A tumor vaccine based on traditional Chinese medicine nanovesicles, its preparation method and application

Abstract

The present invention relates to the field of biomedicine technology, and provides a tumor vaccine based on Chinese herbal medicine nanovesicles, and a preparation method and application thereof. The preparation method comprises mixing Chinese herbal medicine nanovesicles with tumor antigens, and encapsulating them after mixing to obtain a tumor vaccine; the tumor antigen comprises a polypeptide or a protein. The present invention utilizes the characteristics of Chinese herbal medicine nanovesicles targeting lymph nodes, being specifically engulfed by migratory dendritic cells in lymph nodes, and promoting dendritic cell maturation through TLR4, thereby increasing the level of tumor antigen presentation, and finally the mature antigen presenting cells activate cytotoxic T lymphocytes, forming specific anti-tumor immune memory, effectively inhibiting tumor recurrence, and prolonging the survival of tumor-stricken organisms.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the preparation and application of a tumor polypeptide / protein vaccine based on Chinese herbal medicine nanovesicles. Background Art

[0002] Malignant tumors are the leading cause of death among patients. Efficient tumor treatment and delayed tumor recurrence are crucial to improving global life expectancy. Immunotherapy (including immune checkpoint inhibitors, adoptive cell therapy, cytokines and cancer vaccines) has become the trend and mainstream of tumor treatment. In particular, tumor vaccines have inherent advantages in preventing and treating tumor recurrence and tumor metastasis, and have the characteristics of preventing tumor recurrence and forming long-term immune memory. Slightly different from traditional vaccines, tumor vaccines stimulate specific cellular immune responses to prevent tumor growth and ultimately eradicate tumor cells. Tumor vaccines have always been considered a potential high-value tool for tumor treatment.

[0003] Currently, there are only two FDA-approved preventive vaccines for the prevention of viral malignancies (hepatitis B virus and human papillomavirus). In addition, a dendritic cell-based vaccine (Sipuleucel-T) was successfully used to treat prostate cancer and was approved in 2010. Although therapies based on immune checkpoint inhibitors, immune checkpoint blockers, and chimeric antigen T cells have achieved good clinical treatment benefits in different tumors and have been partially approved for marketing, there are still problems such as small clinical scale, low survival rate, and low patient applicability, which hinder progress in this field.

[0004] Antigen selection is a key process in the design of peptide vaccines. Antigens identified from surgically removed tumor tissues can be divided into tumor-associated antigens (TAAs) and tumor-specific antigens (TSAs). Tumor antigens recognized by T lymphocytes are the core of the effectiveness of tumor vaccines. The ideal antigen for peptide vaccines should be highly immunogenic, clearly expressed in all cancer cells (but not normal cells), and required for the survival of cancer cells. However, TAAs have universal adaptability in tissue cells and can be applied to different patients. TSAs are individual-specific non-self proteins produced due to tumor cell mutations, and TSAs are only expressed by tumor cells; at the same time, TAAs and TSAs are derived from the patient's tumor tissue itself, have low immunogenicity, cannot induce a strong immune response, and thus cannot effectively exert the efficacy of tumor vaccines. Therefore, most tumor vaccines are still in the preclinical and clinical research stages, and a suitable vaccine delivery system is needed to deliver tumor antigens to promote a stronger immune response and improve clinical treatment effects.

[0005] Tumor vaccines are mainly based on specific antigens to produce specific anti-tumor immune responses and immune memory effects. Peptide / protein vaccines are a form of vaccine that directly delivers tumor antigens. By using short peptide antigen fragments, they can induce highly targeted immune responses, which can reduce or avoid allergic reactions caused by vaccination. Peptide vaccines are highly safe, can be synthesized on a large scale, and are easy to purify and apply. However, due to shortcomings such as low cell uptake rate, high rapid clearance rate, and poor stability, the effects of clinical trials are relatively limited.

[0006] The stability, immunogenicity and targeting of antigen presentation of peptide vaccines can be improved by designing appropriate delivery systems to assist vaccine delivery. At present, the carriers used to deliver peptide vaccines mainly include liposomes, metal nanoparticles, virus-like nanoparticles, emulsions, immunostimulatory complexes, nanoparticle polymers, self-assembled peptide / protein nanoparticles and microneedles. Since most of the carriers used are artificially synthesized or pathogenic, they are affected by many factors during the in vivo delivery process, which limits their application, such as allergic reactions, safety hazards, easy oxidation and degradation, poor preparation repetition rate and high development cost. These problems further limit the clinical application of peptide / protein vaccines, and there is an urgent need to develop new carriers for peptide vaccine delivery. Summary of the invention

[0007] In order to solve the defects and shortcomings of the above-mentioned existing related technologies, the present invention uses Chinese herbal medicine nanovesicles as a vaccine delivery platform targeting lymph nodes, and provides a tumor polypeptide / protein vaccine based on plant-derived nanovesicles and its preparation method and application.

[0008] The first aspect of the present invention provides a method for preparing the tumor vaccine, comprising mixing Chinese herbal medicine nanovesicles with tumor antigens, encapsulating them after mixing, and obtaining the tumor vaccine; the tumor antigens include polypeptides or proteins.

[0009] Optionally, the tumor antigen includes a natural polypeptide, a recombinant fusion polypeptide, a natural protein, or a recombinant fusion protein.

[0010] Optionally, the tumor antigen includes a tumor-associated antigen or a tumor-specific antigen.

[0011] Optionally, the mixing ratio of the Chinese herbal medicine nanovesicles and the tumor antigen is 10:(1-5) (w / w).

[0012] Optionally, the Chinese herbal medicine in the Chinese herbal nanovesicles includes ginseng; and the tumor antigen includes a pattern antigen.

[0013] Optionally, the method for preparing the Chinese herbal medicine nanovesicles comprises: squeezing fresh Chinese herbal medicine to obtain Chinese herbal medicine juice, collecting the supernatant after centrifugation, and collecting the precipitate after centrifugation of the supernatant to obtain the Chinese herbal medicine nanovesicles.

[0014] Optionally, the encapsulation method includes: extrusion method, low-intensity ultrasonic treatment, electroporation treatment, freeze-thaw encapsulation or pH gradient method.

[0015] Furthermore, the extrusion method includes: sucking the mixed liquid into a booster and repeatedly extruding it using an extrusion membrane; the low-intensity ultrasonic treatment includes: placing the mixed liquid in an ice bath and treating it with 50-100 W ultrasonic waves for 10-15 min; the electroporation treatment includes using an electroporator to repeatedly electroporate the mixed liquid; the freeze-thaw packaging includes using liquid nitrogen to freeze-thaw the mixed liquid for a specific time and then restoring it to room temperature.

[0016] The second aspect of the present invention provides a tumor vaccine prepared by the above preparation method.

[0017] Optionally, the types of Chinese herbal medicines in the Chinese herbal nanocapsule in the above-mentioned tumor vaccine include any one or more combinations of ginseng, American ginseng, ginger, coptis root, Panax notoginseng, angelica, black bean, patchouli, perilla, polygonatum, polygonatum, mint, dendrobium, astragalus, orange or turmeric.

[0018] The third aspect of the present invention provides the above-mentioned tumor disease prevention or treatment drug, including the above-mentioned tumor vaccine, and pharmaceutically acceptable adjuvants or excipients.

[0019] The fourth aspect of the present invention also provides the use of the above-mentioned tumor vaccine in the preparation of drugs for preventing or treating tumor diseases.

[0020] Optionally, for the application, the tumor disease includes melanoma, colorectal tumor, breast tumor, bladder tumor, lung cancer, liver cancer, pancreatic cancer, esophageal cancer, gastric cancer, cervical cancer, bile duct cancer or testicular cancer.

[0021] The fifth aspect of the present invention provides the use of Chinese herbal nanovesicles in the preparation of tumor polypeptide or protein vaccines; it also provides the use of Chinese herbal nanovesicles in the preparation of tumor disease prevention or treatment drugs, including using the Chinese herbal nanovesicles as vaccine delivery vectors, immune adjuvants or targeted preparations in the above-mentioned tumor disease prevention or treatment drugs.

[0022] The present invention prepares a Chinese herbal medicine nanovesicle chimeric tumor polypeptide / protein vaccine based on Chinese herbal medicine nanovesicles and tumor polypeptide / protein antigens. The preparation method is simple to operate. The prepared tumor vaccine has the characteristics of a carrier and an adjuvant, and has good characteristics of polypeptide antigen lymph node targeted delivery. The polypeptide antigen can be taken up by dendritic cells with migration and presentation functions in the lymph nodes, and the presentation level of the polypeptide antigen by the dendritic cells is increased, and finally a specific adaptive immune response and anti-tumor immune memory are generated. The tumor vaccine of the present invention can activate a specific anti-tumor immune response, has a low operating threshold, is low in cost, and has good safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The results of nanoparticle tracking analysis and transmission electron microscope images of 16 kinds of fresh Chinese herbal medicine vesicles separated and extracted in the examples of the present invention are shown in the figure. As can be seen from the figure, the particle sizes of the fresh Chinese herbal medicine nanoparticles are all within a suitable fluctuation range, and the extracted fresh herbal medicine nanovesicles all have a double-layer lipid membrane structure.

[0024] Figure 2 The ratio of the 16 kinds of fresh Chinese herbal medicine nanocapsules listed in the examples of the present invention in promoting the maturation of dendritic cells in vitro. As can be seen from the figure, the fresh ginseng nanocapsules have the highest activation degree for dendritic cells.

[0025] Figure 3 The nanoparticle size tracking analysis of five batches of ginseng nanovesicles extracted in the examples of the present invention shows that the ginseng nanovesicles extracted from different batches have good extraction stability.

[0026] Figure 4 These are transmission electron microscope images of ginseng nanovesicles extracted from five batches in the examples of the present invention. The results show that ginseng nanovesicles extracted from different batches have good structural stability and uniform distribution.

[0027] Figure 5 The nanoparticle size tracking analysis of the ginseng nanocapsules extracted from the same batch in the embodiment of the present invention after being placed at room temperature for different time periods. The results show that the ginseng nanocapsules have good stability at room temperature.

[0028] Figure 6 The small animal imaging instrument in the embodiment of the present invention analyzed the lymph node accumulation of fluorescently labeled ginseng nanocapsules after different administration methods. The results showed that ginseng nanocapsules were significantly accumulated in the spleen after intravenous and intraperitoneal injections.

[0029] Figure 7 The small animal imaging instrument was used to analyze the spleen accumulation of fluorescently labeled ginseng nanovesicles after different administration methods. The results showed that there was no significant difference in fluorescence intensity between the subcutaneous injection group and the blank group.

[0030] Figure 8 The organ distribution of fluorescently labeled ginseng nanoparticles after subcutaneous injection was analyzed using a small animal imaging device. The results showed that ginseng vesicles mainly accumulated in the lymph nodes after subcutaneous injection.

[0031] Fig. 9 Flow cytometry analysis of the phagocytosis of fluorescently labeled ginseng nanocapsules by lymph node immune cells after subcutaneous injection. The results showed that ginseng nanocapsules were more phagocytosed by dendritic cells with migratory functions in the lymph nodes.

[0032] Fig.10 Transmission electron microscopy (above) and cryo-scanning electron microscopy (below) images of the ginseng nanovesicle peptide vaccine after preparation. The results show that the ginseng nanovesicle peptide vaccine still has a double-layer membrane structure after preparation, and the particle size is maintained in the range of 100-200 nm.

[0033] Fig.11 These are the SDS-PAGE images of ginseng nanovesicles, peptide vaccines (i.e., antigen peptide (OVA)) and ginseng nanovesicle peptide vaccines. The ginseng nanovesicle peptide vaccine has characteristic protein bands of both ginseng vesicles and antigen peptides.

[0034] Fig.12 The chimeric rate of the chimeric ginseng nanovesicle peptide vaccine prepared by fluorescently labeled ginseng nanovesicles and fluorescently labeled antigenic peptides was analyzed by flow cytometry. The results showed that the chimeric rate of the ginseng vesicle antigenic peptide was 52.9%.

[0035] Fig.13 This is the nanoparticle tracking analysis of ginseng nanovesicle peptide vaccine after being stored at 4°C for different time periods. The results show that ginseng nanovesicle peptide vaccine still has good stability after being stored at 4°C for different time periods.

[0036] Fig.14 Flow cytometry was used to analyze the phagocytosis of fluorescently labeled ginseng nanovesicles, peptide vaccines (antigenic peptides), and ginseng nanovesicle peptide vaccines after incubation with dendritic cells in vitro. The results showed that ginseng nanovesicle peptide vaccines can enhance the phagocytosis of antigenic peptides by dendritic cells.

[0037] Fig.15 Tumor recurrence and growth after subcutaneous inoculation of melanoma cells B16F10 stably expressing OVA antigens with ginseng nanovesicles, peptide vaccines (antigen peptides) and ginseng nanovesicle peptide vaccines, including tumor volume growth curve, overall survival rate and body weight. The results showed that ginseng nanovesicle peptide vaccination had no significant effect on the weight of mice, but could significantly inhibit tumor growth and prolong the survival of mice.

[0038] Fig.16Tumor volume, survival rate and body weight of melanoma B16F10 cells stably expressing OVA antigen after vaccination with ginseng nanovesicles, peptide vaccine (antigenic peptide) and ginseng nanovesicle peptide vaccine.

[0039] Fig.17 Serum biochemical indexes were tested after vaccination with ginseng nanovesicles, peptide vaccines (antigenic peptides) and ginseng nanovesicle peptide vaccines. The results showed that there was no significant difference in serum biochemical indexes among the groups after vaccination. DETAILED DESCRIPTION

[0040] The present invention provides a tumor vaccine based on Chinese herbal medicine nanocapsule, and the preparation method thereof comprises the following steps: mixing the Chinese herbal medicine nanocapsule with tumor antigens, and then encapsulating the mixed antigens to obtain the tumor vaccine.

[0041] Among them, the tumor antigens described in the present invention include polypeptides or proteins, such as natural polypeptides, recombinant fusion polypeptides, natural proteins, recombinant fusion proteins, etc., that is, the tumor vaccine of the present invention is a tumor polypeptide / protein vaccine. The types of tumor antigens include tumor-associated antigens (TAAs) or tumor-specific antigens (TSAs). That is, the types of tumor antigens applicable to the scheme of the present invention include but are not limited to pattern antigens, tumor-associated polypeptide antigens, tumor-associated protein antigens, tumor-specific polypeptide antigens, tumor-specific protein antigens, tumor-associated fusion polypeptide antigens, tumor-associated fusion protein antigens, tumor-specific fusion polypeptide antigens, tumor-specific fusion protein antigens, etc.

[0042] As for Chinese herbal medicine nanovesicles, they are a type of plant-derived extracellular vesicle-like particles (P-EVLPs). The Chinese herbal medicines described in the present invention include but are not limited to ginseng, American ginseng, ginger, coptis root, notoginseng, angelica, black bean, patchouli, perilla, polygonatum, polygonatum, mint, dendrobium, astragalus, orange and turmeric, etc.

[0043] The preparation method of Chinese herbal medicine nanovesicles includes: squeezing fresh Chinese herbal medicine to obtain Chinese herbal medicine juice, centrifuging the Chinese herbal medicine juice at high speed, taking the supernatant and ultracentrifuging it again, and obtaining the precipitate, which is the Chinese herbal medicine nanovesicle. In some schemes, for ease of use and storage, the precipitate is resuspended with a PBS solution for standby use. In some schemes, in order to further purify the Chinese herbal medicine nanovesicles, sucrose density gradient centrifugation is further used to obtain Chinese herbal medicine nanovesicles with high purity and less impurity content. In some schemes, the preparation method of Chinese herbal medicine nanovesicles is: squeezing fresh Chinese herbal medicine to obtain Chinese herbal medicine juice, using a high-speed centrifuge and an ultracentrifuge, combined with sucrose density gradient centrifugation, the Chinese herbal medicine juice is centrifuged to obtain Chinese herbal medicine nanovesicles. Taking ginseng as an example of Chinese herbal medicine, the preparation method of ginseng nanovesicles includes: taking fresh ginseng, washing it with distilled water, squeezing it with broken walls to obtain crude juice, collecting the supernatant by high-speed centrifugation, collecting the precipitate by ultracentrifugation, and resuspending it with PBS buffer to obtain fresh ginseng-derived vesicle-like nanoparticles, namely ginseng nanovesicles. Among them, high-speed centrifugation is: centrifugation at 200 rpm for 10 to 20 minutes; centrifugation at 2000 rpm for 10 to 30 minutes; and then centrifugation at 10000 rpm for 15 to 30 minutes, and this step is repeated 3-5 times; ultracentrifugation is: centrifugation at 100000 rpm for 30 to 60 minutes; sucrose density gradient centrifugation method: using 5 concentration gradients of 8%, 15%, 30%, 45% and 60%, slowly adding the sample, centrifuging at 100000 rpm for 60 minutes, and taking the yellow band in the 30% sucrose concentration area, which is the required ginseng nanovesicles.

[0044] The Chinese herbal medicine nanovesicles of the present invention, as P-EVLPs, can achieve better therapeutic effects due to their natural ingredients and combined with standard tumor treatment. P-EVLPs are natural nano-scale lipid particles with a lipid bilayer membrane, containing nucleic acids, proteins, lipids and specific small molecule components. They are ideal carriers for drug delivery, and can further enhance the therapeutic effect on diseases while maintaining their original properties. P-EVLPs have superior pharmacological and immunomodulatory abilities, and are easy to structurally modify and fill with contents.

[0045] After obtaining the Chinese herbal medicine nanocapsule, it is mixed with the tumor antigen, mixed evenly and then encapsulated to obtain a tumor peptide / protein vaccine.

[0046] Preferably, the mixing ratio of the Chinese herbal medicine nanovesicles to the tumor antigen is 10:(1-5). The mixing ratio may also be 10:(1-3) or 10:(2-5), or 10:2.5, 10:4 or 10:5, etc.

[0047] The specific operation methods for encapsulation include extrusion method, low-intensity ultrasonic treatment, electroporation treatment, freeze-thaw encapsulation or pH gradient method. The extrusion method includes: sucking the mixed liquid into a booster, using an extrusion membrane with a specific pore size, such as a 400 nm, 200 nm polycarbonate extrusion membrane, repeatedly extruding multiple times, such as 11 times, and finally obtaining a tumor / peptide vaccine chimeric with Chinese herbal medicine nanovesicles. The low-intensity ultrasonic treatment includes: placing the mixed liquid in an ice bath and using 50~100 W ultrasonic treatment for 10~15 min. The electroporation treatment refers to using an electroporator to repeatedly electroporate the mixed liquid. The freeze-thaw encapsulation method is: using liquid nitrogen to freeze and thaw the mixed liquid for a specific time, such as 5min, and then returning to room temperature to achieve encapsulation. The pH gradient method refers to establishing a sufficient pH transmembrane gradient difference between the inside and outside of the lipid membrane by adjusting the acidity and alkalinity of the aqueous phase and the external aqueous phase in the liposome, so that the drug enters the inner aqueous phase and is encapsulated in the lipid membrane cavity in an ionic state. In some schemes, the specific operation method includes: adjusting the pH of the mixed solution to 4-5, gradually adding alkaline solution to adjust the pH to 9-10, and then adding acid solution to restore the pH to 6.8-7.2.

[0048] After encapsulation, the tumor polypeptide / protein vaccine based on Chinese herbal nanovesicles of the present invention is obtained. The tumor vaccine of the present invention uses Chinese herbal nanovesicles as the encapsulation of tumor antigens, making the Chinese herbal nanovesicles a carrier of immunogenic substances such as polypeptides and proteins; at the same time, the Chinese herbal nanovesicles can be used as immune adjuvants and targeted preparations for lymph node cells, promoting tumor antigens to target lymph nodes and antigen-presenting cells, enhancing the phagocytosis and antigen presentation of tumor antigens, and promoting the maturation of antigen-presenting cells. In some schemes, fresh ginseng nanovesicles are encapsulated with specific antigens to prepare antigen-encapsulated vaccines for subcutaneous vaccination. After vaccination, they can target lymph nodes and tend to be engulfed by dendritic cells with migratory functions in the lymph nodes, inducing dendritic cell maturation, producing more efficient antigen-specific immune responses and immune memory, and enhancing the efficacy of the vaccine.

[0049] Therefore, the tumor vaccine of the present invention can be used as a preventive drug or a therapeutic drug; in addition, the present invention also provides the use of the above-mentioned tumor vaccine in the preparation of tumor disease prevention or treatment drugs. The components of the above-mentioned medicine, in addition to the tumor antigen substance encapsulated by the Chinese herbal medicine nanovesicles, also include pharmaceutically acceptable excipients or excipients. When using the tumor vaccine of the present invention, the administration method includes subcutaneous injection, intramuscular injection, oral administration, intravenous injection, mucosal administration or intraperitoneal injection. The types of tumors targeted by the present invention include melanoma, colorectal tumors, breast tumors, bladder tumors, lung cancer, liver cancer, pancreatic cancer, esophageal cancer, gastric cancer, cervical cancer, bile duct cancer or testicular cancer.

[0050] It can be seen that the present invention also provides an application of Chinese herbal medicine nanovesicles in the preparation of tumor disease prevention or treatment drugs, that is, the Chinese herbal medicine nanovesicles are used as the above-mentioned tumor disease prevention or treatment drugs or the above-mentioned tumor vaccines. The embodiment scheme of the present invention has discovered and confirmed the functional role of Chinese herbal medicine nanovesicles in tumor polypeptide / protein vaccines in promoting tumor antigen targeting lymph nodes and antigen presenting cells, enhancing tumor antigen phagocytosis and antigen presentation, and promoting the maturation of antigen presenting cells, so that it can be used as an ideal delivery carrier for tumor vaccines and play the role of immune adjuvant or targeting agent.

[0051] Example

[0052] In this embodiment, a ginseng nanovesicle chimeric tumor peptide / protein vaccine is prepared based on ginseng nanovesicles and tumor peptide / protein antigens. The tumor antigen takes the mature model antigen ovalbumin (OVA) as an example. The ginseng nanovesicle peptide / protein vaccine is prepared by combining fresh ginseng-derived nanovesicles with the specific model antigen OVA. The ginseng nanovesicles are targeted to lymph nodes, specifically phagocytosed by migratory dendritic cells in the lymph nodes, and promote the maturation of dendritic cells through TLR4, thereby increasing the presentation level of OVA antigen-specific MHC-I-antigen peptides. Finally, the mature antigen-presenting cells activate cytotoxic T lymphocytes, form specific anti-tumor immune memory, effectively inhibit tumor recurrence, and prolong the survival of mice.

[0053] Firstly, fresh Chinese herbal medicines were squeezed to obtain herbal juice. High-speed centrifuge and ultracentrifuge were used in combination with density gradient centrifugation to obtain herbal nanovesicles. Transmission electron microscopy and nanoparticle tracking instrument (NTA) were used to determine the morphology and size of the herbal nanovesicles.

[0054] Secondly, in vitro experiments were used to explore the effect of Chinese herbal medicine nanovesicles on dendritic cell maturation, and to determine the biological activity advantage of fresh ginseng nanovesicles in promoting dendritic cell maturation. The Chinese herbal medicines involved include but are not limited to ginseng, American ginseng, ginger, coptis root, notoginseng, angelica, black bean, patchouli, perilla, polygonatum, polygonatum, mint, dendrobium, astragalus, orange and turmeric.

[0055] Subsequently, the ginseng nanocapsules and tumor antigens were mixed and extruded or ultrasound-assisted encapsulation was used to prepare ginseng nanocapsule chimeric tumor vaccines, and the stability and chimeric efficiency of ginseng nanocapsule tumor vaccines were observed. The targeting performance of ginseng nanocapsule tumor vaccines on the lymphatic system and dendritic cells, as well as their properties for adaptive immune activation, were verified in vivo and in vitro.

[0056] Finally, the prepared ginseng nanovesicle chimeric tumor vaccine was screened for multiple vaccination methods, and the distribution of ginseng nanovesicle chimeric tumor vaccine in different administration methods was observed. The efficacy of ginseng nanovesicle chimeric tumor vaccine was verified by establishing a tumor recurrence / treatment model, and its safety was evaluated.

[0057] More specific experimental plans and experimental results are as follows.

[0058] The following are explanations of the abbreviations of experimental groups that may be involved in the specific embodiments:

[0059] Control group: a complete blank without any experiments; Peptide vaccine: taking OVA (chicken ovalbumin) as an example, only OVA peptide was used for stimulation, that is, only OVA antigen was present and no adjuvant was contained; Ginseng nanovesicles (ginseng vesicles): only ginseng nanovesicles were used for stimulation, that is, only the effect of adjuvant was present and no tumor antigen was contained; Ginseng vesicle peptide vaccine, ginseng nanovesicle chimeric OVA peptide was used for stimulation, that is, OVA antigen and adjuvant were delivered simultaneously.

[0060] The above-mentioned control group, peptide vaccine group and ginseng nanovesicle group are all control groups of the ginseng nanovesicle peptide vaccine group.

[0061] Example 1: Extraction and separation of 16 fresh Chinese herbal medicine nanovesicles and in vitro activation of dendritic cells

[0062] The specific process is as follows:

[0063] ① Obtaining crude fresh Chinese herbal medicine extracts: Take fresh Chinese herbal medicine, wash it with double distilled water, add it into a wall breaking machine to squeeze out the crude juice of fresh Chinese herbal medicine, use a high-speed centrifuge to centrifuge at 200 rpm for 15 min; 2000 rpm for 30 min; and finally centrifuge at 10000 rpm for 30 min, and repeat this step 3-5 times.

[0064] ② Extraction of fresh Chinese herbal medicine nanovesicles: Collect the supernatant after centrifugation, centrifuge at 100,000 rpm for 60 min, collect the precipitate and resuspend it in 1× PBS solution, and use BCA protein quantitative kit to determine the concentration of fresh Chinese herbal medicine nanovesicles. The fresh Chinese herbal medicine nanovesicles obtained above are stored at -80°C.

[0065] A total of 16 kinds of fresh Chinese herbal medicine nanovesicles were extracted, including ginseng, American ginseng, ginger, coptis root, Panax notoginseng, angelica, black bean, Patchouli, Perilla, Polygonatum, Polygonatum, Mint, Dendrobium, Astragalus, Tangerine and Turmeric. The particle size of the nanovesicles was analyzed by nanoparticle tracking (NTA), and the morphology of the nanovesicles was analyzed by transmission electron microscopy. The results showed that the 16 kinds of fresh Chinese herbal medicine nanovesicles all met the characteristics of plant nanovesicles and had a double-layer membrane structure ( Figure 1 ).

[0066] Bone marrow-derived dendritic cells (BMDCs) were extracted, and the 16 fresh Chinese herbal medicine nanovesicles extracted above were co-incubated with BMDCs for 24 hours (at a dosing concentration of 10 μg / mL), and then the expression level of BMDCs activation marker CD86 was analyzed by flow cytometry. The results showed that ginseng nanovesicles had the highest relative activation level on BMDCs ( Figure 2 ).

[0067] Example 2: Stability study of ginseng nanovesicles extracted from different batches

[0068] Five batches of ginseng nanovesicles were extracted according to the extraction method of Example 1, and the particle sizes of the five batches of ginseng nanovesicles were analyzed using a nanoparticle size tracker (NTA). The results showed that there was no significant difference in the particle size distribution of the five batches of ginseng nanovesicles extracted ( Figure 3 ); The morphology of ginseng nanovesicles extracted from five batches was analyzed by transmission electron microscopy. The results showed that all the ginseng nanovesicles extracted from the five batches had a double-layer membrane structure ( Figure 4 ). That is, the extraction of ginseng nanovesicles has good batch-to-batch stability.

[0069] One batch of ginseng nanocapsules was taken and stored at room temperature for different periods of time. The particle size of ginseng nanocapsules was analyzed using a nanoparticle size tracking instrument (NTA). The results showed that there was no difference in the particle size distribution of ginseng nanocapsules after being stored at room temperature for different periods of time, indicating that ginseng nanocapsules have good room temperature stability ( Figure 5 ).

[0070] Example 3: In vivo distribution of ginseng vesicles and phagocytosis detection and analysis of lymph node immune cells

[0071] Fresh ginseng nanocapsules were labeled with fluorescent molecules DiR to prepare fluorescently labeled fresh ginseng nanocapsules. The fluorescently labeled fresh ginseng nanocapsules were administered to mice by oral administration, intravenous injection, intraperitoneal injection, and subcutaneous injection. Lymph nodes and spleens were removed 24 hours later, and small animal imaging was used to analyze the accumulation of lymph nodes and spleens, and the fluorescence intensity of the above organs was counted. The results showed that the fluorescence intensity of the lymph nodes after subcutaneous injection of ginseng vesicles was higher than that of other groups, that is, subcutaneously injected ginseng nanocapsules mainly tended to lymphatic tissue ( Figure 6 ); At the same time, it showed that the fluorescence intensity of the spleen after intravenous and intraperitoneal injection of ginseng nanocapsules was higher than that of other groups, that is, intravenous and intraperitoneal injection of ginseng nanocapsules mainly tended to spleen tissue ( Figure 7 ).

[0072] Fresh ginseng nanovesicles were labeled with fluorescent molecules DiR to prepare fluorescently labeled fresh ginseng nanovesicles. The fluorescently labeled fresh ginseng nanovesicles were administered to mice by subcutaneous injection. After 24 hours, the organs were analyzed by small animal imaging and the fluorescence intensity of each organ was counted. The results showed that after subcutaneous injection of nanovesicles, the accumulation of ginseng nanovesicles in the lymph nodes was significantly higher than that in the control group, indicating that ginseng nanovesicles have good lymph node targeting ( Figure 8 ). Single cells from lymph nodes were separated and prepared into single cell suspensions. Flow cytometry was used to analyze the phagocytic efficiency of ginseng nanovesicles by different immune cells in the lymph nodes. The results showed that ginseng nanovesicles were mainly phagocytosed by migratory dendritic cells in the lymph nodes ( Fig. 9 ).

[0073] Example 4: Preparation and detection of ginseng nanovesicle peptide / protein vaccine

[0074] Preparation of ginseng nanovesicle peptide vaccine with OVA (chicken ovalbumin) as model antigen and fresh ginseng nanovesicles encapsulating OVA peptide: The prepared fresh ginseng nanovesicles and OVA peptide were mixed in a ratio of 10:3. The above ratio can reduce the amount of OVA peptide antigen and achieve the maximum OVA peptide antigen encapsulation rate. The mixed solution was encapsulated by extrusion using an extruder, that is, 400 nm and 200 nm extrusion membranes were repeatedly extruded 11 times, and finally a tumor vaccine, namely, ginseng nanovesicle peptide vaccine, was prepared. The prepared tumor vaccine was finally quantified by BCA for protein.

[0075] Characterization of ginseng nanovesicle peptide vaccine: The particle size and ζ potential of ginseng nanovesicle peptide vaccine were detected using dynamic light scattering (DLS). Transmission electron microscopy and cryo-scanning electron microscopy were used to detect the morphology of ginseng nanovesicle peptide vaccine and verify its bilayer lipid membrane structure. The characteristic protein bands of ginseng nanovesicles and peptides were compared (SDS-PAGE method).

[0076] Table 1 Particle size and zeta potential of tumor vaccines

[0077]

[0078] Table 1 shows the dynamic light scattering (DLS) and zeta potential data of ginseng nanovesicles and ginseng nanovesicle peptide vaccines. The particle size of ginseng nanovesicles is about 168.2 nm, and its zeta potential is about -14.1 mV. The particle size of ginseng nanovesicle peptide vaccine is about 163.7±4.1 nm, and the zeta potential is about -15.3±1.5 mV. Transmission electron microscopy and cryo-scanning electron microscopy results show that ginseng nanovesicle peptide vaccine has a relatively uniform cup-shaped or spherical structure, and has an obvious double lipid membrane structure ( Fig.10The SDS-PAGE results showed that the ginseng nanovesicle peptide vaccine had protein electrophoresis bands with obvious characteristics of ginseng nanovesicles and antigenic peptides ( Fig.11 ).

[0079] The particle size distribution of ginseng nanovesicle peptide vaccine at different time points stored at 4°C was analyzed using a nanoparticle tracking instrument (NTA). The results showed that there was no significant difference in the particle size distribution of ginseng nanovesicle peptide vaccine within 21 days ( Fig.13 ), indicating that the ginseng nanovesicle peptide vaccine has good storage stability when stored at 4°C.

[0080] Ginseng nanovesicles were labeled with fluorescent molecules DIR, and then OVA antigen peptides were labeled with fluorescent molecules Cy5. Ginseng nanovesicle peptide vaccines with fluorescent labels were prepared according to the above method, and the encapsulation rate of fluorescently labeled ginseng nanovesicle peptide vaccines was analyzed by flow cytometry. The results showed that the encapsulation rate of ginseng nanovesicle OVA peptide was 52.9% ( Fig.12 ).

[0081] Example 5: In vitro analysis of dendritic cell phagocytosis of ginseng nanovesicle peptide vaccine

[0082] Ginseng nanovesicles were labeled with fluorescent molecules DIR, and then OVA peptides were labeled with fluorescent molecules Cy5. Ginseng nanovesicle peptide vaccines with fluorescent labels were prepared according to the above method. Bone marrow-derived dendritic cells (BMDCs) were extracted, and fresh ginseng nanovesicle tumor vaccines were co-incubated with BMDCs. The uptake of CD11C+ BMDCs was analyzed by flow cytometry. The results showed that BMDCs could take up a large amount of ginseng nanovesicles and ginseng nanovesicle peptide vaccines, and the uptake level of OVA peptide by BMDCs increased after treatment with ginseng nanovesicle peptide vaccines ( Fig.14 ), which shows that ginseng nanovesicle peptide vaccine can promote BMDCs to phagocytize tumor antigens.

[0083] Example 6: Evaluation of in vivo anti-tumor immune response of ginseng nanovesicle peptide vaccine (preventive administration)

[0084] C57BL / 6 mice were raised freely with sufficient food and water. The mice were randomly divided into a blank group (PBS, 200 μL / mouse), a ginseng nanovesicle group (200μg / 200μL / mouse), a peptide / protein vaccine (OVA 20μg / 200μL / mouse) and a ginseng nanovesicle peptide vaccine (calculated as OVA 20μg / 200μL / mouse) group. The vaccines were given on Day -7, -11, and -14 for prevention, and the abdominal fur of C57BL / 6 mice was removed on Day 0. 5×10 5B16F10-OVA tumor cells (0.05 mL / mouse) were subcutaneously inoculated into the left abdomen of mice. The tumor size was recorded every day after tumor loading. When the tumor volume reached about 1800 mm 3 The animals were killed by dislocating the cervical vertebrae at 1:00 pm and the samples were collected after the experiment. The tumor volume of mice was calculated as follows: tumor volume = length × width × width / 2. The weight of mice was recorded every other day during the experiment.

[0085] The results showed that the ginseng nanovesicle peptide vaccine had no significant effect on the weight of mice, and could significantly inhibit the growth of B16F10-OVA tumors, prolong the survival of mice, and increase the complete response rate of mice to tumors (i.e., the tumor did not grow at all). After vaccination, the infiltration of T cells in the B16F10-OVA tumors increased significantly, and the levels of inflammatory cytokines in the tumors increased significantly ( Fig.15 ).

[0086] Example 7: Evaluation of in vivo anti-tumor immune response of ginseng nanovesicle peptide vaccine (therapeutic administration)

[0087] C57BL / 6 mice were fed freely with sufficient food and water. The mice were randomly divided into a blank group (PBS, 200 μL / mouse), a ginseng nanovesicle group (200 μg / 200 μL / mouse), a peptide / protein vaccine (OVA 20 μg / 200 μL / mouse) and a ginseng nanovesicle peptide vaccine (calculated as OVA 20 μg / 200 μL / mouse) group, and 5×10 5 B16F10-OVA tumor cells (0.05 mL / mouse) were subcutaneously inoculated into the left abdomen of mice. The tumor size was recorded every day after tumor loading. When the tumor volume reached about 1800 mm 3 The animals were killed by cervical dislocation at 1:00 pm and samples were collected after the experiment. The vaccine was given on Day 10, 13, and 17 for treatment. The tumor volume of mice was calculated as follows: tumor volume = length × width × width / 2. The weight of mice was recorded every other day during the experiment.

[0088] like Fig.16 As shown, the results show that in addition to its significant efficacy in preventive (recurrent) tumor treatment, the ginseng nanovesicle peptide vaccine also has excellent therapeutic efficacy. After vaccination, the tumor volume of mice was significantly suppressed, and the overall survival of mice was prolonged. The number of mice with completely disappeared tumor volume was also significantly increased. More importantly, the infiltration of OVA-specific CTLs in non-regressed tumors increased significantly, indicating that the therapeutic efficacy of the vaccine can be further enhanced in combination with immune checkpoint inhibitors.

[0089] Example 8: Detection of main serum biochemical indicators after vaccination with ginseng nanovesicle peptide vaccine

[0090] The serum of the mice after vaccination was tested for serum biochemical indicators, and the results showed that there was no significant change in serum biochemical indicators. This indicates that the ginseng nanovesicle peptide vaccine has good biocompatibility and tissue and organ safety ( Fig.17 ).

[0091] It can be seen from the experimental results of the embodiment that the tumor vaccine prepared in this embodiment, namely the ginseng nanovesicle peptide vaccine, utilizes the characteristics of ginseng nanovesicles targeting lymph nodes, being specifically phagocytosed by migratory dendritic cells in the lymph nodes, and promoting the maturation of dendritic cells through TLR4, thereby increasing the presentation level of OVA antigen-specific MHC-I-antigen peptides. Ultimately, mature antigen-presenting cells activate cytotoxic T lymphocytes to form specific anti-tumor immune memory, which can effectively inhibit tumor recurrence and prolong the survival of mice.

[0092] The present invention has been described in detail above in conjunction with specific implementations and exemplary examples, but these descriptions cannot be understood as limiting the present invention. Those skilled in the art understand that, without departing from the spirit and scope of the present invention, a variety of equivalent substitutions, modifications or improvements may be made to the technical solution of the present invention and its implementation methods, all of which fall within the scope of the present invention. The scope of protection of the present invention shall be subject to the attached claims.

Claims

1. A method for preparing a tumor vaccine, characterized in that: The method comprises mixing Chinese herbal medicine nanocapsules with tumor antigens, and then encapsulating them to obtain a tumor vaccine; the Chinese herbal medicine in the Chinese herbal medicine nanocapsules is ginseng; the tumor antigen is a natural polypeptide or a recombinant fusion polypeptide; and the encapsulation method comprises: extruder extrusion method, low-intensity ultrasonic treatment, electroporation treatment, freeze-thaw encapsulation or pH gradient method.

2. A method for preparing a tumor vaccine, characterized in that: The method comprises mixing Chinese herbal medicine nanocapsules with tumor antigens, and then encapsulating the mixture to obtain a tumor vaccine; the Chinese herbal medicine in the Chinese herbal medicine nanocapsules is ginseng; the tumor antigen is a natural protein or a recombinant fusion protein; The encapsulation method includes: extrusion by an extruder, low-intensity ultrasonic treatment, electroporation, freeze-thaw encapsulation or pH gradient method.

3. The preparation method according to claim 1, characterized in that: The tumor antigens include tumor-associated antigens or tumor-specific antigens.

4. The preparation method according to claim 1, characterized in that: The mixing ratio of the Chinese herbal medicine nanocapsule and the tumor antigen is 10: (1-5) w / w.

5. The preparation method according to claim 1, characterized in that: The tumor antigens include pattern antigens.

6. The preparation method according to claim 1, characterized in that: The preparation method of the Chinese herbal medicine nanocapsule comprises: squeezing fresh Chinese herbal medicine to obtain Chinese herbal medicine juice, collecting supernatant after centrifugation, collecting precipitate after centrifugation of the supernatant, and obtaining the Chinese herbal medicine nanocapsule.

7. The preparation method according to claim 1, characterized in that: The extrusion method comprises: sucking the mixed liquid into a booster and repeatedly extruding it multiple times using an extrusion membrane; the low-intensity ultrasonic treatment comprises: placing the mixed liquid in an ice bath and treating it with 50-100 W ultrasonic waves for 10-15 min; the electroporation treatment comprises using an electroporator to repeatedly electroporate the mixed liquid multiple times; the freeze-thaw packaging comprises using liquid nitrogen to freeze-thaw the mixed liquid for a specific time and then restoring it to room temperature.

8. The tumor vaccine prepared by the preparation method according to any one of claims 1 or 3-7.

9. A drug for preventing or treating tumor diseases, characterized in that: It comprises the tumor vaccine according to claim 8, and pharmaceutically acceptable excipients.

10. The drug for preventing or treating tumor diseases according to claim 9, characterized in that: The auxiliary materials include excipients.

11. Use of the tumor vaccine according to claim 8 in the preparation of drugs for preventing or treating tumor diseases.

12. The use according to claim 11, characterized in that: Neoplastic diseases include melanoma, colorectal cancer, breast cancer, bladder cancer, lung cancer, liver cancer, pancreatic cancer, esophageal cancer, gastric cancer, cervical cancer, bile duct cancer or testicular cancer.

13. Use of ginseng nanovesicles in the preparation of a drug for preventing or treating tumor diseases as claimed in claim 9, characterized in that: Ginseng nanocapsules are used as vaccine delivery carriers, immune adjuvants or targeted preparations for the tumor disease prevention or treatment drugs.

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