Nano vaccine carrying transmembrane polypeptide and antigen polypeptide as well as preparation method and application of nano vaccine

Abstract

The invention relates to a nano vaccine carrying tumor specific antigen polypeptide, a preparation method and application thereof, and relates to the technical field of nano vaccines. The nano vaccine is prepared from a polylactic acid (PLA) derivative PLA-Porphyrin-Co < 2 + >, an antigen polypeptide, a vaccine adjuvant and lipid, and the cell-penetrating polypeptide and / or Me [beta] CD are / is included. PLA-Porphyrin-Co < 2 + > is a hydrophobic polymer, the lipid and the vaccine adjuvant are both amphiphilic polymers, stable nanoparticles are formed through the self-assembly characteristic of the two amphiphilic polymers and the hydrophobic polymer, and the antigen polypeptide and the transmembrane polypeptide contain a plurality of histidine tag polypeptides, so that the histidine tag polypeptides can be used for preparing the vaccine. The tag polypeptide can form a coordinate bond with divalent cobalt ions in hydrophobic macromolecules, so that the polypeptide can be automatically adsorbed on nano-particles, and a nano-vaccine is finally formed. The nano vaccine provided by the invention can penetrate through a lymph node barrier, efficiently infiltrate lymph node parenchyma, can efficiently deprive cell membrane cholesterol of lymph node DC cells, and has good prevention and treatment effects in tumors expressing specific antigens.

Description

Technical Field

[0001] This application relates to the technical field of nano-vaccines, and particularly to a nano-vaccine carrying a cell-penetrating peptide and an antigenic peptide, a preparation method thereof, and an application thereof. Background Art

[0002] The good curative effects of anti-tumor personalized vaccines based on tumor cell-associated antigens or specific antigens in pancreatic cancer and melanoma have promoted their rapid development in anti-tumor immunotherapy. The main factors determining the efficacy of vaccines include the efficient antigen presentation of dendritic cells (DCs) and the ability to domesticate naive T cells; currently, most studies focus on enhancing the anti-tumor immune efficacy of vaccines by activating TLR receptors, RIG1 receptors, or the cGAS-STING pathway, as well as by promoting the expression of the second co-stimulatory signal or the third stimulatory signal based on cytokines, but there are few reports on how to enhance the antigen presentation effect of DCs or the function of domesticating cellular immunity by regulating the metabolic pathways or metabolites of DCs.

[0003] The lysosomal degradation of antigens and subcapsular macrophages in lymph nodes may also hinder the efficient infiltration of nano-vaccines into lymph nodes and the cross-presentation of antigens in antigen-presenting cells, thereby limiting the efficacy of anti-tumor nano-vaccines. How to effectively promote the antigen cross-presentation in parenchymal DCs of lymph nodes is a prerequisite for nano-vaccines to exert anti-tumor effects. In recent years, most studies have mainly focused on promoting the cytoplasmic release of antigens by regulating the lysosomal pH value, disrupting the lysosomal membrane, or through the protonation effect, but the stability of lysosomes will also affect the activation and survival of DC cells. Therefore, how to promote the cytoplasmic release of antigens on the premise of protecting lysosomes from being damaged is worthy of exploration. Summary of the Invention

[0004] This application provides a nano-vaccine carrying an antigenic peptide, a preparation method thereof, and an application thereof, so as to solve the following technical problems: how to effectively infiltrate lymph nodes, promote the activation of antigen-presenting cells, and induce the activation of cellular immunity.

[0005] In a first aspect, an embodiment of this application provides a nano-vaccine carrying a tumor-specific antigenic peptide, and the nano-vaccine includes: poly(lactic acid) (PLA) derivative PLA-Porphyrin-Co 2+ , an antigenic peptide, a vaccine adjuvant, and a lipid, and further includes a cell-penetrating peptide and / or MeβCD.

[0006] Optionally, the mass ratio of the poly(lactic acid) (PLA) derivative PLA-Porphyrin-Co 2+ , the antigenic peptide, the vaccine adjuvant, the lipid, the cell-penetrating peptide, and the MeβCD is: 60:(0-60):3:(30-60):(0-60):(0-30).

[0007] Optionally, the antigen polypeptide is OT-I polypeptide and / or OT-II polypeptide. The amino acid sequence of the OT-I polypeptide is shown in SEQ ID NO.1 (SEQ ID NO.1: SIINFEKLHHHHHH), and the amino acid sequence of the OT-II polypeptide is shown in SEQ ID NO.2 (SEQ ID NO.2: ISQAVHAAHAEINEAGRHHHHHH).

[0008] Optionally, the transmembrane polypeptide is TAT polypeptide, and the amino acid sequence of the TAT polypeptide is shown in SEQ ID NO.3 (SEQ ID NO.3: YARKAARQARAHHHHHH).

[0009] Optionally, the adjuvant is one of CpG adjuvant, other agonists related to TLR receptors, aluminum adjuvant, and plant saponins.

[0010] Optionally, the other agonists related to TLR receptors include TLR7 agonist and TLR8 agonist. Among them, the TLR7 agonist can be Resiquimod (R848), Imiquimod (R857), or Vesatolimod (GS-9620), and the TLR8 agonist can be Motolimod or Selgantolimod (GS-9688).

[0011] Optionally, the aluminum adjuvant mainly includes aluminum hydroxide gel, aluminum phosphate, etc.

[0012] Optionally, the adjuvant is CpG-ODN, and the CpG-ODN is a CpG adjuvant modified with cholesteryl oleate.

[0013] Optionally, the lipid is PEG-phospholipid.

[0014] Optionally, the lipid is DSPE-PEG2000.

[0015] In a second aspect, an embodiment of the present application provides a preparation method of the nano-vaccine described in the first aspect, including the following steps:

[0016] Obtain polylactic acid PLA derivative PLA-Porphyrin-Co 2+ ;

[0017] Mix and dissolve the polylactic acid PLA derivative PLA-Porphyrin-Co 2+ , lipid, and vaccine adjuvant according to a mass ratio to obtain a first mixture;

[0018] Add the first mixture to a buffer solution to obtain a nano-carrier solution;

[0019] Mix the nano-carrier solution with a polypeptide to obtain a nano-vaccine.

[0020] Optionally, adding the first mixture to a buffer solution to obtain a nano-carrier solution specifically includes: adding the first mixture to a buffer solution, removing the organic solvent to obtain a second mixture;

[0021] Filter the second mixture to obtain a nano-carrier solution.

[0022] Optionally, the first mixture further includes MeβCD, and the mass ratio of MeβCD to PLA derivative PLA-Porphyrin-Co 2+ , lipid and vaccine adjuvant is (0-30):60:(30-60):3.

[0023] Optionally, the polypeptide includes an antigen polypeptide.

[0024] Optionally, the polypeptide includes a transmembrane polypeptide and an antigen polypeptide.

[0025] Optionally, the antigen polypeptide is OT-I polypeptide and / or OT-II polypeptide, the amino acid sequence of the OT-I polypeptide is as shown in SEQ ID NO.1 (SEQ ID NO.1: SIINFEKLHHHHHH), and the amino acid sequence of the OT-II polypeptide is as shown in SEQ ID NO.2 (SEQ ID NO.2: ISQAVHAAHAEINEAGRHHHHHH).

[0026] Optionally, the transmembrane polypeptide is TAT polypeptide, and the amino acid sequence of the TAT polypeptide is shown in SEQ ID NO.3 (SEQ ID NO.3: YARKAARQARAHHHHHH).

[0027] In a third aspect, an embodiment of the present application provides the use of the nano-vaccine described in the first aspect in the preparation of a preparation for preventing or treating tumors.

[0028] The above technical solutions provided by the embodiments of the present application have the following advantages compared with the prior art:

[0029] An embodiment of the present application provides a nano-vaccine carrying a tumor-specific antigen polypeptide, and the nano-vaccine includes: polylactic acid PLA derivative PLA-Porphyrin-Co 2+ , antigen polypeptide, vaccine adjuvant and lipid; it also includes a transmembrane polypeptide and / or MeβCD. PLA-Porphyrin-Co 2+The hydrophobic polymer, lipid, and vaccine adjuvant are all amphiphilic polymers. Through the self-assembly characteristics of the two amphiphilic polymers and the hydrophobic polymer, stable nanoparticles are formed. The antigen polypeptide and the transmembrane polypeptide contain multiple histidine-tagged polypeptides, which can form coordination bonds with divalent cobalt ions in the hydrophobic polymer. Through the affinity of the tagged polypeptide, the polypeptide containing the histidine tag can be automatically adsorbed onto the nanoparticles, and finally a nano-vaccine containing the antigen polypeptide is formed.

[0030] The nano-vaccine provided in this application can cross the lymph node barrier, efficiently infiltrate the lymph node parenchyma, and efficiently deprive the cell membrane cholesterol of lymph node DC cells, showing good preventive and therapeutic effects in tumors expressing specific antigens. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The accompanying drawings here are incorporated into the specification and form a part of this specification, showing embodiments consistent with this application, and are used together with the specification to explain the principles of this application.

[0032] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the following will briefly introduce the accompanying drawings required for use in the description of the embodiments or the prior art. Obviously, for those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0033] Figure 1 It is a schematic diagram of the synthesis routes and physicochemical properties of the four nano-vaccines provided in Experimental Example 1. Among them, Figure A is the synthesis schematic diagram, Figure B is the zeta potential distribution of different combinations of nano-vaccines, Figure C is the dynamic light scattering result, and Figure D is the transmission electron microscopy result;

[0034] Figure 2 It is the experimental results of the statistics on the targeting and activating DC cell characteristics of the four nano-vaccines provided in Experimental Example 2. Among them, Figure A is the statistics of the targeting ability of different combinations of nano-vaccines on DC by flow cytometry, and Figure B is the identification of the activating ability of different combinations of nano-vaccines on DC;

[0035] Figure 3 It is the identification result of the ability of the four nano-vaccines provided in Experimental Example 3 to regulate the cholesterol in the DC cell membrane. Among them, Figure A is the statistics of the cholesterol content on the surface of DC cells co-incubated with different combinations of nano-vaccines by flow cytometry, and Figure B is the identification of the cholesterol content on the surface of DC cells co-incubated with different combinations of nano-vaccines by laser confocal microscopy;

[0036] Figure 4Experimental results of the proliferation and activation characteristics of antigen-specific T cells induced by the four nano-vaccines provided in Experimental Example 4. In Figure A, the effect on T cell proliferation is shown when the number of DCs incubated with different combinations of nano-vaccines and the number of OT-I T cells are in a 1:1 ratio; in Figure B, the effect on T cell activation is shown when the number of DCs incubated with different combinations of nano-vaccines and the number of OT-I T cells are in a 1:1 ratio.

[0037] Figure 5 Experimental results of verifying the characteristics of the nano-vaccines provided in some embodiments of the present application to cross macrophages in vitro through the transwell experiment. In Figure A, it is a schematic diagram of the experimental design; in Figure B, it is a statistical chart of the intensity of nano-vaccine uptake by DC cells in the lower chamber counted by flow cytometry; in Figure C, it is a statistical chart of the intensity of nano-vaccine uptake by macrophages in the upper chamber counted by flow cytometry.

[0038] Figure 6 Experimental results of verifying the characteristics of the four nano-vaccines to efficiently target lymph node parenchymal DC cells in vivo in Experimental Example 6. In Figures A and B, the ability of different combinations of nano-vaccines to target CD11c-positive DC cells in the lymph node parenchyma is statistically analyzed; in Figures C and D, the ability of different combinations of nano-vaccines to target macrophages in the lymph node parenchyma is shown.

[0039] Figure 7 Experimental results of verifying the preventive effect of the nano-vaccine carrying OT-I and OT-II on B16-OVA tumors or the preventive effect of the nano-vaccine carrying Trp polypeptide on B16 tumors in Experimental Example 7. In Figure A, it is a schematic diagram of the experimental design; in Figure B, it is a statistical chart of the B16-OVA tumor growth curve; in Figure C, it is a statistical chart of the survival rate of B16-OVA mice; in Figure D, it is a statistical chart of the B16 tumor growth curve; in Figure E, it is a physical picture of the B16 tumor growth in different groups.

[0040] Figure 8 Experimental results of verifying the therapeutic effect of the nano-vaccine carrying Trp polypeptide on the lung metastasis of B16 tumors in Experimental Example 8. In Figure A, it is a schematic diagram of the experimental design; in Figure B, it is a physical picture of the lung tumor growth in different groups.

[0041] Figure 9 Schematic diagram of the nano-vaccine synthesis route and experimental design diagram provided in the present application. Detailed implementation manners

[0042] To make the objectives, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present application. Apparently, the described embodiments are only a part rather than all of the embodiments of the present application. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present application without creative efforts shall fall within the scope of protection of the present application.

[0043] The various embodiments of the present application may exist in the form of a range; it should be understood that the description in the form of a range is only for convenience and brevity and should not be construed as a rigid limitation on the scope of the present application; therefore, it should be considered that the described range description has specifically disclosed all possible sub-ranges and single values within that range; for example, it should be considered that the range description from 1 to 6 has specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as single numbers within that range, such as 1, 2, 3, 4, 5, and 6, and this applies regardless of the range; in addition, whenever a numerical range is indicated herein, it means including any cited number (fraction or integer) within the indicated range.

[0044] In this document, the term "including" and the like means "including but not limited to". Relative terms such as "first" and "second" are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. "And / or" describes the associated relationship of associated objects and indicates that three relationships may exist. For example, A and / or B may represent: A exists alone, A and B exist simultaneously, and B exists alone; where A and B may be singular or plural. "At least one" means one or more, and "a plurality" means two or more; "at least one kind", "at least one of the following items (pieces)" or similar expressions refer to any combination of these items, including any combination of single items (pieces) or plural items (pieces); for example, "at least one of a, b, or c", or, "at least one of a, b, and c" can both represent: a, b, c, a - b (i.e., a and b), a - c, b - c, or a - b - c, where a, b, and c can be single or plural respectively. The "parts representation method" such as parts by weight and parts by mass represents the proportional relationship between components. In the proportional relationships involved in this document, the parameters that need to be described by proportion should be understood as the antecedents of the proportion formula in the order of description, and the proportional numbers should be understood as the consequents of the proportion formula. For example, if the mass ratio of substance A, substance B, and substance C is 1:2:3, then substance A, substance B, and substance C should correspond one by one with the proportional numbers in the proportion formula in the order of description, that is, the mass of substance A: the mass of substance B: the mass of substance C = 1:2:3.

[0045] Unless otherwise specified, various raw materials, reagents, instruments, and equipment used in this article can be obtained through market purchases or can be prepared by existing methods.

[0046] In a first aspect, an embodiment of the present application provides a nano-vaccine carrying a tumor-specific antigen polypeptide, and the nano-vaccine includes: poly(lactic acid) PLA derivative PLA-Porphyrin-Co 2+ an antigen polypeptide, a vaccine adjuvant, and lipids, and also includes a cell-penetrating peptide and / or MeβCD.

[0047] In the above embodiment, PLA-Porphyrin-Co 2+ is the end product after the covalent connection of poly(lactic acid) PLA and a porphyrin ring chelating cobalt ions through esterification, and is a hydrophobic polymer. Lipids and vaccine adjuvants are both amphiphilic polymers. Through the self-assembly characteristics of the two amphiphilic polymers and the hydrophobic polymer, stable nanoparticles are formed. The antigen polypeptide and the cell-penetrating peptide contain multiple histidine-tagged polypeptides, and this tagged polypeptide can form a coordination bond with the divalent cobalt ions in the hydrophobic polymer. Through the affinity of the tagged polypeptide, the polypeptide containing the histidine tag can be automatically adsorbed on the nanoparticles, and finally a nano-vaccine containing the antigen polypeptide is formed. The nano-vaccine includes methyl-β-cyclodextrin (MeβCD). Methyl-β-cyclodextrin has a hydrophilic outer surface and a lipophilic central cavity, has relatively high surface activity, and can deprive cell membrane cholesterol by inserting into the cell membrane lipid raft. The cell-penetrating peptide can efficiently infiltrate the lymph node parenchyma, target and activate DC cells, and play an anti-tumor role by enhancing cellular immunity.

[0048] In the above embodiment, three nano-vaccines are included, and the schematic diagram of the preparation principle is as Figure 1 shown in A, which are respectively denoted as NPCM-O, NPC-OT, and NPCM-OT. Specifically:

[0049] 1) Nano-vaccine NPCM-OT: poly(lactic acid) PLA derivative PLA-Porphyrin-Co 2+ an antigen polypeptide, a vaccine adjuvant, lipids, a cell-penetrating peptide, and MeβCD.

[0050] 2) Nano-vaccine NPC-OT: poly(lactic acid) PLA derivative PLA-Porphyrin-Co 2+ an antigen polypeptide, a vaccine adjuvant, lipids, and a cell-penetrating peptide.

[0051] 3) Nano-vaccine NPCM-O: poly(lactic acid) PLA derivative PLA-Porphyrin-Co 2+ an antigen polypeptide, a vaccine adjuvant, lipids, and MeβCD.

[0052] As an alternative embodiment, the polylactic acid (PLA) derivative PLA-Porphyrin-Co 2+ The mass ratio of the antigen polypeptide, the vaccine adjuvant, the lipid, the cell-penetrating polypeptide, and the MeβCD is: 60:(0 - 60):3:(30 - 60):(0 - 60):(0 - 30).

[0053] In the above embodiment, the reason for controlling the mass ratio of the polylactic acid (PLA) derivative PLA-Porphyrin-Co 2+ , the antigen polypeptide, the vaccine adjuvant, the lipid, the cell-penetrating polypeptide, and the MeβCD to be 60:(0 - 60):3:(30 - 60):(0 - 60):(0 - 30) is that the maximum loading amount of the vaccine adjuvant is one-twentieth of that of PLA-Porphyrin-Co 2+ . To ensure the loading effect, the total mass of the cell-penetrating polypeptide and the antigen polypeptide needs to be the same as that of PLA-Porphyrin-Co 2+ , that is, the total amount of the cell-penetrating polypeptide and the antigen polypeptide is the same as that of PLA-Porphyrin-Co 2+ . However, the ratio of the cell-penetrating polypeptide to the antigen polypeptide can be adjusted; at the same time, the total content of the lipid and the MeβCD also needs to be the same as that of PLA-Porphyrin-Co 2+ , but the ratio between the two can be adjusted. Depending on the adjustment of different ratios, different targeting and therapeutic effects are achieved, and the optimal effect ratio is 60:30:3:30:30:30.

[0054] As an alternative embodiment, the antigen polypeptide is OT-I polypeptide and / or OT-II polypeptide. The amino acid sequence of the OT-I polypeptide is shown in SEQ ID NO.1, and the amino acid sequence of the OT-II polypeptide is shown in SEQ ID NO.2.

[0055] As an alternative embodiment, the cell-penetrating polypeptide is TAT polypeptide, and the amino acid sequence is shown in SEQ ID NO.3.

[0056] The TAT polypeptide is derived from a specific amino acid sequence of the trans-activator (Tat) protein of HIV-1. It is a polypeptide with cell-penetrating ability, and this penetrating ability mainly benefits from its cation-rich region, allowing it to interact with the negatively charged phospholipid layer on the cell surface, thereby promoting the entire fusion protein or the carried load to cross the cell membrane and enter the interior of the target cell.

[0057] As an alternative embodiment, the adjuvant is one of CpG adjuvant, other agonists related to TLR receptors, aluminum adjuvant, and plant saponins.

[0058] As an alternative embodiment, other agonists related to TLR receptors include TLR7 agonists and TLR8 agonists, where the TLR7 agonist can be Resiquimod (R848), Imiquimod (R857), or Vesatolimod (GS-9620), and the TLR8 agonist can be Motolimod or Selgantolimod (GS-9688).

[0059] As an alternative embodiment, the aluminum adjuvant mainly includes aluminum hydroxide gel, aluminum phosphate, etc.

[0060] As an alternative embodiment, the adjuvant is a CpG adjuvant. The CpG adjuvant is a synthetic oligodeoxynucleotide that can mimic exogenous DNA (such as bacterial DNA) to activate immune cells, thereby enhancing the intensity of the immune response.

[0061] As an alternative embodiment, the adjuvant is CpG-ODN, and the CpG-ODN is a CpG adjuvant modified with cholesteryl oleate.

[0062] In the above embodiment, the reason for using the CpG adjuvant modified with cholesteryl oleate is that CpG-ODN adds a cholesterol CO molecule to the CpG, which facilitates the loading of CpG into the nanocarrier; pure CpG is a hydrophilic molecule and cannot be loaded onto the carrier, while the addition of the CO molecule can promote the loading of CpG onto the nanocarrier.

[0063] As an alternative embodiment, the lipid is PEG-phospholipid.

[0064] As an alternative embodiment, the lipid is DSPE-PEG2000.

[0065] In the above embodiment, the reason for selecting DSPE-PEG2000 as the lipid is that DSPE-PEG2000 is a conjugate of DSPE (distearoylphosphatidylethanolamine) and PEG (polyethylene glycol), which has both hydrophilic and hydrophobic properties. This combination makes DSPE-PEG2000 a functionalized polyethylene glycol preparation that can be used for drug delivery, gene transfection, and vaccine delivery. Polyethylene glycolated phospholipids can significantly improve blood circulation time and stably encapsulate drugs, and can also be used for targeted drug delivery by modifying with target surface ligands such as antibodies and polypeptides. In addition, a molecular weight of 2000 is beneficial for forming nanoparticles.

[0066] As an alternative embodiment, in order to label the nano-vaccine, the nano-vaccine further comprises a lipophilic long-chain cyanine dye DiD, and the obtained nano-vaccine is denoted as DiD@NPCM-OT, DiD@NPC-OT and DiD@NPCM-O. In the nano-vaccine, the lipophilic long-chain cyanine dye DiD, poly(lactic acid) PLA derivative PLA-Porphyrin-Co 2+ The mass ratio of the antigen polypeptide, the vaccine adjuvant, the lipid, the cell-penetrating polypeptide, and the MeβCD is: (0 to 3):60:(0 to 60):3:(30 to 60):(0 to 60):(0 to 30).

[0067] In a second aspect, an embodiment of the present application provides a method for preparing the nano-vaccine according to the first aspect, comprising the following steps:

[0068] Obtain the poly(lactic acid) PLA derivative PLA-Porphyrin-Co 2+ ;

[0069] Mix and dissolve the poly(lactic acid) PLA derivative PLA-Porphyrin-Co 2+ , the lipid, and the vaccine adjuvant according to the mass ratio to obtain a first mixture;

[0070] Add the first mixture to a buffer solution to obtain a nano-carrier solution;

[0071] Mix the nano-carrier solution with the polypeptide to obtain the nano-vaccine.

[0072] In the above embodiment, the nano-vaccine is prepared by the rotary evaporation method. The method is simple and easy to operate, and can be used for large-scale production. First, the poly(lactic acid) PLA derivative PLA-Porphyrin-Co 2+ is prepared, and then the poly(lactic acid) PLA derivative PLA-Porphyrin-Co 2+, the lipid and the adjuvant are mixed and dissolved by mass ratio to fully mix the raw materials, obtaining a first mixture. Here, organic solvents can be used to fully dissolve the raw materials, such as organic solvents like chloroform, dichloromethane, absolute ethanol, etc. Any organic solvent that can fully dissolve the above raw materials is within the protection scope of this application. Adding the first mixture to the buffer may help adjust the pH value and ionic strength of the organic solution, thereby stabilizing the formation of the nanocarrier. The mixing process may need to promote the dispersion and stability of the nanocarrier through stirring, ultrasonic waves or other physical methods. As an alternative implementation, the first mixture can be added dropwise to the buffer, which helps to form more uniform nanoparticles. Finally, the antigen polypeptide is mixed with the nanocarrier. The antigen polypeptide contains multiple histidine-tagged polypeptides, and this tagged polypeptide can form a coordination bond with the divalent cobalt ions in the hydrophobic polymer. Through the affinity of the tagged polypeptide, the polypeptide containing the histidine tag can be automatically adsorbed onto the nanoparticles, and finally a nano-vaccine containing the antigen polypeptide is formed.

[0073] As an alternative implementation, the temperature of the buffer is 35°C to 45°C. Within this temperature range, it can ensure the uniform dispersion of the nanocarrier and can directly mix with the polypeptide to prevent protein denaturation. Exemplarily, the temperature of the buffer can be 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C or 45°C.

[0074] As an alternative implementation, adding the first mixture to the buffer to obtain the nanocarrier solution specifically includes: adding the first mixture to the buffer and removing the organic solvent to obtain a second mixture;

[0075] Filtering the second mixture to obtain the nanocarrier solution.

[0076] In the above implementation, after the first mixture is mixed with the buffer, the organic solvent needs to be removed to form nanoparticles; filtering the second mixture to filter out large-sized nanoparticles and leaving nanoparticles that meet the size requirements as the nanocarrier. The filtering method can be achieved by using an aqueous filter membrane with a set size, and this set size is usually 0.45 microns.

[0077] As an alternative implementation, the first mixture further includes MeβCD, and the mass ratio of MeβCD to PLA derivative PLA-Porphyrin-Co 2+ , lipid and vaccine adjuvant is (0 - 30):60:(30 - 60):3.

[0078] In the above-described embodiment, MeβCD is added to the first mixture. Since MeβCD has a hydrophilic outer surface and a lipophilic central cavity, it has relatively high surface activity. After being mixed with the PLA derivative PLA-Porphyrin-Co 2+ , lipids, and vaccine adjuvant, stable nanoparticles can be formed.

[0079] As an alternative embodiment, the buffer solution can be physiological saline.

[0080] As an alternative embodiment, the polypeptide includes an antigen polypeptide.

[0081] Mixing the nano-carrier solution with the antigen polypeptide can prepare a nano-vaccine. The types of antigen polypeptides can be antigen polypeptides related to tumors, including but not limited to OT-I polypeptide and OT-II polypeptide. The amino acid sequence of the OT-I polypeptide is shown as SEQ ID NO.1, and the amino acid sequence of the OT-II polypeptide is shown as SEQ ID NO.2.

[0082] As an alternative embodiment, the polypeptide includes a transmembrane polypeptide and an antigen polypeptide.

[0083] In the above-described embodiment, mixing the transmembrane polypeptide and the antigen polypeptide with the nano-carrier solution simultaneously can load the transmembrane polypeptide and the antigen polypeptide onto the nano-carrier at the same time. The obtained nano-vaccine has better penetrability, can cross the lymph node barrier, and efficiently infiltrate the lymph node parenchyma.

[0084] As an alternative embodiment, the mass ratio of the antigen polypeptide to the transmembrane polypeptide is 1:1.

[0085] As an alternative embodiment, the preparation method further includes: using an ultrafiltration method to remove free PLA-Porphyrin-Co 2+ , DSPE-PEG2000, MeβCD, CpG-ODN, and polypeptide to obtain a purer nano-vaccine.

[0086] As an alternative embodiment, the ultrafiltration is achieved using a 30KD ultrafiltration tube.

[0087] In a third aspect, an embodiment of the present application provides the use of the nano-vaccine described in the first aspect in the preparation of a tumor prevention or treatment preparation.

[0088] The nano-vaccine provided in the present application has good penetrability, can cross the lymph node barrier, and efficiently infiltrate the lymph node parenchyma; promotes the penetration of antigens through the lysosomal membrane and enhances the cross-presentation of antigens; efficiently deprives the cell membrane cholesterol of lymph node DC cells, targets and activates DC cells, and exerts an anti-tumor effect by enhancing cellular immunity. Therefore, it can be used in the preparation of a tumor prevention or treatment preparation.

[0089] The present application will be further described below in conjunction with specific embodiments. For the experimental methods without specific conditions indicated in the following embodiments, they are generally determined in accordance with national standards / industry standards; if there are no corresponding national standards / industry standards, they are carried out in accordance with general international standards, conventional conditions or the conditions recommended by the manufacturer.

[0090] Example 1

[0091] This example provides a nano-vaccine NPCM-OT carrying tumor-specific antigen polypeptides. The nano-vaccine includes a poly(lactic acid) (PLA) derivative, PLA-Porphyrin-Co 2+ , antigen polypeptides (OT-I polypeptide and OT-II polypeptide), CpG-ODN, DSPE-PEG2000, TAT polypeptide, and MeβCD, wherein the mass ratio of the poly(lactic acid) (PLA) derivative, PLA-Porphyrin-Co 2+ , antigen polypeptides, CpG-ODN, DSPE-PEG2000, TAT polypeptide, and MeβCD is: 60:30:3:30:30:20.

[0092] The preparation method includes the following steps:

[0093] S1. Covalently link poly(lactic acid) (PLA) with a porphyrin ring chelated with cobalt ions through esterification to obtain PLA-Porphyrin-Co 2+ ;

[0094] S2. Dissolve PLA-Porphyrin-Co 2+ , DSPE-PEG2000, MeβCD, and CpG-ODN in absolute ethanol according to the set mass ratio and mix to obtain a first mixture; the set mass ratio is 60:30:3:30:30:20.

[0095] S3. Drop the first mixture into physiological saline at 40°C and rotary evaporate the absolute ethanol to obtain a second mixture;

[0096] S4. Filter the large particles in the second mixture with a 0.45-μm aqueous filter membrane to obtain a clear nano-carrier solution;

[0097] S5. Add the antigen polypeptides and TAT polypeptide to the nano-carrier solution, and mix evenly to obtain the nano-vaccine NPCM-OT.

[0098] Example 2

[0099] This example provides a nano-vaccine NPCM-O carrying tumor-specific antigen polypeptides. The nano-vaccine NPCM-O includes a poly(lactic acid) (PLA) derivative, PLA-Porphyrin-Co 2+ , antigen polypeptides (OT-I polypeptide and OT-II polypeptide), CpG-ODN, DSPE-PEG2000, and MeβCD. The mass ratio of the poly(lactic acid) (PLA) derivative, PLA-Porphyrin-Co 2+ , antigen polypeptides, CpG-ODN, DSPE-PEG2000, and MeβCD is: 60:60:3:30:20.

[0100] The preparation method includes the following steps:

[0101] S1, covalently linking poly(lactic acid) (PLA) with a porphyrin ring chelated with cobalt ions through esterification to obtain PLA-Porphyrin-Co 2+ ;

[0102] S2, dissolving PLA-Porphyrin-Co 2+ , DSPE-PEG2000, MeβCD, and CpG-ODN in anhydrous ethanol according to the mass ratio and mixing them to obtain a first mixture;

[0103] S3, dropping the first mixture into physiological saline at 40 °C and rotary evaporating the anhydrous ethanol to obtain a second mixture;

[0104] S4, filtering the large particles in the second mixture using a 0.45-μm aqueous filter membrane to obtain a clear nano-carrier solution;

[0105] S5, adding the antigen polypeptides to the nano-carrier solution, mixing evenly to obtain the nano-vaccine NPCM-O.

[0106] Example 3

[0107] This example provides a nano-vaccine NPC-OT carrying tumor-specific antigen polypeptides. The nano-vaccine NPC-OT includes a poly(lactic acid) (PLA) derivative, PLA-Porphyrin-Co 2+ , antigen polypeptides (OT-I polypeptide and OT-II polypeptide), CpG-ODN, DSPE-PEG2000, and TAT polypeptide. The mass ratio of the poly(lactic acid) (PLA) derivative, PLA-Porphyrin-Co 2+ , antigen polypeptides, CpG-ODN, DSPE-PEG2000, and TAT polypeptide is: 60:30:3:30:30.

[0108] The preparation method includes the following steps:

[0109] S1. Covalently link polylactic acid (PLA) and porphyrin ring chelated with cobalt ions through esterification to obtain PLA-Porphyrin-Co 2+ ;

[0110] S2. Dissolve PLA-Porphyrin-Co 2+ , DSPE-PEG2000 and CpG-ODN in absolute ethanol according to the mass ratio and mix them to obtain the first mixture;

[0111] S3. Drop the first mixture into physiological saline at 40°C and rotary evaporate the absolute ethanol to obtain the second mixture;

[0112] S4. Filter the large particles in the second mixture with a 0.45-μm aqueous filter membrane to obtain a clear nano-carrier solution;

[0113] S5. Add antigen polypeptide and TAT polypeptide to the nano-carrier solution to obtain the nano-vaccine NPC-OT.

[0114] Experimental Example 1

[0115] This experimental example provides preparation methods for four nano-vaccines, namely NPC-O, NPCM-O, NPC-OT and NPCM-OT. As Figure 1 shown in A, it specifically includes the following steps:

[0116] 1) Dissolve 1 mg of PLA-Porphyrin-Co 2+ , 0.66 mg of DSPE-PEG2000, 0.33 mg of MeβCD and 0.05 mg of CpG-ODN in 500 μL of absolute ethanol solution and mix well to obtain the first mixture (when MeβCD is not added, 1 mg of DSPE-PEG2000 needs to be added);

[0117] 2) Gradually drop the above first mixture into 20 mL of physiological saline, control the temperature at 37°C, and rotary evaporate for 2 hours by adding a magnetic stirrer;

[0118] 3) Filter the above solution through a 0.45-μm aqueous filter membrane to remove large particle components and obtain a clear solution;

[0119] 4) Use a syringe to add 0.5 mg of TAT transmembrane peptide, 0.25 mg of OT-I polypeptide and 0.25 mg of OT-II polypeptide to the above clear solution respectively, mix well and seal, and place it at 4°C overnight (if TAT transmembrane peptide is not added, 0.5 mg of OT-I polypeptide and 0.5 mg of OT-II polypeptide need to be added);

[0120] 5) The next day, the sample was concentrated using an ultrafiltration tube (30KD, Merck), centrifuged at 2500 rpm at 4 °C to remove free polypeptides, MeβCD, and CpG-ODN, and the NPC-O, NPCM-O, NPC-OT, and NPCM-OT nano-vaccines were obtained.

[0121] In the above preparation method, due to the amphiphilicity of DSPE-PEG2000 and MeβCD, they can be used to encapsulate PLA-Porphyrin-Co 2+ , to obtain a nanocarrier; then the tumor antigen polypeptides (OT-I polypeptide and OT-II polypeptide) and the cell-penetrating polypeptide (TAT cell-penetrating peptide) are coordinately bonded to cobalt ions through histidine tags and loaded on the surface of the nanocarrier to form a nano-vaccine.

[0122] The average particle size and zeta potential of the NPC-O, NPCM-O, NPC-OT, and NPCM-OT nano-vaccines were measured respectively, and detected using a nanoparticle size and potential analyzer Zetasizer Nano-ZS90 and a transmission electron microscope. The results are shown in Figure 1 B, Figure 1 C, and Figure 1 D. It can be seen from the figure that the average particle sizes of the four different NPC-O, NPCM-O, NPC-OT, and NPCM-OT nano-vaccines are all 132 ± 12.3 nm, and the zeta potential is mainly distributed between -20 mV and -15 mV. There are no significant differences in the average particle size and zeta potential.

[0123] Experimental Example 2

[0124] This experimental example was used to determine the characteristics of the four nano-vaccines prepared in the experimental example in targeting and activating DC cells, specifically including:

[0125] 1) Obtaining DC cells: Take a 6-week-old C57BL / 6 mouse and sacrifice it by cervical dislocation. Immerse it in 75% alcohol for 3 - 5 minutes. Under sterile operating conditions, use surgically sterile instruments to dissect the tibia and femur. After cutting open the bone marrow cavity, repeatedly rinse with sterile PBS until all bone marrow cells are flushed out to obtain a cell suspension. Filter the collected cell suspension through a 70μm filter into a 15mL centrifuge tube, centrifuge at 500g for 5 minutes at 4°C, discard the supernatant, add 1mL ACK red blood cell lysate to resuspend the cells. Incubate the resuspended cell suspension at room temperature for 1 minute, then add 10mL PBS to terminate the reaction to obtain lysed cells. Centrifuge the lysed cells at 500g for 5 minutes at 4°C, discard the supernatant, wash once with PBS, and plate-culture the obtained cell pellet with the prepared DC medium. The DC medium contains 10% heat-inactivated fetal bovine serum + 1% double-antibody RPMI 1640 medium + 20ng / mL GM-CSF.

[0126] Perform semi-medium replacement on the cells on the 3rd and 5th days of culture. Culture until the 6th day to obtain DC cells for subsequent experiments.

[0127] 2) Obtaining DC cells with different treatment methods: Divide the DC cells obtained in step 1) into 11 portions. Among them, 2 portions are used as negative controls without any treatment; add NPC-O nano-vaccine, NPCM-O nano-vaccine, NPC-OT nano-vaccine, and NPCM-OT nano-vaccine with the same DiD dye concentration to 4 portions of DC cells respectively. After incubating for 3 hours, obtain DC cells after the first treatment; add 1μg / mL lipopolysaccharide LPS and NPC-O nano-vaccine, NPCM-O nano-vaccine, NPC-OT nano-vaccine, and NPCM-OT nano-vaccine with equal absorbance to 5 portions of DC cells respectively. After incubating for 24 hours, obtain DC cells after the second treatment;

[0128] Among them, the preparation method of NPC-O nano-vaccine, NPCM-O nano-vaccine, NPC-OT nano-vaccine, and NPCM-OT nano-vaccine with the same DiD dye concentration includes the following steps:

[0129] Dissolve 0.05mg of lipophilic long-chain cyanine dye, 1mg of PLA-Porphyrin-Co 2+ , 0.66mg of DSPE-PEG2000, 0.33mg of MeβCD, and 0.05mg of CpG-ODN in 500μL of absolute ethanol solution, and mix well to obtain a first mixture (when MeβCD is not added, 1mg of DSPE-PEG2000 needs to be added);

[0130] The above first mixture was added dropwise to 20 mL of physiological saline, the temperature was controlled at 37 °C, and it was rotary evaporated for 2 hours by adding a magnetic stirrer;

[0131] The above solution was filtered through a 0.45 μm aqueous filter membrane to remove large particle components and obtain a clear solution;

[0132] Using a syringe, 0.5 mg of TAT cell-penetrating peptide, 0.25 mg of OT-I polypeptide and 0.25 mg of OT-II polypeptide were respectively added to the above clear solution. After mixing, it was sealed and placed at 4 °C overnight (if TAT cell-penetrating peptide is not added, 0.5 mg of OT-I polypeptide and 0.5 mg of OT-II polypeptide need to be added);

[0133] 5) The next day, an ultrafiltration tube (30KD, Merck) was used to concentrate the sample, and it was centrifuged at 2500 rpm at 4 °C to remove free polypeptides, MeβCD and CpG-ODN, thus obtaining NPC-O nano-vaccine, NPCM-O nano-vaccine, NPC-OT nano-vaccine and NPCM-OT nano-vaccine containing the same DiD dye concentration.

[0134] 3) Characterization test of targeting and activating DC cells: The first-treated DC cells and the second-treated DC cells were washed twice with phosphate buffered saline (PBS) respectively, and flow cytometry was used to identify the fluorescence intensity of the DC cells in the first-treated DC cells and the negative control group. The results are as Figure 2 shown in A; Flow cytometry combined with CD86 and CD80 antibodies was used to count the proportion of activated DCs in the second-treated DC cells and the DC cells in the negative control group. The results are as Figure 2 shown in B.

[0135] 4) Result analysis: As can be seen from Figure 2 A, compared with the negative control, the four groups of DC cells after the first treatment all had a certain fluorescence intensity, and the fluorescence intensity of the DC cells treated with NPCM-OT nano-vaccine was much higher than that of other groups. It can be seen that the four nano-vaccines all entered the interior of DC cells, indicating that the four nano-vaccines all had a certain targeting ability to DC cells, and the targeting ability of NPCM-OT nano-vaccine was the strongest. Figure 2 The CD80 and CD86 double-positive DC cells in B are activated DC cells. As can be seen from Figure 2 B, compared with the negative control, the four groups of DC cells after the second treatment all had a certain degree of activation, but the ability of NPCM-OT to activate DC cells was the strongest.

[0136] Therefore, from Figure 2It can be seen that compared with NPC-O, NPCM-O, and NPC-OT, the NPCM-OT nano-vaccine loaded with both MeβCD and TAT cell-penetrating peptide has the most efficient DC targeting ability and induces a higher frequency of CD80 + , CD86 + mature DC cells. The NPCM-O nano-vaccine loaded only with MeβCD and the NPC-OT nano-vaccine loaded only with TAT cell-penetrating peptide have relatively strong DC targeting ability and DC cell ability, while the NPC-O nano-vaccine without MeβCD and TAT cell-penetrating peptide has the worst DC targeting ability and DC cell ability.

[0137] Experimental Example 3

[0138] In this experimental example, the ability of four different nano-vaccines to regulate cholesterol in the DC cell membrane was identified, specifically including:

[0139] 1) Obtain the DC cells obtained in step 1) of Experimental Example 2, divide them into 7 portions. Add 5 mM cholesterol to one portion of the DC cells, and add 5 mM MeβCD to another portion of the DC cells. Incubate these two portions for 2 hours. Use one portion of the DC cells as a negative control and add an equal volume of physiological saline. Add the NPC-O nano-vaccine, NPCM-O nano-vaccine, NPC-OT nano-vaccine, and NPCM-OT nano-vaccine with equal absorbance to the other four portions of the DC cells respectively, incubate at 37 °C for 24 h, and take samples at 3 h, 6 h, 12 h, and 24 h respectively. Then co-incubate the sampled DC cells with 1640 complete medium containing 10 μg / mL Flipin-3 dye for 30 minutes. After washing twice with PBS, identify and test the cholesterol content on the surface of the DC cells by flow cytometry. The results are as Figure 3 shown in

[0140] A. Figure 3 2) Co-incubate all 7 portions of the DC cells with 1640 complete medium containing 10 μg / mL Flipin-3 dye and 10 μg / mL PKH26 dye for 30 minutes, then wash twice with PBS, capture the fluorescence intensity of Flipin-3 on the surface of the processed DC cell membrane by confocal microscopy. The excitation wavelength used is 405 nm, and the receiving band is 440 - 460 nm. The obtained images are as shown in

[0141] Figure 3 3) From Figure 3As can be seen from Figure B, for DC cells treated with different nano-vaccines, the longer the culture time, the weaker the fluorescence intensity on the cell membrane. After culturing DC cells treated with NPCM-OT for 24 hours, the largest decrease in fluorescence on the cell membrane was obtained. Among them, the DC cells incubated with cholesterol had the strongest surface fluorescence intensity and served as the positive control group. The DC cells incubated with 5 mM MeβCD had the weakest surface fluorescence intensity and served as the negative control group. The DC cells incubated with PBS had the surface fluorescence intensity as the parallel control group. Compared with these groups, we concluded that the cholesterol on the cell membranes of DC cells treated with the four nano-vaccines was all stripped, and the amount of cholesterol stripped from the cell membranes of DC cells treated with NPCM-OT was the largest.

[0142] Experimental Example 4

[0143] This experimental example was used to determine the proliferation and activation characteristics of antigen-specific T cells induced by four different nano-vaccines in vitro, specifically including:

[0144] 1) Obtain OT-I antigen-specific T cells stained with CFSE proliferation dye: Based on CD8 + T cell magnetic beads (MojoSort TM Mouse CD8a Selection Kit, Biolegend) were used to sort CD8 + T cells in the spleens of OT-1 specific TCR T cell mice (C57BL / 6-Tg(TcraTcrb)1100Mjb / J from Jackson labratory) at 6-8 weeks old. The OT-1 T cells were stained with 5 μM CFSE fluorescent dye (Beyotime) in serum-free 1640 medium for 15 minutes, and the staining was terminated with 1640 medium containing 10% FBS. The obtained T cells were washed twice with PBS.

[0145] 2) Obtain the DC cells obtained in step 1) of Example 2 and divide them into 5 parts. Four of them were co-incubated with NPC-O, NPCM-O, NPC-OT, and NPCM-OT for 24 hours respectively. After washing twice with PBS, the treated DC cells and the CFSE-stained OT-1 specific CD 8+ T cells obtained in step 1) were co-incubated in 1640 complete medium at a cell number ratio of 1:1 for 48 hours. Then, flow cytometry was used to detect the ratio of T cell proliferation and activation, and the results are as Figure 4 shown.

[0146] 3) Through the ratio of T cell proliferation ( Figure 4 A) and the ratio of T cell activation ( Figure 4B) Reflecting the strength of the antigen presentation ability of DC vaccines, as can be seen from Figure 4 Among them, it can be seen that the antigen presentation abilities of DC cells treated with the four nano-vaccines have all been improved. Among them, the DC cells treated with the NPCM-OT nano-vaccine promote the proliferation of antigen-specific T cells best and effectively promote the activation of antigen-specific T cells, showing significant differences compared with the treatment results of other combined nano-vaccines.

[0147] Experimental Example 5

[0148] This experimental example uses the Transwell experiment to verify the characteristics of the four nano-vaccines crossing macrophages in vitro, specifically including:

[0149] 1) Replace the tumor antigen polypeptide added in Experimental Example 1 with a tumor antigen polypeptide containing the TRAMA dye for real-time tracking of the distribution of the antigen polypeptide. At the same time, add the DiD dye to the nano-vaccines such as NPC-O, NPCM-O, NPC-OT, and NPCM-OT. The nano-vaccines in Experimental Example 1 are added and synthesized according to the mass ratio of DiD, TAT polypeptide, PLA derivative, phospholipid, CpG-ODN, and tumor antigen polypeptide of 3:30:60:60:3:30 to obtain NPC-O, NPCM-O, NPC-OT, and NPCM-OT nano-vaccines with equal absorbance carrying DiD fluorescence.

[0150] 2) Obtain a 24-well plate with an upper chamber gap of 0.8 μm. Seed 100,000 Raw264.7 macrophages in the upper chamber and 100,000 DC2.4 cells in the lower chamber, and then culture them with 1640 complete medium for 48 hours; add the NPC-O, NPCM-O, NPC-OT, and NPCM-OT nano-vaccines with equal absorbance carrying DiD fluorescence obtained in step 1) to the upper chamber medium, and then co-incubate the upper chamber medium and the lower chamber medium for 24 hours, as shown in Figure 5 A; Take out the DC2.4 cells in the lower chamber and use flow cytometry to detect the fluorescence of the DiD dye and the TRAMA dye. The results are shown in Figure 5 B and Figure 5 C.

[0151] 3) As can be seen from Figure 5 B, the fluorescence intensity of the DiD dye is detected in the DC cells in the lower chamber. It can be seen that all four nano-vaccines have the effect of crossing macrophages. Among them, the effect of NPC-OT is significantly stronger than that of NPC-O, and the effect of NPCM-OT is the strongest, indicating that the transmembrane peptide TAT can efficiently promote the effect of nano-vaccines crossing macrophages, and combined with MeβCD can further enhance the penetration effect of nano-vaccines; from Figure 5As can be seen from Figure B, the DiD fluorescence carried by DC cells was enhanced synchronously with the TRAMA fluorescence, and the enhancement was most obvious in the NPCM-OT group. Compared with other groups, there were significant differences, which proved that the NPCM-OT nano-vaccine could still maintain its integrity after crossing the macrophage layer, while other nano-vaccines were damaged after crossing the macrophage layer.

[0152] Experimental Example 6

[0153] This experimental example was used to determine the characteristics of four different nano-vaccines for highly targeting DC cells in the lymph node parenchyma, specifically including:

[0154] 1) Obtain the four nano-vaccines loaded with DiD dye and TRAMA-labeled tumor antigen polypeptides simultaneously obtained in step 1) of Experimental Example 5; select C57 / BL6J mice at 6-8 weeks of age and divide them into five groups, including four experimental groups and one control group. The mice in the experimental groups were injected with four nano-vaccines with equal absorbance at the root of the tail, and the mice in the control group were injected with an equal amount of normal saline. After 24 hours of injection, the lymph nodes of each group of mice were collected, and single-cell suspensions of lymph nodes were obtained by grinding and filtering respectively.

[0155] 2) Use antibodies such as CD45, CD11b, CD11c, and F4 / 80 in combination with flow cytometry to label and gate the single-cell suspensions of lymph nodes in each group to distinguish macrophages and DC cells. In addition, ZoomNIR dye needs to be stained to distinguish live and dead cells. All relevant flow antibodies were purchased from Biolegend Biotechnology Co., Ltd. By statistically analyzing the fluorescence intensities of DiD dye and TRAMA dye in DC cells and macrophages in the single-cell suspensions of lymph nodes, the targeting characteristics of the four nano-vaccines to these cells were judged. The relevant results are shown as Figure 6 shown.

[0156] 3) Figure 6 The flow fluorescence intensity distribution diagrams A and C of the results respectively show that the fluorescence intensities of DiD dye and TRAMA dye carried by nano-vaccines such as NPC-O, NPCM-O, NPC-OT, and NPCM-OT in DC cells ( Figure 6 A) and macrophages ( Figure 6 C) were enhanced synchronously. At the same time, the statistical result diagrams of the fluorescence intensities of DC cells and macrophages in each group also show that the fluorescence intensities of DiD dye and TRAMA dye carried by nano-vaccines such as NPC-O, NPCM-O, NPC-OT, and NPCM-OT in DC cells ( Figure 6 B) and macrophages ( Figure 6The synchronous enhancement in (D) demonstrated the in vivo stability of NPC-O, NPCM-O, NPC-OT, and NPCM-OT nano-vaccines; in addition, NPCM-OT showed the most efficient targeting effect on macrophages and DC cells, with significant differences from other groups.

[0157] Experimental Example 7

[0158] This experimental example was used to verify the in vivo preventive characteristics of four different nano-vaccines against B16-OVA and B16 growth, specifically including:

[0159] 1) Five groups of 6-8-week-old C57 mice were randomly obtained, with one group as the control group and the other four groups as experimental groups; the nano-vaccines such as NPC-O, NPCM-O, NPC-OT, and NPCM-OT obtained in Experimental Example 1 were used to immunize the C57 mice in the four experimental groups by tail base injection, immunizing twice in total, with a one-week interval between the two times. The relevant experimental flow chart is as shown in Figure 7 A. The control group was injected with an equal amount of normal saline each time;

[0160] 2) On the 7th day after the second vaccination, the five groups of mice were respectively subcutaneously injected with B16 cells expressing OVA antigen (immunizing OT-I and OT-II polypeptides) or pure B16 cells (immunizing Trp polypeptide), and the injection dose was 2×10 6 cells / mouse. On the 6th day after tumor inoculation, the tumor sizes of different groups were statistically analyzed to evaluate the in vivo tumor growth of B16-OVA or B16. The results are as shown in Figure 7 B and Figure 7 D. At the same time, the survival rates of B16-OVA mice were statistically analyzed, and the results are as shown in Figure 7 C. The physical pictures of the B16 tumor sizes of different treatment groups on the 18th day after tumor inoculation are as shown in Figure 7 E.

[0161] 3) It can be seen from Figure 7 that compared with the control group, all four nano-vaccines had a certain effect of inhibiting tumor growth, and NPCM-OT had the best inhibitory effect and the longest survival time of mice; the preventive effects of NPCM-O and NPC-OT against B16-OVA or B16 were similar, without significant differences, but compared with the NPC-O group, these two had a better effect of inhibiting tumor growth; therefore, the nano-vaccine NPCM-OT carrying both the cell-penetrating peptide and MeβCD had the best preventive effect against B16-OVA or B16 growth, and the efficiency of preventing tumor growth reached 90%, and this preventive effect was independent of the selected antigen and was directly related to the vaccine carrier itself.

[0162] Experimental Example 8

[0163] This experimental example is used to compare the characteristics of four different nano-vaccines in the in-vivo treatment of lung metastasis of B16 tumor cells, specifically including:

[0164] 1) C57 mice aged 6 - 8 weeks were selected and 500,000 B16 tumor cells were injected through the tail vein. And on the 5th day after the injection of tumor cells, they were divided into 7 groups. One group was not treated at all and served as the negative control, and an equal amount of physiological saline was injected; one group was injected with PD-1 antibody through the tail root and served as the positive control. The other 5 groups were immunized through the tail root with NPC-O, NPCM-O, NPC-OT, NPCM-OT nano-vaccines and the mixture of NPCM-OT nano-vaccine and PD-1 antibody with the same absorbance as the PD-1 antibody in the positive control group. Then, on the 20th day after tumor injection, the lungs of the 7 groups of mice were dissected to observe the growth of B16 cell lung metastasis. The schematic diagram of the experimental procedure is as Figure 8 shown in A.

[0165] 2) The physical objects of lung metastasis of mice in different groups were obtained as Figure 8 shown in B. It can be seen from the figure that in the negative control group without immunization treatment and the positive control group immunized only with PD-1 antibody, the lung tumors were huge and had caused the death of the mice. While in the mice immunized with the four nano-vaccines, the volume of lung tumors was significantly smaller than that in the negative control group, indicating that the four nano-vaccines all had the effect of inhibiting lung metastasis. And compared with other nano-vaccines, NPCM-OT had the best effect of inhibiting the formation of lung metastasis.

[0166] In summary, the nano-vaccine provided by this application has at least the following advantages:

[0167] 1) Good penetrability: It can penetrate the lymph node barrier and efficiently infiltrate the lymph node parenchyma; 2) Good targeting: It can efficiently target dendritic cells (DCs) in the lymph node parenchyma; 3) Strong lysosomal escape ability: It can promote the penetration of antigens through the lysosomal membrane and enhance the cross-presentation of antigens; 4) Strong cholesterol deprivation ability: It can efficiently deprive the cell membrane cholesterol of lymph node DCs; 5) Excellent physicochemical properties: The average particle size of the nano-vaccine measured by dynamic light scattering method and transmission electron microscopy is about 120 nm; 6) Good biocompatibility: The raw materials used to prepare this nano-liposome have been used in clinical or clinical trials respectively, with good biocompatibility; 7) Simple preparation process and convenient for large-scale production; 8) Antigen loading rate is greater than 80%; 9) Good prevention and treatment effects: It has good prevention and treatment effects in tumors expressing specific antigens. The nano-vaccine carrying tumor antigens and MeβCD can efficiently infiltrate the lymph node parenchyma using cell-penetrating peptides, target and activate DCs, and play an anti-tumor role by enhancing cellular immunity; 10) Low toxicity and side effects: For the nano-vaccine carrying MeβCD and cell-penetrating peptides, all of its materials are common in clinical applications. The results of animal experimental studies show that the prevention group and treatment group of the nano-vaccine carrying MeβCD and cell-penetrating peptides can significantly inhibit the occurrence and development of tumors and promote the recovery of the body weight of mice; 11) Function can be extended: The nano-vaccine carrier carrying MeβCD and cell-penetrating peptides can present different tumor antigens through the carried polypeptides. At the same time, relevant dye molecules can be loaded in the core for imaging, or other relevant drugs can be co-loaded to achieve the purpose of synergistic treatment, realizing the synergistic targeting or synergistic treatment effect of diseases.

[0168] The above are only specific embodiments of the present application, enabling those skilled in the art to understand or implement the present application. Various modifications to these embodiments will be obvious to those skilled in the art. The general principles defined in the present application can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to these embodiments shown in the present application, but will conform to the widest scope consistent with the principles and novel features claimed in the present application.

Claims

1. A nano vaccine carrying a tumor-specific antigen polypeptide, the nano vaccine comprising: PLA-Porphyrin-Co 2+ , antigenic polypeptides, vaccine adjuvants and lipids, and also include membrane-penetrating polypeptides and / or MeβCD.

2. The nanovaccine according to claim 1, characterized in that The PLA derivative PLA-Porphyrin-Co 2+ The mass ratio of the antigen polypeptide, the vaccine adjuvant, the lipid, the membrane-penetrating polypeptide and the MeβCD is: 60:(0-60):3:(30-60):(0-60):(0-30).

3. The nanovaccine according to claim 1 or 2, characterized in that The antigenic polypeptide is an OT-I polypeptide and / or an OT-II polypeptide. The amino acid sequence of the OT-I polypeptide is shown in SEQ ID NO.1, and the amino acid sequence of the OT-II polypeptide is shown in SEQ ID NO.

2.

4. The nanovaccine according to claim 1, characterized in that The membrane-penetrating polypeptide is a TAT polypeptide, and the amino acid sequence of the TAT polypeptide is shown in SEQ ID NO.

3.

5. The nanovaccine according to claim 1, characterized in that The adjuvant is one of CpG adjuvant, other TLR receptor-related agonists, aluminum adjuvant and plant saponin; and / or the lipid is PEG-phospholipid.

6. A method for preparing the nanovaccine according to any one of claims 1 to 5, comprising the following steps: Obtained PLA-Porphyrin-Co 2+ ; The PLA derivative PLA-Porphyrin-Co 2+ , the lipid and the vaccine adjuvant are mixed and dissolved according to a mass ratio to obtain a first mixture; adding the first mixture into a buffer to obtain a nanocarrier solution; The nanocarrier solution is mixed with the polypeptide to obtain a nanovaccine.

7. The preparation method according to claim 6, wherein the step of adding the first mixture to a buffer to obtain a nanocarrier solution specifically comprises: adding the first mixture to a buffer solution and removing the organic solvent to obtain a second mixture; The second mixture is filtered to obtain a nanocarrier solution.

8. The preparation method according to claim 6 or 7, characterized in that: The first mixture also includes MeβCD, and the MeβCD is mixed with PLA derivative PLA-Porphyrin-Co 2+ The mass ratio of lipid and vaccine adjuvant is (0~30):60:(30~60):

3.

9. The preparation method according to claim 6, characterized in that: The polypeptide comprises an antigenic polypeptide; or, The polypeptides include membrane-penetrating polypeptides and antigenic polypeptides.

10. Use of the nanovaccine according to any one of claims 1 to 5 in the preparation of a preparation for preventing or treating tumors.

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