Method for overcoming tumor immune desert-type drug resistance by nano-carrier delivery vaccine

Abstract

The invention discloses a method for overcoming tumor immune desert-type drug resistance by using a nano-carrier delivery vaccine. The method comprises the following steps: realizing targeted immune activation and microenvironment remodeling by constructing a three-stage structure collaborative delivery platform; the system comprises core-shell lipid nanoparticles (LNP) constructed based on a liposome self-assembly technology, mesoporous silica nanoparticles (MSNs) are adopted as a slow release module, and a manganese dioxide (MnO2) nano-cluster is anchored by a surface modified amino column [6] arene-lactose pyridine host-guest complex through a pi-pi accumulation effect; according to the present invention, through the dual targeting modification, the tumor cell membrane camouflage (retaining EGFR, CD44 and other membrane antigens) and the ApoA1 mimic peptide (22A peptide) synergistically act, the enrichment amount of the nanometer vaccine in the tumor tissue is 5.2 times of the normal liver tissue, and the problems that the traditional vaccine difficultly penetrates through the compact matrix of the immune desert type tumor and the lymph node activation efficiency is low are solved.

Description

Technical Field

[0001] The present invention relates to a method for overcoming tumor immune desert-type drug resistance by using a vaccine, and specifically relates to a method for overcoming tumor immune desert-type drug resistance by using a nanocarrier to deliver a vaccine. Background Art

[0002] (I) Bottlenecks and drug resistance challenges in tumor immunotherapy: Tumor immunotherapy activates the body's immune system to attack tumors, becoming the fourth most popular cancer treatment after surgery, chemotherapy, and radiotherapy. However, approximately 30%-50% of solid tumors exhibit an "immune desert" microenvironment, characterized by minimal immune cell infiltration within the tumor (especially CD8+ cytotoxic T lymphocytes, CTL <5%), dendritic cell (DC) maturation disorders (MHC-II class I molecule expression <30%), and abnormal enrichment of regulatory T cells (Tregs) / myeloid-derived suppressor cells (MDSCs). This results in a response rate of less than 20% to immune checkpoint inhibitors (ICBs) and a high risk of drug resistance. Traditional vaccine delivery systems are difficult to break through the "immune barrier" of this type of tumor due to poor targeting (tumor enrichment <2 times that of normal tissue), low adjuvant activation efficiency (DCs co-stimulatory molecule CD80 / CD86 positivity <40%), and the inability to reverse the immunosuppressive microenvironment, making it difficult to break through the "immune barrier" of this type of tumor, becoming a core problem in current clinical treatment.

[0003] (II) Research progress and limitations of nanocarrier technology: The development of nanomaterials has provided a new path for tumor immunotherapy. Carriers such as lipid nanoparticles (LNPs), mesoporous silica (MSNs), and metal oxides (such as MnO2) have shown potential in antigen delivery, adjuvant loading, and microenvironmental response.

[0004] LNP: LNPs based on high-density lipoprotein (HDL) biomimetic design can efficiently encapsulate hydrophobic adjuvants (e.g., R837 encapsulation efficiency >80%), but single LNPs lack lymph node targeting (lymph node enrichment is only 1.5 times that of the spleen) and cannot achieve sustained release of antigens;

[0005] MSNs: mesoporous silica with high specific surface area (>800m 2 / g) and controllable pore size (2-50nm) make it an ideal sustained-release platform. However, when used alone, it is easily cleared by the reticuloendothelial system (serum half-life <2 hours) and lacks tumor microenvironment responsiveness.

[0006] MnO2 nanoclusters: They can induce immunogenic cell death (ICD) by producing reactive oxygen species (ROS) through degradation, but traditional MnO2 carriers have poor targeting (tumor accumulation <10% of the injected dose) and cannot synergize with immune activation components.

[0007] Existing technologies mostly rely on a single carrier and have the defect of separating the functions of "targeted delivery-immune activation-microenvironment regulation". It is difficult to form a cascade immune response in immune desert-type tumors. There is an urgent need to develop a multimodal synergistic nanovaccine delivery system.

[0008] (3) Technical requirements and innovation directions of the present invention: In response to the above-mentioned deficiencies, the present invention proposes a multimodal nanocarrier system integrating lipid nanoparticles, mesoporous silica and manganese dioxide. Summary of the Invention

[0009] The technical problem to be solved by the present invention is to overcome the defects of the above-mentioned technology and provide a method for delivering vaccines with nanocarriers to overcome tumor immune desert-type drug resistance.

[0010] To solve the above technical problems, the present invention provides a technical solution for overcoming tumor immune desert-type drug resistance by delivering vaccines using nanocarriers:

[0011] include:

[0012] Targeted immune activation and microenvironment remodeling are achieved by constructing a tertiary structure coordinated delivery platform. The system includes:

[0013] Core-shell lipid nanoparticles (LNPs) constructed based on liposome self-assembly technology, whose core is encapsulated by a biodegradable phospholipid bilayer containing at least two lipid components selected from DSPC, DOPC, and DOTAP, and whose surface is modified with polyethylene glycol (PEG) derivatives to prolong circulation time;

[0014] Mesoporous silica nanoparticles (MSNs) are used as sustained-release modules. The MSNs are covalently linked to the LNP surface via a silanized coupling agent. The pore size of the MSNs is 2-50 nm and the specific surface area is ≥800 m 2 / g, the pores are loaded with the phosphorothioate-modified CpGODNs1826 sequence and tumor-associated antigen polypeptide, wherein the antigen polypeptide comprises an MHC-I class restricted epitope and an MHC-II class restricted epitope;

[0015] The surface-modified amino-pillar[6]arene-lactose pyridine host-guest complex anchors manganese dioxide (MnO2) nanoclusters via π-π stacking, wherein the nanoclusters have a particle size of 5-20 nm and are loaded with a hydrazone linker of an isoniazid prodrug and a dihydrochlorin e6 (Ce6) photosensitizer, and the complex dissociates below pH 6.5;

[0016] The outer surface of the system is further fused with ultrasound-fragmented tumor cell membrane vesicles, which retain the intact membrane protein domain and are modified with ApoA1 mimetic peptide (22A peptide) by click chemistry at a molar ratio of 1-5 22A peptide molecules per 100 phospholipid molecules.

[0017] As an improvement, the lipid nanoparticles (LNP) were prepared using microfluidic mixing technology. The specific process parameters were: aqueous phase flow rate 0.5-2 mL / min, organic phase flow rate 1-5 mL / min, mixing channel diameter 50-200 μm, and lipid concentration (5-20 mg / mL) and shear force (10 4 -10 6 s -1 ) achieves a particle size distribution of 80-100 nm, PDI ≤ 0.15, and R837 encapsulation efficiency ≥ 85%.

[0018] As an improvement, the functional modification of the mesoporous silica nanoparticles (MSNs) includes:

[0019] Surface amination: Silanization reaction was carried out by 3-aminopropyltriethoxysilane (APTES), with an amino group density of 0.5-2 mmol / g;

[0020] Targeting group grafting: modification of mannose receptor ligands or integrin targeting peptides by EDC / NHS chemical coupling;

[0021] Drug loading: CpGODNs and GEM-rAgs were loaded by vacuum impregnation method, with loading amounts of 10-50 μg / mg and 50-200 μg / mg, respectively. The release curves were consistent with the Higuchi kinetic model, and the cumulative release amount at pH 5.5 within 24 hours was ≥70%.

[0022] As an improvement, the preparation method of the manganese dioxide (MnO2) nanoclusters is as follows: in the presence of amino pillar [6] aromatic hydrocarbons, potassium permanganate and a reducing agent (ascorbic acid or citric acid) are used to generate in situ the nanoclusters, wherein the nanoclusters have a layered structure with an interlayer spacing of 0.7-0.9 nm. 3+ / Mn 4+ The ratio is 0.3-0.8, and the degradation rate is ≥90% within 6 hours in the presence of 10 mM glutathione. 3+ When the ion concentration reaches 10-50 μM, it can induce tumor cells to produce ≥50% carbon-centered free radicals.

[0023] As an improvement, the tumor cell membrane is an immune desert type tumor cell membrane selected from liver cancer, pancreatic cancer, and triple-negative breast cancer, and is prepared by the following steps:

[0024] Cell lysis: cells were disrupted using a hypotonic buffer (10 mM Tris-HCl, pH 7.4, containing 1 mM EDTA) combined with mechanical homogenization;

[0025] Gradient centrifugation: Separate cell membranes by centrifugation at 300 g (10 min), 2000 g (15 min), and 10,000 g (30 min).

[0026] Membrane protein enrichment: Membrane vesicles were collected by ultracentrifugation (100,000 g, 1 hour) with a protein content ≥5 mg / mL and retention of ≥80% of membrane surface antigens (such as PD-L1, EGFR, HER2).

[0027] As an improvement, the amino acid sequence of the ApoA1 mimetic peptide (22A peptide) is DWFKAFYDKVAEKLKEAF, which is modified on the carrier surface in the following manner:

[0028] The thiol-modified 22A peptide was coupled to the maleimide groups on the surface of PEGylated liposomes using maleimide-thiol click chemistry, with a coupling efficiency of ≥90%.

[0029] The modification density was 0.1–1 nmol 22A peptide per square centimeter of lipid membrane surface;

[0030] The modification increased the accumulation of the nano vaccine in the lymph nodes by 3-5 times compared with the unmodified group, and the DCs uptake rate was ≥40% within 24 hours after injection.

[0031] As an improvement, the mechanism by which the nanovaccine activates innate immunity through a dual adjuvant effect is:

[0032] R837 activates TLR7 / 8 through the endosomal pathway, induces the MyD88-dependent signaling pathway, phosphorylates IRF7 and translocates it to the nucleus, promoting IFN-α / β secretion, with the secretion amount ≥10 ng / mL;

[0033] CpGODNs activated TLR9, triggered the NF-κB signaling pathway, and upregulated the expression of cytokines such as IL-12p70 and TNF-α, with the expression levels increasing by ≥10 times compared with the control group;

[0034] The synergistic effect increased the expression of MHC-I molecules (HLA-A / B / C) on the surface of DCs by ≥2 times, the expression of MHC-II molecules (HLA-DR) by ≥3 times, and the positive rate of co-stimulatory molecules CD80 / CD86 by ≥70%.

[0035] As an improvement, the general structural formula of the isoniazid prodrug is R-CO-NH-NH-R, wherein R is an isonicotinyl group and R is a tumor microenvironment responsive group (such as pinacol terephthalate). Its mechanism of action is:

[0036] In the tumor microenvironment, expressed hydrogen peroxide (H2O2, ≥10 μM) triggers the cleavage of the boronate ester bond, releasing free isoniazid with a release rate of ≥80%;

[0037] Isoniazid inhibits IDO1 enzyme activity, reducing the concentration of tryptophan metabolite kynurenine by ≥50%, thereby reducing Tregs differentiation;

[0038] It works synergistically with the Ce6-mediated photodynamic effect to increase tumor vascular permeability by ≥3 times, promote CD8+ T cell infiltration, and increase the infiltration density by ≥2 times compared with the single-drug group.

[0039] As an improvement, the nanovaccine, when used in combination with immune checkpoint inhibitors (ICBs), overcomes drug resistance through the following synergistic mechanisms:

[0040] Nanovaccine pretreatment increased the CD8+ T cell / Tregs ratio in the tumor microenvironment from ≤0.5 to ≥2.0;

[0041] PD-1 / PD-L1 inhibitors block the binding of PD-1 to its ligand PD-L1, reducing the PD-1 positivity rate on the surface of T cells from ≥60% to ≤20%;

[0042] The expression of perforin in tumor tissues of the combined treatment group increased by ≥3 times compared with the single-drug group, and the proportion of granzyme B-positive cells increased by ≥2 times;

[0043] In PDX models, combination therapy reduced tumor volume by ≥70% and prolonged survival by ≥60 days without significant weight loss (≤10%).

[0044] As an improvement, the preparation method of the nano vaccine comprises the following steps:

[0045] (1) LNP preparation: DSPC, DOPC, cholesterol, and PEG-DMG were dissolved in ethanol at a molar ratio of 50:20:25:5 and mixed with an aqueous phase containing R837 using a microfluidic device. LNPs were formed under a pressure of 1000-5000 psi. After dialysis to remove the alcohol, the LNPs were modified with the 22A peptide by a thin film dispersion method.

[0046] (2) MSNs functionalization: through improved MSNs were synthesized by the method of removing the template with NH4NO3 / ethanol solution, loading CpGODNs and GEM-rAgs, and then amino-modified with APTES, and then coupled to the LNP surface via glutaraldehyde crosslinker;

[0047] (3) MnO2 modification: In the presence of LNP-MSNs complex, potassium permanganate and ascorbic acid were mixed in a molar ratio of 1:2 and reacted for 1-2 hours to form MnO2 nanoclusters, which were stabilized by amino-pillar[6]arene-lactose pyridine complex and loaded with isoniazid prodrug and Ce6;

[0048] (4) Cell membrane encapsulation: Tumor cell membrane vesicles were mixed with the above-mentioned complex at a protein:lipid ratio of 1:10 (w / w), treated with ultrasound (20 kHz, 100-200 W) for 30-60 seconds, and purified by density gradient centrifugation to obtain a biomimetic nanovaccine with an average particle size of 150-250 nm and a zeta potential of -15 to -25 mV.

[0049] The advantages of the present invention compared with the existing technology are: 1. Targeted delivery and precise enrichment, breaking through the immunosuppressive barrier: dual targeted modification: tumor cell membrane camouflage (retaining membrane antigens such as EGFR and CD44) and ApoA1 mimetic peptide (22A peptide) work synergistically to make the nanovaccine enriched in tumor tissue 5.2 times that of normal liver tissue, and the lymph node enrichment is 3.5 times that of the spleen, realizing the dual-path delivery of "tumor targeted colonization-lymph node activation", solving the problem that traditional vaccines are difficult to penetrate the dense matrix of immune desert-type tumors and the lymph node activation efficiency is low.

[0050] 2. Multiple adjuvants synergistically activate innate immunity and reshape antigen presentation function: Dual TLR pathway stimulation: Lipid core-loaded R837 (TLR7 / 8 agonist) and mesoporous silica-released CpGODNs (TLR9 ​​agonist) work synergistically to increase DCs secretion of IL-12 and TNF-α by 2.1 times and 2.3 times, respectively, activate the MyD88-dependent IRF7 / NF-κB signaling pathway, upregulate the expression of MHC-I / II class molecules and CD80 / CD86 co-stimulatory molecules (positive rate ≥72%), and increase the CD8+ T cell proliferation rate from 12% to 35%, completely breaking the functional inhibition of DCs in immune desert-type tumors.

[0051] 3. Microenvironment-responsive killing, reversing the immunosuppressive microenvironment: Mn 3+ Mediated ICD enhancement: MnO2 nanoclusters degrade in the low pH environment of tumors, releasing Mn 3+ Ions (25 μM) and Ce6 photosensitizers produce reactive oxygen species and carbon-centered free radicals through the Fenton reaction, inducing an early apoptosis rate of tumor cells of 32%, and increasing the release of DAMPs such as HMGB1 and ATP by 5 times and 3.8 times, respectively, thereby enhancing the cross-presentation of tumor antigens by DCs. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 This is a schematic diagram of the method of the present invention for delivering vaccines using nanocarriers to overcome tumor immune desert-type drug resistance. DETAILED DESCRIPTION

[0053] To facilitate understanding of the present application, the present application will be described more fully below with reference to the accompanying drawings. The accompanying drawings provide embodiments of the present application. However, the present application may be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to make the disclosure of the present application more thorough and comprehensive.

[0054] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application pertains. The terms used herein in the specification of this application are for the purpose of describing specific embodiments only and are not intended to limit this application.

[0055] It will be understood that spatial relational terms such as "under," "beneath," "beneath," "under," "above," "above," etc., may be used herein to describe the relationship of an element or feature shown in the figures to other elements or features. It will be understood that in addition to the orientations shown in the figures, spatial relational terms also include different orientations of the device in use and operation. For example, if the device in the drawings is turned over, the element or feature described as "under" or "beneath" or "beneath" the other elements will be oriented as "above" the other elements or features. Thus, the exemplary terms "under" and "under" may include both the above and below orientations. In addition, the device may also include alternative orientations, such as, rotated 90 degrees or other orientations, and the spatial descriptors used herein are to be interpreted accordingly.

[0056] It should be noted that when an element is considered to be "connected" to another element, it can be directly connected to the other element or connected to the other element through an intermediate element. In the following embodiments, "connection" should be understood as "electrical connection", "communication connection", etc., if the connected circuits, modules, units, etc. can transmit electrical signals or data to each other.

[0057] When used herein, the singular forms "a", "an", and "the" may also include the plural forms, unless the context clearly indicates otherwise. It should also be understood that the terms "include / comprise" or "have" and the like specify the presence of stated features, integers, steps, operations, components, parts, or combinations thereof, but do not preclude the possibility of the presence or addition of one or more other features, integers, steps, operations, components, parts, or combinations thereof.

[0058] In conjunction with the accompanying drawings, the method for nanocarrier-delivered vaccines to overcome tumor immune desert-type drug resistance includes:

[0059] Targeted immune activation and microenvironment remodeling are achieved by constructing a tertiary structure coordinated delivery platform. The system includes:

[0060] Core-shell lipid nanoparticles (LNPs) constructed based on liposome self-assembly technology, whose core is encapsulated by a biodegradable phospholipid bilayer containing at least two lipid components selected from DSPC, DOPC, and DOTAP, and whose surface is modified with polyethylene glycol (PEG) derivatives to prolong circulation time;

[0061] Mesoporous silica nanoparticles (MSNs) are used as sustained-release modules. The MSNs are covalently linked to the LNP surface via a silanized coupling agent. The pore size of the MSNs is 2-50 nm and the specific surface area is ≥800 m 2 / g, the pores are loaded with the phosphorothioate-modified CpGODNs1826 sequence and tumor-associated antigen polypeptide, wherein the antigen polypeptide comprises an MHC-I class restricted epitope and an MHC-II class restricted epitope;

[0062] The surface-modified amino-pillar[6]arene-lactose pyridine host-guest complex anchors manganese dioxide (MnO2) nanoclusters via π-π stacking, wherein the nanoclusters have a particle size of 5-20 nm and are loaded with a hydrazone linker of an isoniazid prodrug and a dihydrochlorin e6 (Ce6) photosensitizer, and the complex dissociates below pH 6.5;

[0063] The outer surface of the system is further fused with ultrasound-fragmented tumor cell membrane vesicles, which retain the intact membrane protein domain and are modified with ApoA1 mimetic peptide (22A peptide) by click chemistry at a molar ratio of 1-5 22A peptide molecules per 100 phospholipid molecules.

[0064] As an improvement, the lipid nanoparticles (LNP) were prepared using microfluidic mixing technology. The specific process parameters were: aqueous phase flow rate 0.5-2 mL / min, organic phase flow rate 1-5 mL / min, mixing channel diameter 50-200 μm, and lipid concentration (5-20 mg / mL) and shear force (10 4 -10 6 s -1 ) achieves a particle size distribution of 80-100 nm, PDI ≤ 0.15, and R837 encapsulation efficiency ≥ 85%.

[0065] As an improvement, the functional modification of the mesoporous silica nanoparticles (MSNs) includes:

[0066] Surface amination: Silanization reaction was carried out by 3-aminopropyltriethoxysilane (APTES), with an amino group density of 0.5-2 mmol / g;

[0067] Targeting group grafting: modification of mannose receptor ligands or integrin targeting peptides by EDC / NHS chemical coupling;

[0068] Drug loading: CpGODNs and GEM-rAgs were loaded by vacuum impregnation method, with loading amounts of 10-50 μg / mg and 50-200 μg / mg, respectively. The release curves were consistent with the Higuchi kinetic model, and the cumulative release amount at pH 5.5 within 24 hours was ≥70%.

[0069] As an improvement, the preparation method of the manganese dioxide (MnO2) nanoclusters is as follows: in the presence of amino pillar [6] aromatic hydrocarbons, potassium permanganate and a reducing agent (ascorbic acid or citric acid) are used to generate in situ the nanoclusters, wherein the nanoclusters have a layered structure with an interlayer spacing of 0.7-0.9 nm. 3+ / Mn 4+ The ratio is 0.3-0.8, and the degradation rate is ≥90% within 6 hours in the presence of 10 mM glutathione. 3+ When the ion concentration reaches 10-50 μM, it can induce tumor cells to produce ≥50% carbon-centered free radicals.

[0070] As an improvement, the tumor cell membrane is an immune desert type tumor cell membrane selected from liver cancer, pancreatic cancer, and triple-negative breast cancer, and is prepared by the following steps:

[0071] Cell lysis: cells were disrupted using a hypotonic buffer (10 mM Tris-HCl, pH 7.4, containing 1 mM EDTA) combined with mechanical homogenization;

[0072] Gradient centrifugation: Separate cell membranes by centrifugation at 300 g (10 min), 2000 g (15 min), and 10,000 g (30 min).

[0073] Membrane protein enrichment: Membrane vesicles were collected by ultracentrifugation (100,000 g, 1 hour) with a protein content ≥5 mg / mL and retention of ≥80% of membrane surface antigens (such as PD-L1, EGFR, HER2).

[0074] As an improvement, the amino acid sequence of the ApoA1 mimetic peptide (22A peptide) is DWFKAFYDKVAEKLKEAF, which is modified on the carrier surface in the following manner:

[0075] The thiol-modified 22A peptide was coupled to the maleimide groups on the surface of PEGylated liposomes using maleimide-thiol click chemistry, with a coupling efficiency of ≥90%.

[0076] The modification density was 0.1–1 nmol 22A peptide per square centimeter of lipid membrane surface;

[0077] The modification increased the accumulation of the nano vaccine in the lymph nodes by 3-5 times compared with the unmodified group, and the DCs uptake rate was ≥40% within 24 hours after injection.

[0078] As an improvement, the mechanism by which the nanovaccine activates innate immunity through a dual adjuvant effect is:

[0079] R837 activates TLR7 / 8 through the endosomal pathway, induces the MyD88-dependent signaling pathway, phosphorylates IRF7 and translocates it to the nucleus, promoting IFN-α / β secretion, with the secretion amount ≥10 ng / mL;

[0080] CpGODNs activated TLR9, triggered the NF-κB signaling pathway, and upregulated the expression of cytokines such as IL-12p70 and TNF-α, with the expression levels increasing by ≥10 times compared with the control group;

[0081] The synergistic effect increased the expression of MHC-I molecules (HLA-A / B / C) on the surface of DCs by ≥2 times, the expression of MHC-II molecules (HLA-DR) by ≥3 times, and the positive rate of co-stimulatory molecules CD80 / CD86 by ≥70%.

[0082] As an improvement, the general structural formula of the isoniazid prodrug is R-CO-NH-NH-R, wherein R is an isonicotinyl group and R is a tumor microenvironment responsive group (such as pinacol terephthalate). Its mechanism of action is:

[0083] In the tumor microenvironment, expressed hydrogen peroxide (H2O2, ≥10 μM) triggers the cleavage of the boronate ester bond, releasing free isoniazid with a release rate of ≥80%;

[0084] Isoniazid inhibits IDO1 enzyme activity, reducing the concentration of tryptophan metabolite kynurenine by ≥50%, thereby reducing Tregs differentiation;

[0085] It works synergistically with the Ce6-mediated photodynamic effect to increase tumor vascular permeability by ≥3 times, promote CD8+ T cell infiltration, and increase the infiltration density by ≥2 times compared with the single-drug group.

[0086] As an improvement, the nanovaccine, when used in combination with immune checkpoint inhibitors (ICBs), overcomes drug resistance through the following synergistic mechanisms:

[0087] Nanovaccine pretreatment increased the CD8+ T cell / Tregs ratio in the tumor microenvironment from ≤0.5 to ≥2.0;

[0088] PD-1 / PD-L1 inhibitors block the binding of PD-1 to its ligand PD-L1, reducing the PD-1 positivity rate on the surface of T cells from ≥60% to ≤20%;

[0089] The expression of perforin in tumor tissues of the combined treatment group increased by ≥3 times compared with the single-drug group, and the proportion of granzyme B-positive cells increased by ≥2 times;

[0090] In PDX models, combination therapy reduced tumor volume by ≥70% and prolonged survival by ≥60 days without significant weight loss (≤10%).

[0091] As an improvement, the preparation method of the nano vaccine comprises the following steps:

[0092] (1) LNP preparation: DSPC, DOPC, cholesterol, and PEG-DMG were dissolved in ethanol at a molar ratio of 50:20:25:5 and mixed with an aqueous phase containing R837 using a microfluidic device. LNPs were formed under a pressure of 1000-5000 psi. After dialysis to remove the alcohol, the LNPs were modified with the 22A peptide by a thin film dispersion method.

[0093] (2) MSNs functionalization: through improved MSNs were synthesized by the method of removing the template with NH4NO3 / ethanol solution, loading CpGODNs and GEM-rAgs, and then amino-modified with APTES, and then coupled to the LNP surface via glutaraldehyde crosslinker;

[0094] (3) MnO2 modification: In the presence of LNP-MSNs complex, potassium permanganate and ascorbic acid were mixed in a molar ratio of 1:2 and reacted for 1-2 hours to form MnO2 nanoclusters, which were stabilized by amino-pillar[6]arene-lactose pyridine complex and loaded with isoniazid prodrug and Ce6;

[0095] (4) Cell membrane encapsulation: Tumor cell membrane vesicles were mixed with the above-mentioned complex at a protein:lipid ratio of 1:10 (w / w), treated with ultrasound (20 kHz, 100-200 W) for 30-60 seconds, and purified by density gradient centrifugation to obtain a biomimetic nanovaccine with an average particle size of 150-250 nm and a zeta potential of -15 to -25 mV.

[0096] Example 1: Preparation of multimodal nanovaccine (MPL@R837 / LNP-MSNs-MnO2):

[0097] Lipid-based core module (LNP-R837-22A peptide) preparation:

[0098] Material:

[0099] Lipid components: distearoylphosphatidylcholine (DSPC, 50 μmol), dioleoylphosphatidylethanolamine (DOPE, 20 μmol), cholesterol (25 μmol), PEG-distearoylglycerol (PEG-DMG, 5 μmol);

[0100] Hydrophobic adjuvant: Imiquimod (R837, 10 mg);

[0101] Targeting peptide: ApoA1 mimetic peptide (22A peptide, sequence DWFKAFYDKVAEKLKEAF, purity ≥95%).

[0102] Preparation method:

[0103] (1) Lipids and R837 were dissolved in anhydrous ethanol (1 mL) to form an organic phase; the aqueous phase was a buffer solution (9 mL) containing 10 mM Tris-HCl (pH 7.4), preheated to 50°C;

[0104] (2) Through a microfluidic chip (channel inner diameter 100 μm), the organic phase: aqueous phase is mixed at a flow rate of 1:9 and rapidly emulsified under a pressure of 1500 psi to form colostrum;

[0105] (3) Colostrum was dialyzed through a 300 kDa ultrafiltration membrane (Millipore) for 4 h to remove ethanol and obtain core LNPs;

[0106] (4) 22A peptide (final concentration 10 μM) was added to the LNP suspension, followed by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, 5 mM) and N-hydroxysuccinimide (NHS, 10 mM). The mixture was reacted at room temperature for 2 h to couple the 22A peptide to the LNP surface via an amide bond.

[0107] Parameter control:

[0108] Particle size: 92 ± 5 nm as measured by dynamic light scattering (DLS), polydispersity index (PDI) 0.12;

[0109] Encapsulation efficiency: The R837 encapsulation efficiency was 88.6% as determined by high performance liquid chromatography (HPLC);

[0110] Surface potential: Zeta potential -18±2mV;

[0111] Functionalization of mesoporous silica sustained-release module (MSNs-CpG-GEM-rAgs).

[0112] Material:

[0113] Mesoporous silica nanoparticles (MSNs, pore size 15 nm, specific surface area 900 m 2 / g, pore volume 1.2cm 3 / g);

[0114] Immune agonist: phosphorothioate-modified CpGODNs1826 sequence (5'-TCCATGACGTTCCTGATGCT-3');

[0115] Tumor antigen: recombinant antigen GEM-rAgs (containing MHC-I epitope SI INFEKL and MHC-II epitope ISQAVHAAHAEINEAGR).

[0116] Preparation method:

[0117] (1) Surface amination of MSNs: MSNs (100 mg) were dispersed in toluene (50 mL), 3-aminopropyltriethoxysilane (APTES, 2 mL) was added, and the mixture was refluxed at 60 °C for 4 h. The mixture was washed by centrifugation and freeze-dried. The amino group density was 1.2 mmol / g.

[0118] (2) Drug loading: MSNs were mixed with CpGODNs (50 μg / mg) and GEM-rAgs (150 μg / mg) in PBS (pH 7.4) using the vacuum impregnation method, vacuum dried for 24 h, and then lyophilized and dispersed in MES buffer (pH 5.5) containing EDC / NHS;

[0119] (3) Coupling to the LNP surface: The functionalized MSNs were mixed with the LNPs obtained in step 1 at a mass ratio of 1:2, and ultrasonicated (20 kHz, 30 min) for covalent coupling via amide bonds.

[0120] Characterization data:

[0121] Loading rate: UV spectrophotometry determined the loading capacity of CpGODNs to be 48 μg / mg, and that of GEM-rAgs to be 145 μg / mg;

[0122] Sustained-release properties: Under pH 5.5 conditions, the cumulative release of CpGODNs was 75% and the antigen peptide was 68% within 24 hours, which was consistent with the Higuchi sustained-release model.

[0123] Microenvironment response module (MnO2-isoniazid-Ce6) modification.

[0124] Material:

[0125] Host-guest complex: aminopillar[6]arene (1 mM), lactose pyridine (2 mM);

[0126] Manganese dioxide precursors: potassium permanganate (KMnO4, 5 mM), ascorbic acid (10 mM);

[0127] Loaded drugs: isoniazid prodrug (hydrazone bond linker), dihydrochlorin e6 (Ce6, 10μM).

[0128] Preparation method:

[0129] (1) Synthesis of MnO2 nanoclusters: KMnO4 aqueous solution and ascorbic acid aqueous solution were mixed in equal volumes at room temperature, and amino pillar[6]arene-lactose pyridine complex was immediately added. The mixture was stirred for 1 hour to generate MnO2 nanoclusters with a particle size of 12±3 nm.

[0130] (2) Drug loading: Isoniazid prodrug and Ce6 were dissolved in N,N-dimethylformamide (DMF), and MnO2 suspension was added under ultrasonic treatment. The mixture was incubated at 60°C for 2 h, washed by centrifugation, and then freeze-dried. The loading amounts were 50 μg / mg (isoniazid) and 20 μg / mg (Ce6), respectively.

[0131] (3) Anchoring to the carrier surface: MnO2 clusters were connected to the surface of the LNP-MSNs complex through host-guest interaction (π-π stacking), with 50 μg MnO2 bound per mg of carrier.

[0132] Response characteristics:

[0133] pH response: Under pH 6.0, the degradation rate of MnO2 was 60% in 2 hours, and the release of Mn 3+ Ion concentration 25 μM;

[0134] Photosensitizer release: Under 660nm laser irradiation, the release rate of Ce6 is 40%, and the efficiency of generating reactive oxygen species (ROS) is 2 times higher than that of free Ce6.

[0135] Tumor cell membrane camouflage modification:

[0136] Material:

[0137] Hepatocellular carcinoma cells (HepG2, HB-8065 TM );

[0138] Hypotonic buffer: 10 mM Tris-HCl (pH 7.4) containing 1 mM EDTA.

[0139] Preparation method:

[0140] (1) Cell membrane extraction: HepG2 cells were trypsinized and centrifuged (1000 g, 5 min). The cells were lysed in a hypotonic buffer for 30 min and then centrifuged at 300 g (10 min), 2000 g (15 min), and 100,000 g (1 h) to collect cell membrane vesicles. The protein concentration was 5 mg / mL.

[0141] (2) Membrane fusion modification: The cell membrane vesicles were mixed with the complex obtained in step 3 at a protein: lipid ratio of 1:10 (w / w), and ultrasonic treatment (100W, 40 seconds) was used to promote membrane fusion. The biomimetic nanovaccine (MPL@R837 / LNP-MSNs-MnO2) was purified by 20% sucrose density gradient centrifugation.

[0142] Targeting properties:

[0143] Membrane protein retention rate: BCA assay showed a membrane protein retention rate of 85%, and Western blot showed intact expression of membrane antigens such as EGFR and CD44;

[0144] Cell uptake efficiency: Flow cytometry determined that the vaccine uptake rate of HepG2 cells (45%) was 3 times higher than that of the non-membrane modified group (15%).

[0145] 6. Data Verification

[0146] Experiment 1: Characterization of the physicochemical properties of nanovaccines:

[0147] Particle size and distribution: DLS determined the average particle size of the final vaccine to be 180 ± 10 nm, PDI 0.14, and the core LNP particle size (92 nm) was consistent with the 80-100 nm range described in claim 2. The size after assembly met the requirements for targeted delivery;

[0148] Zeta potential: -22 ± 3 mV, balancing serum stability (avoiding rapid clearance) and cellular uptake efficiency (negative charge promotes endocytosis);

[0149] Encapsulation and loading efficiency: HPLC verified that the R837 encapsulation efficiency was 88.6%, and UV spectrophotometry determined that the CpGODNs loading capacity was 48 μg / mg, GEM-rAgs was 145 μg / mg, and the MnO2 binding capacity was 48 μg / mg, all within the range defined in claims 3-4.

[0150] Experiment 2: Targeted delivery and lymph node enrichment ability:

[0151] In vitro cellular uptake:

[0152] DC2.4 dendritic cells were co-incubated with the vaccine for 4 hours. Flow cytometry showed an uptake rate of 45%, significantly higher than the groups without membrane modification (18%) and without 22A peptide modification (25%), demonstrating that dual targeting modifications synergistically enhance APC recognition.

[0153] Fluorescence imaging showed that ApoA1 mimetic peptide modification increased the fluorescence intensity of popliteal lymph nodes by 230% compared with the control group, confirming the lymph node tropism.

[0154] Distribution in the body:

[0155] The DiR-labeled vaccine (5 mg / kg) was injected into the tail vein of HepG2 tumor-bearing mice. 24 hours later, the fluorescence intensity of the tumor tissue was 5.2 times that of the normal liver tissue, and the lymph node enrichment was 3.5 times that of the spleen, verifying the dual-targeting characteristics of tumors and lymph nodes.

[0156] Experiment 3: Immune activation and DCs maturation effects:

[0157] Adjuvant synergy:

[0158] ELISA tests showed that the vaccine stimulated DC2.4 cells to secrete IL-12 (250 pg / mL) and TNF-α (180 pg / mL), which were 2.1 times and 2.3 times that of the R837 group alone and 1.7 times and 1.8 times that of the CpG group alone, respectively, confirming the dual activation of TLR7 / 8 and TLR9 pathways;

[0159] Western blot showed that the expression levels of p-IRF7 (TLR7 / 8 pathway) and p-NF-κB (TLR9 ​​pathway) in DCs increased by 3 times and 4 times respectively compared with the control group.

[0160] DCs mature phenotype:

[0161] Flow cytometry showed that the positive rate of MHC-I class molecules (H-2Kb) increased from 30% to 75%, the positive rate of MHC-I class I molecules (I-Ab) increased from 25% to 68%, and the double positive rate of costimulatory molecules CD80 / CD86 reached 72%.

[0162] In the mixed lymphocyte reaction, the proliferation rate of CD8+ T cells (35%) increased by 2.9 times compared with the control group (12%), and the secretion of IFN-γ increased by 4.2 times, indicating that CTL activation was enhanced.

[0163] Experiment 4: Tumor microenvironment remodeling and ICD induction:

[0164] Immunogenic Cell Death (ICD) Validation:

[0165] Annexin V / PI staining showed that the early apoptosis rate of HepG2 cells after vaccine treatment was 32%, significantly higher than that of the control group (8%) and the single-drug group (R837 or CpG treatment groups were both <15%).

[0166] Western blot detected that the release of HMGB1 in the cell culture supernatant increased by 5 times, and ELISA assayed that ATP secretion increased by 3.8 times, confirming the release of ICD characteristic molecules.

[0167] Regulation of the immunosuppressive microenvironment:

[0168] Flow cytometry analysis of tumor-infiltrating immune cells showed that the proportion of MDSCs decreased from 22% to 8%, the proportion of Tregs decreased from 15% to 6%, and the IDO1 enzyme activity inhibition rate reached 65%. Isoniazid prodrug effectively reversed metabolic inhibition;

[0169] Photodynamic effect: 660nm laser irradiation (200mW / cm 2 , 5 minutes), Ce6-mediated ROS caused the apoptosis rate of tumor vascular endothelial cells to reach 40%, the CD31+ blood vessel density to decrease by 60%, and the CD8+ T cell infiltration density to increase by 2.5 times.

[0170] Experiment 5: In vivo antitumor efficacy in combination with ICB:

[0171] Drug-resistant tumor models:

[0172] Establishment of MC38 drug-resistant mouse model (5 consecutive passages, high expression of PD-L1, CD8+

[0173] T cell infiltration <5%), 1×10 6 Cells / cells.

[0174] Treatment options:

[0175] Control group: PBS (100 μL, iv, twice a week);

[0176] Single-drug group: MPL@R837 / LNP-MSNs-MnO2 (5 mg / kg, iv, twice a week);

[0177] Combined group: vaccine + anti-PD-L1 antibody (10 mg / kg, ip, once a week).

[0178] Efficacy evaluation:

[0179] Tumor growth: Tumor volume on day 21 in the combined group (210 ± 30 mm 3 ) was significantly smaller than that of the single-drug group (450±50mm 3 , p<0.01) and control group (820±80mm 3 , p<0.001), the inhibition rate reached 74%;

[0180] Survival: The median survival time was 65 days in the combination group, 45 days in the monotherapy group, and 30 days in the control group (Log-rank test p < 0.01);

[0181] Immune infiltration: Immunohistochemistry showed that the density of CD8+ T cells in the tumor of the combined group (25 cells / high-power field) was 5 times higher than that of the control group (5 cells / high-power field), the CD8+ T cell / Tregs ratio increased from 0.3 to 2.2, the proportion of granzyme B-positive cells increased 3 times, and the proportion of PD-1-positive T cells decreased from 65% to 22%, verifying the synergistic effect with ICB therapy.

[0182] Experiment 6: Safety evaluation:

[0183] Hematological indicators:

[0184] Routine blood test showed that the white blood cell count (6.2±0.8×10 9 / L), red blood cells (7.5±0.5×10 12 / L), platelets (250±30×10 9 / L) were within the normal range of C57BL / 6 mice;

[0185] Liver and kidney function: ALT (25±3U / L), AST (30±4U / L), BUN (8±1mM), Cr (50±5μM) were not significantly different from those in the control group (p>0.05).

[0186] Histopathology:

[0187] HE staining of heart, liver, spleen, lung, and kidney sections showed no inflammatory cell infiltration, cell necrosis, or tissue structure destruction, demonstrating that the nanovaccine had good biocompatibility and met the safety requirements of claim 10.

[0188] Example 2: Adaptation of pancreatic cancer drug resistance model;

[0189] Adjustment parameters:

[0190] Tumor cell membrane: replaced with Panc-1 pancreatic cancer cell membrane, membrane extraction steps are the same as in Example 1;

[0191] MnO2 loading: increased to 60 μg / mg, enhances ROS-mediated activation of matrix metalloproteinases (MMPs), degrading the abundant collagen fiber matrix of pancreatic cancer;

[0192] Effect verification:

[0193] The CTL infiltration rate in the tumor increased from 10% to 25%, and the tumor growth inhibition rate increased by 15% compared with the unadjusted group, confirming the adaptability of the model (protection scope of claim 1).

[0194] Example 3: Preparation of lyophilized formulation (formulation optimization):

[0195] Preparation method:

[0196] 5% sucrose and 1% trehalose were added to the vaccine suspension as protective agents, and the suspension was pre-frozen (-80°C, 2 hours) and then freeze-dried (-50°C, 10 mTorr, 24 hours);

[0197] Quality parameters:

[0198] After reconstitution, the particle size change was less than 10% (DLS determination: 185±12 nm), the R837 encapsulation efficiency was retained at 92%, and the shelf life was extended to 12 months (stored at 4°C), meeting the scalability of the preparation process described in claim 10.

[0199] VII. Conclusion

[0200] The above examples fully demonstrate the technical advantages of the multimodal nanocarrier vaccine system described in the claims in overcoming tumor immune desert-type drug resistance through precise process parameter control, multi-dimensional characterization methods, and rigorous in vitro and in vivo experiments. Experimental data show that this system significantly improves the treatment efficacy of drug-resistant tumors through targeted delivery, dual adjuvant activation, microenvironment-responsive killing, and ICB synergy, while also demonstrating good biosafety, providing a solid experimental foundation for subsequent clinical translation.

[0201] Advantages: Targeted delivery and precise enrichment, breaking through the immune suppression barrier:

[0202] Dual targeting modification: Tumor cell membrane camouflage (retaining membrane antigens such as EGFR and CD44) and ApoA1 mimetic peptide (22A peptide) work synergistically to make the nanovaccine enriched in tumor tissue 5.2 times that of normal liver tissue, and the lymph node enrichment 3.5 times that of the spleen, realizing the dual-path delivery of "tumor targeted colonization-lymph node activation", solving the problem that traditional vaccines are difficult to penetrate the dense matrix of immune desert-type tumors and the low efficiency of lymph node activation.

[0203] Optimization of particle size and surface potential: The core LNP particle size is controlled at 80-100nm, the average particle size after assembly is 180nm, and the Zeta potential is -22mV, which balances serum stability (circulation time extended by 40%) and cellular uptake efficiency (DCs uptake rate reaches 45%), avoiding rapid clearance by the reticuloendothelial system.

[0204] Multiple adjuvants synergistically activate innate immunity and reshape antigen presentation function:

[0205] Dual TLR pathway stimulation: The lipid core-loaded R837 (TLR7 / 8 agonist) and the mesoporous silica-released CpGODNs (TLR9 ​​agonist) act synergistically to increase the secretion of IL-12 and TNF-α by DCs by 2.1 times and 2.3 times, respectively, activate the MyD88-dependent IRF7 / NF-κB signaling pathway, upregulate the expression of MHC-I / II class molecules and CD80 / CD86 co-stimulatory molecules (positive rate ≥72%), and increase the CD8+ T cell proliferation rate from 12% to 35%, completely breaking the functional inhibition of DCs in immune desert-type tumors.

[0206] Antigen gradient release: The controlled sustained-release properties of mesoporous silica (≥68% antigen / adjuvant release in 24 hours at pH 5.5) ensure that the antigen presentation process lasts for more than 72 hours. Compared with the short-acting stimulation of free antigens (<12 hours), it significantly enhances the T cell memory effect and achieves long-term immune surveillance.

[0207] Microenvironment-responsive killing, reversing the immunosuppressive microenvironment: Mn 3+ Mediated ICD enhancement: MnO2 nanoclusters degrade in the low pH environment of tumors, releasing Mn 3+ Ions (25 μM) and Ce6 photosensitizers produce reactive oxygen species and carbon-centered free radicals through the Fenton reaction, inducing an early apoptosis rate of tumor cells of 32%, and increasing the release of DAMPs such as HMGB1 and ATP by 5 times and 3.8 times, respectively, thereby enhancing the cross-presentation of tumor antigens by DCs.

[0208] Dual regulation of metabolism and blood vessels: Isoniazid prodrug inhibits IDO1 enzyme activity (inhibition rate 65%) and reduces Tregs differentiation (the proportion drops from 15% to 6%); Ce6-mediated photodynamic effect destroys tumor blood vessels (CD31+ blood vessel density decreases by 60%), increases the CD8+ T cell infiltration density by 2.5 times, and completely reverses the immunosuppressive microenvironment enriched with myeloid-derived suppressor cells (MDSCs).

[0209] Synergistically enhances efficacy with ICB therapy to overcome drug resistance: Increased density of infiltrating T cells: Monotherapy increased the CD8+ T cell / Tregs ratio in the tumor from 0.3 to 1.5, and further increased to 2.2 after combination with anti-PD-L1 antibodies. The proportion of PD-1-positive T cells decreased from 65% to 22%, restoring the T cell killing function.

[0210] Doubled anti-tumor efficacy: In the MC38 drug-resistant model, the tumor volume inhibition rate of the combination treatment group reached 74%, and the median survival time was extended to 65 days (30 days in the control group), which was significantly better than the single-drug group (45 days), confirming a breakthrough in the treatment of tumors resistant to immune checkpoint inhibitors.

[0211] Advantages of biosafety and process scalability: Low immunogenicity and biocompatibility: Using degradable lipids such as DSPC and DOPE, mesoporous silica and natural peptides (22A peptide), hematological indicators (leukocytes, liver and kidney function) were no different from those of the control group, no damage was found in pathological sections of major organs, and body weight fluctuation after intravenous injection was <10% (Experiment 6).

[0212] Platform preparation and indication expansion: By replacing tumor cell membranes (such as liver cancer, pancreatic cancer, and triple-negative breast cancer) and adjusting the MnO2 loading (50-60μg / mg), it can quickly adapt to different immune desert-type tumors; freeze-drying preparation technology extends the vaccine's effectiveness to 12 months, and the particle size change after reconstitution is <10%, meeting clinical storage and transportation needs.

[0213] Following the trend of interdisciplinary studies, we lead technological innovation: Multi-carrier collaborative delivery platform: integrating the efficient encapsulation of liposomes (R837 encapsulation rate of 88.6%), the controlled sustained release of mesoporous silica (Higuchi model fitting), and the microenvironmental response of manganese dioxide (pH 6.0 degradation rate of 60%), to solve the industry problem of the single function of traditional single carriers and form a universal technology platform that can flexibly load multiple antigens / drugs.

[0214] Multi-mechanism linkage treatment model: Constructing a cascade reaction system of "targeted delivery-immune activation-microenvironment remodeling-combined synergy" to provide a systematic solution for the treatment of drug-resistant tumors, and promote the upgrade of tumor immunotherapy from "single drug and single mechanism" to "multimodal synergy" model, with significant scientific foresight and clinical translation value.

[0215] In summary, the present invention has successfully broken through the core bottleneck of tumor immune desert-type drug resistance through the deep intersection of nanomaterials and immunology, and has demonstrated unparalleled advantages over traditional therapies in terms of targeting, immune activation efficiency, microenvironment regulation and combined treatment compatibility, providing a new path for the treatment of refractory tumors such as liver cancer and pancreatic cancer.

[0216] The present invention and its embodiments are described above. This description is not restrictive. The drawings show only one embodiment of the present invention, and the actual structure is not limited thereto. In short, if a person skilled in the art is inspired by this and, without departing from the purpose of the present invention, designs structures and embodiments similar to this technical solution without inventiveness, they shall fall within the scope of protection of the present invention.

Claims

1. A method for overcoming tumor immune desert-type drug resistance by delivering vaccines using nanocarriers, characterized by: include: Targeted immune activation and microenvironment remodeling are achieved by constructing a tertiary structure coordinated delivery platform. The system includes: Core-shell lipid nanoparticles (LNPs) constructed based on liposome self-assembly technology, whose core is encapsulated by a biodegradable phospholipid bilayer containing at least two lipid components selected from DSPC, DOPC, and DOTAP, and whose surface is modified with polyethylene glycol (PEG) derivatives to prolong circulation time; Mesoporous silica nanoparticles (MSNs) are used as sustained-release modules. The MSNs are covalently linked to the LNP surface via a silanized coupling agent. The pore size of the MSNs is 2-50 nm and the specific surface area is ≥800 m 2 / g, the pores are loaded with the phosphorothioate-modified CpGODNs 1826 sequence and tumor-associated antigen polypeptides, wherein the antigen polypeptides comprise MHC class I restricted epitopes and MHC class II restricted epitopes; The surface-modified amino-pillar[6]arene-lactose pyridine host-guest complex anchors manganese dioxide (MnO2) nanoclusters via π-π stacking, wherein the nanoclusters have a particle size of 5-20 nm and are loaded with a hydrazone linker of an isoniazid prodrug and a dihydrochlorin e6 (Ce6) photosensitizer, and the complex dissociates below pH 6.

5. The outer surface of the system is further fused with ultrasound-fragmented tumor cell membrane vesicles, which retain the intact membrane protein domain and are modified with ApoA1 mimetic peptide (22A peptide) by click chemistry at a molar ratio of 1-5 22A peptide molecules per 100 phospholipid molecules.

2. The method for overcoming tumor immune desert-type drug resistance by delivering vaccines with nanocarriers according to claim 1, characterized in that: The lipid nanoparticles (LNP) were prepared by microfluidic mixing technology. The specific process parameters were: aqueous phase flow rate 0.5-2 mL / min, organic phase flow rate 1-5 mL / min, mixing channel diameter 50-200 μm, and lipid concentration (5-20 mg / mL) and shear force (10 4 -10 6 s -1 ) achieves a particle size distribution of 80-100 nm, PDI ≤ 0.15, and R837 encapsulation efficiency ≥ 85%.

3. The method for overcoming tumor immune desert-type drug resistance by delivering vaccines with nanocarriers according to claim 2, characterized in that: The functional modification of the mesoporous silica nanoparticles (MSNs) includes: Surface amination: Silanization reaction was carried out by 3-aminopropyltriethoxysilane (APTES), with an amino group density of 0.5-2 mmol / g; Targeting group grafting: modification of mannose receptor ligands or integrin targeting peptides by EDC / NHS chemical coupling; Drug loading: CpG ODNs and GEM-rAgs were loaded by vacuum impregnation method, with loading amounts of 10-50 μg / mg and 50-200 μg / mg, respectively. The release curves conformed to the Higuchi kinetic model, and the cumulative release amount at pH 5.5 within 24 hours was ≥70%.

4. The method for overcoming tumor immune desert-type drug resistance by delivering vaccines with nanocarriers according to claim 3, characterized in that: The preparation method of the manganese dioxide (MnO2) nanoclusters is as follows: in the presence of amino pillar [6] aromatic hydrocarbons, potassium permanganate and a reducing agent (ascorbic acid or citric acid) are used to generate the nanoclusters in situ, wherein the nanoclusters have a layered structure with an interlayer spacing of 0.7-0.9 nm. 3+ / Mn 4+ The ratio is 0.3-0.8, and the degradation rate is ≥90% within 6 hours in the presence of 10 mM glutathione. 3+ When the ion concentration reaches 10-50 μM, it can induce tumor cells to produce ≥50% carbon-centered free radicals.

5. The method for overcoming tumor immune desert-type drug resistance by delivering vaccines with nanocarriers according to claim 4, characterized in that: The tumor cell membrane is an immune desert type tumor cell membrane selected from liver cancer, pancreatic cancer, and triple-negative breast cancer, and is prepared by the following steps: Cell lysis: cells were disrupted using a hypotonic buffer (10 mM Tris-HCl, pH 7.4, containing 1 mM EDTA) combined with mechanical homogenization; Gradient centrifugation: Separate cell membranes by centrifugation at 300 g (10 min), 2000 g (15 min), and 10,000 g (30 min). Membrane protein enrichment: Membrane vesicles were collected by ultracentrifugation (100,000 g, 1 hour) with a protein content ≥5 mg / mL and retention of ≥80% of membrane surface antigens (such as PD-L1, EGFR, HER2).

6. The method for overcoming tumor immune desert-type drug resistance by delivering vaccines with nanocarriers according to claim 5, characterized in that: The amino acid sequence of the ApoA1 mimetic peptide (22A peptide) is DWFKAFYDKVAEKLKEAF, and it is modified on the carrier surface in the following manner: The thiol-modified 22A peptide was coupled to the maleimide groups on the surface of PEGylated liposomes using maleimide-thiol click chemistry, with a coupling efficiency of ≥90%. The modification density was 0.1–1 nmol 22A peptide per square centimeter of lipid membrane surface; The modification increased the accumulation of the nano vaccine in the lymph nodes by 3-5 times compared with the unmodified group, and the DCs uptake rate was ≥40% within 24 hours after injection.

7. The method for overcoming tumor immune desert-type drug resistance by delivering vaccines with nanocarriers according to claim 6, characterized in that: The mechanism by which the nanovaccine activates innate immunity through the dual adjuvant effect is: R837 activates TLR7 / 8 through the endosomal pathway, induces the MyD88-dependent signaling pathway, phosphorylates IRF7 and translocates it to the nucleus, promoting IFN-α / β secretion, with the secretion amount ≥10 ng / mL; CpG ODNs activated TLR9, triggered the NF-κB signaling pathway, and upregulated the expression of cytokines such as IL-12p70 and TNF-α, with the expression levels increasing by ≥10 times compared with the control group; The synergistic effect increased the expression of MHC-I molecules (HLA-A / B / C) on the surface of DCs by ≥2 times, the expression of MHC-II molecules (HLA-DR) by ≥3 times, and the positive rate of co-stimulatory molecules CD80 / CD86 by ≥70%.

8. The method for overcoming tumor immune desert-type drug resistance by delivering vaccines with nanocarriers according to claim 7, characterized in that: The general structural formula of the isoniazid prodrug is R-CO-NH-NH-R, wherein R is an isonicotinyl group and R is a tumor microenvironment responsive group (such as pinacol terephthalate). Its mechanism of action is: In the tumor microenvironment, expressed hydrogen peroxide (H2O2, ≥10 μM) triggers the cleavage of the boronate ester bond, releasing free isoniazid with a release rate of ≥80%; Isoniazid inhibits IDO1 enzyme activity, reducing the concentration of tryptophan metabolite kynurenine by ≥50%, thereby reducing Tregs differentiation; It works synergistically with the Ce6-mediated photodynamic effect to increase tumor vascular permeability by ≥3 times, promote CD8+ T cell infiltration, and increase the infiltration density by ≥2 times compared with the single-drug group.

9. The method for overcoming tumor immune desert-type drug resistance by delivering vaccines with nanocarriers according to claim 8, characterized in that: When used in combination with immune checkpoint inhibitors (ICBs), the nanovaccine overcomes drug resistance through the following synergistic mechanisms: Nanovaccine pretreatment increased the CD8+ T cell / Tregs ratio in the tumor microenvironment from ≤0.5 to ≥2.0; PD-1 / PD-L1 inhibitors block the binding of PD-1 to its ligand PD-L1, reducing the PD-1 positivity rate on the surface of T cells from ≥60% to ≤20%; The expression of perforin in tumor tissues of the combined treatment group increased by ≥3 times compared with the single-drug group, and the proportion of granzyme B-positive cells increased by ≥2 times; In PDX models, combination therapy reduced tumor volume by ≥70% and prolonged survival by ≥60 days without significant weight loss (≤10%).

10. The method for overcoming tumor immune desert-type drug resistance by delivering vaccines with nanocarriers according to claim 9, characterized in that: The preparation method of the nano vaccine comprises the following steps: (1) LNP preparation: DSPC, DOPC, cholesterol, and PEG-DMG were dissolved in ethanol at a molar ratio of 50:20:25:5 and mixed with an aqueous phase containing R837 using a microfluidic device. LNPs were formed under a pressure of 1000-5000 psi. After dialysis to remove the alcohol, the LNPs were modified with the 22A peptide by a thin film dispersion method. (2) MSNs functionalization: through improved MSNs were synthesized by the method of removing the template with NH4NO3 / ethanol solution, loading CpG ODNs and GEM-rAgs, amino-modifying with APTES, and then coupling to the LNP surface with glutaraldehyde cross-linker; (3) MnO2 modification: In the presence of LNP-MSNs complex, potassium permanganate and ascorbic acid were mixed in a molar ratio of 1:2 and reacted for 1-2 hours to form MnO2 nanoclusters, which were stabilized by amino-pillar[6]arene-lactose pyridine complex and loaded with isoniazid prodrug and Ce6; (4) Cell membrane encapsulation: Tumor cell membrane vesicles were mixed with the above-mentioned complex at a protein:lipid ratio of 1:10 (w / w), treated with ultrasound (20 kHz, 100-200 W) for 30-60 seconds, and purified by density gradient centrifugation to obtain a biomimetic nanovaccine with an average particle size of 150-250 nm and a zeta potential of -15 to -25 mV.

Images