CN113171450A - Construction and application of nano-carrier for regulating adaptive cell and humoral immunity - Google Patents

Construction and application of nano-carrier for regulating adaptive cell and humoral immunity Download PDF

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CN113171450A
CN113171450A CN202110425658.7A CN202110425658A CN113171450A CN 113171450 A CN113171450 A CN 113171450A CN 202110425658 A CN202110425658 A CN 202110425658A CN 113171450 A CN113171450 A CN 113171450A
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antigen
endoplasmic reticulum
nano
carrier
lipid
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游剑
施莹莹
李青坡
姜新东
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Zhejiang University ZJU
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Zhejiang University ZJU
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Abstract

The invention provides construction and application of a nano-carrier for regulating and controlling adaptive cell and humoral immunity of an organism. The nano carrier of the invention modifies micromolecules or polypeptides with endoplasmic reticulum tropism on the surface of the nano particle loaded with the antigen, and changes the total intake amount, the transport path and the processing and presenting mode of the exogenous antigen in dendritic cells, thereby regulating and controlling the specific cellular immunity and humoral immunity of the body antigen. The nano-carrier can be a cationic drug-carrying system such as nano-emulsion, liposome or lipid nanoparticle, and the antigen can relate to specific antigen protein/polypeptide of tumor, virus or plasmodium. The nano drug-loaded system mobilizes the specific immune response of resisting tumor, virus or parasite infection by improving the adaptive immunity of organisms, particularly the participation degree of cellular immunity, and has the characteristics of novel scheme, simple preparation and wide application range.

Description

Construction and application of nano-carrier for regulating adaptive cell and humoral immunity
Technical Field
The invention belongs to the field of pharmacy, relates to a nano drug delivery system capable of sensitizing protein/polypeptide subunit vaccine efficacy and regulating adaptive cell and humoral immunity of an organism, and particularly relates to construction of a nano carrier capable of regulating adaptive cell and humoral immunity and application of the nano carrier in preparation of anti-tumor, anti-virus and anti-parasitic infection drugs.
Background
The development of human society is seriously stricken by tumors, viruses and infection for many times, and the emergence of vaccines is an important turning point for artificially and actively coping with the invasion of pathogenic substances, and has the advantages of high controllability, easy popularization, capability of repeatedly inoculating and enhancing specific immunological memory so as to provide long-term protection and the like. Protein/polypeptide-based subunit vaccines are representative of traditional immunization techniques and are one of the mainstream vaccine types in the present day due to safety, production cost, and the like. Through screening the antigen epitope specific to the pathogen, purifying or constructing protein/polypeptide containing the epitope information, and then selecting a proper delivery carrier and a proper administration mode, single or interval multiple vaccine immunization is carried out on potentially susceptible people (preventive vaccines) or individuals (therapeutic vaccines) affected by the pathogen, so that Antigen Presenting Cells (APCs) in vivo are activated, specific T/B lymphocyte response is started, the synergistic mediation of cellular and humoral immunity is induced, and the recognition, killing and memory of the pathogen are amplified.
Dendritic Cells (DCs) are APCs with the strongest functions of the organism and have the double potentials of inducing adaptive cellular immunity and humoral immunity of the organism. Antigens undergo cleavage processing within DCs followed by assembly with major histocompatibility complex Molecules (MHC), forming antigenic peptide-MHC class I or class II molecule complexes (p-MHC I or p-MHC II). Among them, endogenous antigens (located in the cytoplasm, including viral proteins synthesized in virus-infected cells, some intracellular self-antigens, etc.) are usually taken into the MHC class I presentation pathway, forming p-MHC I and CD8+Specific recognition by T cell surface receptor (TCR), thereby inducing CD8+T cell activation, promotion of Cytotoxic T Lymphocyte (CTLs) effector differentiation and target cell killing, and initiation of cellular immunity; exogenous antigens (obtained by phagocytosis, including phagocytosed cells, bacterial or protein antigens, etc.) often form p-MHC II, the latter in combination with CD4+TCR binding of T cells, inducing helper CD4+Differentiation of T cells (Th), promotion of B cell activation, plasma cell-like differentiation and production of specific neutralizing antibodies, elicitation of humoral immunity. Cellular and humoral immunity complement each other in an organism, and the effectiveness and the durability of immune response are determined by the cooperation of the cellular and humoral immunity.
At present, the application of protein/polypeptide antigens is mainly limited by the following: 1) exempt fromLow immunogenicity, often requiring adjuvant assistance; 2) the molecular weight is large, the electronegativity is weak, and the absorption by APCs is less; 3) as an exogenous "isohexide" component, after internalization by APCs, it is mostly presented in the MHC class II pathway to induce humoral immunity, and is dependent on MHC I, CD8+T cell-dominated mobilization of cellular immunity is insufficient. The lack of cellular immunity seriously affects the exertion of the body's antiviral, antitumor and anti-infectious immunity. E.g. CD8+Depletion of T cells attenuates photodynamic-photothermal therapy-induced anti-tumor effects nat]And the protective effect of the recombinant adenovirus vector vaccine on Ebola virus infection is limited [ nat. Med.,2011,17:1128-]Promotes the reactivation and replication of latent HIV or HSV virus induced by interleukin 15 superagonist N-803 [ Nature,2020,578: 154-159-]. The synergistic mobilization of cellular and humoral immunity is realized by improving cellular immune response of an organism, and the resistance of the organism to the invasion of pathogens can be enhanced. E.g., Thakur et al [ J Immunother, 2011,34(5):457-]By combining granulocyte-macrophage colony stimulating factor (GM-CSF) to induce systemic cellular and humoral immune responses, the anti-tumor effect of the percutaneous cryotherapy is enhanced; cao et al [ nat. Commun.,2018,9:3695]Non-invasive radio frequency is introduced into a subcutaneous vaccine of a protein antigen to induce local inflammation, promote the maturation of DCs at the skin and drainage lymph nodes, improve antigen specific body fluid and cell response, and have certain application potential in resisting influenza virus invasion; wilson et al nat. mater, 2019,18:175-]A sugar adjuvant p (Man-TLR7) with DCs targeting and activation inducing properties is synthesized and coupled with antigen protein related to plasmodium falciparum, so that the cytoplasmic release of antigen in DCs is increased, the antigen presentation of DCs to T cells is promoted, and the linkage of anti-malaria cells and humoral immunity is realized.
Regulation of the type and extent of involvement of MHC molecules is critical in determining antigen-induced dominant lymphocyte subpopulations and immune responses (cellular or humoral immunity). At present, the enhancement of the specific cellular immune response of the body to protein/polypeptide antigens is mainly achieved by enhancing the MHC class I presentation of such exogenous antigens by DCs, including the following means: 1) facilitating cytoplasmic release of antigen in DCs, e.g., modification of an antigen delivery vehicle (or direct modification of antigen) with pH-responsive materials, cell-penetrating peptides or membrane fusion peptides, facilitating cytoplasmic diffusion of exogenous antigen in DCs to obtain endogenous features for entry into the MHC class I presentation pathway [ Expert Opin Drug del, 2015.13(3):373 ]; 2) prolonging the intracellular stability of antigens in DCs, mainly by regulating the endosomal acidity or lysosomal acidity of DCs, limiting excessive degradation of antigens, maintaining the integrity of important epitopes required for MHC class I presentation and reducing lysosome-mediated MHC class II antigen presentation [ PNAS,2009,106(16):6730-6735 ]; 3) stimulating factors are added to induce maturation and differentiation of DCs, such as adjuvants or certain cytokines are introduced to improve the quantity and quality of DCs, thereby increasing the overall efficacy of adaptive immune responses [ ACS appl. Mater. interfaces, 2018,10(39):33532-33544 ]; 4) the introduction of appropriate targeting properties to antigens, and thus the improvement of antigen availability and p-MHC I presentation potential, mainly includes lymph node targeting of antigens [ Biomaterials,2016,98: 171-.
In DCs, ER is a key site for MHC molecule synthesis, further processing of antigenic peptides, and p-MHC I assembly, and targeting exogenous antigens to ER of DCs can utilize endoplasmic reticulum-associated degradation mechanisms to facilitate antigen splicing processing, p-MHC I assembly, and expression, thereby enhancing antigen-specific cellular immune responses. At present, the role of exogenous antigen delivery to the endoplasmic reticulum of DCs in enhancing the immune effect of protein/polypeptide subunit vaccines is gradually recognized, and related cellular and molecular mechanisms are continuously revealed. However, this strategy has very limited applications in anti-tumor, anti-viral and anti-infection due to the lack of effective targeting means and suitable antigen delivery vehicles. Specifically, the structure of the antigen is crucial to induce specific immune response, and the direct modification of the antigen by endoplasmic reticulum tropism molecules easily destroys the natural structure of the antigen, affecting the effectiveness and specificity of the immune response; meanwhile, the direct coupling puts high requirements on intracellular dissociation and release of the antigen; in addition, aiming at different antigens, the targeted modification modes and degrees are different, the preparation cost is high, the difficulty is high, and the amplification production is not facilitated. In contrast, the nano-carrier has the advantages of surface modification diversity, drug loading compatibility, drug release controllability and the like, and is more suitable for targeted delivery of protein/polypeptide antigens.
Based on the introduction, the invention constructs an endoplasmic reticulum targeted antigen delivery carrier, takes lipid with high biological safety, weak immunogenicity, easy obtainment and low production cost as a main carrier material, modifies a lipid material by micromolecules or polypeptides with endoplasmic reticulum tropism and exposes the lipid material on the surface of the carrier, and realizes the internal wrapping and/or surface adsorption of tumor, virus or plasmodium specific antigen protein/polypeptide by utilizing the cationic property and hydrophilic lipophilicity of the carrier. The carrier has excellent antigen loading and endoplasmic reticulum targeting capability, is convenient to prepare, low in cost and stable in property, can improve the cellular uptake of the antigen and change the intracellular transport and presentation fate of the antigen so as to regulate and control the cellular and humoral immune response, and has good production and application prospects.
Disclosure of Invention
One of the objectives of the present invention is to provide a method for constructing a nanocarrier capable of regulating adaptive cellular and humoral immunity, which is achieved as follows.
The oil-in-water (O/W) cationic nanoemulsion is prepared by taking phosphatidylcholine, vitamin E, endoplasmic reticulum targeted modified lipid and cationic lipid as oil phases and taking an aqueous solution of antigen protein/polypeptide as a water phase. Wherein the cationic lipid accounts for 1-60% of the total mass of the carrier structure lipid. The oil phase may also contain or be replaced by other oily components, such as soybean oil, olive oil, medium chain fatty acid ester, stearin, sucrose fatty acid ester, and squalene, in addition to vitamin E. The cationic lipid accounts for 1-60% of the total mass of the carrier structure lipid. The cationic lipid may be a positively charged lipid such as (2, 3-dioleoyl-propyl) trimethylammonium chloride (DOTAP), dimethyldioctadecylammonium (DDA), 1, 2-dimethyl-3-trimethylammonium-propane (DMTAP), 1, 2-stearoyl-3-trimethylammonium-propane (DSTAP) or 3 β - [ N- (N ', N' -dimethylaminoethyl) carbamoyl ] cholesterol (DC-Chol); it may also be an ionizable lipid, such as 4- (N, N-dimethylamino) butyric acid (dilinoleyl) methyl ester (DLin-MC3-DMA, pKa ═ 6.44).
The method comprises the following specific steps:
(1) construction and charging properties of the nanocarrier:
the nano-carrier of the invention has cationic property. The oil-in-water (O/W) cationic nanoemulsion is prepared by taking phosphatidylcholine, vitamin E, endoplasmic reticulum targeted modified lipid and cationic lipid as oil phases and taking an aqueous solution of antigen protein/polypeptide as a water phase.
The oil phase may also contain or be replaced by other oily components, such as soybean oil, olive oil, medium chain fatty acid ester, stearin, sucrose fatty acid ester, and squalene, in addition to vitamin E.
The cationic lipid accounts for 1-60% of the total mass of the carrier structure lipid. The cationic lipid may be a positively charged lipid such as (2, 3-dioleoyl-propyl) trimethylammonium chloride (DOTAP), dimethyldioctadecylammonium (DDA), 1, 2-dimethyl-3-trimethylammonium-propane (DMTAP), 1, 2-stearoyl-3-trimethylammonium-propane (DSTAP) or 3 β - [ N- (N ', N' -dimethylaminoethyl) carbamoyl ] cholesterol (DC-Chol); it may also be an ionizable lipid, such as 4- (N, N-dimethylamino) butyric acid (dilinoleyl) methyl ester (DLin-MC3-DMA, pKa ═ 6.44).
The nano-carrier is not limited to cationic nano-emulsion, can be expanded to cationic liposome, lipid nano-particles and the like, and can be prepared by a rotary evaporation-probe ultrasonic method and a micro-fluidic chip technology respectively.
(2) Endoplasmic reticulum targeted surface modification of the nano-carrier:
the nano-carrier provided by the invention has endoplasmic reticulum targeting capability. The selective accumulation of the nanoparticles in the endoplasmic reticulum of the subcellular structure is realized by modifying small molecules or polypeptides with endoplasmic reticulum tropism or positioning capacity on the surface of the nano carrier.
Endoplasmic reticulum-targeted small molecules or polypeptides include: sulfonamide or sulfonylurea synthetic small molecule compounds that interact with the endoplasmic reticulum receptor, or polypeptide molecules containing endoplasmic reticulum localization/retention sequences, such as Pardaxin polypeptides.
The endoplasmic reticulum targeted surface modification is realized in the assembly process of the nano carrier by doping the small molecules or the polypeptide chemically bonded with lipid in the preparation process of the nano carrier: the target head part with certain hydrophilicity is exposed on the surface of the carrier and is used for the endoplasmic reticulum selective guidance of the carrier; the lipophilic long-chain alkyl moiety is inserted into the core structure of the carrier and serves to stabilize the colloidal structure of the carrier. Wherein the endoplasmic reticulum targeted modification compound accounts for 1-30% of the total lipid mass.
The invention also aims to provide the application of the nano-carrier in preparing anti-tumor, anti-virus and anti-parasitic infection medicines.
The constructed nano-carrier is used for delivering tumor-associated antigens or specific antigens, such as melanoma-associated antigens, Wilms tumor protein and prostate cancer antigen, which are used as specific antigen protein/polypeptide, so as to prepare the antitumor drugs.
(1) Antigen loading of nanocarriers:
the nano-carrier constructed by the invention can be used for loading tumor, virus or plasmodium specific antigen protein/polypeptide, such as model antigen chicken Ovalbumin (OVA), melanoma related antigen (gp100 or TRP-2), Wilms tumor protein (WT1), prostate cancer antigen (PSA or PSCA), neocoronavirus SARS-CoV-2 spike protein (S protein) and plasmodium falciparum derived circumsporozoite protein (CSP).
The carrier carries the antigen, for example, the negative electric property of the antigen and the positive electric property of the cation carrier are utilized to carry out electrostatic interaction, so as to carry the antigen protein/polypeptide, and the antigen can be wrapped in the carrier and/or adsorbed on the surface of the carrier.
(2) The molecular basis for regulating the cellular and humoral immunity of the body is as follows:
the antigen loaded nano-carrier constructed by the invention has the capability of regulating and controlling the cell immunity and the humoral immunity of the antigen specificity of an organism. The nano-carrier delivers the antigen to the endoplasmic reticulum of dendritic cells in a targeted mode, degrades and assembles the antigen to a major histocompatibility complex class I molecule (MHC I) by means of an endoplasmic reticulum-related degradation mechanism, and then realizes antigenic peptide-MHC I division by utilizing an endoplasmic reticulum-related secretion pathwayCell surface presentation of seeds for increasing CD8+Cytotoxic T Cell (CTLs) -mediated cellular immunity; antigens that fail to enter the endoplasmic reticulum, on the other hand, are degraded in lysosomes and assemble into major histocompatibility complex class II molecules (MHC II), which are presented to the cell surface for activation of Th cells to amplify the humoral immune response.
The degree to which antigen-loaded nanocarriers modulate adaptive cellular and humoral immunity is largely determined by the efficiency of targeting the endoplasmic reticulum of the nanocarriers.
The nano-carrier provided by the invention has endoplasmic reticulum targeting capability. The selective accumulation of the nanoparticles in the endoplasmic reticulum of the subcellular structure is realized by modifying small molecules or polypeptides with endoplasmic reticulum tropism or positioning capacity on the surface of the nano carrier. Endoplasmic reticulum-targeted small molecules or polypeptides include: sulfonamide or sulfonylurea synthetic small molecule compounds that interact with the endoplasmic reticulum receptor, or natural polypeptide molecules containing endoplasmic reticulum localization/retention sequences, such as Pardaxin polypeptides. The endoplasmic reticulum targeted surface modification is realized in the assembly process of the nano carrier by doping the small molecules or the polypeptide chemically bonded with lipid in the preparation process of the nano carrier: the target head part with certain hydrophilicity is exposed on the surface of the carrier and is used for the endoplasmic reticulum selective guidance of the carrier; the lipophilic long-chain alkyl moiety is inserted into the core structure of the carrier and serves to stabilize the colloidal structure of the carrier. Wherein the endoplasmic reticulum targeted modification compound accounts for 1-30% of the total lipid mass.
The nano-carrier constructed by the invention can be used for loading tumor, virus or plasmodium specific antigen protein/polypeptide, including, for example, model antigen chicken Ovalbumin (OVA), melanoma associated antigen (gp100 or TRP-2), Wilms tumor protein (WT1), prostate cancer antigen (PSA or PSCA), neocoronavirus SARS-CoV-2 spike protein (S protein), plasmodium falciparum derived circumsporozoite protein (CSP) and the like. The carrier carries the antigen, for example, the weak electronegativity of the antigen is utilized to perform electrostatic interaction with the electropositivity of the cation carrier, so as to realize the load of the antigen protein/polypeptide, including the internal wrapping and/or adsorption of the antigen on the surface of the carrier.
The antigen loaded nano-carrier constructed by the invention has the capability of regulating and controlling the cell immunity and the humoral immunity of the antigen specificity of an organism, and has the following mechanism characteristics: the antigen protein/polypeptide is selectively targeted and delivered to the endoplasmic reticulum of dendritic cells through targeted modification and carried by nano-carriers, the antigen is degraded and assembled in major histocompatibility complex class I molecules (MHC I) by means of an endoplasmic reticulum-related degradation mechanism, and then the cell surface presentation of antigen peptide-MHC I molecules is realized by utilizing an endoplasmic reticulum-related secretion pathway, so that the cellular immunity mediated by CD8+ cytotoxic T Cells (CTLs) is improved; antigens that fail to enter the endoplasmic reticulum, on the other hand, are degraded in lysosomes and assemble into major histocompatibility complex class II molecules (MHC II), which are presented to the cell surface for activation of Th cells to amplify the humoral immune response. The degree to which antigen-loaded nanocarriers modulate adaptive cellular and humoral immunity is largely determined by the efficiency of targeting the endoplasmic reticulum of the nanocarriers.
The nano-carrier provided by the invention utilizes the advantage of MHC I class processing presentation of DCs endoplasmic reticulum on antigen, and changes the total intake amount, the transport path and the processing presentation mode of exogenous antigen in dendritic cells by modifying small molecules or polypeptide with endoplasmic reticulum tropism on the surface of the nano-particle loaded with antigen, thereby regulating and controlling the cellular immunity and humoral immunity of the antigen specificity of an organism and constructing the endoplasmic reticulum targeting nano-carrier loaded with protein/polypeptide antigen. The nano-carrier can be a cationic drug-carrying system such as nano-emulsion, liposome or lipid nanoparticle, and the antigen can relate to specific antigen protein/polypeptide of tumor, virus or plasmodium. The nano drug-carrying system can improve the adaptive immunity of organisms, particularly the participation degree of cellular immunity, so as to mobilize the specific immune response of resisting tumors, viruses or parasitic infection. The carrier of the invention is used for increasing the cellular uptake of the antigen and changing the intracellular transport and processing presentation of the antigen, thereby regulating and controlling the specific cellular immunity and the humoral immunity of the organism, and being used for resisting tumors, viruses and parasite infection. The construction method of the invention has novel design thought, reasonable and simple preparation method and wide application range.
Drawings
FIG. 1 shows the particle size, polydispersity index (PDI) and surface potential of the emulsion during screening of blank cationic nanoemulsion formulations (formulations 1-8 from left to right).
Fig. 2 is the morphology of the cationic nanoemulsion (formula 8 in table 1) under Transmission Electron Microscopy (TEM).
Figure 3 is a graph of particle size versus potential change for a cationic nanoemulsion (formula 8) stored at 4 degrees for two months.
FIG. 4 is a graph of the effect of different doses of cationic nanoemulsion (formula 8) on cell viability of different cell lines after 24h of treatment, as measured by the CCK-8 cell viability kit.
FIG. 5 shows the particle size and potential changes before and after loading of cationic nanoemulsion (formula 8) with antigen OVA, including fluorescein DiD-labeled nanoemulsion and FITC-labeled OVA (CNEs: cationic nanoemulsion).
FIG. 6 shows the ability of cationic emulsions to encapsulate OVA protein using 10% SDS-PAGE coupled rapid silver staining ( sample 1,2, 3 and 4, first and last columns are protein loading markers prepared by incubating 70. mu.L of aqueous OVA (5mg/mL, MW:. about.44 kDa) with 0, 100, 250 or 500. mu.L of cationic emulsion).
FIG. 7 is a graph of HEK293 as a model cell to examine the effect of different agents on protein antigen uptake by cells (scale: 100 μm) including Image processing software Image J on the pictures for semi-quantitative fluorescence analysis.
FIG. 8 is a co-localized picture (scale: 200 μm) of antigen (green), nucleus (blue) and emulsion (red) to show that antigen is indeed taken up into the cell by the cell.
FIG. 9 is the uptake of OVA-loaded cationic emulsions over 18h in DC2.4 and BMDCs.
FIG. 10 shows the localization of endoplasmic reticulum-targeted antigen-loaded cationic nanoemulsion in the intracellular subcellular structure of DCs (endoplasmic reticulum: red, liposome: pink, OVA: green, nucleus: blue) (target group: target; non-target group: non-target; control group: control).
FIG. 11 is an endoplasmic reticulum-targeted antigen delivery vector promoting MHC class I and MHC class II molecule expression of DCs.
FIG. 12 is induction of DCs stimulation of CD4 by endoplasmic reticulum-targeted antigen delivery vehicle+And CD8+Proliferation of T lymphocytes.
FIG. 13 is an endoplasmic reticulum-targeted antigen delivery vehicle that facilitates antigen-specific MHC class I presentation by DCs.
Figure 14 is an endoplasmic reticulum-targeted antigen delivery vehicle that promotes DCs induced proliferation of antigen-specific T cells.
Detailed Description
The invention is further illustrated by the accompanying drawings and examples.
Example 1 prescription screening, antigen Loading and cellular uptake of cationic nanoemulsion
(1) Prescription screening and physicochemical property characterization of blank nanoemulsion
Table one: prescription composition of nano-emulsion
Figure BDA0003029467050000071
Firstly, preparing blank nano-emulsion by an emulsification ultrasonic method, and screening the prescription. By changing the types of PC (PL100M and E-80), the ratio of the lipid components and the content of the aqueous phase, a formulation with higher stability, namely formulation 8(E80: VE: DOTAP ═ 1.45%: 1.45%: 0.323%, w/w), was screened out for subsequent studies (see FIG. 1). The nano-emulsion under the prescription has the particle size of about 200nm and the potential of about 60mV (detected by a dynamic light scattering method); typical emulsion morphology under Transmission Electron Microscopy (TEM) (see fig. 2); the stability is high, the storage is easy, and the particle size and the potential do not change obviously after the storage at 4 ℃ for two months (see figure 3); has better biological safety, and still has no significant cytotoxicity to most cells under the treatment of higher administration dose for 24 hours (see figure 4).
(2) Antigen loading of cationic nanoemulsions
The chicken Ovalbumin (OVA) is a common model antigen for researching immune response, and has the characteristics of low production cost, strong immunogenicity and the like. Due to species differences, strong immune responses can be triggered after the OVA is inoculated to individuals such as mice, monkeys and humans; and the composition structure of the OVA is completely analyzed, and related immunodetection means and detection products are rich. In view of the above, the present invention first uses OVA as a model protein antigen and loads the OVA with the above formula 8 cationic nanoemulsion. Through simple vortex with an aqueous solution of OVA protein and incubation for 30min at 37 ℃, the cationic emulsion can realize the loading of the OVA protein through electrostatic interaction and hydrophilic-hydrophobic interaction, the particle size is increased, the potential is reduced, DiD is doped into the oil phase of the emulsion to fluorescently label a carrier, or FITC-covalently coupled OVA (FITC-OVA) is used for labeling an antigen, so that the basic physicochemical property of the preparation is not obviously influenced, and the cationic emulsion can be used for subsequent in vitro research (see figure 5). The results of the 10% SDS-PAGE coupled rapid silver staining experiments show that the encapsulation efficiency of OVA protein by the cationic emulsion is gradually improved along with the increase of the mass ratio of the emulsion (formula 8) to the antigen, and the grey value analysis of OVA protein bands on the electrophoresis gel is carried out by the Image processing software Image J, and the encapsulation efficiency of the semi-quantitative OVA is found to be close to 67% when the mass ratio of the emulsion to the OVA is 47.6 (see FIG. 6).
(3) Cellular uptake of antigen-loaded cationic nanoemulsions
The effect of different preparations on protein antigen uptake by cells was examined by using HEK293 as a model cell, and it was found through the imaging result of a fluorescence inverted microscope that free OVA is rarely taken up within 18h, while the introduction of a cationic emulsion significantly improves the cell uptake of antigen without changing the time-dependent trend of the antigen uptake (see FIG. 7), and the co-localization picture shows that the antigen is actually taken up into cells by cells rather than being simply adsorbed on the cell surface (see FIG. 8). Then, the uptake conditions of the cationic emulsion carrying the OVA in immortalized mouse dendritic cells DC2.4 and immature mouse myeloid dendritic cells BMDCs (obtained by taking mouse bone marrow cells and inducing the cells for 5-6 days by GM-CSF and interleukin 4(IL-4) in vitro stimulation) are considered, and the finding shows that the OVA can be well absorbed into cells by the DCs, and the uptake of the OVA reaches a peak at about 8-18h (see figure 9), thereby showing that the uptake and utilization of antigen protein/polypeptide by APCs can be improved by introducing the cationic nanocarrier, and the cationic nanocarrier has the effect of simultaneously amplifying the antigen specific cellular immunity and the humoral immunity.
Example 2 preparation of cationic nanoemulsion loaded with model antigen OVA, surface-modified p-dodecylbenzenesulfonamide and characterization of endoplasmic reticulum targeting
(1) Endoplasmic reticulum targeted surface modification of antigen-loaded cationic nanoemulsion
Composition of endoplasmic reticulum targeted antigen-loaded cationic nanoemulsion
Egg yolk lecithin E809 mg
Vitamin E (VE)9mg
(2, 3-dioleoyl-propyl) trimethylammonium chloride (DOTAP) 1-28 mg
1 mg-8 mg of p-dodecyl benzene sulfonamide
0.6mL of an aqueous solution of OVA (5 mg/mL).
To better achieve antigen loading and targeted accumulation of endoplasmic reticulum, further adjustments to the prescription 8 in example 1 were performed: the aqueous solution of OVA is directly used as an aqueous phase, and p-dodecyl benzene sulfonamide with endoplasmic reticulum tropism is added into an oil phase of the cationic nanoemulsion. The endoplasmic reticulum targeted modification compound accounts for 1-30% of the total lipid mass, and the cationic lipid accounts for 1-60% of the total mass of the carrier structure lipid. Preparing an O/W type cationic emulsion by an emulsification ultrasonic method, wherein a long-chain alkyl part of dodecyl benzene sulfonamide is inserted into a lipid core of the emulsion, and a benzene sulfonamide part is exposed on the surface of the emulsion to play a role in targeted modification of endoplasmic reticulum of a carrier; the electropositive DOTAP and the electronegative OVA protein generate electrostatic interaction, and the internal entrapment and the external adsorption of the antigen are realized in the assembling process of the nano-emulsion; VE and E80 are used as the constituent lipid of the emulsion, respectively play the role of resisting lipid oxidation and surface active ingredients to stabilize the colloid structure of the nanoemulsion.
(2) Endoplasmic reticulum targeting antigen-loaded cationic nanoemulsion for positioning intracellular subcellular structures of DCs
Inoculating DC2.4 cells to a cell slide, after the cells are stabilized, giving a proper amount of endoplasmic reticulum targeting antigen-loaded cationic nanoemulsion to enable the final concentration of the antigen to be 2-5 mu g/mL, treating for 8-12h, and preparing the antigen-loaded cationic nanoemulsion without targeting molecules as a non-targeting control. Wherein, the antigen OVA is coupled with fluorescein FITC, and the lipid part of the emulsion is marked by DiD for visual observation and analysis. The cells treated by the preparation are washed by PBS to remove free preparation, the endoplasmic reticulum and the cell nucleus are respectively dyed by endoplasmic reticulum fluorescent dyes ER-tracker and Hochest 33342, then the free nuclear dye is removed by PBS washing, the cells are fixed by 4 percent paraformaldehyde solution, and the cell slide is mounted and observed under a laser confocal microscope. The results are shown in FIG. 10 (target group (target): dodecylbenzenesulfonamide targeted modification, accounting for 8% of the total mass of the carrier lipid; non-target group (non-target): no dodecylbenzenesulfonamide targeted modification, the remaining carrier components were the same as the target group; control group (control): free OVA alone was added). Confocal pictures show that (endoplasmic reticulum: red, liposome: pink, OVA: green, nucleus: blue), the coincidence of the endoplasmic reticulum fluorescence signal of the targeted group and the liposome fluorescence signal is better, and the endoplasmic reticulum outline is displayed, while the coincidence of the liposome and the endoplasmic reticulum of the non-targeted group is lower and the distribution is disordered, which shows that the targeted modification strategy can indeed promote the targeted accumulation of the vector and the antigen loaded by the vector in the endoplasmic reticulum.
Example 3 verification that endoplasmic reticulum-targeted antigen-loaded cationic nanocarriers improve cellular and humoral immunity
The antigen protein is directly dissolved in the water phase of the emulsion, which is favorable for realizing internal entrapment and external adsorption of the antigen in the assembly process of the carrier, and can further improve the drug-loading rate of the antigen and promote the cellular uptake of the antigen, thereby up-regulating the overall responsiveness of the cell immunity and the humoral immunity mediated by DCs. The endoplasmic reticulum targeting characteristic of the carrier is beneficial to the selective accumulation of the antigen in the endoplasmic reticulum of DCs, and the mechanism of endoplasmic reticulum related degradation and the like can be utilized to promote the MHC I presentation of the exogenous antigen, thereby improving the participation degree of cellular immunity.
To verify the superiority of this endoplasmic reticulum-targeted antigen delivery vehicle in the regulation of cellular and humoral immunity, C57BL/6 mouse-derived BMDCs were treated with nanocarriers (no fluorescein labeling) as in example 2, cells were harvested after 8-12h, washed 3 times with PBS, resuspended in cold PBS, and appropriate amount of fluorescein-labeled flow antibody was added, and after incubation at 37 ℃ or room temperature for 45min-90min, the expression of MHC I and MHC II molecules in CD11C + cell subpopulation (i.e., DCs cells) was analyzed by flow cytometry, orAnalysis of the proportion of 25-D1.16 positive cells in the CD11c + cell subset (antibody 25-D1.16 specifically recognises binding to H-2K)bOVA epitope of MHC I: OVA257-264And can be used for OVA-specific MHC class I antigen presentation). The results show that the targeting group significantly up-regulates the expression of MHC class I molecules and MHC class II molecules of DCs (see figure 11), which indicates that the strategy has a certain promotion effect on the overall strength of cellular immunity and humoral immunity; formulation-treated BMDCs were co-incubated with lymphocytes from syngeneic mice (C57BL/6) and then flow-tested for CD4+T cells and CD8+The number and the proportion of T cells are found, compared with a control group, the two types of cells in the targeted treatment group are increased (see figure 12), and the promotion effect of the nano drug delivery system on humoral and cellular immunity is verified; furthermore, antigen-specific MHC class I presentation of DCs under targeted group treatment was also significantly improved (see fig. 13), indicating that upregulation of such MHC molecules is accompanied by enhanced antigen presentation, and is an antigen-specific immune response, rather than being caused by simple cell-non-specific activation.
To further verify the antigen-specific CD8 of DCs under the nano-carrier treatment+Activation of T cells, formulation-treated BMDCs and OT-I mice (CD 8 thereof)+TCR specificity of T cells recognizes epitope OVA of OVA257-264) Co-incubation of lymphocytes (BMDCs: lymphocyte ═ 1: 5) followed by flow assay for CD3 in lymphocytes+CD8+Proportion of T lymphocytes. As a result, it was found (see FIG. 14) that CD8 was targeted to lymphocytes in the treated group+The highest proportion of T lymphocytes indirectly proves that the preparation induces the highest antigen-specific MHC class I presentation, and DCs still have good functions of activating T cells under the treatment of the preparation, thereby demonstrating the effectiveness and safety of the endoplasmic reticulum targeted drug-loaded nano system in promoting cellular immunity.
Example 4 preparation and application of cationic liposome loaded with melanoma-associated antigen gp100 and surface-modified ER targeting Pardaxin.
Composition of endoplasmic reticulum targeted antigen-loaded cationic liposome
(2, 3-dioleoyl-propyl) trimethylammonium chloride (DOTAP) 0.1-2.5 mg
Dioleoyl phosphatidylethanolamine (DOPE)1.5mg
N-distearoylphosphatidylethanol-PEG (DSPE-PEG)2000)0.1mg
DSPE-PEG2000-Pardaxin 0.1mg～0.7mg
2mL of gp100 in water (1 mg/mL).
Melanoma is a highly malignant tumor derived from melanocytes, lacks specific treatment and has a poor prognosis, and the incidence and mortality rate have been increasing year by year in recent years. gp100 is an early embryonic differentiation antigen, usually overexpressed in the development of melanoma, and thus a melanoma-associated antigen. Since gp100 is an autodifferentiation antigen, immunogenicity is weak, and it is often difficult to induce an effective anti-tumor effect when administered alone as a vaccine component. The research shows that the CD8 is improved+Antigen-specific immune responses of T cells to melanoma inhibit tumor development, metastasis and recurrence [ Cancer res, 2010,70(21)]Therefore, the improvement of the specific immune response of the tumor-associated antigen in the body of a melanoma patient, especially the cellular immunity, has a high significance for treating or preventing melanoma.
The invention prepares the endoplasmic reticulum targeted antigen-loaded cationic liposome by a rotary evaporation-probe ultrasonic method. Fully dissolving and dispersing the lipid related to the formula into a chloroform solution, and removing the organic solvent of the lipid solution by reduced pressure rotary evaporation in a water bath at 45 ℃ to obtain a uniform and complete lipid film. The lipid film is fully hydrated by aqueous solution of OVA at room temperature, and then the aqueous solution is placed in an ice bath probe for ultrasonic treatment to obtain the endoplasmic reticulum targeted modified antigen-loaded liposome with uniform particle size and stable property, wherein part of the antigen is wrapped in the liposome in the ultrasonic process, and part of the antigen is adsorbed on the surface of the liposome through electrostatic interaction. Wherein the endoplasmic reticulum targeted modification compound accounts for 1-30% of the total lipid mass, and the cationic lipid accounts for 1-60% of the total mass of the carrier structure lipid. The gp 100-loaded ER targeting cationic liposome has the capability of up-regulating specific cell and humoral immunity of an organism antigen, particularly cellular immunity, and can be used for enhancing the prevention and treatment effect of melanoma.
Example 5 preparation and application of cationic nanoemulsion loaded with melanoma-associated antigen (tyrosinase-associated protein 2, TRP-2) and surface-modified ER-targeting dodecyl-4-methylbenzenesulfonamide.
Composition of endoplasmic reticulum targeted antigen-loaded cationic nanoemulsion
Dipalmitoylphosphatidylcholine (DPPC)9mg
Soybean oil 9mg
Dimethyl Dioctadecyl Ammonium (DDA)1 mg-28 mg
Dodecyl-4-methylbenzenesulfonamide 1-8 mg
0.5mL of an aqueous solution (1mg/mL) of TRP-2.
Tyrosinase-related protease (transmembrane signal protein), which is involved in melanin biosynthesis, TRP-2 is a common melanoma-associated antigen, and both mouse and human melanoma-reactive CTLs recognize TRP-2 peptide (amino acid 180-188 of TRP2 protein, SVYDFFVWL) and thus exert antitumor effects. The invention prepares the endoplasmic reticulum targeting antigen-loaded cationic nanoemulsion by a high-pressure homogenization method. Weighing the formula amounts of DPPC, soybean oil, DDA and dodecyl-4-methylbenzenesulfonamide, dissolving the DPPC, the soybean oil, the DDA and the dodecyl-4-methylbenzenesulfonamide in a proper amount of ethanol according to a certain concentration to be used as an oil phase, dropwise injecting the ethanol solution into a water phase containing TRP-2 under a vortex condition by using an injector, forming primary emulsion by utilizing shearing force, and homogenizing by using a homogenizer to obtain the endoplasmic reticulum targeting TRP-2-loaded cationic nanoemulsion with uniform particle size and controllable quality. Wherein the endoplasmic reticulum targeted modification compound accounts for 1-30% of the total lipid mass, and the cationic lipid accounts for 1-60% of the total mass of the carrier structure lipid.
Example 6 preparation and application of cationic lipid nanoparticles loaded with SARS-CoV-2 spike protein (S protein) and surface modified ER targeting dodecyl-4-methylbenzenesulfonamide.
Composition of endoplasmic reticulum targeted antigen-loaded cationic lipid nanoparticles
0.1 mg-2.5 mg of 4- (N, N-dimethylamino) butyric acid (dilinoleyl) methyl ester (DLin-MC3-DMA)
Dioleoyl phosphatidylethanolamine (DOPE)1.5mg
N-distearoylphosphatidylethanol-PEG (DSPE-PEG)2000)0.1mg
Dodecyl-4-methylbenzenesulfonamide 0.08-0.7 mg
2mL of an aqueous solution of S protein (0.2 mg/mL).
The body antiviral immunity comprises innate immunity and adaptive immunity. As the first line of defense against viral infection, innate immunity is mediated primarily by interferons, macrophages and NK cells, which act rapidly but are nonspecific. The adaptive immunity is a firmer defense line for eliminating virus infection and preventing reinfection, has high efficiency and strong specificity and has memory. Thus, the primary goal of immunization with an antiviral vaccine is to mobilize the cellular and humoral immune response of the body. However, the research of people focuses mainly on the research of the action mechanism and effect of antiviral humoral immunity, and the design and application of related vaccines mainly surround the antibody-dependent humoral immunity, so that the research and the mobilization of cellular immunity are insufficient, and the full development of vaccines is hindered. T cell immunity plays an important role in vaccines against Human Immunodeficiency Virus (HIV), Hepatitis C Virus (HCV) or herpes virus [ Immunology,2012,135:19-26 ]. A novel coronavirus (SARS-CoV-2) outbreak at the end of 2019 poses serious threat to the global human health, and researches prove that the specific cellular Immunity and humoral Immunity of an organism to the S protein of the novel coronavirus can be up-regulated to inhibit the infection of the virus [ Immunity,2020,53: 724-1295 ], wherein CTL-mediated cellular Immunity probably plays an important role in inducing high-efficiency and long-lasting antiviral protection [ Immunity,2020,53(6):1281-1295 ].
The invention prepares endoplasmic reticulum targeting cationic lipid nanoparticles by a microfluidic chip technology, and realizes the internal encapsulation of the antigen carrier by the means. Namely: the one-step synthesis of the S protein cationic liposome is realized by utilizing a three-order flow focusing type micro-fluidic chip: mixing S protein water solution as a dispersion phase in the first step, DLin-MC3-DMA ethanol solution as a continuous phase, forming reverse micelles by utilizing the interaction of positive and negative charges of S protein and DLin-MC3-DMA, introducing the reverse micelles into the second step as the dispersion phase, introducing deionized water as the continuous phase to perform flow focusing of the second step, taking the formed liquid flow as the continuous phase of the third step, and introducing DLin-MC3-DMA, DOPE, DSPE-PEG2000And dodecyl-4-methylbenzenesulfonamide ethanolThe solution is used as a dispersed phase, and the cationic lipid nanoparticles carrying the S protein are obtained after full mixing. Wherein the endoplasmic reticulum targeted modification compound accounts for 1-30% of the total lipid mass, and the cationic lipid accounts for 1-60% of the total mass of the carrier structure lipid. The nano-carrier can improve the presentation efficiency and the presentation mode of the antigen through the self targeting advantage, thereby playing a role in resisting the new coronavirus infection.
Example 7 preparation and application of cationic nanoemulsion loaded with plasmodium falciparum-derived circumsporozoite protein (CSP) and surface-modified ER-targeting dodecyl-4-methylbenzenesulfonamide.
Composition of endoplasmic reticulum targeted antigen-loaded nanoemulsion
Egg yolk lecithin E809 mg
Vitamin E (VE)7mg
Medium chain fatty acid ester (MCT)2mg
1-28 mg of 3 beta- [ N- (N ', N' -dimethylaminoethyl) carbamoyl ] cholesterol (DC-Chol)
1 mg-8 mg of p-dodecyl benzene sulfonamide
0.5mL of an aqueous solution of CSP (1 mg/mL).
Vaccines effective against complex infections (malaria, HIV, tuberculosis, hepatitis C, etc.) must induce large antibody repertoires to clear blood-borne pathogens (humoral immunity) while simultaneously activating CD8+T cells respond to eliminate infected cells (cellular immunity). Clinical-stage subunit vaccines induce neutralizing antibody production, but induce CD8+T cells have a weak ability to respond, even with little [ Vaccine,2015,33:40-43 ]]This lack of cellular immune response is due to DCs to CD8+Insufficiency of T cells in MHC class I presentation [ nat. Mater.,2011,10:243-]. Research shows that promoting MHC class I presentation of plasmodium falciparum-derived circumsporozoite protein (CSP) on DCs can inhibit invasion and proliferation of plasmodium in human liver cells and play a good immune protection role [ nat. Mater.,2019,18:175-]。
The invention prepares the endoplasmic reticulum targeting antigen-loaded cationic nanoemulsion by an emulsification ultrasonic method. Dissolving lipid components in a formula in a small amount of ethanol to serve as an oil phase, taking a water solution of the CSP as a water phase, dropwise adding the water phase into the oil phase in a high-speed vortex state to obtain colostrum, then carrying out ultrasonic redispersion on the colostrum, removing an organic solvent, and obtaining the endoplasmic reticulum targeting CSP-loaded cationic nanoemulsion with uniform particle size and controllable quality. Wherein the endoplasmic reticulum targeted modification compound accounts for 1-30% of the total lipid mass, and the cationic lipid accounts for 1-60% of the total mass of the carrier structure lipid. The nano drug-carrying system can improve the adaptive immune response of organisms, particularly the specific cellular immunity of pathogens, and can be used as a vaccine for resisting the infection of the malignant malaria.
Example 8 preparation and application of cationic nanoemulsion loaded with Wilms tumor protein (WT1) and surface-modified ER targeting p-dodecylbenzenesulfonamide.
Composition of endoplasmic reticulum targeted antigen-loaded nanoemulsion
Soybean lecithin S1009 mg
Squalene 9mg
1 mg-28 mg of 1, 2-dimethyl-3-trimethylammonium-propane (DMTAP)
1 mg-8 mg of p-dodecyl benzene sulfonamide
0.5mL of an aqueous solution (1mg/mL) of WT 1.
Wilms tumor protein (WT1) is a transcription factor (the presence of nuclear localization sequences) that is involved in cell development and survival. Tissue distribution of WT1 after birth was limited to the urogenital, central nervous, and hematological systems, but did not elicit WT 1-specific CTLs challenge. WT1 was overexpressed in some tumors and was thought to be involved in tumor formation. Tumors overexpressing WT1 include: leukemia cells, kidney cancer, ovarian cancer, breast cancer, etc. Therefore, WT1 belongs to broad-spectrum tumor-associated antigen, and the preparation of WT 1-loaded endoplasmic reticulum targeting nanocarriers may play an important role in the prevention and treatment of these tumors.
The endoplasmic reticulum targeted antigen-loaded cationic nanoemulsion is prepared by an emulsification ultrasonic method. Dissolving lipid components in the formula in a small amount of ethanol to obtain an oil phase, taking an aqueous solution of WT1 as an aqueous phase, dropwise adding the aqueous phase into the oil phase under a high-speed vortex state to obtain colostrum, and then carrying out ultrasonic redispersion on the colostrum to obtain the ER targeted cationic nanoemulsion loaded with WT 1. Wherein the endoplasmic reticulum targeted modification compound accounts for 1-30% of the total lipid mass, and the cationic lipid accounts for 1-60% of the total mass of the carrier structure lipid.
Example 9 preparation and application of cationic nanoemulsion loaded with prostate cancer antigen (PSA) and surface-modified ER-targeting Pardaxin polypeptides.
Composition of endoplasmic reticulum targeted antigen-loaded nanoemulsion
Hydrogenated Soybean lecithin (HSPC)9mg
Cholesterol 9mg
1 mg-28 mg of 1, 2-stearoyl-3-trimethylammonium-propane (DSTAP)
DSPE-PEG2000-Pardaxin 1mg～8mg
0.5mL of an aqueous solution of PSA (1 mg/mL).
Prostate cancer refers to an epithelial malignancy that occurs in the prostate gland, has a high incidence in men, and has great damage to the urogenital system. Prostate specific antigen (serine protease PSA)/Prostate Stem Cell Antigen (PSCA), is expressed only in the cytoplasm of human prostate acinar and ductal epithelial cells, and is highly expressed in prostate cancer. PSA-specific CD8+The number of T ℃ cells correlated with cells expressing PSA, and studies showed intratumoral CD8 in prostate cancer+Infiltration of T cells affects the efficacy and prognosis of prostatectomy [ Prostate,2021,81(1): 20-28)]。
Preparing the cationic nanoemulsion loaded with PSA in an endoplasmic reticulum targeting manner by a high-pressure homogenization method. Weighing prescription amounts HSPC, cholesterol, DSTAP and DSPE-PEG2000Pardaxin, dissolving the Pardaxin in a proper amount of ethanol according to a certain concentration to serve as an oil phase, using an injector to inject the ethanol solution into a water phase containing PSA (pressure swing adsorption) dropwise under a vortex condition, using shearing force to form primary emulsion, and then using a homogenizer to homogenize to obtain the endoplasmic reticulum targeting PSA-loaded cationic nanoemulsion with uniform particle size and controllable quality. Wherein the endoplasmic reticulum targeted modification compound accounts for 1-30% of the total lipid mass, and the cationic lipid accounts for 1-60% of the total mass of the carrier structure lipid.

Claims (10)

1. The construction of the nano-carrier for regulating and controlling adaptive cell and humoral immunity is characterized by being realized by the following scheme:
(1) construction and charging properties of the nanocarrier:
preparing an oil-in-water type cationic nanoemulsion by using phosphatidylcholine, vitamin E, endoplasmic reticulum targeted modified lipid and cationic lipid as oil phases and using an aqueous solution of antigen protein/polypeptide as a water phase, wherein the cationic lipid accounts for 1-60% of the total mass of the carrier structure lipid;
(2) endoplasmic reticulum targeted surface modification of the nano-carrier:
and (2) incorporating a small molecule with an endoplasmic reticulum targeting effect or a polypeptide chemically bonded with lipid into the cationic nanoemulsion obtained in the step (1) to obtain a small molecule or polypeptide modified nano-carrier with the endoplasmic reticulum tropism or positioning capacity, wherein the compound with the endoplasmic reticulum targeting effect accounts for 1% -30% of the total lipid mass.
2. The construct of claim 1, wherein the vitamin E in the oil phase of step (1) can be replaced by soybean oil, olive oil, medium-chain fatty acid ester, glyceryl stearate, sucrose fatty acid ester, and squalene.
3. The construct of claim 1, wherein the cationic lipid in the oil phase of step (1) is a positively charged lipid selected from (2, 3-dioleoyl-propyl) trimethylammonium chloride, dimethyldioctadecylammonium, 1, 2-dimethyl-3-trimethylammonium-propane, 1, 2-stearoyl-3-trimethylammonium-propane (DSTAP) or 3 β - [ N- (N ', N' -dimethylaminoethyl) carbamoyl ] cholesterol (DC-Chol); the ionizable lipid is 4- (N, N-dimethylamino) butyric acid (dioleyl) methyl ester.
4. The construct of claim 1, wherein the nano-carrier of step (1) is not limited to cationic nanoemulsion and comprises cationic liposome and lipid nanoparticle.
5. The construct of claim 1, wherein the endoplasmic reticulum-targeted small molecule or polypeptide of step (2) comprises: sulfonamide or sulfonylurea synthetic small molecule compounds that interact with the endoplasmic reticulum receptor, or polypeptide molecules containing endoplasmic reticulum localization/retention sequences.
6. The construct of claim 5 wherein the polypeptide molecule comprising an endoplasmic reticulum localization/retention sequence is selected from the group consisting of Pardaxin polypeptides.
7. Use of the nanocarrier constructed according to claim 1 for the preparation of a medicament for the treatment of tumor, viral and parasitic infections, wherein the nanocarrier is used for carrying tumor-, viral-or plasmodium-specific antigenic proteins/polypeptides.
8. The use of claim 7, wherein the constructed nanocarrier is used for delivering tumor-associated antigens or specific antigens, such as melanoma-associated antigens, Wilms tumor protein, prostate cancer antigen as specific antigen protein/polypeptide, for preparing anti-tumor drugs.
9. The use as claimed in claim 7, wherein the constructed vector is used to deliver the spike protein of the new coronavirus SARS-CoV-2 as specific antigenic protein/polypeptide for the preparation of antiviral drugs.
10. The use according to claim 7, wherein the vector is constructed to deliver Plasmodium falciparum-derived circumsporozoite protein as a specific antigenic protein/polypeptide for the preparation of a medicament against parasitic infections.
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