, the fusion and escape of the inner aqueous phase occur simultaneously during the evolution process. The fusion of the inner aqueous phase forms a porous structure inside the microspheres, while the escape of the inner aqueous phase forms a porous morphology on the surface of the microspheres. In a preferred embodiment of the present invention, the polymer matrix is ​​a biodegradable polymer blend matrix, and the polymer blend contains a hydrophobic polymer and an amphiphilic block copolymer.

[0065] In a preferred embodiment of the present invention, the hydrophobic polymer is a lactide polymer (commonly referred to as PLA), a glycolide polymer, a lactide-glycol copolymer (commonly referred to as PLGA), polycaprolactone, polyorthoester and / or polyanhydride, preferably having a molecular weight average molecular weight of 5,000-100,000 Daltons, more preferably 10,000-50,000 Daltons.

[0066] In a preferred embodiment of the present invention, the hydrophilic block copolymer comprises polyethylene glycol, polyacrylic acid, polyoxyethylene, or polyvinyl alcohol, and the hydrophobic block comprises lactone copolymer or homopolymer; preferably, the hydrophilic block is polyethylene glycol or monomethoxyethylene glycol, and the hydrophobic block is polylactic acid (PLA) or a copolymer of lactic acid and glycolic acid (PLGA); the hydrophilic block copolymer has a weight-average molecular weight of 500-30,000 Daltons for the hydrophilic block and a weight-average molecular weight of 500-50,000 Daltons for the hydrophobic block; preferably, the weight-average molecular weight of the hydrophilic block is 1,000-20,000 Daltons, for example, 4,000-10,000 Daltons, and preferably, the weight-average molecular weight of the hydrophobic block is 5,000-20,000 Daltons.

[0067] In a preferred embodiment of the present invention, the amphiphilic block copolymer is a copolymer of polyethylene glycol or monomethoxyethylene glycol with lactide and / or glycolide (commonly referred to as PLGA), preferably having a molecular weight average molecular weight of 5,000-10,000 Daltons, more preferably 10,000-50,000 Daltons.

[0068] In a preferred embodiment of the present invention, the amphiphilic block copolymer can be used to stabilize the primary emulsion, thereby enabling the preparation of porous microspheres with a multi-chamber structure. The amphiphilic block copolymer accounts for at least 5% by weight of the polymer blend matrix in the microcapsules, preferably at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, or at least 50%. PLA is less used in the field of controlled drug release due to its strong local inflammatory immune response. PELA, as a material with better biocompatibility, has gained more application and can help prepare open-pore microspheres with higher porosity. By changing the ratio of the two, the hydrophobic properties and porosity of the microspheres can be improved, while the inflammatory response of the carrier can be modulated, thus improving biocompatibility.

[0069] In a preferred embodiment of the present invention, the organic solvent in step (1) is a volatile organic solvent that is not miscible with water, such as n-butanol, methyl ethyl ketone, diethyl ether, chloroform, tetrachloromethane, toluene, etc., including volatile organic solvents that are partially soluble in water, such as ethyl acetate, phenol, etc., and is further preferably one or a combination of at least two of alcohols, ketones, esters, ethers, alkylbenzenes, haloalkanes, and haloaromatics that are not miscible with water. Typical but not exhaustive examples of such combinations include: combinations of alcohols and ketones, combinations of alcohols, ketones, esters and ethers, combinations of esters, alkylbenzenes and haloalkanes, combinations of esters, alkylbenzenes, haloalkanes and haloaromatics, etc., and is particularly preferred to be one or a combination of at least two of alcohols, esters, alkylbenzenes, chloroalkanes and chloroaromatics.

[0070] In a preferred embodiment of the present invention, an immunostimulatory enhancer, such as monophosphate A, is added to the organic solvent described in step (1) during microcapsule preparation.

[0071] In a preferred embodiment of the present invention, an immunostimulatory enhancer, such as cytosine-guanine oligodeoxynucleotide and / or polyinosinic acid, is further loaded when loading the antigen.

[0072] In a preferred embodiment of the present invention, the solvent removal method in step (3) is solvent extraction, or it can be allowing the solvent to evaporate by standing, stirring to evaporate the solvent, or other methods for removing the solvent.

[0073] In a preferred embodiment of the present invention, during the curing process described in step (3), the inner aqueous phase and the outer aqueous phase fuse to form a through hole.

[0074] In a preferred embodiment of the present invention, the open-pore microsphere with internal and external through-holes described in step (3) has a porous structure.

[0075] In a preferred embodiment of the present invention, the perforated microsphere is a surface-porous microsphere.

[0076] In a preferred embodiment of the present invention, the porosity of the open-pore microspheres is at least 40%, for example at least 50%, preferably at least 60%, more preferably at least 70%, more preferably at least 80%, and preferably has channels with a pore size of, for example, 1-5 μm. Preferably, the pore size inside the microspheres varies from 800 nm to 5 μm, with an average pore size of about 1 μm.

[0077] In a preferred embodiment of the present invention, in step (3), after the oil phase is solidified, the residual surfactant is removed, particularly preferably by sieving or centrifugal washing.

[0078] In a preferred embodiment of the present invention, the sealing process of the open-cell microspheres preferably includes solvent swelling, irradiation, and heating annealing. Those skilled in the art can also seal the open-cell microspheres based on their professional knowledge / new technologies.

[0079] In this invention, those skilled in the art will understand that "sealing" means embedding, fixing, or retaining antigens and / or other substances, such as chemokines, inside the microcapsule. In this invention, sealing the microcapsule does not mean that the microcapsule surface has no openings, but simply that antigens and / or other substances, such as chemokines, can be partially embedded, fixed, or retained inside the microcapsule through the sealing process. The antigens and / or other substances, such as chemokines, may be released from the microcapsule through degradation, for example.

[0080] In a preferred embodiment of the present invention, the temperature-heating annealing method is a relatively ideal sealing method. For example, the unique self-healing sealing properties of biodegradable polymer blends such as polylactic acid can be utilized. This method involves using irradiation or heating to cause the molecules on the surface of the microspheres to absorb energy and rearrange, thereby healing and sealing the pores on the surface. For example, by slowly heating open-pore microspheres carrying antigens to near the glass transition temperature of the microspheres, preferably 1-2°C below the glass transition temperature, and then slowly cooling them after a period of time, the pores on the surface of the open-pore microspheres are closed, thus preparing sealed porous microcapsules. At the same time, the antigen is effectively encapsulated in the microcapsules, and the loading rate and embedding rate are stable. The temperature required for the entire heating process is close to the glass transition temperature of the polylactic acid material. To achieve the healing and sealing of the microspheres, the heating conditions must be controlled, while ensuring that the morphology of the microspheres is not significantly affected.

[0081] In other embodiments of the invention, open-pore microspheres can be prepared by phase separation of the polymer and the porogen. During the porogenization process, the solvent is often referred to as the porogen, and is typically an oil-soluble small molecule. Phase separation of the polymer and solvent can occur during polymer chain growth, where, due to the increase in polymer molecular weight or intermolecular cross-linking, the macromolecules gradually precipitate from the small molecule solvent, forming a solid polymer. It can also occur in polymer solution systems, where changes in environmental conditions, such as temperature variations, removal of a good solvent, or addition of a poor solvent, reduce the solvent's solubility in the polymer, causing the polymer to precipitate into a solid phase.

[0082] One object of the present invention is also a method for inducing a cytotoxic cellular response against tumor cells or tumors in a patient. This method includes administering an effective amount of the vaccine of the present invention, preferably an anti-tumor vaccine, to the patient, particularly via intravenous, injection, or infusion, preferably via infusion. The aim of this method is particularly to induce activation of the patient's dendritic cells and CD8+. + Cytotoxic cell response, acquisition of specific CD4 + Support and CD8 + Cytotoxic response.

[0083] The vaccine of the present invention can be used for the prevention and treatment of diseases such as cancer or hepatitis such as hepatitis B, and more specifically for immunotherapy. The present invention provides a method for the preventive or therapeutic treatment of cancer or hepatitis such as hepatitis B, comprising administering a preventive or therapeutically effective amount of vaccine to a mammalian subject in need of such treatment.

[0084] In this invention, the term "immunization" refers to active immunization, that is, the induction of a specific immune response by administration of a small amount of antigen, for example via subcutaneous, intradermal, intramuscular, oral, or nasal routes, which is recognized as exogenous by the inoculated individual and is therefore immunogenic in a suitable formulation. Thus, the antigen is used as a "trigger" for the immune system to establish a specific immune response against that antigen.

[0085] According to the present invention, immunization can be therapeutic or preventative. For example, preventative protection against the development of cancer may be achieved by immunizing individuals who do not have cancer. Examples of individuals who may be eligible for such preventative vaccination include those at increased risk of developing cancer, although the application is not limited to such individuals. Patients at risk of cancer may already have a tumor, whether as a primary tumor or a metastasis, or show a tendency towards cancer.

[0086] In this invention, the term "effective amount" refers to the amount of the antigenic / immunogenic composition that, when applied to humans or animals, induces an immune response. Those skilled in the art can readily determine the effective amount using conventional procedures.

[0087] The vaccine or pharmaceutical composition provided by this invention can be prepared into a sterile powder form. For example, the sterile powder contains a vaccine and mannitol and can be prepared by the following method: microcapsules are taken, rinsed with water for injection, transferred to a lyophilization tray, mannitol and an appropriate amount of water for injection are added, and the mixture is lyophilized in a freeze dryer; the lyophilized product is sieved, mixed evenly, aseptically dispensed, and capped to obtain the sterile powder. Before administration to a patient, the sterile powder is suspended in an acceptable dispersion solvent, which consists of one or more of a suspending agent, a pH adjuster, an isotonic adjuster, and a surfactant, and water for injection. The suspending agent may be one or more of sodium carboxymethyl cellulose, polyvinyl alcohol, polyvinylpyrrolidone, sodium alginate, and glycerin. The isotonic adjuster may be one or more of sodium chloride, glucose, mannitol, and sorbitol. The surfactant is a nonionic surfactant, such as a polysorbate series (e.g., polysorbate 80, polysorbate 60, etc.).

[0088] The pharmaceutical compositions provided by this invention, such as vaccine microcapsules, can be administered to patients using methods well known to those skilled in the art, such as intra-arterial, intravenous, percutaneous, intranasal, intrabronchial, intramuscular, or oral administration. Dosage and administration method vary depending on the patient's weight and age, and can be selected as needed by those skilled in the art.

[0089] Liquid pharmaceutical compositions are typically formulated to have a pH between about 3.0 and 9.0, more preferably between about 4.5 and 8.5, and still more preferably between about 5.0 and 8.0. The pH of the composition can be maintained by using a buffer such as acetate, citrate, phosphate, succinate, Tris, or histidine, typically in the range of about 1 mM to 50 mM. The pH of the composition can be further adjusted by using physiologically acceptable acids or bases.

[0090] The pharmaceutical composition is administered to an individual in a preventative or therapeutically effective amount (as the case may be, although prevention can be considered treatment) sufficient to demonstrate benefit to the individual. Typically, this will result in therapeutically beneficial activity for the individual. The actual amount of the compound administered, as well as the rate and timing of administration, will depend on the nature and severity of the condition being treated. Prescribing treatments, such as dosage determination, falls within the responsibility of general practitioners and other physicians, and generally takes into account the condition being treated, the individual patient's condition, the delivery site, the method of administration, and other factors known to the physician.

[0091] The specific embodiments disclosed herein are intended to illustrate only a few aspects of the invention.

[0092] Comparative Example 1: Open-pore microspheres prepared from PLA

[0093] 100 mg of racemic carboxyl-terminated polylactic acid (PLA, Jinan Daigang Bioengineering Co., Ltd.) with a molecular weight of 20,000 Daltons was dissolved in 2 mL of ethyl acetate (oil phase, O). 0.5 mL of 0.05% sodium chloride aqueous solution (inner aqueous phase W1) was added, and a primary emulsion (water-in-oil, W1 / O) was prepared using an ultrasonic homogenizer. The primary emulsion was then poured into 15 mL of an aqueous solution containing PVA (purchased from Kuraray, Japan) (outer aqueous phase W2), and emulsified using a homogenizer to prepare a secondary emulsion (water-in-oil, W1 / O / W2). The mixture was then suspended in a vertical suspension apparatus for 25 min to allow for secondary emulsion evolution. The secondary emulsion was then poured into 500 mL of ultrapure water and magnetically stirred at room temperature for 10 min (500 rpm) to solidify the microspheres. Finally, the microspheres were centrifuged multiple times (500 g, 5 min), the supernatant was discarded, and the bottom microspheres were collected. 1 mL of ultrapure water was added to the microspheres, and the mixture was stored at 4 °C.

[0094] Take 50 μL of PLA microsphere suspension and drop it onto aluminum foil, then allow it to air dry at room temperature. Use conductive adhesive to attach the aluminum foil containing the sample to the sample preparation stage. After gold sputtering, observe the microsphere morphology using SEM. The prepared PLA microspheres are shown below. Figure 1 As shown, microspheres prepared solely from PLA do not have a distinct porous structure and are not suitable for use as porous microspheres in this invention to further develop the sealing microspheres of this invention.

[0095] Example 1: Preparation of open-pore microspheres from PLA and PELA

[0096] 95 mg of racemic carboxyl-terminated polylactic acid (PLA, Jinan Daigang Biotechnology Co., Ltd.) with a molecular weight of 20,000 Dalton and 5 mg of a copolymer of polyethylene glycol (mPEG, Mw: 2000) and racemic carboxyl-terminated polylactic acid (PLA, Mw: 36000) with a molecular weight of 38,000 Dalton (PELA, purchased from Jinan Daigang Biotechnology Co., Ltd.) was dissolved in 2 mL of ethyl acetate (oil phase, O). 0.5 mL of 0.05% sodium chloride aqueous solution (inner aqueous phase W1) was added, and a primary emulsion (water-in-oil, W1 / O) was prepared using an ultrasonic emulsionizer. The primary emulsion was then poured into 15 mL of an aqueous solution containing PVA (outer aqueous phase W2), and emulsified using a homogenizer to prepare a secondary emulsion (water-in-oil-in-water, W1 / O / W2). The secondary emulsion was then subjected to vertical suspension for 25 min for further emulsion evolution. The double emulsion was then poured into 500 mL of ultrapure water and magnetically stirred at room temperature for 10 min (500 rpm) to solidify the microspheres. Finally, the microspheres were centrifuged multiple times (500 g, 5 min), the supernatant was discarded, and the bottom microspheres were collected. 1 mL of ultrapure water was added to the microspheres again, and they were stored at 4 °C.

[0097] Take 50 μL of the above microsphere suspension and drop it onto aluminum foil, then allow it to air dry at room temperature. Use conductive adhesive to attach the aluminum foil containing the sample to the sample preparation stage. After gold sputtering, observe the surface morphology of the microspheres using SEM. Figure 2 As shown, the surface pore size is 1-2 μm, and it has a structure with surface openings. Its particle size distribution is as follows. Figure 3 As shown, the average particle size of the microspheres is 60 μm. To observe the internal structure of the microspheres, the dried microspheres were shredded using an ultrathin blade. The samples were then adhered to conductive adhesive, sputtered with gold, and observed using SEM. Figure 4 As shown, the microspheres also have an internal porous structure that is interconnected, with internal pore diameters of about 1-5 μm.

[0098] 30 mg of lyophilized porous microspheres were measured using a mercury porosimeter. The measurements were performed in triplicate, and the porosity and average pore size distribution of the microspheres were calculated. The porosity of the microspheres was 82%. Figure 5 As shown, the pore sizes inside the microspheres range from 800 nm to 5 μm, with an average pore size of about 1 μm.

[0099] Example 2: Preparation of sealed microcapsules loaded with antigen

[0100] Take 1 mL of the 30 mg dry weight open microsphere suspension prepared in Example 1 into a 1.5 mL centrifuge tube, centrifuge to remove the supernatant, add 500 μL of 50 mg / mL or 10 mg / mL antigen OVA (purchased from Sigma) or 500 μL of 8 mg / mL MUC1 peptide (purchased from Jier Biochemical Shanghai Co., Ltd.) and mix with the microspheres. Place the mixture in a vertical suspension apparatus and suspend for 4 h (300 rpm) to allow the protein or peptide to fully enter the cavity of the microspheres through the pores. Then place the vertical suspension apparatus and microspheres in a 39 ℃ constant temperature incubator for heating and sealing. During the process, the suspension speed is 100 rpm to ensure that the microspheres are heated evenly throughout the heating process and that no sedimentation occurs. After 2 h of reaction, sealing is completed. Centrifuge (500 g, 5 min) to remove the supernatant to obtain sealed microcapsules loaded with protein and peptide molecules.

[0101] Take 50 μL of the sealed microcapsule suspension containing the OVA antigen, drop it onto aluminum foil, and allow it to air dry at room temperature. Use conductive adhesive to attach the aluminum foil containing the sample to the sample preparation stage, sputter-coated with gold, and observe the surface morphology of the microcapsules using SEM. Figure 6 As shown, the surface is porous and completely sealed, forming a sealed microcapsule. To observe the internal structure of the sealed microcapsule, the dried microcapsules were shredded using an ultra-thin blade. The sample was then adhered to conductive adhesive, sputtered with gold, and observed using SEM. Figure 7 As shown, although the microcapsule still has an internal porous structure with a pore size of about 1-5 μm, the internal through-porous structure has become a closed and independent porous structure.

[0102] After sealing the microspheres, the loading rate and encapsulation rate of the antigen OVA or peptide MUC1 in the sealed microcapsules were further evaluated. The methods for determining the content of OVA protein and MUC1 peptide are as follows.

[0103] Protein OVA extracted from sealed microcapsules: Weigh 5 mg of lyophilized microspheres loaded with OVA (n=3), add 1 mL of 0.1 M sodium hydroxide aqueous solution, react overnight at 4 °C for 12 h. After the microcapsules are completely degraded, centrifuge and collect the supernatant. Titrate with 0.1 M hydrochloric acid to restore the pH of the solution to neutral. Take 100 μL of the supernatant and use a BCA kit to detect the protein content.

[0104] Extraction of peptide MUC1 from sealed microcapsules: Weigh 5 mg of lyophilized microcapsules loaded with MUC1 peptide (n=3), first add 300 μL of acetonitrile to completely dissolve the material, and after the system becomes clear, add 1.7 mL of 0.015 M HCl solution. Finally, remove insoluble matter and impurities from the system using a 0.45 μm filter membrane. The concentration of the MUC1 peptide was then determined by RP-HPLC. Experimental conditions are as follows:

[0105] Detection conditions: 0.1% trifluoroacetic acid in deionized water (mobile phase A); 0.1% trifluoroacetic acid in high performance liquid grade acetonitrile (mobile phase B); elution gradient: mobile phase B: 0%-60%, 0-25 min; flow rate: 1.0 mL / min; detection wavelength: 220 nm.

[0106] The final drug loading rate (%) of the microcapsules was calculated by dividing the mass of the antigen (OVA / MUC1) in the sealed microcapsules by the mass of the microspheres (dry weight) and then multiplying by 100%. The encapsulation rate (%) of the microcapsules was calculated by dividing the mass of the antigen (OVA / MUC1) in the sealed microcapsules by the mass of the antigen (OVA / MUC1) before loading (concentration multiplied by volume) and then multiplying by 100%.

[0107] like Figure 8 and Figure 9 As shown, the microcapsule loading rate increases linearly with increasing initial antigen OVA protein and MUC1 peptide concentrations. When the antigen concentration is 100 mg / mL, the microcapsule loading rate reaches as high as 20%. Simultaneously, the encapsulation efficiency of the sealed microcapsules remains relatively stable with increasing antigen concentration, indicating that the encapsulation efficiency depends only on the cavity volume of the microcapsules. This linear increase in loading rate and relatively stable encapsulation efficiency will facilitate the control of the antigen-to-microcapsule ratio, thereby meeting the needs of different loading systems.

[0108] Example 3: Preparation of sealed microcapsules loaded with antigen and MPLA

[0109] First, dissolve 1 mg of MPLA (purchased from Sigma) in 500 μL of chloroform (CHCl3). Then, add 100 μL of chloroform to 1.9 mL of oil-phase ethyl acetate. Next, add 95 mg of racemic terminal carboxyl polylactic acid (PLA, purchased from Jinan Daigang Bioengineering Co., Ltd.) with 5... A copolymer of polyethylene glycol (mPEG, Mw: 2000) and racemic carboxyl-terminated polylactic acid (PLA, Mw: 36000) (PELA, purchased from Jinan Daigang Biotechnology Co., Ltd.) with a molecular weight of 38000 Daltons was dissolved in 2 mL of the above-mentioned oil phase containing chloroform and MPLA. 0.5 mL of 0.05% sodium chloride aqueous solution (inner aqueous phase W1) was added, and a primary emulsion (water-in-oil, W1 / O) was prepared using an ultrasonic disruptor. The primary emulsion was then poured into 15 mL of an aqueous solution containing PVA (outer aqueous phase W2), and emulsified using a homogenizer to prepare a secondary emulsion (water-in-oil, W1 / O / W2). The secondary emulsion was then suspended in a vertical suspension apparatus for 25 min for emulsification. Finally, the secondary emulsion was poured into 500 mL of ultrapure water, and the microspheres were solidified at room temperature using magnetic stirring (500 rpm). Finally, centrifuge multiple times (500g, 5min), discard the supernatant and collect the bottom microspheres, add 1mL of ultrapure water to the microspheres again, and store at 4℃.

[0110] The preparation process of sealed microcapsules loaded with antigen and MPLA is as follows: 1 mL of MPLA-loaded microsphere suspension was pipetted into a 1.5 mL centrifuge tube, centrifuged to remove the supernatant, leaving a microsphere volume of 500 μL. Then, 500 μL of 10 mg / mL OVA antigen (purchased from Sigma) or 500 μL of 8 mg / mL MUC1 peptide (purchased from Jier Biochemical Shanghai Co., Ltd.) was added and mixed with the microspheres. The mixture was then suspended in a vertical suspension apparatus for 4 h (300 rpm) to allow the peptide to fully enter the microsphere cavity through the pores. Afterwards, the microcapsules were heated to 39℃ and sealed after 2 h. The supernatant was removed by centrifugation (500 g, 5 min) to obtain sealed microcapsules loaded with antigen and MPLA. Calculations showed that the actual loading rate of OVA in the microcapsules was 2%, the actual loading rate of MUC1 peptide was 1.7%, and the actual loading rate of MPLA was 1‰.

[0111] Example 4: Preparation of sealed microcapsules loaded with chemokines

[0112] A 30 mg dry weight suspension of the open microspheres prepared in Example 2 was transferred to a 1.5 mL centrifuge tube. The supernatant was removed by centrifugation, leaving a 500 μL volume of microspheres. 500 μL of 1 mg / mL GM-CSF chemokine (purchased from Peprotech) solution was added to the tube, allowing it to mix with the microspheres. The mixture was then suspended in a vertical suspension apparatus for 4 hours (300 rpm) to ensure the chemokine fully penetrated the microsphere cavity. The tubes were then placed in a 39°C incubator for two hours to allow for healing and sealing. The supernatant was removed by centrifugation to obtain the sealed microcapsules containing the chemokine. The actual loading rate of GM-CSF in the microcapsules was calculated to be 2.4‰.

[0113] Example 5: Synergistic effect of antigen release and cell recruitment in sealed microcapsules loaded with antigens

[0114] First, female C57BL / 6 mice (n=4, purchased from Beijing Vital River Laboratory Animal Co., Ltd.) aged 6-8 weeks were subcutaneously injected with different vaccine formulations in their backs. The groups included: a group receiving only fluorescent antigen (OVA labeled with Cy5 dye, OVA-Cy5); a group receiving a mixture of fluorescent antigen and porous microspheres; and a group encapsulated with fluorescent antigen in sealed microcapsules. The injection doses of microspheres and antigen were 3 mg and 60 μg for all groups, respectively. At different metabolic stages (1h, 2h, 4h, 8h, 0.5d, 1d, 2d, 4d, 7d, 10d, 14d, 17d, 21d, 25d, and 30d), the different metabolic behaviors of the antigen in porous and sealed microspheres were observed using a small animal in vivo imaging system.

[0115] like Figure 10As shown, the local fluorescence intensity of the pure OVA antigen almost completely disappeared on day 3, indicating that the antigen metabolism rate was very fast and essentially complete. The burst release of the blended group within 24 hours was 50%, because in the early stages of metabolism, the antigen free on the outside of the microspheres was rapidly metabolized by the tissue, resulting in a relatively fast metabolic rate. Subsequently, the antigen inside the porous microspheres was further released; however, strong fluorescence intensity was still observed on day 5. The figure shows that 10% of the antigen remained unmetabolized, due to the local retention of the microspheres and the delaying effect of the pores, significantly slowing down antigen metabolism and extending the total metabolic time to approximately 7 days. In contrast, the embedded group maintained strong fluorescence intensity throughout the two weeks of detection. Quantitative analysis revealed no significant burst release effect in the early stages of metabolism; the release within 24 hours was only 10%. This was because the pores on the surface of the sealed microcapsules meant the antigen could only be released slowly from the nanopores on the material surface, resulting in only 20% antigen being metabolized after 3 days. As metabolism progressed, changes occurred in the surface and internal structure of the carrier, leading to a large release of antigen from the microcapsules; approximately 50% of the antigen was released after 7 days. Subsequently, the metabolic rate slowed further, and strong fluorescence intensity was still observed after 14 days of metabolism. Figure 10 As shown, 35% of the antigen remains unmetabolized.

[0116] Along with the release of antigens, the in situ retention effect after microcapsule injection leads to an inflammatory response, thereby recruiting inflammation-related cells to phagocytose the carriers and release the antigens. To further observe the cell recruitment phenomenon, blank sealed microcapsules from Example 2 were injected into the backs of female 6-8 week old C57BL / 6 mice (n=4, purchased from Beijing Vital River Laboratory Animal Co., Ltd.). Mice were sacrificed at different metabolic times in vivo. The injection site on the back of the mice was dissected, and the local subcutaneous tissue containing the carrier was removed with forceps. The tissue was then cut off with scissors and soaked in 4% formalin solution for 24 hours. Subsequently, it was paraffin-embedded, sectioned, stained with hematoxylin and eosin (H&E), and mounted to prepare H&E-stained samples. Finally, the inflammatory response at the injection site was observed using a panoramic scanning and analysis system for pathological sections (Vectra 3.0, purchased from PerkinElmer). Mice were sacrificed at different metabolic times. The local subcutaneous tissue containing the carrier was removed, and H&E-stained samples were prepared. Finally, the inflammatory response at the injection site was observed using a panoramic scanning and analysis system for pathological sections.

[0117] like Figure 11As shown in Figure a, on day 3, cells (dark dots) were gradually recruited to the vicinity of microcapsules (light circles). With prolonged degradation time, the number of cells around the microspheres increased, the inflammatory response intensified, and infiltration of inflammatory cells was even observed within some microcapsules. The number of dots and microcapsules was statistically analyzed using Inform software, and the number of cells recruited to each microcapsule was quantitatively calculated. Analysis showed that... Figure 11 As shown in b, each microsphere could recruit an average of about 20 cells at 14 days. Therefore, although the local inflammatory response gradually increased due to the accumulation of degradation products during the entire metabolic process, no redness, swelling, granulation tissue, or purulent discharge was found in the local tissues during mouse dissection, indicating that the inflammatory response generated by the sealed microcapsules is safe.

[0118] Three groups were set up: a simple antigen group containing Cy5-labeled OVA (Cy5-OVA), a blend group containing Cy5-OVA and porous microspheres, and an embedded group containing Cy5-OVA in sealed microcapsules. These were injected into the thigh muscles of 6-8 week old female C57BL / 6 mice (n=4, purchased from Beijing Vital River Laboratory Animal Co., Ltd.). Five days later, the mice were sacrificed, and the muscle tissue containing the microspheres was removed, cut into small pieces, ground into a suspension, resuspended in PBS at 4°C, and centrifuged to remove the supernatant. Then, 1 mL of a mixed enzyme solution (1 mg / mL collagenase D and 100 U / mL recombinant DNase I dissolved in RPMI 1640 medium) was added, and the mixture was reacted at 37°C for 30 min. Afterward, the cells were washed with PBS, filtered through a 20 μm cell sieve to obtain a single-cell suspension. Following the flow cytometry sample staining procedure, fluorescent antibodies against mouse FITC-CD11c and eFlour450-F4 / 80 were added to the samples to label dendritic cells (DCs) and macrophages, respectively. OVA was further detected using a BD LSRFortessa flow cytometer. + Cells, CD11c + OVA + Cells and F4 / 80 + OVA + Cells were analyzed and processed using Flowjo software.

[0119] like Figure 12 As shown, the simple antigen group (control group) and OVA + The cell number and intracellular OVA fluorescence intensity were both very weak, indicating that the local antigen was almost completely metabolized by day 5. The retention effect of porous microspheres in the blend group significantly improved antigen utilization, and the OVA... +The number of cells increased by about 10 times, the amount of OVA endocytosis by cells increased by 2 times, and the corresponding antigen utilization rate increased by about 20 times; due to its excellent antigen retention effect and effective antigen release ability, the encapsulated group showed a significant improvement in OVA utilization. + The number of cells increased by approximately 50-fold, the internalization volume of OVA increased by more than 4-fold, and the antigen utilization rate ultimately increased by more than 200-fold. These results indicate that sealed microcapsules can more effectively regulate antigen release and cell recruitment behavior in the microcapsule system, enabling both to work synergistically to further improve antigen utilization. The antigen utilization rate is calculated using the formula: OVA + Cell number × fluorescence intensity was normalized using the antigen utilization rate of the OVA group alone. This synergistic effect not only encourages more cells to phagocytose antigens but also increases the amount of antigen internalized by cells, maximizing antigen utilization.

[0120] Example 6: The effect of the acidic microenvironment of sealed microcapsules loaded with antigens on cell recruitment.

[0121] Lactic acid, a degradation product of polylactic acid microcapsules, can lead to acidification of the local microenvironment. The degree, changes, and duration of acidification may affect the subsequent immune response. Therefore, it is necessary to accurately monitor the acidification process and changes of the local microenvironment. This is a pH-sensitive probe (monitoring range 6-9), excited at 488 nm and received at both 640 nm and 580 nm wavelengths. The fluorescence intensity ratio I640 / I580 corresponds to the pH value of the system. The smaller this ratio, the lower the pH of the system. Therefore, by loading microspheres... Laser confocal microscopy can be used to monitor in real time the changes in pH of the local microenvironment caused by lactic acid, a degradation product of polylactic acid microcapsules.

[0122] The specific procedure was as follows: Subcutaneous injection of [a specific substance] was administered into the back of female C57 / BL6 mice (n=6). Mice were sacrificed on days 3, 5, 7, and 14 after being given sealed microcapsules. Subcutaneous tissue containing the microspheres was removed and examined under a laser scanning confocal microscope to observe local pH changes. Fluorescence images were observed, and the fluorescence intensity ratio I640 / I580 was calculated. The actual pH value was then calculated using a standard curve.

[0123] Experiments showed that after 3 days of metabolism, the local microenvironment changed from neutral to acidic. For example... Figure 13As shown, the local pH changed from neutral 7.2 to weakly acidic 6.5. With the continuous metabolism of the material, no further acidification of the local acidic environment was observed; instead, it remained stable at around pH 6.5, meaning that the lactic acid accumulation problem caused by the gradual degradation of ordinary non-porous polylactic acid (PLA) microspheres did not occur. When the internal and external surfaces of ordinary non-porous PLA microspheres degrade simultaneously, the lactic acid produced by internal metabolism accumulates due to impaired mass transfer, resulting in a lower internal pH than the external pH, leading to local lactic acid accumulation and a potential for sudden pH drops or instability. The sealing microcapsules of this invention, due to their unique internal porous permeable structure, effectively avoid the aforementioned problems of lactic acid accumulation and impaired mass transfer, allowing for efficient exchange of lactic acid produced by internal material degradation with the external environment. When the body's metabolism of lactic acid and the lactic acid produced by the degradation of PLA materials achieve a dynamic balance in the body, the local pH remains at a relatively stable level, thus creating a relatively stable microenvironment for local cells to function and facilitating the analysis of the impact of homeostasis on local cells.

[0124] In many physiological inflammatory conditions, a large number of cells are recruited to the inflammatory site, which is also accompanied by acidification. Therefore, acidity should be related to cell recruitment behavior. This study focuses on the effects of neutral microenvironment (NM) and acidic microenvironment (AM) on cell recruitment. By loading a strong-base-weak acid salt, NaHCO3, into polylactic acid (PLA) microcapsules, the acid-base neutralization effect of NaHCO3 with lactic acid, a degradation product of the microcapsules, was utilized to eliminate local acidity, thus constructing a neutral microenvironment locally. This contrasts with the acidic microenvironment of PLA microcapsules.

[0125] An acidic environment enhances antigen utilization by increasing cell recruitment and stimulating endocytosis of antigens. This also demonstrates that the synergistic effect of antigen release and cell recruitment alone can improve antigen utilization; for example, a beneficial microenvironment stimulating large-scale phagocytosis of antigens by immune cells is needed to further improve antigen utilization. Therefore, the combined effect of multiple factors—antigen release, cell recruitment, the number and type of dendritic cells (DCs), and an acidic microenvironment stimulating endocytosis—is required to maximize antigen utilization. Both the blended group and the NaHCO3-loaded encapsulated group demonstrate that any of these factors is indispensable. Only the sealing microcapsules of this invention can simultaneously mobilize all these factors, synergistically exerting their respective functions to ensure the most efficient utilization of antigens.

[0126] After dendritic cells (DCs) engulf antigens, the antigens are processed into antigenic peptides, which are expressed on the surface of antigen-cell progenitor cells (APCs) as antigen peptide-major histocompatibility complex (MHC) for T cell recognition. However, when APCs only engulf antigens without external stimulation, they do not effectively express co-stimulatory molecules (CD80, CD86, CD40) to activate T cells. Furthermore, in most cases, after DCs engulf foreign antigens, their processing and presentation primarily occur via the lysosomal pathway, a process restricted by MHC class II molecules, mainly mediating humoral immune responses in T cells. In certain special cases, exogenous antigens can be presented via the cytosol pathway, i.e., the MHC class I molecule pathway, directly activating cytotoxic T cells (CTLs) and thus killing target cells most directly and effectively. Therefore, for therapeutic tumor vaccines, it is necessary to present antigens primarily as MHC class I molecules to more effectively kill tumor cells.

[0127] An acidic environment can significantly enhance the internalization of antigens by APCs, especially DCs. However, the activation of APCs and the quantity and pathway of antigen presentation require further investigation. The specific procedure is as follows: First, OVA was dissolved in 20mM NaHCO3 and PBS solutions to prepare sealed microcapsules. The mixing and sealing methods are described in the section on antigen loading. These microcapsules were injected into the thigh muscles of 6-8 week old female C57BL / 6 mice (n=6, purchased from Beijing Vital River Laboratory Animal Co., Ltd.). On day 5, tissue containing the healing microcapsules was removed after dissection. The tissue was cut into small pieces and 1 mL of a mixed enzyme solution (1 mg / mL collagenase D and 100 U / mL recombinant DNase I dissolved in RPMI 1640 medium) was added. The mixture was reacted at 37°C for 30 min. The cells were then homogenized in a cell homogenizer to prepare a single-cell suspension. After centrifugation to remove the supernatant, the cells were collected, filtered through a 20 μm cell sieve to remove impurities, and counted using a handheld cell counter. Then, flow cytometry was used to stain and label the surface co-stimulatory molecules and major histocompatibility complex markers of DCs with PE-CD11c, APC-CD80, APC-Cy7-CD86, eFlour 450-MHC-I, and BV 655-MHC-II, respectively, to detect the proportion of locally activated DCs.

[0128] like Figure 14 As shown, DC is significantly activated in an acidic environment. CD86 + The expression level of MHC-I molecules in dendritic cells (DCs) is 40 times higher than that of MHC-II, indicating that DCs present antigens more frequently via MHC-I, thus demonstrating the high efficiency of microcapsules in promoting antigen cross-presentation. However, in a neutral environment, CD86... +The proportion of MHC-I in DCs decreased by more than half, indicating that the environment plays a very important role in the cross-presentation of antigens promoted by microcapsules.

[0129] Example 7: Animal experimental results of sealed microcapsules based on E.G7 lymphoma solid tumors.

[0130] First, E.G7 tumor-bearing mice (purchased from Beijing Vital River Laboratory Animal Co., Ltd.) were constructed. 1*10 6 E.G7 tumor cells (purchased from ATCC) were injected into the axilla of 6-8 week old female C57BL / 6 mice. The tumor-bearing mice were then randomly divided into groups of 6. On day 4, the tumor volume was 50-60 mm. 3 Vaccination was administered at the appropriate time. The vaccine groups were as follows: PBS, multiple low-dose OVA (20 μg, booster immunizations on days 7 and 14), single high-dose OVA (60 μg), a mixture of porous microspheres and OVA (3 mg microspheres, 60 μg OVA), sealed microcapsules loaded with OVA (preparation method see Example 2) (3 mg microspheres, 60 μg OVA), and sealed microspheres encapsulated with OVA and loaded with MPLA (preparation method see Example 3) (3 mg microspheres, 60 μg OVA, 3 μg MPLA). From the date of tumor model establishment, mouse hair, weight, and survival were observed every other day. The length and width of the tumor were measured using calipers, and the tumor volume was calculated using the following formula: V = 1 / 2 × L × W 2 (L: Length; W: Width).

[0131] like Figure 15 As shown in the tumor growth curves, in the antigen-only group, both the low-dose multiple-dose group and the high-dose single-dose group failed to inhibit tumor growth, with a 25-day survival rate (SR) of only 17%. The blended group, with the adjuvant effect of the microspheres, improved the survival rate to 50%, but tumor growth was rapid in the early stages. The encapsulated group showed significant tumor inhibition in the early stages, with a slow growth rate and a 25-day survival rate of 100% in mice. This indicates that the microcapsule system, using only the antigen without adding additional molecular adjuvants, can achieve good therapeutic effects through the full utilization of the tumor antigen. When a trace amount of adjuvant MPLA (3 μg) was loaded into the healing microcapsule material, not only was the survival rate as high as 100%, but the survival rate of tumor-bearing mice was also significantly inhibited. At 26 days, the tumor volume of 50% of the mice was basically the same as at the beginning of treatment, and more than 60% of the mice survived for more than 40 days, further confirming that the compatibility between the microsphere system and the MPLA system can further enhance the tumor inhibition effect.

[0132] Example 8: Animal experimental results of sealed microcapsules based on B16 melanoma solid tumors.

[0133] First, B16 tumor-bearing mice (purchased from Beijing Vital River Laboratory Animal Co., Ltd.) were constructed, with 5*10 mice... 5 B16 melanoma cells (provided by Jilin University) were injected into the axilla of 6-8 week old female C57BL / 6 mice. The tumor-bearing mice were then randomly divided into groups of 6. On day 4, the tumor area was >10 mm. 2 Vaccination was administered. The following groups were established: PBS group, MUC1 peptide group (50 μg), microcapsule-loaded MUC1 peptide group (microspheres 3 mg, peptide 50 μg), and microcapsule-loaded MUC1 and MPLA-loaded group (microspheres 3 mg, peptide 60 μg, MPLA 3 μg). From the date of tumor model establishment, mouse hair, weight, and survival were observed every other day. The length and width of the tumor were measured using calipers, and the tumor volume was calculated using the following formula: V = 1 / 2 × L × W 2 (L: Length; W: Width).

[0134] like Figure 16 As shown in the tumor growth curves, neither PBS nor the high-dose single-dose group effectively inhibited melanoma growth, while the sealed microspheres significantly inhibited tumor growth, with 33% of mouse tumors having a volume of less than 500 mm². 3 Furthermore, the survival rate was 100% within the monitored 22 days. The sealed microspheres loaded with MPLA exhibited the best anti-tumor effect, with almost no tumor growth in 50% of the mice at 22 days, and melanoma even disappearing in some mice.

[0135] Example 9: Animal experimental results of sealed microcapsules based on B16 melanoma metastases.

[0136] First, a B16 transfer model mouse model was constructed (purchased from Beijing Vital River Laboratory Animal Co., Ltd.). 2*10 5 B16 melanoma cells (provided by Jilin University) were injected into the tail vein of 6-8 week old female C57BL / 6 mice. The mice were then randomly divided into groups of six, and vaccinated on day 4. The specific immunization strategy was consistent with that in Example 8. Eighteen days post-vaccination, two mice from each group were sacrificed, and the metastasis of melanoma in the lungs and kidneys was observed, and the number and statistical analysis of metastatic lesions in the organs were performed.

[0137] like Figure 17 As shown, the PBS group exhibited numerous melanoma metastases in the lungs, with approximately 100 metastatic lesions detected in each mouse's lungs. Simultaneously, the kidney tissue grew to a total volume of 100 mm. 3The presence of metastatic lesions confirmed the successful establishment of a melanoma metastasis model. Eighteen days after vaccine immunization, dissection of mice in the sealed microsphere group revealed a reduction in lung metastases from 100 to 15-20 compared to the PBS group, and a decrease in kidney tumor volume from 100 mm. 3 Reduced to 10mm 3 The addition of MPLA to the sealed microspheres further enhanced the anti-tumor metastasis effect. There were almost no obvious metastatic lesions in the lungs and no tumor formation in the kidneys. Therefore, the combination of the microcapsule system and the TLR receptor agonist produced the best anti-tumor metastasis effect.

[0138] Example 10: Animal Experiment Results of Sealed Microcapsules Loaded with Tumor Neoantigen Based on 4T1 Breast Cancer. The selected tumor neoantigen was a combination of eight peptides in equal proportions (the amino acid sequences of the eight peptides are: SPNRSWVSL, HPMYLFLSM, VAVKVNFYVI, KAPHNFQFV, YHYVLNSMV, EYSAMTTRGTI, GSPPRFFYM, and CPQTHAVVL). The preparation process of the peptides is as follows: Tumor mutated genes were screened by comparative sequencing of 4T1 tumor cell lines and normal Balb / c mouse tissues (provided by the National Key Laboratory of Biochemical Engineering). Then, the mutated peptides were predicted by computer algorithms (submitted to Shenzhen Yuce Biotechnology Co., Ltd.), and then the peptides were synthesized (submitted to Genscript Biotech Co., Ltd.).

[0139] The AS04 adjuvant group was prepared as follows: First, MPLA was dissolved in a 0.5% triethanolamine solution, then heated to 65°C and maintained for 5 minutes to promote dissolution. Next, it was sonicated three times at 60 W power for 1 minute each using an ultrasonic probe. After complete dissolution, the pH was adjusted to approximately 7.4 with HCl. Then, the MPLA aqueous solution was mixed with aluminum adjuvant and sonicated for 30 seconds. The tumor neoantigen of this invention was then added and mixed thoroughly, ensuring that the dosage of each component of the vaccine for each mouse was MPLA (3 μg), aluminum adjuvant (100 μg), and tumor neoantigen (200 μg) in 100 μL of physiological saline solution.

[0140] The preparation method of the sealed microcapsule assembly containing embedded tumor neoantigens and loaded MPLA is described in Example 3 of this invention.

[0141] 4T1 orthotopic tumor model mice were constructed (purchased from Beijing Vital River Laboratory Animal Co., Ltd.). 5*10 5 4T1 breast cancer cells (purchased from ATCC) were injected into the right lower mammary fat pad of 6-8 week old female Balb / c mice. The mice were then randomly divided into groups of 6, and vaccinated on day 4.

[0142] The groups were set up as follows: PBS group, tumor neoantigen alone group (200 μg), AS04 adjuvant group loaded with tumor neoantigen (100 μg aluminum adjuvant, 200 μg tumor neoantigen, 3 μg MPLA), and sealed microcapsule group encapsulating tumor neoantigen and loading MPLA (3 mg microspheres, 200 μg tumor neoantigen, 3 μg MPLA).

[0143] From the date of tumor model establishment, mouse hair, weight, and survival were observed every other day. The length and width of the tumor were measured using calipers, and the tumor volume was calculated using the following formula: V = 1 / 2 × L × W 2 (L: Length; W: Width).

[0144] like Figure 18 As shown in the tumor growth curves, neither PBS nor the tumor neoantigen group effectively inhibited breast cancer growth. The AS04 adjuvant group showed some inhibitory effect on tumor growth, exhibiting a significant delay in tumor growth. In comparison, the sealed microcapsule group showed the best anti-tumor effect, with 83% of mice having tumors smaller than 500 mm² at 28 days. 3 , and such Figure 19 As shown in the survival curve, the survival rate was 100% within 60 days of monitoring.

[0145] Example 11: Animal experimental results of tumor neoantigen loaded into sealed microcapsules in the recurrence of 4T1 breast cancer after surgery.

[0146] A 4T1-luc breast cancer recurrence model mouse model was constructed (purchased from Beijing Vital River Laboratory Animal Co., Ltd.). 5*10 mice transfected with Luciferase were used. 5 4T1-luc breast cancer cells (purchased from ATCC) were injected into the right lower mammary fat pad of 6-8 week old female Balb / c mice until the tumor grew to 200 mm. 3 Afterwards, the mice underwent tumor surgical removal. The size of the remaining tumor tissue was controlled to be similar by bioluminescence imaging. The mice were then randomly divided into groups of 6, and the mice were immunized with the vaccine on day 2.

[0147] The specific immunization strategy is consistent with that in Example 10. From the date of tumor model establishment, mouse hair, weight, and survival were observed every other day. Tumor recurrence was monitored using bioluminescence imaging, and the length and width of the tumor were measured with calipers. The tumor volume was calculated using the following formula: V = 1 / 2 × L × W 2 (L: Length; W: Width).

[0148] Example 12: Comparison of the cell recruitment effects of sealed microcapsules loaded with chemokines

[0149] Example 12: Comparison of the cell recruitment effects of sealed microcapsules loaded with chemokines

[0150] Female C57BL / 6 mice (n=4, purchased from Beijing Vital River Laboratory Animal Co., Ltd.) aged 6-8 weeks were injected with either a blank sealed microcapsule containing 3 mg of microspheres or a sealed microcapsule containing 7 μg of the chemokine GM-CSF, respectively, into their backs. The mice were sacrificed after 5 days of in vivo metabolism. The injection sites on the mice's backs were dissected, and the subcutaneous tissue containing the carrier was collected with forceps, then cut away with scissors. The tissue was then immersed in 4% formalin solution for 24 hours. Subsequently, the tissue was embedded in paraffin, sectioned, stained with hematoxylin and eosin (H&E), and mounted to prepare H&E-stained samples. Finally, the inflammatory response at the injection site was observed using a panoramic scanning and analysis system (Vectra 3.0, purchased from PerkinElmer).

[0151] like Figure 21 As shown, after 5 days of in vivo metabolism, only a small number of inflammatory cells (black dots) were recruited around the blank microspheres (light-colored round objects). However, the microcapsules loaded with the chemokine GM-CSF recruited significantly more cells to the material, meaning that the antigens released from the GM-CSF-loaded microcapsules could be more readily phagocytosed by cells, thereby enhancing antigen presentation efficiency and the final immune response.

Claims

1. A vaccine for the prophylactic or therapeutic treatment of tumors or hepatitis, wherein the vaccine for therapeutic treatment is an antitumor vaccine or a therapeutic hepatitis B vaccine, the vaccine comprising an antigen and a biodegradable polymer blend matrix, the polymer blend containing a hydrophobic polymer and an amphiphilic block copolymer, the vaccine being in the form of microcapsules containing a multi-chamber structure, the microcapsules having an average particle size of 10-100 μm. The microcapsules are prepared by the following method: first, open-pore microspheres are prepared from a polymer blend, then mixed with a solution containing an antigen, and then the open-pore microspheres loaded with the antigen solution are sealed to form sealed microcapsules loaded with antigen. The polymer blend matrix metabolizes to produce an acidic microenvironment that enhances cell recruitment. The hydrophobic polymer is a lactide polymer, glycolide polymer, lactide-glycolic acid copolymer, polycaprolactone, polyorthoester and / or polyanhydride, wherein the weight average molecular weight of the hydrophobic polymer is 5,000-100,000 Daltons, and the amphiphilic block copolymer is a copolymer of polyethylene glycol or monomethoxyethylene glycol with lactide and / or glycolide, wherein the weight average molecular weight of the amphiphilic block copolymer is 10,000-50,000 Daltons, and the amphiphilic block copolymer accounts for at least 5% of the weight of the polymer blend matrix in the microcapsules.

2. The vaccine of claim 1, wherein the average particle size of the microcapsules is 30-60 μm.

3. The vaccine of claim 1, wherein the vaccine is used for therapeutic treatment.

4. The vaccine of claim 1, wherein the antigen is a tumor antigen or hepatitis B surface antigen.

5. The vaccine of claim 1, wherein it contains chemokines.

6. The vaccine of claim 1, wherein the antigen-containing solution contains a chemokine, which is loaded into a sealed microcapsule via an open-cell microsphere seal.

7. The vaccine of claim 5 or 6, wherein the chemokine is granulocyte colony-forming factor, macrophage inflammatory protein 3α, and / or monocyte chemotactic protein 1.

8. The vaccine of claim 1, wherein the hydrophobic polymer has a weight-average molecular weight of 10,000-50,000 Daltons.

9. The vaccine of claim 1, wherein the amphiphilic block copolymer accounts for at least 10% by weight of the polymer blend matrix in the microcapsule.

10. The vaccine of claim 1, wherein the amphiphilic block copolymer accounts for at least 15% by weight of the polymer blend matrix in the microcapsule.

11. The vaccine of claim 1, wherein the amphiphilic block copolymer accounts for at least 20% by weight of the polymer blend matrix in the microcapsule.

12. The vaccine of claim 1, wherein it contains an immune stimulant.

13. The vaccine of claim 12, wherein the immunostimulator is monophosphate A, cytosine-guanine oligodeoxynucleotide and / or polyinosinic acid.

14. The vaccine of claim 1, wherein the open-cell microspheres loaded with antigens are sealed by heating them to near the glass transition temperature of the microspheres.

15. The vaccine of claim 1, wherein the open-cell microspheres loaded with antigen are sealed by heating them to 1-2°C below the glass transition temperature of the microspheres.

16. The vaccine of claim 1, wherein the open-pore microspheres are prepared by a double emulsion solvent removal method.

17. The vaccine of claim 1, wherein the porosity of the open-cell microspheres is at least 40%.

18. The vaccine of claim 1, wherein the porosity of the open-cell microspheres is at least 50%.

19. The vaccine of claim 1, wherein the porosity of the open-cell microspheres is at least 60%.

20. The vaccine of claim 1, wherein the porosity of the open-cell microspheres is at least 70%.

21. The vaccine of claim 1, wherein the open-pore microspheres have channels with a pore size of 800 nm to 5 μm.

22. A pharmaceutical composition comprising the vaccine of any one of claims 1-21, and a pharmaceutically acceptable carrier.

23. The use of the vaccine of any one of claims 1-21 in the preparation of an antitumor vaccine or a therapeutic vaccine.

24. The use of the vaccine of any one of claims 1-21 in the preparation of an antitumor drug or a drug for treating hepatitis.

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